Spinal cord injury (SCI) is a devastating disease which poses health problems in human and veterinary medicine. SCI causes neurological disability, with loss of motor, sensory and autonomic function. This study investigated the efficacy of local treatment with IKVAV-peptide on spinal cord regeneration following compression injury at T12 vertebra in Balb-c mice. IKVAV-peptide is a membrane spanning peptide known to have a long half-life and the peptide motif IKVAV. Thirty Blab-c female mice were used. Hemilaminectomy was performed at T12 and spinal cords were compressed using extradural application of a 24 g modified aneurysm clip for 1 min in the treatment groups. After 24 hours mice were treated with one of 4 different treatments including isoleucine-lysine-valine-alanine-valaine(IKVAV), IKVAVpeptide, peptide and mannitol (vehicle). Functional improvement was assessed every day using Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. 28 days later, the mice were euthanized, and spinal cord segments were studied histologically. Statistical analysis, one-way and two-way analysis of variance (ANOVA) and linear regression model were used to measure some parameters and describe the outcome after SCI. Over a 4weeks period, IKVAV-peptide group demonstrated statistical and histological evidence of cellular reconstruction and behavioral improvement. The BBB score in the IKVAV-peptide group increased by 5.4 (25%) points, the IKVAV and peptide groups by approximately 1 point (5%) and the mannitol group by 4 points (19%). The number of protoplasmic astrocytes in the IKVAV-peptide group was significantly increased compared to IKVAV, mannitol and normal groups but not with the peptide group (p<0.001). Neuron and muscle bundle size were also increased significantly (p<0.05 and p<0.007, resp.) in the IKVAV-peptide group compared to other treatment groups. The treated control groups showed cellular and gross damages including neuron inactivation and muscle atrophy, gliosis and inability of movement. / Graduation date: 2013

It is known that muscles possess both intrinsic and reflexive responses to stretch, both of which have been studied extensively. While much is known about heterogenic and autogenic reflexes during XER, these have not been well characterized during locomotion. In this study, we mapped the distribution of autogenic and heterogenic feedback in hindlimb extensor muscles using muscle stretch in the spontaneously locomoting premammillary decerebrate cat. We used natural stimulation and compared stretch-evoked force responses obtained during locomotion with those obtained during XER. The goal was to ascertain whether feedback was modulated between the two states. We found that heterogenic feedback pathways, particularly those emanating from MG, remained inhibitory during locomotion while autogenic feedback specifically in MG increases in gain. Furthermore, increases in MG gain were due to force-dependent mechanisms. This suggests that rather than an abrupt transition from inhibition to excitation with changes in motor tasks, these pathways coexist and contribute to maintaining interjoint coordination. Increases in autogenic gain provide a localized loading reflex to contribute to the completion of the movement. The results of these experiments are clinically significant, particularly for the rehabilitation of spinal cord injured patients. To effectively administer treatment and therapy for patients with compromised spinal reflexes, a complete understanding of the circuitry is required.

Orientador: Alberto Cliquet Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T03:56:05Z (GMT). No. of bitstreams: 1 Cacho_EnioWalkerAzevedo_M.pdf: 5346795 bytes, checksum: 6e74c1ae0171c8a520aa3738fe6e1bc4 (MD5) Previous issue date: 2004 / Resumo: O efeito do treino de marcha através da estimulação elétrica neuromuscular (EENM) em pacientes com lesão da medula espinhal (LME) tem sido bem estabelecido. A medula espinhal humana reconhece apropriadas informações sensoriais e pode modular respostas motoras que facilitam a locomoção através do treinamento de marcha EENM assistido. Neste trabalho, dez pacientes (9 homens e uma mulher; 19 - 40 anos) com LME crônica (7 completas e 3 incompletas, nível neurológico abaixo de TI) foram avaliados no Laboratório de Biomecânica e Reabilitação do Aparelho Locomotor/Unicamp, no início e no fim de um programa de 30 sessões de treinamento de marcha assistido com EENM. Instrumentos utilizados para mensuração foram: American Spinal Injury Association (ASIA), Escala de Capacidade de Deambulação, Medida