RESUMEN
STUDY DESIGN: Experimental, controlled, animal study. OBJECTIVES: To use non-invasive magnetic resonance imaging (MRI) to corroborate invasive studies showing progressive expansion of a hemorrhagic lesion during the early hours after spinal cord trauma and to assess the effect of glibenclamide, which blocks Sur1-Trpm4 channels implicated in post-traumatic capillary fragmentation, on lesion expansion. SETTING: Baltimore. METHODS: Adult female Long-Evans rats underwent unilateral impact trauma to the spinal cord at C7, which produced ipsilateral but not contralateral primary hemorrhage. In series 1 (six control rats and six administered glibenclamide), hemorrhagic lesion expansion was characterized using MRI at 1 and 24 h after trauma. In series 2, hemorrhagic lesion size was characterized on coronal tissue sections at 15 min (eight rats) and at 24 h after trauma (eight control rats and eight administered glibenclamide). RESULTS: MRI (T2 hypodensity) showed that lesions expanded 2.3±0.33-fold (P<0.001) during the first 24 h in control rats, but only 1.2±0.07-fold (P>0.05) in glibenclamide-treated rats. Measuring the areas of hemorrhagic contusion on tissue sections at the epicenter showed that lesions expanded 2.2±0.12-fold (P<0.001) during the first 24 h in control rats, but only 1.1±0.05-fold (P>0.05) in glibenclamide-treated rats. Glibenclamide treatment was associated with significantly better neurological function (unilateral BBB scores) at 24 h in both the ipsilateral (median scores, 9 vs 0; P<0.001) and contralateral (median scores, 12 vs 2; P<0.001) hindlimbs. CONCLUSION: MRI is an accurate non-invasive imaging biomarker of lesion expansion and is a sensitive measure of the ability of glibenclamide to reduce lesion expansion.
Asunto(s)
Gliburida/uso terapéutico , Hemorragia/tratamiento farmacológico , Imagen por Resonancia Magnética , Traumatismos de la Médula Espinal/tratamiento farmacológico , Envejecimiento , Animales , Modelos Animales de Enfermedad , Femenino , Hemorragia/etiología , Hemorragia/patología , Imagen por Resonancia Magnética/métodos , Ratas , Ratas Long-Evans , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/cirugía , Resultado del TratamientoRESUMEN
Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.
Asunto(s)
Diabetes Mellitus/terapia , Neuropatías Diabéticas/terapia , Terapia Genética , Simplexvirus/genética , Animales , Complicaciones de la Diabetes , Diabetes Mellitus/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Neuropatías Diabéticas/genética , Modelos Animales de Enfermedad , Ganglios Espinales/fisiopatología , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones , RatasRESUMEN
Traumatic spinal cord injury (SCI) in mammals causes widespread glial activation and recruitment to the CNS of innate (e.g. neutrophils, monocytes) and adaptive (e.g. T and B lymphocytes) immune cells. To date, most studies have sought to understand or manipulate the post-traumatic functions of astrocytes, microglia, neutrophils or monocytes. Significantly less is known about the consequences of SCI-induced lymphocyte activation. Yet, emerging data suggest that T and B cells are activated by SCI and play significant roles in shaping post-traumatic inflammation and downstream cascades of neurodegeneration and repair. Here, we provide neurobiologists with a timely review of the mechanisms and implications of SCI-induced lymphocyte activation, including a discussion of different experimental strategies that have been designed to manipulate lymphocyte function for therapeutic gain.
