Aus der Abteilung Stereotaktische Neurochirurgie der Neurochirurgischen Universitätsklinik der Albert-Ludwigs-Universität Freiburg i. Br.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation
Present Clinical Protocol and Recent Advances in the use of Adrenal Chromaffin Cells as an Experimental Alternative Tissue Source
INAUGURAL-DISSERTATION
Zur Erlangung des Medizinischen Doktorgrades
der Medizinischen Fakultät
der Albert-Ludwigs-Universität Freiburg i. Br.
Vorgelegt 20.08.2013
von William Omar Contreras Lopez geboren in Bucaramanga/Colombia
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Dekan: Prof. Dr. Dr. h.c. mult. Hubert E. Blum
1. Gutachter: Prof. Dr. Guido Nikkhah 2. Gutachter: Prof. Dr. Andreas Schulze-Bonhage
Jahr der Promotion: 2013
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Erklärung nach § 2 Abs. 2 Nr. 5 und 6
Ich erkläre, dass ich die der Medizinischen Fakultät der Albert - Ludwigs- Universität Freiburg i.Br. zur Promotion eingereichte Dissertation mit dem Titel
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation
Present Clinical Protocol and Recent Advances in the use of Adrenal Chromaffin Cells as an Experimental Alternative Tissue Source
in der Abteilung Stereotaktische Neurochirurgie der Neurochirurgischen Klinik des Universitätsklinikums Freiburg unter der Betreuung von Prof. Dr. Guido Nikkhah ohne sonstige Hilfe durchgeführt und bei der Abfassung der Dissertation keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.
Ich habe diese Dissertation bisher an keiner in - oder ausländischen Hochschule zur Promotion eingereicht. Weiterhin versichere ich, daß ich den beantragten Titel bisher noch nicht erworben habe.
Freiburg, den 20.08.2013
William Omar Contreras Lopez
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Dedicated to Colombia
Life is the window of time; the universe gives us, to try to beat mortality by means of the contributions we left to society once we are gone
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TABLE OF CONTENTS
SUMMARY……………………………………………………………………………………… 9 ZUSAMMENFASSUNG………………………………………………………………………. 11 ABBREVIATIONS…………………………………………………………………………….. 13 LIST OF FIGURES……………………………………………………………………………. 15 LIST OF TABLES …………………………………………………………………………….. 18 1. INTRODUCTION……………………………………………………………………... 19
1.1 Basal ganglia circuitry and function………………………………………….. 19 1.1.1 Nomenclature…………………………………………………………………... 19 1.1.2 Functional organization of the basal ganglia………………………………….21 1.1.3 Cytoarchitecture……………………………………………………………….. 22 1.1.4 Summary……………………………………………………………………….. 30 1.2 Parkinson’s disease (PD)………………………………………………………. 32 1.2.1 Epidemiology of Parkinson’s disease…………………………………………. 32 1.2.2 Etiology and pathogenesis of Parkinson’s disease…………………………… 34 1.2.3 Therapies for Parkinson´s disease…………………………………………….. 40 1.2.4 Cell replacement therapy and its clinical effects…………………………….. 44 1.2.5 The 6•OHDA lesion model…………………………………………………….. 45 1.3 Huntington’s disease (HD)…………………………………………………….. 47 1.3.1 Epidemiology of Huntington’s disease………………………………………… 47
1.3.2 Etiology and pathogenesis of Huntington’s disease………………………….. 47
1.3.3 Therapies for Huntington´s disease………………………………….…………48
1.3.4 Cell replacement therapy and its clinical effects……………………………...49
1.4 Cell Transplantation in the central nervous system…………………………. 51 1.4.1 History………………………………………………………………………….. 51
1.4.2 Lessons learned for successful Grafting …………………………………….. 52
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1.4.3 Sources of CNS for transplantation……………………………………………53
1.4.4 Techniques of transplantation………………………………………………….54
1.4.5 Immunosuppresion and Immune response…………………………………....54
1.4.6 Mechanisms of Graft function………………………………………………….55
1.4.7 Theoretical therapeutic applications…………………………………………………..55
1.4.8 Restoration of the CNS using progenitor cells from adrenal medulla……… 61
2. OBJECTIVES………………………………………………………………………...... 67
3. MATERIALS……………………………………………………………………………68
4. METHODS…………………………………………………………………………...... 78
4.1 Project I “Human Clinical Neurotransplantation Protocol for Huntington’s and Parkinson’s disease” 4.2 Project II “Stereotactic Planning Software for Human Neurotransplantation: Suitability in 22 surgical cases of Huntington’s disease” 4.3 Project III “Experimental Intrastriatal Transplantation of Neural Crest Derived Chromaffin Progenitors cells in Parkinson’s disease” 5. RESULTS………………………………………………………………………………118 5.1 Project I……………………………………………………………………….. 118 5.2 Project II………………………………………………………………………. 140 5.3 Project III………………………………………………………………………148 6. DISCUSSION…………………………………………………………………………. 158
REFERENCES………………………………………………………………………………...161 PUBLICATIONS………………………………………………………………………………194 ACKNOWLEDGEMENTS…………………………………………………………………...195 CURRICULUM VITAE………………………………………………………………………196
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SUMMARY
Since 1987 an estimated 450 patients with Parkinson’s (PD) and Huntington’s (HD) diseases have been transplanted with human fetal mesencephalic tissue, rich in post mitotic primary dopamine (DA) neurons, or with ganglionic eminences harboring precursors of striatal GABA-ergic medium spiny interneurons respectively. Clinical outcome has been variable, ranging from case reports of PD patients dropping out medication following transplantation up to negative results in two prospective randomized double-blinded trials. Analyzing the results across studies, it becomes apparent that procedures used in different centers vary greatly. However few efforts have been made to standardize the procedure, probably due also to the wider range of different results and the lack of expertise, limitation of tissue, and ethical and legal concerns, creating a vicious cycle between lack of standardized protocols and conclusive results. This thesis was elaborated having in mind such concerns; the thesis is composed of three projects. The work described in the first project of this thesis was aimed at reviewing the clinical neurotransplantation protocol for PD and HD. Two detailed step-wise neurotransplantation protocols are presented, outlining strategies facilitating the avoidance of possible procedure-related complications. Some crucial technical factors enabling the execution of a safe and effective neural transplantation trial were delineated. Special emphasis in understanding the anatomical relationships of the human fetal tissue that are relevant for selection of the desired cells population was also addressed. The protocols reviewed here might contribute to further development of the experimental clinical neurotransplantation towards a routine therapeutic procedure. The second project presents self-developed software optimizing the surgical stereotactic planning for bilateral neurotransplantation procedures. It allows close to symmetrical distribution of the stereotactic coordinates in relation to the mid-commissural point (MCP), proposing automatically the planning coordinates for the first hemisphere to be transplanted and mirrored coordinates for the contra- lateral hemispheres. An analysis of its applicability in twenty-two consecutive human HD patients who underwent bilateral stereotactic striatal transplantation was performed. Intra-individual comparison between software given coordinates and final used coordinates was performed as well as a safety analyses. Project number three examined, via in vitro and in vivo studies, the ability of neural crest-derived sympathoadrenal (SA) progenitor cells to reinnervate the striatum, after a stepwise isolation, propagation and transplantation protocol. The cells were enriched and propagated in chromosphere cultures prior to transplantation. Mastering the isolation of these chromaffin progenitor cells and controlling their differentiation might open new avenues for regenerative therapies.
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ZUSAMMENFASSUNG
Seit 1987 wurden etwa 450 Patienten mit Parkinson- bzw. Huntington-Erkrankung mit fötalem menschlichem mesencephalem Gewebe, oder mit Vorläufern der GABA-ergen striatalen Interneuronen der „ganglionic eminence“ transplantiert. Bei genauerer Analyse der Ergebnisse zeigte sich, dass die angewandten Verfahren in den verschiedenen Zentren sehr stark variierten. Bislang wurden nur wenige Anstrengungen unternommen, um diese Verfahren zu standardisieren, vermutlich wegen den großen Unterschieden an Erfahrung, den ethischen Aspekten der Benutzung des nur begrenzt zur Verfügung stehenden fötalen menschlichen Gewebes und den landesspezifischen Rechtsgrundlagen. Unter Berücksichtigung dieser Voraussetzungen wurden in der hier vorliegenden Arbeit drei Themenschwerpunkte behandelt: In dem ersten Projekt wurden die klinischen Neurotransplantations- Protokolle für Patienten mit Parkinson’scher und Huntington’sche -Erkrankung genauer analysiert. Es wurden zwei ausführliche und schrittweise gegliederte Neurotransplantations-Protokolle vorgestellt, welche Strategien aufzeigen, um mögliche verfahrensbedingte Probleme und Komplikationen zu vermeiden. Die hier analysierten Protokolle können dazu beitragen, die sich derzeit noch im experimentellen Bereich befindliche Neurotransplantation in Richtung klinischer Routine weiter zu entwickeln. Das zweite Projekt stellt eine selbst entwickelte Software-Optimierung für die stereotaktisch- chirurgischen Planung der bilateralen Neurotransplantation dar. Es ermöglicht eine optimale annähernd symmetrische Verteilung der stereotaktischen Transplantationskoordinaten in Bezug auf den Mittelpunkt der AC-PC-Linie (MCP), welcher eine halbautomatische Planung der Neurotransplantation für beide Hemisphären mit Hilfe eines Expertensystems erlaubt. Die Analyse der Anwendbarkeit dieses Verfahrens wurde bei 22 aufeinander folgenden Patienten mit Huntington‘scher Erkrankung, bei denen bilaterale stereotaktische Transplantationen fötaler Stammzellen in das Striatum durchgeführt wurden. Hierbei wurden für jede Seite jeweils zwei Trajektorien in den Nucleus caudatus und vier in das Putamen geplant. Der individuelle Vergleich zwischen den von der Software vorgegebenen Koordinaten und den letztendlich tatsächlich verwendeten Koordinaten wurde genauer analysiert. Im Rahmen der Sicherheitsanalyse wurde ein Vergleich der berechneten räumlichen Abweichungen der Koordinaten aller Patienten mit dem Patientenkollektiv durchgeführt. In dem dritten Projekt die Fähigkeit der von der Neuralleiste abgeleiteten sympatho-adrenergen (SA) Vorläuferzellen, unter Anwendung eines Protokolls mit schrittweiser Isolierung, Vermehrung und Transplantation untersucht, das Striatum zu reinnervieren. Die Zellen wurden angereichert und in Chromosphären-Kulturen vor der Transplantation vermehrt. Die Fähigkeit zur Isolierung dieser chromaffinen Vorläuferzellen und die Steuerung ihrer Differenzierung eröffnen neue Wege für die regenerative Medizin.
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ABREVIATIONS
6-OHDA 6-hydroxydopamine AA Ascorbic acid Amphetamine AMPH Acb Nucleus accumbens bFGF Basic fibroblast growth factor BMP Bone morphogenetic proteins BG Basal ganglia CNS Central nervous system CRL Crown–rump length CS Clusters-chromospheres COMT Inhibitor of cathecol-O-methyltransferase DA Dopamine DAT Dopamine transporter DBS Deep brain stimulation DDC Dopa decarboxylase DI Diabetes insipidus DN Dopaminergic neuron Drd2 D2 dopamine receptor EGF Epidermal growth factor ES cell Embryonic stem cell FBS Fetal bovine serum FCS Fetal calcium serum FNC Fetal neural cells GDNF Glial-derived neurotrophic factor GFAP Glial fibrillary acidic protein GFP Green fluorescent protein LCM Laser Capture Microscopy LGE Lateral ganglionic eminence MGE Medial ganglionic eminence
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Nacl Sodium chloride NGF Nerve growth factor NSC Neural stem cell Nurr 1 Nuclear receptor-related factor 1 PFA Paraformaldehyde PD Parkinson’s disease PNS Peripheral nervous system SA Sympathoadrenal SD Sprague-Dawley SEZ Subependymal zone Shh Sonic hedgehog SA Sympathoadrenal progenitors SN Substantia nigra SNCA α-synuclein SNpc Substantia nigra pars compacta SNr Substantia nigra pars reticulata SNS Sympathetic nervous system STN Subthalamic nucleus STR Striatum TH Tyrosine hydroxylase TX Transplantation VM Ventral mesencephalon VTA Ventral tegmental area W Week WGE Whole ganglionic eminence TANSs Tonically active neurons FSIs Fast-spiking interneurons
Abbreviations of the international system of units (SI units) were used in this thesis and are not specified above
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LIST OF FIGURES
FIGURE 1: Andreas Vesalius’s and Thomas Willis’s basal ganglia…………………………… 20
FIGURE 2: Basal ganglia nuclei……….……………………………………………………….. 21
FIGURE 3: Medium spiny GABAergic neurons in the dorsal striatum………………………….22
FIGURE 4: Indirect and Direct motor pathways…………………………………………………23
FIGURE 5: Striatofungal projections…………………………………………………………… 26
FIGURE 6: Indirect and direct pathways receptors………………………………………………27
FIGURE 7: Fiber tracts associated with the subthalamic nucleus………………………………..28
FIGURE 8: Neurotransmitters involved in the direct and indirect pathway……………………..30
FIGURE 9: Neuro-pathological findings in a patient with PD…………………………………...35
FIGURE 10: Updated scheme of the basal ganglia circuit……………………………………….37
FIGURE 11: Schematic summary of the original basal ganglia model in a normal state or parkinsonian state………………………………………………………………………...38
FIGURE 12: 3-D image of a deep brain stimulation electrode for treatment of PD……………..39
FIGURE 13: X-ray after the implantation of a DBS electrode in the subthalamic nucleus……...43
FIGURE 14: Listenmee® Auditory device to improve walking in PD…………………………..44
FIGURE 15: SD Rat brain and 6OHDA lesion…………………………………………………..46
FIGURE 16: Neuro-pathological findings in a patient with HD……………………………….. 48
FIGURE 17: MRI T1w, showing typical views from the STP3 –planning workstation………..82
FIGURE 18: Overview of the stepwise planning approach to cells transplantation……………..84
FIGURE 19: Screenshot of our software for fast model based functional trajectory planning…. 87
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FIGURE 20: Stepwise of stereotactic lesion and transplantation in SD rats…………………….91
FIGURE 21: Intra-peritoneal injection of CsA and rotational behavioral test…………………92
FIGURE 22: Experimental design of project III…………………………………………………93
FIGURE 23: Laser capture microdissection……………………………………………………..96
FIGURE 24: Stepwise of and electrode implantation in the STN for DBS in a SD rat…………97
FIGURE 25: Microtransplantation using glass capillary with diameter approx. 70µm. Scale bar 500µm…………………………………………………………………………………………...100
FIGURE 26: Suprarenal gland isolation………………………………………………………..101
FIGURE 27: Process map for clinical fetal cell therapy of HD/PD...... 111
FIGURE 28: Comparison of a compete CNS vs. a fragmented CNS before dissection………..125
FIGURE 29: Validation of the region-specific dissection………………………………………125
FIGURE 30: Cytospin staining of VM cells. Cell suspension 1x105 cells of 8 week old human embryo…………………………………………………………………………………………..126
FIGURE 31: Cytospin staining of VM cells. Cell suspension 1x105 cells of 8 week old human embryo. DAPI, MAP 3, 10 days differentiation in neurons (βIII Tubulin staining)……………126
FIGURE 32: Dissection of embryos is performed under microscopic view……………………127
FIGURE 33: Dissection of ganglionic eminence of 8 week-old embryo……………………….128
FIGURE 34: Dissection of the WGE (Whole Ganglionic Eminence) from the lateral ventricular cavity of an 8-weeks-old human embryo………………………………………………………..128
FIGURE 35: Dissection of the ventral mesencephalon from 9-weeks human embryo…………129
FIGURE 36: Intraoperative surgical cells deposits……………………………………………..130
FIGURE 37: 11C-Raclopride binding potential images showing striatal binding of D2 receptors 11C-Raclopride in direct comparison between a normal subject (A) and one of our series with HD (B) (Contribution from Freiburg Neuroradiology Department)……………………………131
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FIGURE 38: MRI T1-weighted Axial images 3 days following second transplantation (left) of bilateral fetal neuronal cell graft placement in a HD patient……………………………………131
FIGURE 39: Comparison of the Absolute Value in millimeters, between right and left hemisphere employed entry point coordinates on the left and target points coordinates on the right. Not statistical significant difference was found between the coordinate’s values given by the software……………………………………………………………………………………...132
FIGURE 40: MRI T1w image taken 3 days following second transplantation. B: T2w MRI Image taken 4 weeks following surgery and 3 weeks following mild head trauma, showing bilateral subdural fronto-parietal hygromas……………………………………………………………..134 FIGURE 41: MRI T1W image with Contrast enhancement native Control image A: 3 years after surgery, control image shows abnormal edematous changes on the site of the grafts on the left site, with contrast enhancement…………………………………………………………………137 FIGURE 42: MRI native Control image A: image taken 3 days following second surgery (left transplantation), needle tracts are visible within the caudate and putamen. B: image taken 12 months…………………………………………………………………………………………..137 FIGURE 47-49 Graft histology project III…………………………………………………142-145 FIGURE 50: Laser Capture microscopy……………………………………………………….145 FIGURE 51: Microscopic view of bovine chromaffin cells cell culture at 6 (A) and 12 (B) days after isolation……………………………………………………………………………………145 FIGURE 52: Summaries of Results of Project III………………………………………………146 FIGURE 53: Graft-induced behavioral improvements………………………………………….146 FIGURE 54: AMPH-induced rotations in Tx rats with and without DBS……...……………....147
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LIST OF TABLES
TABLE 1: Disease Potentially Amenable to Grafting…………………………………………...56
TABLE 2: Data of stereotactic procedures in 22 HD patients undergoing HFST…………...... 80
TABLE 3: Mean length of each tract from cortex to target and AC-PC line in millimeters...... 86
TABLE 4: Stereotactic coordinates for human transplantation in HD…………………………...88
TABLE 5: Trials of chromaffin cell grafts in a PD animal model from project III…………….104
TABLE 6: Reagents for immunohistochemistry from project III………………………………108
TABLE 7: Indications and contraindications for fetal neuronal cells transplantation in HD…..114
TABLE 8: Inclusion and exclusion criteria of PD transplantation …………………………...... 115
TABLE 9: Experimental design and resume of results in all 8 studies of Project 3….…...……146
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1. INTRODUCTION
1.1 BASAL GANGLIA CIRCUITRY AND FUNCTION
1.1.1 Nomenclature:
Historically Andreas Vesalius (1514-1564) was the first to detail the basal ganglia (BG), but it was not until 1664 that the English physician Thomas Willis referred to what is known today as the basal ganglia calling them the “corpus striatum” in an attempt to designate the greater BG constituent (Parent 1986). Interestingly at that time it was thought to be the “sensorium commune” previously defined by Aristotle as the site in the brain taking information of the 5 senses and integrating them, the area where our common sense comes from (Figure 1). Consecutive works in the 18th and 19th centuries highlighted cortical properties diminishing BG physiological significance, notably the German anatomist and physician Karl Friedrich Burdach (1776-1847) provided landmarks differentiating for the first time the caudate nucleus from the putamen, which he named Streifenhügel (elongated hillock) and Schale (shell) respectively, the latest he also termed Linsenkern (lens-shaped nucleus) by recommendation of German physician and anatomist Johan Christian Reil (1759-1813). Pioneer anatomical discoveries of Burdach also include the identification of the globus pallidus and its two portions (blasser Klumpen innern und äussern Theil) (Parent 2012). The 20th century brought more detailed explanations and discoveries about the BG function. Detailed descriptions were given by Ramon y Cajal and Vogt in 1911 and Wilson in 1914 started to discover that lesions in the corpus striatum structures would cause motor disorders in humans. The corpus striatum was then defined as the major element of the “extrapyramidal motor system” (Parent 1986). The extrapyramidal motor system by definition demanded an independent motor entity, grouping the corpus striatum with specific brainstem nuclei (Carpenter 1981). This nomenclature was used in the past as an strategy to distinguish this network of structures from pathological conditions affecting the pyramidal system the latest defined like neurons whose axons runs through the corticospinal (conducts impulses from the brain cortex to the spinal cord) and corticobulbar tracts (connecting the cerebral cortex to the brainstem). At present these distinctions are considered misleading (Obeso 2012). The term BG was adopted in a way to define these major subcortical nuclei at the base of the forebrain.
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Nowadays, the BG are defined as a group of subcortical nuclei of different origin in the brain, which normally act together like a unit responsible for motor control, motor learning and motor memory as well as for executive behaviours and emotions. A lesion of such network is the origin of the majority of movement disorders and could also produce speech, impulse, memory and other cognitive problems (Lanciego 2012). The main role of BG deals with posture and movement control (Marsden 1982-1992). They are responsible for the subconscious automatic execution of learned motor plans e.g. walking or drinking water (Brazis 2007).
The term BG in a rigorously refers to nuclei deep-seated in the brain hemispheres (Striatum [caudate-putamen] and globus pallidus), while related nuclei endure nuclei located in the diencephalon (subthalamic nucleus), mesencephalon (substancia nigra) and pons (pedunculopontine nucleus) (Lanciego 2012).
Figure 1 A. Andreas Vesalius’s drawing of the human basal ganglia as appear on plate VII of Book VII of Vesalius’ fabrica (Vesalius 1543 took from Parent 2012). The figure shows its anatomical vision through an axial section of the brain. A right and B. left hemisphere. The basal ganglia are better discriminated on the right side of the figure; Vesalius clearly identified white matter fibres from masses of grey matter. B. On the right Thomas Willis’s anatomical concept of the basal ganglia, the figure shows a dorsal view of the basal ganglia, brainstem and cerebellum
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 20 of a sheep. The figure is a reproduction of plate VIII from Willis’ Cerebri anatome (Willis 1664, took from Parent 2012) Neuroscience & Medicine, 2012, 3, 374-379
1.1.2 Functional organization of the basal ganglia:
BG and related nuclei can be widely classified in relation to how sequences of information run through the BG as (a) Input nuclei, (b) output nuclei, and (c) intrinsic nuclei.
Input nuclei are defined as those structures receiving afferent information. Such information arrives from different sources mostly cortical, thalamic and nigral. Input nuclei are: The caudate nucleus (CN), the putamen (Pu) and the accumbens nucleus (Acb). Output nuclei are defined as those structures that send basal ganglia information to the thalamus. Output nuclei are: the Globus pallidus in its internal segment (GPi) and the substantia nigra pars reticulata (SNr). Intrinsic nuclei are defined as nuclei located between the input and output nuclei in the transfer of information, with antagonistic actions for correct execution of movement. Intrinsic nuclei are the Globus pallidus in its external segment (GPe), the subthalamic nucleus (STN), and the Substantia nigra pars compacta (SNc)
Cortical and thalamic efferent information enters the striatum (CN, Put, and Acb) to be processed within the BG. The output nuclei (GPi and SNr) project mainly to the thalamus (ventral nuclei), that consequently project back to the cerebral cortex (mostly frontal lobe) (Figure 2).
A proper operating of the BG system depends upon dopamine to be discharged at the input nuclei. Several movement disorders e.g. parkinsonian syndrome, dystonia, chorea, tics are related with a dopamine impaired system.
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Figure 2 Basal ganglia nuclei Parasagittal brain section from a monkey showing the major basal ganglia components. CN: caudate nucleus, Put: putamen, Acb: Accumbens, GPe: globus pallidus externo, ac: anterior commissure, GPi: globus pallidus internus, STN: nubthalamis nucleus, SNr: substantia nigra pars reticular, SNc: substantia nigra pars compacta. (Modified from Lanciego, Cold Spring Harb Perspect Med 2012; 2:a009621)
1.1.3 Cytoarchitecture:
INPUT NUCLEI:
1. Striatal neurons
There are two types of neurons in the striatum: a. projection neurons 90% and b. interneurons 10%. Projection neurons also known as striatofugal neurons or medium-sized spiny neurons (MSNs) named after their size (20µm in diameter) and their dentritic processes which are covered by postsynaptic specializations denominated dentritic spines (Figure 3). All striatal MSNs are inhibitory neurons through the use of the neurotransmitter GABA. According to their projection targets striatofugal MSNs could be divided, into those projecting to the output nuclei (GPi and SNr) and those innervating the GPE nucleus. Striatal MSNs innervating the GPe nucleus express the dopamine receptor subtype 2 (D2R) that inhibits trough G-protein signaling the intracellular adenyl-cyclase, giving rise to the denominated indirect pathway (cortex→striato→Gpe→STN→GPi/SNr→thalamus→cortex) (Figure 4)
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Figure 3 A detailed observations of medium spiny GABAergic neurons in the dorsal striatum (scale bar 100 µm). The picture shows Golgi impregnated medium spiny neurons in the tree shrew dorsal striatum, dentrites and dentritic spines details are shown in the boxed area [Scale bar: 25µm] modified from Rice 2011.
Figure 4 Indirect (A) and Direct (B) motor pathways. A. Indirect motor pathway runs: Cortex→striato→Gpe→STN→GPi/SNr→thalamus→cortex B. Direct motor pathway runs: Cortex→striatum →GPi→ thalamus→cortex.
On the other hand in the direct striatopallidal motor pathway, striatal MSNs projecting directly to
GPi and SNr contain dopamine receptor subtype 1 (D1R), such receptor activate adenylcyclase signaling (D1-containing neurons). Additionally the striatal MSNs of the indirect pathway contains the neuropeptide encephalin, while striatal MSNs of the direct pathway expresses the neuropeptides substance P and dynorphin.
Local-circuit neurons (interneurons)
In addition to spiny neurons there are other different classes of interneurons in the striatum; the larger group of interneurons is constitute by large aspiny cholinergic neurons, such neurons use acetylcholine as neurotransmitter. These neurons manifest a continuous and constant electrophysiological firing pattern, due to this nature they are classified as tonically active neurons (TANSs). GABAergic neurons are other type of striatal interneurons containing parvalbumin (calcium-binding protein), electrophysiologically, these neurons are classified as fast-spiking interneurons (FSIs). Another group of GABAergic interneurons but with a different phenotype is one containing calretinin (calcium-binding protein); a third type of GABAergic interneurons is the nitrergic interneuron, using the neurotransmitter: nitric oxide.
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In this intrastriatal microcircuit TAN and FSI interneurons are responsible for modulating the activity of striatofugal neurons and are under dopaminergic domain. TAN and FSI interneurons are innervated by Calretinin-positive and nitrergic interneurons (Lanciego 2012).
Striosomes and Matrix (striatal compartments)
The striatum rather than be a homogeneous structure has two subdivisions: striosomes and matrix compartments. Such pattern could be found thanks to the immunohistochemical pattern of acetylcholineterase (AChE) staining; those areas showing weak AChE activity were denominated striosomes which are encrypted within a more intense background, denominated matrix (Graybiel 1978).
Within the striosomes was also found an enriched expression of the µ opiod receptor and weaker in the matrix (Perth 1976, Desban 1995)
Striosomes have a strong immunoreactivity against encephalin, substance P, GABA, and neurotensin, while the matrix is enriched with calcium-binding proteins like parvalbumin.
Striosomes and matrix subdivision could be defined as independent compartments in terms of the striatal afferent and efferent systems. Striatal MSNs dentritic domains and local axon collaterals mainly remain restricted within the striatal compartment they never enter the matrix and vice versa (Fujiyama 2001).
Matrix MSNs innervate the GPe, GPi and SNR; striosomes MSNs innervate the SNc with axon collaterals to the GPe, GPi and SNr (Gerfen 1984, Fujiyama 2011).
Cerebral cortex motor and sensory areas, thalamus, amygdala and dopaminergic afferents arising from SNc are equally distributed throughout both compartments (striosome and matrix). Cerebral cortex, thalamostriatal projections and dopaminergic neurons from the SNc innervate at most the matrix. Cortical limbic areas, the basolateral amygdala, and ventral parts of the SNc preferentially target striosomes (Graybiel 1984)
Cortex, thalamus, substantia nigra and raphe (striatal afferents)
Ipsilateral and contralateral cerebral cortices projects into the striatum trough glutamatergic projections making synapses with dendritic spines of striatal MSNs. The totality of cortical
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 24 projections spread to the matrix compartment whereas cortical limbic areas innervate the striosomal compartment (Gerfen 1984). The corticostriatal projections are topographically organized (Selemon 1985). Prelimbic cortices input are originated in pyramidal neurons of layer V, reaching then the striosomal compartment. The matrix instead is innervated by pyramidal neurons located in cortical layers III and Va. Thalamostriatal projections represent by itself a major source of glutamatergic afferents embracing the striatum. Thalamostriatal axons were though to contact the dendritic shafts of striatal MSNs , while corticostriatal afferents mostly synapses with dendritic spines, however thalamostriatal afferents originated from the centromedian-parafascicular (CM-Pf) thalamic complex are the only exclusive projections targeting only dendritic shafts, since all other thalamic inputs from different nuclei contact dendritic spine (Raju et al 2006). Such projections can be differentiated also due to the vesicular glutamate transporter expressed. In corticostriatal projections the vesicular glutamate transporter is isoform 1 (vGlut1) whereas in thalamostriatal terminals isoform 2 (vGlut2).
The nigrostriatal system is the most important source of dopaminergic striatum innervation; it originates from SNc A9 group neurons (Dahlström and Fuxe 1964). The retrorubral field (A8) and the ventral tegmental area (A10) participate as well in the mesostriatal and mesolimbic dopaminergic projections.
The SNc can be divided in two territories: dorsal and ventral tier. Ventral tier dopaminergic neurons are organized in columns, they enter the SNr and create from there a topographically nigrostriatal projection (Lynd-Balta 1994). Nigrostriatal axons make synapses with striatal
MSNs. The dopaminergic input is excitatory on D1R-containing MSNs (direct pathway neurons) and inhibitory on D2R-containing MSNs (indirect pathway neurons).
One dopaminergic axon from the nigra, expresses a high number of arborizations. In the rat one axon innervates an average of 75,000 MDNs and one MSN is under the influence of 95-194 dopaminergic neurons (Matsuda et al. 2009). In the primate one dopaminergic neuron make 1 million synaptic contacts onto striatal neurons.
Other inputs received by the striatum are the glutamatergic sources are originated from the amygdala and from the raphe nuclei (mostly dorsal raphe nuclei) the latest being serotoninergic projections (Anden et al 1966; Szabo 1980). Such serotoninergic axons may be involved in graft- induced dyskinesias and levodopa-induced dyskinesias (Lopez et al 2001).
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Striatum-GPe and Striatum-GPi/SNr (Striatal efferents)
Striatofungal projections are: D2R-expressing MSNs innervating the GPe and D1R-expressing MSNs innervating the GPi and SNr (Figure 5, 6).
Figure 5 Striatofungal projections: neuronal afferents reach the BG when attempt to achieve a movement following different circuitry: A. superimposed composite camera lucida of serial sagittal sections showing a single biocytin- injected striatofugal axon in GPe, GPi and SNr. This axon arises from a neuron located in the putamen. B. Camera lucida drawing of a GPe neuron projecting to GPi and STN (Modified from Parent 2001 Parkinsonism & Related Disorders Volume 7, Issue 3, July 2001, Pages 193–198). IC: internal capsule; Th: Thalamus; OT: optic tract
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Figure 6 Indirect and direct pathways. The SN pars compacta uses its dopamine neurons to send signals up to the striatum; striatofungal projections D1R-expressing striatal MSNs project downstream to the GPi and SNr (left).
