THE EFFECTS OF MINOCYCLINE ON SPINAL ROOT AVULSION INJURY IN RODENT MODEL: A HISTOLOGICAL STUDY

By

DR. TAN YEW CHIN MSurg Neurosurgery (Universiti Sains Malaysia)

Dissertation Submitted in Partial Fulfillment of the Requirement for the Degree of Master of Surgery (Neurosurgery)

UNIVERSITI SAINS MALAYSIA

2015

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Abstrak

Pengenalan: Kecederaan saraf brakial adalah sesuatu kecederaan yang

memudaratkan. Kecederaan tersebut melibatkan 5% kes-kes politrauma yang diakibatkan oleh kemalangan trafik. Matlamat utama rawatan kecederaan saraf brakial adalah pemulihan tenaga motor. Perawatan kecederaan saraf brakial secara pemindahan graf saraf boleh memberikan perlindungan kepada saraf motor. Namun begitu kesan rawatan pembedahan adalah sangat terhad. Rawatan secara perubatan perlu diberikan untuk mengekalkan fungsi yang wujud pada saraf motor. Minocycline sudah terbukti untuk membekalkan perlindungan kepada sel-sel saraf dalam penyakit angin ahmar dan penyakit pengelupasan selaput saraf. Minocycline adalah antibiotik yang mudah didapati, ekonomik, dan berupaya untuk merawat radangan dan kerosakan sel sel saraf. Literasi saintifik telah menunjukkan bahawa Minocycline berupaya untuk menghasilkan kesan perlindungan kepada sel-sel saraf motor dengan prencatan sel microglia dan Nitric Oxide Synthase. Kedua-dua kesan terapiutik ini dapat mengurangkan radangan dan degenerasi saraf setelah mendapat kecederaan

‘avulsion’ saraf brakial.

Objektif: Kajian ini adalah untuk mengkaji kesan Minocycline yang selama ini digunakan sebagai antibiotik kepada hayat sel saraf motor dan juga penghasilan sel

Microglia.

Metodologi: Kajian eksperimental ini menggunakan tikus dewasa Sprague Dawley di

mana urat saraf segmen C7 tikus ditarik sehingga putus daripada saraf tunjang.

Transeksi fizikal ini akan dipastikan untuk berlaku sebelum ganglion saraf. Untuk

mendapatkan validasi saintifik, saraf yang terputus telah diperhatikan di bawah

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Mikroskop untuk memastikan akar-akar saraf dicabut bersama-sama dengan tangkainya. Tikus yang mana transeksi saraf berlaku selepas ganglion telah dikecualikan daripada kajian ini. Tikus-tikus yang mempunyai kecederaan saraf brakial telah dirawat dengan ‘intraperitoneal’ dan ‘intrathecal’ Minocycline 50mg/kg untuk seminggu dan 25mg/kg untuk seminggu yang selanjutnya untuk pemulihan sel saraf motor. Selepas enam minggu, saraf tunjang tikus tersebut akan diambil dan ujikaji histokimia dijalankan untuk mengira sel saraf motor dan sel Microglia.

Keputusan: Kajian ini menunjukkan bahawa hayat sel saraf motor telah berkurang dan penghasilan sel Microglia bertambah selepas tikus Sprague Dawley mendapat kecederaan saraf brakial. Kajian ini juga mendapati bahawa Minocycline berupaya untuk mengurangkan bilangan sel Microglia. Rawatan secara ‘intraperitoneal’ telah mendatangkan kesan yang bermanfaat dari segi perlindungan sel saraf motor melalui perencatan aktiviti sel microglia. Namun begitu apabila ubat tersebut telah disuntik secara ‘intrathecal’, bukan sahaja ia mengancam kehidupan sel saraf motor, malah ia juga mendatangkan kesan negatif lain yang memudaratkan.

Kesimpulan: Penggunaan Minocycline mempunyai dua implikasi. Pengurangan sel

Microglia dengan menggunakan Minocyline dalam dos yang optimal dapat mengurangkan kematian sel saraf selepas kecederaan saraf brakial. Namun, apabila

keberkesanan dan konsentrasi ubat Minocycline ditingkatkan, ubat ini boleh meracuni

sel-sel saraf motor melalui perencatan tindak balas sel glia dan degenerasi walerian.

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Abstract

Introduction: Brachial plexus injuries are debilitating injury affecting young population, which comprise 5 % of all polytraumas caused by road traffic accidents.

Currently the primary aim for the management of root avulsion of the brachial plexus is motor recovery. However, immediate repair or nerve grafting offers some degree of protection to the motoneurons but is clinically limited, so there remains a need for medical approaches to maintain the viability of the injured motor neurons.

Minocycline has been proven to show its neuroprotective effect in stroke and demyelination disease. It is a widely available, cost effective antibiotic with anti- inflammatory and anti-apoptotic properties. Literatures have shown that Minocycline exert its neuronal protection effect primarily via Migroglial inhibition and Nitric

Oxide Synthase downregulation. Both of the effects are key therapeutic means to ameliorate neuroinflammatory and degenerative process following secondary traumatic avulsion injury.

Objective: To study the Neuroprotective effect of Minocycline in adult Sprague

Dawley mouse that suffer from Brachial Plexus Injury.

Methods: The C7 nerve root was avulsed via anterior extravertebral approach. The traction force transected the ventral motor nerve roots at the preganglionic level. The avulsed rootles can be seen under the microscope for validation of the avulsion rodent model in which the avulsion is properly done at the preganglionic level. The rodents in which the transection takes place distal to the rootlets were excluded from the study

Intraperitoneal and intrathecal Minocycline 50mg/kg for the first week and (25mg/kg

iv for the seconda week) was administered to promote motor healing. The spinal cord was harvested at 6 weeks after the injury to analyze the structural changes following avulsion injury and the pharmacological intervention.

Results: Motor neuron death and microglial proliferation was observed following traumatic avulsion injury of the ventral nerve root. The administration of Minocycline to treat rodents suffering from avulsion injury was capable to suppress the Microglial proliferation. Intraperitoneal administration of Minocycline shows some degree of beneficial effect to prolong the motor neuron survival by inhibiting the Microglia activation and proliferation, thus hampering apoptosis of the motor neuron. However when Minocycline was administered via intrathecal route to increase the bioavailability of the therapeutic agent, not only that it compromises the motor neuron survival, some other deleterious effect were also demonstrated.

Conclusion: Microglial suppression via Minocycline can have double effect.

Moderate dosage of Minocycline may be beneficial towards motor neuron survival.

On the other hand, high concentration of Minocycline ( similar drug dosage with increased potency via targeted drug delivery) could be neurotoxic by causing impairment of the Glial response and Wallerian degeneration, which is a pre-requisite to regeneration.

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Table of Contents

Abstrak…………………………………………………………………………………… ii Abstract…………………………………………………………………………………... iv Table of Contents………………………………………………………………………... vi List of Tables…………………………………………………………………………….. ix List of Figures……………………………………………………………………………. x List of Abbreviations……………………………………………………………………. xii 1.0 Introduction 1.1 Background of study……………………………………………………………… 1 1.2 Problem Statement………………………………………………………………... 5 1.3 Research Objectives………………………………………………………………. 6 1.4 Research Questions……………………………………………………………….. 7 1.5 Research Hypotheses……………………………………………………………... 8 1.6 Significance of study……………………………………………………………… 9 2.0 Literature Review 2.1 Spinal root avulsion injures…………………………………………………...…. 10 2.2 Brachial Plexus Anatomy in Rat Models………………………………………... 18 2.3 Microglia 2.3.1 Background of microglia………………………………………………………. 25 2.3.2 The effect of microglial cells activation towards motor neuron (axonal 29 growth)………………………………………………………………………………. 2.4 The effect of minocycline – Beneficial or Harmful? 2.4.1 The beneficial effect of minocycline………………………………………….. 31 2.4.2 The Controversial Side of Minocycline……………………………………….. 37 2.5 Nitric Oxide and Inducible Nitric Oxide Synthase (iNOS)…………………….. 39 3.0 Research Methodology 3.1 Research design…………………………………………………………………… 41 3.2 Duration of Study…………………………………………………….…………… 41 3.3 Ethical consideration 41 3.4 Setting……………………………………………………………………………… 41 3.5 Sample 3.4.1Inclusion and exclusion criteria………………………………………………… 42 3.6 Sampling design…………………………………………………………………… 42

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3.7 Sample size………………………………………………………………………… 42 3.8 Outcome variables measurement 3.8.1 Number of Motor Neuron cell…………………………………………………. 42 3.8.2 Number of Microglial cell……………………………………………………... 43 3.8.3 Nitric Oxide expression………………………………………………………... 43 3.9 Experimental methods 3.9.1 Surgical methods………………………………………………………………. 44 3.9.2 Treatment methods……………………………………………………………. 49 3.9.3 Staining methods………………………………………………………………. 50 3.10Instrumentation…………………………………………………………………. 56 3.10 Statistical analysis……………………………………………………………….. 58 4.0 Results 4.1 Motor Neuron Survival after Brachial Plexus Injury………………………….. 60 4.1.1 Motor Neuron Survival in Sham Group……………………………………….. 62 4.1.2 Changes of Motor Neuron Survival after Intraperitoneal Administration of Minocycline………………………………………………………………………….. 63 4.1.3 Changes of Motor Neuron Survival after Inthecal Administration of Minocycline………………………………………………………………………….. 64 4.2 Comparison of the Neuroprotective Strength of Minocycline using Different Route (Microglial inhibition)………………………………………………………… 67 4.2.1 Microglial Activation after Brachial Plexus Injury (Sham group)…………….. 67 4.2.2 Changes of Microglial Activation after Intraperitoneal Administration of Minocycline………………………………………………………………………….. 68 4.2.3 Changes of Microglial Activation after Intrathecal Administration of Minocycline………………………………………………………………………….. 69 4.3 Comparison of the Neuroprotective Strength of Minocycline using Different Route (Nitric Oxide expression)…………………………………………………….. 74 4.3.1 Control group………………………………………………………………….. 74 4.3.2 Minocycline treatment group (Intraperitoneal)………………………………... 75 4.3.3 Minocycline treatment group (Intrathecal)……………………………………. 76 5.0 Discussions 5.1 Cellular Changes following Traumatic Injury………………………………….. 79 5.2 Excitaneurotoxicity. ……………………………………………………………… 82

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5.3 Neuronal Cell Death………………………………………………………………. 83 5.4 Microglial Response to Avulsion Injury of the Motor Root……………………. 86 5.5 Brachial Plexus Root Avulsion Injury and Oxidative Stress Activity…………. 88 5.6 Effect of Minocycline to the Nervous System following Nerve Avulsion……… 91 5.6.1 The Beneficial Effect of Minocycline to the Nervous System………………… 91 5.6.2 The Effect of High Dose, Concentrated Minocycline through Targeted Drug 94 Delivery……………………………………………………………………………… 5.7. Limited Regenerative Capability after Avulsion Injury………………………. 96

5.8. Mitogen Activated Protein Kinase Pathway in Avulsion Injury of Motor 98

Root…………………………………………………………………………………….

5.9 Degenerative Process following Traumatic Avulsion of Ventral Root………… 99

5.10 Factors Affecting Degeneration 5.10.1 Avulsion and transection - proximal vs. distal injury……………………….. 101 5.10.2 Species and strain…………………………………………………………… 102 5.10.3 Gender and age……………………………………………………………….. 102 5.11 Clinical implication and Prospect of Minocycline in Brachial Plexus 103 Injury...... 5.12 Limitation of Clinical Study……………………………………………………. 105 6.0 Conclusion……………………………………………………………………………. 106 7.0 References……………………………………………………………………………. 107

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List of Tables

Table 1. Chuang’s Classification of Brachial Plexus Injury

Table 2. Instruments used in each experimental method

Table 3. Motor neuron survival in three experimental groups (n=21)

Table 4. Comparison of motor neuron survival in three experimental groups by Kruskal-Wallis test (n=21)

Table 5. Microglial activation in three experimental groups (n=21)

Table 6. Comparison of microglial activation in three experimental groups by Kruskal-Wallis test (n=21)

Table 7. Nitric Oxide expression in three experimental groups (n=21)

Table 8. Comparison of motor neuron survival in three experimental groups by Kruskal-Wallis test (n=21)

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List of Figures

Figure 1. Schematic diagram of human brachial plexus

Figure 2. Pre-ganglionic (Avulsion) injury of the brachial plexus root (Top). Post-ganglionic

transection injury of the brachial plexus.

Figure 3. Brachial plexus and the arterial supply in rat

Figure 4. Dorsal and ventral roots of spinal nerves

Figure 5. Histological section of a spinal cord in rat

Figure 6. The relationship between the spine and the brachial plexus.

Figure 7. A schematically image show how activated microglia change phenotype

Figure 8. Surveying microglia transform into activated microglia upon inflammatory stimuli.

