Regeneração da Medula Espinhal: Avaliação da actividade locomotora e dos efeitos da metilprednisolona no rato após lesão medular
Ficheiros
Data
2009
Autores
Pereira, José Eduardo Teixeira
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
A medula espinhal é uma estrutura bastante complexa tanto a nível anatómico e funcional, como em termos da resposta fisiológica quando lesionada. Da manutenção da sua integridade depende o normal funcionamento do organismo e, a severidade das alterações que a sua lesão proporciona depende do nível a que ela ocorre. A esmagadora maioria das lesões da medula espinhal está associada a traumatismos. Até há relativamente pouco tempo, era universalmente aceite que, após lesão, as células do sistema nervoso central eram incapazes de se regenerar. Apesar de, nos dias de hoje, essa realidade já ter sido alterada, esse processo de regeneração continua a ser extremamente complexo e limitado. Estas são as principais razões pelas quais ainda não existe nenhuma terapia que seja totalmente eficaz no tratamento do trauma medular agudo. As lesões da medula espinhal são um dos maiores desafios clínicos da neurologia e das neurociências. Desta forma se percebe que o estudo da recuperação funcional e a aplicação de diferentes terapias, após lesão da medula espinhal, represente um dos aspectos mais fascinantes a explorar no campo das neurociências.
Ao longo da história da investigação em lesões da medula espinhal, o rato tem assumido cada vez mais um papel de maior importância, tornando-se mesmo o modelo animal favorito para testar os diferentes tipos de lesão. Isto deve-se ao facto de ser um modelo animal que envolve um menor investimento financeiro, mas também, e não menos importante, pelo facto de apresentar semelhanças relativamente aos humanos, no que diz respeito à morfologia e patologia da medula espinhal contundida. O desenvolvimento de modelos animais apropriados para as alterações específicas da medula espinhal é um passo importante nesta área de investigação. Idealmente, as alterações promovidas nestes modelos animais devem ser relevantes para os sinais clínicos observados noutros animais e nos humanos. Um dos pontos-chave da investigação de lesões da medula espinhal é a capacidade de avaliar de uma forma fiável, reprodutível e precisa, as alterações na funcionalidade, de forma a caracterizar o tipo de lesão, bem como, ter a possibilidade de aferir a eficácia das intervenções efectuadas. Por estas razões, têm sido centralizados esforços no desenvolvimento e aperfeiçoamento dos diferentes métodos para caracterização da recuperação da função locomotora, após lesão da medula espinhal. Na execução dos trabalhos conducentes à elaboração desta tese, o tipo de lesão da medula espinhal efectuada foi a lesão de contusão na região toraco-lombar (T9-T11) que origina um quadro de paraplegia.
Por outro lado, apesar de estarem a ser desenvolvidos uma série de tratamentos empolgantes, em modelos animais de lesão da medula espinhal, as aplicações clínicas de agentes farmacológicos na lesão aguda da medula espinhal, é relativamente limitada. Actualmente, o succinato sódico de metilprednisolona (MPSS) é o tratamento estandardizado para a lesão aguda da medula espinhal, como consequência dos resultados obtidos nos National Acute Spinal Cord Injury Studies (NASCIS) II e III. Contudo, muitos já questionaram a eficácia do MPSS dados os seus efeitos marginais e às críticas ao desenho dos estudos e interpretação estatística.
Tendo em consideração os factos anteriormente descritos, surge o objectivo principal desta tese: contribuir para um aumento dos conhecimentos acerca do processo de regeneração da medula espinhal, nomeadamente na melhoria dos métodos de avaliação funcional e dos conhecimentos acerca da utilização do succinato sódico de metilprednisolona no tratamento da lesão moderada de contusão da região toracolombar do rato. Paralelamente à utilização dos métodos tradicionais para quantificar a regeneração nervosa, foi desenvolvida e aperfeiçoada uma metodologia específica para análise computorizada do movimento do rato.
Na elaboração da tese de dissertação foram considerados 10 capítulos principais: um primeiro capítulo introdutório, quatro capítulos que compõem a revisão bibliográfica, quatro capítulos de natureza experimental e por fim, um capítulo dedicado às conclusões da investigação realizada e às perspectivas futuras.