Striatofungal projections D2R-expressing striatal MSNs innervate the GPe (Modified by Lopez W.O from Purves neuroscience fourth edition).
It is important to mention that a sharp discrimination of D1R/D2R expression remains controversial; half of striatal MSNs innervating the GPi and SNr also send collaterals to the GPe.
OUTPUT NUCLEI:
The GPi and SNrare the BG output nuclei; they do share similar cyto- and chemoarchitectural attributes. Both are composed by inhibitory GABAergic neurons firing tonically to inhibit their targets. The GPi and SNr receive through the direct pathway inhibition from the striatum, and from the STN (indirect pathway) excitatory glutamatergic projections. Both GABAergic and glutamatergic systems converge into the BG output nuclei (GPi and SNr) which in turn innervate thalamic and brainstem nuclei [Thalamus ventro lateral, pedunculo-pontine nuclei, superior
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 27 colliculus] (Lanciego 2012). There are two pathways by which pallidothalamic projections can gain access to their respective thalamic destination:
1. Projections from lateral GPi neurons form the ansa lenticularis that passes around the internal capsule to finally enter the field H of Forel (prerubral field) and
2. Projections from medial GPi neurons that travel through the internal capsule forming the lenticular fasciculus (Forel’s field H2) which is located between the STN and the zona incerta (Lanciego 2012).
Ansa lenticularis and lenticular fasciculus together enter the thalamic fasciculus (H1 of Forel) at the level of field H of Forel, to finally reach the ventral and intralaminar thalamic nuclei
(Figure 7).
Figure 7 Fiber tracts associated with the subthalamic nucleus: AL = ansa lenticularis; CP = cerebral peduncle; FF = Fields of Forel; GPe = globus pallidus externus; GPi = globus pallidus internus; H1 = H1 Field of Forel (thalamic fasciculus); IC = internal capsule; LF = lenticular fasciculus (H2); PPN = pedunculopontine nucleus; Put = putamen; SN = substantia nigra; STN = subthalamic nucleus; Thal = thalamus; ZI = zona incerta.
(From Hamani 2003; Brain Volume 127, Issue 1Pp. 4-20)
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INTRINSIC NUCLEI:
1. Globus pallidus external
The GPe is surrounded laterally by the putamen (from which is separated by the lateral medullary lamina) and medially by the GPi (from which is separated by the medial medullary lamina). GPi and GPe are made of GABAergic neurons; both structures have an enriched expression of the calcium-binding protein: parvalbumin (Lanciego et al. 2012). The GPe receives a GABAergic inhibitory projection which represents the first relay of the indirect pathway. GPe neurons are reciprocally connected with STN neurons and with GPi neurons. A minor source of glutamatergic afferents arises from the caudal intralaminar nuclei (Kincaid et al 1991).
2. Subthalamic nucleus
The STN is located ventral to the zona incerta and rostral to the SN. It receives the second relay of glutamatergic projections from GPe neurons (indirect pathway), as well as glutaminergic projections from the cerebral cortex on what is known since 2002 as the hyperdirect pathway (Nambu et al. 2002,; Nambu 2004, 2005) the ipsilateral thalamic caudal intralaminar nuclei (Sugimoto and Hattori 1983) and the contralateral thalamic caudal intralaminar nuclei (Gerfen et al. 1982). It is important to mention that the STN also receives a minor dopaminergic projection from the SNcpart of the nigro-extrastriatal projection system (Rommelfanger and Wichman). The cerebral cortex sends direct projections to the STN innervating then the output nuclei. Cortical inputs from the primary motor cortex, supplementary motor area, premotor cortices, frontal eye field, and supplementary eye field area make part of the hyperdirect pathway. Most STN efferent neurons send axons that simultaneously innervate the GPi, GPe, SNr, ipsilaterally ventral thalamic motor nuclei and contralaterally targeting the parafascicular nucleus (Gerfen et al. 1982) (Figure 8).
3. Substantia nigra pars compacta
In the ventral midbrain, reside many groups of tyrosine-positive neurons whose main function is to supply the BG with dopamine. Those dopaminergic neurons can be classified in: A10 neurons (ventral tegmental area), A9 neurons (SNc), and A8 neurons (retrobulbar field). A10 neurons
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 29 innervate mostly the accumbeus nucleus and several limbic related areas, A8 and A9 are in charge of the nigrostriatal projection. Dopaminergic neurons in the SNc degenerate progressively in PD. Those neurons that survive present intracellular aggregates of proteins, mainly alfa- synuclein, adopting circular shape aggregates identified as Lewy bodies (Lanciego et al. 2012)
Figure 8 Neurotransmitters involved in the direct and indirect pathway. Ach, acetylcholine; DA, dopamine; Glu, glutamate; Enk, enkaphalin; SP, substance P. SNc, substantia nigra pars compacta; SNr, substantia nigra pars retriculata; GPe, globus pallidus pars externa; GPi, globus pallidus pars interna; STN, subthalamic nucleus; VL, ventral lateral nucleus; VA, ventral anterior nucleus (Modified From Clinical Motor and Cognitive Neurobehavioral Relationships in the Basal Ganglia, InTech 2012, Leisman 2012)
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1.1.4 Summary:
The BG is a circuitry that influences movement by regulating the activity of upper motor neurons from the cortex and brainstem. The term BG as mentioned before encompasses a set of nuclei localized deeply within the cerebral hemispheres. The nuclei involved on correct motor function includes the caudate, the putamen, the globus pallidus and the STN (in the ventral thalamus) and SN (in the base of midbrain), such subcortical loop links most areas of the cerebral cortex with upper motor neurons in the primary motor and premotor cortex and in the brainstem. This loop neurons modulate activity both in prior and during movement, such effects on upper motor neurons are necessary for the normal execution of voluntary movements; and this is so because when the basal ganglia and associated structures circuitry is altered, the motor system cannot alternate steadily between initiating and terminating the movement. Having in mind such concepts the movement disorders can be understand under the scope of the absence of regulatory control from an impaired BG circuitry leading to abnormal upper motor neuron activity.
The BG is a remarkably systematized circuitry activated according to specific circumstances and demands. The motor network of the BG has two entry points, the striatum and the subthalamic nucleus (STN) and one output the globus pallidus pars interna (GPi), which connects to the cortex through the motor thalamus (Obeso 2008).
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1.2 PARKINSON’S DISEASE
1.2.1 Epidemiology of Parkinson’s disease
Geographical distribution
Prevalence rates of PD are homogenous when comparing developed and developing countries, for 65 years old or older patients such rate is in China 2.1% for both sex, which is similar to Rotterdam (2.1%) and Europe (1.7%) (Zhang 1993)
Sociodemographic factors
PD affects between 100 and 200 per 100,000 people over 40, and over 1 million people in North America alone. The incidence/prevalence rates of PD increases continuously with age (Tison 1994); the average ratio of male to female standardized rate is 1.35 from prevalence studies and 1.31 from incidence studies (Zhang ZX 1993), there is a suggested potential protective estrogen’s effect (Roca 2008).
The socioeconomic position: there is a positive association of PD with higher socioeconomic position or education in the USA (odds ratio 2.0, 95% CI 1.1 to 3.6) for 9 or more years of education (Frigerio 2006), physicians were also found at increased risk (odds ratio 3.7, 95% CI 1.0 to 13.1) Nowadays no evidence exist to prove lower incidence in races. However blacks with PD are at have worse survival than white patients (Ben-Shlomo 2012).
Risk factors
Family history: Families studies suggest low risk for siblings and offspring, recurrence risk is low even for identical twins (Wirdefeldt K, 2008).
Infections: Potential infectious agents have been of interest due to the historical appearance of postencephalitic PD caused by the epidemic of encephalitis letharica or Von Economo’s disease between 1917 and 1928. Encephalitis letharica is a disease characterized by high fever, headache, double vision and lethargy. The cause of this condition is unknown.
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Intra-uterine influenza is one of many infectious agents postulated as potential infectious agents (Mattock 1988), measles (protective exposure) (Sasco 1985) and age at whooping cough infection (De pedro-Cuesta 1996); However Serological and pathological test does not support the hypothesis of any specific viral particle or inclusions or antigens in brain autopsies (Schwartz 1979).
Neurotoxins:
Neurotoxicity was discovered by the human accidental ingestion of MPTP (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine) which caused rapid onset of PD symptoms; by destroying dopaminergic neurons in the substantia nigra of the diencephalon. Since this lesion has also become important as an animal model to study the disease. MPTP may be accidentally produced during the production of MPPP a synthetic opioid drug with similar effects to those of morphine.
Although exposure to MPTP was rare, the finding encouraged strongly, to start searching for a most common neurotoxin. PD patients seem to have less effective detoxification systems (Waring 1989, Smith 1992) suggesting that PD appears as a combination of inherited susceptibility and an acquired environmental toxin. Similarity between MPTP and paraquat (N, N′-dimethyl-4, 4′- bipyridinium dichloride) which is one of the most widely used herbicides in the world. Paraquat acts killing green plant tissue on contact. It is toxic to humans and animals. (Tanner 2011); led to study the relationships between PD and pesticides. PD in Canada appeared to be more common in rural areas, where pesticides are more use; reports from case studies showed an association of the incidence of PD with the use of pesticides (Semchuck 1992, Hubble 1993, Butterfield 1993, Seidler 1996), other studies however did not find such association (Wong GF 1991, Koller 1990, Jimenez 1992, Stern 1991). It is important to consider that such studies and their positive association with pesticides reflect recall bias. More recently a German study addressed exposure to wood preservatives, finding no difference when exposure was classified using a objective “job exposure matrix” (Seidler 1996).
If there is a truly association, it may be related only to occupational pesticide use, and is only around 10% (Semchuck 1992)
Smoking and Personality: An inverse relationship tendency between smoking and PD has been noticed in US and UK studies (Hammond 1966, Doll 1994). Personality studies talk about
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 33 genetic susceptibility associated with some personality traits. Nonsmokers have high levels shyness of and defensiveness, whilst smokers are more likely to be extrovert (Eysenck 1960, Forgays 1993).
Head trauma:
A positive relation between any mild and severe head injury and PD (Stern 1991), but it is difficult to evaluate it due to the problem of recall bias.
Rural residence and Well Water:
While studying association between PD and herbicides, Canadian researchers started to note a relation between patients drinking well water and PD, however any association between well water and PD could reflect selection bias; since studies with population.-based controls fail to find any association (Seidler 1996, Semchuk 1993).
Dietary factors:
A significant protective effect was seen with Vitamin C and manganese consumption were found in a large prospective cohort study of 41,836 women, who were followed up for 6 years, in the same study no association was found between vitamin E and PD whereas vitamin A consumption was associated with PD (Cerhan 1994). High cholesterol or treatment with simvastatin (cholesterol lowering drug) may reduce risk of PD (Alonso 2007, Schwarzschild 2008). Peanut consumption in females and salad with dressing in males seems to have a protective effect (Golbe 1990). Nuts and seeds appear to be harmful (Butterfield 1993).
1.2.2 Etiology and pathogenesis of Parkinson’s disease
The disease was first described by James Parkinson an English apothecary surgeon, geologist, paleontologist, and political activist. His famous publication was released in 1817, An Essay on the Shaking Palsy (Morris 1955). However is interesting to mention of existing evidence about a disease in ancient India, identified as “Kampavata”, composed of shaking (kampa) and loss of movement (vata). Interesting it was treated 1500 years ago with the Mucuna pruriens plant, which later was discovered to contain levodopa (Katzenschlager 2004).
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The pathology of PD was elucidated in 1912 when a German pathologist named Frederick Lewy found and reported neuronal cytoplasmic inclusions in some brain regions. In 1919, a neuropathologist, during that time, a doctorate student named Konstantin Nikolaevitch Tretiakoff and born in Uzbekistan, described in his thesis a degeneration of the substantia nigra pars compacta of the midbrain in associated to PD. In the 1950s the importance of Dopamine depletion in the basal ganglia was discovered a principal factor to understand the pathophysiology of PD (Hornykiewicz 2006) (Figure 9).
Notwithstanding the cause of PD is still unknown, important discoveries have been made in understanding the possible disease developing mechanisms (Marras 2008). Such progress has been mainly achieved on new discoveries about basal ganglia cytoarchitecture and neurochemical profile, improved precise identification of neuropathologic abnormalities in PD, and characterization of genetic forms of PD.
Figure 9 Neuro-pathological findings in a patient with PD The characteristic histopathological changes in PD are the loss of pigmented dopaminergic neurons, particulary in the ventral tier of the substantia nigra pars compacta, and the presence of Lewy bodies in a proportion of the surviving neurons. A, B: The pigment in the substantia nigra is dark because of the presence of brown neuromelanin in neurons; In A and B the susbtantia nigra
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on the left is normal while that on the right is depigmented .C-D Lewy bodies in a substantia nigra dopaminergic neuron in PD. E. neuromelanin (Pictures with contribution from the Institut für Neuropathologie, Universitäts klinikum Freiburg).
Pathophysiology:
Basal ganglia’s Dopamine depletion causes major disruptions in their connections to the thalamus and motor cortex, leading among others to bradykinesia.
The basal ganglia receive a cortical excitatory input from the cortex; such input is originated from the prefrontal supplementary motor area, amygdala and hippocampus, and is mediated by the neurotransmitter glutamate. The basal ganglia contribute to an automatic control of movements (i.e rhythmic limb movements and adjustment of postural muscle tone during locomotion), their role is performed by providing an estimate of the current sensory state and planned motor command (motor preparation) and trough balanced inhibition of motor activity (Takusaki 2004).
On the other hand, neurons in the substantia nigra pars compacta (SNc) innervates the dorsal striatum (caudate-putamen) providing major dopaminergic input to the striatum, exerting both excitatory and inhibitory influences on the striatal output neurons. The synergy between the afferent and efferent pathways is mediated by striatal interneurons which utilize acetylcholine as the main neurotransmitter.
The striatal output system is mediated by the inhibitory neurotransmitter gamma-amino-butyric- acid (GABA). The connection between the STN and the GPi and between STN and the GPe is excitatory, mediated by glutamate (Lanciego 2012, Jankovic 2013) (Figure 10).
Five different dopamine receptors (D1 through D5) have been identified; throughout the basal ganglia and limbic system. The D1 and D2 receptors are mostly established in the dorsal (motor) striatum and play the most important role in the pathophysiology of PD since they are activated by the dopaminergic pathway originating in the SNc. Receptors denominated as D3, D4, and D5 are more abundant in the mesolimbic (emotional) area of the brain (D3, D4) and hippocampus/hypothalamus (D5) (Gerfen 2000).
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Figure 10 Updated scheme of the basal ganglia circuit. The figure shows the main circuits linking the basal ganglia nuclei. It is important to notice several new transverse loops, which have been described in the last few years, most of them with a putative modulatory role. The actual pathophysiology of the basal ganglia associated with PD may be much more complex than indicated by the current models. Existing models are constantly reevaluated as new findings become available. (From Lanciego et al. 2012 From Cold Spring Harb Perspect Med 2012; 2:a009621)
Dopamine deficiency in the nigrostriatal pathway causes denervation hypersensitivity of D1 and D2 receptors (the nigrostriatal dopamine system develops increased sensitivity to dopamine as a consequence of chronic dopamine receptor denervation induced by PD) (Bamford 2004). When compared with normal matched controls, D2 receptors in the dorsal putamen are increased by 15 percent in patients with PD, whereas D3 receptors in the mesolimbic system are decreased by 40 to 45 percent as one could predict (Ryoo 1998).
There are two output pathways from the striatum (figure 4, 8, 9):
The indirect pathway is mediated majorly via dopamine's inhibitory influence on striatal D2 dopamine receptors. In the indirect pathway, the striatum projects to the neurons in GPe utilizing GABA, and the GPe projects subsequently to the STN also utilizing GABA, the STN provides excitatory input via glutamate to the GPi and SNr. GPi neurons are GABAergic and synapse in the ventrolateral nucleus of the thalamus. Finally thalamic input to the cortex is excitatory via
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 37 glutamate. In the direct pathway, the striatum projects directly to the GPi and SNr. The direct pathway is mediated via dopamine's excitatory influence on striatal D1 dopamine receptors.
In PD, the neurodegenerative process leads to a reduction of dopamine-producing neurons from a normal approximately quantity of 550,000 neurons to the adversely low level of 100,000. By the time a patient with PD displays his initial symptoms, approximately 80% of the dopamine producing cells in the substantia nigra would have die, however there is still production of dopamine but it is significant reduce. Neurodegeneration causes a relative over-activity of the indirect pathway, where the SN-striatal dopamine output should be inhibitory. Having less dopamine causes reduce inhibition, then increasing the striatum indirect output, causing increase inhibitory output in the GPe, reducing consequently the corresponding output of this structure (which is normally inhibitory), leading to reduce inhibitory output in the STN functionally disinhibiting the STN, leading to increase excitatory signals from the STN to the GPi, such event increases significantly the output of the GPi which normally is inhibitory to the thalamus (VL), VLPPN and dPPN, causing reduce excitatory output to the motor and premotor cortex, which is ultimately expressed as bradykinesia and other parkinsonian signs. Decreased inhibition of the direct pathway additionally helps to further disinhibit the output nuclei (GPi and SNr) (Figure 11).
Figure 11 Schematic summary of the original basal ganglia model in a normal state vs. parkinsonian state. The motor circuit is composed of a cortico-striatal projection, two major striatofugal projection systems giving rise to the direct and indirect pathways, and the efferent pallido – thalamo – cortical projections to close the motor loop. In the Parkinsonian state there is a reduced inhibitory output in the STN functionally disinhibiting the STN, leading to
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increase excitatory signals from the STN to the GPi, such event increases significantly the output of the GPi which is inhibitory to the thalamus (VL), and thickness of arrows represents the hipo-hiperfunctional state of a given circuit. (From Cold Spring Harb Perspect Med 2012; 2:a009621)
In PD, the development and advance of bradykinesia is highly correlated with the presence of abnormal oscillations within basal ganglia in the beta band frequency (10 to 50 Hz band), synchronized oscillatory activity in the Beta-band is prevalent in the basal ganglia circuit, such oscillations can arise due to intrinsic properties of the STN-GPe circuit, rather than as a result of an external pacemaker from another neural region (i.e. cortex or striatum) and may be important in mediating other parkinsonian features, including tremor,. Beta-band oscillations can be reduced by dopaminergic treatments (Gatev 2006). Therefore, surgical treatments of PD, such as lesion placement within or stimulation of GPi or STN, may act by desynchronizing the oscillatory basal ganglia-thalamo-cortical network activity (Figure 12).
Figure 12 3-D image of a deep brain stimulation electrode for treatment of PD. On the left: Electrode in the posteroventral lateral Gpi. References: Caudate (brown), Nuc accumbens (dark brown), Putamen (blue), GPe (green), GPi (pink), on the right electrode in the STN (clear blue) on red: red nucleus. (Image Courtesy of Medtronic).
Compensatory mechanisms in PD
The brain has the ability to compensate for the presynaptic dopamine depletion by multiple mechanisms: a. increasing the synthesis of dopamine in surviving neurons, b. increasing the afferents to the dendrites of dopaminergic neurons, c. proliferation of D2 receptors, as well as a co-localization of D1 and D2 receptors. Similarly, gap junctions, which allow rapid communications between striatal neurons, increase dramatically after dopaminergic denervation (Calabresi 2000).
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1.2.3 Therapies for Parkinson´s disease
The treatment of PD comprehends separate phases driven by the progression of the disease and the complications developed by cronic drug use. Dopaminergic substitutes are the most effective medications in improving the motor symptoms, they include: levodopa, dopamine agonists, and the monoamine oxidase B (MAO-B) inhibitors.
I Medical management
1. Levodopa In 1967 was the first medication used to chronic replace of dopamine and still remains as “gold standard” (Hornykiewicz 2010), however only 1% of an oral dose of levodopa is absorved into the blood due to major intestinal metabolism, therefore is combined with a dopa decarboxylase (DDC) inhibitor to reduce periferical metabolism, increasing absortion to 10% (Schapira 2012). Dual inhibition of peripheral metabolism is possible combining an inhibitor of cathecol-O- methyltransferase (COMT) and DCC. Levodopa improves quality of life and life expectancy controlling bradykinesia, rigidity and associated pain, it also improves tremor in many patients. Side effects are nausea, vomiting and anorexia which may disappear after 2-3 weeks. Despite the benefits of Levodopa two critical factors diminish its utility, disease progression and its direct cumulative farmacological effects, as 70% of patients develop motor complications after 6 years of comsuption. Including dyskinesias which are develop at a rate of 10% per year, with 70% of the early-onset PD patients, developing dyskinesias within 3 years of levodopa initiation (Schapira 2009).
2. COMT inhibitors The majority of levodopa is metabolized in the intestine by COMT which produces 3-O- methyldopa, therefore COMT inhibition, is a strategy to increase levodopa absorption by widening its bioavailability. Entacapone is a selective reversible COMT inhibitor. The recommenden dose of entacapone is a 200 mg tablet administered with each dose of levodopa/carbidopa up to a maximum of 8 times in Europe. Entacapone most common adverse
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 40 effect is dyskinesia requiring reducing up to a 25% daily levodopa dosage at the time of entacapone introduction (Schapira 2009). In 2003 Stalevo® was introduced, a combination of levodopa, carbidopa and entacapone in one tablet. Tolcapone, unlike entacapone, can cross the blood-brain barrier and produce central COMT inhibition, however this traduces in minimal clinical effects. On the other hand systematically entacapone produces greater COMT inhibition that Entacapone, however its use is restricted by its potential to cause hepatic toxicity. It should not be given to PD patients with liver disfunction and requires regular monitoring in the rest of the patients. Both ntacapone and tolcapone can induce diarrhea.
3. Dopamine agonists They can be classified into two groups: ergot and non-ergot Ergot agonists are: bromocriptine, cabergoline, lisuride, and pergolide. Non-ergot agonists include: apomorphine, piribedil, ropinirole, and pramipexole. Bromocriptine, cabergoline, pergolide, pramipexole, and ropinirole have been used as monotherapy in early PD, showing a significant benefical effect. Their side effect picture includes: nausea, vomiting and postural hypotension which as levodopa are dopaminergic-related symptoms, but goes beyond with a higher rate of peripheral edema, somnolence, and hallucinosis, mainly in the elderly. Dopamine agonists and levodopa may also cause behavioral abnormalities such compulsive shopping, gambling, and hypersexuality, mainly with dopamine agonists. Monotherapy with a dopamine agonist can control symptoms for a period of time. PD patients initiated on pramipexole or ropinirole are still controlled on monotherapy at 1,2,3,4 and 5 years in a percentage of 85%, 68%, 55%, 43% and 34% respectively. Neverthless, patients will require levodopa addition at some point during their disease.
4. Monoamine oxidase B inhibitors This group of medications is represented by two compounds: selegiline (deprenyl) and rasagiline. Selegiline has proved benefits after the DATATOP study over placebo (Parkinson Study group 1998), at the same time delays the introduction of levopoda and its use a lower doses. Rasagiline (N-propargyl-1(R)-aminoindan) is a roughly selective irreversible MAO-B inhibitor according to doses. Rasagiline is approximately 10 to 15 times more potent than selegiline.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 41
Two studies have been published on the efficacy of rasagiline in PD patients already taking levodopa. The PRESTO (Parkinson Study Group 2005) trial and the LARGO study (Rascol et al 2005) which demonstrated that once-a-day rasagiline (1mg) significantly improves PD control in patients on levodopa with or without additional therapy (Schapira 2009).
5. Other drugs Anticholinergics Used before the introduction of levodopa, they may produce a modest benefit in bradykinesia and rigidity but at expenses of impaired cognitive function. Benztropine and clozapine are equivalent on producing a mild improvement in tremor. Amantadine Is able to produce a mild but transitory (6-9 months) improvement in PD symptoms, it’s mostly used as adjunct therapy more than a monotherapy.
II. Non-medical management
1. Surgery Destructive lesions: Thalamotomy may produce a reduction in tremor and bradykinesia; results improve with lesion in the ventrointermediate (VIM) nucleus. Posteroventral pallydotomy can provide long lasting improvement in contralateral dyskinesia and some improvement in bradykinesia and rigidity in PD patients. Subthalamotomy can provide improvement in PD motor abnormalities including dyskinesias, however long lasting motor complications have been reported: dyskinesia and hemiballismus. Deep brain stimulation DBS has advantages over the lesions: it is reversible; parameters can be adjusted to maximize benefits and reduce side effects, can be use bilaterally, and avoids the need to perform a lesion. The precise mechanism of action is unknown, but hypothesis include depolarization blockade, release of inhibitory neurotransmitters, back firing, and/or inhibition of aberrant neuronal signals (Schapira 2009). DBS of the STN or GPi improve all of the cardinal features of PD as well as dyskinesias. VIM DBS improves significantly contralateral tremor with less side effects than lesion. Long-term studies demonstrate that benefits of DBS persist over more than 5 years of follow-up. Adverse effects are mostly related to the surgical procedure: hemorrhage, tissue damage, lead migration, infection and skin erosion which occur in about 2-3% of cases (Figure 13).
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2. Growth factors Glial-derived neurotrophic factor (GDNF) is a potential treatment for PD due to its demonstrated capacity to protect or rescue dopaminergic neurons in experimental trials.
3. Gene and stem cell therapies Gene and stem cells therapies are matter of intense research but until now have not been safe enough or superior to currently available treatments.
4. Co-adjudant new technologies They are mean to improve PD symptoms increasing the arsenal of possibilities. One of the most interesting technologies is visual and auditory cues to improve gait in PD patients. Listenmee® from Brainmee® is one of these technologies with proof of principle on selected patients (Lopez W.O. et al 2013) (Figure 14).
Figure 13 X-ray after the implantation of a DBS electrode in the subthalamic nucleus for the treatment of PD. (Picture Lopez WO)
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Figure 14 Listenmee® Practical use of an auditory device to improve gait in patients with PD. (From Lopez W.O. et al Basal Ganglia Vol3, Issue 1 and Contreras Lopez WO Movement Disorders 2013; 28 Suppl 1 :249)
1.2.4 Cell replacement therapy and its clinical effects
Two double-blind, placebo-controlled studies evaluated fetal nigral transplantation. In the first study 40 patients were randomized to receive transplantation or a placebo (sham surgery) procedure and were followed for 1 year (Olanow 2001). Modest benefits were observed in UPDRS scores in patients younger than 60 years of age. Positron emission tomography (PET) showed significant increase in striatal fluorodopa uptake, survival of implanted cells was modest at postmortem. The procedure was well tolerated, but 15% of the patients developed dyskinesias being a cause of severe disability in some patients. Several of these patients required further surgical intervention with subthalamic DBS to relieve them of these troublesome GIDs. The primary end point which was quality of life was not improved. The second trial used a slightly different implantation protocol (Olanow 2003). The study was a 2-year double-blind, placebo-controlled study. This study found significant increases in striatal fluorodopa uptake on PET and survival of implanted dopaminergic neurons at postmortem; however transplanted patients did not improved significantly when compared with placebo
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 44 patients. In this study transplanted patients also developed off-medication dyskinesias (56%), which were not observed in non-transplanted patients. The precise mechanism responsible for off-medication graft-induced dyskinesias (GIDs) has been since object of study, hypotheses are: 1. Imbalanced DA innervation due to fiber outgrowth from the graft, causing increased DA release (Freed 2001). 2. Failure of the graft to restore DA synaptic contacts with the host striatal neurons, resulting in abnormal signaling and synaptic plasticity (Hagell 2005). 3. Immunological implications which may cause inflammatory responses triggered against the graft (Olanow 2003). 4. Co-grafting of serotonergic (5-HT) neurons, since 5-HT neurons are physiologically able to store and release DA, GIDs can occur as a result of DA level mishandling. Serotonergic neurons are present in the developmental stages of the VM, and therefore they are also transplanted with the graft when the dissector dotn take care of the limits of the transplanted tissue (Politis 2010). Since these two trials international experts has joined forced to answer questions related to the efficient of fetal cell based treatments in order to start a new round of experimental trials.
1.2.5 The 6•OHDA lesion model
6-Hydroxydopamine (6-OHDA) was the first chemical agent discovered to produce specific neurotoxic effects on catecholaminergic pathways (Sachs 1975, Ungerstedt 1968). &-OHDA produces specific degeneration of catecholaminergic neurons since it uses the same catecholamine transport system as dopamine and norepinephrine. &-OHDA when systematically administered is unable to cross the blood-brain barrier, that is why it should be injected stereotactically (Figure 15) into the substantia nigra, the nigrostriatal tract or the striatum. After injection into SN or the nigrostriatal tract, dopaminergic neurons start progressively degenerating within 24 hours until striatal dopamine is finally depleted 2 to 3 days later (Faull 1969, Betarbet 2002). The magnitude of the lesion is dependent on the amount of 6-OHDA injected, site of injection and animal species sensitivity. Most of estudies achieve extensive striatal dopamine loss (80-90%). Mostly 6-OHDA is injected in one hemisphere while the other serves as control. A unilateral 6-OHDA lesion lead to assymetric circling motor behavior after administration of dopaminergic drugs, this is due to physiologic imbalance between the lesioned and the unlesioned striatum. Despite all the advantages of the 6-OHDA model, it doesn’t reproduce
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 45 enoughtly all the clinical and pathological features of PD: 1. It does not affect other brain regions (i.e. locus coeruleus), 2. It doesn’t result in formation of Lewy bodies (cytoplasmatic inclusions) like PD. 3. The acute nature of the lesion differs from the progressive nigral dopaminergic degeneration in PD (Figure 15).
Figure 15 SD Rat brain and 6-OHDA lesion. A. Dopaminergic neurons reside in the substantia nigra pars compacta (SNc), which is located in the ventral midbrain, and send axonal projections to the striatum, which is situated in the forebrain. In the 6-OHDA animal model of Parkinson's disease, the nigral dopaminergic neurons are destroyed unilaterally by means of a stereotactic injection of the toxin, as indicated by the needle in the SNc (directly into the ventral midbrain) or in the medial forebrain bundle or. Some nucleus marked with BrdU (green) colocalize with the cytoplasmatic TH (red). OB: olfactory bulb; V: ventricle; VZ: ventricular zone (the consecutive depletion of dopamine (DA) in the striatum leads to a decreased proliferation of progenitor cells in the SVZ (From PMID: 18471317 International Archives of Medicine 2008, 1:2) C. Intra-striatal cells transplantation (Lund 2012).
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 46
1.3 HUNTINGTON’S DISEASE 1.3.1 Epidemiology of Huntington’s disease
Huntington’s disease (HD) is a fatal disorder present worldwide, the highest prevalence is on Caucasians of European descent families and is calculated to be 5-7.5 in 100.000 people (Warby et al. 2011) Important concentrations of HD patients are seen in Juan de Acosta near Barranquilla in Colombia and in the state of Zulia, on the east coast of Lake Maracaibo in Venezuela, the latest with 700 cases per 100.000 being the highest concentrated population worldwide. The disease is generally of low occurrence in Asian and African populations (Klapan 2012).
HD manifests at any age but, mainly around 30-40 years, death is inevitable in a period of 7 to 20 years after onset of symptoms and generally from complications such pneumonia and heart disease. (Sorensen 1992, Zuccato 2010) the patients die - often by cachexia - in postural rigidity and severe dementia.