Figure 9. Intraperitoneal injection of anaethetic drugs

Figure 10. Skin incision

Figure 11. Surgical approach to brachial plexus in Sprague Dawley rat

Figure 12. Exposure of Brachial Plexus in Sprague Dawley rat

Figure 13. Close up anatomical diagram of a healthy Sprague Dawley rat’s Brachial Plexus

Figure 14. Brachial plexus of Sprague Dawley rat after avulsion injury of brachial nerve root

Figure 15. A micro infusion pump with intrathecar catheter

Figure 16. A cardiovascular perfusion

Figure 17. Fixation of harvested C7 spinal segment

Figure 18. C7 spinal segment was put in the mold for frozen section

Figure 19. Cord section with thickness of 40 µm was cut with cryostat

Figure 20. Collection of cord sections

Figure 21. Survival of motor neurons

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Figure 22. Microglial activation

Figure 23. Motor neuron counts in sham group (normal saline)

Figure 24. Motor neuron counts in minocycline group administered via intraperitonial route

Figure 25. Motor neuron counts in minocycline group administered via intrathecal route

Figure 26 Motor neurons in control group versus minocycline group administered via intraperitoneal route

Figure 27. Microglial cell counts in sham group (normal saline)

Figure 28. Microglial cell counts in intraperitoneal group

Figure 29. Microglial cell counts in intrathecal group

Figure 30. Microglial cell count between the sham group versus the intrathecal group

Figure 31. Nitric oxide expression in sham group (normal saline)

Figure 32. Nitric oxide expression in intraperitoneal group

Figure 33. Nitric oxide expression in intrathecal group

Figure 34. Nitric Oxide Synthase pathway

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List of Abbreviations

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

Anterior root exit zone (AREZ)

Autoimmune encephalomyelitis (EAE)

β-Amyloid (Aβ)

Choline acetyltransferase (ChAT)

Cluster of differentiation (CD)

Compound Muscle Action Potential ( CMAP)

Cyclooxygenase 2 (COX2)

Damage associated molecular patterns (DAMP)

Histocompatibility complex II (MHCII)

Huntington's Disease (HD)

Inducible NO synthase (iNOS), also known as NO synthase 2 (NOS2)

Insulin-like growth factor-1 (IGF-1)

Mitogen activated protein kinase (MAPK)

N-Methyl-D-aspartate (NMDA)

Nerve conduction study ( NCS)

Nitric oxide (NOS)

Pathogen associated molecular patterns (PAMP)

Phosphate buffered saline (PBS)

Prostaglandin (PG)

nNOS (neuronal/Type I/NOS-1/bNOS)

xii iNOS (inducible/Type II/NOS-2) eNOS (endothelial/Type III/NOS-3)

Sensory Nerve Action Potential ( SNAP)

Ventral root avulsion (VRA)

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CHAPTER 1

INTRODUCTION

1.1 Background of study

Brachial plexus injuries comprise 5% polytraumas caused by motor vehicle accidents in European country (Radek et al., 2014). The exact incidence figure of brachial plexus injury in Asia is not available. It is a mutilating injury as it commonly affects young men in their productive age. It cripples the function of one, or even two limbs at rare occasions, leaving a devastating effect on the unfortunate patient. It results in loss of neurological function, dependence in daily living, employment, depression and even suicidal tendency. Brachial plexus injury has a serious socioeconomic impact since they affect the function and quality of regular life to a great extent. Injuries resulting nerve transection only produce minimal or even undetectable motor neuron degeneration (Thatte et al, 2013). In contrast, a ventral root avulsion injury occurred during brachial plexus injury, with the separation of motor axons from the surface of the spinal cord is followed by a progressive and marked loss of the vast majority of axotomized motoneurons over several weeks after the lesion in rat models (Brabizan et al., 2013, Hoang, 2003). Ventral root avulsion injury in the Rhesus Macaque resulted in a marked loss of axotomized motoneurons and an astroglial reaction in the ventral horn (Marcus, 2013).

Non-Astrocyte Glial cells were discovered by a Spanish neuroanatomist called Ramon y Cajal and identified it as the “third element” of the central nervous system in

1 distinction from the “first element” (neurons) and “second element” (astrocytes) (Del

Rio-Hortega, 1919). After a century after his great discovery, recent researches and studies in microglia have centered not only on understanding their basic biological functions but also on their therapeutic potential during pathological conditions. Recent evidence indicates the potential promise of microglia as targets for therapeutic intervention during pathology. Eyo and Dailey (2013) summarized recent evidence suggesting that microglia perform critical functions during development of the nervous system, including roles in angiogenesis, induction of apoptosis, phagocytic clearance of dead cells, and synapse remodeling (Eyo & Dailey, 2013). In cases of Brachial

Plexus Injury, the ventral root avulsion injury triggers an activation of microglia within the motor nuclei (Novikov et al., 2000). A robust inflammatory response, which involves the activation of glia and neurons as well as cerebral accumulation of leukocytes, following traumatic injury is well known and contributes toward the development of the secondary injury. Microglial cells respond to traumatic injury to the nervous system with proliferation and migration toward the affected area.

However, nobody is sure of the microglial response to axonal growth. Several reports suggest that glial cells might act as a double-edged sword being either detrimental or protective depending on the context (Biber, 2007). For instance, activated microglia synthesize potentially neurotoxic molecules such as reactive oxygen species, and inflammatory cytokines. Conversely, microglia release some neuroprotective molecules in specific conditions (Graeber, 2010). They released chemokines that trigger the Glial response, in which the activated astrocytes also release a wide variety of neuroprotective and/or neurotoxic mediators (Sofroniew, 2010). Reactive microglia

2 may affect axonal regeneration in a variety of ways. They produce a proregenerative factors such as insulin-like growth factor-1 (IGF-1) (Aldskogius, 2001) and a neurotoxic compounds such as free radicals (Boje & Arora, 1992) and glutamate

(Giulian et al., 1993).

Minocycline hydrochloride is a semi-synthetic derivative of tetracycline. It is a potent neuroprotective agent, act by inhibiting microglial activation (Stirling et al., 2004).

Minocycline alleviates the degree of neuropathology in animal models in stroke, spinal cord injury, multiple sclerosis, and neurodegenerative diseases (Parkinson’s Disease and ALS) (Yrjanheikki et al., 1998; Arvin et al., 2002). Using a lumbosacral ventral root avulsion model for cauda equina injury, Huong et al. (2008) demonstrated significant protection of the drug to axotomized motoneurons against retrograde degeneration and death.

Minocycline may also attenuate the development of hyperesthesia and allodynia after spinal nerve transection injury in the rat (Raghavendra et al., 2003). By inhibiting the microglial activation, and also recruitment of T-cell, it was proven to reduce graft rejection & promote survival of neuronal transplants in rat model (Michel-Monigadon et al., 2010). Havton and Carlstedt (2009) suggested that minocycline can be used to improve functional recovery after nerve reimplantation following proximal nerve root injury. In some studies, minocycline has failed to show beneficial effect, especially in neurodegenerative diseases (Smith et al., 2003; Fernandez-Gomez et al., 2005).

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Mechanism of Minocycline is not fully understood. However, it crosses blood brain barrier readily. It has a strong anti-inflammatory effect and has a good safety record. It has been approved by FDA to use to suppress apoptotic pathway (Stirling et al., 2004), to reduce the activation of microglia after a peripheral nerve injury, in part by inhibiting the expression of p38 mitogen activated protein kinase (MAPK) (Piao et al.,

2006) and to reduce pro-nerve growth factor in microglia and thereby reduce death of oligodendrocytes after a traumatic spinal cord injury (Yune et al., 2007).

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1.2 Problem Statement

Previous studies showed controversial effect of migroglial cells towards axonal regeneration. Therefore, we are still not sure of the effect of microglial activation to the axonal growth. Previous studies had shown the beneficial effects of minocycline

(Raghavendra et al., 2003; Stirling et al., 2004; Piao et al., 2006; Yune et al., 2007;

Havton & Carlstedt, 2009; Michael-Monigadon et al., 2010). However, there were also few studies that failed to show the beneficial effects of minocycline (Smith et al.,

2003; Fernandez-Gomez et al., 2005). Therefore, the beneficial effects of minocycline on the avulsion injury are still controversial. Study of Chan et al (2001) showed that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases which is another effect of Minocycline but it is still unsure whether Minocycline can be used to promote axonal regeneration in the cervical area.

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1.3 Research objectives

General objective: To determine the neuroprotective effect of minocycline in rodent spinal root avulsion injury.

Specific objectives:

1. To confirm the microglial cell activation following avulsion injury.

2. To observe the effect of microglial cells activation towards motor neuron

(axonal growth).

3. To determine the effect of minocycline towards motor neuron (axonal growth).

4. To determine the effect of minocycline towards microglial cells.

5. To determine the effect of minocycline towards nitric oxide expression.

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1.4 Research Questions

1. Are the microglial cells will be activated following avulsion injury?

2. What are the effects of microglial cells activation towards motor neuron

(axonal growth)?

3. What are the effects of minocycline towards motor neuron (axonal growth)?

4. What are the effects of minocycline towards microglial cells?

5. What are the effects of minocycline towards nitric oxide expression?

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1.5 Research Hypotheses

Hypothesis 1

HO: The number of motor neurons (axonal growth) are same between the treatment

groups (minocycline) and control group (normal saline).

HA: The number of motor neurons (axonal growth) are different between the

treatment groups (minocycline) and control group (normal saline).

Hypothesis 2

HO: The number of microglial cells are same between the treatment groups

(minocycline) and control group (normal saline).

HA: The number of microglial cells are different between the treatment groups

(minocycline) and control group (normal saline).

Hypothesis 3

HO: The number of nitric oxide expression are same between the treatment groups

(minocycline) and control group (normal saline).

HA: The number of nitric oxide expression are different between the treatment groups

(minocycline) and control group (normal saline).

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1.6 Significance of study

By knowing the effect of microglial activation to the axonal growth, a therapeutic agent to promote or inhibit microglial cells can be applied to promote axonal growth following avulsion injury. Data from extensive series of researches showed good regenerative result from nerve reimplantation. If the promotional effect of

Minocycline towards axonal growth can be determine and assured, then a combination of both Minocycline & nerve reimplantation can be performed to achieve better result.

The anti-inflammatory effect of Minocycline can also reduce the rate of graft rejection.

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CHAPTER 2

LITERATURE REVIEW

2.1 Brachial plexus anatomy and spinal root avulsion injures

Brachial plexus is a network of nerves formed by the anterior rami of the lower four cervical nerves (C5-8) and the first thoracic nerve (T1). It exits the spine and descend through the cervicoaxillary canal of the neck to reach the armpit, arm and hands. It is divided into roots, trunks, divisions, cords and branches of peripheral nerves. There are 5 terminal branches, and other collateral branches that leave the plexus at various points. Figure 1 show the schematic diagram of the human brachial plexus anatomy.

Spinal root avulsion injures are the injuries to the axons of motor and sensory neurons at the junction between the spinal cord and the ventral root. Brachial plexus avulsion can present as two main types: upper and lower (Carlstedt, 2009). Upper avulsions involving C5-C7 occurs from excessive lateral neck flexion away from the shoulder, leading to loss of rotation capacity in the shoulder muscles, arm flexors and hand extensor muscles and results in Erb’s palsy. Lower avulsions involving C8 and T1 occurs through sudden traction force on the arm away from the shoulder with subsequent paralysis of intrinsic muscles of the hand and flexors of the wrist and fingers resulting in Klumpke’s palsy.

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Figure 1. Schematic Diagram of human Brachial Plexus

Figure 1 depicts the anatomical structure of human brachial plexus. It is divided into roots, trunks, divisions, cords and branches of peripheral nerves. There are 5 terminal branches, and other collateral branches that leave the plexus at various points.

(Source: Robert, 2013)

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There are 2 types of brachial plexus injuries. In the first type, the nerve may be partially or totally ruptured distal to the intervertebral foramina (postganglionic injury). The second type (avulsion) refers to an injury in which a root or rootlets have been torn from the spinal cord (pre-ganglionic injury). Due to the anatomy, ruptures are more common in the superior part of the plexus (C5-C6) whereas avulsions are more frequent in the inferior part (C7-T1). Because of its proximity to the Ganglion

Stellatum, the latter is often associated with Horner’s sign. An avulsion is one of the most severe nerve injuries that patients can experience, often with permanent loss of function and a majority of the patients reporting chronic and aggravating pain.

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Figure 2a

Figure 2b

Figure 2. Pre-ganglionic (Avulsion) Injury of the Brachial Plexus Root (Figure 2a on top). Post-ganglionic transection injury of the Brachial Plexus ( Figure 2b ). There are two types of injury that can occur: avulsion injuries, as shown in figure 2,

When the injury to the nerve is proximal to the DRG, it is called preganglionic, and when it is distal to the DRG, it is called postganglionic. (Adapted from Moran et al. 2005)

Lesion s proximal to the Dorsal Root Ganglion (DRG) and on the rootlets from the anterior horn cell on the motor nerve are Pre-ganglionic injuries. The lesions distal to those structures are Post-ganglionic Injuries. Figure 2 illustrated the mechanisms that lead to both types of injuries.

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Adult brachial plexus injury remains a dilemma to many surgeons, especially when planning to reconstruct cases of total root avulsion. Different degrees and different levels of injury require different approaches of reconstruction. Chuang (2010) classified brachial plexus injury into 4 levels as shown in Table 1.

Table 1. Chuang’s Classification of Brachial Plexus Injury

Type of Injury Description Level 1 Preganglionic root injury including spinal cord, rootlets, and root injuries.

Level 2 Postganglionic spinal nerve injury limiting the lesion to the interscalene space and proximal to the suprascapular nerve.

Level 3 Preclavicular and retroclavicular BPI including trunks and divisions.

Level 4 Infraclavicular BPI including cords and terminal branches proximal to the axillary fossa.

Doi et al developed new Magnetic Resonance Imaging classification to demonstratestatus of cervical nerve roots in Brachial Plexus Injuries. He divided

Brachial Plexus Injuries into 4 major categories, namely

1. Normal Rootlets

2. Injured Rootlets

3. Avulsion

4. Meningoceles

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The radiological classification provides valuable information to assess the nerve rootlets and decide whether or not to proceed for surgical exploration, primary nerve repair and reconstruction ( Doi et al, 2002).

Electrophysiological tests are essentially important to confirm the diagnosis, site of lesion, and determine the severity of the injury. Both Nerve Conduction Study, and

Electromyography (EMG) should be performed three to four weeks after the injury when the impulse transmission is lost owing to Wallerian degeneration. Compound

Muscle Action Potential (CMAP) is generally low in Brachial Plexus Injury and it is related to the amount of functional muscles. Sensory Nerve Action Potential (SNAP) is usually normal in Pre-ganglionic lesion simply due to its cell body location at the distally located Dorsal Root Ganglion. Therefore a normal SNAP with an insensate dermatome and low CMAP denotes Preganglionic lesion with less favourable outcome. Denervation ( Fibrillation potential) is seen as early as 2 weeks in proximal muscles, and 3-6 weeks in distal muscles. It carries unfavorable prognosis for the patient. Historically, the Seddon classification system has been used to describe the peripheral nerve injuries. The Neurophysiological parameters, i.e. Conduction

Velocity, CMAP, SNAP, and Denervation are incorporated into the Sedon’s system for prognostication and decision making for surgeon. Three groups of Brachial Plexus

Injuries were described, namely Neuropraxia, Axonotmesis and Neurotnomesis.