Capítulo 1 – Introdução
É constituído por uma breve nota introdutória ao tema.
Capítulo 2 – Fisiopatologia da Lesão da Medula Espinhal
Neste capítulo é realizada uma revisão bibliográfica que visa abordar de forma descritiva o processo fisiopatológico que ocorre após uma lesão da medula espinhal.
Capítulo 3 - A Aplicação do Succinato Sódico de Metilprednisolona no Trauma Medular Agudo
Neste capítulo é realizada uma revisão bibliográfica onde se aborda a temática da aplicação do succinato sódico de metilprednisolona (MPSS) no trauma medular agudo, quer do ponto de vista clínico, quer experimental.
Capítulo 4 - Outras Terapias Usadas no Trauma Medular Agudo
Neste capítulo é realizada uma revisão bibliográfica que descreve a maior parte das terapêuticas que foram utilizadas ao longo dos anos no processo de investigação do trauma medular agudo.
Capítulo 5 – Avaliação Funcional e Histológica do Rato Após Lesão da Medula Espinhal
Neste capítulo é realizada a descrição dos métodos de avaliação da função motora que consideramos ser mais importante incluir, quando se constrói uma bateria de testes para avaliar a função motora, após uma lesão medular na região toracolombar do rato, bem como, a descrição dos processos histológicos utilizados para caracterizar a lesão de contusão dos animais utilizados nos nossos trabalhos.
Capítulo 6 - O Efeito do Movimento da Pele na Análise Cinemática do Membro Pélvico Durante a Locomoção dos Ratos, no Tapete Rolante
Com este estudo pretendeu-se determinar o efeito de artefacto do movimento da pele no cálculo da cinemática das articulações do membro pélvico dos ratos, durante a locomoção em tapete rolante. A execução deste trabalho teve sempre subjacente uma dupla finalidade. Em primeiro lugar pretendeu-se expandir a limitada informação existente acerca do efeito do movimento dos tecidos moles do joelho sobre as posições angulares da anca, do joelho e do tornozelo dos ratos, durante a locomoção no tapete rolante. Em segundo, documentar a magnitude da deslocação da pele da articulação do joelho, quando se utilizam marcas coladas à superfície corporal. Para atingir os objectivos, foi proposto um método para estimar a posição da articulação do joelho, que foi desenhado na tentativa de melhorar a qualidade da análise dos dados da passada e, realizada a comparação da cinemática do membro pélvico no plano sagital, durante a locomoção no tapete rolante, determinada pela marca colocada no joelho e quando a marca do joelho foi determinada indirectamente por análise computacional. Os resultados mostraram que existe um considerável artefacto resultante da movimentação da pele, que se propaga para a posição da articulação do joelho e para a estimação da cinemática do membro pélvico.
Uma parte substancial deste capítulo foi já objecto de publicação:
– Filipe, V. M., Pereira, J. E., Costa, L. M., Mauricio, A. C., Couto, P. A., Melo-Pinto, P., Varejão, A. S. P. (2006). Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats. Journal of Neuroscience Methods, 153, pp. 55-61.
Capítulo 7 – Análise Comparativa da Cinemática dos Membros Pélvicos Durante a Locomoção Sobre o Solo e no Tapete Rolante, em Ratos
O trabalho de investigação apresentado neste capítulo, que foi o primeiro estudo comparativo que se centra nas diferenças da cinemática dos membros pélvicos entre a locomoção sobre o solo e no tapete rolante, em roedores. O objectivo desta investigação foi o de determinar a similaridade entre a locomoção dos ratos no tapete rolante e sobre o solo. Especificamente a cinemática angular a duas dimensões para os membros pélvicos, e as medidas espaciais e da passada, foram quantificadas para o andamento sobre solo e comparadas com os valores correspondentes quando os animais andaram sobre um tapete rolante motorizado. Para tal, foram avaliados dez ratos fêmea à mesma velocidade para a locomoção natural sobre o solo e no tapete rolante. A velocidade a que os animais andaram, a duração da fase de suspensão e o comprimento da passada, foram estatisticamente indistinguíveis, entre as duas condições a serem testadas. Foram encontradas diferenças estatisticamente significativas entre a locomoção sobre o solo e no tapete rolante para os parâmetros, duração do ciclo da passada e duração da fase de suporte. Durante a fase de suporte, o movimento angular das articulações da anca, do joelho e do tornozelo foram significativamente diferentes nas duas condições, com a existência de uma maior flexão quando andaram sobre o solo. Apesar disto, na fase de suspensão, os movimentos angulares do membro pélvico, no plano sagital, foram similares entre as duas condições testadas, com apenas três parâmetros a serem significativamente diferentes. Também só foram encontradas diferenças subtis nos parâmetros de altura da anca ao solo e ciclogramas ângulo-ângulo.