de Independência Funcional (MIF), Escala Modificada de Ashworth e o Registro Multicanais da Eletromiografia de superficie (EMG) dos músculos sóleos (SO), gastrocnêmios mediais (GA) e tibiais anteriores (TA) bilaterais. Os resultados demonstraram uma melhora do padrão EMG nos músculos GA e SO, durante a fase de apoio, e uma significante redução na fase de oscilação. A melhora no padrão EMG do TA ocorreu apenas à esquerda. Os resultados sugerem que o treino de marcha assistido com EENM pode induzir mudanças nos centros medulares espinhais / Abstract: The useful effect of locomotion training through neuromuscular electrical stimulation (NMES) in patients with spinal cord injury (SCI) has already been established. The human spinal cord recognizes the appropriate sensorial information and can modulate responses about the motor pool which facilitates walking under NMES training. In this series, ten patients (9 male and 1 female; 19 - 40 yrs-old) with chronic spinal cord injury (seven complete and three incompIete, neurologic leveI beIow T2) were evaluated at the Biomechanics & Rehabilitation Lab./University Hospital-Unicamp: first as soon as the patients joined the Programme and after 30 NMES based gait sessions. Clinical protocoIs of the American Spinal Injury Association (ASIA), Ability Ambulation Scale, Functional Independence Measure (FIM), Modified Ashworth Scale and the multichannel register surface of Electromyography (EMG) of muscIe soIeous (SO), gastrocnemious medialis (GA) and tibialis anterior (TA) were assessed. Results demonstrated an improvement in the EMG pattem of GA and TO muscles, during the stance phase and a significant decrease in the swing phase. Improvement in the EMG pattem in the TA muscle occurs just in the Ieft leg. Results suggest that gait training with NMES does induce changes in the spinal cord neural center, thus triggering the recovering of functional abilities/gait of paraplegics / Mestrado / Pesquisa Experimental / Mestre em Cirurgia

Orientador: José Irineu Gorla / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Educação Física / Made available in DSpace on 2018-08-27T17:57:13Z (GMT). No. of bitstreams: 1 Borges_Mariane_M.pdf: 1121036 bytes, checksum: 9b0d5cc7811b2bb052eac8f395a8074e (MD5) Previous issue date: 2015 / Resumo: Pessoas com Lesão na Medula Espinhal (LME) apresentam importantes alterações na composição corporal (CC), como o aumento da massa gorda e redução da massa magra. O monitoramento destas alterações através da avaliação é preponderante tanto para se atingir o desempenho esportivo desejado quanto para a saúde destes indivíduos. No entanto, ainda não existe um consenso sobre métodos de campo confiáveis de avaliação da CC nesta população e existe uma variedade de técnicas para uso em laboratório, que dificulta a proposta de indicadores referenciais. Desta forma, os objetivos do presente estudo foram verificar os principais métodos e alterações da CC em indivíduos com LME através de uma revisão de literatura; verificar o perfil da CC de atletas de handebol e rugby em cadeira de rodas com LME e analisar a aplicabilidade do método de pregas cutâneas em comparação aos resultados obtidos pela densitometria óssea (DXA). Para se atingir o primeiro objetivo os estudos relevantes para a revisão foram levantados nas bases de dados Pubmed e Science Direct até maio de 2014. Sendo que foram incluídos 38 estudos, destes, 29 estudos utilizaram como método de analise da CC a densitometria óssea, considerado um método válido para a análise da CC nesta população. Quanto aos métodos de campo, os estudos indicam que o IMC e as equações de predição subestimam a gordura corporal nesta população já a circunferência abdominal e a relação cintura-quadril podem predizer a gordura visceral e risco de doenças cardiovasculares. No que tange às alterações na composição corporal os estudos apontaram para um aumento significativo da massa (MG) e diminuição da massa magra (MM) corporal e regional devido à inatividade física e atrofia causada por desuso nos membros paralisados. A fim de verificar o perfil da CC de atletas com LME participaram deste estudo 12 indivíduos sendo, oito atletas de rugby em cadeira de rodas com tetraplegia e quatro atletas de handebol em cadeira de rodas com paraplegia. Desta forma pode-se observar acúmulo de gordura na região do tronco e de membros inferiores, sendo que a massa corporal (r=0,75, p=0,00) e o tempo de lesão (r=0,73, p=0,00) se mostraram influenciadores do acúmulo de gordura na região do tronco. A fim de analisar a aplicabilidade do método de pregas cutâneas verificou-se a relação entre as variáveis antropométricas e da composição corporal (DXA) e em seguida as variáveis com correlação ? 