Asunto(s)
Autoinmunidad/inmunología , Quimiotaxis de Leucocito/inmunología , Linfocitos/inmunología , Mielitis/inmunología , Neuroinmunomodulación/inmunología , Traumatismos de la Médula Espinal/inmunología , Adaptación Fisiológica/inmunología , Animales , Humanos , Activación de Linfocitos/inmunología , Mielitis/fisiopatología , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa/inmunología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Spinal cord injury (SCI) in mammals leads to a robust inflammatory response followed by the formation of a glial and connective tissue scar that comprises a barrier to axonal regeneration. The inbred MRL/MpJ mouse strain exhibits reduced inflammation after peripheral injury and shows true regeneration without tissue scar formation following an ear punch wound. We hypothesized that following SCI, the unique genetic wound healing traits of this strain would result in reduced glial and connective tissue scar formation, increased axonal growth, and improved functional recovery. Adult MRL/MpJ and C57BL/6J mice were subjected to a mid-thoracic spinal contusion and the distribution of axon profiles and selected cellular and extracellular matrix components was compared at 1, 2, 4 and 6 weeks post-injury. Recovery of hind-limb locomotor function was assessed over the same time period. The MRL/MpJ mice exhibited robust axon growth within the lesion, beginning at 4 weeks post-injury. This growth was accompanied by reduced macrophage staining at 1, 2, 4 and 6 weeks post-injury, decreased chondroitin sulfate proteoglycan staining at 1-2 weeks and increased laminin staining throughout the lesion at 2-6 weeks post-injury. Paradoxically, the extent of locomotor recovery was impaired in the MRL/MpJ mice. Close examination of the chronic lesion site revealed evidence of ongoing degeneration both within and surrounding the lesion site. Thus, the regenerative genetic wound healing traits of the MRL/MpJ mice contribute to the evolution of a lesion environment that supports enhanced axon growth after SCI. However, this response occurs at the expense of meaningful functional recovery.
Asunto(s)
Axones/fisiología , Macrófagos/fisiología , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Astrocitos/patología , Axones/patología , Axones/ultraestructura , Conducta Animal , Antígeno CD11b/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Modelos Animales de Enfermedad , Femenino , Gliosis/etiología , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Microscopía Electrónica de Transmisión/métodos , Proteínas de Neurofilamentos/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Factores de TiempoRESUMEN
Spinal cord trauma activates the immune system and elicits leukocyte recruitment to the site of injury. This increase in immunological activity contributes to acute lesion expansion over a period of days to weeks following the initial trauma. At the same time, inflammatory cells and mediators facilitate endogenous repair processes such as axonal sprouting and remyelination. Thus, to be effective, therapies that target the immune system must limit the destructive effects of neutrophil, macrophage and lymphocyte activation, while simultaneously preserving their reparative functions.
Asunto(s)
Mediadores de Inflamación/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Cicatrización de Heridas/fisiología , Animales , Humanos , Mediadores de Inflamación/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacosRESUMEN
This study characterized the proinflammatory cytokines, interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFalpha), the antiinflammatory cytokines, IL-4 and IL-10, autoantibodies specific for GM1 ganglioside (anti-GM1), IgG and IgM, and myelin-associated glycoprotein (anti-MAG), in the sera of infection-free, chronic (>12 months), traumatically injured SCI patients (n = 24). Healthy able-bodied subjects (n = 26) served as controls. The proinflammatory cytokines and anti-GM1 antibodies were of particular interest as they have been implicated in an autoimmune "channelopathy" component to central and peripheral conduction deficits in various chronic neuroinflammatory diseases. Antibody and cytokine titers were established using enzyme-linked immunosorbent assays (ELISA). The mean anti-GM(1) (IgM) titer value for the SCI group was significantly higher (p < 0.05) than controls. The SCI group also demonstrated significantly higher titers (p < 0.05) of IL-2 and TNF alpha than controls. No differences were found between the SCI group and control group mean levels of IL-4 or IL-10. Overall, the serum of 57% of SCI patients contained increased levels of autoantibodies or proinflammatory cytokines relative to control values. These results provide preliminary support for the hypothesis that chronic immunological activation in the periphery occurs in a subpopulation of chronic SCI patients. It remains to be established whether elevated serum titers of proinflammatory cytokines and autoantibodies against GM1 are beneficial to the patients or whether they are surrogate markers of a channelopathy that compounds the neurological impairment associated with traumatic axonopathy or myelinopathy.