Uma parte substancial deste capítulo foi já objecto de publicação:
– Pereira, J. E., Cabrita, A. M., Filipe, V. M., Bulas-Cruz, J., Couto, P. A., Melo-Pinto, P., Costa, L. M., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2006). A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats. Behavioural Brain Research , 172, pp. 212-218.
Capítulo 8 - Comparação das Técnicas Bi-dimensionais e Tri-dimensionais na Determinação da Cinemática dos Membros Pélvicos Durante a Locomoção no Tapete Rolante, em Ratos, Após Lesão da Medula Espinhal
Neste capítulo encontra-se descrito um estudo onde se pretendeu determinar se deveria utilizar uma metodologia em 2D ou em 3D na avaliação da recuperação comportamental, após uma lesão da medula espinhal em ratos. Este, incluiu uma avaliação da locomoção num tapete rolante utilizando uma análise a 2D e a 3D, em ratos neurologicamente normais e após uma lesão de contusão medular moderada ao nível da vértebra T9. Na avaliação 2D foi utilizado o método de estimação da posição do joelho descrito no capítulo 6, para a avaliação 3D foi também desenvolvido um método novo e ligeiramente diferente. Os resultados para os dados cinemáticos em 2D e 3D, antes e depois da lesão da medula espinhal, mostraram que, de forma geral, o movimento angular articular foi muito semelhante. Todavia, observamos diferenças estatisticamente significativas nos valores angulares articulares em pontos selecionados do ciclo da passada, nomeadamente no que diz respeito à magnitude dos ângulos. A análise 3D da progressão do pé nos animais com lesão da medula espinhal mostra que estes apresentam uma rotação externa aumentada.
Uma parte substancial deste capítulo foi já objecto de publicação:
– Couto, P. A., Filipe, V. M., Magalhães, L. G., Pereira, J. E., Costa, L. M., Melo-Pinto, P., Bulas-Cruz, J., Maurício, A. C., Geuna, S., Varejão, A. S. P. (2008). A comparison of two-dimensional and three-dimensional techniques for the determination of hindlimb kinematics during treadmill locomotion in rats following spinal cord injury. Journal of Neuroscience Methods, 173, pp. 193–200.
Capítulo 9 - O Succinato Sódico de Metilprednisolona não Permite Melhorar a Recuperação Funcional e Histológica, em Ratos, Após Lesão da Medula Espinhal
O objectivo deste trabalho de investigação foi o de determinar, num modelo de roedores, os efeitos da administração de succinato sódico de metilprednisolona, após uma lesão de contusão medular moderada ao nível de T10, comparada com a administração de solução salina (SS). A recuperação funcional foi avaliada utilizando a escala locomotora de 21 pontos de Basso, Beattie e Bresnahan (BBB), o plano inclinado, o beam walk, a análise das pegadas e a escada horizontal. De forma a optimizar a precisão e fiabilidade dos resultados funcionais, foi avaliada a locomoção num tapete rolante utilizando uma análise tri-dimensional (3D). A estereologia foi utilizada para estimar a quantidade de tecido danificado. Os resultados dos métodos de avaliação funcional tradicionais mostraram que, a administração da dosagem de MPSS sugerida no National Acute Spinal Cord Injury Study (NASCIS) II, após uma lesão de contusão aguda da medula espinhal, não levou a quaisquer diferenças significativas na recuperação funcional em relação aos animais tratados com SS. É importante realçar que, os resultados da cinemática em 3D mostraram que a administração de MPSS não afectou a flexão/extensão das articulações da anca, joelho e tornozelo, durante o ciclo da passada. Por fim, também os resultados estereológicos revelaram que não existiram diferenças estatisticamente significativas entre os dois grupos experimentais.