0,70 (r) foram incluídas em modelos de regressão linear e as variáveis que apresentaram coeficiente de determinação R² ? 0,70, foram incluídas em gráficos de Bland Altman a fim de verificar a concordância entre os métodos. Desta forma, constatou-se que somente as pregas cutâneas biciptal (r²= 0,71, p= 0,00), subscapular (r²= 0,83, p=0,00) e abdominal (r²= 0,70, p= 0,00) mostraram-se possíveis preditoras da massa gorda do tronco por DXA. Portanto, no que se refere à população estudada, podemos identificar que aparentemente o método antropométrico de pregas cutâneas possui limitações de forma que não estima precisamente os componentes da composição corporal segmentar / Abstract: People with Spinal Cord Injury (SCI) show some changes in body composition (BC) profile, such as the increasing in fat mass and reduced lean body mass. The monitoring of this change by assessing is important both to achieve the desired performance sport for healthy such as individuals. However, there is still no consensus on reliable field assessment methods BC this population and there is a variety of techniques for laboratory use which hinders the proposed reference indicators. Thus, the objectives of this study were to check the main methods and changes of BC in individuals with SCI through a literature review; check the profile of BC handball athletes and rugby wheelchair with SCI and analyze the applicability of the skinfold thickness method of comparing the results obtained by bone densitometry (DXA). To achieve the first objective relevant studies for review were raised in Pubmed and Science Direct until May 2014. Being that were included 38 studies, of these, 29 studies used as BC method of analysis bone densitometry, considered a valid method for the analysis of BC this population. As for field methods, studies indicate that the BMI and the prediction equations underestimate body fat in this population already waist circumference and waist-to-hip ratio may predict visceral fat and risk of cardiovascular disease. Regarding the changes in body composition studies indicated a significant increase in fat mass and decreased lean mass and regional body due to physical inactivity and atrophy caused by disuse in paralyzed limbs. In order to verify the BC profile of athletes with SCI, participated in this study 12 individuals (eight quadriplegic wheelchair rugby athletes and four paraplegic wheelchair handball athletes). Therefore it can be observed accumulation of fat in the trunk and lower limbs and body mass (r = 0.75, p = 0.00) and time of injury (r = 0.73, p = 0.00) proved influencers of fat accumulation in the region trunk. In order to verify the applicability of the skinfold thickness there was a relationship between the anthropometric variables and body composition (DXA), and then the variables with correlation coefficients ? 0.70 (r) were included in the linear regression models and the variables that presented coefficient of determination R² ? 0.70 were included in graphs Bland Altman in order to verify the agreement between the methods. Thus it was found that only the skinfold thickness bicipital (r² = 0.71, p = 0.00), subscapular (r² = 0.83, p = 0.00) and abdominal (r² = 0.70; p = 0.00) were possible predictors of the trunk fat mass by DXA. Therefore, with regard to the study population, we identified that apparently the anthropometric method skinfold thickness has limitations so that not precisely estimates the components of the segmental body composition / Mestrado / Atividade Fisica Adaptada / Mestra em Educação Física

This study examined the relationship between Bandura' s theory of self-efficacy and the rehabilitation outcome of spinal cord injured persons. The study elicited selfefficacy expectations from fifteen subjects on three occasions: admission and two and four weeks later. Patients rated how they expected to perform six weeks after admission on fifteen rehabilitation behaviors. Patients' ratings were compared to actual performance ratings made by the medical staff on the Barthel Index. Results reveal that subjects' predictions two weeks and four weeks after admission were accurate (r = .74, < .01; r = .89, p < .001, respectively). Findings support the limited applicability of Bandura's theory of spinal cord injury rehabilitation. Recommendations for future research include examining variables which enhance self-efficacy and using a larger, more homogeneous sample.