Asunto(s)
Autoanticuerpos/sangre , Interleucina-2/sangre , Traumatismos de la Médula Espinal/sangre , Traumatismos de la Médula Espinal/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Linfocitos B/inmunología , Enfermedad Crónica , Femenino , Gangliósido G(M1)/inmunología , Humanos , Interleucina-10/sangre , Interleucina-4/sangre , Masculino , Persona de Mediana Edad , Glicoproteína Asociada a Mielina/inmunologíaRESUMEN
Activated microglia and macrophages (CNS macrophages) have been implicated in the secondary or "bystander" pathology (e.g. axon injury, demyelination) that accompanies traumatic or autoimmune injury to the brain and spinal cord. These cells also can provide neurotrophic support and promote axonal regeneration. Studying the divergent functional potential of CNS macrophages in trauma models is especially difficult due to the various degradative mechanisms that are initiated prior to or concomitant with microglial/macrophage activation (e.g. hemorrhage, edema, excitotoxicity, lipid peroxidation). To study the potential impact of activated CNS macrophages on the spinal cord parenchyma, we have characterized an in vivo model of non-traumatic spinal cord neuroinflammation. Specifically, focal activation of CNS macrophages was achieved using stereotaxic microinjections of zymosan. Although microinjection does not cause direct mechanical trauma, localized activation of macrophages with zymosan acts as an "inflammatory scalpel" causing tissue injury at and nearby the injection site. The present data reveal that activation of CNS macrophages in vivo can result in permanent axonal injury and demyelination. Moreover, the pathology can be graded and localized to specific white matter tracts to produce quantifiable behavioral deficits. Further development of this model will help to clarify the biological potential of microglia and macrophages and the molecular signals that control their function within the spinal cord.
Asunto(s)
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Quimiotaxis de Leucocito/fisiología , Gliosis/patología , Macrófagos/citología , Microglía/citología , Mielitis/patología , Traumatismos de la Médula Espinal/patología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Basigina , Quimiotaxis de Leucocito/efectos de los fármacos , Desnervación/métodos , Modelos Animales de Enfermedad , Femenino , Trastornos Neurológicos de la Marcha/inducido químicamente , Trastornos Neurológicos de la Marcha/patología , Trastornos Neurológicos de la Marcha/fisiopatología , Gliosis/inducido químicamente , Gliosis/fisiopatología , Inmunohistoquímica , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microinyecciones , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Mielitis/inducido químicamente , Mielitis/fisiopatología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/patología , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Zimosan/farmacologíaRESUMEN
Traumatic injury to the spinal cord initiates a cascade of inflammatory-mediated injury and repair processes within the nervous system. In parallel, spinal injury could influence peripheral mechanisms of host defense (e.g., wound healing, antibody production) by altering lymphocyte phenotype and function. The goal of this study was to evaluate the physiological impact of spinal contusion injury on phenotypic and functional indices of lymphocyte activation. A flow cytometric time-course analysis of lymphocytes isolated from lymph node and spleen revealed an increase in CD4+ and a decrease in CD8+ lymphocytes during the first week post injury. The functional potential of lymphocytes was also evaluated based on their ability to proliferate in the presence of a biologically relevant antigen (myelin basic protein, MBP) or a lymphocyte mitogen. The data revealed increased proliferation to MBP by 3 days postinjury in lymphocytes isolated from lymph node but not spleen. By 1 week postinjury, increased proliferation to mitogen was noted in both the lymph node and the spleen suggesting a general increase in lymphocyte reactivity during this time interval. Circulating corticosterone (CORT), an endogenous glucocorticoid with significant effects on lymphocyte phenotype and function, was elevated within 24 h after spinal cord injury (SCI) and remained above control levels throughout the duration of our studies (up to 1 month postinjury). The present data suggest injury-associated changes in immune cell phenotype and function paralleled by the activation of the hypothalamic-pituitary-adrenal (HPA) axis.