Uma parte substancial deste capítulo foi já objecto de publicação:
– Pereira, J. E., Costa, L. M., Cabrita, A. M., Couto, P. A., Filipe, V. M., Magalhães, L. G., Fornaro, M., Di Scipio, F., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2009). Methylprednisolone sodium succinate fails to improve functional and histological outcome following spinal cord injury in rat. Experimental Neurology, (submetido para publicação).
Capítulo 10 – Conclusões e Perspectivas Futuras
Neste capítulo, são apresentadas as principais conclusões que se podem extrair dos resultados obtidos com os trabalhos experimentais realizados durante o processo de construção desta tese. De forma complementar são delineadas algumas perspectivas futuras respeitantes ao processo fisiopatológico e, às terapêuticas e métodos de avaliação funcional a utilizar no trauma medular agudo, bem como, as perspectivas futuras relativas ao trabalho a realizar pelo nosso grupo de investigação.
The spinal cord is a quite complex structure both on the anatomical and functional level, as well as in its physiological response after lesion. The normal functioning of the entire organism depends upon the maintenance of its integrity and the severity of an injury depends upon which level it takes place. The majority of spinal cord injuries (SCI) are associated to trauma. Until recently, it was universally accepted that, after an injury, the cells of the central nervous system were incapable of regenerating. Although, at the present time, this reality has changed, this regeneration process continues extremely complex and limited. These are the main reasons why an efficient therapy for the treatment of acute SCI does not exist. Spinal cord injuries are one of the greatest clinical challenges for neurology and neurosciences. Therefore, it is comprehensible that the study of functional recovery and the application of different therapies, after SCI, represent one of the most fascinating aspects to explore in the field of the clinical neuroscience. Throughout the history of SCI research, the rat has gained a role of greater and greater importance, becoming the paramount animal model to test the different types of injury. This is due to the fact of being an animal model that involves a low financial investment, but also, and not less important, because of its similarities to humans, regarding the morphology and pathology of the injured spinal cord. The development of appropriate animal models for the specific alterations of the spinal cord is an important step in this research area. Ideally, the alterations promoted in these animal models must be relevant for the clinical signs observed in other animals and in humans. One of the key points in SCI research is the capability to evaluate the functional alterations in a precise, reliable and reproducible way, in order to characterize the type of injury and include the possibility of surveying the effectiveness of interventions performed. For these reasons, efforts have been centralized in the development and improvement of different methods for the characterization of locomotion recovery after spinal cord injury. In the task implementations which lead to the elaboration of this thesis, a spinal contusion injury was performed in the toraco-lombar region (T9-T11), which creates a paraplegia framework. On the other hand, in spite of a large number of fascinating treatments being developed in SCI animal models, the clinical applications of pharmacologic agents in acute SCI is relatively limited. Currently, methylprednisolone sodium succinate (MPSS) is the standard treatment for acute SCI, as a consequence of the results obtained from the National Acute Spinal Cord Injury Studies (NASCIS) II and III. Nevertheless, many people have already questioned the effectiveness of MPSS due to its marginal effects and the criticism to the studies’ design and statistical interpretation. Taking into account the facts described above, the main objective of this thesis is to contribute to an increase of knowledge regarding the spinal cord regeneration process, namely in the improvement of functional evaluation methods and the knowledge concerning the use of methylprednisolone sodium succinate in the treatment of the moderate toraco-lombar contusion injury of the rat’s spinal cord. Simultaneous to the use of traditional methods to quantify the nervous regeneration, a specific methodology was developed and improved for computerized analysis of the rat’s locomotion. In the elaboration of the thesis, ten main chapters were considered: a first introductory chapter, four chapters which compose the bibliographical revision, four chapters of an experimental nature, and, finally, a chapter dedicated to the conclusions achieved by research and to future perspectives. Chapter 1 – Introduction It is composed of a brief introduction to the subject. Chapter 2 – Pathophysiology of the Spinal Cord Injury This chapter carries out a bibliographical revision which aims to describe the physiopatological process that takes place after a spinal