Department of Psychiatry and Clinical Psychobiology
School of Medicine
The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities.
Thesis presented by
Davinia Fernández-Espejo
To obtain the degree of Doctor of Philosophy from the University of Barcelona
In accordance with the requirements of the European PhD Diploma
Supervised by
Dr. Carme Junqué, University of Barcelona, Spain
Dr. Adrian Owen, MRC Cognition and Brain Sciences Unit, Cambridge, UK
Doctoral Programme in Biomedicine
Dr. Carme Junqué i Plaja, Professor at the University of Barcelona, and Dr. Adrian M. Owen, Senior scientist/Assistant director at the MRC Cognition and Brain Sciences Unit (UK),
CERTIFY that they have supervised and guided the PhD thesis entitled “The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities”, presented by Davinia Fernández-Espejo. They hereby assert that this thesis fulfils the requirements to be defended for the Degree of Doctor of Philosophy.
Signature, Signature,
Dr. Carme Junqué i Plaja Dr. Adrian M. Owen
University of Barcelona MRC Cognition and Brain Sciences Unit
Barcelona, November 2010
III
The work reported in this thesis was carried out at the Neuropsychology Group, Department of Psychiatry and Clinical Psychobiology, School of Medicine, University of Barcelona, and the Impaired Consciousness Research Group, Wolfson Brain Imaging Centre, Addenbrooke´s Hospital, and the MRC Cognition and Brain Sciences Unit. The groups belong respectively to the Institut d‟Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the University of Cambridge and Medical Research Council.
The present work, as well as the studies that have been included, have been financially supported by the following grants and institutions: pre-doctoral fellowship (Formación de Profesorado Universitario [FPU]) from the Spanish Ministry for Education (AP2006-00862) to Davinia Fernández-Espejo; SAF2007-66077 from the Spanish Ministry of Science and Innovation and 2009SGR0941 from the Generalitat de Catalunya to Carme Junqué; National Institute for Health Research, Biomedical Research Centre at Cambridge, UK Department of Health Technology Platform, in addition to grants from the U.K. Medical Research Council (U.1055.01.002.00007.01) and the James S. McDonnell Foundation to Adrian Owen.
IV
Agradecimientos
Acknowledgements
Gracias a todos los que, de manera directa o indirecta y sabiéndolo o no, habéis hecho posible esta tesis. En especial:
A mis directores de tesis, la Dra. Junqué y el Dr. Owen:
Carme, por darme la oportunidad de formar parte de este grupo, por compartir conmigo tu pasión por la ciencia, guiarme y apoyarme durante estos años y, sobre todo, por haber hecho que me sienta querida y valorada en todo momento y que sepa que la puerta siempre estará abierta. ¡Moltes gràcies!
Adrian, thanks for all the doors you´ve opened for me, all your help, support and for that ‘magic’ ability you have for finding always the perfect way out of any problem I´ve had. Also for trusting me to be part of the Canadian adventure!
A todos los miembros del Departamento de Psiquiatría y Psicobiología Clínica. En especial al Dr. Pere Vendrell, el Dr. David Bartrés-Faz y Pilar Bouzas. Pere, gracias por hacer que todo esto funcione, estar siempre dispuesto a ayudar, tu capacidad de crítica constructiva y por esas conversaciones en las que intentamos arreglar el mundo de las que tanto he disfrutado. David, por haber tenido siempre palabras de ánimo y porque ha sido un placer colaborar contigo. Pilar, gracias por tu ayuda y por contagiar serenidad en los momentos de ‘crisis de papeleo’.
Y, por supuesto, gracias a todos los compañeros del Grupo de Neuropsicología, sin excepción, a los que ya os habéis ido, los que os acabáis de incorporar, los que nos habéis acompañado sólo unos meses y, sobre todo, a aquellos con los que he compartido cada día. Porque, aunque suene a tópico, entre todos habéis convertido este grupo en una gran familia con la que disfrutar de las alegrías y compartir frustraciones y confidencias. Porque gracias a vosotros estos cuatro años han sido una experiencia inolvidable y porque gracias a vuestro apoyo, aún en los momentos más duros siempre me he levantado cada mañana con ganas de venir al labo. En especial a: Giusi, Naro y Sara, mis tres doctoras favoritas, Eva y Leyre, los dos soletes del labo, Benji, mi compi en los primeros pasos, Blanca, Bárbara, Cris Sánchez, Bea, Eider, Roser, Dídac, Joana, Cleo, Silvia, Julia, Nuria Pujol, Dani, Cris Solé, Xavi y Hugo. Pero sobre todo a los que, de vosotros, habéis cruzado las puertas del labo y pasado a formar parte de mi vida como amigos. Mi niña italiana, mis compis del norte y mis catalanas favoritas, espero que sepáis lo importantes que sois para mí. Gracias por TODO!
Al Institut de Neurorehabilitació Guttmann, en especial a la Dra. Montse Bernabeu y la Dra. Teresa Roig por vuestro trabajo en el proyecto, vuestra colaboración, disponibilidad y amabilidad. A Rocío, la Dra. Sánchez-
V
Carrión, con la que compartí mis inicios en la neuroimagen. También a Marilina y a Gisela, las siempre agradables voces al otro lado. Y por supuesto a todas las familias de los pacientes.
A la Dra. Neus Fábregas y a Eva Rivas del Servicio de Anestesiología y Reanimación del Hospital Clínic.
A la Dra. Maria Antònia Poca y a Eli, del Servicio de Neurocirugía del Hospital Vall d´Hebron.
A todos los profesionales del Centre de Diagnostic per la Imatge, en especial la Dra. Núria Bargalló, César, Santi, Manel y Alicia. Y por supuesto al Dr. Carles Falcón.
To the Impaired Consciousness Research Group, the people at the HSB and the people at the CBU. In particular thanks to Prof. Pickard and Dr. Coleman, for giving me the opportunity of joining the team and for looking after me in Addenbrooke’s, and to Guy Williams, for all your help. Especial thanks to Damian Cruse, Tristán Bekinschtein, Martin Monti, Beth Parkin, Srivas Chennu, Moos Peeters, Louise Goupil, Jess Grahn and Cristina Nombela: you made the months I spent in Cambridge one of the happiest times of my life, both professional and personally. Thank you, gracias, grazie, dhanyavaad, merci, dankzij! I hope our paths will meet again.... in any Continent ;) Tris, gracias por toda tu ayuda, tu apoyo y tu generosidad. Tampoco me olvido de las chicas Bek: Ali y Luna. Los tres tenéis en mí una amiga para siempre. Marie, thanks for being the perfect housemate and friend! Also to Anne, Richard, Pedro, Paula, Anna.… and all the wonderful people I´ve meet during my months in Cambridge. Damian, thanks for all your help with this thesis but, especially thanks for being always there, for your patience and understanding, for knowing how to listen and for the amazing times we always have together.
A mi gente asturiana, mis amigos de siempre. En especial a Lusi, Maru, Pepe, Nuria y Nano. Gracias por aguantar las ausencias, por cuidar de mí y por hacerme saber que, aunque os tenga lejos, siempre estáis ahí para lo que necesite. A Óscar, porque contigo comencé el camino y me has acompañado una gran parte de él. Gracias por todo lo compartido.
Y el mayor agradecimiento de todos a mi familia, porque vosotros, más que nadie, habéis hecho esto posible. En especial: a mis padres, ejemplo de superación y de lucha. Sin vuestra generosidad, vuestro cariño y apoyo incondicional y vuestra capacidad de sacrificio nunca habría llegado hasta aquí. Gracias por estar ahí y por hacerme sentir cada día orgullosa del lugar del que provengo. A Yoli, mi mejor amiga, mi mayor apoyo y la única persona del mundo capaz de hacerme entrar en razón. A Jaime, el mejor cuñado que nadie podría desear. A la abuela y a Yayina, allá donde esté, por el coraje y por la dulzura infinita. Y a Vera, la sonrisa que hace que cualquier problema, por grande que sea, se desvanezca en un segundo. ¡GRACIAS!
Davinia
VI
A mi familia,
CONTENTS
Page
Foreword XI
Glossary of abbreviations XIII
List of figures XV
List of tables XVI
INTRODUCTION 1
1. Consciousness, awareness and wakefulness 3
1.1. Theoretical framework 3
1.2. Anatomy: structures of wakefulness and awareness 4
2. Disorders of consciousness: definition and clinical aspects 8
2.1. Coma, vegetative state and minimally conscious state 8
2.2. Aetiology 9
2.3. Epidemiology: prevalence and incidence 10
2.4. Prognosis 12
3. Neuropathology: post-mortem findings in DOC patients 15
4. Diagnostic challenges: differential diagnosis, behavioural assessment and misdiagnosis 20
5. Neuroimaging the disorders of consciousness 25
5.1. Functional neuroimaging 25
5.1.1. Activation studies: detecting covert cognitive function 25
5.1.2. Brain function at rest 32
5.2 Structural MRI studies in DOC patients 33
5.3. Electrophysiological studies 35
5.4. Contributions to diagnosis and prognosis 36
APPROACH, OBJECTIVES AND HYPOTHESES 39
1. Approach and general objectives 41
2. Specific objectives and hypotheses 43
METHODS 47
1. Participants 49
IX
2. Clinical and behavioural assessment 51
2.1. Clinical assessment 51
2.2. Behavioural assessment 51
3. Magnetic Resonance Imaging acquisition 52
3.1. Functional MRI 52
3.2. T1-weighted structural MRI 52
3.3. Diffusion tensor imaging 53
4. Neuroimaging techniques 54
4.1. fMRI analysis 54
4.2. DTI analysis 54
4.3. Volume and shape analysis 55
5. Statistical analysis 56
6. Methodological pitfalls 57
RESULTS 59
1. Study I: Cerebral response to speech in the vegetative and minimally conscious states after traumatic brain injury 61
2. Study II: Combination of diffusion tensor and functional magnetic resonance imaging during recovery from the vegetative state 71
3. Study III: Diffusion weighted imaging distinguishes the vegetative state from the minimally conscious state 81
4 Study IV: Reductions of thalamic volume and regional shape changes in the vegetative and the minimally conscious states 91
GENERAL DISCUSSION 99
CONCLUSIONS 111
SUMMARY OF THE THESIS 115
1. Resumen de la tesis (Spanish) 117
2. Resum de la tesi (Catalan) 133
REFERENCES 149
X
Foreword
This thesis, presented to obtain the degree of Doctor of Philosophy from the University of Barcelona, is the result of three studies carried out at the Department of Psychiatry and Clinical Psychobiology, School of Medicine, and one study carried out at the Wolfson Brain Imaging Centre, University of Cambridge and MRC Cognition and Brain Sciences Unit, during a 4-year period. During this period, I have obtained the degree of Master‟s in Neuroscience linked to the Doctoral Programme in Biomedicine, University of Barcelona. The following articles have been published in international journals as a result of the work performed, with a global impact factor (IF) of 13.216 (ISI web of knowledge, Journal Citation Reports 2008 for study I and inferred from 2009 for studies II, III and IV):
Study I:
Fernández-Espejo D, Junqué C, Vendrell P, Bernabeu M, Roig T, Bargalló N, Mercader JM. Cerebral response to speech in vegetative and minimally conscious states after traumatic brain injury. Brain Injury 2008; 22(11):882-90. IF = 1.116 (2008).
Study II:
Fernández-Espejo D, Junque C, Cruse D, Bernabeu M, Roig T, Fabregas N, Rivas E, Mercader JM. Combination of diffusion tensor and functional magnetic resonance imaging during recovery from the vegetative state. BMC Neurology 2010; 10:77. IF = 2.109 (inferred from 2009).
Study III:
Fernández-Espejo D, Bekinschtein T, Monti MM, Pickard JD, Junque C, Coleman MR, Owen AM. Diffusion weighted imaging distinguishes the vegetative state from the minimally conscious state. Neuroimage 2011; 54:103-112. IF = 5.739 (inferred from 2009).
Study IV:
Fernández-Espejo D, Junque C, Bernabeu M, Roig-Rovira T, Vendrell P, Mercader JM. Reductions of thalamic volume and regional shape changes in the vegetative and the minimally conscious states. Journal of Neurotrauma 2010; 27:1187–1193. IF = 4.252 (inferred from 2009).
XI
XII
Glossary of abbreviations
A1: Primary Auditory Cortex FMRIB: Oxford Centre for Functional MRI of the
ARAS: Ascending Reticular Activating System Brain
BA: Brodmann Area FOUR: Full Outline of UnResponsiveness
BOLD: Blood Oxygen Level Dependent FSL: FMRIB Software Library
BPV: Brain Parenchymal Volume GCS: Glasgow Coma Scale
CIDC: Centre for Image Diagnosis Clinic GOS: Glasgow Outcome Scale
CNS: Central Nervous System GPi: Globus Pallidus Interna
15 CRS-R: JFK Coma Recovery Scale Revised H2 O: 15O-radiolabelled water
CSF: Cerebro-spinal Fluid HBI: Hypoxic Brain Injury
CT: Computed Tomography HG: Herschel’s Gyrus
CVA: Cerebrovascular Accident HII: Hypoxic-Ischemic Injury
DAI: Diffuse Axonal Injury IBD: Ischaemic Brain Damage
DBS: Deep-Brain Stimulation IFG: Inferior Frontal Gyrus
DL: Dorsal Lateral Nucleus LCFS: Level of Cognitive Functioning Scale
DM: Dorsomedial Nucleus LDT: Lateral Dorsal Tegmental Nucleus
DMN: Default Mode Network LIS: Locked-in Syndrome
DOC: Disorders of Consciousness LNS: Letter-Number Sequencing
DRS: Disability Rating Scale LP: Lateral Posterior Nucleus
DTI: Diffusion Tensor Imaging MCS: Minimally Conscious State
EEG: Electroencephalography MD: Mean Diffusivity
EPI: Echo-planar Imaging MFG: Middle Frontal Gyrus
ERPs: Event-related Potentials MPFC: Medial Prefrontal Cortex
FA: Fractional Anisotropy MP-RAGE: Magnetization Prepared Rapid
FAST: FMRIB's Automated Segmentation Tool Gradient Echo
FDT: FMRIB’s Diffusion Toolbox MRF: Mesencephalic Reticular Formation
FIRST: FMRIB's Integrated Registration and MRI: Magnetic Resonance Imaging
Segmentation Tool MRS: Magnetic Resonance Spectroscopy
FLAIR: Fluid Attenuated Inversion Recovery MSN: Medium Spiny Neurons fMRI: Functional Magnetic Resonance Imaging MTG: Middle Temporal Gyrus
XIII
NAWM: Normal Appearing White Matter STG: Superior Temporal Gyrus
NMDA: N-methyl-D-aspartic acid STS: Superior Temporal Sulcus
PCC: Posterior Cingulate Cortex TAI: Traumatic Axonal Injury
PET: Positron Emission Tomography TBI: Traumatic Brain Injury
PMP: Per Million Population TE: Echo Time
PPT: Pedunculopontine Nucleus TR: Repetition Time
PVS: Persistent/Permanent Vegetative State TTG: Transverse Temporal Gyrus
RAVLT: Rey’s Auditory Verbal Learning Test UK: United Kingdom rCBF: Regional Cerebral Blood Flow US: United States
ROI: Region of Interest V1: Primary Visual Cortex
S1: Primary Somatosensory Cortex VA: Ventral Anterior Nucleus
S2: Secondary Somatosensory Cortex VL: Ventral Lateral Nucleus
SD: Severely Disabled VP: Ventral Posterior Nucleus
SFG: Superior Frontal Gyrus VPL: Lateral Part of the Ventral Posterior
SIENAX: Structural Image Evaluation, using Nucleus
Normalisation, of Atrophy (single point) VPM: Medial Part of the Ventral Posterior
SMA: Supplementary Motor Area Nucleus
SMART: Sensory Modality Assessment and VS: Vegetative State
Rehabilitation Technique WAIS: Wechsler Adult Intelligence Scale
SNC: Signal-correlated Noise WBIC: Wolfson Brain Imaging Centre
SPM: Statistical Parametric Mapping WHIM: Wessex Head Injury Matrix
XIV
List of Figures
Page
Figure 1. Simplified illustration of the two major components of consciousness 3
Figure 2. Simplified scheme of the Ascending Reticular Activating System 4
Figure 3. Schematic circuit mechanism underlying responses to pharmacological agents and central thalamic deep-brain stimulation in severe brain injuries 6
Figure 4. Intrinsically defined anticorrelated processing networks in the brain 7
Figure 5. Outcome for patients in a persistent vegetative state after a traumatic or non-traumatic origin 12
Figure 6. DAI gradation 16
Figure 7. Examples of neuropathological abnormalities reported for VS patients 17
Figure 8. Schematic representation of the thalamic nuclei and their projections 19
Figure 9. Glasgow Outcome Scale 21
Figure 10. Subscales of the JFK Coma Recovery Scale-Revised 22
Figure 11. Mental imagery in a VS patient 27
Figure 12. Willful modulation of brain activity in DOC patients 27
Figure 13. Default network connectivity correlates with the level of consciousness 33
Figure 14. DTI-derived maps 34
Figure 15. Flowchart of the sampling process 50
XV
Index of Tables
Page
Table 1. Criteria for the diagnosis of VS and MCS 9
Table 2. Most common acute causes of the vegetative state 10
Table 3. Probability of recovery of consciousness and function at 12 months in VS patients 13
Table 4. Neuropathology patterns identified in VS patients 15
Table 5. Neuropathological findings in different DOC groups and aetiologies 17
Table 6. The differential diagnosis of the vegetative state 20
Table 7. SMART hierarchical scale for sensory modalities and their comparison to Rancho levels 23
Table 8. Functional neuroimaging studies in DOC: activation studies 28
Table 9. Brief summary of participants and techniques employed in each study 55
XVI
INTRODUCTION
The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
1. Consciousness, awareness and wakefulness
1.1. Theoretical framework
Consciousness is one of the most problematic and still not-well-understood systems of the human cognition. Despite of the rising tide of interest in the past decade and an extensive scientific literature, defining this term and understanding its nature is still a challenge for the neurosciences. Although, at present, there is no universally accepted definition of human consciousness, in the context of the clinical neurosciences it is typically considered a state of full awareness of the self and the environment (James 1980). It would be a complex system with two major dimensions: arousal or wakefulness (i.e. vigilance or level of consciousness) and awareness (i.e. content of consciousness) (Plum & Posner 1982). Understanding these dimensions is crucial to the concept of the vegetative state (VS), characterized by a dissociation between them. While the exact definition and interpretation depends on the clinical, neuroscientific or philosophical approach of the authors, operational definitions that can be employed at the patient‟s bedside are typically accepted in this field (Laureys et al. 2007). In this sense, wakefulness refers to a state in which the eyes are open and there is a degree of motor arousal, while awareness refers to the ability to have, and the having of, experience of any kind (Royal College of Physicians 2003). Thus, awareness requires wakefulness, but wakefulness can be present without awareness.
Conscious ) Wakefulness
Drowsiness wareness REM A Sleep Light Sleep
Deep Sleep
General of consciousness ( consciousness of
Sleepwalking Anaesthesia Absence seizures
Coma Vegetative State Content
Level of consciousness (Wakefulness)
Figure 1. Simplified illustration of the two major components of consciousness: the level of consciousness (i.e., arousal or wakefulness) and the content of consciousness (i.e., awareness). (Modified from Laureys 2005).
Introduction | 3 PhD Thesis
1.2. Anatomy: structures of wakefulness and awareness
Wakefulness and awareness are functions subserved by different cerebral networks. While the former is an autonomic-vegetative function underpinned by upper brainstem, hypothalamus and specific thalamic nuclei, the latter is based on long rate cortical networks and their reciprocal projections to and from subcortical nuclei.
Arousal-related structures integrate the so-called ascending reticular activating system (ARAS), which regulates the electrophysiological activity of the cerebral cortex. As classically described, this system has its origin in the reticular formation, a specific collection of anatomically and functionally different nuclei located in the region that extends from the pons to the upper midbrain and sends projection to the cerebral cortex through two major pathways: a dorsal pathway that synapses with specific and non-specific thalamic nuclei, and a ventral pathway that reaches the basal forebrain, especially the posterior hypothalamus, before reaching the cortex (Vincent 2000; Parvizi & Damasio 2001; Young 2009).
Recently it has been suggested that this circuitry is more complex involving different nuclei and neurotransmitter systems including the classical reticular nuclei, that send glutamatergic ascending projections to the basal ganglia and the intralaminar thalamic nuclei, which project to cortical regions. Also, the locus coeruleus and rostral raphe complex with their monoaminergic projections to basal ganglia, cortical areas and basal forebrain; the laterodorsal and pedunculopontine tegmental nuclei, which send cholinergic projections to several thalamic nuclei and basal forebrain; and finally, the parabrachial nucleus and the periaqueductal grey matter that project to the intralaminar nuclei, basal forebrain and other brainstem nuclei (Parvizi & Damasio 2001).
Petectum Figure 2. Simplified scheme of the Ascending Ventral pathway Locus coeruleus Dorsal pathway Reticular Activating System. The activating Cerebral cortex nuclei in the brainstem are shown in blue. The ventral pathway, represented by an orange line, involves the hypothalamus (in purple) and the basal forebrain while the dorsal pathway, Pons Basal forebrain represented by a green line, activates the cortex Midbrain via thalamus (in red). The scheme is overlaid on a Medulla T1-weighted image from a healthy subject MRF acquired with a 3-Tesla scanner in the Centre for LDT/PPT Image Diagnosis Clinic. LDT: lateral dorsal tegmental nucleus; PPT: pedunculopontine nucleus; MRF: mesencephalic reticular formation.
4 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
The understanding of the neuronal correlates of awareness is still incomplete although it is generally accepted that the thalamus and its reciprocal connections to the cortex play an essential role in supporting this function. The unique anatomical location of the thalamus, its connectivity and the physiological and anatomical specialization of its nuclei suggest the importance that this structure may have in large-scale cerebral dynamics associated with goal- directed behaviours and consciousness (Schiff 2008).
Based on an extensive review of the literature, Schiff proposed a model to understand the contributions of the central thalamus (defined as the anterior and posterior intralaminar nuclei and the paralaminar portions of related thalamic association nuclei: medial dorsalis, ventral anterior, ventral lateral, and inferior pulvinar) to arousal regulation and consciousness (Schiff 2008). As discussed above, the central thalamus is integrated in the ARAS (Kolmac & Mitrofanis 1999; Parvizi & Damasio 2001; Krout et al. 2002), however it is through its projections to the striatum (Macchi & Bentivoglio 1985; Giménez-Amaya & Scarnati 1999; Jones 2007) and bidirectional connections with the prefrontal, premotor, primary sensory, anterior and posterior parietal and cingulate cortices (Sadikot et al. 1992; Groenewegen & Berendse 1994; Sidibé et al. 2002; Van der Werf et al. 2002) that plays its role in the regulation of awareness (Schiff 2008).
According to Schiff´s model, the central thalamus would contribute to the transition to and maintenance of a state in which the membrane potential of the neurons show a depolarized baseline and therefore a faster firing. Such a state has been identified in vitro in animal studies and reflects broad increases in postsynaptic potentials across large networks connecting cortex, thalamus and basal glanglia (McCormick et al. 2003; Shu et al. 2003; Haider et al. 2007 as discussed in Schiff 2008). The implication of the central thalamus in regulating such a state may allow this structure to act on different networks of sensoriomotor integration and to modulate behaviour and attention through its activation by the frontal executive systems (Schiff 2008).
This model may explain some of the mechanisms involved in a still very few but promising number of reported cases of increase of responsiveness or even recovery of consciousness after pharmacological therapies, using classical dopaminergic agents, as L-Dopa, Bromocriptine and the mixed dopamine agonist/NMDA-channel antagonist Amantadine, (Whyte et al. 2005; McMahon et al. 2009; Vargus-Adams et al. 2010) or the sedative agent Zolpidem (Clauss & Nel 2006; Cohen & Duong 2008; Shames & Ring 2008; Whyte & Myers 2009), and central thalamic deep-brain stimulation (DBS) (Schiff et al. 2007) in patients with a disorder of consciousness (DOC). See Pistoia et al. 2010 for a review and Schiff 2008; 2009 for a comprehensive discussion of those mechanisms.
Introduction | 5 PhD Thesis
Frontal Cortex Parietal / Occipital / Temporal Cortex
Striatum (MSN) DBS Central activation Thalamus (Glutamatergic GPi afferents) Dopamine (L-Dopa, Amantadine) Zolpidem? Inhibition Excitation
Figure 3. Schematic circuit mechanism underlying responses to pharmacological agents and central thalamic deep-brain stimulation in severe brain injuries. DBS: deep-brain stimulation; GPi: globus pallidus interna; MSN: medium spiny neurons. (Modified from Schiff 2008; 2009).
Further insights to the structural basis of awareness have been provided by neuroimaging studies in both healthy volunteers and a variety of altered states of consciousness, which have emphasized the role of the so-called „default mode network‟ (DMN) in the genesis of this function. The DMN encompasses a number of cortical regions (i.e. the precuneus, temporo-parietal junctions and medial prefrontal cortex) that show synchronic low-frequency fluctuations in resting-state conditions and reduce their activity when an individual is engaged in a cognitive task (Gusnard & Raichle 2001). Functional impairments and reduced functional connectivity within this network have been observed in states of altered or reduced consciousness such as sleepwalking, absence seizures, deep sleep and anaesthesia (Bassetti et al. 2000; Maquet 2000; Steriade 2001; Kaisti et al. 2002; Salek-Haddadi et al. 2003). Recently, Vanhaudenhuyse et al. (2010a) have gone one step further demonstrating that the medial regions of the DMN are related only with the awareness of the self whereas a different network comprising fronto-parietal regions explains the awareness of the environment in healthy volunteers. This fronto-parietal network comprises regions routinely activated during goal- directed behaviour, and its activity has been found to anticorrelate with the activity in the DMN (Fox et al. 2005), i.e., the time course in each network fluctuates in an opposite way with respect to the other. It has also been demonstrated that activity in this network facilitates conscious perception of somatonsensory stimuli (Boly et al. 2007).
6 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
Disorders of consciousness provide a unique opportunity for the study of the neural bases of awareness. There is also a growing literature relating these two anticorrelated networks to the consciousness impairment in DOC patients. A detailed discussion of the contributions of those works, however, shall be postponed until the appropriate section in this introduction (see section 5.1.2).
Figure 4. Intrinsically defined anticorrelated processing networks in the brain. Positive nodes are significantly correlated with seed regions involved in focused attention and working memory (task-positive seeds) and significantly anticorrelated with seed regions routinely deactivated during attention demanding cognitive tasks (task-negative seeds). Negative nodes are significantly correlated with task-negative seed regions and significantly anticorrelated with task-positive seed regions. (Left) Lateral and medial views of left hemisphere. (Center) Dorsal view. (Right) Lateral and medial views of right hemisphere. (From Fox et al. 2005).
Introduction | 7 PhD Thesis
2. Disorders of consciousness: definition and clinical aspects
2.1. Coma, vegetative state and minimally conscious state
„Disorders of consciousness‟ typically refer to three clinical conditions: coma, vegetative state and minimally conscious state. Coma describes an acute condition that normally lasts for days to weeks after brain injury, in which the patient has complete failure of the arousal system, with no spontaneous eye opening, and is unable to be awakened by application of vigorous sensory stimulation (Plum & Posner 1982).
The term vegetative state was initially coined by Jennett and Plum in their seminal paper to describe a group of patients who, after suffering severe brain damage, retained wakefulness in the absence of any evidence of awareness of self or environment (Jennett & Plum 1972). A person in VS retains autonomic functions with variable preservation of cranial and spinal reflexes, but exhibits no evidence of sustained, reproducible, purposeful or voluntary behavioural responses to multi-sensory stimulation, no evidence of language comprehension or expression, nor response to command (Royal College of Physicians 1996; 2003; The Multi- Society Task Force on PVS 1994a).
A VS is deemed persistent if it is still present one month after acute injury whereas a diagnosis of permanent VS can only be made three or six months after a non-traumatic brain damage (according to the Multi-Society Task Force on PVS or the Royal College of Physicians criteria respectively) and twelve months after traumatic brain injury (TBI). The ambiguous abbreviation „PVS‟ has caused a frequent confusion between these two terms in spite of the critical difference that lies between them; i.e. permanent implies irreversibility. Such a prediction (that awareness will never recover), based on probabilities and not on an absolute certainty like other clinical diagnoses in medicine, has ethical and legal implications regarding the withdrawal of treatments (Jennett 2002a; 2005). Further discussion on this topic can be found in section 2.4. of this introduction.