Asunto(s)
Linfocitos/inmunología , Traumatismos de la Médula Espinal/inmunología , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Corticosterona/sangre , Femenino , Citometría de Flujo , Inmunofenotipificación , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Neuroinmunomodulación/inmunología , Ratas , Ratas Endogámicas Lew , Bazo/citología , Bazo/inmunologíaRESUMEN
Brain and spinal cord inflammation that develops after traumatic injury is believed to differentially influence the structural and/or physiological integrity of surviving neurons and glia. It is possible that the functional dichotomy of CNS inflammation results from the activity of a heterogeneous macrophage population elicited by trauma. Indeed, unique functions have been attributed to macrophages derived from resident microglia versus those originating from infiltrating monocytes. Thus, whether progressive tissue injury or repair is favored could be explained by the disproportionate contributions of one macrophage subset relative to the other. Descriptive neuroanatomical studies are a reasonable first approach to revealing a relationship between microglia, recruited blood monocytes/macrophages, and regions of tissue degeneration and/or repair. Unfortunately, it is not possible to differentiate between CNS macrophage subsets using conventional immunohistochemical approaches. In the present study, we have used radiation bone marrow chimeric rats to definitively characterize the macrophage reaction elicited by experimental spinal contusion injury. In chimeric animals, antibodies raised against unique cell surface molecules expressed on bone marrow-derived cells (BMCs) were used to distinguish infiltrating BMCs from resident microglial-derived macrophages. Our findings indicate that the onset and plateau of macrophage activation (previously shown to be 3 and 7 days postinjury, respectively) is dominated initially by microglial-derived macrophages and then is supplanted by hematogenous cells. While resident macrophages are ubiquitously distributed throughout the injury site, leukocyte-derived monocytes exclusively infiltrate the gray matter and to a lesser extent subpial white matter. Generally, monocyte foci in white matter remain associated with the lumen or abluminal surface of blood vessels, i.e. few cells actually infiltrate the parenchyma. If functional differences exist between CNS macrophage subsets, differences in the time-dependent accumulation and distribution of these cell types could differentially influence the survival of surrounding neurons and glia.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Macrófagos/inmunología , Quimera por Radiación/inmunología , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/patología , Animales , Recuento de Células , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Macrófagos/patología , Masculino , Microglía/patología , Monocitos/patología , Quimera por Radiación/genética , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Degeneración Retrógrada/patología , Traumatismos de la Médula Espinal/genética , Degeneración Walleriana/patología , Heridas no PenetrantesAsunto(s)
Regeneración Nerviosa/fisiología , Traumatismos de la Médula Espinal/terapia , Animales , Muerte Celular/fisiología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Inflamación/patología , Inflamación/fisiopatología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Oligodendroglía/patología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaAsunto(s)
Degeneración Nerviosa/inmunología , Regeneración Nerviosa/inmunología , Traumatismos de la Médula Espinal/inmunología , Factores de Edad , Animales , Autoinmunidad/fisiología , Barrera Hematoencefálica/inmunología , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Humanos , Terapia de Inmunosupresión/efectos adversos , Inflamación/inmunología , Inflamación/patología , Inflamación/fisiopatología , Linfocitos/inmunología , Linfocitos/patología , Macrófagos/inmunología , Macrófagos/patología , Microglía/inmunología , Microglía/patología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Sistemas Neurosecretores/inmunología , Sistemas Neurosecretores/patología , Sistemas Neurosecretores/fisiopatología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Transforming growth factor-beta1 (TGFbeta1) is a cytokine/growth factor found within the pathological central nervous system. TGFbeta1 has been shown to inhibit the release of cytotoxic molecules from microglia and macrophages, decrease astrocyte proliferation, and promote neuron survival. Because of the relevance of these actions to spinal cord injury, we examined TGFbeta1 and its receptors betaRI and betaRII mRNA levels and localization within the contused rat spinal cord using in situ hybridization. At the lesion site, TGFbeta1 mRNA peaked at 7 days postinjury and declined thereafter. Temporal and spatial localization of the betaRI and betaRII receptor mRNA closely mimicked that for TGFbeta1 in the epicenter. TGFbeta1, betaRI, and betaRII mRNAs also were elevated rostral and caudal to the injury, especially in regions known to contain activated microglia and degenerating axon profiles. Immunohistochemical staining of nearby sections confirmed that the highest levels of TGFbeta1 and receptor mRNA corresponded to regions filled with activated microglia and macrophages. The similar expression pattern of TGFbeta1, betaRI, and betaRII mRNA within the injured spinal cord suggests a local site of action. Since TGFbeta1 can act as an immunosuppressant as well as a stimulant for growth factors and neurite sprouting, it likely plays an important role, both temporally and spatially, in orchestrating postinjury events within the spinal cord.