Thesis (MScMedSc (Rehabilitation))--University of Stellenbosch, 2005. / Many South African National Defence Force (SANDF) soldiers have suffered spinal cord injury either in the line of duty or otherwise. This injury affects all spheres of life (physical, emotional, social and psychological). Servicemen are often considered heroes of their country and are often the ones who need to set aside their emotions in order to fulfil their roles as soldiers. However, it is anticipated that a permanent impairment/disability e.g. SCI will have an impact on their quality of life (QOL) and satisfaction with living (SWL). This study aims to explore whether emotional support offered to soldiers with spinal cord injury (SCI) during rehabilitation improve their long-term QOL and SWL. Thirteen soldiers who have been re-employed post injury were asked to complete a selfcompiled, self-administered questionnaire regarding the emotional support offered during rehabilitation and the impact thereof on long-term QOL and SWL. The questionnaire focused on their ratings of the QOL and SWL in different areas of their lives and circumstances and asked what they would advise newly injured individuals regarding SCI and rehabilitation. Data was analysed using a combination of qualitative and quantitative methods. Pearson’s chi-square test and the M-L chi-square tests were used to analyse the data with the Statistica programme. A p-value of < 0.05 were calculated as statistically significant. Emotional support during rehabilitation showed a significant impact on QOL (p=0.0497). Ninety-two percent (n=12) of participants rated their QOL as good or excellent while 77% (n=10) rated their SWL as good. Participants who were older than 26 reported a significantly higher rate of SWL than younger ones (p=0.0292). Furthermore, results showed that the family was the most constant source of support during rehabilitation (54%, n=7). Despite that, 77% (n=10) of participants felt that they received excellent emotional support from the rehabilitation team. The study results are intended to facilitate growth and development in the rehabilitation process and guide professionals in the offering of emotional support.

O presente estudo objetivou a avaliação do potencial regenerativo e trófico das CS e do fator neurotrófico PEDF sobre os neurônios da medula espinal através da cultura destas células in vitro assim como no modelo experimental da lesão medular contusa in vivo. CS foram cultivadas a partir de fragmentos de nervo ciático de ratos adultos jovens. As culturas primárias foram purificadas e utilizadas em experimentos in vitro e in vivo. Para avaliar se a cultura de CS tratadas com o PEDF proporciona o trofismo dos neurônios da medula espinal, estes foram cultivados a partir de fragmentos da medula espinal de ratos Wistar neonatos e tratados com o meio condicionado das CS (MCCS) pré-tratadas ou não com formas específicas do PEDF. A resposta trófica foi estudada através da quantificação do número de células bem como da área dos prolongamentos e dos corpos celulares dos neurônios cultivados sobreviventes no decurso temporal da cultura, utilizando-se método estereológico específico. Em outra série de experimentos, Ratos Wistar adultos foram submetidos à lesão medular contusa no 10º-11° segmento torácico pelo sistema NYU Impactor e tratados com inoculações locais de CS e do seu meio condicionado na presença ou não do PEDF. Ao término do procedimento cirúrgico, os ratos foram submetidos aos testes do comportamento BBB, CBS durante 6 semanas. Os tratamentos aplicados à cultura de neurônios da medula espinal demonstraram que o MCCS, pré-tratado ou não com diferentes formas moleculares do PEDF e os fatores por si só possuem ações tróficas e plásticas em relação aos neurônios da medula espinal. Foi observado que o MCCS exerceu um efeito de arborização e de trofismo sobre o corpo neuronal dos neurônios medulares, sendo que este efeito foi potencializado quando o MCCS foi obtido a partir de CS pré-tratadas com os fatores nPEDF ou coPEDF, e de intensidade ainda maior quando as formas puras dos fatores em questão foram adicionadas à cultura. Os animais submetidos ao trauma contuso da medula espinal que foram tratados com a inoculação das CS, pré-tratadas ou não com o nPEDF, do meio condicionado destas células e dos fatores nPEDF e coPEDF apresentaram melhor recuperação funcional em relação ao grupo controle. A partir dos dados obtidos neste trabalho pode-se dizer que as CS, seu meio condicionado e o PEDF possuem ações importantes sobre os neurônios da medula espinal, possibilitando a modulação do processo de recuperação funcional deste órgão após o trauma. / The present study aimed the assessment of the trophic and regenerative potential of the Schwann cells (SC) and of the neurotrophic factor PEDF on the spinal cord neurons by means of cultures of those cells in vitro as well as in an experimental model of a contused spinal cord lesion in vivo. SC were cultivated from sciatic nerves fragments of young adult rats. The primary cultures were purified and used in in vitro and in vivo experiments. To evaluate if the SC cultures treated with PEDF provide the