1.3.2 Etiology and pathogenesis of Huntington’s disease
HD is an inherited genetic disorder caused by mutation of the gene (IT15) on the chromosome 4p16.3 causing an abnormal number of CAG repeats (>36) within its 5'-end coding sequence (The Huntington's disease Collaborative Research Group 1993).
The presence of mutant IT15 gen (huntingtin) in the cell causes activation of pro-apoptoic processes in early stages of the disease that finally lead to extensive striatal neuronal cell loss (Klapan 2012)
The huntingtin gene was identified in 1993 by the Huntington’s disease Collaborative Research group but until today the molecular pathways that end causing cell death remain poorly elucidated.
Clinical features are consequence of progressive and permanent degeneration of the medium spiny neurons in the striatum causing emotional, cognitive, behavioural, concentration and memory, depression and finally dementia (Foroud et al., 1999). Motor features of the disease are more notorious with loss of motor control, causing involuntary and undesired movements in the face, body and extremities (the disease is also known as Huntington’s Chorea, by its Greek root which means: “dancing”) consequence loss of the inhibition of these medium spiny neurons in
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 47 the thalamus, causing hyper activation of the direct basal ganglia circuit (Lobo, 2009). With progression of the disease cell death affects other anatomical regions such as the basal ganglia, thalamus, and cerebral cortex (Vonsattel and DiFiglia, 1988) (Figure 16).
Mortality appears in a period of 7 to 10 years after onset of symptoms and generally from complications such pneumonia and heart disease (Sorensen and Fenger 1992; Zuccato et al. 2010).
Figure 16 A. Cortical and subcortical atrophy due to severe neuronal loss in a Huntington’s disease patient. B. Microscopic Findings in HD: Neuronal loss and astrocytosis of the caudate. C. Nuclear inclusions in the nucleus caudate (Pictures with contribution from the Institut für Neuropathologie, Universitäts klinikum Freiburg).
1.3.3 Therapies for Huntington’s disease
Nowadays a valid treatment for Huntington’s disease is still not available.
Respecting medication there isn’t any treatment that can cure, stabilize or stop the progression of HD. Notwithstanding several drugs may ameliorate the symptoms. Tetrabenazine can control the chorea (abnormal muscular movement), by decreasing the uptake of dopamine (Hayden et al., 2009)
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 48
Medicaments which function by lowering the dopamine levels can also be prescribe to reduce chorea such as the antipsychotics: olanzapine, clozapine, chlorpromazine and haloperidol, controlling also psychotic features associated with the disease. (Jankovich, 2009; Phillips et al., 2008)
Lastly anticonvulsivant medications (Clonazepan, Valproic Acid, Divalproex, Lamotrigine) an antiparkinsonian medication (Amantadine) also shown control in chorea (Jakovic., 2008; Lucetti 2003). There is a high depression and suicidal tendency in HD therefore anti-depressants can be prescribed (Diazepam, Benzodiazepines, paroxetine and velafaxin)
Lithium, Valproate and Carbamazepine diminish mania and mood changes (Bonelli and Wenning, 2006). Adverse effects form medication can worsen HD symptoms (1)
Three main therapeutic strategies are in development targeting either on controlling dopamine levels, altering the mutant huntingtin mRNA directly or providing cell death protection to the striatum neurons.
Even so, none of these strategies is available today and patients continue to die without an option of treatment that could at least achieve to stop rapidly progression accomplishing a period of stabilization of the disease.
1.3.4 Cell replacement therapy and its clinical effects
One experimental alternative for HD treatment, based on many isolated experimental studies is to perform fetal neuronal cells transplantation into the striatum (Freeman et al. 1995; Shannon and Kordower, 1996; Dunnett, 1999; Lindvall, 1999; Peschanski et al. 1995, 2004). A 5 years clinical pilot-study was carried out in Creteil (France) including 5 patients with encouraging results on 3 of them; achieving a functional metabolic active graft in PET image and improvement of the motor symptoms (Bachoud-Levi, 2000). Other ongoing trials include those in: the United States (Tom Freeman, Tampa), UK (Steve Dunnet, Cardiff), and UK (Rosser et al 2002). Our centre participates in a multicentre trial since 2005, with 22 patients transplanted bilaterally until October 2012.
Treatment comprises the intrastriatal injection of neural tissue originated from the whole ganglionic eminence of legal-aborted fetuses between the 9 to 12 weeks.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 49
Grafting procedure was performed in 2 sessions separated for a period of 3 weeks, first on the right hemisphere and secondly on the left hemisphere. Patients were also transplanted into two main groups, early transplantation group, those transplanted 6 and 7 months after registration and those transplanted 26 and 27 months after initial enrollment (right-left in each case). All this because patients are part of a prospective randomized multicenter study, which will analyzed clinical result of the grafts and compare also early vs. late transplantation in a way to perform randomization.
Rationale behind transplantation in Huntington’s disease comes from the basis that HD causes a loss of neurons in the striatum leading to a severe atrophy up to 60% of the neurons in the caudate nucleus and the putamen, involving especially the type II Golgi-neurons ("medium spiny") and that it may be possible to reverse clinical deficits by implantation of fetal striatal neurons from the Ganglionic eminence [an anatomical structure with predominance of Gaba neurons which will eventually develop to form the striatum] (Rosser 2002, Bachoud-Levi 2000, Gaura 2004, Gallina 2008, Hauser 2002, Kopyov 1998, Reuter 2008)
Studies in humans are still experimental and HFST is not yet a define treatment, that’s why we wanted to describe early assessment of 22 patients transplanted bilaterally in our centre, as part of a multicenter clinical study, to assure that the procedure is safe and could be beneficial and not harmful for an already compromise brain. Even so, none of these strategies is available today and patients continue to die without an option of treatment that could at least achieve to stop rapidly progression accomplishing a period of stabilization of the disease.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 50
1.4 CELL TRANSPLANTATION IN THE CENTRAL NERVOUS SYSTEM
1.4.1 History
The history of cell transplantation in the nervous system is older that one may thing. It starts as early as 1890 and it can be studied according to Dunnett proposed classification in three eras:
Early era (1890-1940): Attempted cells sources came from tumors and model of transplantation site was the anterior eye chamber. This era results established that the adult mammalian brain is immutable and resistant to plasticity, growth or regeneration. It also served to address problems still present today, like the need for precise targets and the need for immunosuppression and its comorbidity.
Middle era (1940-1970): Attempted cell sources came from embryonic donor tissues and models of transplantation targets were sites with effective vascularization into the brain and neuroendocrine systems. Results were better than in the early era achieving some impressive results, such results were not concordant with the idea left from the early era that the brain does not show potential for plasticity and repair and were not totally accepted.
Modern era (since 1970): In this era improved anatomical transplantation techniques together with refined molecular, biological and cellular methods started to slowly bring acceptance that transplantation could eventually turn into a therapeutic modality strategy against injury or degeneration of not only the brain but also the spinal cord and nerves.
The interest in research on neural stem cells (NSC) observed in three last decades is mainly inspired by the hope of future application of the NSC-based technology in clinical therapeutic scenarios aiming at the substitution for neural tissue lost as a result of injury such as trauma, stroke or due to the neurodegenerative disorders, and thereby, the reconstitution of functional neuronal circuits. Although, due to the complexity of the human nervous system the prospect may seem remote, there is much evidence from the experimental animal studies and clinical trials (Ansari et al., 1996; Bjorklund 1992; Borlongan 1994; Brundin 1996; Dunnet 1993; Freed 2001; Isacson1995; Lindvall 1995, Madrazo 1995; Wijeyekoon and Barker 2011; Hauser 2002; Barker and Björklund 2013), showing that efficient neuronal replacement and at least partial regain of lost function are possible.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 51
1.4.2 Lesson learned for successful Grafting
Profit of CNS transplants depends on a. Upgrading host-graft variables b. Technical factors
Variables:
AGE OF DONOR AND HOST
-The optimal developmental age of the donor, is different for each specific neural substance needed according to the disease i.e. Ventral mesencephalon (VM) in PD; whole ganglionic eminence in Huntington’s disease (HD) (Junn and Lozano1996).
Optimal age will be defined as the time window matching proliferation and migration of neuronal and glial populations. It is during this period when tissue preparation for transplant may be least damaging (Jilek 1970).
The grafted fetal neurons apparently do conserve their biological clock proliferating and developing in the host brain as they will do naturally in normal development (Jaeger 1981).
It is estimated that approximately only 10 to 30 percent of grafted fetal cortical neurons survive transplantation in CNS (Björklund and Steveni 1979).
Survival transplantation of neural grafts for peripheral nervous system i.e. vagal nodose ganglia (Zalewski 1971), superior cervical ganglia (Rosenstein 1984), and adrenal glands (Freed 1983) are less influence by donor age.
The role of the host’s age regarding graft survival is not totally clear. Grafts from peripheral nervous system, Fetal cortical (Hallas 1980), hypothalamic (Kaplan 1985), cholinergic and noradrenergic grafts (Sladek 1982) experimentally showed not differences in graft proliferation associated to host age. However is to say that in general, older recipients had less tolerance mainly for more mature grafts.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 52
1.4.3 Sources of CNS for transplantation
Donor tissue possibilities can be separated into homotopic grafts and heterotopic grafts.
Homotopic graft: Tissue transplanted is from the same anatomic and functional area of the donor and the host. i.e embryonic neural fetal tissue.
Heterotopic graft: Tissue transplanted is from a different anatomic area of the donor compared to the host. i.e. Chromaffin cells form medullar adrenal gland.
Extend of cells survival and synapse formation tends to be greater in homotopic grafts.
The source of the CNS can also be distinguished between:
Autologous (autograft): a graft of tissue between individuals of the same species but of disparate genotype; types of donors are cadaveric, living related, and living unrelated
Xenografts: Tissue or organs from an individual of one species transplanted into or grafted onto an organism of another species, genus, or family i.e. use of ventral mesencephalon embryonic cells from pig in humans.
Nowadays, a variety of stem cell types are being tested in laboratories to generate material suitable for transplantation : primary cells: Astrocytes, Schwann cells, olfactory oligodendroglial cells, adrenal chromaffin cells; Genetically altered cells: Astrocytes, fibroblasts IPs Cells; Tumor cell lines: PC 12, B16HC29, ACT-20/hENK and Neural stem cells with none being secure enough yet to run a human trial. However, after decades of research grafts of primary CNS neurons keep on being the one cell lineage to survive when taken from fetal donors, being able to establish extensive reinnervation, neurotransmitters production and functional recovery (Capetian 2009; Olanow 2003; Politis 2012; Döbrössy 2010; Levy Y 2004; Ardvisson A 2002; Bjorklund and Lindvall 2000; Stanley F 2004). In the case of neurodegenerative diseases like Parkinson’s disease (PD) and Huntington’s disease (HD) this substitution has been already amply tested, based on the knowledge that both diseases share a common clearly almost specific loss of a defined neuron type in a certain anatomic structure, giving the opportunity for targeting specific neurons replacement.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 53
In the case of HD cells loss is prevalent on the efferent medium spiny GABAergic striatal interneurons while in PD such losses affects mainly the population of nigrostriatal dopaminergic neurons leading to a progressive reduction of the striatal neurotransmitter dopamine, necessary for motor, cognitive and behave control.
The clinical outcome of cell-based neuronal replacement therapy depends strictly on a multidisciplinary stepwise rigorous protocol including: adequate patient selection, expertise in dissection and preparation of the donor tissue and a precise stereotactic surgical implantation.
1.4.4 Techniques of transplantation
Transplantation techniques have a major impact on cell survival and synapses formation (Junn 1996). First reported surgery placed grafts into cortex achieving poor graft survival (Gash 1985), probably due to inadequate supply of nutrients and neurotrophic factors. Experience form rat models calculate graft revascularization between 3 to 5 days after transplantation. (Svendgaard 1975), One strategy employed to defeat this problem was implanting grafts into a preformed cavity (previously created by the surgeon) (Stenevi 1976). The hypothesis was that the formed cavities will secret neurotrophic substances that may enhance survival.
The ventricular system has been also used as a transplantation site. Injury of the ependyma has been involved in better graft survival (Rosenstein 1978).
This two initial used techniques, two-sateg grafting into a surgically performed cavity and single- sateg ventricular grafting allowed implantation of large grafts, however larger grafts are in high risks of central necrosis and show also less survival than smaller grafts. Intraventricular implantation has the additional risks of graft migration and rough graft placement.
Nowadays Stereotactic intraparenchymal implantation of grafts is the most common accepted safety technique, such technique pioneered by Schmidt et al in 1981,
Tissue pieces are mechanically dissociated into cell suspensions and implanted into the parenchyma. Neurons implanted as suspension show good survival and differentiation (Schmidt 1981), some groups however still use transplantation of small pieces of graft rather than a single cell suspension with very good results (Backlund 1985, Lindvall 1987). Stereotactic transplantation also allows recreating the circuit using multiple grafts in multiple targets.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 54
1.4.5 Immunologic reaction
Tissue rejection may occur in the CNS with allografts and xenografts transplants (Baker 1977, Björklund 1982, Low 1983). Grafts can be rejected when the host immune system recognizes molecules on the cells surface of the transplanted cells as foreign to the individual. Graft rejection could be prevented using immunosuppressive therapy especially during the initial days when the blood brain barrier is open (Newman 2009). The brain previously thought immunologically privileged due to the absence of specialized antigen-presenting cells, inconsiderable lymphatic drainage and the presence of the blood-brain barrier has been recently however proved wrong. There may be microglia cells with an antigen presenting cell capacity, the BBB can be cross by activated lymphocytes and it there is a lymphatic drainage from the brain into the cervical lymph nodes (Krystkowiak 2007). These findings should classify the brain as an organ where immune responses can occur and therefore be aware of the risk of graft rejection.
1.4.6 Mechanisms of Graft function
The rationale behind transplantation is to achieve functional improvement through these strategies: a. Replacement of neurons and glia b. Enhancement of neural repair and regeneration c. Modulation of altered neural activity (Junn and Lozano 1996)
The concept is to replace lost neurons and glia, reestablishing synaptic connections and achieving neurotransmitters secretion i.e. Dopamine in PD using fetal mesencephalic grafts.
The transplanted neurons in order to survive need to receive host innervation. Such innervation can be specific or not specific, the quantity of integration relays on characteristics of both graft an host, for the grafts its nature (origin) and maturity are important in the host the implantation site and it’s features are important. For the latest it has been proved that grafted fetal retinas only send synapses projections to the area receiving normally retinal fibers, (superior colliculi) and that such inervation was higher when the host eye was enucleated (Lund and Hauschka 1976, Ms Loon and Lund 1980). Same specificity in re-innervation is seen in grating of monoaminergic and cholinergic neurons into the denervated host hippocampus (Björklund A 1977). It is important to
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 55 address that there are exceptions to this explicitness: striatal cholinergic neurons can send projections to the hippocampus which is a target where this cells normally do not project (Lewis and Cotman 1983).
Other strategies in CNS transplantation include trying to increase levels of tropic factors and deliver substrates that help axonal regrowth to enhance axonal neural regeneration after injury. (Junn 1998), examples of this cells which have been implanted as a co-graft are: Schwann cells (Doering 1992, Collier 1990); amnion cells (Seigel 1994, Bankiewicz 1994), and genetically altered neurotrophin- secreting fibroblasts (Rosenberg 1988) and astrocytes (Stromberg 1990)
There is evidence that CNS may block regeneration of lessoned axons releasing inhibitory molecules i.e. NI-35, Ni 250, myelin associated glycoprotein (MAG) (Caroni 1988, Mc Kerracher 1994, Mukhopadhyay 1994). Such molecules inhibit neurite outgrowth by triggering a sensitive G-protein pathway, changing intracellular calcium leading to collapse of neuronal growth cones (Banddtlow 1993, Igarashi 1993)
1.4.7 Theoretical therapeutic applications
CNS diseases amenable to transplantation are summarized in Table 1. Transplantation succeed is most likely to happen in limited localized CNS damage rather than in in diverse areas.
Replacement/Regeneration Neurodegenerative diseases: Parkinson’s disease; Huntington’s disease; Alzheimer’s disease; Amyotropic lateral sclerosis Stroke Trauma Brain & spinal cord injury Hormone deficiency Modulation Epilepsy Pain
Table 1 Disease Potentially Amenable to Grafting (Modified from Junn & Lozano 1996) A diffuse disease process affecting multiple anatomic regions and neurons populations may not be achieve a positive clinical result. To restore a neural function the neural circuitry should be precisely rebuild such goal is easier to achieve when grafting non-synaptic mechanisms such
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 56 liberation of a deficient transmitter or trophic factor rather than reconstruction of synapses like in spinal cord injury.
PARKINSON’S DISEASE: It was this disease the one who made the field progress rapidly into clinical trials, once a satisfactory animal model was established. First attempts aimed replacing Dopamine (DA) by transplanting cells that were able to produce it. The animal models have shown proof of principle achieving behavioural improvement using fetal mesencephalic tissue and chromaffin cells (Lindval 1991). Other cell sources have been transplanted, autologous fibroblasts transfected with the gene for tyrosine hydroxylase achieving supporting results (Fischer 1991). Nowadays transplantation of ventral mesencephalic grafts into the putamen and/or caudate is the technique and cell source achieving the most promising results (Freeman 1995). A more detailed review about transplantation in PD will be giving ahead.
HUNTINGTON’S DISEASE: Huntington’s disease (HD) causes death of intrinsic striatal neurons and medium spiny projection neurons, resulting in diminution of some striatal neurotransmitters: gamma-aminobutyric acid (GABA), acetylcholine (Ach) and encephalin. Striatal DA levels remain normal. Animal models of HD lesion the striatum by excitatory amino acid (EAA) in rats (Mason 1978, Divac 1978) and monkeys (Hantraye 1992). Basal ganglia abnormalities in such models improve partially after fetal striatal transplantation (Mason 1978, Divac 1978, Hantraye 1992). The first human transplantation report in HD, date from 1993 when Madrazo and colleagues implanted a HD patient by an open microsurgical approach. Genetic engineer may be possible to tray soon since the gene responsible for HD is already been identified
ALZHEIMER’S DISEASE: Alzheimer’s disease (AD) causes a diffuse brain dysfunction; loss of neurons and depletion of Acetylcholine (Ach) in the basal nucleus of Meynert (bnm) are important events in its, strategies to replace Ach infusing it, as a medicament into the ventricles; have not shown consistent positive results (Harbaugh 1984).
Cholinergic projections from the septal area and noradrenergic projections from the locus caeruleus arrive into the hippocampus and are involved with processes of learning and memory. (Björklund 1976) such input has inspire animal models of AD; the strategy used in many experiments has been sectioning of the fimbria-fornix involving normal septal-hippocampal function by losing septal nucleus neurons in both monkeys (Kordower 1990, Koiatsos 1990, Ridley 1986) and rats (Nilsson 1990, Williams 1986). Fetal grafts implantation in those models
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 57 targeting septal area, locus caeruleus, and raphe have result in proper reinnervation of rat hippocampus, restoring levels of Ach, noradrenaline (NA), and serotonin (Nilsson 1990, Williams 1986, Richter-Levin 1989). Positive results extend to improving cognitive and learning deficits after fimbria-fornix lesions both in rats and nonhuman primates (Nilsson 1990, Low 1982).
One approach in the treatment of AD is prevention of major degeneration once it has already been diagnosed, this may be intent through administration of trophic substances necessary for cell survival (Junn & Lozano 1996). The medial septal cholinergic magnocellular neurons are the ones depending mainly on nerve growth factor (NGF) transported retrogradely from the hippocampus over the fornix (Kordower 1990, Low 1982) making transplantation of living tissues able to secrete NGF a principle logically alternative. Arenas et al. published in 1994 the use of fibroblast genetically modified who were able to produce neurotrophin-3, achieving noradrenergic neural rescue in the locus caeruleus of a 6-hydroxydopamine (6-OHDA) lesion model (Arenas 1994). More of this strategies are being study nowadays since they could theoretically been use to prevent the progression of AD and may have a wider use once specific neurotropic substances for each neuronal population are identified (Junn & Lozano 1996). Transplantation in humans requires further research which is not easy due to inadequate animal’s models that could characterize slow and global progression of AD.
STROKE AND TRAUMA: After stroke or traumatic brain injury a direct loss of neurons is caused in the affected region and a secondary indirect loss of neurons in other brain areas in caused by phenomenon like edema, isquemia, hypoxia and subsequent haemorrhage and anterograde and retrograde neuronal degeneration. Fetal cortical neurons grafted into infarcted basal ganglia areas achieved to reduce thalamic atrophy secondary to cortical ablation, by means of afford the necessary trophic factors (Sharp 1986, Sorenson 1989). Restoration of the circuit between the host thalamus and the transplanted neurons has been also achieved (Grabowski 1992). Animal models employing rats have suggest integration of the grafts by stimulation increasing glucose utilization in the transplanted neurons in cortex. However is still to be told is such approach will work in the human.
HORMONE DEFICIENCIES: Tissue grafts for treatment of hormone disorders emerge as a promising strategy due to two main factors: a. the quantity of cells needed is limited; b. the
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 58 necessity for synapse formation is less critical. The basis for proposing the alternative of neurons transplantation in humans to correct neuroendocrine disorders is mainly inspired on promising results in animal models: the Battleboro rat (Valtin 1967) and the hypogonadal mouse (Cattenach 1977). Battleboro rats are a strain of laboratory rat descended from a litter born in West Brattleboro, which suffer from chronic diabetes insipidus (DI) due to a naturally occurring genetic mutation losing the ability to produce the hormone vasopressin. Experiments grafting these animals with fetal preoptic hypothalamus cells from normal rats consisting mainly of vasopressin-positive cells into the third ventricle improved their DI (Gash 1980). Such effect is also achieved in hypophysectomised rats (Krieger 1982, Gash 1980).
Mice with a genetic inadequate gonadotropin secretion hormone (GnRH) course with hypogonadism and are sterile. Grafts of neurons transplanted from the preoptic nucleus into the hypothalamic region causes the development of secondary sexual characteristics and the ability to reproduce (Krieger 1982, Gibson 1984): Animal experiments have even achieved tissue survival of pituitary gland itself transplantation into the median eminence of hypophysectomised rats with normalization of blood levels of thyroxin, prolactin and luteinizing hormone (Tulpan 1985).
SPINAL CORD: Unfortunately, at the moment there's no way to reverse damage to the spinal cord. But, continue working aims to improve the function of the nerves that remain after injury. Either injury or disease two major factors lead to poor neurological recovery: a. neuronal loss; b. lack of regeneration of interrupted axons. Animal models achieved impressive recovery but nowadays such results have not been achieved in humans. Animal have transplanted embryonic brain stem noradrenergic and serotoninergic neurons in transected spinal cord model looking to achieve extend processes of the descending projections of brain stem monoaminergic neurons which help to regulate the intrinsic spinal neurons responsible for locomotor and autonomic functions, results are extend processes (synapses) up to 2 cm (Björklund 1986, Privat 1989). Such grafts restore levels of monoamines in a transected spinal cord which traduces in recovery of crude reflex locomotive and sexual function (Mas 1985, Nygren 1977) but not discriminatory sensation of fine motor control. Other use of transplants is as a source of trophic factors to rescue axotomized neurons, such strategy intent a reduction of astrocytic scarring (Bregman 1986, Reier 1986, Houle 1988) again results are impressive in the rat model but this has not been translated into the human. Animal models have been experiment also transplanting glia to promote remyelination after a demyelinating process or spinal cord injury. One model used local
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 59 application of ethidium bromide an irradiation (Blakemore 1985) the other employed a genetic mutant deficient in myelin mouse model (Shiverer mouse) both achieved survival and remyelination at variable degree. (Blakemore 1991) A human model employs oligodendrocytes cells from olfactory bulb with not such positive results (Lima 2006)
EPILEPSY: Animal models of epilepsy and human cortical tissue found decreased level of the inhibitor neurotransmitter GABA, increasing GABA transmission in the substantia nigra (SN) of rodents have shown reduced seizure generalization (Iadarola 1982). Therefore, transplants able to achieve production of inhibitory neurotransmitters may be able to control seizure propagation (Loscher 1987)
PAIN: Adrenal chromaffin cells release catecholamines and opioids both substances that can alter the painful sensation in the dorsal horns of the spinal cord. Enkephalins produce an analgesic effect when delivered into the periaqueductal gray matter. Subarachnoid transplantation of chromaffin cells has achieved pain reduction in a rat model of acute pain (tail flick testing) (Segan 1991). B16F1C29 melanoma cells which release catecholamine and ACT-20/hENK cells, which produce proenkephalins are genetic modified cell lines that when transplanted into the lumbar subarachnoid space ameliorate acute pain response (Wu 1994). Bovine chromaffin cells and geneticically altered cells have been pack as a biopolymer capsule for transplantation (Aebischer 1994) running a European human trial.
FUTURE DIRECTIONS: Rationale for neural transplantation is to compensate or replace deficient neural functions caused by degenerative diseases, vascular events or traumatic or genetic loss by means of engrafted cells and the molecules they produce. Primary tissues are considered safe but there are immunosuppressant secondary complications as well as practical and ethical problems of tissue harvesting and handling before implantation (Junn & Lozano 1996). Such inconvenience could overcome by using cell lines. Those cell lines include: neural stem cells, genetically modified cells, and adult modified stem cells. Future improvements will depend on gaining a better understanding of the neuroanatomical and neurophysiological deficits in neurological diseases (Junn & Lozano 1996).
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 60
1.4.8 Restoration of the CNS using progenitor cells from adrenal medulla
On the search of other sources of cell lineages that could avoid the use of fetal cells due to their still important ethic issue together with the difficulty to acquire enough number of embryos for each transplant and the complications derived from immunosuppression, we studied the viability of Progenitor Cells from Bovine and Rat adult adrenal Medulla transplanted into SD rats after an Isolation original protocol in collaboration with the University of Dresden. Such in vitro study demonstrated the presence of progenitor cells in the bovine adult adrenal medulla and recently also in the human adult adrenal medulla, disclosing their potential to become a candidate cell lineage to be use in neuroendocrine and neurodegenerative diseases. (Santana M, Ehrhart- Bornstein M 2012).
A neural crest progenitor cell persist within the adult adrenal medulla, such Sympathoadrenal (SA) progenitor cells are the origin of Chromaffin cells, sympathetic neurons of the dorsal ganglia and the intermediate small intensely fluorescent cells. (Santana et al 2012)
The human (and mammalian) nervous system comprises two parts: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS consists of the brain and the spinal cord. The PNS is composed of: motor neurons, the autonomic nervous system and the enteric nervous system.
There are two kinds of cells in the PNS: sensory nervous cells and motor nervous cells, the first are afferent and carry information from internal organs and external stimuli to the CNS while the motor nervous cells carry efferent information from the CNS to organs, muscles and glands.
The motor nervous system is as well divided into two groups: the somatic nervous system and the autonomic nervous system, the first one is voluntary and controls the external sensory organs (i.e skin) and the second one is the involuntary, (vegetative or autonomic) which controls involuntary muscles and processes as smooth and cardiac muscle.
The autonomic nervous system (ANS) is divided as well into two major parts the parasympatethic division and the sympathetic division, the first one control various processes including inhibiting heart rate, constricting pupils, contracting the bladder. The second one: the sympathetic nervous system (SNS) mobilizes the body's nervous system to fight-or-flight response. It is, however, constantly active at a basic level to maintain homeostasis. Having an
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 61 opposite effect raising heart rate, dilating pupils and relaxing bladder amongst others, it is also responsible for the response to danger increasing heart rate and metabolic rate.
The ANS is a visceral efferent motor system which function is to control and regulate smooth muscle, cardiac muscle and glands.
It consists of two kinds of neurons:
1. Preganglionic neurons
2. Postganglionic neurons
The ANS has three divisions:
1. Sympathetic
2. Parasympathetic
3. Enteric (mesh-like system of neurons commanding the function of the gastrointestinal system)
The ANS has five neurotransmitters:
1. Acetylcholine: neurotransmitter of the preganglionic neurons
2. Norepinephrine: neurotransmitter of the postganglionic neurons (except sweat glands and some blood vessels which receive sympathetic cholinergic innervation)
3. Dopamine: neurotransmitter of the small intensely fluoresecent (SIF) cells (interneurons of the sympathetic ganglia)
4. Vasoactive intestinal polypeptide (VIP): vasodilatador in some postganglionic parasympathetic fibers.
5. Nitric oxide (NO): neurotransmitter that causes relaxation of smooth muscle (ie. penile erection).
Chromaffin cells which are the cells of our interest are neuroendocrine cells like sympathetic neurons. They originate in the neural crest; a transitory structure located at the outer surface of the embryonic neural tube. (Huber K 2009).
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 62
These neuroendocrine cells are located in the adult mammalian both in the adrenal gland (located above the kidneys) and in extra-adrenal paraganglia: the carotid arteries bodies near the sympathetic ganglia, vagus nerve and the organ of Zuckerkandl, smaller concentrations of extra- adrenal chromaffin cells are also found in the bladder wall, prostate, and behind the liver.
These cells were named Chromaffin (“chromium” – “affinity”) by histologist Alfred Kohn (1867- 1959) because they can be visualized by staining with chromium salts.
Chromaffin cells release Catecholamines: ~80% of Epinephrine (Adrenaline) and ~20% of Norepinephrine (Noradrenaline), a little dopamine, enkephalin and enkephalin-containing peptides, and a few other hormones into systemic circulation for systemic effects (Erlich 1994).
By development chromaffin cells are similar to sympathetic neurons, both cells are able to synthesize, reserve, discharge Catecholamines, vesicular monoamine transporters (VMAT) as well as enzymes for synthesis of noradrenaline: tyrosine hydroxylase (TH) and dopamine-ß hydroxylase (DBH) (Huber 2009).
However there are morphological differences between these two cells: the lack of axons and dendrites of chromaffin cells (which are present in sympathetic neurons), big chromaffin granules (~130-230 nm).
Chromaffin cells synthetize mainly adrenaline (epinephrine) while sympathetic neurons noradrenaline (norepinephrine) both however are innervated by pre-ganglionic axons originated from neurons cell bodies located in the intermediolateral column of the spinal cord (Schober and Unsicker 2001)
The sympathetic system employs two neurons to transmit any signal: a pre-ganglionic neuron and a post-ganglionic neuron
Pre-ganglionic neurons originate in the spinal cord at the thoracolumbar region (levels T-1 trough L-3) and travel to a ganglion (mass of nerve cell bodies) –ganglia of sympathetic trunk or paravertebral ganglia where they synapse with the post-ganglionic neuron, such post-ganglionic neurons are long and extent through most of the body (Drake 2005).
Pre-ganglionic neurons release acetylcholine at the ganglia synapses, such neurotransmitter stimulates nicotinic acetylcholine receptors on post-ganglionic neurons, and once post-ganglionic
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 63 neurons are stimulated they release norepinephrine activating adrenergic receptors on the peripheral target tissues (i.e. heart, bronchial tree, blood vessels).