Since the definition of VS in 1972 to 2002, patients with severe alterations in consciousness were considered to be either comatose or vegetative state. Nevertheless, a number of reports of patients who did not meet diagnosis criteria for VS but, at the same time, were not considered fully conscious started to appear in the literature. In 2002, the Aspen Neurobehavioral Conference Workgroup reported definition and diagnostic criteria for entry into and emergence from a new clinical condition: the minimally conscious state (MCS). Patients in an MCS demonstrate inconsistent, but reproducible, evidence of awareness of self or environment (Giacino et al. 2002). MCS is a broader category comprising a spectrum of patients with different severities that range from the evidence of visual pursuit to the capacity to
8 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities follow commands. Emergence from MCS is characterized by functional interactive communication or functional use of objects.
Table 1. Criteria for the diagnosis of VS and MCS.
Vegetative State Minimally Conscious State
There must be: One or more of the following: - No evidence of awareness of self or - Following simple commands environment at any time - Intelligible verbalization - No response to visual, auditory, - Purposeful behaviour (i.e. behaviours tactile or noxious stimuli of a kind that occur in contingent relation to suggesting volition stimuli). Examples: - No evidence of language o Vocalization or gestures in comprehension or meaningful response to questions expression o Reaching for objects in the right direction Usually satisfied (but no obligatory)*: o Touching objects accommodating to the size - Cycles of eye closure and eye and shape opening o Visual pursuit or sustained - Hypothalamic and brainstem fixation function sufficiently preserved to ensure the maintenance of respiration and circulation * as, for example, third nerve palsies prevent eye opening or injuries to the chest or medulla can affect respiratory function Criteria for VS are in accordance to the Royal College of Physicians of London guidelines (2003); Criteria for MCS are in accordance to Giacino et al. (2002).
2.2. Aetiology
The most common acute causes of the vegetative state in both adults and children are TBI and hypoxic-ischemic encephalopathy (The Multi-Society Task Force on PVS 1994a). In the United Kingdom (UK) head injury accounts for 50-70% of acute cases, while cardio- respiratory insult is the commonest non-traumatic cause (Jennett 2005). In a recent review of Spanish statistics similar trends have been reported (Alberdi Odriozola et al. 2009). The clinical course after the acute insult usually begins with coma and progresses to a VS, after the resumption of the brainstem and diencephalic functions. However, in a few patients, the VS might occur directly after the insult. According to the extensive review of the literature performed by the Multi-Society Task Force on PVS (1994a), 1-14% of TBI comatose patients and 12% of non-traumatic comatose patients progress to a VS. Some predictors for such a progression are the presence of ventilatory dysfunction, decorticate posturing and extraneural
Introduction | 9 PhD Thesis trauma, in cases of TBI, and impairment of eye opening, abnormal oculocephalic or motor responses and the inability to obey commands at two weeks, in non-traumatic cases. A few patients with metabolic or end-stage neurodegenerative disorders may also develop a DOC although it is rare for them to progress to a true VS (Volicer et al. 1997). Finally, severe congenital malformations of the nervous system in infants and children may also prevent the development of awareness, although diagnosis of the VS in this population poses several problems related to the immaturity of the developing brain and the ongoing influences of development on the potential for reorganization of structure and function (Ashwal 1992).
Table 2. Most common acute causes of the Vegetative State.
Traumatic Non-traumatic
o Motor vehicle accidents o Hypoxic ischemic encephalopathy: - Cardiorespiratory arrest o Gunshot wound or other form of direct - Perinatal asphyxia cerebral injury - Pulmonary disease - Prolonged hypotensive episode o Nonaccidental injury in children - Near-drowning - Suffocation or strangulation o Birth injury Cerebrovascular injury - Cerebral haemorrhage - Cerebral infarction - Subarachnoid haemorrhage
o CNS infection - Bacterial meningitis - Viral meningoencephalitis - Brain abscess
o CNS tumour
o CNS toxins or poisoning
This table includes only the most common disorders that have been reported to cause a persistent vegetative state in each category. CNS: central nervous system. (Modified from the Multi-Society Task Force on PVS 1994a to include only acute aetiologies).
2.3. Epidemiology: prevalence and incidence
Not long ago, survival of patients in a comatose state after severe brain injury was very rare, with just a few cases of recovery with cerebral and cognitive sequelae of varying severity. The advances in life-sustaining technologies associated with resuscitation and intensive care have reduced mortality after severe acute brain damage. However, some of those rescued patients are left with permanent brain damage, causing an increase in the cases of DOC as a result. Precise official statistics of the incidence (number of new cases per year) and prevalence
10 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
(cases extant at any one time) of severe DOC are unavailable, as these syndromes are not recognized by the International Classification of Diseases.
In spite of that, a number of estimations of the frequency of VS cases can be found in the literature. In 1977, Higashi et al. collected data from 189 hospitals in Japan and estimated a prevalence of 25 cases of patients in VS for at least 3 months per million population (PMP). A few years later, a prevalence of 5 PMP (for cases of VS for at least 6 months) which, by extrapolation would mean 7.6 PMP at 3 months, was reported for the Netherlands based on data from 384 hospitals and nursing homes (Jennett 2002b). More recently a decrease of this prevalence up to 2 PMP at one month has been reported based on data collected from 380 nursing homes (Lavrijsen et al. 2005). Other recent European reports include the study of 92 hospital and nursing homes in Vienna, in which a prevalence of 19 PMP has been estimated (Stepan et al. 2004) or that of all hospitals and nursing homes in a specific county in western France (Maine-et-Loire), in which a prevalence of 28 PMP, this time considering both VS and MCS patients, has been reported, consistently with the overall prevalence of 25 PMP estimated for France (Saoût et al. 2010). Finally, prevalence estimations have ranged from 56 to 140 PMP for the United States (US) (The Multi-Society Task Force on PVS 1994a) for VS cases and tenfold greater for MCS cases (Strauss et al. 2000).
With regard to the incidence, the most systematic and reliable data is based on cases of TBI, as non-traumatic cases comprise several causes and are concern of different specialist. Based on follow-up after episodes of acute brain damage, it has been estimated an incidence of 46 PMP and 14 PMP for cases vegetative 1 month after an acute insult in the US and the UK respectively (Jennett 2005). In Spain, the Institut de Neurorehabilitació Guttmann estimated in 2003 an annual incidence of severe TBI leading to severe disability of 20 PMP (Alberdi Odriozola et al. 2009).
All these estimations reported for different countries can be useful to have a general picture of the epidemiology of DOC. Nevertheless, the comparison between different studies is not straightforward as the estimated values can vary from one country to another because of, among others, differences in end-of-life regulations. There is still a need of a serious comprehensive international study to obtain accurate epidemiologic data from this population. Some of the reasons for the lack of such a study could be related to factors as that the diagnostic criteria for the VS and MCS are still not completely accepted in many centres or the wide distribution of these patients in different types of institutions and medical specialities, which are no coordinated between them. In any case, such information could be critical to raise questions related to the sufficiency and adequacy of the services the medical system is providing to give response to the needs of this population.
Introduction | 11 PhD Thesis
2.4. Prognosis
The prognostic data that is currently accepted as a basis for both medical and legal decision making is those reported by the Multi-Society task force on PVS after an extensive review of the outcome of 754 published cases (The Multi-Society Task Force on PVS 1994b). They defined two dimensions of recovery: recovery of consciousness (i.e. awareness of self and environment) and recovery of function (i.e. communication, ability to learn and to perform adaptative tasks, mobility, self-care and participation in recreational or vocational activities).
The probability of recovery depends on the aetiology and on how long the patient has been in the VS. Thus, 52% of adults and 62% of children who are in a VS one month after a severe TBI recover consciousness within 1 year, while only 15% of adults and 13% or children do so after a non-traumatic injury. This probabilities decay down to 35/7% of adults and 56/3% of children for patients in a VS 3 months after traumatic/non-traumatic injury and down to 16/0% and 32/6% after 6 months.
Figure 5. Outcome for patients in a persistent vegetative state after a traumatic or non- traumatic injury. Fifty-two percent of adults and 62% of children who are in a PVS 1 month after a traumatic injury recover consciousness within 1 year. The majority recover within the first 6 months; recovery after 6 months is unusual. In contrast, for patients in a PVS 1 month after a non- traumatic injury, recovery of consciousness is much less frequent (15% of adults and 13% of children) and is extremely unlikely after 3 months. Approximately 5% of patients in a PVS 1 month after injury were lost to follow-up at 12 months. (From the Multi-Society Task Force on PVS 1994b).
12 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
Recovery of function was classified according to the Glasgow Outcome Scale (GOS) that defines five outcome categories: good recovery, moderate disability, severe disability, persistent vegetative state and death (Jennett & Bond 1975). Among adult traumatic patients 28% had severe disability, 17% moderate disability and 7% a good recovery after one year, being these percentages 35, 16 and 11% respectively for children. Among non-traumatic patients 11, 3 and 1% of adults had severe disability, moderate disability or good recovery respectively, being these percentages 7, 0 and 6% respectively for children. Once again, these numbers decay as time goes along (see Table 3).
Table 3. Probability of recovery of consciousness and function at 12 months in VS patients.
Outcome Adults Children Traumatic Non-traumatic Traumatic Non-traumatic injury injury injury injury (n=434) (n=169) (n=106) (n=45) % of patients (99% confidence interval) Patients in PVS for 3 months†
Death 35 (27-43) 46 (31-61) 14 (1-27) 3 (0-11)
PVS 30 (22-38) 47 (32-62) 30 (13-47) 94 (83-100)
Severe disability 19 (12-26) 6 (0-13) 24 (8-40) 3 (0-11)
Moderate disability or 16 (10-22) 1 (0-4) 35 (15-49) 0 good recovery Patients in PVS for 6 months‡
Death 32 (21-43) 28 (12-44) 14 (0-31) 0
PVS 52 (40-64) 72 (56-88) 54 (30-78) 97 (89-100)
Severe disability 12 (4-20) 0 21 (1-41) 3 (0-11)
Moderate disability or 4 (0-9) 0 11 (0-26) 0 good recovery Probability of recovery of consciousness and function at 12 months in adults and children in a persistent vegetative state (PVS) three or six months after traumatic or non-traumatic injury. The numbers given in parentheses refer to the numbers of patients who were in a vegetative state one month after injury. † A total of 218 adults with traumatic injuries, 77 adults with non-traumatic injuries, 50 children with traumatic injuries and 32 children with non-traumatic injuries. ‡ A total of 123 adults with traumatic injuries, 50 adults with non-traumatic injuries, 28 children with traumatic injuries and 30 children with non-traumatic injuries. (From The Multi-Society Task Force on PVS 1994b).
Based on the above detailed probabilities the Multi-Society Task Force on PVS concluded that the VS should not be declared permanent until 12 months after a TBI, and 3
Introduction | 13 PhD Thesis months after non-traumatic injury. They acknowledged that occasional cases of late recovery may occur beyond these limits but they considered them very rare and at best associated with moderate or severe disability. Indeed, a number of well documented cases of late recovery can be found in the scientific literature (Tanhehco & Kaplan 1982; Steinbock 1989; Levin et al. 1991; Andrews 1993; Kriel et al. 1993; Childs & Mercer 1996; Eilander et al. 2005; Lammi et al. 2005; Avesani et al. 2006; Faran et al. 2006; Fins et al. 2007; Katz et al. 2009; Sancisi et al. 2009) but they are still considered exceptional. The criterion of 12 months for TBI cases was endorsed by the Royal Society of Physicians of London 4 years later but they stated that permanence should not be declared until 6 months in non-traumatic cases (Royal College of Physicians 1996). In any case, as commented before, such a declaration indicates only reasonable medical certainty that further recovery is very unlikely.
In a recent study performed with the specific aim to verify frequency of late recovery in long lasting traumatic and non-traumatic VS patients, it has been confirmed that the outcome tends to be better in traumatic cases although there is still a non negligible probability of recovery after non-traumatic injury (Estraneo et al. 2010). Nevertheless, as reported previously, such cases of recovery had severe to extremely severe disability, regardless of the aetiology.
Very few studies have addressed specifically the prognosis in MCS cases. Luauté et al. (2010) reviewed the clinical courses of 12 patients in a VS and 39 in an MCS and assessed the outcome at 2, 3, 4 and 5 years after injury. A third of patients in MCS, but none of the VS patients, regained consciousness, although they remained severely disabled. This result, along with the review of the literature, led the authors to conclude that the potential for a favourable outcome is greater in cases of MCS than in VS patients. Moreover, they suggested that the emergence from MCS after 1 year is not exceptional and can occur even after long periods of time (Fins et al. 2007) and, therefore, the time cut-off beyond which recovery cannot be expected is not easy to establish. In this sense, currently a diagnosis of permanent MCS cannot be supported by the available data.
Making a prognosis of irreversibility raises debates around decisions related to continuing or withdrawing artificial nutrition and hydration from these patients (Andrews 2004). It is generally accepted that patients have the right to refuse any treatment and make choices regarding their lives however, by definition, VS or MCS patients cannot communicate their wills. This information could be sought from living wills or advance directives or speaking with the patient‟s relatives but final decisions to this regard are usually mediated by a court. A deep discussion of the ethical and legal issues related to the judgment of persistence or, in general, the management of VS and MCS patients is beyond the scope of the present thesis but a detailed review of relevant laws, judicial rulings and ethical attitudes in developed countries can be found in Jennett 2005.
14 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
3. Neuropathology: post-mortem findings in DOC patients
The neuropathology of the DOC has been extensively described at post-mortem. Kinney and Samuels (1994) reviewed eleven reports of neuropathological changes in VS patients and concluded that the most common abnormality after TBI was white matter damage secondary to diffuse axonal injury (DAI), while in cases of hypoxic-ischemic encephalopathy it was laminar necrosis in the cerebral cortex, which is diffuse or multifocal and extensive, followed by selective necrosis of the thalamus and, much less commonly, leukoencephalopathy. In summary, they concluded that VS was associated with three main patterns of brain damage: 1) widespread white matter damage, 2) widespread destruction of the cortical ribbon, 3) thalamic
damage.
Kinney &
damage; minimal
changes in the cortex;
damage in the cortex and
rebellar damage
Other lesions Other
present in the brainstem
ischemic
mage mage presented in addition to the
ischemic
Generalized demyelination; damage in the basal
hippocampal and ce
Focal lesions in cortex, caudate, putamen;
hipocampal damage
Secondary diffuse cortical
Caudate Caudate
White and matter cerebellar damage
thalamus and cerebellum
Damage in the hipoocampus, basal ganglia,
brainstem
ganglia, ganglia, thalamus, hippocampus, cerebellum and
and brainstem
Damage in the basal ganglia, thalamus, cerebellum
Haemorrhages
cerebellum
hippocampus, basal ganglia, thalamus and
secondary lesions in the brainstem; damage in the
Secondary
basal and ganglia cerebellum
brainstem; damage in the hippocampus, thalamus,
Secondary
Focal damage in cortex, thalamus and cerebellum
ischemic injury. (Modified from
-
: laminar
ebral cortex
ion ion in white matter;
anterior nuclei
-
Main findings
rrhage.
rolateral nuclei and ventro
lencephalic neurons; severe gliosis
midline nuclei
Extensive bilateral damage with relative sparing of
dorsomedial and anterior nuclei n=1
Extensive bilateral thalamic damage: all nuclei n=2;
vent
Entire Entire involvement of thalamus; sparing of anterior
te
Almost Almost total destruction and disappearance of
necrosis, infartcionmultifocal or both
Substantial injury to cerebral hemispheres
Severe Severe generalized necrosis incer the
hemo
Diffuse, severe bilateral white mater degeneration;
Diffuse Diffuse axonal injury, corpus callosal lesions
Generalized demyelination, corpus callosal lesions
corpus callosal lesions
Hemorrhages/diffuse degenerat
white white matter
Extensive symmetrical necrotic lesions of central
HII
HII
HII
HII
HII
HII
TBI
TBI
TBI
TBI
HHI
Aetiology
1
3
1
8
2
5
3
N
10
45
20
80
1976
1994
et al.
et al.
Reference
.
Kinney
Relkin et Relkin al. 1990
French 1952French
Ingvar et al. 1978
Dougherty et Dougherty al. 1981
Brierley et Brierley al. 1971
Strich 1956Strich
Adams et Adams al. 1982
1977
Peters & Rothemund
Jellinger 1977
Ginsberg
predominantly white matter
le 4. Neuropathology patterns identified in VS patients. VS in identified patterns Neuropathology 4. le
Pattern
Tab Main Main findings summarize those injuries classified by the authors as severe. In the „other lesions‟ column every da main finding is described regardless of the severity. TBI: 1994) Samuels Traumatic Brain Injury; HII: Hypoxic
Pattern 3: predominantly thalamic damage
Pattern 2: predominantly cortical cerebral damage
Pattern 1:
Introduction | 15 PhD Thesis
DAI is a widespread damage to axons throughout the brain hemispheres, corpus callosum, brainstem and cerebellum, accompanied by hemorrhagic foci in the corpus callosum and rostral brainstem, as a result of shear, tensile, and compressive strains caused by rapid acceleration and deceleration of the brain (Adams et al. 1982; Gennarelli et al. 1982). There are three severity grades of DAI: in grade 1 there is axonal damage diffusely throughout the white matter but no focal lesions in the corpus callosum or brainstem; in grade 2 additional focal lesions appear in the corpus callosum; finally, in grade 3 there are focal lesions in the corpus callosum and the dorsolateral quadrant or quadrants of the rostral brainstem (Adams et al. 1989). On the other hand, the pattern of damage in non-traumatic cases reflects a selective vulnerability of the cerebral cortex and thalamus to hypoxia-ischemia probably related to differences in oxygen requirements and in the density of glutamate receptors, an important component of excitotoxic injury (Adams & Duchen 1992; Kinney et al. 1994). In addition to that, the thalamus can also be damaged by transtentorial herniation of the posterior cerebral artery and its branches (Lindenberg 1955; Kinney et al. 1994; Kinney & Samuels 1994).
DAI 1 DAI 2 DAI 3
Figure 6. DAI gradation. DAI 1: lesions in the cortico-subcortical boundary; DAI 2: additional lesions appear in the corpus callosum; DAI 3: additional lesions appear in the mesencephalon. Images from http://www.alzforum.org/res/adh/hyp/tbi.asp
Adams et al. (1999) described post-mortem findings in a series of 35 TBI patients considered to fulfil the criteria defining the VS at the time of their deaths. They found that DAI and thalamic damage were the most common structural abnormalities, followed by ischaemic brain damage in the cortex and abnormalities in the brainstem. In a subsequent study, further 14 patients who had suffered from a hypoxic-ischaemic injury were included (Adams et al. 2000). The most common abnormalities reported for this sub-group were diffuse damage in the neocortex, variable abnormalities in the basal ganglia and cerebellum, severe thalamic damage, and minor abnormalities in the brainstem. Subsequently, Jennett et al. (2001) compared the histopathological findings from that same group with a group of 30 severely disabled patients, including 12 who were considered to fulfil the criteria defining the MCS. The occurrence of severe DAI (grades 2 and 3) and thalamic damage were much less frequent in the MCS group.
16 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
Table 5. Neuropathological findings in different DOC groups and aetiologies.
Reference N Diagnosis Aetiology DAI 2 or 3 IBD Thalamus Brainstem
Adams et 35 VS TBI (25) 71% (15)43% (28) 80% (5) 14% al. 1999 Adams et +14 VS non-TBI (1) 7% (13) 93% (14)100% (8) 57%** al. 2000 (39) Jennett et +30 30 SD TBI (9) 30% (14) 47% (11) 37% (4)13% al. 2001 (65) [12MCS] [(5)42%] [(6)50%] [(6)50%] [(2)17%] All three studies included the group of 35 TBI VS patients first described by Adams et al. 1999. In this table only those findings reported for the new group of patients included in each of the further two studies are summarized. Number of cases are listed in parentheses. Data in square parentheses refer to the subgroup of MCS patients. DAI: diffuse axonal injury; IBD: ischemic brain damage in the cerebral cortex; VS: vegetative state; TBI: traumatic brain injury; SD: severely disabled; MCS: minimally conscious state. ** Minimal abnormalities identified only microscopically.
A B
C D
Figure 7. Examples of neuropathological abnormalities reported for VS patients. A) 52-year male who survived 2 years and 4 months after TBI following a road traffic accident. The corpus callosum is narrowed because of an old focal lesion. There is also an old shrunken infarct in the left middle cerebral arterial territory. There is enlargement of the ventricular system. B) 56-year male who survived 9 months after TBI following a road traffic accident. There is a shrunken cystic lesion in the dorsolateral part of the rostral brainstem. C) 50-year male who survived 1 year after TBI following an assault. There is an old infarct in the boundary zone between the anterior and middle cerebral arterial territories. D) 48-year male who survived 5 months after an episode of cardiac arrest. The cerebral cortex is thin and granular. The thalamus is small and there is enlargement of the ventricular system. (Modified from Adams et al. 2000).
Introduction | 17 PhD Thesis
Post-mortem cortical changes in this population have recently been further examined in a group of TBI patients (Maxwell et al. 2010). The authors reported a greater loss of large pyramidal and non-pyramidal cells correspondent with a more severe score on the Glasgow Outcome Scale within all four cortical areas studied: ventromedial prefrontal (Brodman area [BA] 11), dorsolateral prefrontal (BA 10), anterior cingulate (BA 24a) and motor (BA 4) cortices. The greatest loss of neurons was found in the prefrontal cortex, leading to a particularly reduced thickness of the cortical ribbon of BA 10 and BA 11 in VS patients with and without signs of DAI respectively. Cortical changes may be related to damage in specific thalamic nuclei, which could be the result of post-traumatic transneuronal or retrograde degeneration or neuronal loss due to hypoxic mechanisms. In any case, the relation between DAI, damage in the neocortex and damage to the thalamus in DOC patients is evident.
Maxwell et al. (2004) examined post-mortem the thalamus of 9 healthy volunteers with no history of head injury and 31 TBI patients (9 moderately disabled, 12 severely disabled (SD) and 10 VS). He focused on the ventral posterior nucleus (VP), lateral posterior nucleus (LP), and dorsomedial nucleus (DM) and found that the DM was the most severely affected nuclei, showing a significant neuronal loss already in moderately disabled patients, which was progressively more pronounced in SD and VS patients. Damage in the VP was much less pronounced and only present in severely disabled and VS cases. Finally, LP showed no changes in the patients as compared to healthy subjects. The DM has reciprocal connections with the prefrontal, orbital and temporal cortices, connections with the amygdala and the limbic system, and connections with other thalamic nuclei and associated areas of the parietal and temporal lobe. The VP is both the major site of termination for nerve fibres forming the medial lemniscus/dorsal column pathway, spinothalamic tract and the trigeminal cranial nerve, and the origin of fibres to the primary somatic sensory areas of the cerebral cortex (Maxwell et al. 2004; Jones 2007). In a subsequent study using the same sample, these authors demonstrated that there is also loss of neurons correlating with the clinical severity in the lateral (central lateral, central medial rostral and para-central nuclei), ventral (rhomboid and central medial caudal nuclei), and posterior (centre median and parafascicular nuclei) intralaminar groups (Maxwell et al. 2006). The lateral intralaminar nuclei receive projections from the reticular formation, tectal and other mid-brain components and cerebral cortex, and send projections to the dorsal anterior cingulate, adjacent secondary motor, and posterior cingulate cortices (Van der Werf et al. 2002). The ventral intralaminar nuclei are interconnected with the ventral thalamic nuclei (Van Der Werf et al. 1999) and send projections to primary and secondary motor, gustatory, visceral and somatosensory cortices (Van der Werf et al. 2002). Finally, the posterior intralaminar nuclei project to motor, premotor and prefrontal cortices (Parent & Parent 2005). All together, those findings give further support to hypotheses related to the specific involvement of particular thalamic nuclei in the human consciousness, as the one previously discussed (see section 1.2.).
18 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
Figure 8. Schematic representation of the thalamic nuclei and their projections. A1: primary auditory cortex; DL: dorsal lateral nucleus; LP: lateral posterior nucleus; S1: primary somatosensory cortex; V1: primary visual cortex; VA: ventral anterior nucleus; VL: ventral lateral nucleus; VPM: medial part of the ventral posterior nucleus; VPL: lateral part of the ventral posterior nucleus. (From Granger & Hearn 2007).
Introduction | 19 PhD Thesis
4. Diagnostic challenges: differential diagnosis, behavioural assessment and misdiagnosis
A diagnosis of VS or MCS is currently made on the basis of the patient´s clinical history and careful bedside behavioural assessments. The main purpose of such observations is to determine whether the patient is aware of the self and the environment and is able to interact with it in a purposeful manner. Analyzing the diagnostic criteria above detailed, it can be noticed that the clinical differentiation between VS and MCS is based on the subtle distinction between reflexive and cognitively mediated behaviours. Such a distinction is necessarily inferential because it is not possible for a person to directly assess or experience the conscious life of another person. Thus, clinicians who attempt to diagnose these states can only systematically apply auditory, visual, somesthetic, noxious, or olfactory stimuli and make a judgment as to the level of awareness on the basis of the patient´s responses (Bernat & Rottenberg 2007). In addition to this biological limitation, there are a number of confounding variables that can make even more difficult this diagnostic process (Shiel et al. 2004). For instance, underlying deficits such as motor impairments or communication deficits as aphasia can preclude the assessment of the residual cognitive function of the patient (Majerus et al. 2009). Additionally, some pharmacological agents may be reducing the arousal and hiding potential for certain behaviours.
Table 6. The differential diagnosis of the vegetative state.
Condition VS MCS LIS Coma Brain death Awareness Absent Present Present Absent Absent
Sleep-wake cycle Present Present Present Absent Absent
Response to +/- Present Present (in eyes +/- Absent noxious stimuli only)
GCS E4, M1-4, V1-2 E4, M1-5, V1-4 E4, M1, V1 E1-2, M1-4, V1- E1,M1-3,V1 2 Motor function No purposeful Some consistent Volitional No purposeful None or only movement or inconsistent vertical eye movement reflex spinal verbal or movements or movements purposeful motor eyeblink behaviour preserved Respiratory Typically Typically Typically Variable Absent function preserved preserved preserved
EEG activity Typically slow Insufficient data Typically normal Typically slow Typically absent wave activity wave activity
Cerebral Severely reduced Insufficient data Mildly reduced Moderately to Severely reduced metabolism severely reduced or absent (PET) Prognosis Variable: if Variable Depends on Recovery, VS or Already dead permanent, cause but full death within persistent recovery unlikely weeks vegetative state or death VS: vegetative state; MCS: minimally conscious state; LIS: locked-in syndrome; GCS: Glasgow coma scale (eye opening, motor and visual responses); EEG: electroencephalography; PET: positron emission tomography. (Modified from Royal College of Physicians 2003). 20 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
When trying to make a diagnosis of VS, this syndrome should not be mistaken with other DOC such as coma, a condition characterized by lack of both wakefulness and awareness (Plum & Posner 1982), or brain death, a condition of irreversible coma with absent brainstem reflexes (Ad Hoc Committee of the Harvard Medical School 1968; Conference of Medical Royal Colleges and their Faculties 1976). Additionally, in the differential diagnosis of the VS and MCS it should also be considered the locked-in syndrome (LIS), which is not a disorder of consciousness but can be confound with one if not assessed properly. LIS is a condition of severe anarthria and quadriplegia with intact cognition (Plum & Posner 1982), typically produced by a pontine haemorrhage of infarction (Patterson & Grabois 1986). Patients in a LIS are awake and fully aware but cannot produce any speech or movement apart of vertical gaze.
