RESUMO
Regeneration after spinal cord injury is a goal of many studies. Although the most obvious target is to recover motor function, restoration of sensation can also improve the quality of life after spinal cord injury. For many patients, recovery of sensation in the perineal and genital area is a high priority. Currently there is no experimental test in rodents for measuring changes in sensation in the perineal and genital area after spinal cord injury. The aim of our study was to develop a behavioural test for measuring the sensitivity of the perineal and genital area in rats. We have modified the tape removal test used routinely to test sensorimotor deficits after stroke and spinal cord injury to test the perineal area with several variations. A small piece of tape (approximately 1â¯cm2) was attached to the perineal area. Time to first contact and to the removal of the tape was measured. Each rat was trained for 5 consecutive days and then tested weekly. We compared different rat strains (Wistar, Sprague-Dawley, Long-Evans and Lewis), both genders, shaving and non-shaving and different types of tape. We found that the test was suitable for all tested strains, however, Lewis rats achieved the lowest contact times, but this difference was significant only for the first few days of learning the task. There were no significant differences between gender and different types of tape or shaving. After training the animals underwent dorsal column lesion at T10 and were tested at day 3, 8, 14 and 21. The test detected a sensory deficit, the average time across all animals to sense the stimulus increased from 1'32 up to 3'20. There was a strong relationship between lesion size and tape detection time, and only lesions that extended laterally to the dorsal root entry zone produced significant sensory deficits. Other standard behavioural tests (BBB, von Frey, ladder and Plantar test) were performed in the same animals. There was a correlation between lesion size and deficit for the ladder and BBB tests, but not for the von Frey and Plantar tests. We conclude that the tape removal test is suitable for testing perineal sensation in rats, can be used in different strains and is appropriate for monitoring changes in sensation after spinal cord injury.
Assuntos
Adaptação Psicológica , Períneo/lesões , Períneo/fisiologia , Animais , Comportamento Animal , Feminino , Genitália/lesões , Masculino , Estimulação Física , Ratos , Ratos Endogâmicos Lew , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Wistar , Transtornos de Sensação/etiologia , Transtornos de Sensação/psicologia , Pele/lesões , Especificidade da Espécie , Traumatismos da Medula Espinal/psicologiaRESUMO
Axon regeneration in the CNS is blocked by inhibitory molecules in the environment and by a developmental loss of regenerative potential in CNS axons. Axon growth is a specialized form of cell migration, and for any cell to migrate there must be an adhesion molecule at the growth tip that recognizes a ligand in the environment, and which is linked to signaling and cytoskeletal mechanisms. The reasons for this loss of regenerative ability in CNS axons are several, but important contributors are the developmental loss of integrins that recognize ligands in the mature CNS environment, and selective trafficking of integrins and other molecules to exclude them from axons and direct them to dendrites. Regeneration of sensory axons in the spinal cord can be achieved by expression of tenascin-binding α9-integrin together with the integrin activator kindlin-1. This works because integrins are transported into sensory axons. Transport of integrins into retinal ganglion cell axons is seen in the retina, but may become more restricted in the optic nerve, with a subset of axons containing expressed integrins. Transduction of ganglion cells with α9-integrin and kindlin-1 should promote regeneration of this subset of axons, but attention to transport may be required for regeneration of the remaining axons.