There are two post-ganglionic neurons that behave different after pre-ganglionic stimulation, the first one are post-ganglionic neurons of sweat glands which instead of releasing norepinephrine after acetylcholine stimulation, liberates acetylcholine to activate muscarinic receptors (more sensitive to muscarine than to nicotine) and the Chromaffin cells of the suprarenal adrenal medulla acting as a modified sympathetic ganglion which when stimulated by the pre-ganglionic neurons release norepinephrine and epinephrine directly into the blood (Silverthorn 2009).
There is a third cell type with similarities to sympathetic neurons and chromafin cells, the “small granule containing” or “small intensely fluorescent (SIF) cell (intense catecholamine specific histofluorescence). SIF cells can be found in the adrenal gland, in paraganglia and sympathetic ganglia (Huber 2009).
Chromaffin cells originate from neural crest (Le Dourain and Teillet, 1974).
The neural crest cells are a transient group of progenitor’s cells that completely delaminate and migrate to specific regions once the neural tube completely separates from the ectoderm during embryogenesis. Although derived from the ectoderm, the neural crest originates at the dorsal region of the neural tube and has been called the fourth germ layer because of its importance. These migratory cell populations generate a prodigious number of differentiated cell types. Including: (a) the neurons and glial cells of the sensory, sympathetic, and parasympathetic nervous systems, (b) the epinephrine-producing (medulla) cells of the adrenal gland, (c) the pigment-containing cells of the epidermis (melanocytes), and (d) many of the skeletal and connective tissue components of the head (craniofacial cartilage and bone) (Gilbert, Sunderland 2000).
Adrenal chromaffin cells derive from crest cells exiting the dorsal tube at somatic levels 18-24 (adenomedullary level) (Gilbert, Sunderland 2000). After delamination neural crest cells migrate through a ventral position. SoxE is expressed by neural crest cells (Cheung et al, 2005). Sox8 and Sox10 are expressed by Neural crest cells migrating to the adrenal gland (Reiprich et al. 2008). The lack of Sox10 causes complete agenesis of the adrenal medulla, secondary to cell death during migration (Reiprich et al 2008).
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Both sympathetic neurons and chromaffin cells originate from catecholaminergic neuronal progenitors denominated: sympathoadrenal progenitors (SA), collecting cell groups in the near of the dorsal aorta forming the primary sympathetic ganglia (Anderson and Axel 1986; Anderson et al, 1991) migrating then to the secondary sympathetic ganglia to differentiate into mature sympathetic neurons and the adrenal medulla, where they differentiate into chromaffin cells (Santana 2012).
SA cells immigrating from the adrenal gland express neuron-specific markers like SCG10 and neurofilament (Anderson and Axel 1986). In the enclosure of the dorsal aorta Bone morphogenetic proteins (BMP) -2/4/7 are synthesized which is fundamental to instruct neural crest cells pointing to a catecholaminergic and neuronal fate (Reissman et al 1996; Shah el atl, 1996; Varley et al,. 1995, Scheneider et al., 1999; Bilodeau et al., 2001).
A network of transcription factors is activated in response to BMPS: Mash 1/Cash1, Phox2A/B, Hand 2, Gata2/3 and Isnm1, which promote further development (Guillemot et al., 1993).
Observations in mouse embryos conclude that many progenitors of chromaffin cells migrate to the adrenal gland as “undifferentiated” neural crest cells, expressing Sox 10 (general neural crest marker). SA markers like TH, Phox2B and Mash1 have been also observed en route to the adrenal gland (Anderson and Axel 1986). Chromaffin progenitors may receive also an instructive BMP-4signal from tissues other than the aorta (periadrenal tissues) (Gut et al., 2005).
Once in the adrenal medulla, chromaffin cells do specialize to produce and secret catecholamines as well as other bioactive substances like peptides, cytokines, enkephalines and neurotrophic factors (Cavadas C 2001, Crivellato 2008).
Chromaffin are special types of cells for two major caractheristics: a. The possibility to proliferate in an adult state b. The possibility to convert in a neural-like phenotype when receives a neurogenic stimulation (Doupe 1985., Sicard 2007., Tischler 1989).
Such properties stimulated the study of chromaffin cells as a possible cell lineage to autologous progenitors-neurons transplantation in the treatment of neurodegenerative diseases and pain. (Backlund EO 1985, Madrazo I 1987, Francis NJ 1999, Lazorthes Y 2000,Drucker-Colin R 2004,
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 65
Jozan S 2007). The first human transplant of medullary tissue was reported in 1985 by Backlund et al, when he transplanted a PD patient with courageous results, starting an enthusiastic boom with different series of cases of different groups showing in some of them clinical improvements that were not sustainable more than 1-2 years after transplantation due to low survical rates of the grafts in the brain (Drucker-Colin R 2004).
Currently molecular biology has open new avenues to try to avoid mistakes from the past improving the protocols for a potential autologous use of adult chromaffin progenitor cells. On that perspective the University of Dresden has isolate chromaffin progenitor cells from bovine adrenal medulla (Chung KF 2009, Vukicevic V 2012). Similar to neural stem cells, they grow as free-floating spheres (chromospheres) and differentiate into functional neurons, which at least in vitro is a new promising strategy for the regenerative treatment of neurodegenerative diseases.
The same strategy demonstrated the presence of progenitor cells in the human adrenal medulla revealing their potential future use in neuroendocrine and neurodegenerative diseases (Santana M, Erhart-Bornstein M 2012).
In this project we aimed to transplant such isolated cells from adult bovine and rat adrenal glands and transplant the cells into a SD rat PD’s model.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 66
2. OBJECTIVES
The concept of transplantation of neuronal cells to treat Huntington’s and Parkinson’s diseases is based on the proven principle that dopaminergic and GABA-ergic progenitor neurons (from the human developing ventral mesencephalon and whole ganglionic eminence) can: survive, differentiate and functionally integrate into an allogenic host brain. However, several donor and host-specific variables play a major role in the safety and outcome of this procedure. The main goal of this thesis was to analyze some of these involved factors seeking to summarize an updated clinical cell transplantation protocol from tissue donation to brain transplantation and follow up, including a detailed description of the human nigral and striatal dissection stages.
This thesis also comprises in vitro and in vivo experimental work based on the search of other sources of cell lineages that could avoid the use of fetal cells; we studied the viability of neural crest derived chromaffin progenitors cells isolated from adult adrenal medulla after grow them in chromospheres to significantly increase the number of TH positive dopaminergic neurons.
The following specific aims were addressed in this thesis:
1. To present a detailed clinical neurotransplantation protocol for PD and HD with special emphasis in understanding the anatomical relationships of the human fetal tissue that are relevant for selection of the desired donor’s cells population.
2. To present self-developed software to optimize the surgical stereotactic planning for human bilateral neurotransplantation procedures. The software allows close to symmetrical distribution of the stereotactic coordinates in relation to the mid-commissural point (MCP).
3. To assess the viability of cultured chromosphere grafts from chromaffin progenitor’s cells isolated from adult adrenal medulla and its effect on behavior.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 67
3. MATERIALS
3.1 Basic Equipment
Equipment Suplier
T75 Culture Flasks Falcon, BD, USA
24 well cell culture plates Falcon, BD, USA
Examination gloves Ansell, Germany
Falcon cell strainer 70µl BD Biosciences, USA
Falcon cell strainer 100µl BD Biosciences, USA
Gilson Pipettes Abimed, Germany
Glass beakers Roth, Germany
Nitrile gloves Nitratex Ansell, Germany
Plastic tubes Greiner, Germany
Pipett tips Biozym, Germany
Plastic Pipettes, sterile Costar, USA
Well plates 12, 24 wells Falcon, Germany
Cell counting chamber Neubauer
(0.0025 mm2; 0.1mm depth) Brand, Germany
Cotton tip applicators 15cm Dalhausen, Germany
Disposable hypodermic needles
0,45x12mm 26Gx1/2 Braun, Germany
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Dry ice and wet ice
Filter papers 210mm diameter Roth, Germany
Food pellets Campden Instruments, UK
Glass vials Rotilabo, 15ml Roth, Germany
Luer-syringes 10ml, 20ml, sterile Braun, Germany
Micro-centrifuge-tubes Greiner, Germany
Microscope slides and cover slips R. Langenbrinck, Germany
Omnifix-syringes 1ml, sterile Braun, Germany
Parafilm PM-996 Pechiney, USA
Pipetboy acu Integra Biosciences, Switzerland
Pipette tips Braun, Germany
Pipettes Gilson: 2µl, 10µl, 20µl, 100µl, 200µl, 1000µl Abimed, Germany
Plastic pipettes, sterile 1, 2, 5, 10, 25, 50 Costar, USA
Polypropylene conical tube: 15ml, 50ml, sterile Greiner, Germany
Rotilabo syrigue filter 0.22 µm Roth, Germany
Safe-lock tubes 0.5ml, 1.5ml and 2.0ml Eppendorf, Germany
Tissue culture dish 0/20MM, sterile Greiner, Germany
Tissue culture flask 250ml Greiner, Germany
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 69
3.2 Technical Equipment
Cell culture clean bench HeraSafe Kendro, Germany
Cell culture incubator 37°c Heraeus Function line Kendro, Germany
Centrifuge Megafuge Heraeus, Germany
Cryotome SM 2000 R Leica, Germany
Ice machine Ziegra, Germany
Magnetic heating plate Heidolph, Germany
Microcentrifuge 5417R Eppendorf, Germany
Micropipette puller P-97 Sutter, USA
Mamocytometer, Neubauer Brand, Germany
Microwave Siemens, Germany
Paxinos and Watson atlas oft he rat brain Paxinos and Watson 1997
PH-Meter pH 320 WTW, Germany
Platform shaker IKA-Vibrax VXR Janke & Kunkel, Germany
Refrigerator +4°C and freezer -20°C Liebherr, Germany
Rotation boxes and paw-reaching boxes University of Hannover, Germany
Thermomixer 5436 Eppendorf, Germany
Vaccum pump n° 02046 Schütt, Germany
Vortex KS250 basic Janke & Kunkel, Germany
Waterbath Haake SWB25 Haake, Germany
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3.3 Dissection, surgery and perfusion Instruments
Watchmaker’s forceps No.5 Dumont, Switzerland
Iridectomy scissors Dumont, Switzerland
Microscissors OC 498 and 010R Aesculap, Germany
Dissecting Scissors BC 561R, BC 103R and BC 140R Aesculap, Germany
Dissecting Forceps BD 215R, BJ 009R and BD 335R Aesculap, Germany
Microforceps BD 333R and BD 335R Aesculap, Germany
Tissue Forceps BD 559R Aesculap, Germany
Suture Clips BN 507R Aesculap, Germany
Glass capillaries, 50µm diameter University of Freiburg, Germany
Hamilton microliter syringes 2µl and 10µl Hamilton Europe, Switzerland
Isoflurane-vaporiser University of Freiburg, Germany
Microdrill: Proxxon Micromot 40 Proxxon, Germany
Perfusion pump Neolab no. 30001921 NeoLab, Germany
Shaving machine Braun, Germany
Stapler Aesculap, Germany
Sterile disposable scalpel no. 10 Feather Safety Razor, Japan
Sterile disposable scalpel no. 11 Feather Safety Razor, Japan
Lab Standard stereotactic frame no. 51600 Stoelting Co., USA
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3.4 Software analySIS Software Imaging System Soft Imaging Systems, Germany
Microcal Origin Microcal, USA
Microsoft Office Microsoft, USA
Rotation behavior analysis software University of Hannover, Germany
StatView data analysis & presentation system Abacus, USA
Stereo Investigator, version 5.05.1 MicroBrightField, USA
3.5 Microscopes
Axiovert 135 Zeiss, Germany
Dialux 22 Leitz, Germany
Leica MS5 dissection microscope Leica, Germany
Leica DMRB with camera Sony DXC 950P Leica, Germany
Operation microscope system Yasargil Studer, Switzerland
Olympus AX70 with camera Olympus C-12 Olympus, Germany
Confocal laser scanning microscope Leica, Germany
3.6 Chemicals
6- OHDA (6- Hydroxydopamine- hydrochloride) Sigma, USA
ABC- standard elite kit
(Avidin- biotinylated horseradish peroxidase complex) Vectasin, USA
Apomorphine hydrochloride Sigma, USA
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Bepanthen eye ointment Roche, Germany
Borgal anibiotic solution 24%
(Sulfadoxin + Trimethoprim) Intervet, Germany
Ciclosporin: Sandimmun®
Immunosuppressant 50mg/ml Novartis, Gemany
DAB buffer tablets (Diamino benzidine) Merck, Germany
D- amphetamine- sulfate Sigma, USA
DAPI (4’, 6- Diamidino- 2- Phenylindole
Dihydrochloride hydrate) Sigma, USA
EDTA (Ethylene diamine tetraacetate) Sigma, Germany
Ethanol 99.9% Baker, Netherlands
Ethylene glycol Merck, Germany
Fluorescent mounting medium S3023 Dako, USA
Forene Isoflurane 250ml Abbott, Germany
Glycerine 3T012 Waldeck, Germany
Glycerol 87% Merck, Germany
HCl 37% (hydrochloric acid) Merck, Germany
Histofluid mounting medium Zitt -Thoma, Germany
Hydrogen peroxide Merck, Germany
KCl (potassium chloride) Sigma, USA
Ketamine 10% Essex, Germany
KH2 PO4 (potassium dihydrogen phosphate) Merck, Germany
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 73
L+ Ascorbic acid Merck, Germany
Methanol Merck, Germany
MgCl (magnesium chloride) Sigma, USA
Na2 HPO4
(Disodium hydrogen phosphate) Merck, Germany
NaCl (sodium chloride) Sigma, USA
NaN 3 (sodium azide) Merck, Germany
NaOH (sodium hydroxide) Baker, USA
PFA (paraformaldehyde) Merck, Germany
Povidon- iodine disinfectant Mundipharma, Germany
Proteinase K Merck, Germany
Rompun 2% Bayer, Germany
Saline (sterile 0.9% sodium chloride solution) Braun, Germany
Sucrose, molecular biology grade Calbiochem, USA
Tissue tec O.C.T. compound Sacura, Netherlands
Tris- HCl Merck, Germany
TritonX- 100 Sigma, USA
Trypan blue solution 0.4% Sigma, USA
Tween - 20 Calbiochem, USA
Xylol Roth, Germany
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 74
Antibodies:
Anti-TH mouse primary antibody T- 1299 Sigma, USA
Rabbit anti-mouse secondary antibody E0464 Dako, USA
Goat anti-mouse secondary antibody with Alexa 568 Sigma, USA
Rabbit anti-5HT primary antibody #11160 MP Biochemicals, Germany
Rabbit anti-TH primary antibody PRD - 515P Covance, USA
Goat anti-rabbit secondary antibody E0432 Dako, USA
3.7 Cell culture
B27 cell- culture supplement Gibco Invitrogen, Germany
Bovine serum albumine Sigma, USA
BSA (bovine serum albumine) Sigma, USA
DMEM (Dulbecco’s modified eagle medium) Gibco Invitrogen, Germany
FCS (Fetal calf serum) Gibco Invitrogen, Germany
HBSS (Hanks’ Balanced Salt Solution, Ca2+ and
Mg2+ free) Gibco Invitrogen, Germany
L - Glutamin 200 mM, sterile Gibco Invitrogen, Germany
N2 cell-culture supplement Gibco Invitrogen, Germany
Neurobasal medium Gibco Invitrogen, Germany
PBS (Phosphate buffered saline) 1x, sterile Gibco Invitrogen, Germany
PenStrep supplement (penicillin/streptomycin) Gibco Invitrogen, Germany
Poly L-ornithine Sigma, USA
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Poly L-lysine hydrobromide Sigma, USA
Trypsine Worthington, USA
20 ng/ml FGF-2 Prepotech, USA
PSA Gibco, USA
AccutaseTM PAA Laboratories GmbH, Germany
Neurobasal Gibco, USA
DNase I Worthington, USA
3.8 Solutions
ABC - Complex
2 drops of substance A and 2 drops of substance B of the ABC-standard elite kit dissolved in 10ml 1xPBS and incubated for 30min at room temperature before use.
Amphetamine solution (0.25%)
2.5mg of D - amphetamine- sulfate were dissolved per 1ml 0.9% sterile saline.
Antifreeze medium
For 1l medium, 400ml 1xPBS, 300ml glycerol and 300ml ethylene glycol were mixed at room temperature.
Apomorphine solution (0.05%)
0.5mg of apomorphine hydrochloride and 2mg of ascorbic acid were dissolved per 1ml
0.9% sterile saline and kept in dark at +4°C.
Borgal solution for antibiotic protection of immuno- suppressed adult rats
4ml Borgal were dissolved per 1l drinking water and given to the immuno-suppressed rats ad libitum.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 76
Blocking solution (BSA 3%)
30mg BSA and 3µl TritonX- 100 dissolved per 1 ml 1xPBS.
Ciclosporin solution
10mg of ciclosporin were dissolved per 1ml sterile 0.9% saline.
DAB buffer solution
1 tablet DAB was dissolved in 10ml 1xPBS. Immediately before use, 1 µl H 2 O2 was added.
6- OHDA solution for MFB- lesion of adult rats (0.36%)
3.6mg 6- hydroxydopamine-hydrochloride and 0.1µg ascorbic acid were dissolved in 1ml sterile saline, resulting in a 1.75x10-2M solution of 6- OHDA HCl.
PBS 10x
80g NaCl, 2g KCl, 7.7g Na 2 HPO4 x H2 O, 2g KH2 P O4 were dissolved up to 1l distilled
H2O. For experimental use diluted 1:10 to 1x PBS with pH=7.2.
PFA 4%
Materials: Parafolmaldehyde powder (Merck #1040051000 1kg/Bessy #5400), NaOH-tablets, 10xPBS solution, 1 x 3l beaker, 2x1l beaker, 3 x 10l containers for 1xPBS and 4%PFA, 1 x larger magnet stir-bar, 1 x 2l measuring cylinder, paper filters, large scale spatula und disposable weighing dish, heat-stirrer, microwave, protection (i.e. appropriated gloves, face mask, goggles), pH-meter . 40g of PFA were added to 800ml 1x PBS at 65°C. The mixture was transferred to a fume hood and maintained on a heating plate at 60°C with stirring, until the PFA was dissolved. The pH was adjusted at 7.2 with NaOH or HCl and the volume was made up to 1l.
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4. METHODS
4.1 Project I “Clinical neurotransplantation protocol for Huntington’s and Parkinson’s disease”
The concept of transplantation of neuronal cells to treat Huntington’s and Parkinson’s diseases is based on the proven principle that dopaminergic and GABA-ergic progenitor neurons (from the human developing ventral mesencephalon and whole ganglionic eminence) can: survive, differentiate and functionally integrate into an allogenic host brain. However, several donor and host-specific variables play a major role in the safety and outcome of this procedure. In this paper, we seek to summarize an updated neural transplant transplantation protocol, based on our institutional experience and many years of collaboration with other neurotransplantation centers aiming to ensure high processes quality, necessary for minimizing unwarranted complications.
We present a detailed clinical neurotransplantation protocol for Parkinson’s (PD) and Huntington’s (HD) diseases emphasizing the anatomical relationships of the human fetal tissue that are relevant for appropriate dissection of desired cell populations.
Author contributions:
The author was responsible for the conception, organization and execution of this project and managing communication between all co-authors. Professor author of this project was Jaroslaw Maciaczyk with important contributions from Prof. Guido Nikkhah. Writing of the first draft was performed by the author with supervision from Jaroslaw Maciaczyk. All dissections and pictures were realized by the author and Jaroslaw Maciaczyk. All co-authors contributed on the writing, review and critic of the manuscript giving input based on their enormous experience on the field and co-affiliations with other experienced transplantation centers. Co-authors of this project were: Guido Nikkhah, Ulf D. Kahlert, Donata Maciaczyk, Tomasz Bogiel, Sven Moellers, Elisabeth Schültke, Máté Döbrössy, and Jaroslaw Maciaczyk.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 78
4.2 Project II “Stereotactic Planning Software for human neurotransplantation: Suitability in 22 surgical cases of Huntington’s disease”
The main technical challenge in surgical neural transplantation is that in order to reconstruct striatal circuits, multiple targets with multiple cell deposits are needed in an atrophied brain. These targets can only be selected based on magnetic resonance anatomy without electrophysiological confirmation. This makes surgical planning a time consuming step during surgery that is well suited for optimization. The aim of this work was to provide an atlas-based coordinates method for identification of the six targets to be used in transplantation treatment planning. The coordinates of putamen and caudate nucleus were identified in Three-Tesla Magnetic Resonance imaging and transformed into mid-commissural point (MCP) coordinates. From these an electronic coordinate’s atlas was built up. For the second contra-lateral transplantation in the same patient, the coordinates were mirrored in order to determine contralateral targets and tracts. An assessment and safety analysis of the transplantation procedure was performed.
Author contributions:
The author altogether with Michael Trippel formulated the problem and hypothesis, structured the experimental design, organized and conducted the statistical analysis and interpreted the results. Writing was performed by the author and Michael Trippel. The author is also responsible for managing communication between all co-authors. The software here presented is an original idea and development from Michael Trippel. All co-authors contributed on the writing, review and critic of the manuscript. Other co-authors of this project were: Guido Nikkhah, Elisabeth Schueltke and Luciano Furlanettil.
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 79
Cortical and subcortical brain atrophy is a common feature in HD, making stereotactic planning a tedious procedure with risk of surgical complications and suboptimal spatial distribution of the grafts. Here we present self-developed software to optimize the surgical stereotactic planning for bilateral neurotransplantation procedures. It allows close to symmetrical distribution of the stereotactic coordinates in relation to the mid-commissural point (MCP), proposing automatically the planning coordinates for the first transplanted hemisphere transplantation and mirrored coordinates to be used in the contra-lateral hemispheres
Twenty two consecutive patients of genetically confirmed HD underwent bilateral stereotactic HFST using six striatal trajectories: Two in the head of the caudate nucleus and four in the putamen, employing two 6mm width burr holes on each hemisphere as entry points.
All patients had a clinically symptomatic and genetically confirmed Huntington’s disease (number of CAG repeats >= 36), were between the ages of 25 and 65 years old, with an early to moderate stage of the disease. All had a UHDRS motor scores >/= 5 on the Unified Huntington's Disease Rating Scale and a largely preserved autonomy in everyday life. The procedure was approved by a local Ethics committee at the Freiburg University Medical Center as part of a European multicentre study: MIG-HD study. Only patients who were aware of the full extent of their illness, and that have appropriate decisional capacity and legal empowerment, were included giving their informed consent to medical care. Patients that had advanced disease, severe comorbidity, psychiatric disturbances, or severe cortical atrophy on the CT and MRI images and/or positive HIV serology (HIV1, HIV2, and AgP24), active hepatitis (B and C) or HTLV 1 and 2 were excluded (Table 2).
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 80
Time Patient Age Sex CAG Symptoms KPS% between Tx #Tracts #Embryos (Years) duration procedures Group (Years) (Weeks) R L R L 1 43 M 44 8 70 9 B 6 6 2 2 2 45 M 46 9 60 10 B 6 6 2 2 3 42 M 44 8 90 4 A 5 5 2 2 4 50 F 46 5 80 1 B 6 6 4 2 5 32 F 51 3 80 14 A 6 6 2 2 6 45 F 47 12 70 2 A 6 6 4 4 7 36 F 45 4 90 10 A 6 6 2 4 8 38 M 43 5 70 3 B 5 6 2 2 9 31 F 41 7 60 4 B 6 5 2 2 10 46 F 50 3 70 5 A 6 6 2 3 11 35 F 50 3 90 3 A 6 6 2 2 12 39 M 42 5 90 7 B 6 6 2 1 13 54 M 46 4 90 7 A 6 6 2 2 14 50 M 44 8 80 9 A 6 6 1 2 15 41 F 43 7 80 3 A 6 6 2 2 16 49 F 43 5 80 7 A 6 4 2 2 17 40 M 45 9 70 3 B 6 6 2 2 18 50 M 42 7 60 3 A 6 6 2 2 19 41 M 44 4 70 3 A 6 6 2 2 20 38 M 51 6 80 3 A 6 6 2 2 21 46 F 46 4 80 4 A 6 6 2 2 22 30 F 46 3 80 7 A 6 6 2 2 M 41.8 45.4 5.8 76.8 5.5 5.9 5.8 2.1 2.1 SD 6.64 2.87 2.43 9.94 3.3 0.2 0.5 0.6 0.6
Table 2 Data of stereotactic procedures in 22 HD patients undergoing HFST with a two entry points approach. CAG: Genetic CAG repeat expansion; M: Mean, SD*: Standard deviation; KPS: Karnofsky index performance scale; Tx group: Transplantation group, early (A) vs. late (B); R: right, L: left.
Patients treated were assessed preoperatively for at least six months and postoperatively by clinical, neuropsychological, psychiatric, neurophysiological and neuroimaging (MRI and PET) tests.
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Transplantation surgery was performed in two separate surgical sessions, with a minimum interval of two weeks. The first transplantation was performed always in the right hemisphere, followed by a second surgery into the left hemisphere.
Transformation of Stereotactic Coordinates to Mid Commissural Point (MCP) Coordinates The MCP coordinates system
For every procedure; the stereotactic coordinates or the mid commissural point ⃗⃗⃗⃗⃗ ⃗⃗⃗ ⃗ were calculated as middle of the two vectors ⃗⃗⃗⃗ ⃗ and ⃗⃗⃗⃗ ⃗ ( ⃗⃗⃗⃗⃗ ⃗⃗⃗ ⃗ = ( ⃗⃗⃗⃗ ⃗ + ⃗⃗⃗⃗ ⃗ ) / 2). The unit vectors defining the MCP coordinate system for each patient were determined as follows: the stereotactic coordinates ⃗ , , ⃗⃗ of the anterior commissure (AC), the posterior commissure (PC), the upper (MU) and lower midline (ML) points were determined forming the vectors ⃗⃗⃗ = ⃗⃗⃗⃗ ⃗ - ⃗⃗⃗⃗ ⃗ and ⃗⃗ = ⃗⃗⃗⃗⃗ ⃗ - ⃗⃗⃗⃗⃗⃗ . The cross vector product ⃗ of and ⃗⃗ was calculated ( ⃗ = x ⃗⃗ ⃗⃗ ) indicating to the right direction. The cross product ⃗ of the vectors ⃗ and gave the third upwards-directed coordinate ( ⃗ = ⃗ x ). These vectors ⃗ , , ⃗ were normalized to the unit vectors , ⃗ , showing right, anterior and upwards ( = ⃗ / |R |; ⃗ = / | |; = ⃗ / | ⃗ |). These three orthogonal unit vectors built up the lines of the matrix TM, based on which the inverse matrix T = TM-1. To transform any stereotactic coordinate point S into the MCP coordinate system the vector difference ⃗⃗ = – ⃗⃗⃗⃗⃗ ⃗⃗⃗ ⃗ was calculated and multiplied with the inverse transformation matrix T to obtain MCP coordinates ( ⃗⃗ = [ ] * ⃗⃗ ).
Thus, the stereotactic coordinates of target and entry points were transformed to MCP – coordinates for all patients. The points on the left side of the head with negative X – MCP - coordinates were mirrored to the right side by inverting the sign of the X coordinate, to become X + MCP - coordinates. For each functional point, the spatial center of the cluster, the distance and its standard deviation were calculated. Points belonging to a cluster with a distance of more than 2.25 standard deviations from the center were eliminated to improve data quality.
The coordinates allow for identification of targets, in which 8 tissue deposits along each trajectory are performed, and can be used in the planning of the transplantation treatment, aiming for a safer and faster target planning. The coordinates of the putamen and caudate nucleus were
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 82 identified in Three-Tesla Magnetic Resonance imaging and transformed into mid-commissural point (MCP) coordinates. From this data an electronic coordinates-base atlas was created. For the second contra-lateral transplantation, the MCP coordinates were mirrored in order to determine contralateral targets and tracts.
The software allows to use either five trajectories, two into the head of the caudate nucleus, two into the pre-commissural putamen, and one into the lateral aspect of the post-commissural putamen (Gallina P. 2008) or six trajectories distributing two in the head of the caudate nucleus and four into the putamen, all within at least 1 mm from each other (Bachoud-Levi A. 2000). The second approach is used in our group (Figure 17). The decision of performing 5 or 6 tracts lays on the individual atrophy of the striatal nuclei, which may reduce the number of possible tracts, avoiding the last post commissural putaminal tract. The number of grafts therefore, is customized for each patient based on the degree of atrophy and accessibility.
Figure 17 MRI T1w, showing typical views obtained from the STP3 –planning workstation. The images correspond to MRI coronal projections with tracts going to the caudate nucleus and to the putamen on the left side. (Picture Lopez WO with contribution of the Neuroradiology Department of Freiburg Medical Center).
Transplantation technique
The grafts consisted of striatal tissue obtained from the striatal primordia. Fetal Striatal Primordia are located bilaterally in the whole ganglionic eminences (WGE) of 7 to 12 weeks-old human fetuses. The tissue was the product of a voluntary interruption of pregnancy and was obtained in accordance with the local ethical committee guidelines and legal regulations of Federal Republic of Germany and the European Union (EU) with informed consent of the donors.
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Under general anaesthesia with additional local anaesthesia, the stereotactic frame was fixed. Stereotactic contrast-enhanced CT (2mm slices-Siemens Somatom plus) was performed and the images transferred to a workstation for fusion with preoperatively acquired MRI (MP-Rage post Gadolinium, 1mm, longitudinal view, TRIO, Siemens Erlangen/Germany) for target planning. Prophylactic antibiotics, i.e. 1,5g cephalosporin i.v., were given half an hour preceding surgery.
The modified Riechert-Mundinger stereotactic frame system was used for the transplantation procedures. This frame system consists of an aiming bow attached to the base ring, fixed to the patient's head (MHT, Koch, Bad Krozingen/Germany). First the stereotactic entry coordinates for each trajectory are set at the aiming attached bow to a phantom base ring on which the target coordinates for each trajectory can be set; the surgeon can verify the correctness of his entry coordinate input. The aiming bow is then transferred onto the base ring on the patient’s head, thus transferring all the verified coordinates of the three-dimensional system. Also, this system allows a second neurosurgeon to set up the coordinates for a subsequent trajectory while the first neurosurgeon is still transplanting cells along the previous trajectory, thus shortening surgery time.
A three-dimensional reconstruction and calculation of the implantation trajectory on the chosen hemisphere was planned using a STP3 workstation (Stryker-Leibinger, Duisburg/Germany). The planning began with the identification of the anterior and posterior commissural points to set up the denominated AC-PC line, allowing more precision and the possibility to use either model based planning or mirrored coordinates obtained from the first surgery, speeding up planning of the second surgery (Figure 18).