In 1993, a team of experts from a rehabilitation centre in the US reviewed the diagnoses of 49 patients considered to be in a coma or a VS when referred for inpatient neurorehabilitation. This team found that 18 (37%) of those patients showed behaviour indicative of cognitive responsiveness (Childs et al. 1993). A few years later, in a further study performed in the UK, 17 (43%) out of 40 patients with a referral diagnosis of the VS were found to retain evidence of awareness when assessed appropriately (Andrews et al. 1996). Some of the factors suggested by both teams as possible causes for this high misdiagnosis rates were related to confusions in the terminology used to describe alterations in consciousness in different centres, or the lack of an extended and systematic observation for behavioural evidence of cognitive awareness by qualified personal. As discussed above, the prevalence of the VS is very low and therefore few clinicians have the necessary experience for appropriate assessment and clinical management of these patients (see Gill-Thwaites 2006 for a review of the factors contributing to misdiagnosis). After those works, it has been accepted that classical bedside assessment is not enough to make a diagnosis of VS or MCS, rather that a careful and repeated standardized assessment, reinforced by the involvement of the family and caregivers, are needed before such a diagnose can be made with confiability (Gill-Thwaites 2006; Bernat 2006) .
Eye opening response (E) Motor response (M) Verbal response (V)
• None: 1 • None: 1 • None: 1 • To pain: 2 • Extends to pain: 2 • Incomprehensible sounds: 2 • To sound: 3 • Abnormal flexion to pain: 3 • Inappropriate words: 3 • Spontaneous: 4 • Flexion/withdrawal to pain: 4 • Confused: 4 • Localises pain: 5 • Oriented: 5 • Obeys commands: 6
Figure 9. Glasgow Outcome Scale
The Glasgow Coma Scale (GCS) was the first validated rating scale developed to monitor the state of head injured patients in the intensive care unit (Teasdale & Jennett 1974). The use of this scale is extended in the clinical routine; nevertheless, it is insufficient for the assessment of
Introduction | 21 PhD Thesis
DOC patients because of its rough measurement of awareness and its omission of relevant neurological functions (Bernat 2006).
Over the last decade, especially after the establishment of the diagnostic criteria for the MCS (Giacino et al. 2002), a number of specialized neurobehavioural rating scales have been developed to provide a reliable means of assessing awareness and neurobehavioural functioning in severely brain-injured patients (Majerus et al. 2005). Some of these scales are the Full Outline of UnResponsiveness (FOUR), which has been developed to replace the GCS (Wijdicks et al. 2005) and has demonstrated its utility in the detection of LIS (Giacino et al. 2009); or the Wessex Head Injury Matrix (WHIM), which was developed to assess patients and monitor cognitive recovery after severe head injury (Shiel et al. 2000). However, the most comprehensive multimodal scales validated in VS and MCS patients are the Sensory Modality Assessment and Rehabilitation Technique (SMART) (Gill-Thwaites 1997) and the JFK Coma Recovery Scale-Revised (CRS-R)(Giacino et al. 2004). The SMART assesses eight modalities including the five sensory domains as well as motor function, functional communication and wakefulness/arousal, and scores the behaviour of the patient according to five hierarchical levels. The CRS-R was specifically developed to differentiate between VS and MCS patients and includes six subscales addressing auditory, visual, motor, oromotor, communication and arousal functions. Apart from slight differences related to their respective origins in an English or American tradition in the study of DOC, both scales have been shown to have high validity and reliability for discriminating awareness in VS and MCS (Gill-Thwaites & Munday 2004; Giacino et al. 2004; Schnakers et al. 2006; Schnakers et al. 2008a). Factors related to a higher complexity and the necessity of a specific formal training with the respective accreditation to use the SMART may have led to the current broader use of CRS-R in the scientific literature.
Auditory Function Scale Visual Function Scale Motor Function Scale • 4-Consistent Movement to Command* • 5-Object Recognition* • 6-Functional Object Use† • 3-Reproducible Movement to • 4-Object Localization:Reaching* • 5-Automatic Motor Response* Command* • 3-Visual Pursuit* • 4-Object Manipulation* • 2-Localization to Sound • 2-Fixation* • 3-Localization to Noxious Stimulation* • 1-Auditory Startle • 1-Visual Startle • 2-Abnormal Posturing • 0-None • 0-None • 0-None/Flaccid
Oromotor/Verbal Function Scale Communication Scale Arousal Scale • 3-Intelligible Verbalization* • 3-Oriented† • 3-Attention* • 2-Vocalization/Oral Movement • 2-Functional:Accurate† • 2-Eye Opening without Stimulation • 1-Oral Reflexive Movement • 1-Non-Functional: Intentional* • 1-Eye Opening with Stimulation • 0-None • 0-None • 0-Unarousable
Figure 10. Subscales of the JFK Coma Recovery Scale-Revised (Giacino et al. 2004). *Denotes MCS; †Denotes emergence from MCS.
22 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
Additionally, there are others scales that, although not specifically developed for the assessment of DOC patients, may be useful for categorizing the level of responsiveness in VS and MCS patients. The Rancho Los Amigos Level of Cognitive Functioning Scale (LCFS) (Malkmuss et al. 1979) is a behavioural rating scale that assesses the level of recovery of severely brain-injuried patient classifying their responses into five functional levels that range from the coma to the reintegration into the community. Finally, the Disability Rating Scale (DRS) (Rappaport et al. 1982), also developed to track the evolution of an individual from coma to community after TBI, assesses the eye opening response, communication ability, motor response as well as the cognitive ability for feeding, toileting, grooming and the general level of functioning (physical, mental, emotional or social function).
Table 7. SMART hierarchical scale for sensory modalities and their comparison to Rancho levels.
SMART LCFS
Level Response Level Response
1 No response: to any stimulus I No response: in deep sleep and unresponsive to stimuli 2 Reflex response: reflexive and generalized II Generalized response: reacting responses to stimuli, i.e. startle, flexor or inconsistently and non-purposefully to extensor pattern stimuli 3 Withdrawal response: may, for example, III Localized response: reacts specifically turn head or eyes away or withdraw limbs but inconsistently to stimuli from stimulus 4 Localizing response: may, for example, turn head or move upper limbs towards stimuli 5 Differentiating response: may, for IV Confused, agitated: exhibits bizarre, example, follow visual or auditory nonpurposeful, incoherent or inappropriate commands or use object appropriately behaviours
LCFS levels V (confused, inappropriate, non-agitated), VI (confused, appropriate), VII (automatic, appropriate), VIII (purposeful, appropriate) are not compared in this table as they are beyond the capabilities of VS and MCS patients and, thereby, not included in the SMART assessement. (Modified from Gill- Thwaites & Munday 2004).
Taking into account the current availability of specific diagnostic criteria for both the VS and the MCS, as well as the behavioural scales specifically developed to assess and diagnose this population, it should be expected a decrease in the misdiagnosis rates previously reported. Nevertheless, in a recent work there has been revealed that 41% of the patients who participated in the study and were referred with a diagnosis of the VS showed signs of awareness when re-assessed by an specialist team using the CRS-R (Schnakers et al. 2009). An
Introduction | 23 PhD Thesis accurate diagnosis is crucial for any decision related to daily management, analgesia procedures, end-of-life decision, etc. Results as the one reported in that study suggest that, despite the recent advances on this topic, there is still a need of a real implementation of the adequate scales in the clinical practice and of a better training in their use for the medical specialists in touch with these patients.
24 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
5. Neuroimaging the disorders of consciousness
The critical importance of using the adequate scales in the behavioural assessment of DOC people has been argued in the previous section. Notwithstanding this use, the ultimate diagnostic decision is based on the clinician´s subjective judgement about the patient´s responses. Considering the intrinsic difficulties of such a judgement the diagnostic process may be prone to error even in cases of experienced teams. Of special consideration here may be, for instance, some severe cases of LIS in which even vertical gaze responses can be abolished. Despite the complete maintenance of awareness, behavioural assessment itself in those patients would lead to a diagnosis of VS. As it will be discussed through this section, neuroimaging techniques can shed light on the diagnostic process in providing complementary information that cannot be obtained from a bedside examination.
5.1. Functional neuroimaging
Over the last decade there has been a rapid increase in the number of studies that have applied functional neuroimaging techniques to the study of the DOC. The two main general aims of this line of research typically are to study the residual cognitive capacities of these patients (activation studies) or the characterization of their brain functioning at resting (resting state studies).
5.1.1. Activation studies: detecting covert cognitive function
The first report of cerebral activation to external stimuli in a VS patient dates from the late nineties, when de Jong et al. (1997) used 15O-radiolabelled water positron emission 15 tomography (H2 O-PET) to assess the activation of a post-TBI VS patient while listening to a story told by his mother as compared to non-word sounds. This revealed activation of cingulate, temporal and premotor cortices, possibly reflecting processing of emotional attributes of the mother´s speech. After this study an increasing number of cases of stimulus-related activations in VS and MCS patients have appeared in the literature. A detailed description of the main findings of each study using functional magnetic resonance imaging (fMRI) or PET can be found in Table 8.
15 H2 O-PET uses the radiolabelled isotope oxygen-15 to measure changes in the regional cerebral blood flow (rCBF) to controlled external stimulation. After that first report, a number of early studies using this technique have also reported appropriate responses (i.e. activations similar to those showed by healthy subjects) to different types of visual, auditory or tactile
Introduction | 25 PhD Thesis stimuli in some VS patients (Menon et al. 1998; Owen et al. 2002; 2005a; Kassubek et al. 2003). Those studies were typically based on small samples of patients (usually limited to just one) and the data analyses were performed at a single-subject level with the purpose of identifying whether individual patients retained some degree of residual cognitive function. 15 Other H2 O-PET studies, with larger cohorts of patients, have employed group-level analysis to compare the activation showed by VS patients to those exhibited by healthy volunteers (Laureys et al. 2000a; Laureys et al. 2002; Silva et al. 2010) or MCS patients (Boly et al. 2004; 2008a). All these works have consistently reported activation restricted to primary areas in VS patients (as a group) and extended to higher order associative areas, similar to healthy subjects, in MCS patients to auditory, somatosensory and noxious stimulation. Such findings seemed to contradict those previously reported in single-subject based works. Nevertheless, they only speak about the behaviour of VS and MCS patients as a group, so they cannot be interpreted either as a confirmation of high order responses in every MCS patient nor the lack of them in every VS patient. In any case, they may suggest a trend for a higher associative function in MCS patients as compared to VS. As can be seen in Table 8, in the last few years the field has experienced a shift towards the use of fMRI, due to its increased statistical power, improved temporal and spatial resolution and the non-involvement of radiation (Owen & Coleman 2008). fMRI is a non-invasive technique to assess brain function related to external stimuli, based on the blood-oxygen-level dependent (BOLD) response (Ogawa et al. 1990). Very simplified, when a neural region increases its function so does its metabolism, and thus it receives additional blood flow in order to supply glucose, through a process called hemodynamic response. This results in a change in the local ratio oxyhaemoglobin/deoxyhaemoglobin, which can be detected by specific magnetic resonance imaging (MRI) sequences. Advances in the development of this technique have allowed a progression from simple tasks assessing activation to passive stimulation to more complex active paradigms or even the use of some of them as a communication tool. Initial reports confirmed previous PET studies, demonstrating different levels of activation in both VS and MCS patients to hearing the own name (Staffen et al. 2006; Di et al. 2007; Qin et al. 2010), speech (Moritz et al. 2001; Bekinschtein et al. 2004; 2005; Owen et al. 2005a; 2006; Schiff et al. 2005; Coleman et al. 2007; 2009a), visual (Moritz et al. 2001; Zhu et al. 2009), or tactile stimuli (Moritz et al. 2001; Schiff et al. 2005).
Demonstrating high-order responses in DOC patients has broad implications for diagnosis, management and treatment planning (see Giacino et al. 2006 for an in-depth discussion). Nevertheless, in 2006 Owen et al. went one step forward in demonstrating that some VS patients are able to perform mental imagery tasks in respond to command. The patient showed activation comparable to healthy volunteers when asked to imagine playing tennis or walking through her house. The authors concluded that this demonstrated her ability to
26 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities understand instructions and respond to them, confirming that, although behaviourally VS, she was aware of herself and her environment. Recently, that same paradigm has been applied to a large cohort of VS and MCS patients (Monti et al. 2010). Five out of the fifty-four patients recruited for that study showed appropriate activation in the supplementary motor area or the parahippocampal gyrus in response to the respective command. This confirmed that, although rare, such capacity for a wilful modulation of the brain activity can be preserved in some DOC patients. Moreover, the authors used their paradigm to communicate with one patient in VS by asking him yes/no questions and instructing him to respond by imagining playing tennis or walking through his house. This finding opens the door to a possible future application of techniques as the fMRI to address important clinical questions regarding pain perception, expression of necessities or even end-of-life decisions.
Figure 11. Mental imagery in a VS patient. SMA activity was observed during tennis imagery in the patient and 12 healthy volunteers. Parahippocampal gyrus, posterior parietal lobe and lateral premotor cortex activity was observed during the spatial navigation imagery. (From Owen et al. 2006).
.
Mental Imagery Tasks Communication scans
Figure 12. Willful modulation of brain activity in DOC patients. Five DOC patients showed activation in the motor imagery as compared with spatial imagery tasks (yellow and red) and vice versa (blue and green). On the right an example of two communication scans obtained from one of
those patients as well as a healthy volunteer is shown. (From Monti et al. 2010). Introduction | 27 PhD Thesis
trauma: trauma:
-
scan: scan: 2
-
years. years.
-
was was able to
Outcome
moths moths post
-
months post
-
the normal range at at 15 range months the normal
cognitive cognitive functioning within
Other patient recovered
communicate at 2 communicate
1 patient
Not reported
sensorimotor functions
recovery recovery of many cognitive and
At At 2
disability, and 1 good recovery 1anddisability, good recovery
recovered recovered to a moderate
patients remained in a VS, 2
At At 3
the study
recognise recognise faces 4 months after
Able Able to communicate to
Not reported
in in
order associative
-
; right extriate cortex
Main Findings
ortex ortex and BA40, anterior cingulate
ular gyrus and middle and inferior
), right premotor cortex (BA 6). (BA premotor cortex right ),
left planum temporale (only tested planum temporale (only patient). one left in
speech speech vs. SNC: bilateral superior temporal plane,
SCN vs. rest: bilateral primary auditory cortex;
(only patient).tested onein(only
Right fusiform gyrus; similar to 1 healthy volunteer
Primary visual cortex (only patient).tested one in (only cortex visual Primary
prefrontal cortices.
premotor, posterior parietal, superior temporal and
Functional disconnections between S1 and: S2,
cortices.
VS less than controls in higher
patients; similar than 15 healthy controls. than15healthy patients; similar
Brainstem, Brainstem, contralateral thalamus and S1
right SMA for tactile. for right SMA
frontal frontal gyrus for auditory; bilateral S1 cortex and
temporal/ang
for for visual; bilateral primary auditory, left posterior
Near Near normal activation for the 3 tasks: bilateral V1
cortex (BA24) (BA24) andhippocampus. cortex
association c
Functional disconnection between auditory
activate incontrols. with patients compared activate as
STS STS contralateral to the stimulation failed to
controls.
TTG TTG (BA41), STG (BA 42) similar to 53 healthy
(BA 18/19);dorsal(BA right cerebellum.
Right fusiform gyrus (BA 37)
24
Right MTG (BA 21), anterior cingulate gyrus (BA
word word
-
.
Paradigm
vs. SNCRest vs. vs.
est
Speech
images
Familiar Familiar faces vs. control
stimuli vs. Rest vs. stimuli
Moving coloured visual
Rest
the median nerve) vs.
(electrical (electrical stimulation of
Noxious stimulation
R
Bilateral Bilateral palm scratch vs.
rest
Listening Listening to speech vs.
Blinking light Rest vs.
rest
Auditory Auditory stimulation vs.
images
Familiar Familiar faces vs. control
sound
mother mother vs. non
Familiar Familiar story told by his
Neuroimaging
tion studies tion
MRI
-
-
-
O PET
O PET
O PET
O PET
O PET
15
15
15
15
15
2
2
2
2
2
PET/f
subject level
Single
H
Group level
H
subject level
Single
fMRI
Group level
H
subject level
Single
H
subject level
Single
H
38)
–
11 11 months
Interval
–
4
36 ± 9 days 369 ±
4 days
(range 3 (range
22 15 days ±
4 months
2 months
Diagnosis/Aetiology
encephalomyelitis
VS / 1 TBI; 1 HBI; 1
monoxide intoxication monoxide
overdose; 1 carbon
encephalitis, 2 drug
VS / 3 TBI; 7 HBI; 2
VS/ TBI
VS/ HBI
/Encephalomyelitis
VS
VS/ TBI
N
3
15
1
5
1
1
a
aureys et
Reference
2002
Owen Owen et al.
al. 2002
L
2001
Moritz et al.
al. 2000
Laureys Laureys et
al. 1998
Menon et
al. 1997
de Jong et
Table 8. Functional neuroimaging studies in DOC: activa DOC: in studies neuroimaging 8.Functional Table
28 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
scan: scan:
scan: scan:
ictus: ictus:
-
-
-
gnitive
months: months:
-
months post
months post
months post
-
-
-
year later year
Remain Remain in a VS until death one
Not reported
Remained VSinRemained
At At 13
of independenceof
functioning functioning with partial levels
Acceptable Acceptable level of co
At At 18
feed himself himself feed
was was able to chat normally and
Regained Regained consciousness and
At At 8
environment environment
1 independent in special
1 moderately dependent, 1 moderately
1 died, 2 markedly dependent,
MCS at 6 patients
Not reported
condary
months.
central central gyrus similar
-
-
responses responses to reversed
familiar: familiar: amygdala and insula
-
non
.
familiar familiar voice vs. rest: TTG, STG, planum
-
volunteers.
Bilateral Bilateral MPFC to own name; similar to 3 healthy
to controls
Tactile: Tactile: bilateral S1 and post
narratives as compared as to narratives controls.
volunteers; reduced
Speech: STG, MTG; similar to 7 healthy
hemisphere.
evidence evidence for inferior frontal gyrus in either
Left Left posterior inferior temporal region, but no
gyri. Results very similar at at 13 similar Results very gyri.
low low vs. high: left superior and middle temporal
Speech vs. rest: bilateral superior temporal plane;
areas and frontal areas areas).
after after recovery reaching higher associative temporal
Speech vs. rest: left TTG and STG. (much stronger
spreading to the inferior frontal gyrus. spreadingthe to inferior
Mother vs.
temporale, insula.temporale,
Non
association cortices in MCS than VS. in associationthan MCS cortices
auditory auditory cortex and temporal and prefrontal
Stronger functional connectivity se between Stronger connectivity functional
BA 41 42BA andbilaterally.
healthy healthy volunteers. In VS: activation restricted to
Bilateral Bilateral STG (BA 41, 42, 22) in MCS and 18
stimuli.
gyrus/S1; cingulate gyrus contralateral to the
Bilateral Bilateral posterior insula/S2 and postenctral
–
matched matched
-
sentences
familiar familiar age
-
est
est
names
Own Own name vs. other
narratives
reversed reversed version of the
by by a familiar person vs.
Auditory: Auditory: narratives told
both hands
Tactile: Tactile: light touch of
sentences
High High vs. low ambiguity
Rest
background noise) vs.
(different (different rates of signal
intelligibility intelligibility
High High vs. medium vs. low
R
Speech vs. white noise vs.
voice vs. Rest vs. voice
non
vs. vs. same story told by a
Story Story told by his mother
R
Auditory Auditory clic stimuli vs.
median nerve) vs. rest vs. median nerve)
(electrical (electrical stimulation
Painful stimulation
-
-
-
-
-
-
le
O PET
O PET
O PET
RI
15
15
15
2
2
2
subject level
Sing
fMRI
subject level
Single
fMRI
subject level
Single
fM
subject level
Single
H
subject level
Single
fMRI
subject level
Single
fMRI
Group level
H
Group level
H
–
4
–
10 months
months
18 and 24
13 months)
(Repeated (Repeated at
4 months
2 months
5 months
124)
(range (range 20
53 41 days ±
years
3 months
y arrest y
Laureys et al. Laureys
VS/ HBI
haemorrhagic haemorrhagic
MCS / 1 TBI; 1
VS/ HBI
VS/TBI
MCS / TBI
1 respirator
encephalopathy; encephalopathy;
1 hypertensive 1 hypertensive
1 encephalitis;
5 MCS / 2 TBI; 5 TBI; / 2 MCS
2002)
(
15 VS / described15 VS / in
VS/ HBI
1
2
1
1
1
20
7
3
005
2006
Staffen Staffen et al.
2005
Schiff Schiff et al.
2005a
Owen Owen et al.
et et al. 2
Bekinschtein
et et al. 2004
Bekinschtein
2004
Boly Boly et al.
al. 200 Kassubek Kassubek et
Introduction | 29
PhD Thesis
, 3
injury: injury:
-
scan: scan: 1
-
scan: scan: the 7
scan: scan: the 3
scan: scan: all the
-
-
-
months months post
-
months post
-
4 VS patients showed no
months post
months post
months post
-
-
-
emerged toMCSemerged an
VS who responded to speech
At At 6
became distinctly independent distinctly became
moderately moderately independent
MCS died, 1 became
At At 12
change
other
speech evolved to evolved speech an MCS. The
VS patients who responded to
At At 6
The rest remained remained VS rest The
activity activity evolved to an MCS.
VS who showed high level
MCS remained MCS; The 2
At At 3
visual fixation visual
able to track only to the right;
At At 11.5
nterior cingulate
ponse.
.
healthy volunteers. healthy
rtex, rtex, and the lateral premotor cortex for
d 22.d
and and 2 MCS temporal lobe activations to
R) at 6 months but not at present. not at present. but R) at 6 months
-
(CRS
fMRI fMRI response correlated with clinical profile
13 VS and 3 MCS noMCS13 VS3 and res
semantic ambiguity semantic
From From those: 2 VS and 2 MCS responses to
7 VS and 12 MCS responses to sound and speech.
only.
2 VS and 4 MCS significant responses to sound
areas was preserved in VS.inaltered MCS areas was and
Functional connectivity between S1 and associative
cortices thanVS. cortices
cortex, cortex, posterior parietal and dorsolateral prefrontal
activation activation in S1, S2, insula, a
deactivation than 15 healthy volunteers. Greater
MCS: similar patterns of activation and
4 VS and 3 MCS no activation to sound.toVS3 no 4activation MCS and
activation to ambiguity contrast. activation to ambiguity
sound and speech. In 2 VS and 1 MCS appropriate
3 VS
all all three contrasts.
SD: similar than reported for healthy volunteers in
BA 21 BA an
VS: 2 none; 3 primary only auditory; 2 primary and
22).
higher order associative auditory cortices (BA 21,
MCS: primary auditory cortex (HG and 41 42), and
observed 34in
spatial spatial navigation. Indistinguishable from those
parietal parietal co
SMA SMA for tennis; parahippocampal gyrus, posterior
(Rodd et al. 2005)(Rodd et al.
equivalent to those observed in healthy volunteers
ambiguity: ambiguity: left inferior frontal region. Both
Speech: bilateral MTG and STG; High vs low
sentences vs. SNC vs. rest SNCrest vs. sentences
High High vs. low ambiguity
Rest
the median nerve) vs.
(electrical (electrical stimulation of
Noxious stimulation
Rest
sentences sentences vs. SNC vs.
High High vs. low ambiguity
voice vs. Rest vs. voice
Own Own name by familiar
spatial spatial Rest navigation vs.
playing playing tennis vs. rest;
Mental imagery tasks:
Rest
sentences sentences vs. SNC vs.
High High vs. low ambiguity
-
-
-
-
O PET
15
ngle
2
MRI
subject level
Single
f
Group level
H
subject level
Single
fMRI
subject level
Single
fMRI
subject level
Si
fMRI
–
48)
122)
122)
–
-
-
(range 2 (range
18±26 months
116)
(range (range 37
57 33 days ±
(range 2 (range
27±39 months
(range 2 (range
10±14 months
5 months
phalitis
m m stroke; 1
stroke
HBI; 2 midbrain
19 MCS / 15 TBI; 2
stroke
stroke; stroke; 1 midbrain
HBI; 1 brainstem
22 VS / 11 TBI; 9
TBI; 1 TBI; ence
5 MCS / 2 HBI; 2
Laureys et al. (2002)Laureys
15 VS / described in
2 TBI SD /
brainstem stroke brainstem
5 MCS / 3 TBI; 2
midbrain stroke
1 brainste
7 VS / 3 HBI; 2 TBI;
5 MCS / 55 / TBI MCS
7 VS / 5 TBI; 2VS TBI; 7 HBI 5 /
VS/ TBI
41
20
14
12
1
al. 2009a
Coleman Coleman et
2008a
Boly Boly et al.
al. 2007
Coleman Coleman et
2007
Di et al.
2006
Owen Owen et al.
30 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
lly lly
scan: scan: The 2
-
post
months
-
MCS
cingulate cingulate cortex evolved to an
activation activation in the anterior
VS patients who showed
At At 3
Not reported
Not reported
Not reported
Not reported
lateral
disabled; MFG: middle frontal gyrus; SMA: supplementarydisabled; middleSMA: MFG: motor frontal gyrus;
related related names.
-
stimulating; stimulating; 3 MCS showed
-
stimulating.
R score at the time of fMRI and 3
-
olunteers.
-
three three regions identified in a group of
ndings‟ column). ndings‟
central central gyrus, cingulate and medial
-
frontal, prefrontal cortex in 6 MCS; similar
-
related and self non
-
onal onal MRI; HBI: hypoxic brain injury; IFG: inferior frontal gyrus; MCS: minima
months later.
with with the CRS
Changes Changes in the anterior cingulate cortex correlated
self
17 healthy volunteers in a paradigm contrasting
in one of the
All All the MCS and 6 VS patients showed activation
in patients.
Functional disconnection between these two areas
contrary contrary to a group of 10 healthy volunteers.
no activation in the inferior parietal lobule (BA40),
Contralateral (left) primary sensorimotor cortex but
conditions.
yes yes / no questions using the two mental imagery
One of the patients accurately answered 5 out of 6
gyrus for spatial tennis.navigation for vs. gyrus
tennis vs. spatial navigation, parahippocampal
wilfully wilfully modulate their brain activity: SMA for
4 VS and 1 MCS patients (all TBI) were able to
volunteers.
cerebellum; cerebellum; thalamus. Similar to 12 healthy
inferior inferior parietal lobule; MTG, planum temporale;
frontal frontal gyri; right supramarginal gyri, bi
IFG, IFG, post
Target Target detection vs. passive listening: SFG, MFG,
that medium to
family family pictures to high
Stimulating pictures:similarStimulating 2than MCS to showed
to 15 healthy v healthy to 15
orbito
Family Family pictures: occipital, parietal, temporal,
est
R
pe of pe of imagery mental
ght index finger vs. Rest vs. finger ght index
familiar rest vs. voice familiar
Own Own name presented by a
ri
Passive Passive extension of the
ty
questions by usingquestions one by
answer answer to yes/no
Communication task:
spatial spatial rest navigation vs.
playing playing tennis vs. rest;
Mental imagery tasks:
listening words to
words) words) vs. passive
Target Target detection (listened
vs. vs.
vs. vs. rest; family pictures
stimulating stimulating new pictures
High High and medium
-
-
-
O PET
15
2
STS: STS: superior temporal sulcus; TBI: traumatic brain injury; TTG: transverse temporal gyrus; SFG: superior frontal gyrus; VS:
fMRI
Group level
H
subject level
Single
fMRI
subject level
Single
fMRI
subject level
Single
fMRI
–
48)
22)
-
-
2 months
revised; revised; CVA: cerebrovascular accident; fMRI: functi
-
-
(range 2 (range
101 month ±
(range 2 (range
7 months ± 6
309)
(range (range 0.4
months
22 ± 45
Not reported
1
is
4 / TBI MCS
7 VS HBI 7 52 TBI; /
VS / 6 HBI; 4 HBI VS HBI; / 4 6
brainstem stroke brainstem
HBI; 2 meningitis, 1
31 MCS / 20 TBI; 8
meningit
HBI; 2 CVA; 1
23 VS / 12 TBI; 8
MCS / HBI
haemorrhagic
MCS / 7 TBI; 2
R: R: coma recovery scale
-
11
10
54
1
9
correlated correlated noise; STG: superior temporal gyrus;
-
2010
Qin et al.
2010
Silva Silva et al.
2010
Monti et al.
2009
Monti et al.
2009
Zhu et al.