Neuropraxia defined presence of conductional dysfunction of the nerve in the absence of macroscopic lesion. Neuronal transmission is temporarily interrupted at site of lesion. Conduction velocity is normal. CMAP can be normal or reduced. SNAP is

15 reduced. Denervation evidence in EMG is absent. Axonotmesis described axon transection with intact perineurium and epineurium. Conduction velocity is normal or reduced. CMAP is reduced. SNAP is reduced. Neuronotmesis is the transection of the axon, together with all covering layers of the nerve (Sedon, 1943). All NCS parameters (Conduction velocity, CMAP and SNAP) are absent with presence of spontaneous fibrillation in EMG denoting denervation.

How motor neurons respond to avulsion injury is not fully understood and there is not a cure for such injury. However, previous study showed that spinal root avulsion but not the distal axotomy results dramatic motor neuron degeneration. Both necrosis and apoptosis are involved in the degeneration of injured motor neurons. Axonal injury of motor neurons also results in changes of intracellular molecules as detected by cDNA microarray.

Chai et al. (2000) studied the morphological and biochemical changes of motor neurons in response to root avulsion injury and also the regeneration and functional recovery of motor neurons after root avulsion. They had found that root avulsion in adult rats results in dramatic motor neuron loss which is coincident with the de novo expression of neuronal nitric oxide synthase (NOS). Avulsion induced motor neuron death occurs between 2-6 weeks post-injury. Chai et al. (2000) had concluded that spinal motor neurons die quickly following root avulsion and re-implantation of avulsed ventral root allowed the regeneration of motor neurons into their axons into the original ventral root after root avulsion. Study conducted by Li et al. (1995) also

16 showed that injuring adult motoneuron axons at the exit point of the nerve from the spinal cord (avulsion) resulted in a 70% loss of motoneurons by 3 weeks following surgery and a complete loss by 6 weeks.

Besides motor neuron degeneration, the ventral root avulsion injury also triggers an activation of microglia within the motor nuclei (Novikov et al., 2000). Microglial cells respond to traumatic injury to the nervous system with proliferation and migration toward the affected area. Wu et al. (2008) studied the late effects of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice on the spinal cord gray matter. Autoimmune encephalomyelitis (EAE) results in inflammatory white matter lesions in the central nervous system. Their findings showed that EAE induced marked astrocytic, microglial, and macrophage activation in the ventral horn gray matter.

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2.2. Brachial Plexus Anatomy in Rat Models

Most of the experimental studies on spinal cord and peripheral nerve injuries were using rats sample. Although there was a clear homology with the elements of the brachial plexus in the rat and in man, the origin of the different terminal and collateral branches were found to be different in these two species (Pais et al., 2010). The rat’s spinal cord is made up of 34 segments: 8 cervical (named C1 to C8), 13 thoracic (T1 to T13), 6 lumbar (L1 to L6), 4 sacral (S1 to S4), and 3 coccygeal (Co1 to Co3). A brachial plexus morphology study in 30 rats by Angelica-Almeida et al. (2013) demonstrated that brachial plexus was composed of branches originating from the ventral aspect of C4 to C8 and T1. In 57% of cases, the ventral aspect of T2 established an anastomosis with the ventral aspect of T1, thus contributing to the formation of the brachial plexus. This branch from T2, as well as the branch from C4 to the brachial plexus, was smaller than the remaining branches that formed the roots of the plexus. The brachial plexus roots emerged between the anterior and middle scalene muscles, forming a flattened plexus below the clavicle. The lateral, medial and posterior cords of the plexus were not clearly seen compared to those in human.

The median nerve was the thickest terminal branch of the brachial plexus in rats, and almost always originated from three different roots. A branch from the second and/or the third intercostal nerve to the medial brachial and medial antebrachial cutaneous nerves was found in 87% of cases. The rat’s brachial plexus anatomy and the arterial supply is demonstrated in Figure 3.

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Figure 3. Right Brachial plexus and the arterial supply in rat

Ventral aspect of a right forepaw dissection showing several of the terminal and collateral branches of the brachial plexus, and their association with several major arterial trunks (4X magnification). 1- Axillary nerve; 2- Musculocutaneous nerve; 3- Radial nerve; 4- Median nerve; 5- Ulnar nerve; 6- Medial brachial cutaneous nerve; 8- Dorsal scapular nerve; 9- Suprascapular nerve; 10- Nerve to subclavius muscle; 11- Upper subscapular nerve; 12- Lower subscapular nerve; 15- Lateral pectoral nerve; 16- Medial pectoral nerve; 18- Axillary artery; 19- Brachial artery; 20- Acromial arterial trunk.

(Angelica-Almeida et al., 2013)

19

Ventral aspect of a right forepaw dissection showing several of the terminal and collateral branches of the brachial plexus, and their association with several major arterial trunks (4X magnification). 1- Axillary nerve; 2- Musculocutaneous nerve; 3-

Radial nerve; 4- Median nerve; 5- Ulnar nerve; 6- Medial brachial cutaneous nerve; 8-

Dorsal scapular nerve; 9- Suprascapular nerve; 10- Nerve to subclavius muscle; 11-

Upper subscapular nerve; 12- Lower subscapular nerve; 15- Lateral pectoral nerve;

16- Medial pectoral nerve; 18- Axillary artery; 19- Brachial artery; 20- Acromial arterial trunk.

The spinal cord is divided into spinal cord segments. Each segment gives rise to paired spinal nerves. Ventra and dorsal spinal roots arise as a series of rootlets (Figure

4). A spinal ganglion is present distally on each dorsal root. Each ventral root (also named the anterior root, radix anterior, radix ventralis, or radix motoria) is attached to the spinal cord by a series of rootles that emerge from the ventrolateral sulcus of the spinal cord. Unlike the dorsal root fibers that are arranged in a neat line at their emergence from the spinal cord, ventral root fibers form an elliptical area named the anterior root exit zone (AREZ). The ventral roots predominantly consist of efferent somatic motor fibers (thick alpha motor axons and medium-sized gamma motor axons derived from nerve cells of the ventral column (Watson et al., 2009).

Each dorsal root (also known as the posterior root, radix posterior, radis dorsalis or radiz sensoria) is attached to the dorsolateral sulcus of the spinal cord by a series of rootlets arranged in a line, the dorsal root entry zone (DREZ). In the experimental

20 study using rat model, the avulsion surgery was done by separating both the ventral and dorsal roots at the junction between their attachment to the spinal cord, which were the AREZ and DREZ (Watson et al., 2009). Figure 4 demonstrated the cut dissection showing the ventral surface of the spinal cord, as well as the ventral and dorsal rootlets. Figure 5 showed the histological section of the rat’s spinal cord. The junction between spinal cord and spinal roots is circled (blue, dorsal root entry zone,

DREZ; red = anterior root exit zone, AREZ). Figure 6 showed the relationship between the spine and the brachial plexus in which every spinal nerve root comes from the intervertebral foramen above its synonymous lamina.

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* *

Figure 4. Dorsal and ventral roots of spinal nerves

(Watson et al., 2009)

This is a dissection showing the ventral surface of the spinal cord and the ventral and dorsal rootlets. Groups of rootlets form the dorsal and ventral roots of each spinal nerve. The dura and arachnoid have been removed to expose the spinal cord. The junction between spinal cord and ventral root (anterior root exit zone, AREZ) is labeled **.

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Figure 5. Histological section of a spinal cord in rat

(Watson et al., 2009)

The junction between spinal cord and spinal roots is circled (blue, dorsal root entry zone, DREZ; red = anterior root exit zone, AREZ).

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Figure 6. The relationship between the spine and the brachial plexus: Every spinal nerve root comes from the intervertebral foramen above its synonymous lamina. The cervical plexus is also illustrated. T, thoracic; C, cervical; a, fifth cervical nerve root; b, sixth cervical nerve root; c, seventh cervical nerve root; d, eighth cervical nerve root; e, first thoracic nerve root; f, subclavicular artery; g, first rib; h, second rib; i, cranium; f, spinal cord.

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2.3 Microglia

2.3.1 Background of microglia

Microglia are the innate immune cells of the central nervous system and play an important role in maintaining and protecting the integrity of the peripheral nervous system or spinal cord. Microglia does not invade the Spinal Cord without any traumatic injury (Kettenmann et al., 2011). Microglia account for approximately 20% of the glial cell population and around 5-20% of the total cells of the adult CNS, depending on the species (Aguzzi et al., 2013).

Microglia, were first named microgliocytes when they were discovered around 1920 by the Spanish neuroscientist Pio del Rio-Hortega, who identified the cells with a silver carbonate staining (Kettenmann et al., 2011). The developmental origin of microglia remains a matter of debate either they are derived from invasion of mesodermal or mesenchymal origin, from neuroectodermal matrix cells together with macroglia (astrocytes and oligodendrocytes), from pericytes, from invading circulating monocytes in early development, or, according to later research, are derived from macrophages produced by primitive hematopoiesis in the yolk sac (Ranshoff &

Cardona, 2010). Despite their origin, there are no fundamental or functional differences between microglia and peripheral macrophages; thus microglia are still classified as the CNS’s own macrophages, expressing several macrophage-associated markers. Microglia cells have high plasticity and mobility as they can rapidly transform from a ramified phenotype, with small body and multiple branches, into a

25 more active amoeboid-like cell (reactive microglia) (Figure 7) with capacity to proliferate or migrate, invade and phagocytose Although the ramified shape was long considered as the “resting state” of microglia waiting for pathology, evidence indicates that ramified microglia actively move their fine processes in the healthy brain

(Kettenmann et al., 2013), making the term “surveying microglia” a more accurate description. Further, the ramified microglia have shown to play critical roles in determination of neuronal fate, axonal growth and synaptic remodeling during central nervous system development (Smith et al., 2012; Kettenmann et al., 2013).

(Schafer DP et al, 2013) Figure 7. A schematically image show how activated microglia change phenotype: From a ramified surveying microglia (a2) towards a more round microphage like (a4), with migrating/invading capacities or into a chronically active microglia (a1).

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Upon a change in the brain micro-environment, stimulation of microglia’s receptors induces signaling cascades that further lead to transcription and expression of new proteins, including cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase

2 (COX2) and major histocompatibility complex II (MHCII). In addition, to clean areas from pathogens, dying cells and their fragments, and to accumulate in areas where neuronal death occurs, microglia migrate or/and invade, proliferate and/or phagocytose cellular debris. Surveying microglia constantly screen for any imbalance in tissue homeostasis. Upon a change in the brain micro-environment, different inflammatory mediators stimulate microglia to become activated by change of morphology, enhanced proliferation, phagocytosis, invasion/migration, or/and induction of transcription and secretion of distinct inflammatory molecules and proteins.

27

Diagram deleted, Drawn after correction

Figure 8. Surveying microglia transform into activated microglia upon inflammatory stimuli.

Inflammatory stimuli including cytokines, and chemokines secreted during stressful condition e.g. infection or trauma change the surveying, branching microglia which is

28 round, hypertrophied without dendritis process. Activated Microglia upregulates many types of cytokines, proliferate and phagocytize the dead cells and debri.

2.3.2 The effects of microglial cells activation towards motor neuron (axonal growth)

Reactive microglia may affect axonal regeneration in a variety of ways. Results from in vitro studies demonstrate that microglia can develop a regeneration supportive phenotype by producing proregenerative factors, such as insulin-like growth factor-1

(Aldskogius, 2001). Microglia may also produce neurotoxic compounds such as free radicals (Boje & Arora, 1992; Giulian et al., 1993). Study of Boje and Arora (1992) had showed that immunostimulated microglia had mediated neurotoxicity by free radicals agents such as nitric oxide (NO), reactive nitrogen oxides, superoxide anion and n-methyl-d-aspartate (NMDA)-like substances. Their findings suggest the role for microglial-produced nitric oxide (NO) and reactive nitrogen oxides as a neurotoxic agent in neurodegenerative disease states. (Barbizan, 2013).

Previous studies had reported that microglia govern the survival of neurons after damage to the central nervous system. Giulian et al. (1993) had examined microglia and astroglia secretion products for effects upon growth of cultured neurons. Activated microglia was found to secrete small neurotoxic factors (< 500 Da), while astroglia constitutively release proteins (> 10 kDa) that promote neuronal growth. Interestingly, these proteins released from astroglia were found to attenuated microglial toxicity.

This suggested that different glial populations have opposing actions on the neuronal survival. They also found that neurotoxins from microglia are heat-stable and

29 protease-resistant molecules with biologic activities blocked by n-methyl-d-aspartate

(NMDA) receptor antagonists (Sanagi et al, 2012). Giulian et al. (1993) had proposed that secretion products from reactive microglia, but not astroglia, endanger surviving neurons after central nervous system injury by release of neuron-killing molecules.

Similarly, Giulian et al. (1993) also reported that microglia was found to secrete neurotoxic agents, while astroglia was a source of neuronotrophic factors. Their observations suggest that microglia and astroglia compete for control of neuronal survival. Giulian et al. (1993) also stated that microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.

Reactive microglia was found to respond within hours to central nervous system ischemic injury. However, it was at least several days after an insult before these activated mononuclear phagocytes reach a peak of secretory activity with the release of neurotoxins. This period of cytotoxin secretion is associated with a delayed neuronal loss seen in tissues neighboring sites of ischemia. Giulian and Vaca (1993) found that microglia-suppressing drugs reduced the tissue production of neurotoxic factors and improve functional outcome after ischemic injury. Therefore, they had suggested that immunosuppressive therapy might offer a means to reduce late neuronal damage associated with ischemic injury ( De Fonceca, 2014).

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2.4 The effect of Minocycline – Beneficial or Harmful?

2.4.1 The beneficial effect of Minocycline

Cell therapy in the brain is limited by the requirement of high dosage of immunosuppressors that may give harmful side effects to the patients. Minocycline shows potential as therapeutic agent that can replace or enable a reduction in the doses of usual immunosuppressors. Minocycline crosses the blood-brain barrier, has good safety records, and exhibits strong antiinflammatory effects. Michel-Monigadon et al.