Assuntos
Axônios/fisiologia , Integrinas/fisiologia , Regeneração Nervosa/fisiologia , Animais , Axônios/metabolismo , Integrinas/metabolismo , Nervo Óptico/fisiologia , Células Ganglionares da Retina/fisiologiaRESUMO
During postnatal development, closure of critical periods coincides with the appearance of extracellular matrix structures, called perineuronal nets (PNN), around various neuronal populations throughout the brain. The absence or presence of PNN strongly correlates with neuronal plasticity. It is not clear how PNN regulate plasticity. The repulsive axon guidance proteins Semaphorin (Sema) 3A and Sema3B are also prominently expressed in the postnatal and adult brain. In the neocortex, Sema3A accumulates in the PNN that form around parvalbumin positive inhibitory interneurons during the closure of critical periods. Sema3A interacts with high-affinity with chondroitin sulfate E, a component of PNN. The localization of Sema3A in PNN and its inhibitory effects on developing neurites are intriguing features and may clarify how PNN mediate structural neural plasticity. In the cerebellum, enhanced neuronal plasticity as a result of an enriched environment correlates with reduced Sema3A expression in PNN. Here, we first review the distribution of Sema3A and Sema3B expression in the rat brain and the biochemical interaction of Sema3A with PNN. Subsequently, we review what is known so far about functional correlates of changes in Sema3A expression in PNN. Finally, we propose a model of how Semaphorins in the PNN may influence local connectivity.
Assuntos
Matriz Extracelular/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Semaforina-3A/metabolismo , Animais , Proteínas da Matriz Extracelular/metabolismo , RatosRESUMO
The extracellular matrix (ECM) is known to regulate important processes in neuronal cell development, activity and growth. It is associated with the structural stabilization of neuronal processes and synaptic contacts during the maturation of the central nervous system. The remodeling of the ECM during both development and after central nervous system injury has been shown to affect neuronal guidance, synaptic plasticity and their regenerative responses. Particular interest has focused on the inhibitory role of chondroitin sulfate proteoglycans (CSPGs) and their formation into dense lattice-like structures, termed perineuronal nets (PNNs), which enwrap sub-populations of neurons and restrict plasticity. Recent studies in mammalian systems have implicated CSPGs and PNNs in regulating and restricting structural plasticity. The enzymatic degradation of CSPGs or destabilization of PNNs has been shown to enhance neuronal activity and plasticity after central nervous system injury. This review focuses on the role of the ECM, CSPGs and PNNs; and how developmental and pharmacological manipulation of these structures have enhanced neuronal plasticity and aided functional recovery in regeneration, stroke, and amblyopia. In addition to CSPGs, this review also points to the functions and potential therapeutic value of these and several other key ECM molecules in epileptogenesis and dementia.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Doenças do Sistema Nervoso , Neurônios/fisiologia , Animais , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Humanos , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/terapia , Neurônios/citologiaRESUMO
BACKGROUND: Loss of urinary control after spinal cord injury increases risk of urinary tract disease and is problematical for owners of affected dogs. OBJECTIVES: To design, implant, and test a sacral nerve stimulating device for controlling urine voiding in paraplegic dogs. ANIMALS: Nine pet dogs with severe thoracolumbar spinal cord injury causing paraplegia, loss of hindquarter sensation, and incontinence for more than 3 months. The procedure was offered prospectively to owners of suitable candidates after the irreversibility of the incontinence had been ascertained. METHODS: Open label clinical study. Surgically implantable electrode "books" were designed for insertion and retention of mixed sacral nerves. Sacral nerves were accessed via laminectomy and stimulated to test their ability to elicit detrusor contraction and then inserted into the electrode book, which was attached to a subcutaneously implanted, externally activated receiver. RESULTS: In 8/9 dogs, S2 nerves elicited the largest increases in intravesicular pressure with minimum stimulation and were placed in electrode books. Voiding efficiency was >90% in 8 of the 9 implanted dogs. No important detrimental effects of the procedure were observed. CONCLUSIONS AND CLINICAL IMPORTANCE: This sacral nerve stimulating implant is a simple and apparently effective neuroprosthetic device that restores urine voiding in paraplegic dogs.