N refers only to the number of patients (healthy volunteers‟ results, if included in the study, are described in the „main fi „main the in are described study, the in included if results, volunteers‟ (healthy of thepatients refers N number to only BA: Brodmann area; CRS medialconsciousMPFC: state; tomography; MTG: cortex; severelymiddleSD: prefrontal temporal emission gyrus; positron PET: area; SNC: signal vegetative state Introduction | 31 PhD Thesis
5.1.2. Brain function at rest
Early PET studies, including some of those discussed above, used fluorine-18 (18F) labeled deoxyglucose, to assess the global cerebral metabolism of DOC patients at rest. They consistently reported a severe cerebral metabolic dysfunction (40-50% of normal values) in VS patients (Levy et al. 1987; DeVolder et al. 1990; Tommasino et al. 1995; Laureys et al. 1999; 2002; Rudolf et al. 1999; Schiff et al. 2002; Kassubek et al. 2003) . More interestingly, when regional metabolism was assessed, a more prominent metabolic dysfunction was identified in areas related to the default mode network (Laureys et al. 1999; Kassubek et al. 2003). Recently, metabolic impairment in the precuneus, a critical node in the DMN (Greicius et al. 2003) has been related to a functional disconnection between this network and the ARAS in VS patients, producing an abnormal hyperactivation of the arousal system at rest in these patients (Silva et al. 2010). On the other hand, hypometabolism in anterior regions of this network and the thalamus have also been related to the degree of consciousness impairment in TBI patients (Nakayama et al. 2006; Lull et al. 2010). Moreover, Laureys et al. identified functional cortico- cortical and thalamo-cortical disconnections within this network, which were restored in one patient when he regained consciousness (Laureys et al. 2000b).
Those findings have been recently extended by three studies using resting-state fMRI. Cauda et al. (2009) reported a marked dysfunction in the DMN with decreased connectivity in several brain regions, including the dorsolateral prefrontal cortex and anterior cingulate cortex, especially in the right hemisphere, for 3 VS patients as compared with a group of healthy volunteers. Furthermore, the impairment in this network seemed to be related with the clinical severity, although this could not be statistically demonstrated because of the small sample size. This qualitative observation was reinforced by Vanhaudenhuyse et al. (2010b), who demonstrated in a group of 14 DOC patients that the connectivity in all the DMN-related areas negatively correlates with the degree of clinical consciousness impairment, ranging from healthy controls and LIS to MCS, VS and finally, coma patients. Although VS patients as a group were found to show a marked connectivity reduction as compared to both MCS patients and healthy volunteers, some of them may retain connectivity in this network (Boly et al. 2009). Nervertheless, although cortico-cortical connectivity was preserved in this VS patient he demonstrated absent thalamo-cortical connectivity, stressing the importance of the thalami in awareness.
Resting state fMRI presents a number of advantages as compared to fMRI paradigms: it is easier to acquire and, as a result, could be more available in routine clinical settings. However, the interpretation of dysfunction or disconnections in resting state networks is still controversial (see Boly et al. 2008b for a review). The authors of the above detailed works suggested that the DMN may have two layers: one that would persist independently of the level
32 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities of consciousness and possible related to underlying anatomical connectivity, and the other being more related to conscious cognitive processes (Vanhaudenhuyse et al. 2010b). However, further studies in this population are needed to clarify this issue.
Figure 13. Default network connectivity correlates with the level of consciousness. (A) Areas showing a linear correlation between default network connectivity and consciousness. Results are thresholded for display at uncorrected P<0.05 and rendered on the mean T1 structural image of the patients. (B) Mean Z-scores and 90% confidence interval for default network connectivity in PCC/precuneus, temporo-parietal junction, medial prefrontal cortex and parahippocampal gyrus across patient populations. Locked-in syndrome patient Z-scores are displayed for illustrative purposes as an additional red circles overlaid on control population data. (From Vanhaudenhuyse et al. 2010b).
5.2 Structural MRI studies in DOC patients
Contrary to the rapid progress in functional studies, and in spite of the fact that post- mortem studies set the grounds for a neuropathological characterization of the DOC, structural studies in this population still very rare. An early MRI report confirmed previous neuropathological findings demonstrating that grades 2 and 3 of DAI are the most common damage in post-traumatic VS patients (Kampfl, et al. 1998a). These authors also identified lesions of basal ganglia, thalamus and parahippocampal gyrus as well as cortical contusions (usually in frontal and temporal lobes) in half of the patients studied. In a subsequent study, they demonstrated that focal lesions in the corpus callosum and the dorsolateral brainstem were predictors of non-recovery in VS patients (Kampfl et al. 1998b). A number of studies with severe TBI patients have reinforced this prediction (see Tshibanda et al. 2010 for a review). MRI acquisitions in acute settings usually included T1-weighted, fluid attenuated inversion recovery (FLAIR), T2-weighted, and T2* acquisitions. The number of lesions detected by those Introduction | 33 PhD Thesis sequences correlated with the GOS in a sample of acute TBI patients (Carpentier et al. 2006). However, only when combined with brainstem magnetic resonance spectroscopy (MRS), a non- invasive technique that provides appreciation of metabolic functions, morphologic measures were able to classify the patients in their GOS category. MRS has previously been able to detect thalamic brain damage in VS patients (Uzan 2003). Despite those encouraging findings, to date, no conventional structural study has been able to identify a clear diagnostic biomarker in this population.
Diffusion Tensor Imaging (DTI) is an emerging technique that is able to characterize brain tissue microstructure through the observation of molecular movement of water, being sensitive to changes that may not be observable with standard MRI sequences (Le Bihan et al. 2001). In an isotropic medium such as the cerebro-spinal fluid (CSF) water molecules move equally in all directions. In white matter, on the contrary, axon bundles restrict this free movement and the water diffuses faster along fibre tracts. DTI allows the computation of indices as the mean diffusivity (MD) or fractional anisotropy (FA), as well as the reconstruction of white matter pathways by means of tractography algorithms. MD measures the overall mean displacements of molecules and reflects the overall presence of obstacles to diffusion. FA provides information of the degree of directionality of water diffusion. In cases of severe TBI, DTI has been identified as providing what may be clinically relevant biomarkers that may serve a prognostic role, as well as a tool for tracking the changes in neural tissue which accompany recovery (Sidaros et al. 2008). With this purpose, DTI has been applied to an exceptional case of recovery of consciousness and expressive language in a patient who was in an MCS for 19 years (Voss et al. 2006). The authors reported an increase in FA values that correlated with a higher metabolic function in a medial posterior region, encompassing cuneus and precuneus. Those values returned to normal range 18 months later and were interpreted as signs of axonal regrowth.
Figure 14. DTI-derived maps. Colours in the colour map and tractographic reconstruction of the fibres represent the principal direction of the tensor: red for left-right, blue for superior-inferior, and green for anterior-posterior. FA, MD and colour maps have been obtained from a healthy volunteer who participated in our study. Tractography figure from: http://www.martinos.org/neurorecovery/technology.htm
34 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
A recent study of a group of acute TBI patients with a DOC has highlighted the potential of DTI for predicting outcome (Tollard et al. 2009). These authors observed that FA decreases in a number of supratentorial and infratentorial regions correlated with an unfavourable outcome. Combining DTI with MRS they were able to predict the outcome with 86% sensitivity and 97% specificity. Similar results have been reported for FA in the inferior longitudinal fasciculus, cerebral peduncule, internal capsule and corpus callosum (Perlbarg et al. 2009). Recently, Newcombe et al. (2010) applied DTI to a sample of 12 VS patients in order to characterize the differences between TBI and hypoxic-ischemic aetiologies. While these two groups or patients were found to share a similar pattern of abnormalities in supratentorial regions, they identified abnormalities in the brainstem only in the TBI group.
5.3. Electrophysiological studies
There is also an extensive electroencephalography (EEG) literature in patients with disorders of consciousness both in resting state conditions and in cognitive tasks. However, such approach is not directly related to the neuroimaging techniques employed in the studies included in the present thesis so only the main contributions of those works will be summarized here. A more extensive review of those studies can be found in (Cruse & Owen 2010).
DOC patients usually undergo EEG recordings as part of their clinical assessment in the acute period. However, it has been recently demonstrated that abnormal EEG patterns in these patients are variable and nonspecific, and thus cannot be used as a diagnostic tool (Kulkarni et al. 2007). The EEG bispectral index, a variable statistically and automatically derived from the EEG as a composite measure of its frequency content, on the contrary, has demonstrated its utility on disentangling the MCS from the VS, as well as on estimating the outcome (Schnakers et al. 2008b).
A number of studies have also investigated the utility of event-related potentials (ERPs) in detecting covert cognitive functions. They have demonstrated responses to hearing the mother´s voice (Machado et al. 2007), the own name (Qin et al. 2008; Fischer et al. 2010) or prosody violations (Kotchoubey et al. 2009) among others. Some studies have also moved towards the use of active paradigms in order to demonstrate awareness in DOC patients. For example, Schnakers, et al. (2008c) demonstrated that some MCS patients were able to follow instructions and being engaged in a task of target detection. Bekinschtein et al. (2009a), demonstrated that DOC patients are able to learn associations, showing anticipatory electromyographic responses to aversive conditioning stimuli. Furthermore, the amount of learning was a good indicator of further recovery. Finally, Bekinschtein et al. (2009b) Introduction | 35 PhD Thesis demonstrated that some MCS patients can respond to violations of complex temporal regularities that were related to attention and awareness in healthy volunteers. Interestingly, these responses were associated with a subsequent recovery of consciousness in those patients who show them, suggesting their possible prognostic value. In this sense, in regards to prognosis, in general, EEG studies have suggested that early evoked responses to sensory and auditory stimuli can successfully predict a negative outcome from acute comatose or vegetative states (Frank et al. 1985; Fischer et al. 2006), while late ERPs (mismatch negativity and P300) predict awakening (Kotchoubey et al. 2005; Daltrozzo et al. 2007).
5.4. Contributions to diagnosis and prognosis
The implications of some the above detailed neuroimaging findings for both diagnosis and prognosis have been extensively discussed in the literature (see, for example, Giacino et al. 2006 or Owen & Coleman 2008). In addition of ethical reasons, disentangling the MCS from the VS has critical implications for daily management of these patients. For instance, it has been demonstrated that patients in MCS show appropriate brain responses to painful stimuli, suggesting that they may perceive pain and thus they would need analgesic treatment (Boly et al. 2008a). Moreover, prognosis is much favourable for MCS than for VS patients (Giacino et al. 1997; Luauté et al. 2010) and thereby an accurate diagnosis should precede any clinical decision based on the expectance of a positive or negative outcome.
One of the diagnostic criteria for the VS is the absence of language comprehension or expression. Thus, demonstrating preserved language comprehension by means of an fMRI in a VS patient should invalidate such a diagnosis. Nevertheless, it could be argued that the presence of brain activation in passive paradigms cannot be so straightforward interpreted as a confirmation of the preservation of the cognition function those cerebral networks subserve. However, is when we speak about active paradigms when implications for diagnosis became more clear. As detailed previously in this introduction, diagnosis of DOC is currently based on behavioural assessment. One of the key behaviours that support a diagnosis of MCS is the command following. Patients as those reported by Owen et al. (2006) or Monti et al. (2010) demonstrated that were able to follow commands by modulating their cerebral activity so, although behaviourally VS, they cannot be considered as unaware.
Regarding to the prognosis, a recent review of 15 PET and fMRI studies including a total of 48 VS patients has shown that activation in high-order associative areas is able to predict good recovery with a 93% specificity and 69% sensitivity (Di et al. 2008). Indeed, in one of the studies including the largest cohorts of DOC patients to date this predictive value has 36 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities been reinforced, in demonstrating that the cerebral response exhibited by the patients were no correlated with their present clinical status but with that showed 6 months after the scan (Coleman et al. 2009a).
Further studies with large cohorts of patients are needed to clarify whether neuroimaging techniques could be employed in the clinical routine to help in diagnostic procedures and prognostic estimations with DOC patients. However, there is increasing accumulation of evidence in the literature that suggest that this could be a not-so-distant possibility.
Introduction | 37
APPROACH, OBJECTIVES AND HYPOTHESES
The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
1. Approach and general objectives
Although there has been a shift in the fMRI literature with DOC patients towards active paradigms with the objective of determining whether a patient, despite of being unable to show behavioural evidence of awareness, is indeed conscious, it is unlikely that all patients with DOC may benefit from these paradigms. Actually, only 5 out of the 54 patients included in (Monti et al. 2010) study were able to wilfully modulate their brain activity in response to the task. Patients in the lower end of the severity spectrum will not be able to engage in active paradigms but they could show evidence of residual brain functioning in more simple passive paradigms. For that reason, the authors of those works argued that fMRI studies in DOC patients should be conducted hierarchically, beginning with the simplest form of processing in a particular domain and then progressing through more complex cognitive functions (Owen et al. 2005b; Owen & Coleman 2008). Furthermore, even in those cases in which the patient demonstrates awareness, it could be still interesting to explore the functionality of different cognitive domains in order to systematically characterize the full range of preserved cognitive functions. Language is one of the central functions of the human cognition and identifying residual linguistic function in a DOC patient may have a critical impact on the motivation of relatives and caregivers. At the inception of the work that led to the present thesis there was only one group-study that specifically explored the residual speech-processing function in a sample of VS and MCS patients (Coleman et al. 2007).
It has been demonstrated that the presence of high-order level responses to speech in VS patients is a predictor for a positive outcome (Coleman et al. 2009a). However, little is known about the neural changes associated with those cases of good recovery from the VS. In a seminal PET study, a restoration of previously impaired functional cortico-thalamic disconnections was found to accompany the recovery of consciousness in a VS patient (Laureys et al. 2000b). Before the second study included in the present thesis there were only two additional published longitudinal studies of recovery from VS and MCS respectively (Bekinschtein et al. 2005; Voss et al. 2006). These studies have provided insights into the mechanisms involved in such recovery. However, they either focused on just functional data, or contrasted a number of scans performed after recovery.
As discussed in the introduction of this thesis, the clinical differential diagnosis of the DOC is based on behavioural differences, sometimes very difficult to assess. The intrinsic difficulties related to the clinical assessment of this group of patients have led to wrong diagnoses of VS in a remarkable number of patients who indeed retained awareness (Childs et al. 1993; Andrews et al. 1996; Schnakers et al. 2009). Despite the growing emergence of neuroimaging studies in DOC patients, no structural or functional work has been able to identify a clear biomarker that could contribute to the diagnostic process in this patient group, helping to
Approach, objectives and hypotheses | 41 PhD Thesis reduce the high misdiagnosis rates. Neuropathological studies have identified grades 2 and 3 of DAI, and necrosis in the neocortex as the most common abnormalities present in traumatic and non-traumatic VS patients respectively, as well as thalamic damage in both aetiologies (Kinney & Samuels 1994; Adams et al. 1999; Adams et al. 2000). Furthermore, in traumatic cases, grades 2 and 3 of DAI and abnormalities in the thalamus were found to be less common in MCS than in VS patients (Jennett et al. 2001). The former findings were subsequently confirmed in- vivo using MRI (Kampfl et al. 1998a; Uzan 2003; Newcombe et al. 2010). Nevertheless, the only structural MRI studies including both VS and MCS patients have focused on the application of this technique to predict the outcome and classified these patients within the same GOS category (Carpentier et al. 2006; Perlbarg et al. 2009; Tollard et al. 2009), thus precluding any attempt to identify differences between the two subgroups of DOC patients.
In order to provide further insights to the above posed questions we developed a number of studies with the general aim to examine the neuroanatomical and neurofunctional bases of the impairment of consciousness in VS and MCS patients. Firstly, we focused on the assessment of brain responses to speech in a group of VS and MCS patients by means of fMRI (study I). Secondly, following claims related to the need of a multimodal assessment to accurately characterize DOC patients (Coleman et al. 2009b), we combined fMRI, DTI and clinical and neuropsychological assessment to study the cerebral and clinical changes of a VS patient from the time of VS through to recovery (study II). Finally, we applied DTI (study III) and high- resolution T1-weighted structural MRI (study IV) to in-vivo characterize the neuropathological patterns of VS and MCS patients and explore possible differences between these two groups of patients. In the third study we focused on the subcortical white matter, brainstem and thalami, following post-mortem works. Based on the results obtained in that study, in the fourth study we focused on the thalamus and its global and regional atrophy.
42 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
2. Specific objectives and hypotheses
Specific objectives and hypotheses were as follows:
- Study I: Cerebral response to speech in vegetative and minimally conscious states after traumatic brain injury. 1) To study preserved residual auditory and linguistic function in VS and MCS patients on basis of their cerebral response to sound and speech stimuli in a hierarchical fMRI paradigm. 2) To assess whether there are differences in the patters of activation shown by these two groups of patients. 3) To explore whether the presence of cerebral activation in an fMRI paradigm could have any prognostic value.
We hypothesized that: 1) Some MCS and some VS patients would show signs of preserved residual auditory and linguistic functioning. 2) Although they may be present in both groups of patients, activation in high- order associative areas would be more frequent in MCS than in VS patients. 3) Cerebral activation in high-order associative areas might be a predictor for a better outcome.
- Study II: Combination of diffusion tensor and functional magnetic resonance imaging during recovery from the vegetative state. 1) To study the evolution of a patient in a VS from one month post-TBI through to the recovery of consciousness, in order to explore the cerebral changes that accompanied the recovery, focussing on the function of the linguistic and the default mode networks and the structural integrity of the white matter. 2) To relate the cerebral activation exhibited by the patient in the fMRI task with the structural integrity of the cerebral networks involved in the linguistic function. 3) To characterize the profile of cognitive sequelae and explore the relation between the cerebral responses to speech showed by the patient when he was in a VS and the subsequent linguistic function assessed by means of a neuropsychological battery when he recovered consciousness.
Approach, objectives and hypotheses | 43 PhD Thesis
We hypothesized that: 1) The linguistic function of the patient after recovery would be related with the functionality of the linguistic network revealed by the fMRI. 2) The patient would be able to recruit higher-order areas in response to speech processing when conscious as compared to when he was in a VS. 3) The patient would show an abnormal function in the default mode network when he was in a VS and a partial restoration with the recovery of consciousness. 4) The extra information gained from the analyses of the default mode network and the DTI data would complement that obtained from the analysis of the fMRI language paradigm, and would therefore contribute to a greater understanding of the patient´s neuropsychological profile following recovery.
- Study III: Diffusion weighted imaging distinguishes the vegetative state from the minimally conscious state. 1) To study the integrity of the subcortical white matter, brainstem and thalami in two groups of patients in a VS or an MCS by means of the mean diffusivity in these regions. 2) To evaluate whether the in-vivo diffusion characteristics of the regions of interest studied match those of previous post-mortem work. 3) To assess whether DTI-derived measures correlate with the behavioural profile of each patient. 4) To determine whether those measures can distinguish between the VS and the MCS patient groups.
We hypothesized that: 1) DTI will allow the identification of signs of DAI and thalamic damage in patients in a VS or an MCS. 2) According to previous post-mortem findings, white matter and thalamic damage would be more severe in VS than in MCS patients, but no differences would appear in the brainstem. 3) The clinical severity, identified by the Coma Recovery Scale-Revised will be related to the severity of the damage in these two regions.
44 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
- Study IV: Reductions of thalamic volume and regional shape changes in the vegetative and the minimally conscious states. 1) To determine whether global as well as regional atrophy of the thalamus could be detected in-vivo in a sample of VS and MCS patients by means of the analysis of its shape on a per-vertex basis. 2) To evaluate whether there are thalamic morphological differences between VS and MCS patients, as well as if these differences correlate with the clinical severity.
We hypothesized that: 1) Global atrophy as well as regional differences would be more prominent in VS than in MCS patients and would partially explain their respective clinical profiles.
Approach, objectives and hypotheses | 45
METHODS
The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
The present thesis consists of four studies which examine the residual cognitive functions preserved in the vegetative state and the minimally conscious state, as well as the neuroanatomical basis of the impairment of consciousness in this population. We studied two different samples and used several MRI acquisition procedures and neuroimaging analyses, as well as a number of behavioural assessment protocols. All the studies were approved by the local ethics committees. Written informed assent was obtained from the appointed „consultee‟ for each patient (in all cases this was the patient‟s next of kin) prior to the participation in the study. All healthy participants gave written informed consent. The specific characteristics of the samples included in each study as well as the methods employed, i.e. behavioural assessment, MRI sequences, analysis procedures, etc., are described in detail in the methods section of the respective paper (see Results section). Nevertheless, the main methodological characteristics are briefly described below.
1. Participants
Patients for studies I, II and IV were recruited from the Institut de Neurorehabilitació Guttmann (Badalona, Spain) and the Hospital Clínic (Barcelona, Spain). From among all patients admitted to these two centres during a three-year period (2007-2009), we first selected those who fulfilled the following criteria:
- Inclusion criteria: i) diagnosis of VS according to the criteria defined by the Royal College of Physicians (2003) or MCS, according to the criteria defined by Giacino et al. (2002); ii) age under 55-years; iii) traumatic brain injury. - Exclusion criteria: i) history of previous severe neurological or psychiatric disease; ii) premorbid intellectual disability; iii) metallic plates incompatible with the MRI scanner; iv) respiratory complications.
Twenty patients fulfilled those criteria and were initially recruited to participate in the study. Five patients had to be excluded because of severe medical complications prior to the scanning session. In two more cases the scanning session had to be interrupted because of severe agitation of the patients. Finally, all images underwent quality control tests prior to the analysis. As a result, some patients had to be excluded from the specific studies because of artefacts and low quality of the images (see methods section of the respective papers).
Twenty healthy volunteers, all right handed, native Spanish speakers and aged 19-49, were recruited to constitute the control group. Inclusion criterion was age under 55-years. Exclusion criteria were: i) history of previous severe neurological or psychiatric disease; ii) premorbid intellectual disability; iii) metallic plates incompatible with the MRI scanner.
Methods | 49 PhD Thesis
For the first study, macrostructural lesions in language related areas were added to the exclusion criteria. Seven right-handed patients, three VS and four MCS participated in this study (age: 13-48 years, time of evolution: 1-12 months). One of the VS patients included in this study regained consciousness and therefore was included in a follow-up protocol in which he underwent a second MRI examination as well as a complete neuropsychological assessment. This led to study II. For the fourth study, as we were interested in structural differences, patients under 18-years of age were also not considered for inclusion. Twelve patients (four VS and eight MCS) took part in this study (age: 19-49 years, time of evolution: 1-12 months).
Patients for study III were recruited from two specialist neurorehabilitation centres in the United Kingdom. Among all patients admitted in those centres during a three-year period (2006-2008), those fulfilling the inclusion and exclusion criteria were first selected to take part in the study. Those criteria were similar to the ones detailed above with the exceptions that there were no restrictions based upon age or aetiology (recruiting patients of both traumatic and non- traumatic origin). Thirty patients were initially recruited to participate in the study. Five patients failed to complete the MRI assessment because of medical complications or severe agitation. The final sample included in this study comprised ten VS patients and fifteen MCS patients (age: 17-68, evolution time: 1 to 19 months). Twelve healthy volunteers, aged 27-40, were recruited to constitute the control group.
Centre Barcelona Cambridge
Fulfilled criteria 20 DOC patients 30 DOC patients Institut de Neurorehabilitació Two specialist neurorehabilitation centres Guttmann and Hospital Clínic in the UK
Excluded because of medical complications or severe agitation MRI assessment 13 25
5 VS 8 MCS 10 VS 15 MCS
Studies Study I
3 VS 4 MCS Study II Study III Study IV
1 VS 4 VS 8 MCS 10 VS 15 MCS
Figure 15. Flowchart of the sampling process 50 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
2. Clinical and behavioural assessment
2.1. Clinical assessment
To further characterize the sample, we recorded the following variables:
- Demographical data: Age, gender, years of education and handedness. - Severity of the injury: score in the Glasgow Coma Scale (Teasdale & Jennett 1974; Wilson et al. 1998) at the time of admission at the hospital after the injury as well as the minimum score reached. - Aetiology. - Lesions on MRI/CT: at the first admission in the acute phase and at the moment of the participation in the study. - Time post injury. - Prescribed medication.
2.2. Behavioural assessment
Behavioural assessment of the patients was performed using the Disability Rating Scale (DRS) (Rappaport et al. 1982), the Rancho Los Amigos Level of Cognitive Functioning Scale (LCFS) (Hagen et al. 1979) and the Coma Recovery Scale-Revised (CRS-R) (Giacino et al. 2004).
The VS patient who went on to regain consciousness (study II) underwent a complete neuropsychological assessment using the following tests: Spanish version of the Boston Diagnosis Aphasia Examination (Goodglass & Kaplan 1986), Albert‟s test (Albert 1973), the Test Barcelona-Revised (Peña-Casanova 1991), Digit span and Letter-Number Sequencing (LNS) subtests of the WAIS-III (Wechsler 1999), Rey‟s Auditory Verbal Learning Test (RAVLT) (Lezak et al. 2004), and a phonetic verbal fluency task (Artiola-i-Fortuny et al. 1999).
Methods | 51 PhD Thesis
3. Magnetic Resonance Imaging acquisition
Images were acquired using two 3T MRI Magnetom Trio Tim scanners (Siemens Medical Systems, Germany) located respectively at the Centre for Image Diagnosis of the Hospital Clinic (CIDC), Barcelona (studies I, II and IV), and the Wolfson Brain Imaging Centre (WBIC), Addenbrooke‟s Hospital, Cambridge, UK (study III).
3.1. Functional MRI
fMRI data comprising 240 volumes of 36 axial slices each were acquired at the CDIC using a gradient-echo echo-planar imaging (EPI) sequence with the following parameters: TR = 2000ms, TE = 1.29ms, matrix size = 128x128, slice thickness = 3mm, interslice gap = 0.75mm, flip angle = 90. This acquisition was used for studies I and II.
The stimuli used for the fMRI task consisted of eight twenty-second long spoken narratives regarding everyday events. Participants heard these narratives played both normally (forward narratives) and reversed (backward narratives). Reversed narratives match the originals in terms of acoustic characteristics but violate several properties of human speech, thus allowing the isolation of those processes specifically related to the processing of language relative to those associated with the processing of simple sounds (Dehaene-Lambertz et al. 2002; Schiff et al. 2005). Eight blocks of a baseline silence condition of the same duration were used to separate the narratives. Stimuli were presented using „Presentation‟ (v.10.1, Neurobehavioural System), running on a Windows XP PC with an MRI-compatible high- quality digital sound system incorporating noise-attenuated headphones (VisuaStim Digital. Resonance Technology, Inc.).
3.2. T1-weighted structural MRI
High-resolution (1x1x1 mm) T1-weighted magnetization prepared rapid gradient echo (MP-RAGE) images were acquired in the sagittal plane with the following parameters:
- CIDC (studies I, II and IV): TR=2300 ms, TE = 2.98 msec, TI = 900, matrix size = 256x256, flip angle = 9. - WBIC (study III): TR = 2250 ms, TE = 2.98ms, TI = 900, matrix size = 256x231, flip angle = 9.
52 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities
3.3. Diffusion tensor imaging
Axial diffusion weighted datasets were acquired using an EPI sequence with the following parameters:
- CIDC (study II): TR = 5600 ms, TE = 89ms, 49 slices, slice thickness = 1mm, gap = 0.6 mm, matrix size = 122x122. Diffusion-weighted images were sensitized in 30 non-collinear directions with a b-value = 1000s/mm2. - WBIC (study III): TR = 8300 ms, TE = 98, 63 slices, slice thickness = 2mm, no gap, matrix size = 96x96. Diffusion-weighted images were sensitized in 12 non- collinear directions using 5 b-values ranging from 340 to 1590s/mm2 and 5 b=0 images.
Methods | 53 PhD Thesis
4. Neuroimaging techniques
4.1. fMRI analysis
The fMRI data were pre-processed and analyzed using SPM5 (http://www.fil.ion.ucl.ac.uk.spm) running in Matlab 7.0 (MathWorks, MA). Pre-processing steps included realignment, co-registration with structural data, spatial normalization (Friston et al. 1995a) and smoothing. The statistical analysis was based on the general linear model (Friston et al. 1995b). Each scan was modeled to belong to the speech, non-speech or silence condition. Low-level auditory processing was assessed in a comparison of the fMRI responses to both auditory conditions with those associated with the silence condition. Speech processing was assessed by a comparison of the fMRI responses to forward narratives with those to backward narratives. Activation maps obtained from the healthy participants were used to obtain normal patterns of auditory and linguistic processing to compare the patients‟ activation patterns with.