(2010) had studied the impact of minocycline on the survival of intracerebral transplant. A total of 400,000 porcine fetal neurons were transplanted into the striatum of rats treated daily with minocycline until sacrifice. Graft survival and immunologic reaction were evaluated by immunohistochemistry. Findings of Michel-Monigadon et al. (2010) showed that all the grafts were rejected at day 63 in the control groups, whereas healthy grafts exhibiting tyrosine hydroxylase neurons were observed in 40% of the treated rats. The low immunoreactivity for ED1 and R73 in treated rats when compared with the control groups suggests that minocycline promotes long-term survival of neuronal xenograft by inhibiting microglial activation and T-cell recruitment. Michel-Monigadon et al. (2010) had proposed the use of minocycline as a safe and efficient immunosuppressive for intracerebral transplantation.

Study of Piao et al. (2006) had shown that use of minocycline as an inhibitor of microglial activation successfully reduced microglial activation by inhibiting p38

Mitogen-activated protein kinase (MAPK) activation in microglia, and significantly

31 attenuated the development of pain hypersensitivity in inferior alveolar nerve and mental nerve transection model.

Raghavendra et al. (2003) determined whether minocycline could attenuate both the development and existing mechanical allodynia and hyperalgesia in an L5 spinal nerve transection model of neuropathic pain. Minocycline (10, 20, or 40 mg/kg intraperitoneally) was administered daily, beginning 1 h before nerve transection. This regimen produced a decrease in mechanical hyperalgesia and allodynia, with a maximum inhibitory effect observed at the dose of 20 and 40 mg/kg. The attenuation of the development of hyperalgesia and allodynia by minocycline was associated with an inhibitory action on microglial activation and suppression of proinflammatory cytokines at the L5 lumbar spinal cord of the nerveinjured animals. The effect of minocycline on existing allodynia was examined after its intraperitoneal administration initiated on day 5 post-L5 nerve transection. Although the postinjury administration of minocycline significantly inhibited microglial activation in neuropathic rats, it failed to attenuate existing hyperalgesia and allodynia. These data demonstrate that inhibition of microglial activation with minocycline treatment attenuated the development of behavioral hypersensitivity in a rat model of neuropathic pain but had no effect on the treatment of existing mechanical allodynia and hyperalgesia.

Minocycline has been reported to be neuroprotective after spinal cord injury. Study of

Yune et al. (2007) showed minocycline improves the functional recovery after spinal

32 cord injury (SCI) by reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia. After spinal cord injury, the stress-responsive p38 mitogen- activated protein kinase (p38MAPK) was activated only in microglia, and proNGF was produced by microglia via the p38MAPK-mediated pathway. Minocycline treatment significantly reduced proNGF production in microglia in vitro and in vivo by inhibition of the phosphorylation of p38MAPK. Minocycline treatment also inhibited p75 neurotrophin receptor expression and RhoA activation after injury. Yune et al. (2007) concluded that minocycline treatment inhibited oligodendrocyte death and improved functional recovery after SCI and suggested that minocycline may represent a potential therapeutic agent for acute SCI in humans ( Tao et al, 2013).

Stirling et al. (2004) had examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract dieback, and lesion size. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract and descending corticospinal tract in segments (3-7 mm) both proximal and distal to the injury site. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daily for 2 days. Stirling et al. (2004) had reported a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the ascending sensory tract after minocycline treatment. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both corticospinal tract dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed

33 improved functional outcome after minocycline treatment. They had concluded that minocycline ameliorates multiple secondary events after spinal cord injury.

Study of Lee et al. (2003) also demonstrated beneficial effects of minocycline treatment on improvement in locomotor behavior and reduced histopathological changes after spinal cord injury in animal models. However, Pinzon et al. (2008) had repeated the study of Lee et al. (2003) and reported that administration of minocycline after spinal cord injury (SCI) in rats did not lead to significant behavioral or histopathological improvement. Although positive effects with minocycline have been reported in several animal models of injury with different drug administration schemes, Pinzon et al. (2008) argued that the use of minocycline following contusive

SCI still requires further investigation.

Stirling et al. (2005) had suggested that minocycline may inhibit several aspects of the inflammatory response and prevent cell death through the inhibition of the p38 mitogen-activated protein kinase pathway, an important regulator of immune cell function and cell death. Study conducted by Yrjanheikki et al. (1998) showed that doxycycline and minocycline have an antiinflammatory effect which protects the hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 hours before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and post-treatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the

34 ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1beta-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Their study results suggested that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Arvin et al. (2002) showed evidence that minocycline reduces brain injury after neonatal hypoxic-ischemic brain injury. They assessed the minocycline's neuroprotective effects after insults to the neonatal brain in a rodent model. They administered minocycline either immediately before or immediately after a hypoxic- ischemic insult substantially blocks tissue damage. Their findings showed that minocycline treatment prevents the formation of activated caspase-3, a known effector of apoptosis, as well as the appearance of a calpain cleaved substrate, a marker of excitotoxic/necrotic cell death. Arvin et al. (2002) suggest that minocycline or a related neuroprotective tetracycline may be a candidate to consider in human clinical trials to protect the developing brain against hypoxic-ischemic-induced damage.

Hoang et al. (2008) had investigated whether minocycline may rescue degenerating autonomic and motor neurons after ventral root avulsion (VRA) injury in a rodent

35 lumbosacral VRA injury model. The injury models were design with both autonomic and motor neurons that progressively die over several weeks. Adult female rats underwent lumbosacral ventral root avulsion injuries followed by a 2-week treatment with either minocycline or vehicle injected intraperitoneally. The sacral segment of the spinal cord was studied immunohistochemically using choline acetyltransferase

(ChAT) and activated caspase-3 at 4 weeks post-operatively. Minocycline was found to increase the survival of motoneurons but not preganglionic parasympathetic neurons

(PPNs). Their investigations also demonstrated that a larger proportion of motoneurons expressed activated caspase-3 compared to preganglionic parasympathetic neurons after ventral root avulsion injury and indicated an association with minocycline's differential neuroprotective effect. Hoang et al. (2008) had suggested that minocycline may protect degenerating motoneurons and expand the therapeutic window of opportunity for surgical repair of proximal root lesions affecting spinal motoneurons.

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2.4.2 The Controversial Side of Minocycline

Many other evidence indicates that minocycline does not always present beneficial actions. Study of Fernandez-Gomez et al. (2005) which analyzed the effect of minocycline on malonate-induced cell death in rat showed that, the pretreatment for 4 h with minocycline (10-100 microM) did not present cytoprotective actions and minocycline failed to block reactive oxygen species (ROS) production and to abrogate malonate-induced oxidation of GSH and cardiolipin. Additional experiments revealed that minocycline was also unsuccessful to prevent the mitochondrial swelling induced by Malonate. Furthermore, Malonate did not induce the expression of the iNOS, caspase-3, -8, and -9 genes which have been shown to be up-regulated in several models where minocycline resulted cytoprotective. In addition, malonate-induced down-regulation of the antiapoptotic gene Bcl-2 was not prevented by minocycline, controversially the mechanism previously proposed to explain minocycline protective action. Fernandez-Gomez et al. (2005) suggested that the minocycline protection observed in several neurodegenerative disease models is selective, since it is absent from cultured cerebellar granular cells challenged with malonate.

Smith et al. (2003) had studied the effect of tetracycline and minocycline, on the disease phenotype in the R6/2 mouse model of Huntington's Disease (HD). They found that tetracyclines are potent inhibitors of Huntingtin aggregation in a hippocampal slice culture model of HD at an effective concentration of 30 microM.

However, despite achieving tissue levels approaching this concentration by oral treatment of R6/2 mice with minocycline, there was no observed clear difference in

37 their behavioral abnormalities, or in aggregate load postmortem when using minocycline as treatment. Smith et al. (2003) stated that caution should be exercised in proceeding into human clinical trials of minocycline.

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2.5 Nitric Oxide and Inducible Nitric Oxide Synthase (iNOS)

Nitric Oxide is synthesized from Nitric Oxide synthase (NOS). Nitric oxide synthase

(NOS) oxidizes a guanidine nitrogen of L-arginine and produce Nitric Oxide in the form of free radical and citrulline. Nitric oxide (NO) is both a signaling and an effector molecule in diverse biological systems. Inducible NO synthase (iNOS), also known as

NO synthase 2 (NOS2), is normally expressed in the brain by neurons. They are involved in several physiological processes including smooth muscle relaxation, inflammation, vasodilatation, neurogenesis, synaptic plasticity, long-term potentiation, and nociceptive transmission. Low level of Nitric Oxide plays a role to prevent apoptosis. On the other hand, sustained and elevated Nitric Oxide can react with superoxide to form peroxynitrite, which pose a direct cytotoxicity to neurons and oligodendrocytes (Wen et al., 2011).

After traumatic avulsion injury, cellular death causes release of cytoplasmic content including Glutamate. Glutamate triggers influx of Calcium, which is mediated by

NMDA receptor. The Calcium concentration surge will trigger the constitutively expressed Nitric Oxide Synthase ( c-NOS) production, leading to a brief peak of Nitric

Oxide level at the injured site. 2 NOS isoforms that are expressed constitutively are the Neuronal Nitric Oxide Synthase ( Type 1 or n-NOS) and Epithelial Nitric Oxide

Synthase (Type 3 or e-NOS). Traumatic injury related vasospam would cause local tissue ischemia, rendering oxidative stress for the affected cells. Nitric Oxide and

Reactive Oxygen Species are produced. The c-Nitric Oxide Synthase depletes with time. Downregulation of the c-Nitric Oxide Synthase is associated with upregulation

39 of inducible Nitric Oxide Synthase ( i-NOS). i-NOS is associated with a second wave of Nitric Oxide surge. Massive amount of Nitric Oxide expression induce detrimental effect to the neuronal tissue.

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CHAPTER 3

RESEARCH METHODOLOGY

3.1 Research design

Randomized laboratory based experimental study.

3.2 Duration of Study

March 2012 until July 2014

3.3 Ethical considerations and Grant

This study was approved by the Animal Ethics Committee, Universiti Sains Malaysia with reference number of USM/Animal Ethics Approval/2011/(73)(346). The study was funded by Exploratory Research Grant Scheme (ERGS) (grant numbered 203/PPSP/6730023).

3.4 Setting

• Neurosciences Teaching Laboratory, School of Medical Sciences, Health

Campus of Universiti Sains Malaysia.

• Central Research Laboratory, School of Medical Sciences, Health Campus

of Universiti Sains Malaysia.

• Maxillofacial Science Laboratory, School of Dental Sciences, Health

Campus of Universiti Sains Malaysia.

3.5 Sample 41

Adult female Sprague-Dawley rats are used in the experiment.

3.5.1 Inclusion and exclusion criteria

Inclusion criteria is weight of 200-300g whereas exclusion criteria is weight less than

200g.

3.6 Sampling design

Sampling design that will be used is a randomized controlled trial. For randomization, the study subjects will be allocated into treatment group and control group. Subjects for this study are adult female Sprague-Dawley rats, which will be randomised into 3 groups: Treatment groups will be given minocycline (via intraperitoneal and intrathecal) and control group will be given normal saline.

3.7 Sample size

The total subjects used in this study are 63 Sprague-Dawley rats. A total of 21 rats were used to study each outcome (motor neurons, microglial cells and nitric oxide expression). Each study comparing three treatment groups consisted of 7 subjects: normal saline group (n=7), minocycline group administered via intraperitoneal (n=7) and minocycline group administered via intrathecal (n=7).

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3.8 Outcome variables measurement

3.8.1 Number of Motor Neuron cell

• The number of motor neuron cell in the lesioned site and normal site will be

calculated by an appointed laboratory technologist.

• It will be measured as a ratio of the motor neuron cell at the lesioned site over

the normal site.

• The mean will be obtained by averaging up the ratio obtained from 10 cross

section measurement.

3.8.2 Number of Microglial cell

• The number of microglia in the lesioned site and normal site will be calculated.

• It will be measured as a ratio of the microglial number at the lesioned site over

the normal site.

• The mean will be obtained by averaging up the ratio obtained from 10 cross

section measurement.

3.8.3 Number of nitric oxide expression

• The number of nitric oxide expression in the lesioned site and normal site will

be calculated.

• It will be measured as a ratio of the nitric oxide expression at the lesioned site

over the normal site.

• The mean will be obtained by averaging up the ratio obtained from 10 cross

section measurement.

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3.9 Experimental methods

3.9.1 Surgical methods

• The seventh cervical segment (C7) spinal roots of all adult Sprague-Dawley

rats (200-300 g) will be avulsed by an extravertebral extraction procedure.

• Rats will be anesthetized with an intramuscular injection of ketamine (80

mg/kg) and xylazine (8 mg/kg).

• The rats will be placed on the surgical table in a supine position and the right

brachial plexus will be exposed by separating the scalene muscles with fine

forceps.

• The C7 nerve will be identified and isolated from the surrounding adipose or

connective tissue to the point where it exits the vertebral foramen.

• With microhemostat forceps, an extravertebral avulsion will be carried out by a

steady moderate traction applied away from the intervertebral foramen.

• The avulsed ventral and dorsal roots as well as dorsal root ganglia (DRG) will

be cut away from peripheral nerve and examined to confirm the success of the

avulsion (the avulsed DRG, ventral rootlets, and dorsal root can be easily

observed with a dissecting microscope).

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Figure 9. Intraperitoneal injection of anaethetic drugs

Each rat was given intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg)

Figure 10. Skin incision

45

After shaving the chest hair and cleaned with antiseptic solution, a linear skin incision fashioned over the chest below the clavicle.

Figure 11. Surgical Approach to Right Brachial Plexus in Sprague Dawley rat

The skin was retracted away, exposing the Pectoris Major muscle and the Pectoral

Nerve. The Pectoral nerve is an important superficial anatomical hallmark to be identified. Muscular dissection following the Pectoral nerve will lead the operator to the Extra-vertebral Brachial Plexus

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Figure 12. Exposure of Right Brachial Plexus in Sprague Dawley rat

Retraction of the Right Pectoris Major Muscles revealed the Right Brachal Plexus, in which the branches fan out as it exits the Neural Foramen of the spinal cord.

Figure 13. Close up anatomical diagram of a healthy Sprague Dawley rat’s

Brachial Plexus.

All segments of the Rodent’s Brachial Plexus is located below the clavicle. This is distinctly different from the adult Brachial Plexus.