Assuntos
Doenças do Cão/terapia , Terapia por Estimulação Elétrica/veterinária , Eletrodos Implantados/veterinária , Paraplegia/veterinária , Bexiga Urinaria Neurogênica/veterinária , Retenção Urinária/veterinária , Animais , Doença Crônica , Cães , Próteses Neurais , Reflexo , Raízes Nervosas Espinhais , Bexiga Urinaria Neurogênica/terapia , Retenção Urinária/terapiaRESUMO
Chondroitin sulfate is a glycosaminoglycan composed of N-acetylgalactosamine and glucuronic acid. It attaches to a core protein to form chondroitin sulfate proteoglycan (CSPG). Being a major component of the brain extracellular matrix, CSPGs are involved in neural development, axon pathfinding and guidance, plasticity and also regeneration after injury in the nervous system. In this review, we shall discuss the structure, the biosynthetic pathway, its functions in the nervous system and how we can improve regeneration in the nervous system by modulating its structure and binding properties.
Assuntos
Encéfalo/metabolismo , Sulfatos de Condroitina/metabolismo , Animais , Encéfalo/fisiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/química , Regulação da Expressão Gênica , Humanos , Plasticidade Neuronal , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
STUDY DESIGN: Retrospective, longitudinal analysis of sensory, motor and functional outcomes from individuals with thoracic (T2-T12) sensorimotor complete spinal cord injury (SCI). OBJECTIVES: To characterize neurological changes over the first year after traumatic thoracic sensorimotor complete SCI. METHODS: A dataset of 399 thoracic complete SCI subjects from the European Multi-center study about SCI (EMSCI) was examined for neurological level, sensory levels and sensory scores (pin-prick and light touch), lower extremity motor score (LEMS), ASIA Impairment Scale (AIS) grade, and Spinal Cord Independence Measure (SCIM) over the first year after SCI. RESULTS: AIS grade conversions were limited. Sensory scores exhibited minimal mean change, but high variability in both rostral and caudal directions. Pin-prick and light touch sensory levels, as well as neurological level, exhibited minor changes (improvement or deterioration), but most subjects remained within one segment of their initial injury level after 1 year. Recovery of LEMS occurred predominantly in subjects with low thoracic SCI. The sensory zone of partial preservation (ZPP) had no prognostic value for subsequent recovery of sensory levels or LEMS. However, after mid or low thoracic SCI, ≥3 segments of sensory ZPP correlated with an increased likelihood for AIS grade conversion. CONCLUSION: The data suggest that a sustained deterioration of three or more thoracic sensory levels or loss of upper extremity motor function are rare events and may be useful for tracking the safety of a therapeutic intervention in early phase acute SCI clinical trials, if a significant proportion of study subjects exhibit such an ascent.
Assuntos
Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Vértebras Torácicas/lesões , Adulto , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Regeneração Nervosa/fisiologia , Estudos Retrospectivos , Transtornos de Sensação/diagnóstico , Transtornos de Sensação/fisiopatologia , Transtornos de Sensação/reabilitação , Traumatismos da Medula Espinal/reabilitação , Adulto JovemRESUMO
STUDY DESIGN: Retrospective, longitudinal analysis of motor recovery data from individuals with cervical (C4-C7) sensorimotor complete spinal cord injury (SCI) according to the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI). OBJECTIVES: To analyze the extent and patterns of spontaneous motor recovery over the first year after traumatic cervical sensorimotor complete SCI. METHODS: Datasets from the European multicenter study about SCI (EMSCI) and the Sygen randomized clinical trial were examined for conversion of American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade, change in upper extremity motor score (UEMS) or motor level, as well as relationships between these measures. RESULTS: There were no overall differences between the EMSCI and Sygen datasets in motor recovery patterns. After 1 year, up to 70% of subjects spontaneously recovered at least one motor level, but only 30% recovered two or more motor levels, with lesser values at intermediate time points. AIS grade conversion did not significantly influence motor level changes. At 1 year, the average spontaneous improvement in bilateral UEMS was 10-11 motor points. There was only moderate relationship between a change in UEMS and a change in cervical motor level (r(2)=0.30, P<0.05). Regardless of initial cervical motor level, most individuals recover a similar number of motor points or motor levels. CONCLUSION: Careful tracking of cervical motor recovery outcomes may provide the necessary sensitivity and accuracy to reliably detect a subtle, but meaningful treatment effect after sensorimotor complete cervical SCI. The distribution of the UEMS change may be more important functionally than the total UEMS recovered.