trophism of the spinal cord neurons, these were cultivated from spinal cords fragments of new born rats and treated with SC conditional medium (SCCM), of cells pre-treated or not with specifics isoforms of PEDF. The trophic responses were analyzed by the quantification of the cell number, and the area of sprounting and perycaria of the survived neuronal cells in the culture time course using a specific stereological method. In another series of experiment, adults Wistar rats were submitted to a contuse spinal cord lesion in the 10º-11º thoracic level by means of the NYU Impactor and treated with local injection of SC and your CM with the presence or not of the PEDF. In the end of the surgical procedure, the rats were submitted to behaviors testes as the BBB and the CBS during the 6 follow weeks .The spinal cord neuron treatments show that the SCCM, prétreated or not with specifics molecular isoforms of PEDF and this factors by themselves demonstrated trophic and plastic actions with the spinal cord neurons. It was observed that the SCCM exerted a sprouting and trophic effect on the neuronal body of the spinal cord neurons, and that this effect was enhanced when the SCCM was achieved from SC pre-treated with PEDF, and even better when the pures forms of the PEDF was added to the culture. The animals that received the spinal cord lesion and were treated with injection of SC, pre-treated or not with nPEDF, or with the conditioned medium of those cells and the factors nPEDF or coPEDF showed a better functional recovery when compared to the control groups. The results of this study showed that the SC, your conditioned medium and the PEDF play an important role in the spinal cord neurons, allowing the modulation of the functional recovery process of this organ after the trauma.

A utilização experimental de CS é apontada como uma perspectiva benéfica no tratamento da lesão medular devido estas células produzirem fatores neurotróficos e citocinas inflamatórias que influenciam o ambiente do sistema nervoso. Ratos machos foram submetidos à lesão medular contusa de intensidade moderada (25mm) ao nível do 11o segmento torácico. As CS cultivadas a partir do nervo ciático foram pré-tratadas ou não com as citocinas inflamatórias TGF-1 (5ng/ml) ou TNF- (30ng/ml) e inoculadas no local da lesão imediatamente após o trauma. O grupo controle e o grupo lesão receberam inoculação do meio de cultura. Após a cirurgia os ratos foram submetidos à análise do comportamento motor durante 8 semanas. O BBB mostrou melhora do comportamento motor ao longo do período analisado para o grupo tratado com as CS. Este efeito não foi potenciado pelo pré-tratamento das CS com as citocinas inflamatórias. As medulas espinais foram processadas e receberam coloração pelo Violeta de Cresilo ou imunomarcação com a GAP-43, o glutamato, o NPY, a substância P, a GFAP, o FGF-2 e do NG2. Os tratamentos realizados neste desenho experimental não alteraram as respostas neuroprotetivas. Segundo análise estereológica não foram encontradas diferenças no número estimado de neurônios remanescentes e no volume de tecido preservado entre os grupos que receberam lesão medular. A lesão promoveu uma diminuição da imunorreatividade da GAP-43 no corno anterior, que foi revertida quando a lesão recebeu tratamento com as CS prétratadas ou não com a citocina TGF-1. O resultado do tratamento com as CS foi o de aumento na imunorreatividade do glutamato nas células no corno anterior e no funículo lateral quando comparado com o grupo lesão. Para o NPY no corno anterior houve aumento da imunorreatividade do grupo tratado com as CS apenas em comparação ao grupo controle. Já no funículo lateral a lesão aumentou a imunorreatividade NPY em comparação ao grupo controle, inclusive quando houve tratamento com as CS, mas este deixou de existir quando houve prétratamento das CS com as citocinas. Referente ao FGF-2, no funículo lateral e no fascículo grácil houve aumento da imunorreatividade no grupo tratado com as CS, acrescidas ou não de TGF-1, apenas em comparação ao grupo controle. Porém, o tratamento com as CS aumentou a imunorreatividade do FGF-2 na região do fascículo grácil. A imunorreatividade do FGF-2 aumentou na região de epicentro da lesão, e este efeito foi mantido nos grupos tratados com as CS, acrescidas ou não de TGF-1. Houve significante aumento da imunorreatividade da GFAP no corno anterior e no funículo lateral quando os grupos lesados foram tratados com as CS e/ou citocinas comparado ao grupo controle. Na região do epicentro, a lesão promoveu aumento da imunorreatividade da GFAP, que foi potencializado quando houve tratamento com as CS. A lesão promoveu aumento da imunorreatividade do NG2 no epicentro, inclusive quando houve tratamento com as CS. O melhor desempenho motor observado no grupo que recebeu inoculação de CS provavelmente está relacionado a fatores como respostas plásticas e neuroquímicas no tecido preservado da medula espinal. / The experimental use of Schwann cells is indicated on spinal cord treatment due to the ability of these cells to produce neurotrophic factors and inflammatory cytokines, influencing nervous system environment. Male rats were