Asunto(s)
Contusiones/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Axones/metabolismo , Axones/patología , Femenino , Inmunohistoquímica , Macrófagos/metabolismo , Macrófagos/patología , Microglía/metabolismo , Microglía/patología , Ratas , Ratas Endogámicas Lew , Receptores de Factores de Crecimiento Transformadores beta/genética , Médula Espinal/patología , Factor de Crecimiento Transformador beta/genética , Regulación hacia Arriba/genéticaRESUMEN
Previous work from this laboratory has described a rat spinal cord injury (SCI) model in which the mid-thoracic spinal cord is subjected to a single rapid and calibrated displacement at the site of a dorsal laminectomy. Injury is initiated at the tip of a vertical shaft driven by an electromagnetic shaker. Transducers arranged in series with the shaft record the patterns of displacement and force during the impact sequence. In the present study, this device and the relevant surgical procedures were adapted to produce a spinal contusion injury model in laboratory mice. The signal generator for the injury device has also been converted to a computer-controlled interface to permit extension of the model to other laboratories. Mice were subjected to SCI across a range of severities by varying the amplitude of displacement and the magnitude of measured preload force on the dural surface. A moderate injury produced by displacement of 0.5 mm over 25 msec resulted in initial paralysis and recovery of locomotion with chronic deficits in hindlimb function. The magnitude of the peak force, impulse, power, and energy generated at impact were correlated with behavioral outcome at 1 day postinjury, while peak displacement and impulse were the best predictors of behavioral outcome at 28 days postinjury. The shape of the force recording proved to be a highly sensitive measure of subtle variations in the spinal compartment that were otherwise difficult to detect in this small species. The results demonstrate that the electromagnetic spinal cord injury device (ESCID) can be used to produce a well-controlled contusion injury in mice. The unique features of controlled displacement and monitoring of the biomechanical parameters at the time of impact provide advantages of this model for reducing outcome variability. Use of this model in mice with naturally occurring and genetically engineered mutations will facilitate understanding of the molecular mechanisms of pathophysiology following traumatic spinal cord injury.