Assuntos
Avaliação da Deficiência , Movimento/fisiologia , Quadriplegia/fisiopatologia , Quadriplegia/reabilitação , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Adulto , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Traumatismos da Medula Espinal/patologiaRESUMO
Age-related macular degeneration is the leading cause of blindness in the developing world. Retinal pigmented epithelium (RPE) transplantation in subretinal space, has been assessed in various animal models of age-related macular degeneration and in humans as a potential technique to preserve the visual function. However, the RPE cell survival posttransplantation is limited because of lack of attachment of the transplanted cells to the pathological Bruch's membrane and also partly because of iatrogenic removal of adhesive elements in the membrane during the removal of choroidal new vessels before transplantation procedure. Although pathological Bruch's membrane is well studied, there is still much debate as to why and how changes in the structure and components of this membrane leads to loss of RPE cells and disruption of their function and subsequent death of photoreceptors leading to visual loss. Integrins on RPE cells have been characterized and shown to be important for attachment of cells to Bruch's membrane. Considering the essential role of integrins in functions such as cell migration and adhesion, it is plausible that lack of attachment of RPE cells posttransplantation can be overcome by improving integrin function. Here, we have focused on some of the recent findings on the use of integrins and modulation of their function to improve the adhesion of RPE cells to normal and pathological Bruch's membrane. This work also aims at elucidating a potential mechanism by which accumulating inhibitory molecules in the Bruch's membrane in the pathological state, interferes with integrin function.
Assuntos
Lâmina Basilar da Corioide/patologia , Degeneração Macular/cirurgia , Epitélio Pigmentado Ocular/transplante , Animais , Lâmina Basilar da Corioide/metabolismo , Adesão Celular/fisiologia , Humanos , Integrinas/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologiaRESUMO
The International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) supported an international panel tasked with reviewing the methodology for clinical trials in spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the first of four papers. Here, we examine the spontaneous rate of recovery after SCI and resulting consequences for achieving statistically significant results in clinical trials. We have reanalysed data from the Sygen trial to provide some of this information. Almost all people living with SCI show some recovery of motor function below the initial spinal injury level. While the spontaneous recovery of motor function in patients with motor-complete SCI is fairly limited and predictable, recovery in incomplete SCI patients (American spinal injury Association impairment scale (AIS) C and AIS D) is both more substantial and highly variable. With motor complete lesions (AIS A/AIS B) the majority of functional return is within the zone of partial preservation, and may be sufficient to reclassify the injury level to a lower spinal level. The vast majority of recovery occurs in the first 3 months, but a small amount can persist for up to 18 months or longer. Some sensory recovery occurs after SCI, on roughly the same time course as motor recovery. Based on previous data of the magnitude of spontaneous recovery after SCI, as measured by changes in ASIA motor scores, power calculations suggest that the number of subjects required to achieve a significant result from a trial declines considerably as the start of the study is delayed after SCI. Trials of treatments that are most efficacious when given soon after injury will therefore, require larger patient numbers than trials of treatments that are effective at later time points. As AIS B patients show greater spontaneous recovery than AIS A patients, the number of AIS A patients requiring to be enrolled into a trial is lower. This factor will have to be balanced against the possibility that some treatments will be more effective in incomplete patients. Trials involving motor incomplete SCI patients, or trials where an accurate assessment of AIS grade cannot be made before the start of the trial, will require large subject numbers and/or better objective assessment methods.