submitted to a moderate spinal cord contusion (25mm) at 11th thoracic level. Culture Schwann cells were obtained from sciatic nerves and pre-treated or not with inflammatory cytokines TGF-1 (5ng/ml) or TNF- (30ng/ml) and then were inoculated in lesion site just after trauma. Sham and lesion groups received culture medium inoculation. After surgery, rats were submitted to behavior analyses during 8 weeks. BBB showed motor recovery in the Schwann cell group. This effect was not potentiated by pretreated of Schwann cells with inflammatory cytokines. The spinal cords were processed for Cresil Violet or immunolabeling to GAP-43, glutamate, NPY, substance P, GFAP, FGF-2 and NG2. Stereological analyses showed no differences in the estimated number of remaining neurons and in the volume of the preserved tissue among the lesioned groups. The injury reduced GAP-43 immunoreactivity in ventral horn, which was reverted when injury was treated with Schwann cells pre-treated or not with TGF-1. Schwann cell treatment enhanced glutamate immunoreactivity in the ventral horn and lateral funiculus compared with lesioned group. In ventral horn, NPY immunoreactivity was enhanced in Schwann cells group compared with sham group. In lateral funiculus, the injury or the treatment with Schwann cells increased NPY immunoreactivity, comparing with sham group. FGF-2 of lateral funiculus and gracile fasciculus enhanced in Schwann cells group. Treatment with Schwann cells enhanced FGF-2 immunoreactivity in gracile fasciculus. Injury enhanced FGF-2 immunoreactivity in epicenter region, also observed in the groups treated by Schwann cells. In ventral horn and lateral funiculus when the lesioned groups were treated by Schwann cells and/or cytokines there was significant increase in GFAP immunoreactivity compared with sham group. In epicenter, the injury promoted GFAP immunoreactivity increase that was potentiated by Schwann cells treatment. The injury produced NG2 increase in epicenter, included by Schwann cells treatment. The motor recovery showed in Schwann cell group is probably related to neurochemical plasticity in spinal cord preserved tissue.

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury (SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal regeneration and promote functional recovery when delivered at the site of injury. However, chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity. Maintaining these infusion systems is invasive and clinically problematic. In this dissertation, three studies are reported that demonstrate our strategy to overcome current limitations of using chABC and develop a delivery system for facilitating chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo. Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational change of the enzyme was measured via circular dichroism (CD) with and without trehalose. When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4 weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a sustained release and showed that chABC released from the delivery system is still functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-microtube system was used to locally deliver chABC over two weeks at the lesion site following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and chABC loaded lipid microtubes was implanted at the top of the lesion site immediately following injury. To determine effectiveness of topical delivery of thermostabilized chABC, animal groups treated with single injection or gel scaffold implantation of chABC and penicillinase (P'ase) were included as controls. Two weeks after surgery, the functionality of released chABC and the cellular responses were examined by immunohistological analysis with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results demonstrated that thermostabilized chABC was successfully delivered slowly and locally without the need for an indwelling catheter by using the hydrogel-microtube delivery system in vivo. The results demonstrated that released chABC from the gel scaffold effectively digested CSPGs, and therefore, there were significant differences in CSPG digestion at the lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and controls. Third, a long term in vivo study (45 days) was conducted to examine axonal sprouting/regeneration and functional recovery with both a single treatment each of microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the hydrogel-microtube delivery system. Over the long term study period, the treated animals showed significant improvement in locomotor function and more sprouting of cholera toxin B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers around the lesion site. We demonstrated that this significant improvement of chABC thermostability facilitates the development of a minimally invasive method for sustained, local delivery of chABC that is potentially a useful and effective approach for treating SCI. In addition to that, we demonstrated that combinatorial therapy with chABC and neurotrophic factors could provide a synergistic effect on axonal regrowth and functional recovery after SCI.

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.