Asunto(s)
Traumatismos de la Médula Espinal/fisiopatología , Animales , Calibración , Modelos Animales de Enfermedad , Campos Electromagnéticos , Femenino , Laminectomía , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Actividad Motora , Ratas , Traumatismos de la Médula Espinal/patología , Factores de Tiempo , Heridas no Penetrantes/patología , Heridas no Penetrantes/fisiopatologíaRESUMEN
Traumatic injury to the spinal cord initiates a series of destructive cellular processes which accentuate tissue damage at and beyond the original site of trauma. The cellular inflammatory response has been implicated as one mechanism of secondary degeneration. Of the various leukocytes present in the spinal cord after injury, macrophages predominate. Through the release of chemicals and enzymes involved in host defense, macrophages can damage neurons and glia. However, macrophages are also essential for the reconstruction of injured tissues. This apparent dichotomy in macrophage function is further complicated by the overlapping influences of resident microglial-derived macrophages and those phagocytes that are derived from peripheral sources. To clarify the role macrophages play in posttraumatic secondary degeneration, we selectively depleted peripheral macrophages in spinal-injured rats during a time when inflammation has been shown to be maximal. Standardized behavioral and neuropathological analyses (open-field locomotor function, morphometric analysis of the injured spinal cord) were used to evaluate the efficacy of this treatment. Beginning 24 h after injury and then again at days 3 and 6 postinjury, spinal cord-injured rats received intravenous injections of liposome-encapsulated clodronate to deplete peripheral macrophages. Within the spinal cords of rats treated in this fashion, macrophage infiltration was significantly reduced at the site of impact. These animals showed marked improvement in hindlimb usage during overground locomotion. Behavioral recovery was paralleled by a significant preservation of myelinated axons, decreased cavitation in the rostrocaudal axis of the spinal cord, and enhanced sprouting and/or regeneration of axons at the site of injury. These data implicate hematogenous (blood-derived) macrophages as effectors of acute secondary injury. Furthermore, given the selective nature of the depletion regimen and its proven efficacy when administered after injury, cell-specific immunomodulation may prove useful as an adjunct therapy after spinal cord injury.
Asunto(s)
Ácido Clodrónico/farmacología , Macrófagos/fisiología , Traumatismos de la Médula Espinal/terapia , Médula Espinal/patología , Animales , Axones/efectos de los fármacos , Axones/patología , Axones/fisiología , Ácido Clodrónico/administración & dosificación , Ácido Clodrónico/uso terapéutico , Portadores de Fármacos , Femenino , Miembro Posterior , Inflamación , Liposomas , Macrófagos/efectos de los fármacos , Macrófagos/patología , Actividad Motora , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Ratas , Ratas Endogámicas Lew , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
We have studied temporal mRNA expression patterns for interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and transforming growth factor-beta1 (TGF-beta1) in two rat injury paradigms with very different cellular inflammatory reactions: contussion of the spinal cord and axotomy of the facial nerve. Our comparative analyses using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) show an early and robust upregulation of IL-1beta, TNF-alpha, IL-6, and M-CSF mRNAs in spinal cord after contusion injury. Peak expression of these mRNAs was transient and returned to control levels by 24 h postinjury. In contrast, expression of IL-1beta and TNF-alpha mRNAs in the axotomized facial nucleus was minimal and delayed, and levels of M-CSF mRNA remained unaltered. Similar to injured spinal cord, the axotomized nucleus showed a dramatic and early upregulation of IL-6 mRNA, but unlike spinal cord, IL-6 mRNA levels subsided only gradually. Both injury paradigms showed gradually increasing levels of TGF-beta1 mRNA which were maximal at 7 days postinjury. RT-PCR analyses were also performed on isolated blood-borne mononuclear cells and neutrophils. The results showed that these cells contain high levels of IL-1beta and M-CSF mRNAs, moderate levels of TGF-beta and TNF-alpha mRNAs, and minimal levels of IL-6 mRNA. The RT-PCR analyses together with histological observations indicate that expression of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 is short-lived and self-limited after contusion injury, and that it occurs primarily within endogenous glial cells. Transient expression of these molecules likely triggers secondary events which may be beneficial to wound repair and regeneration.