Assuntos
Ensaios Clínicos como Assunto/normas , Recuperação de Função Fisiológica/fisiologia , Projetos de Pesquisa/normas , Traumatismos da Medula Espinal/terapia , Ensaios Clínicos como Assunto/métodos , Guias como Assunto , Humanos , Remissão Espontânea , Traumatismos da Medula Espinal/fisiopatologia , Resultado do TratamentoRESUMO
The International Campaign for Cures of Spinal Cord Injury Paralysis established a panel tasked with reviewing the methodology for clinical trials for spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the fourth of four papers. Here, we examine the phases of a clinical trial program, the elements, types, and protocols for valid clinical trial design. The most rigorous and valid SCI clinical trial would be a prospective double-blind randomized control trial utilizing appropriate placebo control subjects. However, in specific situations, it is recognized that other trial procedures may have to be considered. We review the strengths and limitations of the various types of clinical trials with specific reference to SCI. It is imperative that the design and conduct of SCI clinical trials should meet appropriate standards of scientific inquiry to insure that meaningful conclusions about efficacy and safety can be achieved and that the interests of trial subjects are protected. We propose these clinical trials guidelines for use by the SCI clinical research community.
Assuntos
Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/normas , Projetos de Pesquisa/normas , Traumatismos da Medula Espinal/terapia , Humanos , Avaliação de Resultados em Cuidados de Saúde/normasRESUMO
The International Campaign for Cures of Spinal Cord Injury Paralysis established a panel tasked with reviewing the methodology for clinical trials for spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the third of four papers. It examines inclusion and exclusion criteria that can influence the design and analysis of clinical trials in SCI, together with confounding variables and ethical considerations. Inclusion and exclusion criteria for clinical trials should consider several factors. Among these are (1) the enrollment of subjects at appropriate stages after SCI, where there is supporting data from animal models or previous human studies; (2) the severity, level, type, or size of the cord injury, which can influence spontaneous recovery rate and likelihood that an experimental treatment will clinically benefit the subject; and (3) the confounding effects of various independent variables such as pre-existing or concomitant medical conditions, other medications, surgical interventions, and rehabilitation regimens. An issue of substantial importance in the design of clinical trials for SCI is the inclusion of blinded assessments and sham surgery controls: every effort should be made to address these major issues prospectively and carefully, if clear and objective information is to be gained from a clinical trial. The highest ethical standards must be respected in the performance of clinical trials, including the adequacy and clarity of informed consent.
Assuntos
Ensaios Clínicos como Assunto/ética , Ensaios Clínicos como Assunto/normas , Seleção de Pacientes/ética , Projetos de Pesquisa/normas , Traumatismos da Medula Espinal/terapia , HumanosRESUMO
An international panel reviewed the methodology for clinical trials of spinal cord injury (SCI), and provided recommendations for the valid conduct of future trials. This is the second of four papers. It examines clinical trial end points that have been used previously, reviews alternative outcome tools and identifies unmet needs for demonstrating the efficacy of an experimental intervention after SCI. The panel focused on outcome measures that are relevant to clinical trials of experimental cell-based and pharmaceutical drug treatments. Outcome measures are of three main classes: (1) those that provide an anatomical or neurological assessment for the connectivity of the spinal cord, (2) those that categorize a subject's functional ability to engage in activities of daily living, and (3) those that measure an individual's quality of life (QoL). The American Spinal Injury Association impairment scale forms the standard basis for measuring neurologic outcomes. Various electrophysiological measures and imaging tools are in development, which may provide more precise information on functional changes following treatment and/or the therapeutic action of experimental agents. When compared to appropriate controls, an improved functional outcome, in response to an experimental treatment, is the necessary goal of a clinical trial program. Several new functional outcome tools are being developed for measuring an individual's ability to engage in activities of daily living. Such clinical end points will need to be incorporated into Phase 2 and Phase 3 trials. QoL measures often do not correlate tightly with the above outcome tools, but may need to form part of Phase 3 trial measures.