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Figure 14. Brachial Plexus of Sprague Dawley rat after avulsion injury of

Brachial nerve root

After extravertebral avulsion, the nerve root is transected at the Preganglionic level.

Under the Microscope, the avulsed nerve root can be seen to be stretched and thinned out.

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3.9.2 Treatment methods

• Minocycline (50mg/kg) or saline injections will be administered

intraperitoneally (i.p.) daily for 7 days post-operatively. The first dose will be

given on the day of surgery in connection with the root injury procedure. The

minocycline dose will be decreased in half (25mg/kg i.p. daily) for days 8–14

post-operatively, providing a total of 2 weeks of treatment.

• Another treatment group of Sprague-Dawley rats received implantation of a

micro infusion pump connected to a intrathecal catheter which was placed at

the avulsion site. In the Treatment group, the rats would receive intrathecal

infusion of Minocycline. The dosage of the Minocycline is similar to what was

described for treatment via intraperitoneal administration.

Figure 15. A micro infusion pump with intrathecar catheter

The infusion pump was programmed to deliver drug. The dosage of the Minocycline is similar to what was described for treatment via intraperitoneal administration.

49

Intrathecal Minocycline administered to the avulsion site using Micro infusion intrathecal pump, bypassing the blood- spinal cord barrier, and therefore increase its bioavailability and drug efficacy.

3.9.3 Staining methods

• At 4 weeks after surgical avulsion of brachial nerve root, under deep

anesthesia, the rats will be intravascularly perfused with 100 ml of phosphate

buffer followed by 500 ml of 4% paraformaldehyde (4°C).

• The cervical spinal cord segments will be removed and postfixed for 6 hr in the

perfusion fixative (4°C)

• Then the cross section is cryoprotected in 30% sucrose in phosphate buffered

saline (PBS), frozen overnight, and serially sectioned (40 μm thick) with

cryostat microtome in the transverse plane.

• The spinal segments of each animal was incubated at 37°C for 1 hour in 10 ml

of 0.1 M Tris-HCL (pH 8.0) containing 0.2% Triton X100, 10 mg NADPH, 2.5

mg nitroblue tetrazolium and then washed with 0.1M PBS for three times. The

stained sections were then mounted onto slides and counterstained with 1%

neutral red. The surviving motoneurons would be stained red with the

presence of neutral red which stained the Nissl substance.

• Spinal cord cross sections will be placed on slides for immunocytochemical

detection for Rabbit IBA1

• Sections were rinsed in PBS, and incubated overnight with Rabbit IBA1 in

0.3% Triton X-100/ PBS at room temperature in the well.

50

• This primary antibody for IBA1 detection will be left to cross-reacts with an

anti rabbit Alexa 594 Rhodamine ( red) conjugated secondary antibody.

• The sections will be rinsed the next day, incubated in solution containing a

secondary antibody for 1 hour, rinsed, and placed on slides. Antifading

solution will be applied and cover-slided.

• Physical disector method was used for obtaining stereological counts of

motoneurons and IBA1 labeled microglial cells.

• For this purpose, 10 sections from the C7 spinal cord segment in each animal

will be analyzed.

• The cell count will be expressed as a ratio of the number of neurons on the

avulsed side divided by the number of neurons on the contralateral, non-

lesioned side.

Figure 16. Cardiovascular perfusion

The rodents were perfused with paraformaldehyde at 6 weeks after injury.

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Intravascular perfusion of Sprague Dawley rat under deep sedation with 100 ml of phosphate buffer followed by 500 ml of 4% paraformaldehyde (4°C) 6 weeks after operation.

Figure 17. Fixation of harvested C7 spinal segment

The C7 spinal segment was cut in a bloc and fixed with 4% PFA.

The spinal segment was first fixed with 4% PFA at 4o for 3 hours, and followed by fixation in 30% sucrose at room temperature overnight.

52

Figure 18. C7 spinal segment was put in the mold for frozen section

Careful placement of the spinal segment is needed to get an even surface for each of the sections obtained.

Figure 19. Cord section with thickness of 40 µm was cut with cryostat

53

Every third section of the slices was used for NADPH-d histochemistry to avoid double-counting of the same motoneurons.

Figure 20. Collection of cord sections in PBS

For each rat, 10 sections from the C7 spinal segment were collected for the NADPH-d histochemistry.

54

3.10 Instrumentation

Table 2. Instruments used in each experimental method

Types of instruments Instruments Surgical instruments 1. Anaesthetic agents:

• ketamine (80 mg/kg)

• xylazine (8 mg/kg)

2. Microhemostat forceps Fine forceps (FST 18200-50)

3. Dissecting microscope (6×-40×)

4. Vannas-Tübingen spring scissors (FST 15003-08)

5. Micro knife (FST10056-12)

Treatment instruments 1.Minocycline (Minocin, Intravenous preparation, Wyeth,

100mg) (50mg/kg i.p daily) for 7 days: D1-D7

2. Minocycline (Minocin, Intravenous preparation, Wyeth,

100mg) (25mg/kg i.p. daily) for 7 days: D8-D14

3. Normal saline

Staining instruments 1. 100 ml of phosphate buffer

2. 500 ml of 4% paraformaldehyde (4°C)

3. Perfusion fixative (4°C)

4. 30% sucrose in phosphate buffered saline (PBS)

5. Cryostat microtome

6. NADPH-d histochemistry plus neutral red (Sigma)

55

7. Goat ChAT (Santa Cruz Biotechnology, Santa Cruz)

8. Rabbit IBA-1 (Santa Cruz Biotechnology, Santa Cruz)

9. Anti rabbit Rhodamine conjugated secondary antibody

10. 0.3% Triton X-100

11. Antifading solution

12. Anti-rabbit Alexa 594 red (Santa Cruz Biotechnology,

Santa Cruz)

13. Anti-goat Alexa 488 green conjugated secondary

antibodies (Santa Cruz Biotechnology, Santa Cruz)

56

3.11 Statistical analysis

Descriptive analysis was performed to obtain descriptive statistics. Outcome variables

(motor neuron cells, microglia cells and nitric oxide expression) were assessed for its normality distribution. All outcome variables have skewed distribution. Therefore, median and its interquartile range (IQR) were reported in the statistical test.

Non-parametric tests were applied in this study as data were non-normally distributed.

Kruskall-Wallis test was used to compare:

i. The number of motor neuron cells between the three treatment groups

(normal saline, minocycline admisnistered via intraperitoneal and

minocycline administered via intrathecal).

ii. The number of microglial cells between the three treatment groups (normal

saline, minocycline admisnistered via intraperitoneal and minocycline

administered via intrathecal).

iii. The number of nitric oxide expression between the three treatment groups

(normal saline, minocycline admisnistered via intraperitoneal and

minocycline administered via intrathecal).

The level of significance will be set at α=0.05. Result of the statistical testing will be reported as P value. P value of less than 0.05 will be considered as significant and the null hypothesis will be rejected.

57

For significant P value following Kruskal-Wallis test, further pairwise tests were conducted using Mann-Whitney test to determine which pairs of the treatment group was significant. Bonferonni correction was applied for multiple testing correction to reduce making type 1 error (wrongly rejecting the null hypothesis). Bonferonni correction was performed by multiplying the P value from the test with number of test to obtain the adjusted P values. The adjusted P values were then compared to the level of significance (0.05) to determine whether to reject or accept the null hypothesis.

58

CHAPTER 4

RESULTS

4.1 Motor Neuron Survival after Brachial Plexus Injury

A quantitative analysis of the motor neurons was performed over the Rexed Lamina

IX at the ventral horn of the spinal cord’s grey matter. Two independent observers who are blinded to the treatment groups performed data quantification and analysis.

Kappa score for inter-observer bias was 0.7. The survival is expressed as a ratio between the number of survived motor neurons at the avulsed site over the number of motor neurons at the contralateral site, where no injury was induced. After the traumatic avulsion of the nerve root, chromolytic response in association with nuclear eccentricity, nucleolar enlargement and cellular edema takes place, which eventually lead to cellular death. The maximal motor neuron loss typically occurs at 2 to 6 weeks.

The number of the survived motor neuron was therefore recorded after 6 weeks, which mark the point of maximal neuronal damage. Figure 21 demonstrated the survived motor neurons that have pinkish appearance after NADPH staining at the ventral horn of the spinal cord’s grey matter. The non-viable motor neurons undergo chromolytic response in association with nucleolar swelling, soma oedema and Nissl substance dispersion. The loss of Nissl substance within the dying motor neurons causes them to fail to take up the NADPH stain.

59

Figure 21. Survival of motor neurons: This figure demonstrated the survived motor neurons that have pinkish appearance after NADPH staining at the ventral horn of the spinal cord’s grey matter. The non-viable motor neurons undergo chromolytic response in association with nucleolar swelling, soma oedema and Nissl substance dispersion. The loss of Nissl substance within the dying motor neurons causes them to fail to take up the NADPH stain. (x40)

Figure 22. Microglial activation: Using IBA-1 immunofluorescent staining technique, activated Microglia undergo morphological transformation and proliferate following injury. ( x40)

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4.1.1 Motor Neuron Survival in Sham Group

The sham group was treated with intraperitoneal saline after traumatic root avulsion injury. After 6 weeks the motor neurons were found to deplete in number. There was

86% neuronal loss in the sham group. Figure 23 showed the number of viable motor neurons in sham group, ranging from 10 to 17.

18 17 16 16 15 14 14 14 12 12 10 10 8 6 4 Motor Neuron Counts 2 0 1 2 3 4 5 6 7 Rodents

Figure 23. Motor neuron counts in sham group (normal saline)

61

4.1.2 Changes of Motor Neuron Survival after Intraperitoneal Administration of

Minocycline

The intraperitoneal group was treated with 50mg/kg/day of intraperitoneal

Minocycline for one week after traumatic root avulsion injury, followed by

25mg/kg/day of intraperitoneal Minocyline for another week. After 6 weeks the motor neuron counts also depleted, but to a lesser extent as compared to the sham group.

There was only 81% of neuronal loss, as compared to 86% in the sham group. Figure

24 showed the number of viable motor neurons in minocycline group administered via intraperitonial route, ranging from 17-21.

25 21 20 19 19 20 18 18 17

15

10

5 Motor Neuron Counts

0 1 2 3 4 5 6 7 Rodents

Figure 24. Motor neuron counts in minocycline group administered via

intraperitonial route

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4.1.3 Changes of Motor Neuron Survival after Intrathecal Administration of

Minocycline

The intrathecal group was treated with 50mg/kg/day of intrathecal Minocycline for one week after traumatic root avulsion injury, followed by 25mg/kg/day of intrathecal

Minocyline for another week. The similar drug dosage delivered via targeted drug delivery aims to achieve a higher therapeutic potency to promote motor neuron survival. After 6 weeks the motor neuron counts depleted to the greatest extent. There was only 88% neuronal loss, as compared to 86% and 81% in the sham and intraperitoneal group respectively. Figure 25 showed the motor neuron counts in minocycline group administered via intrathecal route, ranging from 10-14.

16 14 14 13 13 12 12 12 11 10 10 8 6 4 Motor Neuron Counts 2 0 1 2 3 4 5 6 7 Rodents

Figure 25. Motor neuron counts in minocycline group administered via intrathecal route.

63

Table 3 showed the count of the survived motor neuron in three experimental groups

(n=21). The mean number of survived motor neurons was 14 (sham group), 18.6

(intraperitoneal group), and 12.1 (intrathecal group). Table 4 showed the comparison of motor neuron survival in three experimental groups using Kruskal-Wallis test.

Subjects in the intraperitoneal group have significantly higher survived motor neuron cells (median 19 IQR 2) as compared to sham group (median 14 IQR 4) (P=0.006) and intrathecal group (median 12 IQR 2) (P=0.005). No difference was shown between the intrathecal group and sham group (P=0.279). Figure 26 showed a distinct difference of the survived, NADPH stained motor neuron cells in the control group ( Figure 26A) and intraperitoneal group ( Figure 26B).

Figure 26A

64

Figure 26B

Figure 26 Motor neurons in control group versus minocycline group administered via intraperitoneal: This figure demonstrates the differences between the number of NADPH stained motor neuron in control group (Figure 26A) versus Intraperitoneal Minocycline treatment group (Figure 26B). The Intraperitoneal Minocycline treatment group (Figure 26B) has more NADPH stained, pinkish motor neuron at the ventral horn 6 weeks after avulsion injury. (x40)

Table 3. Motor neuron survival in three experimental groups (n=21).

Experimental subject Motor Neuron Counts In sham Group After After intrathecal (n=7) intraperitoneal administration of administration Minocycline of Minocycline (n=7) (n=7) 1 16 20 11 2 14 18 10 3 15 21 13 4 14 19 12 5 12 17 14 6 10 18 12 7 17 19 13 Mean 14 18.6 12.1

65

Table 4. Comparison of motor neuron survival in three experimental groups by Kruskal-Wallis test (n=21) Experimental groups Variables Sham group Intraperitoneal Intrathecal Chi-square P value* (n=7) group group (df) (n=7) (n=7) Median (IQR) Median (IQR) Median (IQR) Motor Neuron 14 (4) 19 (2) 12 (2) 14.395 (2) 0.0007 Counts *Kruskall-Wallis tests

Post hoc test with Bonferonni corrections by Mann-Whitney test:

Sham group vs Intraperitoneal group, P=0.0021 x 3=0.0063 (<0.05);

Sham group vs Intrathecal group, P=0.0930 x 3=0.279 (>0.05);

Intraperitoneal group vs Intrathecal group, P=0.0017 x 3=0.0051 (<0.05)

4.2 Comparison of the Neuroprotective Strength of Minocycline using Different

Route (Microglial inhibition)

A quantitative analysis of the microglia was performed over the Rexed Lamina IX at the ventral horn of the spinal cord’s grey matter. Two independent observers who are blinded to the treatment groups performed data quantification and analysis. Kappa score for inter-observer bias was 0.7. The survival is expressed as a ratio between the number of activated microglia at the avulsed site over the number of microglia at the contralateral site, where no injury was induced. Figure 22 demonstrated microglial activation following avulsion injury. The activated microglia underwent morphological transformation, and proliferated in number. Their existence was easily recognized by their strikingly pinkish appearance as they took up the IBA immunofluorescent stain.