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Figure 18 The figure shows an overview of the stepwise planning approach to transplantation as well as a post- operative image control in the same patient. A: MRI T1w contrast-enhancement sagittal image, planning begins by identification and registration of AC-PC line (yellow) and midline (purple line) starting from the posterior part of the brainstem (corresponding colored starts help to identify the lines in the picture). B: Axial view of a Contrast MRI T1w sequence image, showing Stereotactic planning of the 6 targets (2 in the head of the CN –yellow arrow and 4 into the Pu -short red arrow) with two independent entry points for CN and Pu targets. C: Sagittal MRI T1w sequence image showing planned tracts and clear necessity of two entry points. D: Picture shows the exact moment of a deposit implantation, it is possible to identify in the picture the Stereotactic Riechert-Mundinger frame with its aiming bow, the patient is under general anesthesia and the transplantation is performed with a Hamilton syringe attached to the aiming bow (Inomed, Medizintechnik GmbH Emmendingen, Germany); Transplantation is performed delivering 5μl of tissue suspension in 8 deposits per each tract. E: Postoperative MRI (T1w, Siemens TRIO) of the same patient three days after left striatal transplantation. The yellow line shows the deposits into the head of the left CN and the red arrow the deposits into the Pu; this patient was previously transplanted on the right side where it is possible to identify the grafted area. F: MRI (T1w, TRIO, Siemens) of the same patient in a sagittal view 3 days after left transplantation, the yellow arrow shows a characteristic local hyperintensity on the deposits sites. (Picture Lopez WO with contribution of the Neuroradiology Department of Freiburg Medical Center).
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The software was developed for a fast model based functional trajectory planning, giving a prediction for the possible coordinates, allowing where required individual or multiple adjustments through movement of a whole group of target coordinates according to the atrophy of the patient’s brain. A functional database of stereotactic coordinates of target and entry points was created, together with the coordinates of the AC-PC line and mid-line of the patients, which were obtained from the stereotactic planning system and patient records (Table 3). Next, all stereotactic coordinates were transformed into MCP coordinates and mirrored to one side to increase the total number of tracts for statistical analysis. The typical MCP - coordinates of all target and entry points were calculated using cluster analysis excluding patients with a spatial distance of more than two standard deviations from the centre of the corresponding cluster. After registration of a new patient, the patient’s coordinates were included in the database improving statistics for all succeeding patients. Thus a self-learning functional database of coordinates was created. The patient’s stereotactic coordinates of the first transplanted hemisphere were used for planning of the second transplantation procedure on the contralateral hemisphere, by first transforming stereotactic standard coordinates to MCP coordinates, then mirroring the X - Coordinates in the Y-Z plane, and by finally transforming the MCP coordinates back into the actual stereotactic coordinates system of the second transplantation (Figure 19). In this way the target and entry points must only be adjusted to avoid blood vessels on the surface of the brain and in the path of the trajectories (Table 4). In some of the patients, with a far advanced state of striatal degeneration, it was impossible to make a plan for all four-target points in the putamen. In these cases the most posterior trajectory was omitted.
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Mean Name length of the tract PCP2 63,95 mm PCP1 63,63 mm CP 63,22 mm PP 62,71 mm CC 58,92 mm PC 58,17 mm AC-PC 25,15 mm
Table 3 Mean length of each tract from cortex to target and AC-PC line in millimeters PCP2 (Putamen posterior), PCP1 (Putamen posterior), CP (Putamen anterior), PP (Putamen anterior), CC (Caudate anterior), PC (Caudate posterior), AC-PC line (anterior commissure-posterior commissure) connecting anterior and posterior commissure.
A first entry point was chosen frontal pre-coronal with a 6 mm burr hole for the caudate nucleus tracts, and a second entry point was chosen more lateral, to accommodate the four trajectories to the putamen (Table 4). The aim was to perform: two separate trajectories in the head of the caudate nucleus; two trajectories in the pre-commissural and commissural region of the putamen and two trajectories in the post-commissural region of the putamen bilaterally.
50 µl of tissue suspension were aspirated into a Hamilton syringe through a 0.6 mm diameter needle (Hamilton™ Company- Bonaduz, Switzerland). The tip of the needle was inserted in the target of each planned tract, and then 5 µl of tissue suspension were deposited. After a waiting period of 2 minutes, the needle was retracted 1 mm, repeating the deposit each time over a distance of 7 mm (Figure 18). Thus eight grafts were deposited along each trajectory
All patients received bilateral HFST in two separate procedures under general anaesthesia. Transplantation was always performed first in the right hemisphere, and after a minimum of two weeks (mean: 5.5, SD: 3.3) transplantationin the left hemisphere was performed. For the second surgery, the software automatically provided the coordinates based on the previous contralateral
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 87 surgery, proposing mirrored coordinates for intra-individual planning. In total a number of 94 WGE from 47 embryos were obtained and transplanted. The number of embryos transplanted per side ranged between one and four, with a mean of 2.15 embryos, for each procedure (SD: 0.6).
Figure 19 Screenshot of our software for fast model based functional trajectory planning, giving a prediction for the possible coordinates, and allowing the individual adjustment through movement of a whole group of target coordinates according to the atrophy of the patient’s brain. (Compensation offset) MRN: medical record number; PC: pre-commissural caudate; CC: commissural caudate; PP: pre-commissural putamen; CP: commissural putamen; PCP1: post-commissural putamen; PCP2: post-commissural putamen 2. To create the functional database, the stereotactic coordinates of all functional target and entry points were obtained, together with the coordinates of the AC-PC line and mid-line of all patients from our stereotactic planning system and patient records. Next all stereotactic coordinates were transformed to MCP coordinates and mirrored to the left side to increase the total number of tracts for statistical analysis. The patient’s stereotactic coordinates of the first transplanted side were used for planning of the second transplantation on the opposite side, by transforming the stereotactic coordinates to MCP coordinates, mirroring the X - coordinates in the Y-Z plane, and by transforming the MCP coordinates back into the new stereotactic coordinates system of the second transplantation. (Picture Lopez WO, Trippel M with contribution of the Neuroradiology Department of Freiburg Medical Center).
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Target tx ty tz sdx sdy sdz sdt ex ey ez sdx sdy sdz sdt PCP2 21,34 13,78 -0,66 1,81 2,39 1,73 3,47 33,84 42,67 54,94 3,74 5,36 7,83 10,20 PCP1 20,92 16,12 -1,41 1,97 2,00 1,66 3,26 34,03 41,56 55,27 3,65 6,35 7,90 10,77 CP 20,22 19,00 -2,22 1,55 1,59 1,30 2,57 34,02 41,52 55,05 3,69 6,24 7,80 10,65 PP 19,57 21,84 -2,73 1,56 1,52 1,38 2,57 34,07 41,40 54,95 3,72 6,21 7,56 10,46 CC 12,44 21,84 2,10 1,79 1,56 1,27 2,69 47,83 37,28 46,41 6,82 7,39 8,01 12,86 PC 13,16 24,63 0,88 1,84 1,48 1,18 2,63 47,61 37,51 46,01 6,25 7,31 7,82 12,40 Table 4 Stereotactic coordinates. On the left: MCP-Coordinates (tx, ty, tz) of the target (t), in millimetres. Four targets in the putamen, two posterior: PCP2 and PCP1, two anterior: CP and PP and two targets in the caudate nucleus: CC and PC.SD (standard deviation). On the right: MCP-Coordinates (ex, ey, ez) of the entry points (e) in millimetres, posterior putamen: PCP2 and PCP1, Anterior: CP and PP and caudate nucleus: CC and PC. sdx, sdy, sdz, sdt (standard deviation for the target and entry points in the x, y, z direction and total).
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4.3. Project III “Intrastriatal Transplantation of Neural Crest Derived Chromaffin Progenitors Cells in a Rat Model of Parkinson’s Disease”.
Chromaffin cells are neuroendocrine cells found mostly in the medulla of the suprarenal glands. They develop from a neural crest-derived sympathoadrenal (SA) progenitor cell. The SA cell lineage is an important descent of the neural crest (NC) differentiating into sympathetic neurons, chromaffin cells of the adrenal medulla, extra-adrenal chromaffin cells, and the denominated intermediate small intensely fluorescent cells of sympathetic ganglia and paraganglia (Landis 1981, Anderson 1993, Unsicker 1993, 2002). Recent studies have revealed the presence of neural crest-derived self-renewing sympathoadrenal progenitor cells within the adult adrenal medulla (Ehrhart-Bornstein M 2010): due to their close relation of SA progenitor cells to sympathetic neurons (acetylcholine, norepinephrine and dopamine), isolation and differentiation of these cells in culture might provide a cell source for the treatment of neurodegenerative diseases (Ehrhart- Bornstein 2010). In this study we aimed to investigate the in vivo survival and functional restorative capacity of neural crest-derived self-renewing sympathoadrenal progenitor cells after a stepwise isolation, propagation and transplantation protocol. In particular, we investigated whether and to what degree the cultured progenitor cells differentiate into dopaminergic cells by means of motor improvement in a Parkinson animal model and posterior immunohistochemistry. The adrenal medulla progenitor cells were isolated from either bovine or rat donors and transplanted into a rat host in vivo animal model of Parkinson’s disease. The project is composed of 8 studies, one exclusively in vitro and seven in vivo prior in vitro works. For the in vivo studies, the isolation protocol included the enrichment and propagation of adrenal cromaffin cells in chromosphere cultures prior to transplantation in six of the studies with one left avoiding cell culture transplanting the cells right after isolation.
Author contributions:
This Study was a collaboration of the Department of Molecular Endocrinology of the University Clinic Dresden and the Stereotaxy Interventional Neuroscience Laboratory of the Department of Stereotactic and Functional Neurosurgery, University Medical Center Freiburg.
The project was composed by 8 studies; experimental work was performed by the author and supported by some collegues. Studies 1-4 include isolation of bovine adrenal cells, which were
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 90 performed by Monika Ehrhart-Bornstein, Vladimir Vukicevik and Christiana Ossig in Dresden with participation of the author following in studies 3 and 4. The lesion and transplantation surgeries in studies 1 to 7 were performed by the author in collaboration with Vladimir Vukicevik, Christiana Ossig, Luciano Furlanettil and Mate Dobrossy in studies 1-4. Study 3 included the implantation of deep brain stimulation electrodes which was performed by Luciano Furlanettil with close collaboration from the author. Study 8 is an in vitro study, isolation of the glands was performed by me and the in vitro work was performed by Johanna Wessolleck, further work of these study included cell culturing which was performed by me. Animal care including everyday immunosuppression was performed by the author. Immunohistochemistry was performed by author with important contribution from Ryan Mclean. Writing and statistical analyses were performed by the author.
MATERIALS AND METHODS
1. Animals
A total of 70 adult female Sprague-Dawley (SD) rats (225-250 g at purchase, Charles River Laboratories research models and services Germany, GmbH, Sandhofer Weg 797633 Sulzfeld) were used in the present study. The animals were housed on a 12 h light/dark cycle (light on 7:00 to 19:00) with free access to food and water at all stages of the experiment. All animal works were performed in accordance with regulations set by the Ethical Committee for the use of laboratory animals at Freiburg University, Germany. Since appropriate labelled donor cells were essential in order to study differentiation and integration of grafted cells. Green fluorescent protein (GFP) lewis transgenic rats GFP were used as donors of chromaffin cells in some studies. Studies were always compared to sham surgery.
2. Animal model
The animal model used in this in-vivo experiment was the 6-hydroxydopamine (6-OHDA) model. This model requires the unilateral 6-OHDA intracerebral neurotoxic injection that produces the specific degeneration of catecholaminergic neurons leading to a degeneration of TH-immunoreactive neurons in SNc. This model is accepted for preclinical testing of therapies designed to protect or replace dopaminergic cells (Betarbet 2002). 6-OHDA was injected stereotactically, due to the fact that it is unable to cross the blood-brain barrier. In this lesion
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 91 model dopaminergic neurons start degenerating within 24 hours after the surgery and striatal dopamine is depleted 2 to 3 days later. All rats received stereotactic injections unilaterally into the medial forebrain bundle (MFB) to target the nigrostriatal tract. A basic assumption of the model is that three to four weeks afte the lesion, the unilateral destruction of the nigrostriatal pathway provoques a characteristic ipsilaterally rotation in the behavioral test, tipically to the lesioned side once the animal is exposed to the influence of IP amphetamine (α- methylphenethylamine) a post-synaptically acting dopaminergic agonist (Kehinde LO 1984).
The 6-OHDA lesion was produced using a two tracks protocol: Tooth Bar: [TB]: -2.3/[AP]:- 4.4/[L]:-1.2/[DV]:-7.8 and [TB]: +3.4/[AP]:-4.0/[L]:-0.8/[DV]:-8.0. Total Volume injected per animal was 5.5µl (2.5µl in the first track and 3µl in the second track) the dosage was 3.6µg/µl (19.8µg per animal) (Figure 20). Transplantation of progenitor’s cells to test striatal reinnervation was performed 1 month later. For transplantation surgery, a Hamilton syringe was fitted to a glass capillary (outer diameter of 50–70 mm). Animals received an intrastriatal injection of 300,000 cells at the following coordinates: AP: + 0.2 mm, ML: -3.5 mm, tooth bar: 0.0 mm (Carlsson et al., 2006). Two 0.5 m l deposits were injected at DV -5.0 mm and -4.0 mm and the needle was kept in place for an additional 4 min before it was slowly retracted.
Figure 20 The figure shows the stepwise of stereotactic lesion. A. Chamber with animal for anesthesia with Isoflurane and O2, B. Surgical table including all needed instruments and the stereotactic frame, C. SD rat under
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general anesthesia ready for surgery, D. Tooth bar, E cranial sutures: coronal and sagittal, bregma is localized in their intersection, 2 craniotomy bur holes. F. Hamilton siringe employed for lesioning (Pictures by Lopez WO). The rotational behavior model was tested three to four weeks later. Animals were injected IP with 2.5 mg/kg of amphetamine for amphetamine (AMPH)-induce rotational behavior. The test allowed to distinguish the animals with a complete dopamine depletion vs. those partially lesioned.The rotational behavior was measured in an automated spherical rotometer for 60 minutes and consequently repeated one week after. One full rotation is defined as 4 consecutive 90° turns in the same direction (Robinson 1983). Only rats exhibiting an average of minimum 6 turns per min were used in this experiment (Figure 21).
Figure 21 A. intra-peritoneal injection of AMPH. Rotational behavioral test, in B-E the animal gives a complete turn to the right (Pictures by Lopez WO)
3. Experimental design
Well lesioned animals were divided into two groups (Group I and Group II).
Group I [studies 1 to 4]: A total of 36 SD rats received intrastriatal transplantation of neural crest derived chromaffin progenitor’s cells from adult bovine adrenal medulla. All animals received immunosuppression one day prior to surgery and until perfusion. Based on previous studies on isolation and characterization of chromaffin progenitors from adult adrenals (Chung et al., 2009; Vukicevic 2012)., we used special protocols for isolation, culture and transplant in a PD in-vivo model using SD rats, aiming to investigate the survival of adult chromaffin progenitor cells in vivo.
Group II [studies 5 to 8]: A total of 14 SD rats received intra-cerebral allografting of neural crest derived Chromaffin progenitors cells, 12 animals from green fluorescent protein (GFP +)
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Lewis rats donors and 4 from SD rats donors. The purpose of these studies was to test adult chromaffin progenitor’s cells as a valuable source for replacement therapies in Parkinson’s disease (Figure 22, Table 5).
Figure 22 Experimental design of project III. After the unilateral 6-hydroxydopa mine (6-OHDA) lesion of the medial forebrain bundle (MFB) and selection of animals according to performance in amphetamine-induced rotation and cylinder test, animals received stereotactic implantation of adrenal medulla cells in 7 individual studies with different isolation and incubation periods for enrichment and propagation of the cells in chromosphere cultures prior to transplantation. Studies were always compared to sham surgery.
Study 1:
Bovine adrenal glands were acquired from the local slaughterhouse in Dresden, initially trimmed free of adipose tissue and stored in ice-cold phosphate-buffered saline (PBS) to be transported to the laboratory of molecular endocrinology in Dresden. The donors were 1-3 year-old freshly slaughtered cattle. Adult adrenals are usually >5 cm long (Sicard 2007). After arrival to the laboratory, all work was performed in an aseptic biological safety cabinet; initially the glands were put into 70% ethanol for 10 seconds for sterilization and minimization of contaminations. Two steps need to be performed in order to obtain the medullary adreno-chromaffin cells, washing and digest. For washing, the intact glands were then perfused with PBS through the central vein several times; using a syringe to flush away blood. For digestion the central vein was
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 94 again infused injecting 0.3% collagenase (Sigma–Aldrich, Munich, Germany) and 30 units/ml DNase I (Sigma–Aldrich) at 37°C every 15 minutes for 1 hour. The glands were finally cut in half in order to identify the digested medullary cells which contrast easily from the undigested cortex (Haidan 1998). Cells from the adrenal medulla were separated by mechanical dissociation, sieved through 100µm cell strainers and washed twice with PBS.
Adrenal medulla cells were then cultured at 37°C in a humidified atmosphere (95% O2, 5% CO2) for 12 hours in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY) supplemented with 10% steroid-free fetal bovine serum (FBS, charcoal/dextran treated; Hyclone, South Logan, UT) and 1% antibiotic-antimycotic solution (Gibco) in DMEM/F12 medium.
Chromaffin cells were then purified by differential plating, a method that takes advantage of the different on adhesiveness between chromaffin (floating and slow adhering cells) and non chromaffin cells (they precipitated) (Sicard 2007, Ehrhart-Bornstein 2010) to do so the cell suspension was moved to a new flask three times every 1.5 hours, thereby removing most non- chromaffin cells achieving a 97.5% purity of the isolated medullary chromaffin cells.
Isolated cells were then cultured in Neurobasal medium. containing 2% B27-Supplement (both Gibco, Grand Island, NY), 1% penicillin–streptomycin solution, 2mM L-glutamine (PAA Laboratories, Cölbe, Germany), 20 ng/ml epidermal growth factor, 20 ng/ml bovine fibroblast growth factor (bFGF), and 10 ng/ml leukemia inhibitory factor (all Sigma–Aldrich, St. Louis, MO) in ultra-low-attachment culture flasks where cells formed non-adherent spheroid clusters.
After 16 days of cultivation, chromospheres were collected and stored frozen in 10% DMSO and neurobasal medium for transportation to the Molecular Laboratory at the University Medical Center, Freiburg im Breisgau. The approximate number of cells at this point was 50x106 cells, and they were kept frozen in a liquid nitrogen tank. At day of transplantation, frozen cells were removed from the liquid nitrogen tank and were thawed by holding vial in hand; cells were then pipetted from the vial to a Falcon tube for thawing in 18 µL Dimethyl Sulfoxide (DMSO) 5 mM stock; prior to transplantation cells were centrifuged at 1000 rpm/min for 5 minutes and then the pellet resuspend in Accumax (Chemicon-Millipore) for 10 minutes at 37°C. Cells were periodically shaken during incubation as they tend to precipitate. PBS was added and cells were pushed through 70 µm strainer (Becton Dickinson),centrifuged at 1000 rpm/ min for 5 minutes
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 95 and the pellet re-suspended in few mililiters of neurobasal medium. Cells were counted, and viability assessed using trypan blue method. Two µl of a cell-tracing reagent was added: CFSE (CellTrace™) per ml of Neurobasal. Cells were incubated at 37°C for 10 minutes in a waterbath, then washed 3 times by centrifugation at 1000 rpm/ min for 5 minutes and re-suspend in an appropriate volume for transplantation: 100,000 cells/µl, transplanting 10 SD rats (~300,000 cells per animal). Stereotactic coordinates of the targets according to Bregma position were: [TB]: 0.0/[AP]:+0.2/[L]:-3.5/[DV]:-5.0 and -4.0. Animals received immunosuppression daily with intra-peritoneal: Cyclosporine A (CsA), and until perfusion.
Study 2
In this study cells were not transplanted on the same day as they were thawed; but instead, keep in culture for 7 more days prior to transplantation.
Isolation of adult bovine chromaffin cells was performed following the same protocol as in Study 1. Cells were frozen in 10% DMSO and Neurobasal medium and transported from Dresden to Freiburg. Cells were thawed in 18 µL DMSO (5 mM stock), centrifuged at 1000 rpm/min for 5 minutes, the pellet re-suspend in Neurobasal medium (Life technologies #21103-0499) supplemented with 2% B27 (Life technologies), L-glutamine (Sigma-Aldrich) and Pen/Strep + 20ng/ml bovine fibroblast growth factor (bFGF) and cultured in three cell suspension flasks (20ml medium per flask) [CELLSTAR® Cell Culture Flasks]. Two days later, 5 ml fresh medium were added to each flask and cultured in a 37°C incubator. Three days later cells were centrifuged at 1000 rpm/min for 5 minutes and re-suspended in fresh medium. Seven days upon arrival transplantation surgery was performed. Prior to transplantation cells were centrifuged at 1000 rpm/min for 5 minutes, the pellet re-suspended in Accumax (Chemicon-Millipore) for 10 minutes incubation at 37°C in water bath, cells were periodically shaken during incubation because they tended to precipitate. Cells were filtered through a 70 µm strainer (Becton Dickinson), and then centrifuged at 1000 rpm/5 min and the pellet re-suspended in few milliliters of Neurobasal medium. Cells were counted and 2 µl of CFSE (CellTrace™) per ml was added. Cells were incubated with a cell-tracing reagent CFSE at 37°C for 10 minutes. Cells were washed 3 times by centrifugation at 1000 rpm/min for 5 minutes and re-suspended in appropriate volume for transplantation at 100,000 cells/µl. 300,000 cells were injected unilaterally per animal in 2 tracts in the right striatum in 10 lesioned SD rats. Stereotactic coordinates of the targets according
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 96 to Bregma position were: [TB]: 0.0/[AP]:+0.2/[L]:-3.5/[DV]:-5.0 and -4.0. Animals received immunosuppression daily with intra-peritoneal CsA until perfusion. In this series four animals were sacrificed and their brain freeze to perform laser capture microscopy (LCM) analyses. All animals were sacrificed with an intra-peritoneal overdose injection of Ketamine/Rompun (0.7 Ketamin + 0.1 Rompun-Bayern for adult animals between 300-350g) by decapitation with a guillotine. The protocol includes: careful brain removal; snap freezing with 2-methylbutan and dry ice (see materials section); The brains were cut in a cryostat (Leica 2800E Frigocut microtome Cryostat) fixing the brain with tissue tek on the tissue holder; cuts were made at 30µm and 20µm in the striatum sections; then the slices were thaw mounted on membrane slides (4 brain slices per slide), fixed by sinking the slides in acetone for 3 minutes transported on dry ice for Dapi staining; then slides were taken in dry ice to perform micro-dissection by LCM. LCM technique permits the rapid and reliable procurement of pure populations of cells from tissue sections, in one step, under direct microscopic visualization (Currant 2000) [Figure 23].
Figure 23. Laser-capture microdissection. The laser beam partially melts the film coating the cap, causing the film to attach to the choosen tissue. On removal from the slide, the tiddue (in this case graft) remains adherent to the cap (From Curran 2000)
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Study 3
A total of 6 animals following the same protocol of Study 2 were divided and compared as follows: 3 animals (6-OHDA unilateral lesion + transplant [300.000 cells x 2 tracts in the right striatum] + deep brain stimulation (DBS) in the subthalamic nucleus (STN) 200 µA) vs. 3 controls (6-OHDA unilateral lesion + transplant [300.000 cells x 2 tracts in the right striatum]). Animals received immunosuppression daily with intra-peritoneal CsA until perfusion (Figure 24).
Figure 24 Stepwise of and electrode implantation in the STN for DBS in a SD rat. The stereotactic coordinates used were STN (A 3.2–3.8 mm from bregma, L 2.2–2.4 mm, H 7.5–8.2 mm). The stimulation parameters used in this study (frequency, 130 Hz; pulse width 60 μs; current intensity 200 μA) Stimulation parameters started from 100µA achieving not improvements on rotations; 200 µA the best answer, improving significantly on rotations; until 300 to 400 µA with the animal having secondary effects with freezing (Pictures by Lopez W.O)
Study 4
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In this experiment the same isolation protocol as in Study 1 was followed, however we decided not to freeze the cells but instead to transport them at room temperature from Dresden to Freiburg. This way 4 days after isolation, the cells were taken out the incubator and transported during 8 hours in culture flashes. Once in Freiburg the cells were moved to new 250 flasks with fresh medium and cultured. Three days after the cells were put into 50 ml Falcon tubes under sterile conditions and centrifuged at 1000 rpm/ min for 5 minutes, supernatant was removed and the pellet re-suspended in Accumax (Chemicon-Millipore) for 10 minutes incubation at 37°C. Cells were periodically shaken to avoid precipitation. Cells were then filtered through a 70 µm strainer (Becton Dickinson) centrifuged at 1000 rpm/5 min and pellet re-suspend in few milliliters of Neurobasal medium. The cells were transplanted in 10, previously lesioned (6- OHDA) SD rats at 6µl per animal x 2 tracts in the right striatum at a concentration of 100,000 cells/µl. Stereotactic coordinates of the targets according to Bregma position were: [TB]: 0.0/[AP]:+0.2/[L]:-3.5/[DV]:-5.0 and -4.0. Animals received immunosuppression daily with intra-peritoneal CsA until perfusion.
Group II: A total of 14 SD rats received autologous intra-cerebral transplantation of neural crest derived Chromaffin progenitors cells, 12 animals from Green fluorescent protein (GFP +) Lewis rats donors and 4 from SD rats donors.
Study 5
In this experiment a total of 8 SD rats received intra-cerebral adrenal medulla cells allotransplantation. For this the suprarenal gland of 10 green fluorescent protein (GFP +) Lewis rats were bilaterally dissected, the adrenal medulla was resected as a unit under microscopy view and cut into 1 mm pieces which were transferred into 1,5 ml dissociation medium. Cell suspension was prepared in 10% FCS, 1% Pen/Strep and 1% L-Glutamine in DMEM for one night after isolation. The next day we exposed the cells to pro-neural conditions for few days using 20ng/ml FGF, 10% B27, 1% Pen/Strep, 1% L-Glutamine in neurobasal medium. Three days later, the cells were centrifuged at 1000 rpm/5 min and re-suspend in fresh medium. After 7 days, the cells were grafted into previously lesioned (6-OHDA) SD rats at a dose of 100,000 cells/µl. The total cell count was 11 x 106 cells with a viability of 76%. Eight animals were transplanted into three different targets to compare regional differences in survival: A.) Striatum (2 deposits x 1.5µl and a total of 300,000 cells); B.) Hippocampus (1 deposit x 1,0µl and a total
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 99 of 100,000 cells); C.) Substantia nigra (1 deposit x1,0µl and a total of 100,000 cells) for a total number of 500,000 cells per animal. Coordinates: Striatum (L (lateral) -3.5, AP (anterior- posterior) +0.2 and DV (dorsal-ventral) -5.0/-4.0, i.a. 0.0]; SN [L (lateral) -1.9, AP (anterior- posterior) -6.3 and DV (dorsal-ventral) -7.0/-6.7, i.a. -3.3]; Hippocampus [L (lateral) -2.0, AP (anterior-posterior) -3.6 and DV (dorsal-ventral) -2.8, i.a. -2.3]. Animals received immunosuppression daily with intra-peritoneal CsA and were perfused at 4 weeks (n=2), 8 weeks (n=3) and 12 weeks (n=2) (Figure 25)
Figure 25 Microtransplantation using glass capillary with diameter approx. 70µm. Scale bar 500µm. Three different targets were chosen to deposit 500,000 cells per animal: A. Striatum 2x1.5µl 300,000 cells, B. Hippocampus 1x1.0µl 100,000 cells, C. Substantia Nigra (SN) 1x1.0µl 100,000 cells. Coordinates: Striatum [X (medial-lateral) -3.5, Y (anterior-posterior) +0.2 and Z (dorsal-ventral) -5.0/-4.0, i.a. 0.0]; SN [X (medial-lateral) -1.9, Y (anterior-posterior) -6.3 and Z (dorsal-ventral) -7.0/-6.7, i.a. -3.3]; Hippocampus [X (medial-lateral) -2.0, Y (anterior-posterior) -3.6 and Z (dorsal-ventral) -2.8, i.a. -2.3] (Figures adaptations from Paxinos rat atlas 2006, Jiang 2011). Method used to dissect the adrenals
1. SD and (GFP +) Lewis rats 4 and 10 months old were used as donors.
2. A lethal dose of anesthesia (0,7 ml Ketamin and 0,1ml Rompun) was administered to the animals before dissection.
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3. A skin incision was made with scissors through the abdomen on a V shape 2.5 cms below the rib cage in the midline to open the peritoneum and expose the liver and kidneys. The area of interest was the adrenal glands (also known as suprarenal glands) which in the rats (as in humans) are sited at the top of the kidneys; the glands can be identified by naked eye having a round to triangular shape.
4. A traction surgical separator allowed for see the surgical field, Liver and kidney were separated achieving a visual identification of the gland, surrounding fat was grasp with forceps and then released with scissors.
5. The gland is then liberated using scissors, the gland were kept in Hibernate-E medium until microscopic dissociation.
6. Under microscope view, excess of fat was further performed, grasping fat with forceps and removing it with scissors; to isolate medulla from cortex; cortex was grasped with forceps and cut it away with scissors, exposing medulla. Right adrenal were excise and then cut into small pieces [~1mm diameter] with scissors and placed in Hibernate-E medium.
7. Left adrenal was expose and excise following the same stepwise protocol (Figure 26).
Study 6
In this study we aimed to compare two protocols for isolation of rat adrenal chromaffin cells for autologous transplantation purposes using 6 SD rats as hosts;
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Figure 26. Suprarenal gland isolation. A-B neon light evidence fluorescence in donors GFP lewis rats. Note natural position of the gland after skin incision through the abdomen on a V shape 2.5 cms below the rib cage in the midline to open the peritoneum and expose the liver and kidneys (C, D); size of the suprarenal glands were in average 5 mm (Pictures by Lopez W.O.).
Comparison between two rats’ adrenal medulla isolation protocols
For each protocol 2x adrenal medullas were dissected from SD rats; glands were kept in Hibernate-E medium until dissociated. The glands were cut into small pieces ~1mm diameter in Hibernate-E medium.
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Protocol 1 (Dresden University)
Medulla pieces were transferred into 1,5 ml dissociation medium, then incubated with enzymes for a total of 15 minutes at 37°c. After 5 min pieces were triturated first with blue tip of 1000µl pipette ten times (10x) and after 10 min with yellow tip of 200µl pipette twenty times. Cells were passed through a 100µl cell strainer. 5ml 1xPBS+1%PSA were added to wash. Cells were passed through 70µm cell strainer and centrifuged at 600rpm for 5 min. Supernatant was cultured with 10ml propagation medium (same as previous experiments-see Experiment 1) for further survival analyses. Pellet was re-suspended in 500µl 1xPBS+1%PSA solution and final volume check:
Protocol 2 (Freiburg University)
After surgical dissection, medulla pieces were transferred into 1,5ml dissociation medium (Freiburg)
Incubation was the performed at 20 min at 37°C inverted every 5-7 min, enzyme solution (Accutase) was carefully removed with cannula in order not to disturb medulla pieces, medulla pieces were washed 4x with 0.05% DNAse solution: waiting for pieces to settle and supernatant removed with cannula. After last wash pieces were re-suspend in 1000µl 0.05% DNAse solution. Pieces were triturated first with blue tip of 1000µl pipette then with yellow tip of 200µl pipette until pieces have dissolved into mostly singular cells Blood vessels and bigger pieces were strained away by passing suspension through a 100µm cell strainer. Then Centrifuged at 600 rpm for 5 min. Pellet was re-suspend in 500µl 0.05% DNAse solution and check final volume. Then, it was attemped to induce differentiation of chromaffin cells by 7 days cultivation in DMEM/F12 medium (5 mM HEPES, 1% penicillin–streptomycin solution, 2 mM L -glutamine, 7.5% sodium bicarbonate, 5 µg/ml heparin, 5 µM retinoic acid, and 10 µM ascorbic acid) supplemented with 1% N2-Supplement and 20 ng/ml bFGF. Cells were kept for 5 more days in differentiation medium DMEM/F12 medium supplemented with 2% B27 supplement and 20 ng/ml EGF. Neurons were identified with green, stained with anti-tyrosine hydroxylase (TH) antibody and counterstained with DAPI. Slides were examined by fluorescence microscopy.