Assuntos
Ensaios Clínicos como Assunto/normas , Avaliação de Resultados em Cuidados de Saúde/normas , Recuperação de Função Fisiológica/fisiologia , Projetos de Pesquisa/normas , Traumatismos da Medula Espinal/diagnóstico , Atividades Cotidianas , Ensaios Clínicos como Assunto/métodos , Avaliação da Deficiência , Humanos , Avaliação de Resultados em Cuidados de Saúde/métodos , Qualidade de Vida , Traumatismos da Medula Espinal/terapia , Resultado do TratamentoRESUMO
Oligodendrocyte precursor cells recognized with the NG2 antibody respond rapidly to CNS injuries with hypertrophy and upregulation of the NG2 chondroitin sulfate proteoglycan within 24 h. These cells participate in glial scar formation, remaining around the injury site for several weeks. After injury, reactive oligodendrocyte precursor cells increase their production of several chondroitin sulfate proteoglycans, including NG2: this cell type thus represents a component of the inhibitory environment that prevents regeneration of axons in the injured CNS. This study analyzes factors that activate oligodendrocyte precursor cells. Both microglia and astrocytes become reactive around motor neurons following peripheral nerve lesions. We show that oligodendrocyte precursor cells do not hypertrophy or increase NG2 levels after these lesions. Those lesions that cause an oligodendrocyte precursor cell reaction generally open the blood-brain barrier. We therefore opened the blood-brain barrier with microinjections of vascular endothelial growth factor or lipopolysaccharide to the rat and mouse brain, and examined oligodendrocyte precursor cell reactivity after 24 h. Both treatments led to increases in NG2 and hypertrophy of oligodendrocyte precursor cells. Of directly injected blood components serum and thrombin were without effect, while platelets and macrophages activated oligodendrocyte precursor cells. We tested the effects of a range of injury-related cytokines, of which tumor necrosis factor alpha; interleukin-1; transforming growth factor beta; interferon gamma had effects on oligodendrocyte precursor cells. Oligodendrocyte precursor cell chemokines, and mitogens did not increase NG2 levels.
Assuntos
Plaquetas/fisiologia , Citocinas/farmacologia , Macrófagos/fisiologia , Oligodendroglia/metabolismo , Neuropatia Ciática/patologia , Células-Tronco/metabolismo , Animais , Antígenos/metabolismo , Axotomia/métodos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Antígeno CD11b/metabolismo , Linhagem Celular , Doenças do Nervo Facial/metabolismo , Doenças do Nervo Facial/patologia , Feminino , Lateralidade Funcional , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Camundongos , Microinjeções/métodos , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/fisiopatologiaRESUMO
The oligodendrocyte precursor cell (OPC) has until recently been regarded as a lineage-restricted precursor cell. Considerable interest has been generated by reports suggesting that OPCs may possess a wider differentiation potential than previously assumed and thus be considered a multipotential stem cell. This study examined the neuronal differentiation potential of rat, postnatal cortical OPCs in response to extracellular cues in vitro and in vivo. OPCs did not exhibit intrinsic neuronal potential and were restricted to oligodendrocyte lineage potential following treatment with the neural precursor mitogen fibroblast growth factor 2. In contrast, a postnatal hippocampal astrocyte-derived signal(s) is sufficient to induce functional neuronal differentiation of cortical OPCs in vitro in population and single cell studies. Co-treatment with Noggin, a bone morphogenetic protein antagonist, did not attenuate neuronal differentiation. Following transplantation to the adult rat hippocampus, cortical OPCs expressed doublecortin, a neuroblast-associated marker. The present findings show that hippocampal, astrocyte-derived signals can induce the neuronal differentiation of OPCs through a Noggin-independent mechanism.