66

4.2.1 Microglial Activation after Brachial Plexus Injury

The sham group was treated with intraperitoneal saline after traumatic root avulsion injury. After 6 weeks the activated microglia were found to increase in number. There was 14.4% increment of Microglial count as compared to the non-lesioned site. Figure

27 showed the count of activated microglial cells in sham group, ranging from 12 to

16.

18 16 16 15 15 15 14 14

14 12 12 10 8 6

MicrogliaCounts 4 2 0 1 2 3 4 5 6 7 Subjects

Figure 27. Microglial cell counts in sham group (normal saline)

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4.2.2 Changes of Microglial Activation after Intraperitoneal Administration of

Minocycline

The intraperitoneal group was treated with 50mg/kg/day of intraperitoneal

Minocycline for one week after traumatic root avulsion injury, followed by

25mg/kg/day of intraperitoneal Minocyline for another week. After 6 weeks the activated microglial cell increased in number, but to a lesser extent as compared to the sham group. There was only 12.9% of Microglial count increment as compared to

14.4% in the sham group. Figure 28 showed the count of activated microglial cells in intraperitoneal group, ranging from 10 to 14.

16 15 14 14 14 13 12 12 12 10 10 8 6

MicrogliaCounts 4 2 0 1 2 3 4 5 6 7 Subjects

Figure 28. Microglial cell counts in intraperitoneal group

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4.2.3 Changes of Microglial Activation after Intrathecal Administration of

Minocycline

The intrathecal group was treated with 50mg/kg/day of intrathecal Minocycline for one week after traumatic root avulsion injury, followed by 25mg/kg/day of intrathecal

Minocyline for another week. The similar drug dosage delivered via targeted drug delivery aims to achieve a higher therapeutic potency to inhibit microglial activation.

After 6 weeks activated microglial cell increased in number, but to the least extent as compared to the sham and intraperitoneal group. There was only 9.7% of Microglial count increment as compared to 14.4% in the sham group and 12.9% in the intraperitoneal group. Figure 29 showed the count of activated microglial cells in intrathecal group, ranging from 8 to 12.

14 12 12

10 10 10 10 9 9 8 8

6

4 MicrogliaCounts

2

0 1 2 3 4 5 6 7 Subjects

Figure 29. Microglial cell counts in intrathecal group

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Figure 30 A

Figure 30B

Figure 30 showed comparison of the activated microglia between Sham group

(Figure 30A) and Intrathecal group ( Figure 30B). (x40)

70

There are lesser number of activated microglia in the intrathecal group (Figure 30B) as

compared to the sham group (Figure 30A).

Table 5 showed the count of the activated microglial cells in three experimental groups (n=21). The mean number of activated microglia was 14.4 (sham group), 12.9

(intraperitoneal group), and 9.7 (intrathecal group). Table 6 showed the comparison of activated microglial count in three experimental groups using Kruskal-Wallis test.

Subjects in intrathecal group have significantly lower microglia cells (median 10 IQR

1) as compared to sham group (median 15 IQR 1) (P=0.006) and intraperitoneal group

(median 13 IQR 2) (P=0.019). However, no difference was shown between intraperitoneal group and sham group (P=0.198).

After avulsion injury, there was upregulation of IBA-1 in activated Microglia, which give rise to a pinkish appearance under Fluorescent microscope. The activated

Microglia lost their dendritic processes and became ameboid in shape. Without any inhibiting factor the activated Microglia is seen to proliferate and infiltrate the area within the vicinity of the avulsion site (Figure 30A).

After administration of Minocycline, activation of Microglia is suppressed. Only view pinkish, round, ameboid activated Microglia is seen as compared to the control group.

(Figure 30B).

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Table 5. Microglial activation in three experimental groups (n=21)

Experimental subject Microglia Counts In sham Group After After intrathecal (n=7) intraperitoneal administration of administration Minocycline of Minocycline (n=7) (n=7) 1 15 10 12 2 14 12 10 3 15 13 10 4 14 12 9 5 12 14 10 6 16 15 9 7 15 14 8 Mean 14.4 12.9 9.7

Table 6. Comparison of microglial activation in three experimental groups by Kruskal- Wallis test (n=21)

Experimental groups Variables Sham Intraperitoneal Intrathecal Chi- P value* group group group square (n=7) (n=7) (n=7) (df) Median (IQR) Median Median (IQR) (IQR) Microglia 15 (1) 13 (2) 10 (1) 13.054 0.0015 cell counts (2) *Kruskall-Wallis tests

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Post hoc test with Bonferonni corrections by Mann-Whitney test:

Sham group vs Intraperitoneal group, P=0.0661 x 3=0.1983 (>0.05);

Sham group vs Intrathecal group, P=0.0019 x 3= 0.0057 (<0.05);

Intraperitoneal group vs Intrathecal group, P=0.0063 x 3=0.0189 (<0.05)

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4.3 Comparison of the Neuroprotective Strength of Minocycline using Different

Route (Nitric Oxide expression)

The following figures show the number of neuronal nitric oxide synthase (nNOS) positive motoneurons at injured ventral horn in control group and Minocycline treatment groups (after 6 weeks after avulsion in mean percentage).

4.3.1 Nitric Oxide Expression in Control group

Figure 31showed nitric oxide expression in sham group, ranging from 28 to 34. There was a 31.6% of nitric oxide expression over the lesioned side of the spinal cord, as compared to the normal side (table 7).

40 34 34 35 33 31 32 29 30 28 25 20 15 10

Nitric Oxide Nitric Oxide Expression 5 0 1 2 3 4 5 6 7 Subjects

Figure 31. Nitric oxide expression in sham group (normal saline)

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4.3.2 Nitric Oxide Expression in Minocycline treatment group (Intraperitoneal)

Figure 32 showed nitric oxide expression in intraperitoneal group, ranging from 25 to

29. There was only a 27% of nitric oxide expression, as compared to 31.6% in the sham group (table 7).

30 29 29 28 28 27 27 27 27 26 26 25 25

Nitric Oxide Nitric Oxide Expression 24

23 1 2 3 4 5 6 7 Subjects

Figure 32. Nitric oxide expression in intraperitoneal group

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4.3.3 Nitric Oxide Expression in Minocycline treatment group (Intrathecal)

Figure 32 showed nitric oxide expression in intrathecal group, ranging from 28 to 31. There was 30.6% nitric oxide expression, as compared to 27% in the intraperitoneal group, and 31.6% in the sham group (table 7).

34 33

33 32 32 31 31 31 30 30 29 29 28 28 27 Nitric Oxide Nitric Oxide Expression 26 25 1 2 3 4 5 6 7 Subjects

Figure 33. Nitric oxide expression in intrathecal group

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Table 7. Nitric Oxide expression in three experimental groups (n=21)

Experimental subject Nitric Oxide Expression In sham Group After After intrathecal (n=7) intraperitoneal administration of administration Minocycline of Minocycline (n=7) (n=7) 1 29 28 29 2 31 27 31 3 33 27 30 4 32 26 32 5 34 25 28 6 34 27 33 7 28 29 31 Mean 31.6 27 30.6

Table 8. Comparison of motor neuron survival in three experimental groups by Kruskal-Wallis test (n=21) Experimental groups Variables Sham Intraperitoneal Intrathecal Chi- P-value group group group square (n=7) (n=7) (n=7) (df) Median (IQR) Median Median (IQR) (IQR) Nitric 32 (5) 27 (2) 31 (3) 11.688 0.0029 Oxide (2) Expression *Kruskall-Wallis tests

Post hoc test with Bonferonni corrections by Mann-Whitney test:

Sham group vs Intraperitoneal group, P=0.0038 x 3=0.0114 (<0.05);

Sham group vs Intrathecal group, P=0.3331 x 3=0.9993 (>0.05);

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Intraperitoneal group vs Intrathecal group, P=0.0038 x 3=0.0114(<0.05)

Table 8 showed the comparison of motor neuron survival in three experimental groups by Kruskal-Wallis test (n=21). Subjects in intraperitoneal group have significantly lower nitric oxide expression (median 27 IQR 2) as compared to sham group (median 32 IQR 5) (P=0.011) and intrathecal group (median 31 IQR 3) (P=0.011). However, no difference was observed between sham group and intrathecal group (P=0.999).

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CHAPTER 5

DISCUSSION

5.1 Cellular Changes following Traumatic Avulsion Injury

5.1.1 Primary Injury secondary to Traumatic Avulsion Injury

In our experimental research, we created an animal Preganglionic avulsion injury by separating the motor neuron from the anterior horn of the spinal cord grey matter. It is performed by applying a balance traction force at the cervical nerve root in an outward direction after grasping its most proximal aspect with a blunt forceps. The avulsed rootlets were examined under the microscope to ensure that separation is performed at its origin located at the Central Nervous System (CNS)-Peripheral Nervous System

(PNS) interface. Avulsed models without motor rootlets seen at its proximal segment are excluded from sampling. The impact of this type of avulsion injury involves both the peripheral nerve as well as the spinal cord. The segment proximal to the lesion, i.e. the spinal cord and the anterior horn cells undergo extensive changes, from the primary injury largely contributed by the mechanical trauma created by the avulsion force The mechanical injury sacrificed the local motor neurons at the affected level. It also transected the axons of the ascending and descending tracts travelling across the lesioned side. Occasionally local micro-hemorrhage can be appreciated under the microscope as soon as the injury takes place. The hematoma is created by local disruption of the vascular endothelia of the epidural (Batson’s) plexus and rarely, radiculo-medullary vessels on the spinal cord. The hemorrhage can interrupt the

79 delivery of oxygen and nutrient to the injury site, leading to Hemorrhagic necrosis.

Continuous hemorrhage can spread and affect adjacent spinal segment, leading to local mass effect and compression to the cord and its nerve roots. Since the neurons have critically high demand for glucose and oxygen for metabolism, the ischemic state resulted from the injury process can cause defective metabolism, which eventually die off. (Juan et al, 2014).

5.1.2 Secondary Injury secondary to Traumatic Avulsion Injury

We observed atrophic and degenerative changes within the dorsal horn of the spinal cord after the injury. It is localized at the beginning but spread to the adjacent area as time progresses. These histological changes are pertaining to the secondary damage, which starts minutes after the primary injury. It is a complex cascade of secondary response with inflammatory elements. Our experiment demonstrated a progressive and disastrous consequence of the secondary injury. Within a timeframe of 6 weeks period, there was a neuronal loss as high as 85%. An injury to axons of motor neurons in the adult peripheral nervous system induces a series of structural and metabolic alterations. Anatomically, the dorsal horn of the spinal cord grey matter developed a small focal area of fluid filled cavity. The formation of the cavity is related to traumatic lysis, ischemia, hemorrhage, excitotoxicity, and marked inflammatory response. The complement system is activated causing granulocytes, lymphocytes, and macrophages infiltration over the lesioned site. They release chemotactic agents such as Cytotokines, Inteleukines and Tumour Necrosis Factor- α, which trigger a complex cascade of inflammatory response. The disruption of spinal cord blood barrier

80 facilitates the process. The injured neurons undergo morphological changes, which includes swelling of the soma, decentralization of the perikarya and dispersal of Nissl substance “chromatolysis”. This phenomenon is known as Retrograde Axonal

Degeneration. Beside the motor neurons, there are also a number of structural changes in the immediate vicinity to the affected motor neurons, including the glial cell response, “gliosis”. Six weeks after the injury, microscopic inspection of the histological specimen revealed an increase of IBA11 staining to 14.4%. IBA1 is an

Ionizing Calcium-binding adaptor molecule that is specifically expressed on activated

Microglia, especially in pathological condition. Synaptic ‘stripping’ is another phenomenon occurred as a consequence to secondary traumatic injury from root avulsion. These retrograde degenerative changes in the central nervous system happens at the same time with the Anterograde “Wallerian” degeneration, which is marked by disintegration of the transected distal portion of the axon, Schwann cell proliferation followed by sprouting and regeneration. The ultimate outcome of the inflammatory process following the avulsion injury is apoptosis of the motor neurons as a result from the detrimental effect from free radicals and oxidative stress mediated by cytokines (Barbizan and Oliveira, 2010).

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5.2 Excitatoxicity

Excitotaxicity is deemed to be a major pathogenesis of the secondary damages occurring in our experimental avulsed nerve models. Accumulation of Excitatory

Amino acid such as Aspartate and Glutamate are major causes leading to secondary injury secondary to avulsion injury. They play a very prominent role for over expression of AMPA, Kainate, and NMDA receptor. These amino acids trigger intracellular Sodium influx, leading to cellular edema, abnormal depolarization and neuronal death. The Glutamate reach its excitatoxic level almost immediately after traumatic avulsion injury. This phenomenon is related to the release of Glutamate from the lysed, injured and necrotic cells. The Glutamate level continues to rise due to dysfunctional Glutamate re-uptake. The Glutamate induced excitatoxicity can contribute to the death of motor neurons at the anterior horn of the spinal cord’s grey matter, as well as the loss of supportive cells at the white matter, namely

Oligodendrocytes, leading to axonal dysfunction and degeneration. A similar toxic concentration of Glutamate found in traumatic avulsion model introduced to a non- traumatic brachial plexus can be equally neurotoxic ( Rao et al, 2014, Chuang, 2005).