We decided to base on Freiburg protocol for subsequence rat to rat experiments, since it achieved higher cell survival: 90.5% vs. 78% viability.
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Study 7
Isolation of chromaffin cells for transplantation
Step 1: Isolation and enrichment of chromaffin progenitor cells
10x adrenal medullas were dissected from 5x GFP (+) Lewis rats. Glands were kept in Hibernate- E medium until dissociated. Glands were cut into small pieces ~2 mm diameter in Hibernate-E medium. Glands were divided into two portions (A+B)* and each was incubated in 8ml collagenase + DNAse solution for 15min at 37°c: a. After 5 min pieces were triturated first with blue tip of 1000µl pipetted 10x, b. After 10 min triturate with yellow tip of 200µl pipette 20x. Debris was removed by using 70µm strainer and cells were washed through using 20ml 1xPBS + 1% PSA. Centrifugation 5min 300g and 5min 600g as no pellet was seen: suspension culture as is*. Re-suspend pellet in 1000µl 1xPBS+1%PSA solution and check final volume:
Each portion was cultivated in-vitro for 48h in propagation medium prior to transplantation
Step 2: Preparation of chromaffin progenitors cells for transplantation
Portion A and B were pooled and cells cultured * in suspension medium and centrifuged at 1000g for 5 min. Pellet was incubated (=neurospheres) for 10 min with 1.5ml Accutase at 37°C. 8.5ml of medium + 0.05% DNAse was added and suspension triturated using blue tip around 15 times. And centrifugated at 1000g 5min. Cells were re-suspended in 500µl medium + DNAse.
Study 8
Isolation of chromaffin cells for transplantation. Transplantation of prepared cells without in- vitro selection
The aim of this experiment was to test the viability and graft survival in vivo of allografted cells transplanted the same day of isolation. For that 7 SD rats were used as donors and 3 SD rats previously effectively lesioned with 6-OHDA served as recipient.
Fourteen adrenal medullas were isolated from 7x SD rats. Glands were kept in Hibernate-E medium until dissociated. Glands were isolated and incubated intact in 20 ml Collagenase + DNAse solution for 15min at 37°C for 5min, then triturated into pieces by pippeting, first with blue tip of 1000µl pipette ten times and after 10min with yellow tip of 200µl pipette twenty
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 104 times. Removal of debris Debris was performed using a 70µm strainer and cells were washed through using 20ml 1xPBS + 1%PSA. Cells were Centrifuged 6min 600g. Cells were washed with 5ml PBS+1%PSA.
Study Tissue Host n animals n days Frozen n tx Cells Survival Markers/analyses Incubation cells cells/animal viability times
1 Bovine SD/PD 10 16 Yes 500,000 86% 8 w CFSE, CHGA, TH, adrenal model ED1 2 Bovine SD/PD 10 4 23 Yes 300,000 78% 8 w CFSE, CHGA,TH, adrenal model LCM ,ED1 Laser C Microscopy 3 Bovine SD/PD 6 3 23 Yes 300,000 78% 12 w CHGA,TH,CFSE,E Adrenal model DBS D1 4 Bovine SD/PD 10 7 No 600,000 72% 8 w CHGA, TH, Nurr1 adrenal model 5 Lw rat SD/PD 8 7 No 500,000 76% 4,8,12 CHGA,TH, ED1 GFP ad model w 6 In vitro comparison of two isolation protocols of rat adrenal medulla cells DAPI, TH
7 Lw rat SD/PD 3 2 No 293,333 91,8% 8 w CHGA, TH, Nurr1, GFP ad model LMX1A, GFP, ED1 8 SD rat SD/PD 3 0 No 300,000 98,5% 8 w CHGA, TH, Nurr1, adrenal model LMX1A, GFP, ED1
Table 5 Trials of chromaffin cell grafts in a PD animal model from project III. SD/PD: Sprague Dawley/Parkinson’s disease. LCM: laser captured microscopy. DBS: deep brain stimulation. CFSE: Carboxyfluorescein diacetate succinimidyl ester (Cell tracerTM). CHGA: chromogranin A. TH: thyroxine hydroxylase. ED1: Anti Macrophages/Monocytes Antibody. Nurr 1: Nur-related factor 1 (expressed in developing and mature dopamine neurons). LMX1A (LIM homeobox transcription factor 1). GFP: anti-Green Fluorescent Protein.
4. Behavioral test
Amphetamine induced rotation was used to evaluate graft-derived motor recovery by comparing post-transplantation scores with pre-transplantation lesion deficits. Unilateral 6- hydroxydopamine (6-OHDA) lesion of the nigrostriatal bundle, has been reported to cause complete (> 95%) dopamine depletion in the unilaterally lesion striatum causing a range of motor deficits (Torres E.M., Dunnett S. 2007). It has been demonstrated that the administration of 2.5
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 105 mg/kg of amphetamine induces an ipsilateral rotational response of 6 to 20 turns a minute, for a period up to 2 h following injection in unilateral lesioned rats.
Implants of embryonic dopamine cells derived from E14 ventral mesencephalon (VM) a region rich in dopaminergic progenitor cells have been prove to ameliorate the amphetamine induced rotational deficit, reducing the amplitude of rotation and classically, achieving an over compensatory response in which the direction of rotation changes from ipsilateral to contralateral, at rates of up to 25 turns a minute (Bjorklund et al., 1980; Dunnett et al., 1983).
5. Perfusion
At the end of the experiment (at 4-8 and 12 weeks depending on the group) animals received i.p. injection of Rompum (20 mg/ml. Bayer-Germany) 0.1 ml and ketamine (Parke-Davis (India) Ltd.
0.9 ml 50mg/1ml) and were trans-cardiac perfused with 100 ml of 0.9% saline followed by 250 ml of ice-cold paraformaldehyde (4% in phosphate buffered saline, pH 7.4 Gibco® -Life
Technologies). The brains were removed and leaved for cryo-protection in 25% sucrose in PBS overnight at 4°C.
7. Sectioning Cryo-protected brains were cut coronally at 40µm thickness in 12 series using a freezing slide- microtome (Leica). Sections were incubated stored in antifreeze medium in a 24 wells plate (Life
Science Products, Inc. Denver CO USA).
Fixed brains were sectioned using a sliding cryostat microtome (Frigomobil Leica, Nussloch, Germany). The used sliding microtome has a platform on which the brain is held, the stage is connected by tubing to a CO2 cylinder, which is used to generate dry ice around the brain. The microtome is connected to a cooling apparatus to maintain the stage and brain frozen. The freezing process lasted around 15 minutes. The brains were cut at 40µm and divided into 12 series, sliding the microtome knife across the surface of the frozen brain. Sections were carefully
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 106 transferred with a brush to plates containing anti-freezing solution and stored in a 24 well cell culture plate, using consecutively 12 well per brain and kept at -20°c until immunohistochemistry.
8. Immunohistochemistry
At 4, 8 or 12 weeks after grafting and according the study, series of sections of the rats brains grafted were processed for immunohistochemistry using primary antibodies listed in table 6. All steps of staining were performed in 15 ml glass vials. Free-floating sections were first rinsed in
PBS. 2 wells per animal were put in a vial with 1xPBS, 2ml of solution was the constant approximately volume per vial, each vial was washed 3x with 1xPBS à 5min, then a 10min incubation with 3%H2O2/10% Methanol (10ml: 1ml H2O2 + 1ml Methanol + 8ml 1xPBS) washed 3x with 1xPBS à 5min , incubated with blocking solution for 2h (10ml: 300mg BSA +
30µl Triton X ad 10ml 1xPBS) added primary antibody and leaved it a room temperature for overnight incubation. The next day all pots (vials) were washed 3x with 1xPBS à 5min and incubate with secondary antibody for 1h, after that washed again 3x with 1xPBS à 5min. 30 minutes before the end of that period ABC-complex was prepared prior to used (for 10ml: 1xPBS
+ 2 drops A + 2 drops B). ABC-complex was added and incubated for 1h.at the end of that period all pots were washed again 3x with 1xPBS à 5min. DAB solution was prepared (10ml: 1 tablet
DAB + 10ml 1xPBS – filter + 1µl H2O2 and added only before use!). Time of incubation with
DAB was individually accessed according to a positive microscopic or macroscopic staining. All pots were again washed 3 times (3x) with 1xPBS à 5min. Sections were and mounted on glycerine coated glass slides. The slides were left to dry overnight at room temperature. Tissue was hydrated by passing each glass slice in Ethanol: 2x 70% - 2x 96% - 2x 100% - and 2x Xylol
1 time per 3min; mounting medium was placed on coverslip and brought slide down on slide, taking care to avoid without bubbles, clean slices. Dry slices 2-3 days to leave them ready for
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 107 microscopic analyses. For immunofluorescence Unspecific binding was blocked for 2 hours in a solution consisted of 3% bovine serum albumin (BSA, Sigma), 0,3% Triton X-100 (Sigma) in
PBS (RT) and then the sections were incubated with the appropriate primary antisera for 36 hours in 4°C. Following multiple washes in PBS, appropriate secondary antibodies conjugated with fluorescent dyes Alexa 488 or Alexa 568 (1:200, Molecular Probes, USA) were applied for
2 hours in dark and RT. Sections were then mounted on glycerin-coated slides and cover-slipped with DAKO fluorescent mounting medium (DAKO, Denmark).
9. Counting
Graft survival was identified counting positive stained cells one by one by, using an Olympus
AX70 generation SZX2 stereo microscope, allowing fluorescence analysis on fixed examples, the software employed is named Cell P. To count the number of cells per graft, a Leica microscope
DMRB with the program Stereo Investigator, version 5.05.1 Windows 98/ME/NT 2000/XP
(MicroBrightField, Williston, USA) were analysis of cells was also performed.
10. Statistical analyses
For statistical evaluation the data was subjected to One-way ANOVAs in excel 2010. A value of p < 0.05 was considered significant in all tests. Studies were always compared to sham surgery.
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Antibody Specificity Clonality Dilution Source TH mouse Monoaminergic neurons Monoclonal 1:2000 Sigma, St. Louis, MO, USA 1:10000 ED1 mouse anti-rat Tissue macrophages Monoclonal 1:50 Millipore, Temecula, CA, 1:300 USA Chromogranin A Adrenal medulla Polyclonal 1:200 Abcam, Cambridge, UK. rabbit 1:1000 GFP rabbit GFP cells Polyclonal 1:200 Invitrogen Cat No A11122 1:2000 Nurr 1 Immature dopaminergic marker Rabbit polyclonal 1:500 Santa cruz, Biotech Dallas, texas, USA LMX1A Immature dopaminergic marker Goat polyclonal 1:100 Santa Biotech cruz, Dallas, texas, USA
Table 6 Datasheet Immunocytochemistry antibodies from project III
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5. RESULTS
5.1 Project I
Here, we present a comprehensive guide of the necessary pre-operative preparation, and a detailed surgical protocol with state-of-the–art technical modalities for HD and PD neuronal transplantation; based on our experimental and clinical experience (Pauly 2012, Capetian 2011, Döbrössy 2011, Jiang 2010, Hackl 2010, Lepski 2010, Nikkhah 1994, 2001, 2009; Falkenstein 2009, Maciaczyk 2009, Köllensperger 2009, Steiner 2007, Klein 2007, Singec 2007, Timmer 2004). The clinical outcome of cell-based replacement therapy strictly depends on a multidisciplinary, stepwise and rigorous procedure including adequate patient selection, appropriate handling of the tissue, expertise in dissection and careful preparation of the graft as well as precise stereotactic surgical implantation (Lopez et al. 2013)
STRATEGICAL PLANNING
The transplantation of human fetal-derived neural precursor cells (hFNPCs), either as a single cell suspension or as tissue pieces, into the brain of selected patients aims at replacing the functional regeneration of lost neuronal cells populations. Therefore, cells to be transplanted have to be chosen accordingly to the function they are expected to fulfill in the host brain. This requires a precise micro-dissection of the brain region of interest from human fetuses obtained from the voluntary termination of pregnancy during the first trimester of gestation. The crucial steps of an optimally designed clinical trial are summarized in (Figure 27) and discussed in the following sections.
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Figure 27 Process map for clinical fetal cell therapy of HD/PD. The clinical cell therapy using fetal cells can be divided into three main processes: (1) Donation and dissection of fetal tissue, is in Germany regulated by §20b AMG (German national health act). (2) Preparation of the graft, is regulated by §20c AMG. HD and PD preparation protocols differ in the key process steps, each being optimized to the biological requirements of the graft tissues; (3) Implantation of the graft in clinical trials, generally controlled by Good Clinical Practice (GCP). Once the grafts for HD and PD are prepared, the implantation procedure occurs in a similar way for both trials. The full preparation of one graft can imply up to 43 individual documents (6 embryos pooled), all allowing for full traceability of the graft manufacturing (Modified from Lopez et al 2013).
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PATIENT SELECTION
Patient selection in HD
Currently there is no curative medical treatment for HD. Nonetheless, in vivo studies using rodent animal models have generated promising results of partial reversal of neurological deficits when fetal neuronal grafts were transplanted stereotactically into affected striata (Döbrössy and Dunnett 2001, 2005, 2006, 2008). These and other results led to a clinical pilot trial (Bachoud-Lévi 2000) showing a viable graft, as revealed by PET examination, and a clear clinical benefit for a duration of at least 2 years after transplantation. Based on these observations, a Phase II multi-centric European study for the treatment of HD with intracerebral fetal neuron transplantation was initiated in 2001. This ongoing study is designed to assess the clinical results of neuroreplacement in a total of 80 HD patients; of which, about half of the transplantations have already been performed with 22 patients transplanted in our center. The prerequisite of a successful clinical HD transplantation program is the thorough selection of patients and detailed pre-transplantation work up, including the observation of disease progression for at least six months, after inclusion into the transplantation program (day 0). In our institution, all transplanted patients were recruited from the cohort registered and treated within the European Huntington’s Disease Network (Euro-HD). According to the trial guidelines, only patients with a minimum one-year of clinical history (motor, neuropsychiatric, neuropsychological symptoms), Unified Huntington Disease Rating Scale (UHDRS) >/=5 motor score and genetically confirmed HD in an early-moderate stage are eligible for participation in the transplantation program. Patients either in a very early stage or in an advanced stage of the disease with severe comorbidity, psychiatric or personality disturbances that might compromise the compliance as well as patients with severe cortical atrophy on diagnostic images (CT, MRI), which hinders the surgical circuit reconstruction due to lack of striatal space for proper cell deposits and the absence of enough number of host cells to establish connections and give support to donor cells were excluded from the study. On the other hand the lack of some degree of disease-specific changes in T2-weighted MRI (volume loss and increased level of abnormal signal changes within the white matter), positive HIV serology (HIV1, HIV2, and AgP24), active hepatitis (HBV and HCV) as well as HTLV-1 and HTLV-2 infections also exclude the
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 112 participation in the program. All inclusion and exclusion criteria of the HD transplantation trial are summarized in Table 7. b. Patient selection in PD
Our Department is member and partner of the TRANSEURO - a European FP7 research consortium with the principal objective to develop an efficient and safe treatment method for Parkinson’s disease suffering patients using fetal cell based transplants, starting a new human transplantation trial. In accordance with the TRANSEURO PD transplantation program, selected patients should be aged between 30 and 60 years, be in an early to moderate disease’s stage (course duration greater than 4 years but less than 10 years) with a Dopa or Apomorphine significant positive motor response and no serious co-morbidities (Freed 2001, Olanow 2003, Kordower 1995, 1996, 1998). Exclusion criteria comprise atypical parkinsonism, poor or no response to Dopamine or Apomorphine challenge, age under 30 or over 60 years, more than 10 years since diagnosis, a concurrent major medical or psychiatric disorder and significant drug induced dyskinesias (increasing the risk of graft induce dyskinesias). A comprehensive list of criteria can be found in Table 8 at the same time further information can be found in transeuro.org.uk
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INCLUSION CRITERIA-HD EXCLUSION CRITERIA-HD Symptomatic disease for a minimum of one year Severe cognitive function deterioration or (motor, neuropsychiatric, neuropsychological neuropsychiatric disorder, MATTIS score < to 120 symptoms), UHDRS motor score 5
Positive genetic testing (CAG 36) in the 1st exon of Associated illness with neurological compromise, such the huntingtin gene as structural cerebral lesion in MRI Functional Independence of the patient, FCT Absence of HD specific changes in T2-weighted (Functional Capacity Test) 10 sequence of MRI or non-typical morphological brain abnormalities, in MRI or CT scans suggesting other, concomitant brain pathology Age between 25 and 65 years Serious systemic progressive disease Patient’s informed consent Mental disorders such as, spontaneous or medication- induced hallucinations; depression or suicide attempts Very early minor stage of disease Positive HIV serology (HIV1, HIV2, AgP24), active hepatitis (B and C), HTLV 1 and 2 syphilis-positive serology (except vaccine profile or old and cured infection)
Table 7 Indications and contraindications for fetal neuronal cells transplantation in HD (From Lopez et al 2013)
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INCLUSION CRITERIA-PD EXCLUSION CRITERIA-PD PD as defined using PDSUKBB criteria (UKBiobank) Atypical or secondary parkinsonism with typical F- DOPA PET Disease duration of greater than 4 years and less than Disease duration of less than 4 years or more than 10 10 years years Age between 30 and 60 years at the time of grafting Poor or no response to DA agonist and/or apomorphine challenge Hoehn & Yahr stage 2.5 or better when “on” Under 30 or over 60 years of age at the time of grafting Treatment with Levodopa, DA agonists and/or MAOB Mini-Mental State Examination (MMSE) score below inhibitors; anticholinergics or Amantadine 24 or evidence for dementia using DSM-IV criteria Significant motor response (i.e. > 33% improvement Unable to do normal copying of interlocking pentagons on UPDRS part III on 2 occasions) to a DA or and/or semantic fluency score for naming animals of Apomorphine challenge test less than 20 over 90 seconds, as these have recently been associated with earlier onset dementia in PD (Williams Gray, Brain 2007) Ongoing major medical or psychiatric disorder including depression and psychosis Other concomitant significant neurological disorder or major CNS injury/insult in the past Concomitant treatment with neuroleptics (incl. atypical neuroleptics) and cholinesterase inhibitors Significant drug induced dyskinesias Previous neurosurgical procedure F-dopa changes on PET that extends out of the dorsal striatum Disability to be imaged using MRI
Table 8 Inclusion and exclusion criteria of PD transplantation according to TRANSEURO (From Lopez et al. 2013)
2. COLLECTION OF EMBRYOS Human fetal tissue derived from elective surgical terminations of pregnancy (STOPs) was collected according to the local ethical committee guidelines and respective legal regulations, with informed consent of the donors (Lopez et al 2013).
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The age of the fetuses ranged between 6 and 12 weeks of gestation and was documented by pre- surgical ultrasound examination. Abortions should preferentially be performed by a low-pressure aspiration technique (Nauert and Freeman, 1994). The technique involves using a nozzle of Karman assembled to a 60 ml syringe for aspiration, the fetus would be then pushed back immediately into a bottle with appropriate transport medium i.e HBSS, sealed and shipped to the transplantation center. However, dilatation and curettage as well as medical termination could also be suitable. The fetal tissues were then immediately placed into sterile sealed containers: fetal tissue intended for transplantation in HD patients was collected in original flasks from the manufacturer, containing sterile Hanks Balanced Salt Solution (HBSS, Life Technologies), whereas tissue intended for transplantation in PD patients required Hibernate E medium (Life Technologies) as transport medium. Hibernate E medium allowed the storage of neural tissues over several days to collect a sufficient number of ventral mesencephali for transplantation (never recommended for more than 8) (Petersen and Brundin 2000, Grasbon-Frodl 1996). Hibernate medium could be used also in HD tissue storage, however in these cases overnight storage in HBSS is sufficient, taking into account that for each transplant a single 8 weeks old embryo, would usually be enough compared to 3-5 embryos required for PD grafts. Until dissection, the specimen should be stored in HBSS at 4°C but no longer than 24 hours. For longer storage Hibernate E should be used. Region specific dissection of the specimen must then be performed in fresh sterile media (HBSS or Hibernate E) under microscopic view (see paragraph 3).
Labeling and specimen storage It is obligatory to use a consistent anonymous labeling system and to collect all relevant data regarding each fetal specimen (age of the embryo, donor information, data related to the termination procedure etc.). In concordance with state-of-the-art tissue preparation and Good Manufacturing Practice (GMP-standards), traceability sheets ("shuttle book") have to be filled out with detailed information on tissue donors as well as tissue recipients. This information includes all aspects of available data of each individual fetal tissue, using a format and standard of documentation, which allows for tracing back of all information needed for a period of at least 30 years according to national laws. All documentation must be stored in a suitable room with access only to authorized personnel.
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Specific laboratory controls In order to achieve the highest standards of safety for the graft preparation, bacteriological and microbiological tests of the tissue as well as particle monitoring of the preparation facility are indispensable. These are common procedures for cell therapy laboratories within the EU as laid out by directive 2004/23/EC. Two blood samples from the donor, provided with the potential donor tissue, are to be sent for serological testing for HIV 1 and 2, Ag P24 of the HIV, HTLV I and II, Treponema pallidum, Hepatitis B, C, CMV, Rubella, Epstein Barr Virus Toxoplasmosis, according to international standards (i.e. ICH, “The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use”). In addition, depending on the donor case history, testing of HTLV, VZV might be indicated. Positive donor serology for HIV1, HIV2, hepatitis virus B (except from a typical vaccine profile), hepatitis virus C and Treponema pallidum in the donor blood sample will result in immediate exclusion of this donor material from the transplantation process. Donor tissue with positive IgG for CMV and EBV can be accepted after thorough evaluation of the individual benefit/risk ratio, but the patient must be informed and monitored for seroconversion and clinical symptoms for the duration of at least 6 months following transplantation. An additional tube with serum from the donor is preserved in the laboratory for later analysis if needed. About 10 ml of transportation fluid are sent for microbiological analysis (Microbiological sample A). Tissue pieces are then cleaned by successive passages in HBSS baths (2 minutes per bath) in a sterile laminar flow hood at least 5 times. Such dilution washes away possible pathogens. The supernatant of the last washing step is then sent for microbiological assessment (Microbiological sample B). Systematic bacteriological examinations are carried out on the microtube containing the tissue to be transplanted and on the remaining product, after the intervention. The nature of the procedure (transvaginal abortion) and multiple transportation steps facilitate the contamination of the fetal tissue. Our experience showed that 47.7% of the samples taken during human fetal tissue processing were positive for a microbial contamination (Piroth et al. 2013). However, following washing procedures according to proposed protocol no sample exhibited bacterial growth. Therefore, the decontamination described in the current manuscript seems to be very effective and allows, in our opinion, a safe preparation of tissue for transplantation. Furthermore, transplanted patients receive regular antibiotic medication for the immediate perioperative period, according to standard institutional protocols for intracranial interventions.
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In the unexpected case of positive bacteriological test results of the transplant (the final result being available only 14 days after transplantation of the tissue) the antibiotic treatment would be modified according to the antibiogram. However, this has not yet been necessary in any of patients transplanted within the program (22 transplanted patients in Freiburg, 39 in Créteil and 5 in Brussels).
DISSECTION OF THE TISSUE - General remarks
Depending on the performed abortion technique, a high percentage of embryos (average of 70%), could be damaged or fragmented (Figure 28), making the proper region-specific identification of the fetal nervous tissue very challenging due to loss of typical anatomical landmarks. That is why it is very important to prepare a surgeon in the team to become an expert with the anatomy even when some anatomical landmarks are missing. Therefore, training of new surgeons requires several months. In our department, during the training phase a region-specific validation of the tissue takes place (i.e. specific markers expression for nigral precursors) (Figure 29-31). Before dissection, the age of the embryo is determined based on developmental stages i.e. according to Carnegie classification (O’Rahilly and Müller, 2010), crown-rump-length and/or by using the Evtouchenko scale (Evtouchenko et al., 1996; a mathematical model for the estimation of human embryonic and fetal age based on various morphometric parameters measured on a routine basis). The greatest crown-rump-length, the neck-rump length, the foot length, and the proximal and distal arm and leg lengths correlated with the anamnestic and ultrasonographically estimated age should be determined (Evtouchenko 1996). For this purpose, the embryo or the available body parts are positioned in a dish with fresh Hibernate E medium for photographic documentation, with a scale bar below the dish and a calibrated scale bar within the optic computer system (Figure 32, 33). Fetal tissue should be dissected in a lateral position if possible, under sterile conditions and microscopic view, using previously sterilized micro instruments i.e. two Dumont watchmakers’ ® forceps nº5 and one pair of iridectomy scissors (Cat. Nr. 11254-20, Fine Science Tools GmbH Heidelberg, Germany). Fiber optic illumination allows the identification of the CNS within the translucent skull. This enables us to perform the separation of neuro- and viscerocranium with a sharp single incision to be made above the eye at the level of the skull base to the level of the
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 118 mesencephalic curve (using micro-scissors and fine forceps). The anterior fontanelle can also be used as a reference starting point. The skull is opened (in younger embryos is still open) by carefully incising the skin and underlying skull and then gently peeling it back. The fetal cerebral tissue should be handled very carefully; lifting and turning of the embryo should be performed by manipulating the extremities, or meninges rather than the area of interest (Björklund, 1992). Upon identification of the CNS and its structures, the meninges (transparent system of membranes which envelops CNS), and adhering tissue should be very carefully removed to assure the purity of the future graft and optimal identification of all anatomical structures. Separating the meninges from the area of interest by teasing it carefully away might be very challenging in embryos of a younger age but is always necessary; such separation is most easily achieved by grasping a corner of the meningeal sheet with a pair of forceps (Dumont watchmakers’ forceps nº5) and teasing it through the partly open jaws with a second pair of forceps.
TISSUE DISSECTION FOR TRANSPLANTATION IN HD
In the context of HD, the graft material consists of fetal whole ganglionic eminence (WGE), corresponding to the striatal primordium (which will eventually develop into the caudate and putamen) of embryos from the first trimester of gestation (between 15 and 40 mm of cranio- caudal length - crown-rump length [CRL]) (Rosser et al. 2003). The WGE develops during embryogenesis as two elevations in the rostral floor of the lateral ventricles, later divided into the medial and lateral ganglionic eminence (Figure 34.2). The WGE can be identified under the microscope, after opening the prominent dorsal part of the telencephalic vesicle using iridectomy scissors and a Dumont watchmakers’ forceps n°5. The WGE borders laterally to the cortical lamina separated inside from the diencephalon by a deep caudo-rostral depression containing the plexus choroideus. For dissection, the brain should be positioned on its ventral surface, with the dorsal cortex upward. The telencephalic vesicle should be opened by a longitudinal cut through the medial part of the cortex as shown in (Figure 34). If the embryo is between 6 to 8 weeks this step is not necessary since the telencephalic vesicle is still open. The telencephalic vesicle is then folded aside by holding it with forceps and reflecting it posteriorly to expose the striatal primordium in the floor of the lateral ventricle. Using the same instruments, the WGE can be separated from the adjacent structures, first medial
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 119 from diencephalon, then lateral from basal forebrain resulting into a heart-shaped structure (for stepwise description see Figure 34). After dissecting the contralateral ganglionic eminence, the pooled tissue should be rinsed in 5 successive baths of fresh HBSS, for 2 minutes each. The tissue is then stored in sterile HBSS in a dedicated fridge for up to 24 hrs. Roughly two hours before the transplantation procedure begins, the tissue is washed again through 5 washing steps as previously described. The supernatant of the last washing step is sent for microbiological analysis (Microbiological sample C). The tissue is then cut into small pieces with a size of approximately half a square millimeter; we do not use enzymes to treat the tissues.
TISSUE DISSECTION FOR TRANSPLANTATION IN PD
The area of interest for neural transplantation in PD is the ventral part of the developing mesencephalon, containing the A9 group of dopaminergic nigral precursor cells (Rosser et al. 2003). In a lateral view, once the CNS is identified, the dopaminergic cells from the substantia nigra are dissected from the mesencephalon with anterior and posterior cuts, as a wedge from the base of the neural tube at the level of the mesencephalic flexure. The cuts are performed anterior to the junction of midbrain and forebrain and posterior to the junction between midbrain and hindbrain. Depending on the developmental stage, the mesencephalon can assume a tubular aspect after closure of the tectum. It should then be opened with iridectomy scissors making a longitudinal cut along the dorsal midline or the superficial lateral segment of the tube bilaterally allowing the ventral mesencephalon to be folded out, giving rise to the classical butterfly-shaped tissue of VM (Figure 35). This step is not necessary in younger embryos where the tectum is still open. After dissecting the ventral mesencephalon bilaterally, the pooled tissue should be placed in 5 successive rinsing baths in fresh washing solution, 3 minutes each (same as in HD dissection).The tissue is then submitted to a combination of enzymatic and mechanical dissociation) in order to create near single cell suspension for implantation. VM Tissue is incubated for 15-20 minutes in the TrypLE® Express (Life Technologies) supplemented with 20U/ml Dornase alpha (Pulozyme, Genetech) to avoid re-aggregation of the cells and triturated gently several times with fire polished blue and yellow pipette tips. Cells are eventually re- suspended in DMEM/Pulmozyme solution and transferred for transplantation.
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The fetal tissue should be implanted within 24 hours following the collection when stored in HBSS. It can also be stored according to our validated hibernation protocol for up to 4 days prior to use in Hibernate E.
The amount of tissue to be grafted per hemisphere should approximately correspond to VMs dissected from 3-5 fetuses of 8-10 weeks post-conception. In general fetal tissue can be transplanted either as a cell suspension of dissociated VM as practiced in our Department or as a solid graft, since experimental studies in PD show comparable survival graft with both tissue preparation techniques (Kordower 1995, 1996, 1998, Olanow 2003, Lindvall and Björklund 2004). Currently, the tendency is to use so-called “near”-single cells suspension instead of solid pieces. The transplantations should be performed in a staged fashion; both sides of the brain should be grafted in a period of 4-6 weeks.
TISSUE TRANSFER INTO THE OPERATING ROOM
The preparation of the tissue suspension is done in a cellular laboratory EU GMP-Clean Room inside the hospital. All steps of the preparation are carried out in a laminar flow hood, with sterile equipment. The tissue pieces are transferred into an individual sterile microtube which is introduce in a bigger bottle closed under sterile conditions, such microtube contains HBSS for transplantation in HD and DMEM/Pulmozyme for transplantation in PD and brought to the operation room, where they are stored in the fridge until the transplantation procedure commences. Before the transfer, a final check is made whether all information from the donor, including the results from the virological tests is available, and whether any of those results constitutes an exclusion criterion for the use of the donor material.