Assuntos
Astrócitos/citologia , Diferenciação Celular/fisiologia , Neurônios/fisiologia , Oligodendroglia/fisiologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Transplante de Células/métodos , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura/métodos , Meios de Cultivo Condicionados/farmacologia , Diagnóstico por Imagem/métodos , Proteína Duplacortina , Fator 2 de Crescimento de Fibroblastos/farmacologia , Citometria de Fluxo/métodos , Gangliosídeos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Hipocampo/transplante , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Transfecção/métodosRESUMO
The cortical stab injury has been widely used for biochemical analysis of molecular changes following CNS injury. However, the cellular responses to this injury have not been accurately quantified. In order to provide a baseline for biochemical studies and future experiments on the manipulation of the CNS injury response we have undertaken a quantitative analysis of this injury. The proliferative and reactive responses of oligodendrocyte precursor cells, astrocytes and microglia were measured, using antibodies to NG2, glial fibrillary acidic protein (GFAP) and the cd11-b clone OX-42 to characterise these cell types at 2, 4, 7 and 14 days post-injury. Oligodendrocyte precursors and microglia proliferated rapidly during the first week, mostly within 0.3 mm of the lesion. Of the dividing cells over 60% were oligodendrocyte precursor cells with microglia making up the balance of the dividing cells. Minimal numbers of astrocytes divided in response to the lesion. Large cells with one or two short processes that were both NG2 and OX-42 positive were identified very close to the lesion at 2 and 4 days post-lesion but not thereafter. They are likely to be blood-derived cells that express NG2 or have ingested it. NG2 immunohistochemistry and platelet-derived growth factor alpha receptor (PDGFalpha-R) in situ hybridisation on neighbouring sections was performed. In the lesioned area only 12% of NG2 positive (+ive) cells were PDGFalpha-R +ive (a ratio of 1:8 for PDGFalpha-R +ive cells: NG2 +ive cells) compared with 33% in the unlesioned cortex and an almost 100% overlap in the spinal cord.
Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Córtex Cerebral/lesões , Microglia/citologia , Oligodendroglia/citologia , Células-Tronco/citologia , Animais , Astrócitos/química , Diferenciação Celular/fisiologia , Córtex Cerebral/química , Proteína Glial Fibrilar Ácida/análise , Microglia/química , Oligodendroglia/química , Ratos , Ferimentos Penetrantes/patologiaRESUMO
The MRL/MpJ mouse has a greatly enhanced healing response and an absence of scarring compared with other mouse strains. Following lesions to the CNS mammals show a scarring response known as reactive gliosis, and this CNS scar tissue blocks regeneration of cut axons. We have therefore compared reactive gliosis in the MRL/MpJ mouse and the Swiss Webster mouse, which exhibits normal scarring in the periphery. The lesion model was a stab lesion to the cortex, in which reactive gliosis has previously been quantified. Axon regeneration was examined following a cut lesion to the dopaminergic projection from the substantia nigra to the striatum used in previous regeneration experiments. In the MRL/MpJ following the lesion compared with Swiss Webster mice there was greater cell loss around the lesion followed by greater and more widespread and more prolonged cellular proliferation. Early after the lesion there was a greater loss of glial fibrillary acidic protein (GFAP)-positive astrocytes around the injury site in the MRL/MpJ, and an enhancement and prolongation of the microglial inflammatory response. This was accompanied by greater and more widespread blood-brain barrier leakage following injury. RNA levels for the matrix metalloproteinases (MMP)-2 and MMP-9 as well as for the thrombin receptors PAR-1 and PAR-4 were also greater at the MRL/MpJ injury site. All of these differences were transient and by 14 days post-injury there were no differences observed between MRL/MpJ and control mice. No axonal regeneration was observed following axotomy to the nigrostriatal pathway of the MRL/MpJ or the Swiss Webster mice at any time point.