cord injury. Chapter 3 – The Application of the Methylprednisolone Sodium Succinate in the Acute Medullar Trauma In this chapter, a bibliographical revision is carried out which deals with the application of the methylprednisolone sodium succinate (MPSS) in acute SCI, in a clinical and experimental point of view. Chapter 4 – Other Therapies Used in Acute Spinal Cord Trauma In this chapter, a bibliographical revision is done which describes most of the therapeutics used, throughout the years, in the process of SCI research. Chapter 5 – Functional and Histological Evaluation of the Rat after Spinal Cord Injury This chapter carries out the description of the evaluation methods of the motor function which were considered to be important to mention, subsequent to a battery of tests built to assess motor function following a toraco-lombar injury in the rat, as well as, the description of the histological processes used to characterize the spinal cord contusion injury performed in the animals used in our studies. Chapter 6 – Effect of Skin Movement on the Analysis of Hindlimb Kinematics during Treadmill Locomotion in Rats This study intended to determine the effect of skin movement artefact on the calculation of hindlimb joint kinematics during treadmill locomotion in rats. The purpose of this study was two-fold. The first objective was to expand the limited pool of information describing the effect of soft tissue movement of the knee upon the angular positions of the hip, the knee and the ankle of rats during treadmill locomotion. Secondly, it was a goal of this study to document the magnitude of skin displacement when using markers which were attached superficially to the knee joint. In order to achieve these objectives, a method was proposed to estimate the position of the knee joint by comparing the hindlimb kinematics in sagittal plane during treadmill locomotion determined by a marker attached to the knee and its position determined indirectly by computer analysis. Results showed that there is a considerable skin movement artefact which propagates to knee joint position and hindlimb kinematics estimates. A substantial part of the material presented in this chapter has already been published: – Filipe, V. M., Pereira, J. E., Costa, L. M., Mauricio, A. C., Couto, P. A., Melo-Pinto, P., Varejão, A. S. P. (2006). Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats. Journal of Neuroscience Methods , 153, pp. 55-61. Chapter 7 – A Comparison Analysis of Hindlimb Kinematics during Overground and Treadmill Locomotion in Rats The research presented in this chapter was the first comparative study focused on the differences of hindlimb kinematics between overground and treadmill locomotion in rodents. The objective of this investigation was to determine the similarity between overground and treadmill locomotion in rats. Specifically, twodimensional (2D) angular kinematics of the hindlimb and the temporal and stride measurements were quantified for overground walking and compared to the corresponding values when walking on a motor-driven treadmill. Therefore, ten female rats were evaluated at the same speed for natural overground and treadmill walking. The walking velocity, swing duration and stride length were statistically indistinguishable between the two testing conditions. Significant differences were found between overground and treadmill locomotion for step cycle duration and stance phase duration parameters. During the stance phase of walking, the angular movement of the hip, knee and ankle joints were significantly different in the two conditions, with greater flexion occurring on overground. Despite this, the sagittal joint movements of the hindlimb were similar between the two walking conditions, with only three parameters being significantly different in the swing. Hip height and angle–angle cyclograms were also only found to display subtle differences. A substantial part of the material presented in this chapter has already been published: – Pereira, J. E., Cabrita, A. M., Filipe, V. M., Bulas-Cruz, J., Couto, P. A., Melo-Pinto, P., Costa, L. M., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2006). A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats. Behavioural Brain Research , 172, pp. 212-218. Chapter 8 – A Comparison of Two-dimensional and Three-dimensional Techniques for the Determination of Hindlimb Kinematics during Treadmill Locomotion in Rats Following Spinal Cord Injury In this chapter, a study which was intended to determine whether a 2D or 3D approach should be considered for the assessment of behavioural recovery following SCI in rats is described. The study includes an examination of locomotion on a treadmill using 2D and 3D analysis, in neurologically intact animals and following moderate T9 contusion injury. During 2D assessment, the method of knee joint estimation was used as described in chapter 6; as to 3D evaluation, a new and slightly different method was developed. The results of 2D and 3D kinematic data before and after SCI revealed that the general shapes