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5.3 Neuronal Cell Death

Our study clearly demonstrated neuronal death at the deformed C7 spinal cord segment at 6 weeks following avulsion injury. The C7 nerve root is extracted by applying traction force directed parallel to the nerve trajectory with a blunt toothless

Adson forcep via Extravertebral avulsion technique. A preganglionic injury is confirmed via direct inspection of the stump of the rootlets, as well as cerebrospinal fluid egression from the torn Meninges on the lesioned side. Occasionally bleeding occurred during the process of avulsion, which required hemostasis using Micro-

Fibrilla. If the transection takes place distal to the rootlets, the changes of spinal cord atrophy and degeneration is nil or minimal. Only Anterograde “Wallerian” degeneration was observed. Those specimens were excluded in our study. In a rodent whose C7 ventral root is properly avulsed, one can appreciate the distally transected rootlets under the microscope, which appeared to be edematous, as immediate response to the mechanical traction injury. The rootlets are most fragile at their segment within the subarachnoid cistern. This is the site where most of the rootlets getting transected. At 6 weeks after the avulsion injury, the proximal avulsed rootlets can be inspected after cardiovascular perfusion. At this stage the proximal segment of the avulsed rootlet can be visualized to have an unhealthy, dystrophic, pale, stretched and thinned out appearance under microscope. Edema subsided at this stage. Adhesion and fibrosis is seen between the meninges and the spinal cord at the injury site, indicating that the secondary traumatic avulsion injury involves an inflammatory

83 response. This injury process results in significant and progressive loss of motor neuron over time. Inspection of the C7 spinal segment, defined as the spinal segment between the lower edge of C6 rootlets and upper edge of C7 rootles, demonstrated

85% neuronal loss after 6 weeks. Within the injured spinal segment, the surviving motoneurons were stained red colour with NADPH-d histochemistry plus neutral red counterstaining. They can be observed and identified at the Rexed’s lamina IV of the affected ventral horn. The affected motor neurons underwent early degeneration and chromatolysis, with death occurring by necrosis or apoptosis. The size of the motor neurons at the injured ventral horn also appear to be smaller as compared to those at the contralateral, uninjured ventral horn. This phenomenon resembles significant post traumatic atrophic changes. Our extravertebral avulsion technique has a villainous consequence. It created maximal tensile stress on the motor neurons at the ventral horn of the spinal cord prior to the rupture of the ventral rootles. The mechanical loading of pressure on the grey matter of spinal cord can induce contusion, microhemorrhages, local ischemia, which will trigger cascades of excitatoxicity and inflammatory response at the vicinity of the injury site. Occasionally hematoma was also created following inadvertent laceration of the microvessels along the nerve roots. They are the Radiculo-medullary branches of arteries and veins. Some have connection with the

Batson’s plexus at the epidural space. Vessels injuries compromise oxygen and glucose delivery to the adjacent spinal cord segments. An avulsion injury at a single level is therefore said to have their disastrous effect at few other neighboring level or segments. The extent of the injury depends on the mechanical loading force and the extent of vessel injury. In other study, the motor neuron loss can be detected from the

84 first week after the injury. The rate of motor neuron loss increases in second and third week, then finally reach a plateau at the fourth to sixth week. The motor neuron response to avulsion injury reported by the previous studies is consistent to our finding

(Huong, 2003, Rao, 2014). The degree of cell death has been suggested to be underestimated since the atrophy of these cells can be substantial and long lasting, and thus hard to find in the tissue in cell counting experiments. How motor neurons die is also to some extent unclear and controversial but it is more or less concluded to be by both programmed, apoptotic, and by means of necrotic death depending on the severity and/or type of the lesion (Noguchi, 2013). Excitotoxicity, Mitogenic

Activated Protein Kinase pathway signalling are among the important mechanisms involved in the process (Park et al., 2007). Nitric Oxide and Microglial activation are two elements that are closely associated with the aforementioned signalling pathways.

Neutrotrophic factors, targetting at modifying the above signalling pathway, added on to the implantation surgery have been shown to have various effect on root avulsion injuries ( Daniels et al,2014, Eggers et al, 2010).

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5.4 Microglial Response to Avulsion Injury of the Motor Root

Microglial response to avulsion injury is certainly related to the process of secondary injury. In the present study, Immunohistological study of the anterior horn of the spinal cord labeled with anti-Iba1 was performed 6 weeks after injury to assess the degree of microglial reactivity after root avulsion. The normal, uninjured Microglial neither expressed nor upregulated IBA-1. The dormant Microglia was visualized to have a uniform, and ramified morphology. It has a cell body with its dendritic processes radiating outward in all directions.

After injury the Microglia gets activated at the injury site. Activated Microglia express

IBA-1, which is a ionizing Calcium-binding Adaptor molecules, which appeared to be pinkish under Fluorescent Microscope after antibody staining. The activated Microglia rapidly proliferate and infiltrate the lesioned side. The supportive cells also take on a distinctly interesting morphology when they get activated. It loses the dendritic processes and become round or ameboid.

The microglial response is the main “player” in a cascade of neuroinflammatory events. Cellular response of the spinal cord following any traumatic injury including a proximally located nerve root avulsion always ends up in glial scar formation. Glial response in rodents after ventral nerve root avulsion causes proliferation of activated

Microglia, Astrocytes, Fibroblast, Microphages, Granulocytes towards the

86

Perineuronal area of the affected neurons. The magnitude of the cellular inflammatory response differs with time. For instance, the microglial responses tend to peak within days whereas astrocytic reactions show the strongest reactivity at later stages after a traumatic spinal cord injury in rats. Recent study on the immunological response of rats after trauma is sustained even after a long period of time as evidenced by persistence of reactive Microglia even after 6 months following trauma (Beck, 2010).

The role of the activated Microglia as the immune cells within the nervous system has been heatedly debated over the century. Recent literatures unveiled the Janus face of the activated Microglia. Some of the biomolecular changes seen after Microglial activation includes induction of MHC class I and II antigen, synthesis of key factors in the complement system as well as synthesis and release of free radicals (Min et al,

2011, Svensson & Aldskogius, 1993). The activated Microglia is known to release

Excitotoxic substance such as Glutamate, Nitric Oxide, Reactive Oxygen Species

(ROS) and Proinflammatory Cytokines. The Chemotactic agents trigger inflammatory process, which recruits the Leukocytes and T cells to migrate and proliferate over the injured site, and eventually lead to apoptosis of the injured motor neuron. The detection of Glial response at the time of motor neuron loss following avulsion injury reflects a direct response to motor neuron death. On the other hand many other literatures showed a totally opposite role of Microglia in orchestrating the

Neuroinflammatory response. Microglia is also claimed to have neuroprotective role in traumatic injury. It is known to protect the neurons from Nitric Oxide Neurotoxicity

(Toku, 1998) as well as ischemia induced cell death (Neuman, 2006). Results from in

87 vitro studies demonstrate that microglia can develop a regeneration supportive phenotype by producing proregenerative factors, such as insulin-like growth factor. It is also known to secrete cytokines to trigger glial response that is associated with collateral reinnervation after the nervous system suffer from traumatic injury.

(Barbizan 2010).

5.5 Brachial Root Avulsion Injury and Oxidative Stress Activity

Expression of Nitric Oxide in injured rat spinal motoneurons has been shown to be closely associated with avulsion injury and neuronal death (Wang et al., 2011). This fact is clearly demonstrated and proven in the present study. Nitric Oxide Synthase

(NOS) produces Nitric Oxide by oxidizing L-Arginine in pathological and stressful condition. There are three distinct forms of NOS:

1. nNOS (neuronal/Type I/NOS-1/bNOS)

2. iNOS (inducible/Type II/NOS-2)

3. eNOS (endothelial/Type III/NOS-3)

The iNOS is expressed almost immediately following injury to produce the first wave of Nitric Oxide surge. The iNOS depletes with time. iNOS depletion triggers the constitutive Nitric Oxide Synthase ( comprises nNOS and eNOS) expression, leading to the second wave of Nitric Oxide surge.

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We only measure nNOS since the spinal cord is only harvested six weeks after the injury. iNOS has returned to its baseline concentration by this time. In normal, non- injured rat spinal cord, neuronal nitric oxide synthase (nNOS) positive neurons were located mainly in the dorsal horn (Rexed’s lamina I to IV) and the area immediately surrounding the central canal, but none in the ventral horn. However, root avulsion induced nNOS-positive motoneurons were detected in the ventral horn (Rexed’s lamina IV) of the injured site. Expression of nNOS is responsible for the generation of the free radical gas nitric oxide (NO), which may exert toxic effects on the neurons.

The overproduction of NO will react with the superoxide anion to produce the potent oxidant, peroxynitrite (ONOO-). Peroxynitrite has direct cytotoxicity. It could inhibit

DNA synthesis, liberate iron from the iron storage protein ferritin, and influence iron metabolism at the post transcription level (Penas et al., 2009). Damage to DNA by NO and ONOO- induced activation of the nuclear enzyme poly ADP-ribose synthetase

(PARS) and causes cell death by rapid depletion of cell energy. Howoveer on the other hand, many literatures also reported the positive role of Nitric Oxide Synthase pathway in synaptic plasticity and neuronal signalling, as well as neurogenesis ( Li,

2009).

The Nitric Oxide Synthase pathway and its biological effect discussed above can be summarized as below,

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Nitric Oxide Synthase Pathway

Fig 31 Nitric Oxide Synthase Pathway.

(Valdes et al, 2009)

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Upregulation of Nitric Oxide Synthase oxidizes Arginine to form Nitric Oxide. At low levels, they are critical proteins to mediate synaptic plasticity and neuronal signaling.

At concentrated level, it changes from a physiological modulator to a cytotoxic chemofactor.

One of the key steps to promote neuroprotection by Minocycline is to downregulate

Nitric Oxide Synthase and hamper its oxidative stress response. Overzealous suppression of the Nitric Oxide Synthase, much like the Microglial suppression, may hamper neurogenesis ( Huang, 2013, Valdes et al, 2009).

5.6 Effect of Minocycline to the Nervous System following Nerve Avulsion

5.6.1 The Neuroprotrective Therapeutic Effect of Minocycline to the Nervous

System

Experimental group in our study that were treated with Intraperitoneal (IP)

Minocycline have significantly higher motor neuron cells (median 19 IQR 2) as compared to sham group (median 14 IQR 4) (P=0.006). The hypothesized pathway in which Minocycline exerts its biochemical effect has a prominent role in neuroprotection. Pharmacological pathways of Minocycline that involve the inhibition of p38-MAPK phosphorylation and microglial reactivity, blockade of caspase, metallomatrix proteins and endocannabinoid production play a crucial anti- inflammatory role to reduce Microglia activation and motility, hence glial scar formation, which serve as both microanatomical and functional barrier to nerve regeneration.(Kobayashi et al, 2013, Amy and Ping, 2014). Hoang et al. (2008)

91 discovered that Minocycline exerts an anti-apoptotic effect toward degenerating motor neurons by interfering the Caspase pathway and microglial inhibition. In his study, he introduced Minocycline to the rodents via peritoneal injection. In this manner the peritoneal cavity works as a slow release depot, which keeps the compound to be maintained at steady state in the circulation for 8 hours. The therapeutic effect of

Minocycline is concentration and time dependant. Intraperitoneal route of administering Minocycline as therapeupeutic agent is considerably effective and convenient albeit having a lower bioavailability in theory as compared to the more costly and technically demanding targeted drug delivery via intrathecal route. Other potential systemic side effects of Minocycline were not seen in all rodents clinically when it was administered intraperitoneally. The positive result prompted us to attempt a more potent mode of drug delivery via intrathecal route. An opposing result is obtained .The motor neuron count in the intrathecal group is slightly less than the control group (12 versus 14). The difference is not statistically significant. At this point we still cannot deny the beneficial effect of Intrathecal Minocycline completely after taking some technical issue into consideration. Insertion of intrathecal pump in rodents requires a second surgery and this could be technically challenging. The operative time for the electronic implant insertion is also longer as compared to the avulsion surgery. We experienced wound dehiscence secondary to infection in up to

57% of the rats that received implantable intrathecal pump. On top of that, two rodents expired owing to general anesthesia complication. Infection and stress response after the second surgery could be among the important factors that impair the regenerating capability of the nervous system. Benefit of doubt should be given to the

92 fact that a favourable outcome could be obtained if the intrathecal pump is inserted with better microsurgical techniques or into larger sized experimental subjects. In terms of modulation of Oxidative stress, the experimental group in our study that were treated with Intraperitoneal (IP) Minocycline have significantly lower Neuronal Nitric

Oxide Synthase expression (median 27 IQR 2) as compared to sham group (median 32

IQR 5) (P=0.011). The finding proved that Minocycline is capable of downregulating the Nitric Oxide Synthase (NOS), which is the catalyst enzyme for the production of

Nitric Oxide. The subsequent cascade of interaction between Nitric Oxide and other

Reactive Oxygen Species is therefore interrupted. Correlating both the results of the

Motor Neuron count and the Nitric Oxide Synthase expression in the rodents after

Intraperitoneal Minocycline administration, we can conclude that Nitric Oxide

Synthase downregulation is an important pathway used by Minocycline to protect the motor neurons. (Cheng et al, 2010). Our present study demonstrated that Minocycline exerts neuroprotection towards motor neurons. The effect is dose dependant. At higher concentration or potency it can exert neurotoxicity. The primary mechanism implicated in Minocycline neuroprotection is the drug’s highly potent inhibitory effect on Microglial activation as well as apoptosis via downregulation of Nitric Oxide

Synthase. Other pathways described in other literatures include inhibition of P38 phosphorylation as well as Mitogen Activated Protein Kinases pathway signaling. As a result, inflammatory cytokines production leading to cell death and microglial action for debri clearance is prevented. Minocycline can also induce change in regulation and transportation of neurofilaments. (Huntington Study Research group, 2010) The drug has rich lipophilic property. It can easily penetrate the nervous system when it is

93 introduced via peritoneal route. Other advantages of Minocycline apart from its neuroprotective potential include antimicrobial effect and anti-inflammatory property, which reduces graft rejection related problems, especially if brachial plexus surgery involving nerve transfer and implantation is done. It is also widely available, affordable and generally tolerable without much serious side effects.

5.6.2 The Effect of High Dose, Concentrated Minocycline through Targeted Drug

Delivery

We demonstrated that Minocycline strongly suppressed the Microglia when it was introduced intrathecally. Rodents in the intrathecal group have significantly lower microglia cells (median 10 IQR 1) as compared to sham group (median 15 IQR 1)

(P=0.006), and intraperitoneal group (median 13 IQR 2) (P=0.019). The Intrathecal group also has lesser, but non-statistically significant motor neurons count than the sham group. Besides, Nitric Oxide expression is also higher than the intraperitoneal group. The present study demonstrated that the intrathecal administration of

Minocycline, which is deemed to have higher bioavailability, might exert adverse effect towards the motor neuron survival. The destructive effect toward motor neuron could be a direct effect from the Microglial inhibition or indirectly via other mechanisms. Scar formation, which is a natural response following injury hindered by the Minocycline, is demonstrated to have beneficial effect by many other literatures.