Assuntos
Córtex Cerebral/lesões , Córtex Cerebral/metabolismo , Camundongos Endogâmicos MRL lpr/fisiologia , Cicatrização/fisiologia , Animais , Sistema Nervoso Central/lesões , Sistema Nervoso Central/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr/genética , Microglia/metabolismo , Especificidade da Espécie , Cicatrização/genética , Ferimentos Penetrantes/genética , Ferimentos Penetrantes/metabolismoRESUMO
The effects of corticosterone (CORT) and dehydroepiandrosterone (DHEA) on the expression of growth factor mRNA in either primary hippocampal cultures or astrocyte-enriched cultures from E18 CD rats was studied. In mixed primary cultures, 1 microM CORT up-regulated basic fibroblast growth factor (bFGF; FGF2) after 6 h of exposure, but down-regulated nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were unchanged. A 100 microM NMDA alone decreased NT-3, increased BDNF, but had no effect on NGF or FGF2. Concurrently administered CORT had no additional effect on either NGF, BDNF or NT-3, but up-regulated FGF2. In astrocytic cultures, 1 microM CORT increased FGF2 and NT-3, but decreased BDNF and NGF. A dose-response study confirmed these results. DHEA (100 nM) up-regulated NGF after 3 h, but not at other time points (6, 12, 24, 48 h). It had no effect on the other growth factors in mixed primary cultures. In astrocytic cultures, there was no effect of DHEA. Adding DHEA or its sulphate (up to 1 microM) to CORT did not alter the latter's action on growth factor mRNA expression. These results show that CORT has a selective action on growth factor expression, which was greater in astrocytic than in mixed cultures, that CORT amplifies or moderates activity-induced expression following NMDA, but that DHEA does not influence the effects of CORT on growth factor mRNA expression under these conditions.
Assuntos
Astrócitos/efeitos dos fármacos , Corticosterona/farmacologia , Desidroepiandrosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Fatores de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Fatores de Crescimento Neural/genética , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
It is well established that axonal regeneration in the adult CNS is largely unsuccessful. Numerous axon-inhibitory molecules are now known to be present in the injured CNS, and various strategies for overcoming these obstacles and enhancing CNS regeneration have been experimentally developed. Recently, the use of chondroitinase-ABC to treat models of CNS injury in vivo has proven to be highly beneficial towards regenerating axons, by degrading the axon-inhibitory chondroitin sulphate glycosaminoglycan chains found on many proteoglycans in the astroglial scar. This enzyme has now been shown to restore synaptic plasticity in the visual cortex of adult rats by disrupting perineuronal nets, which contain high levels of chondroitin sulphate proteoglycans (CS-PGs) and are expressed postnatally around groups of certain neurons in the normal CNS. The findings suggest exciting prospects for enhancing growth and plasticity in the adult CNS; however, some protective roles of CS-PGs in the CNS have also been demonstrated. Clearly many questions concerning the mechanisms regulating expression of extracellular matrix molecules in CNS pathology remain to be answered.
Assuntos
Axônios/fisiologia , Sistema Nervoso Central/lesões , Sulfatos de Condroitina/fisiologia , Regeneração Nervosa/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Condroitina ABC Liase/uso terapêutico , Cicatriz/metabolismo , HumanosRESUMO
Following a CNS lesion many glial cell types proliferate and/or migrate to the lesion site, forming the glial scar. The majority of these cells express chondroitin sulphate proteoglycans (CS-PGs), previously shown to inhibit axonal growth. In this study, in an attempt to diminish glial scar formation and improve axonal regeneration, proliferating cells were eliminated from the lesion site. Adult rats received a continuous infusion of 2% cytosine-D-arabinofuranoside (araC) or saline for 7 days over the lesion site, immediately following a unilateral transection of the right medial forebrain bundle. Additional groups of rats that received subdural infusions prior to the lesion, and lesioned rats which received no infusion, were also compared in the analyses. Animals were killed at 4, 7, 12 or 18 days post-lesion (dpl) and immunohistochemistry was used to determine the effects of these treatments on tyrosine hydroxylase (TH)-lesioned axons, and on the injury response of glial cells. Almost complete elimination of NG2 oligodendrocyte progenitor cells from the lesion site was seen up to 7 dpl in araC-infused animals; reduced numbers of reactive CD11b microglia were also seen but no effects were seen on the injury response of GFAP astrocytes. Significantly more TH axons were seen distal to the lesion in araC-treated brains, but these numbers dwindled by 18 dpl.