Additionally, in study II, the default mode network integrity was assessed by means of task-induced deactivations (contrast between silence and forward narratives) as well as a „resting state‟ functional connectivity analysis of the interleaved resting state blocks from our fMRI task, following the method proposed by Fair et al. (2007). For this particular analysis, we applied a temporal band-pass filter (0.009 Hz 4.2. DTI analysis DTI data were preprocessed using the FMRIB Software Library (FSL, v4.1.0; http://www.fmrib.ox.ac.uk/fsl). Pre-processing steps included eddy-current correction and skull and non-brain tissue removal (Behrens et al. 2003; Smith 2003). Fractional anisotropy and mean diffusivity maps were obtained using FMRIB Diffusion Toolbox (FDT). Images were segmented to obtain white matter binary masks using FMRIB's Automated Segmentation Tool (FAST) (Zhang et al. 2001). Macrostructural lesions were then manually outlined and removed from the masks to obtain normal-appearing white matter (NAWM) masks. For study III, we focused on three regions of interest (ROI): subcortical (supratentorial) white matter, brainstem and thalamus. An in-house script running on Matlab was used to generate FA and MD histograms from the particular ROIs studied. Additionally, in study II we assessed the integrity of the arcuate fasciculus in order to relate it to the linguistic function assessed by fMRI. 54 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 4.3. Volume and shape analysis T1-weighted images were analyzed to assess thalamic volume and shape using FSL tools. SIENAX was applied to estimate brain parenchymal volume (BPV), normalized for head size (Smith et al. 2002). Thalamus segmentation was performed using FMRIB's Integrated Registration and Segmentation Tool (FIRST) running a two-stage affine registration to standard space and an automatic segmentation to produce both mesh and volumetric outputs from this structure in native space (Patenaude 2007). Shape differences between groups as well as correlations with clinical variables were assessed on a per-vertex basis using F-statistics and BPV as a covariate. The statistics were rendered on the shape surface providing a map of the regions where the displacements of the mean vertex location was significantly different between groups, or correlated with the clinical variables, respectively. Table 9. Brief summary of participants and techniques employed in each study. Study number Sample Aetiology Lesions MRI and design characteristics Study I 4 MCS TBI 71% TAI, focal lesions fMRI Prospective, 3 VS mainly in the frontal and cross-sectional 19 healthy volunteers temporal lobes N=26 Study II 1 VS TBI Right parenchymal insular fMRI /DTI Prospective, 19 healthy volunteers hematoma with subdural single-case, collection longitudinal Study III 10 VS TBI/ non- 20% HBI, 32% TAI, focal DTI Prospective, 15 MCS TBI lesions mainly in the cross-sectional 12 healthy volunteers frontal lobe, one case of N=37 brainstem stroke Study IV 4VS TBI 67% TAI, focal lesions T1-weighted MRI Prospective, 8MCS mainly in the frontal and (Shape analysis) cross-sectional 20 healthy volunteers temporal lobes affecting N=32 also basal ganglia and thalamus MCS: minimally conscious state; VS: vegetative state; TBI: traumatic brain injury; HBI: hypoxic brain injury; TAI: traumatic axonal injury; fMRI: functional magnetic resonance imaging; DTI: diffusion tensor imaging; MRI: magnetic resonance imaging. Methods | 55 PhD Thesis 5. Statistical analysis Group comparisons and correlations using clinical and demographical variables as well as neuroimaging derived indices (i.e. FA and MD histogram indices, volumes, etc.) were performed using SPSS v.14 and v.16 in studies III and IV respectively. Statistical graphs were produced using SigmaPlot 10. 56 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 6. Methodological pitfalls Processing of neuroimaging data in DOC patients presents a number of challenging problems that should be addressed, in order to reduce their impact in the analysis, and considered when interpreting the results. On the one hand, the quality of MRI sequences is very susceptible to motion. Spontaneous movements of the patient during the scan may thereby cause in the images major artefacts that may preclude the analysis or minor artefacts that may incur in biases in the results. On the other hand, the presence of large morphological abnormalities, such as severe hydrocephalus, craniotomies or large focal lesions, in the brains of these patients may lead to errors when trying to corregister images of different modalities for a patient (e.g. structural image and functional data), or to normalize these images to a standard template. The latter precludes statistical comparisons of the maps at a group level and localization of results in terms of standard stereotaxic coordinates. Finally, MRI-compatible metallic plates or cranial shunts may lead to a loss of signal in the regions surrounding the implant precluding any analysis involving this area. In the studies included in this thesis special care was taken to minimize the effects of these problems in the results. Specifically, to minimize the effects of head motion we took the following measures: Firstly, before the scan the procedure was explained to the patient and the scan was not started until the patient was calm. As we acquired structural and functional data during the same scanning session we could not use any anaesthetic to sedate the patients and prevent them to move. Nevertheless, we used foam pads to minimize head motion and should the patient get agitated the scan was stopped and initiated again after the patient was calm. Secondly, after the scanning session all images underwent quality control tests prior to being introduced in any analysis. The author of this thesis visually inspected all the raw images for the presence of any artefacts, and should any be present in the images, these were discarded as a result. Finally, for further confirmation, the results from the registration steps performed as part of the pre- processing of fMRI and DTI data were also checked. It is generally accepted in the literature that levels of movement below 1.5 mm and 0.035 radians have non significant effects in the statistical analysis of fMRI data. In cases of motion beyond those levels the correspondent scans were eliminated from the analysis by introducing them into the subject matrix as specific regressors. Moreover, to further control possible slight effects, motion parameters were included as covariates of no interest in the statistical analysis. In regard to the DTI data, there is not yet a convention regarding the maximum levels of displacements tolerable for an accurate analysis. Nevertheless, the movement detected in the datasets that passed the first visual quality control was very low and thus it seems plausible that it had no important effects on the results. Regarding the coregistration between two modalities for the same subject (only applicable to studies I, II and III), results of this step were carefully inspected to ensure their Methods | 57 PhD Thesis accuracy. The specific algorithms used (see methods section of the papers for more details) performed well in the patients studied. Because of the particularities of the analyses performed in studies III and IV images were assessed in native space. In studies I and II, however, we normalized the images to standard space. Again, the result of this step was carefully inspected and we confirmed that no spatial errors were introduced in the images. Finally, focal lesions or regions artefacted due to metallic implants were manually masked out and excluded of the statistical analysis. 58 | Davinia Fernández-Espejo RESULTS The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 1. Study I Results | 61 PhD Thesis 62 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 63 PhD Thesis 64 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 65 PhD Thesis 66 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 67 PhD Thesis 68 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 69 The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 2. Study II Results | 71 PhD Thesis 72 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 73 PhD Thesis 74 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 75 PhD Thesis 76 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 77 PhD Thesis 78 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 79 PhD Thesis 80 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 3. Study III. Results | 81 PhD Thesis 82 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 83 PhD Thesis 84 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 85 PhD Thesis 86 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 87 PhD Thesis 88 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 89 PhD Thesis 90 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 4. Study IV Results | 91 PhD Thesis 92 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 93 PhD Thesis 94 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 95 PhD Thesis 96 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Results | 97 GENERAL DISCUSSION The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities This thesis comprises four studies developed to contribute to a better understanding of the neuroanatomical and neurofunctional bases of the impairment of consciousness in vegetative and minimally conscious patients. By means of different functional and structural neuroimaging approaches we attempt to shed some light into the study of convert linguistic function in these patients, the understanding of the structural and functional mechanisms involved in cases of recovery, and into the structural abnormalities that characterize the brains of DOC patients. Disorders of consciousness are defined by dissociations between arousal and awareness, which appears absent in the VS and fluctuates in the MCS and, in this sense, they also provide a unique opportunity to study the neural mechanisms that underpin normal awareness. In our first study we focused on the assessment of speech processing by means of the study of the cerebral responses in an fMRI paradigm. Previous works reported different levels of activation to auditory linguistic stimuli in single cases of DOC patients (de Jong et al. 1997; Moritz et al. 2001; Bekinschtein et al. 2004; 2005; Owen et al. 2005a; 2006; Schiff et al. 2005), demonstrating that some of these patients may retain residual cognitive function. However, group studies were needed to further explore the frequency of such responses in this population. In our study, we found evidence of cerebral responses to auditory stimuli comparable to those obtained by a group of healthy volunteers in four out of six patients. Two of these patients, one in VS and one in MCS, showed significant activation only in the contrast between the two types of narratives and the silence baseline, which reflects cerebral processing of complex sound. The remaining two, once more one in VS and one in MCS, also demonstrated activation in the contrast between the narratives played normally and reversed, which reflects speech processing. These ratios were similar to those reported by the only previous group study using a similar task (Coleman et al. 2007) and suggested that there is not a clear relationship between the patients‟ response to linguistic stimuli and their diagnosis of VS or MCS. On the contrary, in a more recent study of a larger cohort of patients, Coleman et al. (2009a) found that both types of responses (i.e. to sound and to speech) were almost twice as frequent in the MCS than in the VS groups. This could explain results such as those reported in PET studies comparing VS and MCS patients with healthy volunteers at a group level (Laureys et al. 2000a; Boly et al. 2004), which found that only MCS patients activated high-order associative areas in response to auditory stimuli, whereas this activation was restricted to primary areas in VS patients. In our study, contrary to our predictions, the four VS and MCS patients who showed responses to sound were able to engage high-order associative areas, such as the middle and superior temporal gyri, in the contrast between the two auditory conditions and the silence baseline. The mid and anterior parts of the superior temporal gyrus (which posterior portion corresponds to Wernicke´s area in the left hemisphere) are known to be involved in unimodal auditory processing of acoustic-phonetic features of speech (Galaburda & Sanides 1980; Creutzfeldt et al. 1989; Binder et al. 1994; 1996). The middle temporal gyrus, on the contrary, is General discussion | 101 PhD Thesis a multimodal associative area known to be involved in linguistic and semantic processing (Mesulam 1998; Cabeza & Nyberg 2000). The fact that some of our patients activated both areas may be suggesting a preservation of some high-order integrative processes at least in some VS and MCS patients. In addition to responses to sound, two of our patients also showed evidence of speech processing in temporal regions. However, they failed to show any activation in the left inferior frontal gyrus, which is known to be involved in phonological as well as lexical/semantic functions (Poldrack et al. 1999; Friederici et al. 2006). Davis et al. (2007) demonstrated that temporal lobe responses to sentences compared with signal-correlated noise (a similar contrast than the one performed in our study) are preserved at deep levels of sedation in healthy volunteers. Frontal areas, on the contrary, were only engaged when the individuals were awake and showed a marked effect of semantic ambiguity, i.e. they showed specific responses to sentences containing ambiguous words versus matched sentences that lacked equivalent ambiguities. This suggested that frontal areas may play a specific role in semantic processes necessary for language comprehension. In this sense, our results provided evidence of preserved speech processing to some extent in our patients, but cannot be interpreted as evidence of preserved comprehension or, even less, of any conscious experience associated with this function. In fact, the maintenance of responses to speech versus signal-correlated noise at deep levels of sedation invalidates their value as correlates of awareness. This notwithstanding, our results illustrate the discrepancy that may exist in some DOC patients between their ability to show behavioural responses and the functionality of underlying cerebral processes, and support the value of the fMRI to detect covert preserved functions in these patients. A recent review of fMRI and PET studies highlighted the potential of the identification of high-order responses in VS patients to predict a positive outcome (Di et al. 2008). Consistently with this trend, the only VS patient showing appropriate responses to speech in our study regained consciousness and was able to communicate when assessed twelve months later. To further characterize the mechanisms involved in the recovery of this patient, in our second study, we combined fMRI and DTI data and contrasted the images acquired at one month post- ictus, when he was in a VS, with those acquired one year later when the patient had regained consciousness. There are just three previously published studies of recovery from a VS (Laureys et al. 2000b; Bekinschtein et al. 2005) or an MCS (Voss et al. 2006). However, they focused on either functional or structural data, or contrasted a number of scans performed after recovery. Our study, therefore, is the first time that data from fMRI, DTI and neuropsychological assessment have been combined in the study of a case of recovery from a DOC. We described a patient who suffered from a severe TBI, which caused a large damage in the right hemisphere, after falling from a ladder one month prior to the first MRI 102 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities examination, when he fulfilled criteria for the VS. As reported in study I, in this scanning session, the contrast between narratives and reversed narratives revealed activation in the posterior region of the superior and middle temporal gyri (Wernicke´s area), demonstrating that some aspects of speech processing were preserved in this patient. Nevertheless, in the second scan, when he had regained consciousness, responses to speech extended to more anterior regions in these gyri as well as to the parieto-temporal junction, engaging Brodmann area 40. This heteromodal area is known to be involved in the association of visual and auditory linguistic information, and so activation may reflect a recovery of high-level integrative functioning (Stoeckel, et al. 2009; Graves et al. 2010). The DTI data supported and complemented the information obtained from the fMRI. We studied the structural integrity of the arcuate fasciculus, the main fibre tract of the linguistic network, which stems from the caudal part of the superior temporal gyrus, arches around the caudal end of the Sylvian fissure and extends to the lateral prefrontal cortex (Makris et al. 2005). This fasciculus appeared to be relatively preserved in the acute phase, showing only a very mild reduction of FA values and MD values within the normal range, and did not show any significant changes in the second scanning session. Demonstrating preserved integrity of this fasciculus along with maintenance of the capacity of processing speech reinforces the predictions that can be made on the basis of the fMRI alone. In this sense, it could be expected a good recovery of linguistic functions following the resumption of consciousness in this patient. The good deal of functional abilities achieved by the patient allowed us to study his neuropsychological profile relating specifically to his linguistic functioning. As predicted, the patient recovered language comprehension ability at ten months post-ictus being able to communicate reliably. Two months later, at the moment of the second MRI assessment, he showed a recovery of all language functions assessed. Whereas the relationship between high- order cerebral responses to stimuli and a subsequent recovery is still not completely understood and could be argued, it seems plausible to hold that the integrity of a specific cerebral network and the subsequent functionality of the cognitive processes that it subserves can be related more straightforwardly. In this sense, the structural and functional information obtained from the combination of neuroimaging techniques in patients who are not able to demonstrate overt responses may serve as guidance for designing interventions in order to maintain the integrity of the cognitive functions that appear preserved, once the utility of methods as the one reported here has been demonstrated in larger cohorts of patients. A second focus of this study was placed on the functioning of the default mode network, which was assessed by means of the task-related deactivations in the linguistic paradigm and a resting state functional connectivity analysis. In the first scan, the patient showed a reduced pattern of deactivations relative to the second scan, which was restricted to the left parieto-temporal junction. When the patient regained consciousness, this pattern became General discussion | 103 PhD Thesis more similar to the pattern classically reported in healthy volunteers as being part of the DMN (Gusnard & Raichle 2001), engaging parietal and frontal areas in the non damaged hemisphere. Functional connectivity analysis confirmed and complemented these results as the patient showed absent parieto-frontal functional connectivity in the first scan and restored functional connectivity within these regions in the second. These findings are in agreement with early metabolic studies with VS patients (Laureys et al. 2000b) and more recent work with healthy subjects which have suggested that the activity in this fronto-parietal network may be related to conscious perception (Boly et al. 2007). Furthermore, recently it has been demonstrated that functional connectivity within the DMN may be partially preserved in some VS patients, though significantly reduced compared to healthy volunteers (Boly et al. 2009). This reduction has also been related to the level of consciousness experienced in states of altered consciousness (Vanhaudenhuyse et al. 2010b). The authors of these works have proposed two different levels of functional connectivity to explain their findings: one that would persist independently of the level of consciousness, and another that is more related to the presence of conscious cognitive processes. The presence of deactivations within parietal areas only in the initial scan, along with the outcomes of the functional connectivity analysis showing connectivity restricted to these regions, may suggest that they are involved in the lower level processes they describe, while the more frontal areas observed in the second scan may be more related to the higher conscious processes. This proposal, however, is necessarily speculative. In any case, it seems reasonable to assume that the dysfunctional pattern of deactivations exhibited by the patient in the first scan is a reflection of some level of structural or functional disorganization associated with a post-acute state. A recent report of a post-traumatic VS patient who recovered some degree of consciousness following the stimulation of the dorsolateral prefrontal cortex has stressed the importance of the maintenance of the fronto-parietal network in the recovery of consciousness (Canavero et al. 2009). The current findings of a greater fronto-parietal network of deactivations observed when consciousness had been regained relative to those observed when the patient was in the VS, are consistent with this notion. As for the DTI data, the global analysis of the normal appearing white matter (after masking out the large right parieto-temporal lesion) revealed an absence of any major impairment in the white matter tissue, without any outstanding changes between the first and second scans. As discussed in the introduction, one of the most common abnormalities in post- traumatic vegetative brains is grades 2 and 3 of DAI (Kinney & Samuels 1994; Adams et al. 1999; 2000). However, the mechanisms commonly involved in this damage, i.e. stretching and shearing of white matter fibres due to rotational forces normally related to acceleration- deceleration effects (Adams et al. 1982; Gennarelli et al. 1982), did not come into play in the current case, as the cause of the TBI was a fall. The accident caused an extensive concussive lesion restricted to the right parieto-temporal area but it did not seem to affect areas far away 104 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities from the lesion boundaries. DAI can disrupt critical cortical-subcortical pathways, leading to severe cognitive dysfunction and precluding an effective reorganization of the preserved structures (Povlishock 1992). The absence of structural evidence of DAI in our patient revealed by the DTI analysis may highlight the potential for this as a measure of the extent to which recovery made on the basis of functional re-organization can take place, once the large haemorrhage and post-evacuation complications have subsided. In this sense, a number of studies have recently reinforced the potential of DTI measures for predicting outcome in acute DOC patients (Perlbarg et al. 2009; Tollard et al. 2009). However, to our knowledge no previous study has specifically assessed its applicability for detecting differences between VS and MCS patients. With that purpose, in study III, we applied DTI to assess the integrity of white and grey matter regions in a group of 25 DOC patients in vivo. In accordance with previous post-mortem findings, significant tissue damage in the subcortical white matter, brainstem and thalami were observed in the patients when compared to healthy volunteers (Kinney & Samuels 1994; Adams et al. 1999; 2000). More interestingly, the analysis of the distribution of mean diffusivity values within these three regions revealed differences between VS and MCS patients in subcortical white matter and thalami but not in the brainstem. These differences pointed to a more severe damage in the VS patients in those two regions, consistently with the only neuropathological study that compared these two groups (Jennett et al. 2001). Nevertheless, the exact pathological correlate of changes in MD values has not been clearly established and, thereby, the interpretation of our findings in terms of neuropathological processes can only be speculated upon. In any case, it seems plausible to hold that the reported changes in the histograms reflect a greater vulnerability of the fibres typically associated with lower MD values along with a larger proportion of voxels with higher MD values. Increases in MD are associated with a greater displacement of water molecules, presumably related to axonal injury and demyelination in the white matter and with a loss of healthy neurons related to ischaemic necrosis or transneuronal degeneration affecting the thalamic grey matter. The cohort of patients included in this study comprised patients of both traumatic and non-traumatic aetiologies, in an attempt to increase the potential clinical applicability of our methods. It has been recently demonstrated that abnormalities in supratentorial grey and white matter regions do not differ between VS patients of traumatic and of hypoxic-ischemic aetiologies (Newcombe et al. 2010). This finding provides further validation to our results and supports conclusions related to the applicability of our methods to different aetiologies. Although supratentorial areas seem not to differ, previous post-mortem studies identified other differences in the patterns of damage between these two groups of patients (Kinney & Samuels 1994; Adams et al. 2000). Consistently, Newcombe et al. (2010) reported differences in the infratentorial compartment, with DTI abnormalities in the brainstem confined to the TBI group. General discussion | 105 PhD Thesis Although we also studied the brainstem, the number of patients with non-traumatic brain injuries included in our study was small and thus, any meaningful statistical comparison between them and the TBI patients was preclude as a result. Interestingly, we found that the severity of the damage in the subcortical white matter and the thalami, as revealed by the histogram-derived indices, predicted the clinical severity of the consciousness impairment as defined by the CRS-R score (Giacino et al. 2004), consistent with Newcombe et al. (2010). Clinical challenges aside, disorders of consciousness also provide a valuable opportunity for studying the neural mechanisms that underpin normal awareness. In study II, we provide further evidence to support the important role that the functional connectivity between parietal and frontal areas in the default mode network could be playing in human consciousness. As discussed above, the degree of functional disconnection within this network has been found to correlate with the level of consciousness in DOC patients (Vanhaudenhuyse et al. 2010b). Functional connectivity analysis can provide partial information about structural connectivity, but precise relationships between regions cannot be determined as functional correlations can be observed between regions where there is little or no structural connectivity because of third regions that act as a link (Damoiseaux & Greicius 2009). Our results complement those reported in functional studies and provide further support to studies suggesting that DOC are disconnection syndromes characterized by long-range cortico- cortical and thalamo-cortical disconnections (Laureys et al. 1999; 2000b; Boly et al. 2009). Nevertheless, the central question under investigation in this study was whether these clinically defined sub-groups could be reliably distinguished purely on the basis of DTI-derived measures. Taken together the highest effect size measures summarising the changes in subcortical white matter and thalami we were able to classify VS and MCS patients into their appropriate diagnostic category with 95% accuracy. To our knowledge, this is the first time that in vivo pathological indices have been identified that are able to correctly differentiate between VS and MCS with a high level of accuracy. Such measures thereby can provide additional information which may be combined with behavioural indices to aid the diagnostic decision- making process. Moreover, they may help to resolve what are often subjective and ambiguous behavioural markers in these patient groups, thereby reducing the high migdiagnosis rates. In keeping with the study of the neuropathological basis of the DOC and guided by the findings of our third study, which reinforced the scientific evidence suggesting the important role that the thalamus may be playing in human consciousness and its disorders (see the introduction section of this thesis for an in-depth discussion of these works), in study IV, we evaluated thalamic global and regional patterns of atrophy and their clinical correlates in a group of TBI patients in a VS or an MCS. We found that the total thalamic volume was reduced in the patient group as compared to a group of healthy volunteers. The shape analysis performed 106 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities revealed that the dorsomedial body was the most affected region. Its inward deformation can be explained by atrophy in both the dorsomedial nucleus and the internal medullar lamina, consistent with previous neuropathological findings (Adams et al. 1999; Maxwell et al. 2004; 2006). The dorsomedial nucleus has reciprocal connections with the prefrontal, orbital, and temporal cortices, connections with the amygdala and the limbic system, and connections with other thalamic nuclei and associated areas of the parietal and temporal lobes (Maxwell et al. 2004). Intralaminar thalamic neurons are part of an ascending pathway that originates in the midbrain reticular formation, and has a primary role in maintaining wakefulness (Schiff 2008). Interestingly, atrophy in these two regions was also correlated with a greater clinical severity, as assessed by means of the DRS (Rappaport et al. 1982), reinforcing theories that claim about the involvement of the dorso-medial and intrallaminar nuclei in the support of human consciousness. These findings may also suggest a potential value of the damage in these nuclei for predicting the outcome after severe TBI. Moreover, they also provided further insights into the mechanisms which underlied some cases of recovery after interventions with pharmacological agents (see Pistoia et al. 2010 for a review) or deep-brain stimulation of the central thalamus (Schiff et al. 2007) and they suggest that morphometric analyses based on high-resolution MRI could prove a powerful tool for identifying patients who might retain enough structural substrate to benefit from those neuromodulatory interventions. To our knowledge, this is the first time that a relationship between thalamic local changes and clinical profile has been reported in DOC patients. Furthermore, when the two subgroups of patients were compared independently with the group of healthy volunteers, we identified differences in their respective patterns of atrophy. Specifically, VS patients showed a more widespread atrophy, which affected not only the dorsal and dorsomedial bodies but also, bilaterally, the left anterior body and the medial posterior body, and caused