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For example Cui et al. (2001) described the deleterious effect of Glial cells removal, causing reduction of Glutamate transporters, which help to evacuate Glutamate out of the synapse. Low level of Glutamate transportor will lead to accumulation of

Glutamate and subsequently excitotoxic neuronal cell death. The reactive Microglia phagocitize the debri which contain neuronal growth inhibitory molecules such as

Myelin associated Glycoprotein and Myelin Component Nago A (Kiegerl, 2009).

Microglia inhibition removes the motor neurons’ shield from injury by the Nitric

Oxide and the Reactive Oxygen Species. The microglial motility is reduced by

Minocyline, thus hamper glial scar formation. The literature described the neuroprotective effect of the Glial scar via a Glutathion dependant mechanism. It also inhibits Astroglial motility, migration and coupling neutrotrophic effect. The

Astrocytes play a crucial role to repair blood brain barrier, regulate the immune response, and promote neuronal survival and axonal outgrowth. Minocycline thereby prevent the creation of microenvironment at site of injury. Ironically, Minocycline is also proven to have deleterious effect, which is largely dose dependant. For instance it enhances 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced damage in dopaminergic neurons in mice. Under conditions of oxygen glucose deprivation and ischemic injury, high concentration of minocycline enhances the cell death of neurons and astrocytes. In the peripheral nerve system (PNS), Wallerian degeneration, a prerequisite of regeneration, was significantly impaired. The overzealous Microglia suppression could have remove the Microglial protection on the Motor Neurons against oxidative stress (Tanaka et al, 2013).

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5.7. Limited Regenerative Capability after Avulsion Injury

Avulsion injury compromising the anterior horn cell body and its peripheral nerve has pronounced limitation for regenerative process to restore their connectivity. The Pre- ganglionic avulsion created a loss of axonal continuity and internal neuronal damage.

Externally scarring and dystrophic changes around the nerve stumps form a formidable barrier within the frank gap to prevent re-connection of the previously transected neuronal structure. These changes are the consequence of the neuroinflammatory response and degeneration secondary to the secondary traumatic injury. It is also a prominent extrinsic limiting factor that restricts the neuronal regeneration. The glial scar, a scar tissue formed primarily by the Microglial cell,

Astrocytes, Fibroblasts, Oligodendrocytes and the Meningeal Fibroblast is dense, firm and becomes impermeable to the regenerating axons. As discussed before, the neuroinflammatory and degenerative process can be long lasting with apoptosis or death as the ultimate outcome. The anti-inflammatory and anti-‘apoptotic’properties of

Minocycline is thus employed by the author to promote regeneration by mediating the inflammatory response (Casha et al, 2012).

There are also intrinsic factors which play an equally important role as the extrinsic factor to orchestrate the regeneration process. Unlike the peripheral nerve, which is well equipped with local proteins and structures such as Ribosomes, Golgi-like

Structures, mRNAs which are essential for Regeneration associated Proteins production, the spinal segment in the vicinity of the anterior horn cell is lacking those

96 vital proteins, which transcribes supportive cytoskeletons such as Actin, Tubulin,

Growth Associated Proteins and Cytoskeleton associated Proteins. Few Neutrotrophic factors identified at the CNS-PNS interface are Oncomodulin, Brain Derived

Neutrotrophic Factor (BDNF), GAP-43. (Carrier et al, 2014). These regeneration associated molecules could be downregulated by Minocycline in the process of

Neuroinflammatory modulation.

Another important intrinsic factor contributing to the limited regeneration in avulsion injury as compared to peripheral nerve transection is the microtubule response to the injury. In peripheral nerve injury the microtubules are well organized and dynamic at the axonal stump, aiding in growth cone formation and axonal extension. The microtubules at the avulsion site within the spinal segment is disorganized and depolymerized. The awkward microanatomical arrangement at the axonal stump does not help to propel axonal elongation but rather form the identical dystrophic, retracted end bulb. Having a poorer intrinsic regenerative potential does not mean that the spinal motor neurons do not grow at all, provided that the local microenvironment is permissive. For instances, the spinal motor neuron will continue to grow if the adjacent myelin is intact. If the Myelin is damage, the growth capability is compromised as well. An important Neurotrophic factor expressed during remyelination phase of neuronal recovery is Ciliary Neurotrophic factor (CNTF). The neuroprotective protein is produced by both Astrocytes and Microglia. On the contrary there are also important Myelin associated inhibitory proteins notorious for the limitation of regeneration are NOGO, Myelin-associated Glycoprotein and

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Oligodendrocyte Myelin Glycoprotein. Administration of Minocycline can downregulate the Ciliary Neurotrophic Factor and trigger the expression of the Myelin associated Inhibitory Proteins. (Tanaka et al, 2013, Kiegerl, 2009).

The environment of CNS-PNS interface already has limited regenerative capability.

Introduction of Minocycline, with the intention of immunomodulation for neuroprotection, can compromise the neuroregeneration, especially at high, concentrated dosage.

5.8. Mitogen Activated Protein Kinase Pathway in Avulsion Injury of Motor Root

Mitogen Activated Protein Kinases are Serine Threonine Kinases that mediate intracellular signals, which regulate cellular differentiation, proliferation, transformation, survival and death. The family of Mitogen Activated Protein Kinases consist of Extracellular signal Regulated Kinases ( ERK), P38 and JNK/SAPK (C-

June/NH2 Kinases/Stress Oxidative Associated Protein Kinases. Each MAPK signaling axis comprises at least three components: a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. MAP3Ks phosphorylate and activate

MAP2Ks, which in turn phosphorylate and activate MAPKs.

Stimuli → MAP3k→ MAP2K→MAPK→Cellular response

The P38 and SAPK pathway are triggered by cellular injury or oxidative stress, which release chemotactic cytokines such as Tumor Necosis Factor Alpha and Interleukin 1.

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Oxidative Stress, Cytokines etc →P38 and JNK→Inflammatory response→Cellular proliferation, apoptosis.

The Extracellular Regulated Kinases pathway signalling is ignited from the Kinases

Suppresor Proteins namely K-ras, N-ras and H-ras (activated Members of RAS family). They play an important role to transmit the extracellar signal into the cells, which trigger cellular proliferation, differentiation and migration.

RAS →ERK →cellular proliferation, differentiation and migration

The Mitogen Activated Protein Kinases pathways are activated as a result from binary interaction between the kinases or a signal cascade involving multiple kinases. The later is guided by scalfold proteins including ras and JNK. The MAPK pathway was previously thought to have a negative and opposing effect on neural regeneration.

However recent studies proved the otherwise ( Kato et al, 2013).

5.9 Degenerative Process following Traumatic Avulsion of Ventral Root

Upon traumatic avulsion of ventral root, the anterior horn of spinal cord undergoes atrophic changes. This phenomenon reflects that the peripheral nervous system fails to produce trophic support on its on and up-regulate the regeneration associated genes.

Synthesis of important supportive cytoskeleton such as Tubulin and Actin diminishes

99 after traumatic avulsion, hampering further neuronal regeneration and growth capability. The distal segment of the avulsed site thus undergoes ‘Wallerian

Degeneration’. At the proximal end, the injured axons retracted from the injury site and form dystrophied end bulbs. Further cellular edema, lysis and necrosis release chemical substances that have detrimental effect towards the cells surround the injury site. Many adjacent neurons at the peri-lesional area die off. Others might be remaining intact structurally but biochemically and functionally impaired. Ionic imbalance secondary to axonal membrane damage can produce large amount of

Caspase, which will compound the pre-existing damage done by axotomy. Axonal injury only produces neuronal damage to the surviving neuron to some extent. Another damage of greater extent is done by the spread of the injury to the supporting glial cells, especially Oligodendrocytes. Loss of the Myelin producing Glia produces focal demyelination at the site of lesion, contributing to conduction blockade and increased latency. Not only demyelination compromises the neurological function, it also catalyses further degeneration.

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5.10 Factors Affecting Degeneration

The severity of the injury and outcome of a peripheral nerve injury depend on several factors. When analyzing data from different studies concerning e.g. neuronal cell death following axotomy it is important not only to identify type and level of injury, but also several other confounding parameters such as nerve model, species, gender and age of experimental animals which may have significant impact on the results.

5.10.1Avulsion and transection - proximal vs. distal injury

The distance of the injury site to the cell body is a crucial factor that affects neuronal survival. This is related to the amount of trophic support by Schwann cells at the proximal remnant of the nerve. Avulsion injury (tearing) usually causes proximal breaking of axons near interface between the Central and Peripheral Nervous System or even at the Central Nervous System side close to the cell bodies and should thus be regarded as injury at the Central Nervous System. Such injury cause massive cell death as a result of the extreme proximity of the axotomy with total disconnection to the periphery (Carlstedt, 2011). A transection injury (nerve cut) of a peripheral motor nerve leads to disruption of all of the components of the nerve, nonetheless such an injury is followed by only a low number of motor neuronal loss as long as the two nerve endings are in close contact, although the more proximal the injury is to the site of the localization of the motor neurons, and the greater the distance between the proximal and distal ends of the transected nerve, the more severe consequences will follow (Svensson & Aldskogius, 1993). There are thus profound differences in-

101 between the transection and avulsion injuries with regards to regeneration capacity and re-growth of axons.

5.10.2 Species and strain

Immune response and inflammatory changes after injury might vary according to animal type and species. The differences have to be considered when evaluating and comparing results (Popovich et al., 1997; Sroga et al., 2003). Variations in genetic factors between the same species but different strains can also influence outcome. In rats these strain differences are also observed after ventral root avulsion (Swanberg et al., 2006; Piehl et al., 2007).

5.10.3 Gender and age

The age of the animal is strongly influencing the level of motor neuron cell loss and for example it is shown that most of the hypoglossal motor neurons survive in adult animals after a transection injury, while such an axotomy during development and early in postnatal life instead leads to massive depletion of motor neurons (Snider &

Thanedar, 1989; Fukuyama et al., 2006).

There are also significant differences reported in male and female adult rats in the hypoglossal and facial nuclei, where females were shown to have a twofold greater neuronal cell loss after axotomies (Yu, 1988). Furthermore, depletion of sex hormones in male animals elevates cell loss while administration of testosterone to females after injury reduces neuronal loss (Yu, 1988; Yu, 1989).

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5.11 Clinical Implication and prospect of Minocycline in Brachial Plexus

Avulsion Injury

This exploratory animal model study has shown satisfactory results in which

Minocycline poses the potential neuroprotective effect in the prevention of spinal motoneurons death following brachial root avulsion injury. The mechanisms of minocycline action appear to result from microglial inhibition and anti-apoptotic functions. Microglial inhibition results in the downregulation of MHC II expression, the inhibition of the p38 MAPK pathway, decrease in cell motility, reduction in β- integrin and Kv1.3 potassium channel expression, reduction in prostaglandin E synthase expression or reduction in the level of matrix metalloproteinases . Anti- apoptotic mechanisms include a decrease in Ca2+ uptake under stress conditions, reduction in reactive species formation and the inhibition of the p38 MAPK pathway.

Further studies are needed to understand the molecular mechanisms in this context.

Axonal regeneration in nerve root re-implantation following brachial plexus avulsion injury has been studied in rats and primates. The regeneration potentials between ventral root and dorsal root are different. Regeneration in the ventral root contains more myelinated axons than that could be found in the dorsal root for the same period of time. For successful regeneration to occur after a dorsal root avulsion injury, sensory neurons must regenerate from the dorsal root ganglion and elongate their axons across CNS tissues into the spinal cord to reach their target regions in the dorsal horn of the spinal cord. On the other hand after a ventral root avulsion injury,

103 motoneurons must regenerate in the spinal cord before extension of axons into the ventral root is possible. These animal model studies demonstrated a large number of retrograde marker labelled neurons were found in the ventral horn of the spinal cord following reimplantation of avulsed spinal root.

By looking at these positive experimental findings, one of the future possible approaches in managing brachial plexus avulsion injury could be re-implantation of the avulsed nerve root together with Minocycline. Being a antibiotic, it can be used as a decent antimicrobial prophylaxis for the surgical procedure. Nevertheless, in animal study, the re-implantation of spinal nerve root was carried out immediately after the avulsion injury under a controlled environment. This is completely different in the actual clinical setting. For example, a patient who sustained brachial plexus injury following a motor vehicle accident, accumulative time spent for pre-hospital transportation, in-hospital stabilization, radio-imaging procedure and preparation for surgical repair should be taken into consideration. Thus a well-designed experimental animal model considering the appropriate timing of Minocycline delivery after the initial brachial root avulsion injury is needed in order to apply all these experimental findings into such a complex clinical situation.

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5.12 Limitations and Recommendations of Clinical Study

The sample size of 7 rats per group is maybe too small to yield better result in this study. Since this is an experimental animal-based study, the sample size used was based on previous studies of similar method and aims. For future study a larger sample size should be used to get better result.

The outcome of this study is assed via histopathological examination only. Even though there is significant survival of motoneuron in the implanted group and reduction of minocycline, the clinical outcome of the animal is not assessed. Clinical outcome of the animal can be assessd in few parameters such as paw grasping power, paw walking pressure and animal self-grooming. Having a positive survival of motoneuron in the cervical spinal cord should be correlated to improvement of the clinical symptom as well to achieve better meaning of this study. Thus for future study, animal behavioural of the studied animal should be done to observed the clinical improvement aspect of the test animals.

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CHAPTER 6

CONCLUSION

Following traumatic avulsion injury of the ventral nerve root, motor neuron death and microglial proliferation was observed. Findings showed that administration of

Minocycline to treat rodents suffering from avulsion injury was capable to suppress the Microglial proliferation. Intraperitoneal administration of Minocycline shows some degree of beneficial effect to prolong the motor neuron survival by inhibiting the

Microglia activation and proliferation, thus hampering apoptosis of the motor neuron.

However when Minocycline was administered via intrathecal route to increase the bioavailability of the therapeutic agent, this compromised the motor neuron survival.

Therefore, we conclude that microglial suppression via Minocycline can have double effect. Moderate dosage of Minocycline may be beneficial towards motor neuron survival. However for high concentration of Minocycline, it could be neurotoxic by causing impairment of the Glial response and Wallerian degeneration, which is a pre- requisite to regeneration.

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