of the joint angular motion were very similar. However, statistically significant differences in joint angle values at selected points of the step cycle were observed. The 3D analysis of the foot progression in animals with SCI shows an increased external rotation. A substantial part of the material presented in this chapter has already been published: – Couto, P. A., Filipe, V. M., Magalhães, L. G., Pereira, J. E., Costa, L. M., Melo-Pinto, P., Bulas-Cruz, J., Maurício, A. C., Geuna, S., Varejão, A. S. P. (2008). A comparison of two-dimensional and three-dimensional techniques for the determination of hindlimb kinematics during treadmill locomotion in rats following spinal cord injury. Journal of Neuroscience Methods , 173, pp. 193–200. Chapter 9 – Methylprednisolone Sodium Succinate Fails to Improve Functional and Histological Outcome Following Spinal Cord Injury in Rats The purpose of this investigation was to determine, in the rodent model, the effects of MPSS following moderate T10 contusion injury compared to saline solution (SS). Functional recovery was evaluated by applying the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale, the inclined plane, the beam walk, foot print analysis and the horizontal ladder. In order to optimize the precision and accuracy of functional results the locomotion on a treadmill using three-dimensional (3D) was analysed. Stereology was used to estimate the amount of damaged tissue. The results of traditional assessment functional methods showed that the administration of the NASCIS II dosage of MPSS following acute spinal cord contusion did not lead to any significant differences in the functional recovery of MPSS vs. SS treated animals. It is important to emphasise that the results of 3D kinematic revealed that MPSS administration did not affect the flexion/extension of the hip, knee and ankle joints during the step cycle. Finally, stereological results also exposed that there were no statistically significant differences between the two experimental groups. A substantial part of the material presented in this chapter was submitted for publication: – Pereira, J. E., Costa, L. M., Cabrita, A. M., Couto, P. A., Filipe, V. M., Magalhães, L. G., Fornaro, M., Di Scipio, F., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2009). Methylprednisolone sodium succinate fails to improve functional and histological outcome following spinal cord injury in rats. Experimental Neurology, (submitted for publication). Chapter 10 –Conclusions and Future Perspectives In this chapter, the main conclusions which could be brought together as a result of the research at hand are presented, performed during the construction process of the present thesis. Afterwards, future perspectives are described regarding the pathophysiology, the therapeutic interventions and the functional evaluation methods used in SCI, as well as, the future working perspectives of our research group.
The spinal cord is a quite complex structure both on the anatomical and functional level, as well as in its physiological response after lesion. The normal functioning of the entire organism depends upon the maintenance of its integrity and the severity of an injury depends upon which level it takes place. The majority of spinal cord injuries (SCI) are associated to trauma. Until recently, it was universally accepted that, after an injury, the cells of the central nervous system were incapable of regenerating. Although, at the present time, this reality has changed, this regeneration process continues extremely complex and limited. These are the main reasons why an efficient therapy for the treatment of acute SCI does not exist. Spinal cord injuries are one of the greatest clinical challenges for neurology and neurosciences. Therefore, it is comprehensible that the study of functional recovery and the application of different therapies, after SCI, represent one of the most fascinating aspects to explore in the field of the clinical neuroscience. Throughout the history of SCI research, the rat has gained a role of greater and greater importance, becoming the paramount animal model to test the different types of injury. This is due to the fact of being an animal model that involves a low financial investment, but also, and not less important, because of its similarities to humans, regarding the morphology and pathology of the injured spinal cord. The development of appropriate animal models for the specific alterations of the spinal cord is an important step in this research area. Ideally, the alterations promoted in these animal models must be relevant for the clinical signs observed in other animals and in humans. One of the key points in SCI research is the capability to evaluate the functional alterations in a precise, reliable and reproducible way, in order to characterize the type of injury and include the possibility of surveying the effectiveness of interventions performed. For these reasons, efforts have been centralized in the development and improvement of different methods for the