a significant reduction of the global volume. On the contrary, the atrophy in the MCS group was focused on the dorsal body, with a relative preservation of anterior and posterior regions and no significant differences in global volumes as compared to the healthy volunteers group. In our third study we demonstrated that the thalamus was less severely damaged in MCS than in VS patients, consistent with previous neuropathological findings (Jennett et al. 2001). Here we went one step forward demonstrating for the first time local differences in the thalamus, related to specific nuclei, between these two groups of patients. Several mechanisms may explain the thalamic atrophy seen in cases of severe TBI. It has been suggested that it may reflect DAI or ischaemia, arising as a result of post-traumatic transneuronal and retrograde degeneration, excitotoxicity, or as a result of neuronal loss due to hypoxic mechanisms (Adams et al. 1999). The exact pathological processes that came into play in the damage present in our patients can only be speculated upon. However, considering the relative heterogeneity of the lesions identified in the clinical MRI and CT, our results may be General discussion | 107 PhD Thesis indicating a combination of both types of damage in our patients. In any case, thalamic atrophy could be interpreted as a surrogate marker of more diffuse damage that interrupts thalamo- cortical connectivity and, in that sense, the analysis of this structure could speak about the global integrity of those pathways. Thereby, our findings support the importance of thalamo- cortical disconnections in the neural bases of the VS (Laureys et al. 2000b; Boly et al. 2009). Following this idea, the less severe pattern of atrophy exhibited by MCS patients may be reflecting the potential availability of cerebral networks that can allow a better outcome (Giacino et al. 1997; Luauté et al. 2010). Moreover, along with the results of our third study, they may provide further support to the concept of MCS itself in suggesting a structural substrate for this behaviourally defined syndrome. In summary, taken as a whole the four studies included in this thesis contribute to a better characterization of the cerebral mechanisms that construct awareness and the structural and functional bases of the DOC. They further support the use of fMRI to detect residual cognitive function that may remain unobservable in bedside examinations of these patients. They also highlight the potential of the combination of structural and functional neuroimaging techniques in their study as well as their possible future use in the estimation of outcome. Furthermore, our results provide for the first time evidence that VS and MCS patients can be reliably distinguished in vivo in base of structural measures, suggesting that MRI techniques could be combined with other measures in the clinical field to help in the solution to the problem of misdiagnosis. There are a number of caveats related to the studies that led to this thesis that should be mentioned. The main limitation is the sample size. Disorders of consciousness are rare, and it is extremely difficult to accumulate larger cohorts for the inclusion in research studies. This is a common problem in the neuroimaging literature with this population, and most of the studies have been performed with similar or smaller number of patients than the studies included here (see Table 8, Tshibanda et al. 2010 and Cruse & Owen 2010). Notwithstanding this, the use of small samples reduces the statistical power of the comparisons performed and the magnitude of the correlations that can be identified. Moreover, it may affect the generalizability of the findings reported here to the general population of DOC patients. In this sense, our results, although promising, should be taken as preliminary. Nevertheless, the consistency of the findings reported in study I with other published functional studies with DOC patients, and of those reported in studies III and IV with previous neuropathological findings partially mitigates this limitation and supports the reliability of our results. Special mention, however, should be made about study II. The low incidence of cases of good recovery from a DOC makes cases as the one reported in that study exceptional opportunities to explore the mechanisms associated with this process. Nevertheless, conclusions extracted from single-case studies and the interpretation of the results in relation to the wider population should be made with care. 108 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities On the other hand, there are multiple ways to address the analysis of neuroimaging data and each technique counts with a number of advantages and disadvantages. An in-depth discussion of this topic would lead to methodological questions that are beyond of the scope of this thesis. Nevertheless, two specific examples deserved to be mentioned. Firstly, the functional connectivity analysis performed in study II, was based on the resting state blocks included in our fMRI linguistic paradigm. Although such a method has been validated before (Fair et al. 2007) our resting state data was significantly shorter than those used in previous resting state studies. We were unable to acquire resting state data for a more robust functional connectivity analysis due to time demands of the other scanning protocols on the patient. Nevertheless, the consistency of the findings derived from this analysis with those obtained in the analysis of task related deactivations in this study, along with the previously demonstrated consistency in the anatomy of the DMN across different methodological approaches (McKiernan et al. 2003; Harrison et al. 2008), further validates our connectivity findings. Secondly, shape analysis methods are relatively new and their applications to the study of pathological brain abnormalities have not been sufficiently explored in the literature. Nevertheless, similar approaches to the one performed in our fourth study have been previously carried out in Parkinson´s disease (McKeown et al. 2008), schizophrenia (Harms et al. 2007; Kang et al. 2008; Coscia et al. 2009; Qiu et al. 2009a), and Alzheimer´s disease (Qiu et al. 2009b; Zarei et al. 2010). This, along with the consistency of our results with previous neuropathological findings with DOC patients, provides support to their reliability. Considering the above mentioned questions, future work would benefit from the development of multicenter studies that allow the recruitment of larger cohorts including patients with different aetiologies, ages, and times of evolution. Such studies would allow the isolation and subsequent study of the effects of those variables in results as those reported in this thesis. One important field that should be further explored is the potential of the combination of different structural and functional neuroimaging modalities, along with behavioural assessments, in the study of DOC patients. This could lead to a better understanding of the cerebral bases of these clinical categories as well to a reliable validation of their potential diagnostic and prognostic value. General discussion | 109 CONCLUSIONS The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities The main conclusions of this thesis, derived from the four studies developed, can be summarized as follows: I. Some vegetative and minimally conscious patients may retain aspects of auditory and linguistic processing in absence of overt responses to those stimuli. Responses to both auditory and linguistic stimuli can engage high-order associative areas, activated in healthy volunteers, in both groups of patients. Such presence of high-order responses to stimuli in vegetative state patients may have predictive value for a good outcome. The fMRI may have an important role in the identification of residual cognitive function that may remain unobservable in a bedside examination in these patients. II. Dysfunctions and disconnections between areas related to the default mode network may have a role in explaining disorders of consciousness. In the absence of significant structural damage to long-range connections, the functional recovery of this network may accompany the restoration of consciousness. III. Patients with a disorder of consciousness have cerebral abnormalities in the subcortical white matter, brainstem and thalamus, which can be identified in vivo with diffusion tensor imaging. These abnormalities correlate with the level of consciousness as assessed by means of the Coma Recovery Scale-Revised and thus may explain the clinical profile of these patients. IV. Vegetative state patients show a more severe damage in the subcortical white matter and the thalami than patients in a minimally conscious state. The analysis of diffusion tensor imaging indices from these two regions allows an accurate classification of the patients into their respective diagnostic category. That suggests the utility of this technique for providing useful information that may complement the behavioural assessment to inform the diagnostic decision making process. V. Patients with a disorder of consciousness show a significant reduction of thalamic volume as compared to healthy volunteers. This atrophy is specially marked in the dorsomedial body, presumably affecting the dorsomedial nucleus and the internal medullar lamina. Atrophy in these two nuclei is also related with the clinical severity, suggesting their relevance in the disorders of consciousness. Conclusions | 113 PhD Thesis VI. Vegetative state and minimally conscious state patients show different patterns of regional thalamic atrophy. Vegetative patients show a global volumetric reduction caused by a more widespread atrophy that affects not only dorsomedial bodies but also anterior and posterior adjacent areas. This result suports the utility of morphometric analyses based on high-resolution magnetic resonance imaging for noninvasively detecting subtle thalamic shape changes in patients with a disorder of consciousness. 114 | Davinia Fernández-Espejo SUMMARY OF THE THESIS The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 1. Resumen de la tesis: El estado vegetativo y el estado de mínima conciencia: función cerebral, conectividad y anormalidades estructurales Glosario de abreviaciones AF: Anisotropía Fraccional LCFS: Level of Cognitive Functioning CDIC: Centre de Diagnòstic per la Imatge Clínic RM: Resonancia Magnética CRS-R: Coma Recovery Scale-Revised RMf: Resonancia Magnética Funcional DM: Difusividad Media TC: Tomografía Computarizada DRS: Disability Rating Scale TCE: Traumatismo Craneoencefálico EMC: Estado de Mínima Conciencia TEP: Tomografía por Emisión de Positrones EV: Estado Vegetativo WAIS: Wechsler Adult Intelligence Scale FDR: False Discovery Rate WBIC: Wolfson Brain Imaging Centre ITD: Imagen con Tensor de Difusión Introducción A pesar del interés que a lo largo de los años la conciencia humana ha despertado en la literatura científica aún no existe una definición universalmente aceptada de la misma. Sin embargo, en el contexto de la neurociencia clínica se considera un sistema complejo que incluye dos elementos básicos: wakefulness, alerta o nivel de conciencia y awareness, contenido de la conciencia, o conciencia per sé (James 1980). Ambos componentes están mediados por distintas redes cerebrales. Mientras que el arousal sería una función autonómica o vegetativa mediada por tronco encefálico, hipotálamo y determinados núcleos talámicos, como parte del llamado sistema activador reticular ascendente, el awareness depende de complejas redes corticales, entre las que destaca la llamada red neuronal por defecto, y sus conexiones recíprocas con los núcleos subcorticales (particularmente con el tálamo). El estado vegetativo (EV), descrito por Jennett & Plum (1972), se caracteriza por una disociación entre estos dos componentes. A diferencia del coma, definido por una completa ausencia de arousal (y por lo tanto de conciencia), el estado vegetativo se define por la preservación del arousal, manifestada por la Summary | 117 PhD Thesis presencia de ciclos de sueño y vigilia, en ausencia de evidencia comportamental de conciencia de uno mismo o el medio. Se trata de pacientes incapaces de reaccionar de modo intencional a la estimulación y que no manifiestan ninguna capacidad comunicativa (Royal College of Physicians 2003). El estado vegetativo se considera persistente si el paciente continúa en dicho estado un mes después del daño cerebral que lo causó. Sin embargo, no será considerado permanente hasta pasados tres o seis meses, dependiendo respectivamente de si seguimos criterios americanos o ingleses, en caso de daño cerebral no traumático y hasta pasados doce meses en caso de traumatismo craneoencefálico (TCE). En la evolución del EV algunos pacientes progresan a un estado de mínima conciencia (EMC), comenzando a mostrar signos de conciencia de sí mismos o del medio, aunque de manera fluctuante e inconsistente (Giacino et al. 2002). El diagnóstico diferencial entre estos subtipos de pacientes con pérdida de conciencia se basa en la evaluación clínica exhaustiva y repetida del repertorio de comportamientos espontáneos y elicitados que el paciente muestra, con el objetivo de determinar si es o no consciente del medio y capaz de interactuar con él de manera intencional. Sin embargo, en muchos casos determinar si un comportamiento es reflejo o proposicional es difícil y esto hace el diagnóstico de estos pacientes extraordinariamente problemático. De hecho, se estima que un tercio de los pacientes diagnosticados como EV conservan signos de conciencia y en algunos casos son incluso capaces de comunicarse con su entorno cuando disponen de las herramientas adecuadas (Andrews et al. 1996; Childs et al. 1993; Schnakers et al. 2009a). Realizar un diagnóstico correcto es esencial para planificar un buen abordaje terapéutico así como para tomar cualquier decisión clínica basada en la estimación del pronóstico, dado que se ha demostrado que éste es mucho más favorable en los casos de EMC que en pacientes en (Giacino & Kalmar 1997; Luauté et al. 2010). En los últimos años se ha experimentado un rápido desarrollo en el campo de la aplicación de la neuroimagen al estudio de estos pacientes con el objetivo de abordar las bases de la alteración de conciencia así como de aportar nuevas herramientas que puedan facilitar el diagnóstico. Por un lado, las técnicas de neuroimagen funcional permiten obtener información acerca de la actividad cerebral de un paciente en respuesta a una determinada tarea cognitiva o estimulación o en reposo, de manera independiente a la capacidad del paciente para emitir respuestas externas. Mediante la tomografía por emisión de positrones (TEP) varios autores demostraron la existencia de respuestas comparables a las encontradas en voluntarios sanos en casos aislados de pacientes en estado vegetativo expuestos a diferentes tipos de estimulación visual, auditiva o táctil (Menon et al. 1998; Owen et al. 2002; 2005a; Kassubek et al. 2003). Sin embargo, las ventajas relacionadas con un mayor poder estadístico, una mejorada resolución espacial y temporal y el hecho de no implicar la aplicación de radioisótopos, han contribuido a un desplazamiento hacia el uso de la resonancia magnética funcional (RMf) en la evaluación de 118 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities las funciones cognitivas residuales en pacientes en EV y EMC. Mediante el uso de esta técnica, diferentes autores han confirmado en muestras más amplias de pacientes los hallazgos preliminares de los estudios de TEP. Así, con RMf se han identificado evidencias de procesamiento visual, táctil, o lingüístico en varios pacientes en EV y EMC (Moritz et al. 2001; Bekinschtein et al. 2004; 2005; Schiff et al. 2005; Owen et al. 2005a; 2006; Staffen et al. 2006; Di et al. 2007; Coleman et al. 2007; 2009a; Zhu et al. 2009; Qin et al. 2010). Recientemente se ha constatado que la presencia de activación en áreas asociativas en pacientes en EV predice una evolución favorable con una alta especificidad (Di et al. 2008). Sin embargo, no es un indicador de conciencia y, por ello, se han desarrollado paradigmas activos con el objetivo de evaluar si un paciente es capaz de interactuar de manera intencional con el medio modulando su actividad cerebral en respuesta a órdenes. En 2006, Owen et al. Publicaron el caso de una paciente en EV tras 5 meses de evolución que mostró respuestas comparables a las de sujetos sanos cuando le pidieron que se imaginara jugando al tenis o recorriendo las habitaciones de su casa. Este hallazgo tuvo un alto impacto en la comunidad científica. Recientemente, estos autores aplicaron este paradigma a un grupo de cincuenta y cuatro pacientes. El 9% de ellos mostró respuestas comparables a sujetos sanos ante las órdenes confirmando que, aunque es poco frecuente, esta capacidad puede estar presente en algunos pacientes en EV y EMC. A pesar de las enormes posibilidades que estos resultados abren para ambos grupos de pacientes, sólo aquellos que se encuentran en la parte menos grave del espectro se beneficiarían de este tipo de técnicas. En cualquier caso, incluso en los pacientes que muestran evidencias de conciencia, sigue siendo de interés el estudio de la respuesta cerebral mediante RMf para caracterizar el perfil de funciones preservadas en los diferentes dominios cognitivos. Entre ellos, el lenguaje es una de las funciones centrales de la cognición humana y demostrar evidencia de procesamiento lingüístico preservado puede tener importantes implicaciones en la motivación de familiares y cuidadores. En el momento del planteamiento del trabajo que condujo a esta tesis únicamente había un estudio grupal que exploraba específicamente el procesamiento lingüístico en pacientes en EV y EMC (Coleman et al. 2007). Un segundo foco de interés de la neuroimagen funcional ha sido el estudio de la función cerebral en reposo. Los primeros trabajos de TEP demostraron una importante reducción global del metabolismo cerebral en pacientes en EV y EMC (Levy et al. 1987; Laureys et al. 1999; 2002; Schiff et al. 2002; DeVolder et al. 1990; Tommasino et al. 1995; Rudolf et al. 1999; Kassubek et al. 2003), que era más pronunciada en áreas relacionadas con la red neuronal por defecto (Laureys et al. 1999; Kassubek et al. 2003). Dicha red incluye una serie de regiones corticales (precuneus, encrucijada temporo-parietal y corteza prefrontal medial) que muestran fluctuaciones sincrónicas de baja frecuencia en condiciones de reposo y reducen su actividad ante tareas cognitivas (Gusnard & Raichle 2001). Varios autores han demostrado alteraciones en el funcionamiento y la conectividad de esta red en estados de conciencia alterada o reducida Summary | 119 PhD Thesis como el sonambulismo, ausencias epilépticas, sueño profundo o anestesia (Bassetti et al. 2000; Maquet 2000; Steriade 2001; Kaisti et al. 2002; Salek-Haddadi et al. 2003). Mediante estudios de RMf se ha demostrado que la conectividad en esta red se encuentra reducida en pacientes en EV y EMC y que dicha reducción correlaciona con el nivel de conciencia (Cauda et al. 2009; Vanhaudenhuyse et al. 2010b). El papel de la resonancia magnética estructural en el estudio de estos pacientes está menos explorado. Los estudios morfométricos han confirmado los hallazgos de trabajos anatomopatológicos previos (Kinney & Samuels 1994; Adams et al. 1999; 2000; Jennett et al. 2001), al identificar en las imágenes signos de lesión axonal difusa grados 2 y 3 así como anomalías en el tálamo como las dos características neuropatológicas más frecuentes en pacientes en EV tras TCE (Kampfl et al. 1998a). Dentro de las técnicas estructurales, la imagen con tensor de difusión (ITD) permite caracterizar la microestructura del tejido cerebral mediante el estudio del movimiento de las moléculas de agua. Se trata de una técnica especialmente sensible a las alteraciones en la sustancia blanca y en núcleos grises subcorticales como el tálamo, y que permite detectar cambios sutiles que pasan desapercibidos en secuencias de resonancia magnética convencionales. Esta técnica ha demostrado un alto potencial en la estimación del pronóstico en pacientes agudos con alteración de la conciencia tras TCE (Perlbarg et al. 2009; Tollard et al. 2009). Sin embargo, hasta la fecha, ningún estudio estructural ha conseguido encontrar diferencias entre pacientes en EV y en EMC. Objetivos e hipótesis La presente tesis doctoral consta de cuatro estudios cuyo objetivo general de partida era examinar las bases neuroanatómicas y neurofuncionales de la alteración de la conciencia en el EV y el EMC. En primer lugar nos centramos en la evaluación de las respuestas cerebrales al lenguaje mediante la RMf en un grupo de pacientes en estos dos estados (estudio I). En segundo lugar, combinamos técnicas de RMf, de ITD y exploraciones clínicas y neuropsicológicas para estudiar los cambios cerebrales y clínicos que acompañaron la recuperación de la conciencia en un paciente en EV (estudio II). Por último, nos centramos en el estudio de ITD (estudio III), así como imágenes estructurales de alta resolución potenciadas en T1 (estudio IV) para caracterizar in vivo los patrones neuropatológicos presentes en pacientes en EV y EMC y explorar posibles diferencias entre estos dos subgrupos. En el tercer estudio investigamos las alteraciones de la sustancia blanca subcortical, tronco cerebral y tálamo, basándonos en trabajos post mortem 120 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities previos. En base a los resultados obtenidos en dicho estudio, en el cuarto estudio nos centramos en el tálamo y sus patrones de atrofia global y regional. Los objetivos e hipótesis de los cuatro estudios se concretan en los siguientes puntos: - Estudio I: Respuesta cerebral al lenguaje en el estado vegetativo y el estado de mínima conciencia tras traumatismo craneoencefálico. Objetivos: 1) Estudiar la posible preservación del procesamiento auditivo y lingüístico preservado en pacientes en EV y EMC en base a sus respuestas cerebrales al sonido y al lenguaje oral en un paradigma jerárquico de RMf. 2) Evaluar si existen diferencias en los patrones de activación mostrados por estos dos grupos de pacientes. 3) Explorar el posible valor pronóstico de la presencia de activación cerebral en un paradigma de RMf. Hipótesis: 1) Algunos pacientes en EV o en EMC mostrarán respuestas cerebrales específicas ante estímulos auditivos y lingüísticos. 2) Aunque puede aparecer en ambos grupos de pacientes, la activación de áreas de alto nivel asociativo será más frecuente en pacientes en EMC que en pacientes en EV. 3) La activación cerebral en áreas de alto nivel asociativo tendrá valor predictivo para un pronóstico más favorable. - Estudio II: Combinación de imágenes con tensor de difusión y resonancia magnética funcional durante la recuperación del estado vegetativo. Objetivos: 1) Estudiar la evolución de un paciente en EV desde un mes tras el accidente hasta la recuperación de la conciencia, con el propósito de explorar los cambios cerebrales que acompañaron dicha recuperación, centrándonos en la función de la red lingüística y la red neuronal por defecto. 2) Relacionar las respuestas cerebrales identificadas mediante RMf con la integridad estructural de las redes cerebrales implicadas en la función lingüística. 3) Caracterizar el perfil de secuelas cognitivas y explorar la relación entre las respuestas cerebrales al lenguaje mostradas cuando estaba en EV y la función lingüística subsecuente evaluada mediante una batería neuropsicológica. Summary | 121 PhD Thesis Hipótesis: 1) La función lingüística del paciente tras la recuperación de la conciencia estará relacionada con la funcionalidad de la red lingüística evaluada mediante el paradigma de RMf. 2) El paciente será capaz de activar aéreas de mayor nivel asociativo en respuesta al lenguaje cuando haya recuperado la conciencia. 3) El paciente mostrará disfunciones en la red neuronal por defecto cuando se encuentra en EV y una parcial recuperación de la misma acompañando a la recuperación de la conciencia. 4) La información extra obtenida del análisis de la red neuronal por defecto y las imágenes de ITD complementarán aquella obtenida del análisis del paradigma lingüístico de RMf y, por lo tanto, en conjunto contribuirán a un mejor entendimiento del perfil neuropsicológico del paciente tras la recuperación de la conciencia. - Estudio III: El estudio de imágenes por tensor de difusión diferencia el estado vegetativo del estado de mínima conciencia. Objetivos: 1) Estudiar la integridad de la sustancia blanca subcortical, tronco encefálico y tálamo en dos grupos de pacientes en EV y EMC mediante el análisis de la difusividad media en dichas regiones. 2) Evaluar si las características detectadas in vivo mediante ITD en estas regiones son consistentes con las encontradas previamente en estudios post mortem. 3) Evaluar si las medidas derivadas de la ITD correlacionan con el perfil clínico de los pacientes. 4) Determinar si dichas medidas pueden diferenciar entre pacientes en EV y EMC. Hipótesis: 1) El análisis de ITD permitirá la identificación de signos de lesión axonal difusa y anormalidades en el tálamo en pacientes en EV o EMC. 2) De acuerdo con estudios post mortem previos, las alteraciones en la sustancia blanca y el tálamo serán más graves en los pacientes en EV que en los pacientes en EMC, pero no se encontrarán diferencias en el tronco cerebral. 3) La gravedad clínica, identificada mediante la Coma Recovery Scale-Revised, estará relacionada con la gravedad de las lesiones en esas dos regiones. 122 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities - Estudio IV: Reducciones en el volumen talámico y cambios regionales de forma en el estado vegetativo y el estado de mínima conciencia. Objetivos: 1) Determinar si es posible detectar en vivo atrofia talámica global y regional en pacientes en EV y EMC mediante un análisis de su forma basado en vértices. 2) Evaluar si hay diferencias morfológicas en el tálamo entre estos dos grupos de pacientes, así como si dichas diferencias correlacionan con la gravedad clínica. Hipótesis: 1) Tanto la atrofia global como la regional serán más marcadas en los pacientes en EV que en los pacientes en EMC y explicarán parcialmente sus respectivos perfiles clínicos. Método Para los estudios incluidos en la presente tesis doctoral hemos contado con dos muestras diferentes y se han aplicado distintas técnicas de análisis de neuroimagen así como diferentes pruebas de exploración clínica y neuropsicológica. Los estudios fueron aprobados por los comités de ética correspondientes (Hospital Clínic de Barcelona, Universidad de Barcelona y Addenbrooke´s Hospital, Cambridge) y todas las familias de los pacientes, así como todos los voluntarios sanos, entregaron consentimiento informado por escrito previamente a la participación en el estudio. Cada artículo contiene una descripción detallada de las características de los respectivos participantes, así como de las técnicas de neuroimagen y de evaluación clínica empleadas. No obstante, las principales características de cada estudio se resumirán a continuación. La primera muestra (estudios I, II y IV) fue recogida a partir de pacientes del Institut de Neurorehabilitació Guttmann y el Hospital Clínic de Barcelona. De entre todos los pacientes ingresados entre 2007 y 2009 seleccionamos aquellos que cumplían criterios de inclusión y exclusión. Los primeros fueron: etiología traumática, diagnóstico de estado vegetativo (Royal College of Physicians 2003) o de mínima conciencia (Giacino et al. 2004) y edad inferior a 55 años. Como criterios de exclusión se contemplaron: historia de alteraciones neurológicas o psiquiátricas previas, discapacidad intelectual premórbida, implantes metálicos incompatibles con la resonancia magnética, complicaciones respiratorias graves. Veinte pacientes cumplieron Summary | 123 PhD Thesis dichos criterios. De ellos, cinco tuvieron que ser excluidos por complicaciones médicas graves en los días anteriores a la resonancia. En otros dos casos la prueba de resonancia tuvo que interrumpirse por agitación del paciente. Finalmente, todas las imágenes se sometieron a un control de calidad y, como resultado, varios pacientes tuvieron que ser excluidos de los respectivos estudios debido a la presencia de artefactos de movimiento. En el primer estudio participaron siete pacientes (tres EV y cuatro EMC). Uno de los pacientes en EV incluidos en este estudio recuperó la conciencia y fue incluido en un protocolo de seguimiento, dando lugar al estudio II. En el estudio IV participaron cuatro pacientes en EV y ocho en EMC. El grupo control consistió en veinte voluntarios sanos de edades comprendidas entre los 19 y los 49 años. El único criterio de inclusión fue la edad menor a 55 años y los criterios de exclusión fueron los mismos que en el caso de los pacientes. La segunda muestra (estudio III) fue recogida a partir de dos centros de neurorrehabilitación del Reino Unido. Entre todos los pacientes ingresados en dichos centros entre 2006 y 2008 se seleccionaron aquellos que cumplían criterios de inclusión y exclusión (similares a los utilizados en la primera muestra con la excepción de no haber restricciones relativas a la etiología). Inicialmente se incluyeron treinta pacientes en el estudio. Cinco de ellos no llegaron a completar el estudio debido a complicaciones médicas o agitación. La muestra final del estudio III incluyó diez pacientes en EV y quince en EMC. El grupo control estaba formado por doce voluntarios sanos. Para la caracterización clínica de los pacientes se recogieron las siguientes variables: datos demográficos (edad, género, años de escolarización y lateralidad manual), gravedad del daño cerebral mediante la puntuación inicial y mínima en la Glasgow Coma (Teasdale & Jennett 1974; Wilson et al. 1998), etiología, lesiones identificadas en TC y resonancia, tiempo de evolución y medicación. La exploración clínica se realizó mediante las escalas Disability Rating Scale (DRS) (Rappaport et al. 1982), Rancho Los Amigos Level of Cognitive Functioning (LCFS) (Hagen et al. 1979) y Coma Recovery Scale-Revised (CRS-R) (Giacino et al. 2004). Así mismo, el perfil cognitivo del paciente que recuperó la conciencia (estudio II) fue evaluado mediante la versión española del Test de Boston para el diagnóstico de la afasia (Goodglass & Kaplan 1986), el test de Albert (Albert 1973), el test Barcelona-revisado (Peña-Casanova 1991), los subtests dígitos y números y letras del WAIS-III (Wechsler 1999), el test auditivo verbal de Rey (Lezak et al. 2004), y las fluencias fonéticas (Artiola-i-Fortuny et al. 1999). Las imágenes de resonancia se adquirieron en el Centre de Diagnòstic per la Imatge Clínic (CDIC) del Hospital Clínic de Barcelona, y el Wolfson Brain Imaging Centre (WBIC) del Addenbrooke´s Hospital, Cambridge, Reino Unido. En ambos centros se utilizó un escáner de resonancia magnética de 3 Tesla (Magnetom Trio Tim, Siemens). Se adquirieron imágenes de resonancia magnética funcional, aplicando un paradigma de procesamiento lingüístico, imágenes con tensor de difusión e imágenes estructurales de alta definición potenciadas en T1. Para el pre-procesamiento y posterior análisis de las imágenes de RMf se empleó un protocolo 124 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities estándar con el software SPM5 (http://www.fil.ion.ucl.ac.uk/spm/). El procesamiento auditivo fue evaluado mediante el contraste entre una serie de narraciones reproducidas normalmente e invertidas y la línea de base, mientras que el procesamiento lingüístico se evaluó