TRANSPLANTATION PROCEDURE
The transplantation technique is very similar for both HD and PD, with some small but rather crucial differences. On the day of surgery the patient receives regular perioperative antibiotic treatment with intravenous cephalosporin (i.e. cefuroxime). In the operating room, the stereotactic frame is fixed with a positive ring mount to the skull of the patient, who is in a supine position and under general anesthesia. Additional local anesthesia is given to the site of the skin
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 121 incision. We use a Riechert-Mundinger stereotactic system with phantom; computed tomography images of 2 mm thickness are acquired after contrast-agent administration, which are then merged with the preoperative MRI scans (MP-Rage post Gadolinium and T2 space, 1 mm thickness). After three-dimensional reconstruction, the implantation trajectories are planned on a computerized stereotactic planning workstation (Leibinger, Striker). This step is crucial during the intervention and needs a well-equipped facility manned by surgeons with comprehensive and profound knowledge in stereotactic planning (Figure 36).
Transplantation in HD
The original protocol proposed here has been approved by the Comité Consultatifs de Protection des Personnes se prêtant à des Recherches Biomédicales (CCPPRB) in the Hospital Henri-Mondor in Creteil France on August 8, 2001, as part of an on-going phase II trial (Euro- HD phase II trial) run within the Euro-HD Network. In total, two grafting procedures should be carried out; the first into the right striatum, and the second, after a post-surgical recovery period of at least two weeks, into the left striatum. Small tissue cube fragments of less than 0.5 mm3 are placed in a sterile glass recipient or in a microtube with 300 μl of Hank’s solution. The required volume per implantation side is at least 240 µl of suspension with tissue pieces corresponding to what is gained from the dissection of two WGEs of an eight-week-old fetus. In case more fetuses are available, the WGEs of the donors can be pooled in order to increase suspension volume and therefore, optimize the transfer of the tissue during further handling. Subsequently two 6 mm burr hole craniotomies should be performed. The first of two entry points should be localized frontal, pre-coronal, to enable two trajectories into the caudate nucleus. The second entry point localized medially and anteriorly, delivers the fetal tissue via four trajectories into the putamen. For deposit positioning, the neurosurgeon introduces a 0.8 mm external diameter needle assembled to a 50 µl Hamilton syringe with a Luer tip (SIM products® UK) into the microtube and aspirates 50 µl of tissue suspension. Through a burr hole, the neurosurgeon guides the needle along the two separate trajectories into the caudate nucleus and along four trajectories into the putamen. The latter, are divided into two trajectories in the pre-commissural and commissural region, and further two trajectories into the post-commissural region of the putamen. Along each trajectory, with the maximum distance of 7 mm, a total of 8 graft deposits are placed. The
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 122 neurosurgeon will purge the needle at the end of each track before charging again new tissue from the microtube. In case of significant cortical/subcortical atrophy of the patient, it is advisable to carry out 5 trajectories instead of 6 omitting the last posterior commissural target of the putamen (Bachoud-Levi 2000).
Transplantation in PD
In PD the putamen is targeted with 4-5 trajectories as described previously (Molina 1992, Mendez 2000). The protocol uses one burr hole (entry point) at the level of the coronal suture (2 cm laterally to the midline). The stereotactic coordinates for targets in the post-commissural putamen are calculated using T1- T2-weighted and Flair MRI images fused with CT images performed on the day of surgery; applying computerized stereotactic planning workstation as described above.
5. a. POST-TRANSPLANTATION ASSESMENT AND FOLLOW UP IN HD All patients need to be enlisted for transplantation at least 1 year prior to surgery, and be followed up closely at least for the first 2 years post-grafting. Such follow up consists of regular diagnostic imaging studies and clinical evaluations of the patients, as well as close monitoring of immunosuppressive therapy. Control MRI is performed 3 days post-operative to assess possible surgery-dependent complications and 18 months after operation for the evaluation of transplant development. In case of a new neurological deficit a CT has to be performed immediately at any time-point (Lopez et al., 2013)-
Concomitant Therapy Immunosuppressive therapy is initiated three days prior to the first surgical procedure and given as follows: •Cyclosporine (300 mg /d for 6 months, in order to achieve a blood level around 100 and 150 ng/ml); •Azathioprine 1 mg/kg/d; •Prednisolone 0.3 mg/kg/d;
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When the human leukocyte antigen-Antibody (HLA-Ab) is negative, Cyclosporine should be withdrawn one year after second transplantation and Azathioprine after 18 months, parallel with slow tapering of prednisolone.
Primary measurements
1. Progression on the motor UHDRS scale
2. 11C-Raclopride-PET and 18F-FDG-PET preoperative and 18 months post-transplantation to explore graft survival and striatal-like tissue within the grafts (Figure 37)
Secondary measurements
1. Clinical assessments of postoperative cognitive functions, VMA and functional scales (UHDRS, dependence scale, TFC, functional ability).
2. MRI preoperative, 3 days postoperative and 18 months after operation for assessment of tissue growth or graft rejection (Figure 38).
5. b. POST-TRANSPLANTATION ASSESMENT AND FOLLOW UP IN PD
All patients will receive triple immunosuppression
Primary outcome
-Safety and feasibility as assessed using standard surgical, neurological, behavioral and psychiatric testing, including incidence of “off” graft induced dyskinesias.
Secondary outcomes
-Change in motor UPDRS in a defined “OFF” period at 2 years.
-Presence or absence of dyskinesias at 2 years post intervention.
-Quality of life assessed by PDQ-39 2 years after transplantation.
-Changes in F-DOPA PET 2 years after transplantation.
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Figure 28 Comparison of a compete CNS (A) vs. a fragmented CNS before dissection. Thalamus (thal); Mes (mesencephalon); Cer (Cerebellar anlage); MedOb (medulla oblonga) (From Lopez WO et al. Restor Neurol Neurosci. 2013)
Figure 29 Validation of the region-specific dissection. TH+neurons found in ventral midbrain of 7 week old human embryo within fetal substantia nigra (A, B) giving projections mostly to the ventricular eminence (fetal striatum) forming the nigro-striatal pathway (SN-substantia nigra, NSP-nigrostriatal pathway, MAP2/TH/DAPI) (From Lopez WO et al. Restor Neurol Neurosci. 2013)
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Figure 31 Cytospin staining of VM cells. Cell suspension 1x105 cells of 8 week old human embryo. A, B Nestin, C, D Human VM 7 weeks Nestin [C] (green) and Nestin-TH [C,D] stainings(Lopez WO et al 2013 not published)
Figure 32 Cytospin staining of VM cells. Cell suspension 1x105 cells of 8 week old human embryo. A. DAPI, B MAP 3, [C,D] 10 days differentiation in neurons βIII Tubulin (Lopez WO et al 2013 not published)
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Figure 32 Dissection of embryos is performed under microscopic view. Illumination allows to outlining the CNS. On the right human embryo of 8 weeks post-conception with crown-rump-length of 30 mm; on the left: dissected CNS still with meninges. F (Forebrain); M (Mesencephalon); H (Hindbrain); SC (Spinal Cord); TV (Telencephalic Vesicle); VM (Ventral Mesencephalon) (From Lopez WO et al. Restor Neurol Neurosci. 2013)
Figure 33 Dissection of ganglionic eminence of 8 week-old embryo. The striatal primorium develops as two elevations in the rostral floor of the lateral ventricles, designated the medial and lateral ganglionic eminences. (MGE-LGE) together they form the whole ganglionic eminence (WGE) (B). C: The WGE is dissected (cut) into cube shape pieces smaller than 0.5 mm and processed for transplantion (From Lopez WO et al. Restor Neurol Neurosci. 2013)
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Figure 34 Dissection of the WGE (Whole Ganglionic Eminence) from the lateral ventricular cavity of an 8- weeks-old human embryo. On the upper left picture, sagittal view, on the right superior picture coronal view identifying the WGE, which then is separated from cortex and can subsequently be divided into MGE and LGE. TV (Telencephalic vesicle); D (Diencephalom); M (Mesencephalom); Th (Thalamus); CX (Cortex); MGE (Medial Ganglionic Eminence); LGE (Lateral Ganglionic Eminence) (From Lopez WO et al. Restor Neurol Neurosci. 2013)
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Figure 35 Stepwise dissection of the ventral mesencephalon from 9-weeks human embryo. A: Measurement of the crown-rump length, to confirm stage of gestation. B. Dissection and identification of the VM (Ventral Mesencephalon) F (Forebrain); M (mesencephalon); H (Hindbrain). Meninges should be gently removed. C: Dissection of the VM: first two coronal cuts through the neural tube are performed as a wedge, the first at the junction of the forebrain and midbrain (1), and the second at the border of mesencephalon and hindbrain, at the caudal bend of the flexure (2). D: opening of the VM. E: Opening of the VM in its dorsal portion. Exposure the inner surface of the target tissue, identification of the “butterfly” shape VM ready for transplantation. Before transplantation the “Butterfly” will be cut into small pieces of less than 1 mm (Lopez WO et al 2013 not published)
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Figure 36 Intraoperative surgical cells deposits. Tissue arrives into the operating room from cell therapy lab in sterile vial (A) and is mounted into a special designed glass syringe (B, Hamilton); two targets are chosen from the Caudate nucleus and four from within the Putamen in case of HD and 4 targets exclusively in Putamen for PD transplantation (C, D) (From Lopez WO et al. Restor Neurol Neurosci. 2013).
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Figure 37 11C-Raclopride binding potential images showing striatal binding of D2 receptors 11C-Raclopride in direct comparison between a normal subject (A) and one of our series with HD (B) (Contribution from Freiburg Neuroradiology Department)
Figure 38 MRI T1-weighted Axial images 3 days following second transplantation (left) of bilateral fetal neuronal cell graft placement in a HD patient, with trajectories projecting on both sides of the caudate head and the anterior putamen (From Lopez WO Restor Neurol Neurosci. 2013).
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5.2 Project II
The intended number of targets was six (two targets into the head of the caudate nucleus and four into the putamen). All patients were planned using the software for that purpose, however, that was not always possible. Four patients received transplantation along five trajectories, and one patient four trajectories only in one hemisphere. In two cases this was due to a reduced quantity of suspension, and in three cases due to increased cerebral atrophy required reduction in the number of trajectories. Those results agree with from Gallina report (Gallina 2008). The extend of brain atrophy was assessed in 8 consecutive HD patients before human fetal striatal transplantation, giving an atrophy score based on the visual rating of the shape of the lateral wall of the frontal ventricular horn and of the size of the caput nuclei caudate. The score slightly differed between the two hemispheres only in two patients, being equal in the other six.
For all patients/target points: Two caudate nucleus tracks were performed in all the 22 patients bilaterally; two pre-commissural putamen tracks were performed bilaterally in 21 patients. One patient received two pre-commissural tracks on the right hemisphere and one on the left. Eighteen patients received two post-commissural tracks bilaterally; three patients received only one. Two of these were on the right side and one on the left side. One patient (due to atrophy) received only one post-commissural track bilaterally.
Grafted volume in microliters (μl): Seventeen patients received the standardized grafted suspension volume (8 deposits of 5 μl in all the targets) with every injection followed by a 2 minutes waiting period, between deposits. Three patients received a volume of 4 μl x each of 8 deposits x 6 targets bilaterally. Two patients received 4 μl x each of 8 deposits x 6 targets on one side (one patient right and the other patient left), and 5 μl x each of 8 deposits x 6 targets on the contra lateral hemisphere.
In all cases MRI was performed within 1-4 days after HFST, detecting no complications and showing the needle tracks and appropriate location of the pre-planned targets. It is important to mention that MRI may show a hyperintensity signal on the sites of the grafts, mainly interpreted as local signs of an inflammatory reaction, as well as an increased glial reaction in the region surrounding the grafts. This is not regarded as indicator of an ongoing rejection process but may be related to an unspecific reaction of the host tissue to the surgical trauma (krystkowiak P. 2007).
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Total surgical time: The duration of the first transplantation procedure in the right hemisphere was slightly longer than the duration for transplantation in the left hemisphere (400 minutes vs. 373 minutes). This difference was not statistically significant, however, there was an obvious trend shortening the duration of the procedure by the use of the mirrored MCP proposed by the software. Thus, the coordinates were definitely useful in shortening overall procedure time (Figure 39).
In this patient series no intra or early peri-operative complications were reported, neither was any brain infection or acute rejection of the graft within the first 6 months post-transplantation. No bleeding occurred following transplantation indicating, that though the procedure evolved multiple targets and deposits, the bleeding incidence remained similar to a regular stereotactic procedure, (i.e. stereotactic biopsy). In a case series of 3805 patients operated in the same institution, the bleeding incidence was 0.9% (Gilsbach J 1987).
Figure 39 Comparison of the Absolute Value in millimetres, between right and left hemisphere employed entry point coordinates on the left and target points coordinates on the right. Not statistical significant difference was found between the coordinate’s values given by the software (black bars) and the final used entry and target points (grey bars).
We have however three special conditions that are worth to be mentioning in this series
The first one, a non-direct procedure related complication was presented in a 42 years old male patient; his disease onset was at 35 years old, with minor motor disturbances as well as
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 133 depressive mood and insomnia. Genetic diagnostic reported 19/44 trinucleotide CAG repeats, enlisted and transplanted 7 years after onset and 27 months after enlisted (later transplant group).
Seven days after transplantation the patient presented an accidental fall from his own height, with no loss of consciousness but secondary mild head trauma, developing 15 days after a chronic bilateral frontal parietal subdural hematoma, which, 35 days later, required a subdural-atrial left shunt (Figure 40).
Figure 40 A: MRI T1w image taken 3 days following second transplantation. B: T2w MRI Image taken 4 weeks following surgery and 3 weeks following mild head trauma, showing bilateral subdural fronto-parietal hygromas. Size of the hygroma was bigger on the left side (18 x 47 mm) with midline shift to the right of 2 mm. Patient received a subduro-atrial permanent shunt (Contribution from Freiburg Neuroradiology Department).
The second special condition involved a 42-year-old male patient (CAG 44 repeats) who died through suicide 6 months after the first and 5 months after the second fetal striatal transplantation respectively. The patient received striatal grafts on the early group at M7 into the right and at M8
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 134 into the left caudate nucleus and putamen. He had a very difficult social problem and an immobilized leg and foot after a car accident, last clinical examination was performed 1 month before the event with a Karnofsky performance score of 80% and Concentration and memory unremarkable. The patient denied hallucinations and suicidal thoughts. Due to the pronounced bilateral atrophy of the putamen a second trajectory into the post-commissural putamen was omitted during both surgical interventions.
Post-operative MRI scans revealed a proper location of all transplants within the target structures and a lack of surgery-related unwanted side effects. Autopsy identified clearly All 10 grafts (5 in each hemisphere) correctly placed in the planned position. This was the earliest time point when a human, brain specimen receiving neural fetal tissue transplants was examined (Capetian 2009). In comparison with other morphological data from autopsies, from other two reported series, where three HD patients’ specimens were analysed, 18 and 74-79 months after transplantation (Bakay 1998, Freeman 1997, Keene 2007), identifying the presence of surviving grafts populated by some classes of phenotypically mature neurons and the absence of considerable immunological and glial reaction at implantation sites, our case from this series assessed instead immature graft components like neural precursors that are multipotent and thus able to differentiate into neurons, astrocytes and oligodendroglial cells. Deleterious effects on engrafted cells were not found and the patient was under continuous triple immunosuppressive medication as is indicated by the protocol (Bachoud-Levi 2000).
Late postoperative extensive edematous changes in the grafts of a Huntington’s disease patient who received bilateral striatal neural transplantation
The third special condition occurred to a 45 year old, female patient, with minor motor clinical symptoms at onset and genetic HD diagnosis 2 years after with 19/46 trinucleotide CAG repeats, enlisted and was transplanted 5 years later with HFST neurons bilaterally (late group transplant). The patient had a previous history of a benign essential hypertension.
Regular three year’s post-surgery clinical and image control, showed on a simple brain (Magnetic resonance imaging) MRI: extensive edematous changes in the grafts, bigger on the left side simulating an overgrowth of the transplant (Figure 41). The patient was no longer receiving immunosuppressant. Clinical assessment showed no clinical deterioration or signs that may
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 135 suggest infection process. After ruling out an infection on the basis of the lack of alteration of blood cells count, a graft rejection was hypothesized. Blood tested were normal, laboratory viral, bacterial and fungal testes were negative, a lumbar puncture revealed no evidence of acute rejection and human leukocyte antigen-Antibody (HLA-Ab) was negative; indicating no immune system reaction.
An 18F2-fluoro-2-deoxyglucose (FDG)-PET image, showed no increased uptake at either side (Figure 42). It was decided to perform an additional MRI with gadolinium, finding important contrast medium intake at some areas and vascular edema (Figure 43).
The possibility of stereotactic biopsy was considered mainly for histological diagnostic to discard neoplasia formation, however we decided to wait, in keeping with the patient’s wishes and due to her stable condition (UHDRS score was stable with 63 points). The patient had no acute deficits, nor minor neurological decline, ruling out a rejection; the patient was subsequently closely monitored clinically and with imaging.
3 months later the patient remained in a stable neuropsychological condition. UHDRS score was stable with 64 points. Control MRI showed reduction of the frontal subcortical signal increases and a slight decline in barrier disruption compared to preliminary examination. A lumbar puncture was performed: the cytology revealed normal findings. And a new HLA-Ab was negative. In synopsis of the findings, it was discarded any evidence of malignancy or a rejection in the graft area. Last follow up was at 60 months after transplantation, with a stable clinical condition as well as a normalized MRI image.
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Figure 41 MRI T1W image with Contrast enhancement native Control image A: 3 years after surgery, control image shows abnormal edematous changes on the site of the grafts on the left site, with contrast enhancement. No clinical neurological deficits were present. B: 3 years and 3 months control image with stabilization and reduction of the edema, blood screening and CSF were normal (Contribution from Freiburg Neuroradiology Department)
Figure 42 Metabolic activity imaging using 18F2-fluoro-2-deoxyglucose (FDG)-PET A: 2 prior to surgery; B: 1 year following surgery, C: 3 years following surgery. Uptake ratios on C: striatum left / right 0.68 / 0.78 (for VU A 0.70 / 0.74 and for VU B 0.67 / 0.72). Metabolic analyses concluded a left striatal small diffuse increased of radio- glucose storage (in the voxel-based statistical and ROI analysis) and left dorso-frontal hypometabolism in the cortex above the aforementioned lesion; clear correspondence with the MRI Contrast enhancement. Clinical observation was recommended and performed without further relevant findings (Contribution from Freiburg Neuroradiology Department)
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Figure 43 MRI native Control image A: image taken 3 days following second surgery (left transplantation), needle tracts are visible within the caudate and putamen. B: image taken 12 months following surgery reveals edematous normal changes in the site of the grafts. C: 3 years after surgery, control image shows abnormal edematous changes on the site of the grafts, especially on the left site, no clinical neurological deficits were present. D: 3 years and 3 months control image with stabilization and reduction of the edema, blood screening and CSF were normal (Contribution from Freiburg Neuroradiology Department)
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5.3 Project III
Graft survival and striatal reinnervation
Our results evidence that cultured chromaffin cells survive within the host brain, demonstrate tyrosine hydroxylase-like immunoreactivity and can be identified. Immunohistochemistry for CHGA and TH showed surviving chromaffin and DA cells in the striatum in all in vivo studies however in few numbers (i.e. 0.6% of all implanted cells in study 1) (Figure 44-47).
In Study 1 chromaffin cells from bovine adrenal glands were isolated, propagated and cultured for 16 days prior to transplantation; these cells were then frozen and transported from Dresden to Freiburg. Once in Freiburg they were thawed and transplanted the same day into 10 SD rats. Cells were counted, and viability assessed using Trypan blue (Gibco®) [total cell number: 15x 106, with a viability of 86%]. Transplanted cells were ~500,000 cells x animal, unilaterally using 2 tracts in the right striatum (higher number than previous studies due to not ideal pre-transplant cells viability value). All animals received immunosuppression daily with intra-peritoneal CsA until perfusion. Immunohistochemistry showed surviving CHGA-positive cells, TH-positive cells and ED1-positive cells in the striatum in all grafted animals. Cell numbers were estimated at 2595±2083 CHGA-positive cells [p= 0,029], 30±6,25 TH-positive cells [p= 0,449] and 6422±3388 ED1-positive cells [p=0,00020] (compared to sham surgery).
In study 2 chromaffin cells from bovine adrenal glands were isolated, propagated and cultured for 16 days prior to transplantation; these cells were then frozen and transported from Dresden to Freiburg. In this study we decided not to transplant the cells on the same day they were thawed, but instead, to keep them in culture for 7 more days prior to transplantation. Chromaffin cells were transplanted into 10 SD rats. The total number of cells was 6x106 cells with a viability of 78%. 300,000 cells were injected unilateral per animal in 2 tracts in the right striatum. All animals received immunosuppression daily with intra-peritoneal CsA until perfusion. In this series four animals were sacrificed and brains freeze to perform graft (LCM) analyses. Immunohistochemistry showed surviving CHGA-positive cells, TH-positive cells and ED1- positive cells in the striatum in all grafted animals. Cell numbers were estimated at 2376±1292 CHGA-positive cells [p= 0,1085], 25±16 TH-positive cells [p= 0,1804] and 20993±8403 ED1- positive cells [p=0,0791].
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In Study 3 a total of 6 animals following the same protocol of Study 2 were divided and compared as follows: 3 animals (6-OHDA unilateral lesion + transplant [300.000 cells x 2 tracts in the right striatum] + DBS in the subthalamic nucleus (STN) 200 µA) vs. 3 controls (6-OHDA unilateral lesion + transplant [300.000 cells x 2 tracts in the right striatum]). The total number of cells was 6x106 cells with a viability of 78%. All animals received immunosuppression daily with intra-peritoneal CsA until perfusion. Immunohistochemistry showed surviving CHGA- positive cells, TH-positive cells and ED1-positive cells in the striatum in all grafted animals. Cell numbers were estimated at 1995±2474 CHGA-positive cells [p= 5,896], 35±33 TH-positive cells [p= 0,1263] and 5178±5882 ED1-positive cells [p=0,0684] (compared always to sham surgery).
In study 4 in this experiment the same isolation protocol as in Study 1 was followed, however we decided not to freeze the chromaffin cells but instead to transport them four days after isolation, in cultures flasks at room temperature from Dresden to Freiburg. Once in Freiburg medium was changed and three days later the chromaffin cells were transplanted in 10 SD rats. The total cell count was 11 x 106 cells with a viability of 72%; since the viability of the cells was low, we decided to transplant 600,000 cells, instead of the 300,000 transplanted in Studies 2 and 3. Animals received immunosuppression daily with intra-peritoneal CsA and were perfused at 8 weeks. Immunohistochemistry showed surviving CHGA-positive cells, TH-positive cells and ED1-positive cells in the striatum in all grafted animals. Cell numbers were estimated at 1437±629 CHGA-positive cells [p= 0,496], 160±62 TH-positive cells [p= 0,9706], 5057±3794 ED1-positive cells [p=0,4649], 218±384 LMX1A-positive cells [p= 0,2907], 530±252 GFP- positive cells [p= 0,6748] and 184±104 CFSE-positive cells [p=0,95282].
In study 5 a total of 8 SD rats received intra-cerebral allotransplantation. For this the suprarenal gland of 10 Green fluorescent protein (GFP +) Lewis rats were bilaterally dissected, adrenal medulla cells isolated and exposed to pro-neural conditions, after 7 days the cells were grafted. The total cell count was 11 x 106.cells with a viability of 76%.Eight animals were transplanted into three different targets to compare regional differences in survival: A.) Striatum (2 deposits x 1.5µl and a total of 300,000 cells); B.) Hippocampus (1 deposit x 1,0µl and a total of 100,000 cells); C.) Substantia nigra (1 deposit x1,0µl and a total of 100,000 cells) for a total number of 500,000 cells per animal. Animals received immunosuppression daily with intra-peritoneal CsA and were perfused at 4 weeks (n=2), 8 weeks (n=3) and 12 weeks (n=2). Immunohistochemistry showed surviving CHGA-positive cells, GFP-positive cells, Nurr1-positive cells, LMX1A-
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 140 positive cells, TH-positive cells and ED1-positive cells in the striatum of most animals. Cell numbers were estimated at 1439±442 CHGA-positive cells [p= 7,7E-10], 34±14 TH-positive cells [p= 0,9885], 45395±43843 ED1-positive cells [p=0,1758], 218±234 LMX1A-positive cells [p= 0,2907], 630±252 GFP-positive cells [p= 0,7975] and 211±83 CFSE-positive cells [p=0,95282].
In study 6 we aimed to compare two protocols for isolation of rat adrenal chromaffin cells for autograft transplantation purposes using 6 SD rats as hosts. Results protocol Dresden: Live cells/ml= 7,45x106; total number of live cells in 660 µl=4,9x106. Dead cells/ml= 2,1 x106=78% viability. Findings: plenty of debris, few singular cells visible. Results protocol Freiburg: Live cells/ml= 5,9x106; total number of live cells in 548 µl=3,23x106. Dead cells/ml= 1,15 x106=83,7% viability. Findings: little debris, singular cells visible, hardly any clusters.
In study 7 a total of three SD rats received intra-cerebral allotransplantation. For this 10 suprarenal glands from 5 Green fluorescent protein (GFP +) Lewis rats were bilaterally dissected, adrenal medulla cells isolated and exposed to pro-neural conditions, after 3 days the adrenal medulla cells were grafted transplanted= 293.333 per animal, using two tracts into the right striatum. Animals received immunosuppression daily with intra-peritoneal CsA and were perfused at 8 weeks. Immunohistochemistry showed surviving CHGA-positive cells, TH-positive cells and ED1-positive cells in the striatum in all grafted animals. Cell numbers were estimated at 2056±301 CHGA-positive cells [p= 0,008], 35±4,14 TH-positive cells [p= 0,1804] and 766±188 ED1-positive cells [p=0,894].
In study 8 a total of four SD rats received intra-cerebral autograft transplants. For this 14 adrenal medullas were isolated from seven SD rats donors and transplanted into three SD rats previously effectively lesioned with 6-OHDA served as host. One rat died shortly after surgery, most probably to prolonged use of propofol. Animals received 3 µl each, ~300.000 cells/animal= 2 tracts into the striatum at 1,5 µl each. For transplantation surgery, a Hamilton syringe was fitted to a glass capillary (outer diameter of 50–70 mm). Animals received immunosuppression daily with intra-peritoneal CsA and were perfused at 8 weeks. Immunohistochemistry showed surviving CHGA-positive cells, TH-positive cells and ED1-positive cells in the striatum in all grafted animals. Cell numbers were estimated at 2372±550 CHGA-positive cells [p= 0,6429],
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62±16 TH-positive cells [p= 0,8505], 60289±63589 ED1-positive cells [p=0,2200] and 376±298 Nurr1-positive cells [p=0,280].
Figure 44 Graft morphology A. Immunohistochemistry for TH: the figure evidences an intact striatum when compared with complete loss of TH-positive striatal fibers in the contralateral lesioned and transplanted striatum (E) there are however few but present intrastriatal transplanted TH+ cells. In B control striatum vs transplanted contralateral striatum stained with tissue macrophages Anti-ED1 antibody (F); In C and G control striatum vs transplanted contralateral striatum stained with Anti-Chromogranin A antibody Chromogranin-A. Figures D and H shows intact vs. lesion and transplant striatum with Anti-Nurr-1 antibody. High-power views in panels E– H illustrate single positive staining cells and fibers. The scale bar represents 500 µm.
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Figure 45 Immunohistochemistry Anti-ED-1 in four different animals from 4 different studies, the first two from bovine donor, and the last two from rat donors. A: Study 1; B: Study 2; and C Study 7 and D: Study 8. ED-1 antibody is tissue specificity, highly expressed by blood monocytes and tissue macrophages, also expressed in lymphocytes, fibroblasts and endothelial cells. Scale bar: 500 µm
Figure 46 Graft histology. A representative field of each group with chr-A (A,E), TH (B,F), ED-1(C,G), Nurr1
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(D,H) The scale bar represents 500 µm
Figure 47 Immunohistochemistry Anti-ED1. Findings in multiple brains of ED-1 staining surrounding grafted area, which instead of having the graft, presented a cyst at 12 weeks in the graft site in 4 animals that receiving xenografts
Figure 48 A: Intrastriatal Carboxyfluorescein diacetate succinimidyl ester (CFSE) passively diffuses into cells. CSFE staining + GFP positive staining from study 4. B: Immunofluorescence of TH+ cells including a big neuron extending ist processes into the graft.
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Figure 49 A. Immunohistochemistry TH+staining showing graft with TH (+) cells B. Graft and nurr 1 (+) cells from Study 7. Magnicifcation at 40x.
Figure 50 A. SD intra-striatal graft from Study 2, B. Laser capture micro-dissection (from LCM), green laser contrast Transplantation in Striatum, 63 x magnifications. C: Laser capture micro-dissection (LCM). Green laser contrast Transplantation in Striatum. C: DAPI staining. E: identification of the graft at 1,25x magnifications, laser capture micro-dissection at 46x magnification (E).
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Figure 51 Microscopic view of bovine chromaffin cells cell culture at 6 (A) and 12 (B) days after isolation, cells growth in spheres. Magnification 40 xs (Pictures by Lopez W.O)
Figure 52 Summaries of results of studies 1,3,5 and 7 from Project III. Cell count averages, standard deviations and p through ANOVA simple test are given. CHGA showed poor survival rate, nevertheless, the transplant site was filled with tyrosine hydroxylase immunoreactive fibers. Numerous macrophages filled the transplant site (ED-1).
Graft-induced side effects
Although graft-induced dyskinesia (GID) appears to be a frequent phenomenon in Vm grafted PD patients, in the rat PD model spontaneous GID is more seldom observed and cannot easily be
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 146 quantified ( Lane et al., 2006; Vinuela et al., 2008 ). In all our studies, we did not find any motor complications u observations and neither tumor formation with autologous or allogenic adrenal medulla’s cells transplantation.
Functional recovery in amphetamine-induced rotation
Figure 53 Graft-induced behavioral improvements. Rotational behavior after intra-peritoneal injection of 2.5mg/kg amphetamine (AMPH). Recovery of the motor behavioral test was present but either disappeared on time or was not statistically significant.
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Figure 54 Comparinson between AMPH-induced rotation in SD rats who received chromaffin cells transplantation and STN DBS vs. control SD rats that received transplantation alone. Rotation scores improved significantly in the Tx+DBS group. Tx (transplant).
Amphetamine-induced rotation analyzed at 4-8-12 weeks after grafting indicated not statistically significant rotational reductions in any of the groups.
In study 1 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 6,9; rotation scores 4 weeks after transplant was: 5,4 and rotation scores 8 weeks after transplant was: 5,65; [p=0,1059].
In study 2 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 7,6; rotation scores 4 weeks after transplant was: 8,1 and rotation scores 8 weeks after transplant was: 6,79; [p=0,547].
In study 3 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 7,3; rotation scores 4 weeks after transplant was: 12,1 and rotation scores 8 weeks after transplant was: 11,01; [p=0,1841].
In study 4 average values were: In the control group, rotation average scores, prior to transplant and 4 weeks after 6-OHDA lesion was: 6,8; rotation scores 4 weeks after transplant was: 4,5; rotation scores 8 weeks after transplant was: 6,7; and rotation scores 12 weeks after transplant was: 5,8 [p=0,9381].