characterization of locomotion recovery after spinal cord injury. In the task implementations which lead to the elaboration of this thesis, a spinal contusion injury was performed in the toraco-lombar region (T9-T11), which creates a paraplegia framework. On the other hand, in spite of a large number of fascinating treatments being developed in SCI animal models, the clinical applications of pharmacologic agents in acute SCI is relatively limited. Currently, methylprednisolone sodium succinate (MPSS) is the standard treatment for acute SCI, as a consequence of the results obtained from the National Acute Spinal Cord Injury Studies (NASCIS) II and III. Nevertheless, many people have already questioned the effectiveness of MPSS due to its marginal effects and the criticism to the studies’ design and statistical interpretation. Taking into account the facts described above, the main objective of this thesis is to contribute to an increase of knowledge regarding the spinal cord regeneration process, namely in the improvement of functional evaluation methods and the knowledge concerning the use of methylprednisolone sodium succinate in the treatment of the moderate toraco-lombar contusion injury of the rat’s spinal cord. Simultaneous to the use of traditional methods to quantify the nervous regeneration, a specific methodology was developed and improved for computerized analysis of the rat’s locomotion. In the elaboration of the thesis, ten main chapters were considered: a first introductory chapter, four chapters which compose the bibliographical revision, four chapters of an experimental nature, and, finally, a chapter dedicated to the conclusions achieved by research and to future perspectives. Chapter 1 – Introduction It is composed of a brief introduction to the subject. Chapter 2 – Pathophysiology of the Spinal Cord Injury This chapter carries out a bibliographical revision which aims to describe the physiopatological process that takes place after a spinal cord injury. Chapter 3 – The Application of the Methylprednisolone Sodium Succinate in the Acute Medullar Trauma In this chapter, a bibliographical revision is carried out which deals with the application of the methylprednisolone sodium succinate (MPSS) in acute SCI, in a clinical and experimental point of view. Chapter 4 – Other Therapies Used in Acute Spinal Cord Trauma In this chapter, a bibliographical revision is done which describes most of the therapeutics used, throughout the years, in the process of SCI research. Chapter 5 – Functional and Histological Evaluation of the Rat after Spinal Cord Injury This chapter carries out the description of the evaluation methods of the motor function which were considered to be important to mention, subsequent to a battery of tests built to assess motor function following a toraco-lombar injury in the rat, as well as, the description of the histological processes used to characterize the spinal cord contusion injury performed in the animals used in our studies. Chapter 6 – Effect of Skin Movement on the Analysis of Hindlimb Kinematics during Treadmill Locomotion in Rats This study intended to determine the effect of skin movement artefact on the calculation of hindlimb joint kinematics during treadmill locomotion in rats. The purpose of this study was two-fold. The first objective was to expand the limited pool of information describing the effect of soft tissue movement of the knee upon the angular positions of the hip, the knee and the ankle of rats during treadmill locomotion. Secondly, it was a goal of this study to document the magnitude of skin displacement when using markers which were attached superficially to the knee joint. In order to achieve these objectives, a method was proposed to estimate the position of the knee joint by comparing the hindlimb kinematics in sagittal plane during treadmill locomotion determined by a marker attached to the knee and its position determined indirectly by computer analysis. Results showed that there is a considerable skin movement artefact which propagates to knee joint position and hindlimb kinematics estimates. A substantial part of the material presented in this chapter has already been published: – Filipe, V. M., Pereira, J. E., Costa, L. M., Mauricio, A. C., Couto, P. A., Melo-Pinto, P., Varejão, A. S. P. (2006). Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats. Journal of Neuroscience Methods , 153, pp. 55-61. Chapter 7 – A Comparison Analysis of Hindlimb Kinematics during Overground and Treadmill Locomotion in Rats The research presented in this chapter was the first comparative study focused on the differences of hindlimb kinematics between overground and treadmill locomotion in rodents. The objective of this investigation was to determine the similarity between overground and treadmill locomotion in rats. Specifically, twodimensional (2D) angular kinematics of the hindlimb and the temporal and stride measurements were quantified for overground walking and compared to the corresponding values when walking on a motor-driven treadmill. Therefore, ten female rats were evaluated at the same speed for natural overground and treadmill walking. The walking velocity, swing duration and stride length were statistically indistinguishable between the two testing conditions. Significant differences were found between overground and treadmill locomotion for step cycle duration and stance phase duration parameters. During the stance phase of walking, the angular movement of the hip, knee and ankle joints were significantly different in the two conditions, with greater flexion occurring on overground. Despite this, the sagittal joint movements of the hindlimb were similar between the two walking conditions, with only three parameters being significantly different in the swing. Hip height and angle–angle cyclograms were also only found to display subtle differences. A substantial part of the material presented in this chapter has already been published: – Pereira, J. E., Cabrita, A. M., Filipe, V. M., Bulas-Cruz, J., Couto, P. A., Melo-Pinto, P., Costa, L. M., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2006). A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats. Behavioural Brain Research , 172, pp. 212-218. Chapter 8 – A Comparison of Two-dimensional and Three-dimensional Techniques for the Determination of Hindlimb Kinematics during Treadmill Locomotion in Rats Following Spinal Cord Injury In this chapter, a study which was intended to determine whether a 2D or 3D approach should be considered for the assessment of behavioural recovery following SCI in rats is described. The study includes an examination of locomotion on a treadmill using 2D and 3D analysis, in neurologically intact animals and following moderate T9 contusion injury. During 2D assessment, the method of knee joint estimation was used as described in chapter 6; as to 3D evaluation, a new and slightly different method was developed. The results of 2D and 3D kinematic data before and after SCI revealed that the general shapes of the joint angular motion were very similar. However, statistically significant differences in joint angle values at selected points of the step cycle were observed. The 3D analysis of the foot progression in animals with SCI shows an increased external rotation. A substantial part of the material presented in this chapter has already been published: – Couto, P. A., Filipe, V. M., Magalhães, L. G., Pereira, J. E., Costa, L. M., Melo-Pinto, P., Bulas-Cruz, J., Maurício, A. C., Geuna, S., Varejão, A. S. P. (2008). A comparison of two-dimensional and three-dimensional techniques for the determination of hindlimb kinematics during treadmill locomotion in rats following spinal cord injury. Journal of Neuroscience Methods , 173, pp. 193–200. Chapter 9 – Methylprednisolone Sodium Succinate Fails to Improve Functional and Histological Outcome Following Spinal Cord Injury in Rats The purpose of this investigation was to determine, in the rodent model, the effects of MPSS following moderate T10 contusion injury compared to saline solution (SS). Functional recovery was evaluated by applying the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale, the inclined plane, the beam walk, foot print analysis and the horizontal ladder. In order to optimize the precision and accuracy of functional results the locomotion on a treadmill using three-dimensional (3D) was analysed. Stereology was used to estimate the amount of damaged tissue. The results of traditional assessment functional methods showed that the administration of the NASCIS II dosage of MPSS following acute spinal cord contusion did not lead to any significant differences in the functional recovery of MPSS vs. SS treated animals. It is important to emphasise that the results of 3D kinematic revealed that MPSS administration did not affect the flexion/extension of the hip, knee and ankle joints during the step cycle. Finally, stereological results also exposed that there were no statistically significant differences between the two experimental groups. A substantial part of the material presented in this chapter was submitted for publication: – Pereira, J. E., Costa, L. M., Cabrita, A. M., Couto, P. A., Filipe, V. M., Magalhães, L. G., Fornaro, M., Di Scipio, F., Geuna, S., Maurício, A. C., Varejão, A. S. P. (2009). Methylprednisolone sodium succinate fails to improve functional and histological outcome following spinal cord injury in rats. Experimental Neurology, (submitted for publication). Chapter 10 –Conclusions and Future Perspectives In this chapter, the main conclusions which could be brought together as a result of the research at hand are presented, performed during the construction process of the present thesis. Afterwards, future perspectives are described regarding the pathophysiology, the therapeutic interventions and the functional evaluation methods used in SCI, as well as, the future working perspectives of our research group.
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Medicina veterinária , Traumatismos da medula espinhal , Ratos , Medula espinhal , Regeneração