mediante el contraste entre los dos tipos de narraciones. De manera adicional, en el estudio II se realizó un análisis de las de-activaciones así como un análisis de conectividad funcional para estudiar el funcionamiento de la red neuronal por defecto. Para el procesamiento de las ITD e imágenes potenciadas en T1 se empleó el software FSL (http://www.fmrib.ox.ac.uk/fsl/). Concretamente, para la ITD se emplearon las herramientas, BET (Smith 2003), FDT (Behrens et al. 2003), para obtener los mapas de anisotropía fraccional (AF) y difusividad media (DM), y FAST (Zhang et al. 2001), para la creación de las máscaras. En el estudio III se analizó la distribución de los valores de DM en la sustancia blanca subcortical, tronco encefálico y tálamo. En el estudio II, se estudiaron la AF y DM en sustancia blanca y el fascículo arqueado. Por último, las imágenes potenciadas en T1 se utilizaron para evaluar la atrofia talámica usando, las herramientas SIENAX (Smith et al. 2002), para la estimación del volumen cerebral total, y FIRST (Patenaude 2007), para la segmentación y posterior análisis de forma del tálamo. Los análisis estadísticos y la posterior producción de gráficos se realizaron con el SPSS 14 y 16 y el SigmaPlot 10 respectivamente. Resultados Estudio I: El grupo control mostró activación bilateral en el giro temporal superior en el contraste entre los dos tipos de narraciones tomadas conjuntamente y la línea de base (procesamiento auditivo) (p<0.01 corregido por múltiples comparaciones mediante FDR). En el contraste entre las narraciones reproducidas normalmente y sus equivalentes invertidas (procesamiento lingüístico) este grupo mostró activación en los giros temporal superior y medio izquierdos, el giro temporal medio derecho y el giro frontal inferior izquierdo (p<0.01 corregido por múltiples comparaciones mediante FDR). Dos pacientes en EV y dos en EMC mostraron una activación comparable al grupo control en el contraste de procesamiento auditivo, implicando bilateralmente el giro temporal superior y medio. De ellos, un paciente en EV y un paciente en EMC mostraron además una activación comparable al grupo control en el contraste de procesamiento lingüístico, aunque centrada en los giros temporal superior y medio izquierdos. El único paciente vegetativo que mostró activación al lenguaje recuperó la conciencia en los meses posteriores. Summary | 125 PhD Thesis Estudio II: El análisis de la tarea de RMf mostró activaciones anatómicamente coherentes con las regiones lingüísticas en el contraste de procesamiento lingüístico tanto en la primera exploración, cuando el paciente se encontraba en EV, como en la segunda, cuando había recuperado la conciencia. Sin embargo, en la primera exploración el paciente mostró un patrón anómalo de de-activaciones, centradas exclusivamente en la encrucijada parieto-temporal. En la segunda sesión, este patrón fue muy similar al clásicamente descrito en voluntarios sanos para la red neuronal por defecto. El análisis de la conectividad funcional confirmó la presencia de desconexiones parieto-frontales en la primera exploración, que se normalizaron cuando el paciente recuperó la conciencia. El análisis de ITD mostró una preservación parcial del fascículo arqueado y de la sustancia blanca global, dado que los valores de AF y MD estaban en el rango de los obtenidos en el grupo de voluntarios sanos o sólo ligeramente alterados. Por último, la exploración neuropsicológica realizada tras la recuperación de la conciencia reveló la recuperación del funcionamiento lingüístico receptivo. Se evidenciaron únicamente las alteraciones neuropsicológicas propias de la lesión focal en el hemisferio derecho, es decir, alteraciones de la atención, memoria de trabajo y función visuo-perceptiva. Una segunda exploración realizada dos meses más tarde mostró una mejoría significativa en todas las funciones alteradas. Estudio III: El análisis de los índices extraídos de los histogramas reveló diferencias significativas en la integridad del tejido en la sustancia blanca subcortical, tronco encefálico y tálamo entre los dos grupos de pacientes y el grupo de voluntarios sanos. Así mismo, se encontraron diferencias significativas entre los pacientes en EV y en EMC en sustancia blanca subcortical y tálamo. Los pacientes en EV mostraron un una reducción en la altura del pico (U = 16, p=0.006) y un aumento de su ancho (U = 8, p=0.003) en la máscara de sustancia blanca subcortical, así como una reducción de su altura (U = 4, p<0.001) en el tálamo comparados con los pacientes en EMC. No se encontraron diferencias estadísticamente significativas para el tronco encefálico. El análisis de regresión lineal reveló que la altura del pico para estas dos regiones predecía significativamente la categoría clínica de los pacientes medida mediante la CRS-R (F2,16=13.35, p<0.001). Usando estas dos medidas se realizó un análisis de regresión logística binaria para evaluar la contribución de cada factor en la clasificación diagnóstica de los pacientes. El modelo que incluía los dos factores mostró una capacidad predictiva del 94.7% (χ2(1)=5.74, p=0.017; Nagelkerke-R2=0.88; Hosmer and Lameshow χ2(8)=2.79, p=0.95). 126 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Estudio IV: Los pacientes, considerados globalmente, mostraron una reducción estadísticamente significativa del volumen total del tálamo comparado con los controles (F1,26=9.888; p=0.004). Esta reducción se observó también cuando los pacientes en EV se compararon con los controles (F1,21=12.167; p=0.002) pero no alcanzó la significación estadística cuando los pacientes en EMC se compararon con los controles (F1,21=3.577; p=0.072). En el grupo total de pacientes el volumen del tálamo correlacionó negativamente con la puntuación en la escala DRS (r=0.717; p=0.023) y mostró una correlación marginal con la LCFS (r=0.567; p=0.071). El análisis de vértices reveló que las zonas más afectadas en el grupo de pacientes correspondían con el núcleo dorso-medial y la lámina medular interna. Así mismo, demostró que el patrón de atrofia era mucho más grave en el grupo de pacientes en EV que en el de pacientes en EMC. Por último, la atrofia en las zonas relacionadas con la lámina medular interna y el núcleo dorso- medial correlacionó con las puntuaciones en la DRS. Discusión general La presente tesis comprende cuatro artículos desarrollados para contribuir a una mejor comprensión de las bases neuroanatómicas y neurofuncionales de la alteración de la conciencia en el estado vegetativo y el estado de mínima conciencia. Estos síndromes clínicos se caracterizan por disociaciones entre arousal y awareness, que aparece ausente en el EV y fluctuante en el EMC, y de este modo suponen una oportunidad única para el estudio de los mecanismos neurológicos que subyacen a la conciencia humana. En el primer estudio nos centramos en la exploración del procesamiento lingüístico a través de la resonancia magnética funcional. Cuatro de seis pacientes (dos de ellos en EV y dos en EMC) mostraron respuestas cerebrales ante la estimulación auditiva comparables a las exhibidas por un grupo de voluntarios sanos. Dos de ellos (un paciente en EV y uno en EMC) mostraron además evidencia de procesamiento lingüístico. Estas proporciones fueron similares a las halladas en el único estudio grupal previo en el que se uso una tarea similar (Coleman et al. 2007), y sugieren que no hay una relación clara entre la respuesta cerebral a estimulación auditiva del paciente y su diagnóstico. No obstante, en un estudio más reciente en el que estos autores exploraron una muestra más amplia de pacientes, encontraron que este tipo de respuestas son más frecuentes en pacientes en EMC que en pacientes en EV (Coleman et al. 2009a). Contrariamente a nuestras predicciones, las respuestas al sonido mostradas por los cuatro pacientes implicaron tanto áreas primarias como áreas asociativas uni y multimodales, Summary | 127 PhD Thesis sugiriendo que ciertos procesos de integración podrían estar preservados en ellos. Además de estas respuestas, dos pacientes mostraron evidencia de procesamiento lingüístico, ya que activaban regiones de la red lingüística en el lóbulo temporal. Sin embargo, ninguno mostró respuestas en el giro frontal inferior izquierdo. Davis et al. (2007) demostraron, usando un contraste similar al nuestro, que las respuestas al lenguaje en el lóbulo temporal se mantienen en voluntarios sanos en un nivel de anestesia profunda. Por el contrario, las áreas frontales sólo mostraron activación cuando los participantes estaban despiertos y estaban especialmente implicadas en el procesamiento de ambigüedades semánticas. Así, nuestros resultados no han de ser interpretados como prueba de comprensión o de ningún tipo de experiencia consciente asociada con esta función. En cualquier caso, demuestran la discrepancia entre las respuestas conductuales mostradas por algunos de estos pacientes y la funcionalidad de las redes cerebrales subyacentes. El único paciente en EV que mostró respuestas en el contraste lingüístico, recuperó la conciencia y la capacidad comunicativa en los meses posteriores al estudio de RMf. Con el objetivo de caracterizar los mecanismos implicados en su recuperación, en nuestro segundo estudio combinamos estudios de RMf e ITD en el momento agudo y doce meses más tarde. Los tres únicos estudios longitudinales previos de pacientes que han recuperado tras un EV (Laureys et al. 2000b; Bekinschtein et al. 2005) o EMC (Voss et al. 2006) se centraron únicamente en los datos funcionales o estructurales o contrastaron datos adquiridos en varios momentos temporales sólo tras la recuperación. De este modo, nuestro estudio es el primero en combinar datos de RMf, ITD y evaluaciones clínicas y neuropsicológicas en el estudio de la recuperación tras un EV. El paciente sufrió un TCE grave que le causó una amplia lesión en el hemisferio derecho un mes antes de la primera sesión de RM. En dicho estudio, la RMf mostró respuestas al lenguaje en regiones posteriores del giro temporal superior y giro temporal medio. En la segunda sesión dichas respuestas se extendieron también a regiones más anteriores en estos dos giros y a la encrucijada parieto-temporal, reflejando una recuperación de la capacidad de integración multimodal (Stoeckel et al. 2009; Graves et al. 2010). El análisis de ITD apoyó y complementó la información obtenida del análisis de la RMf. Los valores de AF y DM en el fascículo arqueado, principal fascículo de la red lingüística, estaban dentro de valores normales o sólo ligeramente alterados en ambas sesiones, sugiriendo la preservación de su integridad estructural. Dicha preservación junto con la funcionalidad de esta red demostrada con la RMf llevaría a pensar en una buena recuperación de la función lingüística en caso de recuperación de la conciencia. En efecto, el paciente recuperó la capacidad comunicativa y la exploración neuropsicológica mostró la preservación de todas las funciones lingüísticas evaluadas. En este sentido, la información obtenida de la combinación de técnicas de neuroimagen estructural y funcional podría servir como guía en el diseño de intervenciones destinadas a mantener la 128 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities integridad de las redes cerebrales implicadas en las funciones que aparecen preservadas, una vez que la validez de estos métodos sea demostrada en muestras más amplias de pacientes. El segundo foco de interés en este estudio versaba sobre la funcionalidad de la red neuronal por defecto. En la primera sesión el paciente mostró un patrón anómalo de de- activaciones junto con una desconexión funcional parieto-frontal, que se normalizaron con la recuperación de la conciencia. Este hallazgo es consistente con estudios previos realizados con este tipo de pacientes que demostraron reducciones en la conectividad dentro de esta red, que correlacionaban con el nivel de conciencia (Boly et al. 2009; Vanhaudenhuyse et al. 2010b) y podían normalizarse con la recuperación de la misma (Laureys et al. 2000b). El análisis de la sustancia blanca mediante ITD no mostró evidencia de daños importantes en el tejido (exceptuando las zonas colindantes a la lesión parieto-temporal derecha). Los mecanismos comúnmente implicados en casos de lesión axonal difusa (Adams et al. 1982; Gennarelli et al. 1982), una de las alteraciones más frecuentes en los cerebros de pacientes en EV tras TCE (Kinney & Samuels 1994; Adams et al. 1999), no estuvieron presentes en este caso, dado que la causa fue una caída. La lesión axonal difusa puede provocar disrupciones en tractos cortico- subcorticales impidiendo reorganizaciones en las estructuras preservadas (Povlishock 1992), con lo que su ausencia en este caso puede estar indicando que la recuperación estuvo asociada a fenómenos de reorganización una vez que las complicaciones asociadas con la lesión primaria se resolvieron. En este sentido, varios estudios han mostrado el potencial de la ITD para predecir el pronóstico en casos de alteraciones de la conciencia agudas (Perlbarg et al. 2009; Tollard et al. 2009). Sin embargo, hasta la fecha, no se había explorado su aplicación para detectar diferencias entre pacientes en EV y en EMC in vivo. Con este propósito, en nuestro tercer estudio, aplicamos la ITD para evaluar la integridad estructural de la sustancia blanca subcortical, tronco encefálico y tálamo en un grupo de veinticinco pacientes en EV y EMC. De acuerdo con hallazgos anatomopatológicos previos los pacientes mostraron evidencias de daño estructural en las tres regiones estudiadas (Kinney & Samuels 1994; Adams et al. 1999; 2000). Así mismo, también de manera consistente con hallazgos post-mortem (Jennett et al. 2001), el análisis de ITD identificó diferencias entre estos dos grupos de pacientes en sustancia blanca subcortical y tálamo, relacionadas con una mayor gravedad en el grupo de pacientes en EV. La gravedad del daño en estas dos áreas también correlacionó con la gravedad clínica de la alteración de la conciencia evaluada mediante la CRS-R. Diversos estudios de conectividad funcional previos han sugerido que el EV y EMC son esencialmente síndromes de desconexión cortico-cortical y tálamo-cortical (Laureys et al. 1999; 2000b; Boly et al. 2009; Vanhaudenhuyse et al. 2010b). El análisis de la conectividad funcional puede aportar información sobre la conectividad estructural pero las relaciones entre ambas no siempre son directas, dado que dos regiones pueden estar funcionalmente relacionadas gracias a una tercera que actúa como nexo estructural Summary | 129 PhD Thesis (Damoiseaux & Greicius 2009). Nuestros resultados, a pesar de no estar basados en un análisis de la conectividad per sé, demuestran alteraciones globales en las fibras de sustancia blanca y el tálamo y dan, por tanto, soporte estructural a los hallazgos de estudios funcionales anteriores. Sin embargo, el objetivo principal de nuestro tercer estudio era comprobar si estos dos grupos clínicos podían ser diferenciados de manera fiable en base a medidas derivadas de la ITD. Usando los índices que mostraron el mayor tamaño del efecto en la comparación entre pacientes en EV y EMC fuimos capaces de clasificar a los pacientes en su categoría diagnóstica adecuada con un 95% de exactitud. Según nuestro conocimiento, esta es la primera vez que se ha conseguido diferenciar entre estos dos grupos de pacientes con exactitud in vivo. De confirmarse este hallazgo en muestras más amplias de pacientes, medidas de ITD podrían ser combinadas con exploraciones clínicas para ayudar al diagnóstico de estos pacientes y, quizás, reducir el alto porcentaje de error diagnóstico actual (Schnakers et al. 2009). Continuando con el análisis de las bases neuropatológicas de los estados vegetativo y de mínima conciencia, y guiados por la importancia del tálamo sugerida por numerosos autores así como por nuestros resultados en el estudio III, en el cuarto estudio evaluamos los patrones de atrofia global y regional de esta estructura, así como sus correlatos clínicos en una muestra de pacientes en EV y EMC. El grupo total de pacientes mostró una reducción del volumen global del tálamo con respecto al grupo de voluntarios sanos que correlacionó con la gravedad clínica, evaluada mediante la DRS. El análisis de forma reveló que la región más afectada era el cuerpo dorso-medial, probablemente reflejando la atrofia del núcleo dorso-medial y lámina medular interna subyacentes descrita en estudios neuropatológicos previos (Adams et al. 1999; Maxwell et al. 2004; 2006). Este hallazgo apoya teorías que defienden la importancia de estos dos núcleos en la conciencia humana (Schiff 2008) y su posible papel en la explicación de los efectos positivos de determinadas terapias farmacológicas (ver Pistoia et al. 2010) o de estimulación eléctrica del tálamo (Schiff et al. 2007) en estos pacientes. Así mismo, sugieren que técnicas como la empleada en este estudio pueden ser de utilidad para la identificación de pacientes que cuentan con una preservación suficiente del tálamo como para beneficiarse de este tipo de intervenciones modulatorias. Por otro lado, cuando los dos subgrupos de pacientes fueron comparados de modo independiente con el grupo control encontramos que los pacientes en EV mostraban un patrón mucho más generalizado de atrofia que se extendía a regiones anteriores y posteriores del tálamo, las cuales aparecían preservadas en los pacientes en EMC. El presente estudio no permite aislar los mecanismos exactos que subyacen dicha atrofia aunque, considerando la relativa heterogeneidad de lesiones presente en estos pacientes, cabría pensar que se debe a una combinación de fenómenos de degeneración transneuronal y retrógrada asociada con la lesión axonal difusa y la pérdida neuronal relacionada con la presencia de hipoxia (Adams et al. 1999). 130 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities En cualquier caso, la atrofia del tálamo podría ser considerada un marcador indirecto de una alteración más difusa que afecta a la conectividad tálamo-cortical y, así, los resultados de este estudio se integrarían con los del estudio anterior en su apoyo al concepto de desconexión para definir a estos síndromes. En resumen, tomados en conjunto, los cuatro estudios incluidos en la presente tesis doctoral contribuyen a una mejor caracterización de los mecanismos cerebrales implicados en la conciencia y las bases estructurales y funcionales de sus alteraciones. Nuestros resultados apoyan la utilidad de la RMf para detectar funciones cognitivas residuales en pacientes en EV y EMC que pueden no ser detectadas en las exploraciones clínicas convencionales. Así mismo, destacan el potencial de la combinación de técnicas de neuroimagen funcional y estructural en el estudio de estos pacientes, así como su posible uso futuro en la estimación del pronóstico. Por último, proporcionan por primera vez evidencia de que los pacientes en EV y en EMC pueden ser diferenciados en vivo en base a medidas estructurales, sugiriendo que las técnicas de RM podrían ser combinadas con otras medidas clínicas para ayudar a solucionar los problemas diagnósticos existentes en este grupo de pacientes. Conclusiones I. Algunos pacientes en EV y EMC tienen preservados aspectos del procesamiento auditivo y lingüístico en ausencia de respuestas conductuales a estos estímulos. Las respuestas cerebrales a dichos estímulos pueden implicar áreas asociativas en ambos grupos de pacientes, lo cual se asociada con un mejor pronóstico. La RMf desempeña un papel importante en la identificación de estas funciones cognitivas residuales que no pueden ser detectadas en la exploración clínica convencional. II. Las alteraciones de la conciencia en el EV están relacionadas con deficiencias en la funcionalidad y la conectividad funcional de la red neuronal por defecto. En ausencia de daño estructural significativo ambas se normalizan con la recuperación de la conciencia. III. Los pacientes en EV y EMC presentan alteraciones en la sustancia blanca subcortical, el tronco encefálico y el tálamo, las cuales pueden ser identificadas in vivo con ITD. Dichas alteraciones correlacionan con el nivel de conciencia evaluado mediante la CRS-R. Summary | 131 PhD Thesis IV. Las alteraciones en la sustancia blanca subcortical y el tálamo son de mayor gravedad en los pacientes en EV que en EMC. El análisis de ITD en estas dos regiones permite clasificar a los pacientes en sus respectivas categorías diagnósticas con exactitud. Esto sugiere la utilidad de esta técnica para proporcionar información que puede ayudar en el proceso diagnóstico. V. Los pacientes en EV y EMC muestran una reducción significativa del volumen del tálamo, que es especialmente pronunciada en el cuerpo dorso-medial, probablemente afectando al núcleo dorso-medial y la lámina medular interna. La atrofia en ambos núcleos está relacionada con la gravedad clínica. VI. Los pacientes en EV muestran un patrón de atrofia más generalizado, que afecta también a áreas anteriores y posteriores, preservadas en los pacientes en EMC. Esto apoya la utilidad de técnicas morfométricas en la detección de cambios sutiles que pueden permitir diferenciar estos dos grupos de pacientes. 132 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities 2. Resum de la tesi: L’estat vegetatiu i l'estat de mínima consciència: funció cerebral, connectivitat i anomalies estructurals. Glossari d’abreviacions AF: Anisotropia Fraccional LCFS: Level of Cognitive Functioning CDIC: Centre de Diagnòstic per la Imatge Clínic RM: Ressonància Magnètica. CRS-R: Coma Recovery Scale-Revised RMf: Ressonància Magnètica Funcional DM: Difussivitat mitja TC: Tomografia Computada DRS: Disability Rating Scale TCE: Traumatisme Cranioencefàlic EMC: Estat de Mínima Consciència TEP: Tomografia per Emissió de Positrons EV: Estat Vegetatiu WAIS: Wechsler Adult Intelligence Scale FDR: False Discovery Rate WBIC: Wolfson Brain Imaging Centre ITD: Imatge de Tensor de Difusió Introducció Tot i l‟ interès que al llarg dels anys la consciència humana ha desvetllat en la literatura científica, encara no existeix una definició que s‟accepti de forma universal. Malgrat això, en el context de la neurociència clínica es considera que la consciència és un sistema complex que inclou dos elements bàsics: alerta o nivell de consciència (wakefulness) i contingut de la consciència, o consciència per se (awareness) (James 1980). Per aquests dos components s‟han identificat diferents xarxes cerebrals. Aixi l‟arousal és una funció autonòmica o vegetativa que depèn d‟una xarxa que inclou el tronc encefàlic, l‟hipotàlem i determinats nuclis talàmics, estructures que formen part del clàssicament anomenat sistema reticular activador ascendent. L‟awareness depèn de complexes xarxes corticals, entre les que destaca l‟anomenada “xarxa neuronal per defecte” i les seves connexions recíproques amb els nuclis subcorticals (particularment amb el tàlem). L‟estat vegetatiu (EV), descrit per Jennett & Plum (1972), es caracteritza per una dissociació d‟aquests dos components. Summary | 133 PhD Thesis A diferència del coma, que es defineix com una absència completa d‟arousal (i per tant de consciència), l‟EV es defineix per la preservació de l‟arousal, que es manifesta per la presència de cicles de la son i vigília, i per la absència d‟evidència comportamental de la consciència d‟un mateix o del medi que l‟envolta. Els pacients amb EV, són incapaços de reaccionar amb intencionalitat a l‟estimulació i no manifesten capacitat de comunicació (Royal College of Physicians 2003). L‟EV es considera “persistent” si el pacient continua en estat vegetatiu un mes després de l‟inici del procés patològic que va causar el dany cerebral. Respecte al diagnòstic de “permanent” hi ha diferències de criteris segons els països i l‟etiologia. En cas de dany cerebral no traumàtic els criteris diagnòstics americans proposen tres mesos i els anglesos sis. En el cas de traumatismes cranioencefàlics (TCE) no es considera el diagnòstic de permanent fins passats dotze mesos. En l‟evolució del EV, alguns pacients progressen a un estat de mínima consciència (EMC) de sí mateixos o del medi, tot i que aquesta consciència es mostra deforma fluctuant i inconsistent (Giacino et al. 2002). El diagnòstic diferencial entre aquests subtipus de pacients amb pèrdua de consciència, es basa en l‟ avaluació clínica exhaustiva i repetida del repertori de comportaments espontanis i suscitats que mostra el pacient, amb l‟objectiu de determinar si és o no conscient del medi i de si és capaç d‟interactuar-hi de manera intencional. En molts dels casos però, determinar si un comportament és de caràcter reflex o premeditat és difícil de diferenciar, la qual cosa fa que el diagnòstic d‟aquests pacients en converteixi en una tasca de gran complexitat. De fet, s‟estima que un terç dels pacients diagnosticats d‟EV conserven signes de consciència i en alguns dels casos són fins i tot capaços de comunicar-se amb el seu entorn quan disposen de les eines adequades (Andrews et al. 1996; Childs et al. 1993; Schnakers et al. 2009). Realitzar un diagnòstic correcte, és essencial per planificar un bon abordatge terapèutic així com per prendre qualsevol decisió clínica basada en l‟estimació del pronòstic, ja que s‟ha demostrat que aquest és molt més favorable en els casos d‟EMC que en pacients amb EV (Giacino & Kalmar 1997; Luauté et al. 2010). En els darrers anys s‟ha experimentat un ràpid desenvolupament en el camp de l‟aplicació de les tècniques de neuroimatge en l‟estudi d‟aquests pacients, amb l‟objectiu d‟abordar les bases de l‟alteració de consciència, així com d‟aportar noves eines que puguin facilitar el diagnòstic. Les tècniques de neuroimatge funcional permeten obtenir informació sobre l‟activitat cerebral d‟un pacient en resposta a una determinada tasca cognitiva, durant l‟estimulació o fins i tot en repòs. Aquesta resposta cerebral és independent de la capacitat del pacient per emetre respostes externes. Mitjançant la tomografia per emissió de positrons (TEP), a finals dels anys 90 i a principis del 2000, diversos autors varen demostrar l‟existència de respostes comparables a les trobades en voluntaris sans en casos de pacients en EV exposats a diferents tipus d‟estimulació visual, auditiva o tàctil (Menon et al. 1998; Owen et al. 2002; 2005a; Kassubek et al. 2003). No obstant, els avantatges relacionats amb un major poder 134 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities estadístic, una millorada resolució espacial i temporal, i el fet de no implicar l'aplicació de radioisòtops, han contribuït a substituir l‟ús del TEP per l‟ús de la ressonància magnètica funcional (RMf) per a l‟avaluació de les funcions cognitives residuals en pacients en EV i EMC. Mitjançant l‟ús de la RMf diferents grups de treball han confirmat troballes preliminars dels estudis de TEP i amb mostres més amplies de pacients. Així, amb la RMf, s‟han identificat evidències de processament visual, tàctil o lingüístic en part dels pacients diagnosticats com a EV i/o EMC (Moritz et al. 2001; Bekinschtein et al. 2004; 2005; Schiff et al. 2005; Owen et al. 2005a; 2006; Staffen et al. 2006; Di et al. 2007; Coleman et al. 2007; 2009a; Zhu et al. 2009; Qin et al. 2010). Pel que fa al pronòstic, s‟ha constatat que la presència d‟activació en àrees associatives en pacients en EV prediu una evolució favorable amb una alta especificitat (Di et al. 2008). Això però, no ho converteix en un indicador de consciència i, per aquest motiu, s‟han desenvolupat paradigmes funcionals actius amb l‟objectiu d‟avaluar si un pacient és capaç d‟interactuar de manera intencional amb el medi modulant la seva activitat cerebral en resposta a ordres. A l‟any 2006, Owen et al., publicaren el cas d'una pacient en EV que després de mesos d'evolució del TCE, mostrava respostes comparables amb les de subjectes sans en demanar-li que s‟imaginés jugant al tenis o recorrent les habitacions de casa seva. Aquesta troballa va tenir un alt impacte en la comunitat científica. Recentment, aquests autors varen aplicar aquest paradigma a un grup de cinquanta-quatre pacients en EV i EMC. Varen trobar que el 9% d'ells mostraren respostes comparables als subjectes sans davant d'ordres, confirmant que, tot i que poc freqüent, aquesta capacitat pot estar present en alguns pacients en EV i EMC. Cal advertir, però, que tot i les amplies possibilitats que representen aquestes tècniques, sembla que només els que es troben en la part menys greu de l‟espectre se‟n poden beneficiar. En qualsevol cas, fins i tot en pacients que mostren evidència de consciència, segueix sent d‟interès l‟estudi de la resposta cerebral mitjançant RMf per caracteritzar el perfil de les funcions preservades en els diferents dominis cognitius. Dins les funcions cognitives, el llenguatge és una de les funcions més rellevants i el fet de demostrar evidència de processament lingüístic preservat pot tenir importants implicacions en la motivació dels familiars i cuidadors del pacient. En el moment del plantejament del projecte que va conduir a aquesta tesi, només hi havia un estudi de grup que explorava específicament el processament lingüístic en pacients en EV i EMC (Coleman et al. 2007). Un segon focus d‟interès de la neuroimatge funcional, ha estat l‟estudi de la funció cerebral en repòs. Els primers treballs realitzats amb TEP demostraren una important reducció global del metabolisme cerebral en pacients en EV i EMC (Levy et al. 1987; Laureys et al. 1999; 2002; Schiff et al. 2002; DeVolder et al. 1990; Tommasino et al. 1995; Rudolf et al. 1999; Kassubek et al. 2003), que era més pronunciada en àrees relacionades amb l‟anomenada actualment “xarxa neuronal per defecte” (Laureys et al. 1999; Kassubek et al. 2003). Summary | 135 PhD Thesis L‟esmentada xarxa inclou una sèrie de regions corticals (precuneus, cruïlla temporo-parietal i escorça prefrontal medial) que mostren fluctuacions sincròniques de baixa freqüència en condicions de repòs i redueixen la seva activitat mentre es realitzen tasques cognitives (Gusnard & Raichle 2001). Diversos autors han demostrat alteracions en el funcionament i la connectivitat d‟aquesta xarxa en estats de consciència alterada o reduïda com el somnambulisme, crisis d‟absències en l‟epilèpsia, son profund o anestèsia (Bassetti et al. 2000; Maquet 2000; Steriade 2001; Kaisti et al. 2002; Salek-Haddadi et al. 2003). Mitjançant estudis de RMf, s‟ha demostrat que la connectivitat en aquesta xarxa es troba reduïda en pacients en EV i EMC i que aquesta reducció correlaciona amb el nivell de consciència (Cauda et al. 2009; Vanhaudenhuyse et al. 2010b). El paper de la ressonància magnètica estructural en l‟estudi de pacients amb afectació de consciència està molt menys explorat. Els estudis morfomètrics han confirmat les troballes de treballs anatomopatològics previs (Kinney & Samuels 1994; Adams et al. 1999; 2000; Jennett et al. 2001), que havien descrit que les dues característiques més freqüents en pacients en EV després d‟un TCE eren la lesió axonal difusa graus 2 i 3 i les anomalies en el tàlem, (Kampfl et al. 1998a). Dins de les tècniques de neuroimatge estructural, la imatge de tensor de difusió (ITD) permet caracteritzar la microestructura del teixit cerebral mitjançant l‟estudi del moviment de les molècules d‟aigua. Es tracta d‟una tècnica especialment sensible a les alteracions de la substància blanca cerebral i del nuclis grisos subcorticals com per exemple el tàlem. Permet doncs, detectar canvis subtils que passen desapercebuts en seqüències de ressonància magnètica convencionals. Aquesta tècnica, ha demostrat tenir un alt potencial a l‟hora d‟estimar el valor pronòstic dels pacients aguts amb alteració de la consciència després de patir un TCE (Perlbarg et al. 2009; Tollard et al. 2009). Malgrat això, fins ara, cap estudi estructural no ha aconseguit trobar diferències entre els pacients en EV i els que estan en EMC. Objectius i hipòtesi La present tesi doctoral consta de quatre estudis, que comparteixen l‟objectiu general d‟examinar les bases neuroanatòmiques i neurofuncionals de l‟alteració de la consciència en l‟EV i l‟EMC. En primer lloc ens centrem en l‟avaluació de les respostes cerebrals al llenguatge mitjançant la RMf en un grup de pacients amb els esmentats estats (estudi I). En segon lloc, combinem tècniques de RMf, de ITD i exploracions clíniques i neuropsicològiques per l‟estudi dels canvis cerebrals i clínics que varen menar la recuperació de la consciència en un pacient en EV (estudi II). En darrer lloc, ens centrem en l‟estudi de ITD (estudi III), així com imatges 136 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities estructurals d‟alta resolució potenciades en T1 (estudi IV) per caracteritzar in vivo els patrons neuropatològics presents en els pacients en EV i EMC i explorar les possibles diferències entre aquests dos subgrups. En el tercer estudi, investiguem les alteracions de la substància blanca subcortical, tronc cerebral i tàlem, basant-nos en treballs post mortem previs. Fonamentant-nos en els resultats obtinguts en l‟esmentat estudi, en el quart ens centrem en l‟estudi dels patrons d‟atròfia global i regionals del tàlem. Els objectius i hipòtesis dels quatre estudis s'especifiquen en els següents punts: - Estudi I: Resposta cerebral al llenguatge en l’estat vegetatiu i l’estat de mínima consciència després d’un traumatisme cranioencefàlic. Objectius: 1) Estudiar la possible preservació del processament auditiu i lingüístic en pacients en EV i EMC en base a les respostes cerebrals al so i al llenguatge oral en un paradigma jeràrquic de RMf. 2) Avaluar si existeixen diferències en els patrons d‟activació que mostren aquests dos grups de pacients. 