In the DBS group, rotation average scores, prior to transplant and 4 weeks after 6-OHDA lesion was: 6,3; rotation scores 4 weeks after transplant was: 5,6; rotation scores 8 weeks after transplant was: 6,7; and rotation score 12 weeks after transplant was: 6,1. Rotation score during
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DBS was in average: 2,3 with standard deviation of ± 0.9 at 200 µA STN continuous stimulation.
In study 5 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 7,1; rotation scores 4 weeks after transplant was: 10,4; rotation scores 8 weeks after transplant was: 8,9; and 12 weeks after: 9,2[p=0,265].
In study 7 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 9,9; rotation scores 4 weeks after transplant was: 10,4; rotation scores 8 weeks after transplant was: 10,7; [p=0,9539].
In study 8 average values were: rotation average scores, prior to transplant and 4 weeks after 6- OHDA lesion was: 14,7; rotation scores 4 weeks after transplant was: 13,1; rotation scores 8 weeks after transplant was: 12; [p=0,893].
Study/n Cell Host Behavioral results Cell Survival rate Time of animals source evaluation after graft 1/10 Bovine SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 2595±2083 8 w adrenal (6-OHDA) Not statistically significant reductions Intrastriatal TH: 30±6,25 Intrastriatal ED1: 6422±3388 2/10 Bovine SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 2376± 1292 8 w adrenal (6-OHDA) Not statistically significant reductions Intrastriatal TH: 25 ±16 Intrastriatal ED1: 20993±8403 3/6 Bovine SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 1995±2474 12 w Adrenal (6-OHDA) Not statistically significant reductions Intrastriatal TH: 35±33 Intrastriatal ED1: 5178±5882 4/10 Bovine SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 1437±629 8 w adrenal (6-OHDA) Not statistically significant reductions Intrastriatal TH: 160±62 Intrastriatal ED1: 5057±3794 5/8 Lewis rat SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 1439±442 4, 8, 12 w GFP (6-OHDA) Not statistically significant reductions Intrastriatal TH: 34±14 adrenal Intrastriatal ED1: 45395±43842 6 SD rat In vitro Protocol 1 (Dresden) Live cells/ml= 7,45x106; viability 78% adrenal Protocol 2 (Freiburg) Live cells/ml= 5,9x106; viability 83,7% 7/3 Lewis rat SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 2056 ±301 8 w GFP (6-OHDA) Not statistically significant reductions Intrastriatal TH: 35±4 adrenal Intrastriatal ED1: 766±188 8/3 SD rat SD Uni STX AMPH-induced rotation behavior: Intrastriatal CHGA: 2372±550 8 w adrenal (6-OHDA) Not statistically significant reductions Intrastriatal TH: 62±16 Intrastriatal ED1: 10217±4351
Table 9 Experimental design and resume of results in all 8 studies of Project 3.
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6. DISCUSSION
Over the last decades, significant efforts have been made to improve the standard of care of patients with neurodegenerative diseases, such as Parkinson’s disease (PD). These efforts have led to the development of new generation of therapeutics including nerve growth factor (NGF), subcutaneous apomorphine pumps, Duodopa® pumps, and Deep Brain Stimulation (DBS). All of these therapeutic modalities are focused on the alleviation of the symptoms of the disease and can be used to achieve temporary improvement of the neurological status. This consequently improves the quality of life of the patients. However, the progressive course of the disease is not influenced and the neuronal loss remains unstoppable.
The interest in the research of neural stem cells (NSC) observed in the last decades, is inspired by the hope of the future application of the NSC-based technology in clinical therapeutic treatments. It aims at the replacement of the diseased neural tissue in order to reconstitute functional neuronal circuits lost due to trauma, stroke or neurodegenerative disorders (Lindvall and Kokaia, 2006). Given the complexity of the human nervous system, the prospect may seem remote. Nevertheless, there is a growing body of evidence from experimental animal studies and clinical trials, proving that efficient direct neuronal replacement using human fetal tissue can exert clinical stabilization and in some cases lead to improvement of compromised functions (Brundin 1996, Freed 2001, Hantraye 1992, Isacson 1995, Lindvall and Kokaia 2010, Freeman 2011, Barker and Björklund 2013).
A variety of non-fetal stem cell types have been and are currently being tested in laboratories to generate material suitable for transplantation (Boyer 1995, Barker 2013). However, none of these non-fetal stem cell types are yet considered stable and safe enough to initiate human clinical trials. Since 1987 an estimated 450 patients with PD or HD have been transplanted (Lindvall 2004) with human fetal mesencephalic tissue, rich in post mitotic primary dopamine (DA) neurons, or with ganglionic eminences harboring precursors of striatal GABA-ergic medium spiny interneurons respectively. The transplanted tissue was derived from aborted human fetuses, between 6-12 weeks of gestation. In case of PD patients, they were grafted mainly into the putamen bilaterally, using between three to five donors per hemisphere (Lindvall 1992, Freed 1992). There were also reported cases of caudate nucleus transplantation (Henderson 1991,
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Molina 1991, Lopez-Lozano 1991, Spencer 1992) and of putamen and substantia nigra (SN) simultaneous transplantation (Mendez 2002). Several open-label trials reported clinical benefit and long-term graft survival. In some patients this led to withdrawal of medical treatment during years after transplantation (Hagell 1999, Wenning 1997, Brundin 2000, Piccini 1999). The mean improvement of motor-symptoms achieved as measured by the motor part of the United Parkinson Disease Rating Scale (UPDRS) was between 30% and 40% at 10-24 months post grafting (Lindvall and Björklund, 2004).
In response to the increasing number of reports derived from small trials claiming positive results, the FDA supported two double-blind sham surgery-controlled clinical studies for PD transplantation; using intrastriatal implants of human fetal mesencephalic tissue in an attempt to define, in an prospective randomized manner, the true impact of the cell-based neuroreplacement therapy on cardinal symptoms of the disease (Freed 2001, Olanow 2003). The first study reported 34% improvement in the UPDRS in patients younger than 60 years with modest clinical response for the rest, and no clinical improvement in the sham-surgery group. Histopathological analyses of two transplanted brains, from patients who died independently of the grafting, showed a number of dopaminergic neurons reaching between 7,000 and 40,000 in each putamen years after the transplantation procedure. It is important to note, that this number of dopaminergic neurons was much lower than in previously reported open trials (Kordower 1995, 1996, 1998). The low number of graft-derived dopaminergic cells could be related to the use of only two donors in each putamen. It could also be related to the long tissue storage prior implantation, which took up to 4 weeks impairing, possibly, the viability of the cells and the lack of immunosuppressive treatment after transplantation (Lindvall and Björklund, 2004).
The second double-blind sham surgery-controlled clinical study for PD (Olanow 2003) had a poor clinical outcome. This study was performed in patients at a more advanced stage of the disease as compared to the open-label trials. Furthermore, patients received solid pieces of graft instead of single cell suspension from one to four donors and immunosuppressive therapy with Cyclosporine, but only for 6 months. Positive clinical response, when present, in those two double blind sham surgery-controlled clinical studies appeared between 6 to 12 months after transplantation (Lindvall and Björklund, 2004).
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Human VM from fetuses has not been the only source of dopaminergic tissue tested. Different open-label trials performed during the 90’s used a different cell source: fetal swine mesencephalic neural cells (xenotransplantation) (Pakzaban & Isacson 1994; Deacon 1997). It is important to mention, that most of these trials were not followed by systemic immune suppression with Cyclosporine which presumably had a large impact on the disappointing survival of the transplanted tissue possibly leading to the observed lack of constant clinical improvement of the grafted patients. Only about 0.1% of the total number of transplanted fetal swine dopaminergic cells survived seven month following implantation.
Open PD trials using fetal human cells continued to report encouraging outcomes. Mainly young patients, at an early stage of the disease, showed amelioration of the symptoms. Interestingly, in this subgroup some of the patients presented secondary clinical deterioration developing graft- induced dyskinesias (hyperkinetic, dystonic abnormal involuntary movements, and postural impairments similar to those found in the majority of patients after chronic 5–10 years of L- DOPA treatment). This was a turning point which led to a general discontinuation of the transplantation procedures.
All these trials supported the assumption that grafts of primary neurons were able to survive in the host environment when taken from human fetal donors, having the potential to establish extensive host re-innervation, and initiating neurotransmitter’s release leading to partial functional recovery (Arvidsson 2002, Björklund and Lindvall 2000, Döbrössy 2010).
The clinical experience gained in PD has been translated to Huntington’s disease, another neurodegenerative disease affecting the basal ganglia. HD is an inherited genetic disorder caused by mutation of the gene (IT15) on the chromosome 4p16.3. This mutation causes an abnormal number of CAG repeats (>36) within its 5'-end coding sequence (The Huntington's disease Collaborative Research Group 1993). The presence of this mutant IT15 gene (huntingtin) in the cell causes activation of pro-apoptotic processes in early stages of the disease. This progressively leads to extensive, but not exclusive, striatal neuronal cell loss of the medium spiny neuron population, neuronal type responsible for the production of γ-aminobutyric acid (GABA) (Kaplan 2012). Such loss gives rise to progressive motor, cognitive, and psychiatric deficits (Foroud and Conneally, 1999). Applying fetal cell grafting to replace damage host striatal’s neurons might
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 152 retard or even reverse clinical signs and symptoms during the otherwise unequivocally deathly course of HD (Capetian 2009).
PROJECT I
Here, we present a comprehensive guide of the necessary pre-operative preparation, and a detailed surgical protocol with state-of-the–art technical modalities for HD and PD neuronal transplantation; based on our experimental and clinical experience. The clinical outcome of cell- based replacement therapy strictly depends on a multidisciplinary, stepwise and rigorous procedure including adequate patient selection, appropriate handling of the tissue, expertise in dissection and careful preparation of the graft as well as precise stereotactic surgical implantation.
The clinical success of CNS transplants depends largely on the optimization of the transplantation protocol concerning patient selection, definition of the source of transplanted cells, graft preparation, and the quality of surgical intervention being performed.
The pioneering human neuronal transplantation was carried out in Lund, Sweden in 1987 (Lindvall 1988, Madrazo 1988), using 7-9-week-old human fetal tissue in PD patients. Though initial results demonstrated varying degrees of clinical recovery, many patients showed viable grafts and clinical improvement many years after transplantation (Hagell 1999). Up to now, more than 350 PD and 100 HD patients have been transplanted worldwide (Dyson and Barker, 2011).
Interestingly, two North American clinical trials failed to demonstrate clinical benefit in all patients and also reported severe complication - a graft-induced dyskinesias (GID) (Freed 2001, Kordower 1997, Olanow 2003). As a result of these reports, further trials were stopped for years. However, despite the “disappointing” inconsistent overall result of both studies, a sub-population of patients improved clinically and could reduce or stop their medication for some years (Freed et al. 2001, Olanow 2003). A closer look at the results enabled the identification of factors that can improve the outcome of future neural transplantation trails. Patients younger than 60 years of age, having a less advanced disease status are more suitable candidates to benefit from the transplantation procedure (Olanow 2003). In addition, the most critical point of those trials was the apparent lack of standardized protocols concerning the age of the fetus, the time of transplantation (some embryos were transplanted after 72 days of hibernation, endangering cell
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 153 viability and graft-host-integration capacity), the critical determination of the disease stage as well as previous history of levodopa induces dyskinesias (LID). The role of serotonin neurons in the etiology of GID has also been investigated and the experimental data suggests that as long as sufficient dopaminergic neurons are present, serotoninergic cells from the graft do not need to be excluded (Garcia 2012, Shin 2012, Politis 2011). Therefore, a development of standardized step- by-step protocols as presented here based on a consensus of clinical centers involved in neural transplantation, may lead to reduced procedure-related complications, increasing the chance for potential clinical improvement. Designing a successful clinical neurotransplantation trial relies on the precise orchestration of this multistep endeavor including development of an embryo tissue supply network, establishing good medical practice (GMP) standards in the laboratory, and training of surgical stereotactic and tissue preparation teams.
In our clinical experience fetal brain transplantation is a safe surgical procedure. In our series of patients, no surgery-related complication occurred. Adverse effects were mostly related to prolonged immunosuppression (i.e. systemic repetitive infections). However, potential risks of transplantation therapy for neurodegenerative diseases need to be taken into account when aiming to perform intracranial transplantation studies. These include:
1. Sub-optimal preparation. This risk can be reduced with strategies that include rational patient selection and appropriate dissection of the fetal tissue. Due to the fact that tissue for transplantation is obtained in a multi-step process, the errors influencing clinical success and safety can be made either during transport, the choice of media, anatomical dissection, or during hibernation. Standardization and validation of these process steps allows for reproducible and safe clinical graft applications.
2. Risk of transmission of viral- or bacterial diseases from fetal tissue. Blood tests and further microbiological analyses after transplantation are absolutely indispensable although, no infection has been detected among transplanted patients to date. Nevertheless in blood analyses from donor prior to dissection, fetal tissue bacterial analyses before and after transplantation and conservation of the tissue for eventual tests need to be routinely performed.
3. The surgical risk. This mainly depends on the experience of neurosurgical stereotactic team, and can be significantly minimized by having an adequate surgical planning strategy on a state- of-the-art stereotactic workstation applying pre-operative imaging (CT and MRI). In our
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 154 experience no operative or post-operative surgical complications occurred, and all patients satisfactory tolerated the procedure. There were no adverse events attributable to either anesthesia or neurosurgery and all the grafts could be identified on MRI scans appropriately localized. Previous published data of neural transplantation in HD patients also showed no serious incidents (Bachoud-Levi and Peschanski, 2000). Some minor complications such as subdural hematoma or hygroma have been reported in the past, commonly in later-stage patients with a greater degree of cerebral atrophy (Bachoud-Levi 2000, Hauser 2007).
4. Side effects of immunosuppressant. Some patients develop repetitive infections as a consequence of the long-lasting immunosuppressant regiment. The administration of an immunosuppressant is necessary to avoid graft rejection. To monitor the risk, it is important to perform a regular laboratory analyses including regular blood examinations and give the patient clear instructions concerning alert signs, such as fever, polyuria, etc., as well as recommending to visit the emergency room in case of any acute or progressive changes.
5. Graft rejection and overgrowth of the graft. Without immunosuppression, eventual rejection seems to be inevitable. However, the duration of compulsory immunosuppression remains to be established (Rosser A. et al. 2011). In case of rejection, HLA antibody assessment allows comparing before and after immunological responses to the transplantation, for this it is important to keep part of the transplanted tissue stored at -80°C. Overgrowth of the graft has been hypothesized as a complication in HD transplantation due to the proliferative nature of the cells transplanted from WGE, with a possible unexpected growth (Freeman et al. 2011). Possible complications emphasize the need for proper standardization, documentation, training and validation of tissue dissection protocols, surgery, and follow up.
Immune responses can occur in the brain, thus according to experimental models is not an absolute immunologically privileged site (Barker 2004). Activated lymphocytes can cross the BBB, while certain cells such as microglia may have an antigen presenting cell capacity and further to this there is lymphatic drainage from the brain into the cervical lymph nodes.
Fetal neural cells grafts may provoke a humoral response as in other organ transplants (Hourmant 2005), with specific alloimmunisation to donors’ antigens in a significant proportion of patients. The humoral response has been studied in the pathogenesis of acute or hyperacute vascular rejection and also in chronic rejection were reported that a large proportion of transplanted
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 155 patients exhibited biological signs of alloimmunisation to donors’ antigens, in general a few months after interruption of the immunosuppressive treatment (Krystkowiak 2007).
The screening for immune rejection can be performed by searching for the appearance of HLA antibodies, which in other organ transplants is associated with poor transplant outcome and acute and chronic rejection. HLA control assay is performed before surgery and should be negative for HLA antibodies, in case of an immune rejection a serum sample is taken and should remain HLA ab negative. In case of a positive HLA the patient should receive aggressive acute anti- inflammatory treatment associated to rapidly reinstatement of the original immunosuppressive regimen in the case that is not being delivered.
Limited availability of the primary tissue, ethical concerns and immunosuppression related complications, drive the search for new, alternative, “off-the-shelf” cell sources in cell-based neuroreplacement therapy. Despite promising laboratory results concerning novel cell types, the clinical neurotransplantation today still depends on human fetal tissue. When applied in the frame of a regulated and rigorous transplantation protocol, it has been demonstrated to have the ability to partially restore function, and improve the quality of life in subgroups of patients with neurodegenerative disorders.
PROJECT II
Huntington’s disease (HD) is a fatal inherited genetic disorder caused by mutation of the gene
(IT15) on the chromosome 4p16.3 causing an abnormal number of CAG repeats (>36) within its 5'-end coding sequence. The huntingtin gene was identified in 1993 by the Huntington’s Disease Collaborative Research group but until today the molecular pathways that end causing cell death remain poorly elucidated.
HD is a neurodegenerative disorder present worldwide. The highest prevalence of the disease is on Caucasians descent families and is calculated to be 5-7.5 in 100.000 people (Warby et al., 2011). Important concentrations of HD patients are also found in Juan de Acosta near Barranquilla in Colombia and in the state of Zulia, on the east coast of Lake Maracaibo in Venezuela, with 700 cases per 100.000 being the highest concentrated population reported. The disease is generally of low occurrence in Asian and African populations (Kaplan A., 2012).
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Symptoms appear between the third and fourth decade of life. Clinical features are consequence of severe cell loss (up to 60% of type II Golgi-neurons) and atrophy in the striatum, with consequent loss of thalamus inhibition, and hyper activation of the direct basal ganglia circuit (Lobo, 2009). Such neurodegeneration causes progressive motor, cognitive, and behavioural deterioration, until dementia (Foroud et al., 1999; Rosser A., 2012). Mortality appears unequivocally in a period of 7 to 10 years after onset of symptoms and generally from complications such pneumonia and heart disease. (Sorensen and Fenger, 1992; Zuccato et al., 2010)
At present there are no curative treatments for HD and there is a lack of effective symptomatic therapies. Bilateral stereotactic human fetal striatal micro-transplantation (HFST) is a promising experimental therapy for patients with HD. Neuronal transplantation had demonstrated in open clinical trials it’s feasibility, safety, and proof-of-principle (Peschanski M. 1995; Kopyov OV 1998; Freeman TB. 2000, 2011; Roser A, NEST-UK 2002; Hauser 2002; Bachoud-Levi A 2006; Reuter I.2008; Gallina P 2008; Ruwani W. 2011).
Studies in humans are still experimental and HFST is not yet a standard treatment since clinical outcome has not yet been established conclusively. Safety of the procedure is an essential requirement for a clinical study. However as with any other surgical procedure, complications can occur. Intracranial haemorrhages, symptomatic subdural hygromas, and infections related to donor origin and graft-rejection have been previously reported (Hauser 2002; Krystkowiak P. 2007). Transplanted tissue has little migratory capacity and mostly remains in the surgical implantation site (Bachoud-Levi A. 2000; Gallina P. 2008); making adequate caudate-putaminal cells implantation crucial to increase the chances of a successful transplantation (appropriate donor-host cells connectivity).
The transplantation technique involves the surgical implantation of the tissue by means of a stereotactic procedure. Coordinates of the targets are currently calculated on workstations using direct target visualization on MRI images onto stereotaxic equipment (Gallina P. 2008) or MRI plus contrast medium computed tomography (CCT) image fusion. Such strategy requires time consuming planning sessions, defining the individual targets and tracts, optimizing spatial distribution and safety.
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Hoping to fulfil the need of an easier, faster, and more precise way to achieve a safe and homogeneous intrastriatal graft distribution, we report and propose the use of an original software that allows fast distribution of the stereotactic coordinates in relation to the MCP proposing the user automatically the planning coordinates for transplantation with two entry points and six tracts. Planning can be modified manually, according to anatomy or presence of vessels on direct image visualization and mirrored coordinates to be used in the contra-lateral side. Trajectories proposed by the software move as a whole or as individual tracts. Evaluation of clinical benefit of the transplantation procedure is out of the scope of this manuscript since a minimum of two years is required to evidence the efficacy of the transplant and that analyses will be part of the final results of an on-going multicentre study the MIG-HD trial phase I.
HD is a progressive, fatal and difficult to treat neurodegenerative disorder. Neural transplantation is a promising experimental treatment for HD, attempting to functionally replace lost neurons and improve patient outcomes. Such strategy may be able to offer the patient stabilization of the disease and in some cases even temporarily improve motor and cognitive deficits. The challenge to deposit multiple grafts in an atrophic brain requires, fine-tuning of the stereotactic procedure, beginning with the planning process to assure the safety of the procedure.
The selected targets allow the distribution of grafts in the caudate nucleus, anterior putamen (areas primarily associated with cognitive and neuropsychological function) (Farrer LA 1986; Freeman TB. 1995; Olanow CW 1996) and post-commissural putamen (primary involved in motor circuits) (Hauser RA 2002; Wijeyekoon R. 2011). o date, some series have reported that neural cell replacement therapy is not associated with serious surgical complications (Kopyov OV 1998; Rosser A 2002). Subdural hematoma seems to be the most frequent one, and has been directly related to the degree of cerebral atrophy. It may suggest that surgery performed in an earlier stage of the disease could minimize this problem (Hauser RA 2002). Common post- operative complications such as subdural hygromas (SDHs) are generally the consequence of chronic subdural hematomas, commonly seen in elderly patients. Large hygromas may cause secondary localized mass effect on the adjacent brain parenchyma. SDHs are reported in the literature as a notable adverse event in HD transplantations studies. Intraoperative CSF loss causes brain “shrinkage” and shift in HD patients who had cerebral atrophy, expanding the subdural space, predisposing to postoperative SDHs (Freeman TB. 2011). The risk may be
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 158 directly related to the degree of cerebral atrophy, and could be reduced by covering the burr hole with bone cement or fibrin glue (Hauser RA 2002; Freeman TB 2000).
In the present patient series, no operative or post-operative complications occurred, and the procedure was well tolerated by all the patients. There were no adverse events attributable to either anaesthesia or to the neurosurgical procedure. The grafted areas could be identified on MRI scans as appropriately placed into the caput nuclei caudate and putamen. Neural transplantation in HD patients has been previously performed without serious incidents (Bachoud-Levi A. 2006; Gallina P. 2008) or in some instances with some relevant complications (Hauser R. 2002). However, to the best of our knowledge, this is the largest reported patient series and therefore it is especially suited to create a data base for target coordinates.
The model-based coordinates predicted by the software optimized the surgical planning, which is considered a key part of the surgery. The presented technique can also be applied in another context where bilateral neurotransplantation is aimed for, for instance in patients with Parkinson’s disease. The software presented here, allowed achieving a faster distribution of the stereotactic coordinates in relation to the MCP for the first surgery and providing mirrored coordinates for the contralateral transplantation facilitating an optimized spatial distribution of the grafts. The stereotactic trajectories have to be adapted for anatomical variations, according to the disease-related brain atrophy, and to the presence of blood vessels, by selecting the most suitable entry point. The proposed use of model-based coordinate’s data bank can shorten procedure planning time and thus increase the safety of the overall procedure.
PROJECT III
Adrenal medullary chromaffin cells are derived from the neural crest and belong to the sympathoadrenal cell lineage (Unsicker 1978); such cells were intented as a cell source for transplantation during the first two clinical trials of cell transplanted stereotactically or by microsurgery into the brain of PD patients (Backlund 1985, Madrazo 1987). Initial outcomes reports claimed success on primary outcomes but were in time disappointing on maintaining such improvement. Additionally 40% of 126 patients suffered from side effects (Newman 2009). Autopsy from a patient who died 2 years after transplantation reported that no DA producing chromaffin cells had survived. Bakalay proposed the partial improvement result as the
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 159 consequence of neurotrophic effects on the host surviving DA neurons influenced by neurothropic factors released by the graft. Since that time advances in the understanding of chromaffin cells molecular bases have been important, and they remain as a promising alternative cell source in different pathological states, mainly PD and chronic pain. Chromaffin cells can act as mini-pumps that release amines and peptides. Different strategies are being test to improve the survival and efficacy of the graft, however there is a lack of molecular studies that support behavioral results (Ambriz 2012). Neural crest derived chromaffin progenitors isolated from adult adrenal medulla outline a potential grafting strategy for PD research. Bornstein group from Dresden developed a technique to grow these cells in chromospheres. This protocol significantly increased the number of TH positive dopaminergic neurons and dopamine secretion, and reduced epinephrine and norepinephrine production (Chuung 2009). Project number III was born on the necessity to assess the viability of chromosphere grafts and its effects on behavior.
We achieved a higher number of chromaffin cells survival ~2000 vs. 200 cells in previous reports (Freed et al. 1986), however the AMPH-induced rotation behaviour showed globally not statistically significant reductions. We tried in 8 different studies to modify factors that may affect the outcome i.e avoid to freeze the cells, to perform experimental allograft instead of xenograft, to use a glass capillary in the tip of the siringe for more gentle treatment of the cells achieving an average of 1% in vivo survival of transplanted cells. Fetal nigral cells transplantation has been reported to achieve a 3 to 5% grafted neurons survival remaing as the best cell source until today (Brundin 1998). There is to say that some animals were found to reduce until 40% their circling behavior. TH immunoreactivity increased specially in study 1 (Bovine to rat transplant after 16 days of culture). Some grafts became necrotic with a perigraft halo, which has been reported previously (Bankiewicz 1994, Bohn 1982, Bresjenac 1997) and all have strong presence of macrophages which contributed to poor survival. Interestingly, xenografts (bovine to rat) had a major predisposition to develop this phagocytic reaction with the formation in some animals of intrastriatal cysts. The possibility to increase dopaminergic cell numbers in vitro (dopaminergic population in the adult adrenal medulla is only 1%) remains an important current area of research due to the trophic factors that can promote survival of dopamine neurons in vivo. Extra-adrenal chromaffin cells (paraganglia and Zuckerkandl organ) are other proven source with sustained neurorestaurative action and better survival that the author
Huntington's and Parkinson's disease: Clinical and Experimental Transplantation 160 may test in the future. Studies in human adrenal medulla are also in the scope of the author for future trials.
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PUBLICATIONS
PUBLICATIONS INCLUDED IN THIS THESIS
I. Lopez WO, Nikkhah G, Kahlert UD, Maciaczyk D, Bogiel T, Moellers S, Schültke E, Döbrössy M, Maciaczyk J. (2013) Clinical neurotransplantation protocol for Huntington's and Parkinson's disease. Restor Neurol Neurosci. Jan 1;31(5):579-95.
II. Lopez WO, Trippel Michael. (2013) Atlas-base Coordinates of Targets for Stereotactic Bilateral Neural Transplantation. Stereotact Funct Neurosurg; 91(suppl 1): 1-334 – Page 213.
III. Lopez WO, Nikkhah G, Schültke E, Furlanetti L, Trippel M. (2014) Stereotactic planning software for human neurotransplantation: suitability in 22 surgical cases of Huntington's disease. Restor Neurol Neurosci. 2014;32(2):259-68. doi: 10.3233/RNN-130340.
PUBLICATIONS DURING DOCTORATE PERIOD NOT INCLUDED IN THIS THESIS
III. Reithmeier T, Lopez WO, Doostkam S, Machein MR, Pinsker MO, Trippel M, Nikkhah G.(2013) Intraindividual comparison of histopathological diagnosis obtained by stereotactic serial biopsy to open surgical resection specimen in patients with intracranial tumours. Clin Neurol Neurosurg. Jun 14. [Epub ahead of print] PMID: 23769864
IV. Lopez WO, Trippel M, Doostkam S, Reithmeier T. (2013) Interstitial brachytherapy with iodine-125 seeds for low grade brain stem gliomas in adults: Diagnostic and therapeutic intervention in a one-step procedure. Clin Neurol Neurosurg. Aug; 115(8):1451-6.
V. Lopez WO (2013) Practical Use of an Auditory Device to improve Gait in patients with Parkinson’s disease. Basal Ganglia Volume 3, Issue 1, Page 54.
VI. Lopez WO, Espinoza Martinez JA, Escalante Higuera CA. (2013) Gait improvement in 10 patients with Parkinson's disease by feedback from a new portable auditory device with smartphone applications. Movement Disorders, June Volume 28.
VII. Reithmeier T, Lopez WO, Spehl TS, Nguyen T, Mader I, Nikkhah G, Pinsker MO. (2013) Bevacizumab as salvage therapy for progressive brain stem gliomas. Clin Neurol Neurosurg. Feb; 115(2):165-9.
VIII. Lopez WO, Vialle EN, Anillo CC, Guzmao M, Vialle LR. (2012) Clinical and radiological association with positive lumbar discography in patients with chronic low back pain. Evid Based Spine Care J. Feb; 3(1):27-34
IX. Fonoff ET, Lopez WO, de Oliveira YS, Lara NA, Teixeira MJ. (2011) Endoscopic approaches to the spinal cord. Acta Neurochir Suppl; 108:75-84.
X. Fonoff ET, de Oliveira YS, Lopez WO, Alho EJ, Lara NA, Teixeira MJ. (2010) Endoscopic-guided percutaneous radiofrequency cordotomy. J Neurosurg; Sep:113(3):524-7
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ACKNOWLEDGEMENTS
I deeply thank my family: Himelda Lopez, Pascual Contreras, Carlos Andres y Javier Dario Contreras Lopez as well as Ana Maria and Javier Alejandro, because they were the most directly affected by my absence. I thank the whole Lopez family and Efrain Guerrero, to Carolina Casas for her love and friendship; my very especial thanks to Frederik and the Dresch family. Thanks to Freiburg and all the friends I found here, I thank Liliana Carvajal, Monika Gomez, Sussette Schweigert Katharina Mutter and all the dentist gang, Helena Valerie, Lotte Elmlinger, la familia Mexicana, Johana Quintero, Adriana Walker, Juliana Mendoza, Nathalia Anaya, Tatyana Barbosa, Manuella Caldana, Serena Neri, Carlos Alberto Paez, Carlos Andres Escalante, la Asociacion Colombiana de Neurocirugia, everyone from the Goethe gäste hause, Jairo Espinoza, Hans Carmona, Diana Gomez, Ana Lucia Bastos, Scheila Karam, Eric Vega, Juan Carlos Benedetti, Juan Carlos Oviedo, Gustavo Bohorquez, Richard Wunderlich, Chas Harris, Eric Fonoff, Christian Winkler, Hans dieter-Hoffman, Thomas Freiman and Andreas Schulze- Bonhage.
I deeply thank Prof. Guido Nikkhah for his patience, kindness, support and friendship, to Mate Dobrossy, Thomas Reithmeier, Marcus Pinsker, Jaroslaw Maciaczyk and Michael Trippel, Johanna Wessolleck, Joacir Cordeiro, Ulf-dietrich Kahlert and every single person from the Stereotaxy Interventional Laboratory and the Stereotactic Neurosurgery Department from Freiburg Medical Center
I thank Prof. Volker Coenen for his support. I thank the centers involve in the multicentre study Euro-HD phase II trial, To GE Euro-HD; to the neuroradiology department in Freiburg and the French collaboration group lead by Prof. Dr. A. Bachoud-Levi as well as NECTAR group and TransEuro organizations for permanent close collaboration. Special thanks to DAAD to Iñigo Alonso Aguirre and Luciano Furlanetti. I thank everyone who at some point missed my company, and everyone with whom I shared a moment in the last 4 years, even if it lasted only some minutes. Finally I would like to thank my future wife and kids wherever you are, soon we will meet.