3) Explorar el possible valor pronòstic de la presència d‟activació cerebral en un paradigma de RMf. Hipòtesis: 4) Alguns pacients en EV o en EMC mostraran respostes cerebrals específiques davant estímuls auditius i lingüístics. 5) Tot i que pot aparèixer en ambdós grups de pacients, l‟activació de les àrees d‟alt nivell associatiu serà més freqüent en pacients en EMC que en pacients en EV. 6) L‟activació cerebral d‟àrees d‟alt nivell associatiu tindrà valor predictiu per un pronòstic més favorable. - Estudi II: Combinació d’imatges de tensor de difusió i ressonància magnètica funcional durant la recuperació de l’estat vegetatiu. Objectius: 1) Estudiar l‟evolució d‟un pacient en EV des d‟un mes després de l‟accident fins a la recuperació de la consciència, amb el propòsit d‟explorar els canvis cerebrals que acompanyen l‟esmentada recuperació i centrant-nos en la funció de la xarxa lingüística i la xarxa neuronal per defecte. Summary | 137 PhD Thesis 2) Relacionar les respostes cerebrals identificades mitjançant RMf amb l'integritat estructural de les xarxes cerebrals implicades en la funció lingüística. 3) Caracteritzar el perfil de seqüeles cognitives i explorar la relació entre les respostes cerebrals al llenguatge quan el pacient estava en EV i la funció lingüística subseqüent avaluada mitjançant una bateria neuropsicològica. Hipòtesis: 1) La funció lingüística del pacient un cop realitzada la recuperació de la consciència estarà relacionada amb la funcionalitat de la xarxa lingüística avaluada mitjançant el paradigma de RMf. 2) El pacient serà capaç d‟activar àrees de major nivell associatiu en resposta al llenguatge quan hagi recuperat la consciència. 3) El pacient mostrarà disfuncions de la xarxa neuronal per defecte quan es trobi en EV i s‟observarà una recuperació parcial d‟aquesta xarxa després de la recuperació de la consciència. 4) La informació addicional obtinguda de l‟anàlisi de la xarxa neuronal per defecte i les imatges de ITD complementaran la obtinguda de l‟anàlisi del paradigma lingüístic de RMf i, per tant, en conjunt, contribuiran a una millor comprensió del perfil neuropsicològic del pacient després de la recuperació de la consciència. - Estudi III: L’estudi d’imatges de tensor de difusió diferència l’estat vegetatiu de l’estat de mínima consciencia. Objectius: 1) Estudiar la integritat de la substància blanca cerebral, tronc encefàlic i tàlem en dos grups de pacients en EV i EMC mitjançant l‟anàlisi de la difussivitat mitjana en les esmentades regions. 2) Avaluar si les característiques detectades in vivo mitjançant ITD en aquestes regions són consistents amb les trobades prèviament en els estudis post-mortem. 3) Avaluar si les mesures derivades de la ITD correlacionen amb el perfil clínic dels pacients. 4) Determinar si les mesures de ITD poden diferenciar entre pacients en EV i pacients en EMC. 138 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities Hipòtesis: 1) L‟anàlisi de la ITD permetrà la identificació de signes de lesió axonal difusa i anomalies en el tàlem en pacients en EV o EMC. 2) D‟acord amb els estudis post-mortem previs, les alteracions en la substància blanca cerebral i el tàlem seran més greus en els pacients en EV, malgrat no haver-hi diferències en el tronc cerebral. 3) La gravetat clínica, identificada mitjançant la Coma Recovery Scale-Revised, estarà relacionada amb la gravetat del dany en les dues regions estudiades. - Estudi IV: Reduccions en el volum talàmic i canvis de forma regional en el estat vegetatiu i l’estat de mínima consciència. Objectius: 1) Determinar si és possible detectar in vivo l'atròfia talàmica global i regional en pacients en EV i EMC mitjançant una anàlisi de la seva forma basada en vèrtexs. 2) Avaluar si hi ha diferències morfològiques en el tàlem entre aquests dos grups de pacients, així com si aquestes diferències correlacionen amb la gravetat clínica. Hipòtesis: 1) Tant l‟atròfia global com la regional serà més marcada en els pacients en EV que en els pacients en EMC i aquesta explicarà parcialment els seus respectius perfils clínics. Mètode Pels estudis inclosos en la present tesi doctoral hem comptat amb dos mostres diferents de pacients amb pèrdua de consciència i s‟han aplicat diferents tècniques d‟anàlisi de neuroimatge, així com diferents proves d‟exploració clínica i neuropsicològica. Els estudis varen ser aprovats pels comitès d‟ètica corresponents de les institucions implicades en el treball (Hospital Clínic de Barcelona, Universitat de Barcelona y Addenbrooke´s Hospital, Cambridge). Els familiars dels pacients i els voluntaris sans, varen entregar consentiment informat per escrit prèviament de la participació en l‟estudi. Cada article conté una descripció detallada de les Summary | 139 PhD Thesis característiques dels respectius participants així com de les tècniques de neuroimatge i avaluació clínica emprades. No obstant, les principals característiques de cada estudi es resumeixen a continuació. La primera mostra (estudis I, II, IV) fou recollida a partir de pacients de l‟Institut de Neurorehabilitació Guttmann i l‟Hospital Clínic de Barcelona. Entre tots el pacients ingressats des del 2007 al 2009 vàrem seleccionar els que complien criteris d‟inclusió i exclusió. Els primers foren: etiologia traumàtica, diagnòstic d‟EV (Royal College of Physicians 2003) o EMC (Giacino et al. 2004) i edat inferior a 55 anys. Com criteris d‟exclusió es van tenir en compte: història d‟alteracions neurològiques o psiquiàtriques prèvies, discapacitat intel·lectual premòrbida, implants metàl·lics incompatibles amb la ressonància magnètica i complicacions respiratòries greus. Vint pacients complien aquests criteris. D‟ells, cinc varen haver de ser exclosos per complicacions mèdiques greus en els dies anteriors a la ressonància. En dos més dels casos la prova de ressonància magnètica es va haver d‟interrompre per agitació del pacient. Finalment, totes les imatges es varen sotmetre a un control de qualitat i, com a resultat, es varen excloure alguns pacients dels respectius estudis per la presència d‟artefactes a les imatges relacionats amb el moviment. En el primer estudi varen participar set dels pacients (tres en EV i quatre en EMC). Un dels pacients vegetatius inclosos en aquest estudi va recuperar la consciència i va ser inclòs en un protocol de seguiment, que va donar lloc a l‟estudi II. En l‟estudi IV varen participar quatre pacients en EV i vuit en EMC. El grup control es va estar format per vint voluntaris sans d‟edats compreses entre els 19 i els 49. L‟únic criteri d‟inclusió fou tenir edat menor a 55 anys i els criteris d‟exclusió els mateixos que els descrits pels pacients. La segona mostra (estudi III) fou recollida a partir de dos centres de neurorehabilitació del Regne Unit. Entre tots els pacients ingressats en aquests centres entre els anys 2006 i 2008 es varen seleccionar aquells que complien els criteris d‟inclusió i exclusió (iguals als utilitzats en la primera mostra amb l‟excepció de les restriccions relatives a l‟etiologia). Inicialment es varen incloure trenta pacients a l‟estudi. Cinc d‟ells no varen aconseguir completar l‟estudi degut a complicacions mèdiques o la presència d‟agitació en el moment de l‟adquisició de la RM. La mostra final de l‟estudi III va incloure deu pacients en EV i quinze en EMC. El grup control estava format per dotze voluntaris sans. Per la caracterització clínica dels pacients es recolliren les següents variables: dades demogràfiques (edat, gènere, anys d‟escolarització i lateralitat), gravetat del dany cerebral mitjançant la puntuació inicial i mínima en la Glasgow Coma (Teasdale & Jennett 1974; Wilson et al. 1998), etiologia, lesions identificades en TC i ressonància magnètica, temps d‟evolució i medicació. L‟exploració clínica es va realitzar administrant les escales Disability Rating Scale (DRS) (Rappaport et al. 1982), Rancho Los Amigos Level of Cognitive Functioning (LCFS) (Hagen et al. 1979) i Coma Recovery Scale-Revised (CRS-R) (Giacino et al. 2004). Així mateix, el perfil cognitiu del pacient que recuperà la consciència (estudi II), va ser avaluat mitjançant la 140 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities versió espanyola del Test de Boston pel diagnòstic de la afàsia (Goodglass & Kaplan 1986), el test d‟ Albert (Albert 1973), el test de Barcelona-revisat (Peña-Casanova 1991), els subtests de números i lletres del WAIS-III (Wechsler 1999), el test auditiu verbal de Rey (Lezak et al. 2004), i les fluències fonètiques (Artiola-i-Fortuny et al. 1999). Les imatges de ressonància es varen adquirir en el Centre de Diagnòstic per la Imatge Clínic (CDIC) de l‟Hospital Clínic de Barcelona, i el Wolfson Brain Imaging Centre (WBIC) de l‟Addenbrooke´s Hospital, Cambridge, Regne Unit. En ambdós centres es va utilitzar un escàner de ressonància magnètica de 3 Tesla (Magnetom Trio Tim, Siemens). Es varen adquirir imatges de ressonància magnètica funcional, aplicant un paradigma de processament lingüístic, imatges de tensor de difusió i imatges estructurals d‟alta definició en T1. Pel preprocessament i posterior anàlisi de les imatges de RMf es va emprar un protocol estàndard amb el software SPM5 (http://www.fil.ion.ucl.ac.uk/spm/). El processament auditiu es va avaluar mitjançant el contrast entre una sèrie de narracions reproduïdes normalment i invertides respecte a la línia de base, mentre que el processament lingüístic s‟avaluà contrastant entre els dos tipus de narracions. De manera addicional, en l‟estudi II es realitzà un anàlisi de connectivitat funcional per estudiar el funcionament de la xarxa neuronal per defecte. Pel processament de les ITD i imatges potenciades en T1 es va utilitzar el software FSL (http://www.fmrib.ox.ac.uk/fsl/). Concretament, per la ITD es varen emprar les eines BET (Smith 2003), FDT (Behrens et al. 2003), per obtenir els mapes d‟anisotropia fraccional (AF) i difussivitat mitjana (DM), i FAST (Zhang et al. 2001), per la creació de màscares. En l‟estudi III es va analitzar la distribució dels valors de DM en la substància blanca subcortical, tronc encefàlic i tàlem. En l‟estudi II es va estudiar la AF i DM en la substància blanca i el fascicle arquejat. En darrer lloc, les imatges potenciades a T1 es varen utilitzar per avaluar l‟atròfia talàmica utilitzant les eines SIENAX (Smith et al. 2002), per l‟ estimació del volum cerebral total i el FIRST (Patenaude 2007), per la segmentació i posterior anàlisi de la forma del tàlem. Per les anàlisis estadístiques i realització de gràfics s‟utilitzaren els programes SPSS 14, 16 i el SigmaPlot 10 respectivament. Resultats Estudi I: El grup control va mostrar activacions en el gyrus temporal superiors en el contrast entre els dos tipus de narracions preses conjuntament i la línia de base (processament auditiu) (p<0.01 corregit per múltiples comparacions mitjançant FDR). En el contrast entre les Summary | 141 PhD Thesis narracions produïdes normalment i les seves equivalents invertides (processament lingüístic) aquest grup mostrà activació en els gyrus temporal superior mig esquerres, gyrus temporals mig drets i el gyrus frontal inferior esquerre (p<0.01 corregit per múltiples comparacions amb FDR). Dos dels pacients en EV i dos en EMC mostraren una activació comparable a la del grup control en el contrast del processament auditiu, implicant bilateralment al gyrus temporal superior i mig. D‟ells, un pacient en EV i un pacient en EMC mostraren a més a més una activació comparable al grup control en el contrast del processament lingüístic, encara que centrada en els gyrus temporal superior i mig esquerre. L‟únic pacient vegetatiu que va mostrar activació del llenguatge fou el que va recuperar la consciència en els mesos posteriors. Esstudi II: L‟anàlisi de la tasca de RMf va mostrar activacions anatòmicament coherents amb les regions lingüístiques en el contrast de processament lingüístic tant en la primera exploració, quan el pacient es trobava en EV, com en la segona, quan ja havia recuperat la consciència. Tot i això, en la primera sessió el pacient presentà un patró anòmal de de-activacions, centrades exclusivament a la cruïlla parieto-temporal. A la segona sessió, aquest patró va ser molt similar al clàssicament descrit en voluntaris sans per la xarxa neuronal per defecte. L'anàlisi de la connectivitat funcional confirmà la presència de desconnexions parieto-frontals en la primera exploració, que es normalitzaren quan el pacient recuperà la consciència. L‟anàlisi d' ITD mostrà una preservació parcial del fascicle arquejat i de la substància blanca global, donat que els valors d‟AF i MD estaven en el rang dels obtinguts amb els voluntaris sans o lleugerament alterats. En darrer lloc, l‟exploració realitzada un cop recuperada la consciència del pacient, revelà la recuperació del funcionament lingüístic receptiu. S'evidenciaren només algunes de les alteracions pròpies de la lesió focal en l'hemisferi dret, és a dir, alteracions de l'atenció, memòria de treball i funció visuo-perceptiva. En una segona exploració realitzada dos mesos més tard, el pacient va presentar una millora significativa en totes les funcions cerebrals alterades. Estudi III: L'anàlisi dels índexs obtinguts dels histogrames revelà diferències significatives en la integritat del teixit en la substància blanca subcortical, tronc encefàlic i tàlem entre els dos grups de pacients i el grup de voluntaris sans. Així mateix, es varen trobar diferències significatives entre els pacients en EV i en EMC en la substància blanca subcortical i tàlem. Els pacients en EV mostraren una reducció a l'alçada del pic (U=16, p=0.006) i un augment de l'amplitud (U=4, p<0.001) dels histogrames en el tàlem comparats amb els pacients en EMC. Pel contrari, no es 142 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities varen trobar diferències estadísticament significatives pel tronc encefàlic. L'anàlisi de regressió lineal revelà que l'alçada del pic per aquestes dues regions predeia significativament la categoria clínica dels pacients mesurada amb CRS-R (F2,16=13.35, p<0.001). Utilitzant aquestes dues mesures es va realitzar un anàlisi de regressió logística binària per avaluar la contribució de cada factor en la classificació diagnòstica dels pacients. El model que incloïa els dos factors mostrà una capacitat predictiva del 94.7% (χ2(1)=5.74, p=0.017; Nagelkerke-R2=0.88; Hosmer and Lameshow χ2(8)=2.79, p=0.95). Estudi IV: Els pacients, a nivell global, mostraren una reducció estadísticament significativa del volum total de tàlem comparat amb els controls (F1,26=9.888; p=0.004). Aquesta reducció es va també observar quan els pacients en EV es varen comparar amb els controls (F1,21=12.167; p=0.002), encara que no va assolir significació estadística quan els pacients amb EMC es varen comparar amb els controls (F1,21=3.577; p=0.072). En el grup de pacients, el volum del tàlem correlacionà negativament amb la puntuació de l'escala DRS (r=0.717; p=0.023) i va mostrar una correlació marginal amb la LCFS (r=0.567; p=0.071). L'anàlisi de vèrtexs revelà que les zones més afectades en el grup de pacients corresponien amb el nucli dorso-medial i la làmina medul·lar interna. Així mateix, es demostrà que el patró d'atròfia era molt més greu en el grup de pacients en EV que en el de pacients amb EMC. Per últim, l'atròfia en les zones relacionades amb la làmina medul·lar interna i el nucli dorso-medial correlacionà amb les puntuacions del DRS. Discussió general La present tesi comprèn quatre articles desenvolupats per contribuir a incrementar el coneixement de les bases neuroanatòmiques i neurofuncionals de l'alteració de la consciència en l'EV i l' EMC. Aquests síndromes clínics es caracteritzen per dissociacions entre arousal i awareness, que és absent en l'EV i fluctuant però presents en l'EMC. Aquesta característica fa que proporcionin una oportunitat única per l'estudi dels mecanismes neurològics subjacents a la consciència humana. En el primer estudi ens centrem en l'exploració del processament lingüístic a partir de la RMf. Quatre de sis pacients (dos d'ells en EV i dos en EMC) mostraren respostes cerebrals davant l'estimulació auditiva comparables a les que s‟observen en un grup de controls sans. Summary | 143 PhD Thesis Dos d'ells (un pacient en EV i un en EMC) varen mostrar, a més a més, evidències de processament lingüístic. Les proporcions observades en el nostre estudi són similars a les descrites a un únic estudi grupal previ en el que es va utilitzar una tasca similar (Coleman et al. 2007). Els resultats suggereixen doncs que no hi ha una relació clara entre la resposta cerebral a l'estimulació auditiva del pacient i el seu diagnòstic clínic. Malgrat això, en un estudi més recent, en el que els mateixos autors varen explorar una mostra més àmplia de pacients, varen concloure que aquest tipus de respostes són més freqüents en pacients amb EMC que amb pacients en EV (Coleman et al. 2009a). Contràriament a les nostres prediccions, les respostes al so mostrades pels quatre pacients implicaren tant àrees primàries com àrees associatives uni i multimodals, suggerint que certs processos d'integració podrien estar preservats. A més d'aquestes respostes, dos pacients mostraren evidència de processament lingüístic, ja que activaven regions de la xarxa lingüística en el lòbul temporal esquerre. Tot i això, cap pacient va mostrar respostes en el gyrus frontal inferior esquerre. Davis et al., (2007) demostraren, utilitzant un contrast similar al nostre, que les respostes al llenguatge en el lòbul temporal es mantenen en voluntaris sans en un nivell d'anestèsia profunda. Pel contrari, les àrees frontals només mostraren activació quan els participants estaven desperts i s'implicaven en el processament d'ambigüitats semàntiques. Així, els nostres resultats no han de ser interpretats com una prova de comprensió o de cap tipus d'experiència conscient. En qualsevol cas, demostren discrepàncies entre les respostes conductuals mostrades per alguns d'aquests pacients i la funcionalitat de les xarxes subjacents. L'únic pacient en EV que mostrà respostes en el contrast lingüístic, va recuperar la consciència i la capacitat comunicativa en els mesos posteriors a la RMf. Amb l'objectiu de caracteritzar els mecanismes implicats en la seva recuperació, en el nostre segon estudi varem combinar la RMf i la ITD en el moment agut i dotze mesos més tard. El tres únics estudis longitudinals previs de pacients que han recuperat consciència després d‟un EV (Laureys et al. 2000b; Bekinschtein et al. 2005) o EMC (Voss et al. 2006), es centraren únicament en les dades funcionals o estructurals, o contrastaren les dades adquirides en diferents moments temporals només un cop recuperada la consciència. En resum, el nostre estudi és el primer en combinar dades de RMf, ITD i avaluacions clíniques i neuropsicològiques en l'estudi de la recuperació després d‟un EV. El pacient va patir un TCE greu que li va causar una àmplia lesió en l'hemisferi dret un mes abans de la primera sessió de RM. En aquest estudi, la RMf mostrà respostes al llenguatge en regions posterior del gyrus temporal superior i gyrus temporal mig. En la segona sessió les esmentades respostes es varen estendre també a regions més anterior en els dos mateixos gyrus i en la cruïlla parieto-temporal, mostrant una recuperació de la capacitat d'integració multimodal (Stoeckel et al. 2009; Graves et al. 2010). L'anàlisi d' ITD recolzà i complementà la informació obtinguda en l'anàlisi de la RMf. Els valors d'AF i MD en el fascicle arquejat, principal fascicle 144 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities en la xarxa lingüística, estaven dins dels valors normals o lleugerament alterats en ambdues sessions, suggerint una preservació de la seva integritat estructural. Dita preservació conjuntament amb la funcionalitat d'aquesta xarxa demostrada amb la RMf ens duria a pensar en una bona recuperació de la lingüística en cas de la recuperació de la consciència. En efecte, el pacient va recuperar la capacitat comunicativa i amb l'exploració neuropsicològica es mostrà la preservació de les capacitats lingüístiques avaluades. El segon punt d'interès d'aquest estudi versava sobre la funcionalitat de la xarxa neuronal per defecte. En la primera exploració el pacient mostrà un patró anòmal de de- activacions junt amb una desconnexió funcional parieto-frontal, que es va normalitzar un cop recuperada la consciència. Aquesta troballa es consistent amb estudis previs realitzats amb aquest tipus de pacients que demostraren reduccions en la connectivitat dins d'aquesta xarxa, que a la vegada, correlacionaven amb el nivell de consciència (Boly et al. 2009; Vanhaudenhuyse et al. 2010b) i podien normalitzar-se amb la recuperació de la mateixa (Laureys et al. 2000b). L'anàlisi de la substància blanca mitjançant ITD no va mostrar evidència de danys importants en el teixit cerebral (exceptuant zones properes a la lesió parieto-temporal dreta). Els mecanismes comunament implicats en els casos de lesió axonal difusa (Adams et al. 1982; Gennarelli et al. 1982), una de les alteracions més freqüents en els cervells de pacients en EV després d‟un TCE (Kinney & Samuels 1994; Adams et al. 1999), no varen estar presents en aquest cas, donat que la causa va ser una caiguda. La lesió axonal difusa pot provocar disrupcions en tractes cortico-subcorticals impedint reorganitzacions en les estructures preservades (Povlishock 1992), pel que la seva absència en aquest cas pot estar indicant que la recuperació va estar associada a fenòmens de reorganització una vegada que les complicacions associades a la lesió focal es varen resoldre. En aquest sentit, diferents estudis han demostrat el potencial de la ITD per predir el pronòstic en casos d'alteració aguda de la consciència (Perlbarg et al. 2009; Tollard et al. 2009). No obstant això, fins ara, no s'havia explorat la seva aplicació per detectar les diferències entre pacients en EV i en EMC in vivo. Amb aquest propòsit, en el nostre tercer estudi, varem aplicar l'ITD per avaluar la integritat estructural de la substància blanca subcortical, tronc encefàlic i tàlem en un grup de vint-i-cinc pacients en EV i EMC. D'acord amb les troballes anatomopatològiques prèvies, els pacients mostraren evidència de dany estructural en les tres regions estudiades (Kinney & Samuels 1994; Adams et al. 1999; 2000). Així mateix, també de manera consistent amb les troballes post-mortem (Jennett et al. 2001), l'anàlisi de ITD identificà diferències entre aquests dos grups de pacients en la substància blanca subcortical i el tàlem, relacionades amb una major gravetat en el grup de pacients en EV. La gravetat del danys en aquestes dues àrees també correlacionà amb la gravetat clínica de l'alteració de consciència avaluada mitjançant la CRS-R. Hi ha diversos estudis de connectivitat funcional previs que han suggerit que l' EV i EMC són essencialment síndromes de desconnexió córtico-cortical i Summary | 145 PhD Thesis talàmic-cortical (Laureys et al. 1999; 2000b; Boly et al. 2009; Vanhaudenhuyse et al. 2010b). L'anàlisi de connectivitat funcional pot aportar informació sobre la connectivitat estructural però les relacions entre ambdues no sempre són directes, donat que dues regions poden estar funcionalment relacionades gràcies a una tercera que actua com a nexe estructural (Damoiseaux & Greicius 2009). Els nostres resultats, tot i no estar basats en una anàlisi de la connectivitat per se, demostren alteracions globals en les fibres de substància blanca i tàlem, donant per tant suport estructural a les troballes d'estudis funcionals anteriors. L'objectiu principal del nostre estudi era comprovar si aquests dos grups clínics podien ser diferenciats de manera fiable en base a mesures derivades de la ITD. Utilitzant els índexs que mostraren una major mida de l'efecte en la comparació entre pacients en EV i EMC, varem ser capaços de classificar als pacients en la seva categoria diagnòstica pertinent amb un 95% d'exactitud. Segons el nostre coneixement, aquesta era la primera vegada que s'havia aconseguit diferenciar entre aquests dos grups de pacients amb exactitud in vivo. De confirmar-se aquesta descoberta en mostres més amplies de pacients, les mesures d'ITD podrien ser combinades amb exploracions clíniques per ajudar al diagnòstic d'aquests pacients i així poder reduir l'alt percentatge d'error diagnòstic actual (Schnakers et al. 2009). Tot continuant amb l'anàlisi de les dades neuropatològiques de l'EV i EMC, i guiats per la importància del tàlem suggerida per nombrosos autors així com pels nostres resultats de l'estudi III, en el quart estudi avaluem els patrons d'atròfia global i regional d'aquesta estructura, així com els seus correlats clínics en la mostra de pacients en EV i EMC. El grup total de pacients mostrà una reducció del volum talàmic global respecte al grup de voluntaris sans que correlacionà amb la gravetat clínica, avaluada mitjançant la DRS. L'anàlisi de forma revelà que la regió més afectada era el cos dorso-medial, probablement reflectint atròfia del nucli dorso- medial i de la làmina medul·lar interna subjacents descrita en estudis neuropatològics previs (Adams et al. 1999; Maxwell et al. 2004; 2006). Aquesta troballa recolza les teories que defensen la importància d'aquests dos nuclis en la consciència humana (Schiff 2008) i el seu possible rol en l'explicació dels efectes positius de determinades teràpies farmacològiques (veure Pistoia et al. 2010) o d'estimulació elèctrica de tàlem (Schiff et al. 2007). Així mateix, suggereixen que tècniques com les emprades en aquest estudi poden ser d'utilitat per la identificació de pacients que comptin amb una preservació suficient del tàlem com per beneficiar-se d'aquest tipus d'intervencions modulatòries. Per altra banda, quan els dos subgrups de pacients varen ser comparats de manera independent amb el grup control, trobàrem que els pacients amb EV mostraven un patró molt més generalitzat d'atròfia que comprenia regions talàmiques tant anteriors com posteriors, les quals semblaven estar preservades en els pacients amb EMC. El present estudi no permet aïllar els mecanismes exactes subjacents a aquesta atròfia, tot i que, si considerem la relativa 146 | Davinia Fernández-Espejo The vegetative and the minimally conscious states: brain function, connectivity and structural abnormalities homogeneïtat de lesions presents en aquests pacients, hauríem de pensar que podria ser deguda a una degeneració transneuronal i retrògrada associada amb la lesió axonal difusa i la pèrdua neuronal relacionada amb la presència d'hipòxia (Adams et al. 1999). En qualsevol cas, l'atròfia del tàlem podria ser considerat un marcador indirecte d'una alteració més difusa que afecta a la connectivitat tàlamo-cortical i, així, els resultats d'aquest estudi s'integrarien amb els de l'estudi anterior en suport al concepte de desconnexió per definir aquestes síndromes. En resum, i considerant tot el conjunt, els quatre estudis inclosos en la present tesi doctoral contribueixen a la caracterització dels mecanismes cerebrals implicats en la consciència i les bases estructurals i funcionals de les seves alteracions. Els nostres resultats recolzen la utilitat de la RMf per detectar funcions cognitives residuals en pacients en EV i EMC que poden no ser detectades en les exploracions clíniques convencionals. Així mateix, destaquen el potencial de la combinació de tècniques de neuroimatge funcional i estructural en l'estudi d'aquests pacients, així com el seu possible ús futur en l'estimació del pronòstic. Per últim, proporcionen per primera vegada evidència de que els pacients en EV i EMC poden ser diferenciats in vivo en base a mesures de RM estructurals. Aquests resultats suggereixen que les tècniques de RM podrien ser combinades amb altres mesures clíniques per ajudar a solucionar els problemes diagnòstics existents en aquest grup de pacients. Conclusions I. Alguns pacients en EV i EMC tenen preservats aspectes del processament auditiu i lingüístic en absència de respostes conductuals a aquests estímuls. Les respostes cerebrals als esmentats estímuls poden implicar àrees cerebrals associatives i poden associarse a un millor pronòstic. La RMf exerceix un paper important en la identificació d'aquestes funcions cognitives residuals que no poden ser detectades en l'exploració clínica convencional. II. Les alteracions de la consciencia en l‟EV estan relacionades amb les deficiències en la funcionalitat i la connectivitat funcional i de la xarxa neuronal per defecte. En absència de dany estructural significatiu ambdues es normalitzen amb la recuperació de la consciència. III. Els pacients en EV i EMC presenten alteracions en la substància blanca subcortical, el tronc encefàlic i el tàlem, les quals poden ser identificades in vivo Summary | 147 PhD Thesis amb ITD. Aquestes alteracions correlacionen amb el nivell de consciència avaluat mitjançant la CRS-R. IV. Les alteracions en la substància blanca subcortical i el tàlem són de major gravetat en els pacients en EV respecte als que estan en EMC. L'anàlisi mitjançant ITD en aquestes dues regions permet classificar als pacients en les seves respectives categories diagnòstiques amb exactitud. Aquest resultat suggereix la utilitat d'aquesta tècnica per proporcionar informació necessària pel correcte diagnòstic. V. Els pacients en EV i EMC mostren una reducció significativa del volum del tàlem, que és especialment prominent en el cos dorso-medial i la làmina medul·lar interna. L'atròfia en ambdós nuclis està relacionada amb la gravetat clínica. VI. Els pacients en EV mostren un patró d'atròfia més generalitzat que en els pacients en EMC, que afecta també a àrees anteriors i posteriors. 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