Asunto(s)
Axones/fisiología , Contusiones/metabolismo , Citocinas/biosíntesis , Nervio Facial/fisiología , Gliosis/patología , Inflamación/patología , ARN Mensajero/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Animales , Axotomía , Citocinas/genética , Femenino , Inmunohistoquímica , Leucocitos/metabolismo , Macrófagos/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Regeneración Nerviosa/fisiología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas LewRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) in which clinical neurological signs and histopathologic changes of disease can be suppressed by feeding CNS myelin proteins. Using immunohistochemistry and image analysis, the cellular immune response was quantified over the rostral-caudal axis of the spinal cord in rats with EAE and in animals fed high- or low-dose myelin basic protein (MBP) prior to inducing EAE (tolerized animals). In a subset of rats, MBP was fed 9 days after MBP immunization to examine the effect of oral tolerance on the progression of CNS pathology. In unfed rats or rats fed vehicle only, activated microglia and macrophages were co-localized with T-lymphocytes throughout the spinal cord, but greater cellular reactions were evident in gray matter relative to white matter. In all tolerized animals, the CNS inflammatory response was reduced relative to controls. Subtle pathologic changes were occasionally observed in the CNS of MBP-fed animals, but the distribution of inflammatory cells in the dorso-ventral axis was more polarized in animals fed high-dose MBP. In this group, more T-cells and activated microglia were present in the dorsal spinal cord, specifically in the gray matter. In the group fed MBP after disease induction, clinical disease progressed as in control non-fed rats, but recovery from disease appeared to be accelerated. Thus, the results presented here provide a comprehensive analysis of the distribution and magnitude of inflammatory cells within the spinal cord in EAE and challenge the theory that MBP-induced EAE is only a white matter disease. These data also describe how the activation and distribution of immune effector cells is altered by oral tolerance and may help predict a range of neurological deficits not previously appreciated in EAE, particularly those effected by gray matter pathology.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Proteína Básica de Mielina/uso terapéutico , Médula Espinal/patología , Administración Oral , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Tolerancia Inmunológica , Inmunohistoquímica , Linfocitos/patología , Macrófagos/patología , Microglía/patología , Proteína Básica de Mielina/administración & dosificación , Proteína Básica de Mielina/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7-28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions.
Asunto(s)
Macrófagos/citología , Microglía/citología , Traumatismos de la Médula Espinal/fisiopatología , Linfocitos T/citología , Animales , Inmunohistoquímica , Ratas , Ratas Endogámicas Lew , Ratas Sprague-DawleyRESUMEN
In previous experiments we utilized quantitative autoradiography to temporally describe vascular permeability of a radiolabeled vascular tracer following spinal contusion injury in the rat. In the present report we compare these findings with permeability assessments following fetal grafting in the contused rat spinal cord. At 10 days postinjury, Embryonic Day 14 spinal tissue was grafted into the lesioned spinal cord of Sprague-Dawley rats. At 7, 14, and 28 days postgrafting the alpha-aminoisobutyric acid (AIB) technique was used to assess blood-to-tissue transfer rates in graft and host tissue over several segments of the injured spinal cord. Regional changes in permeability were assessed using four distinct image analysis techniques. Using these methods, we have previously shown that contusion injury alone results in a chronic relapse in vascular permeability. The present data indicate that fetal transplants at 7 days postgrafting have AIB transfer rates that are significantly above uninjured control levels and are similar in magnitude to neighboring host spinal tissue. In addition, permeability in 14- and 28-day intraspinal grafts decreased relative to that of the 7-day transplant group, but remained significantly elevated at and rostral to the injury epicenter. Alternately, graft and host tissue in regions caudal to the injury epicenter (e.g., T10--L2) acquired a functional barrier to AIB as early as 14 days posttransplantation. These experiments suggest that graft development occurs in a different manner or at a different rate in segments of the injured spinal cord rostral and caudal to the injury site. Additionally, it appears that vascular permeability of the injured spinal cord can be influenced by the process of intraspinal transplantation.
Asunto(s)
Barrera Hematoencefálica/fisiología , Trasplante de Tejido Fetal , Médula Espinal/fisiología , Médula Espinal/trasplante , Ácidos Aminoisobutíricos/farmacocinética , Animales , Autorradiografía , Permeabilidad de la Membrana Celular/fisiología , Femenino , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratas , Ratas Sprague-Dawley , Médula Espinal/químicaRESUMEN
Blood-spinal cord barrier (BSB) permeability was measured using quantitative autoradiography following contusion injury to the rat spinal cord. Permeability was assessed by calculating blood-to-tissue transfer constants (Ki values) for the vascular tracer [14C]-alpha-aminoisobutyric acid (AIB) in injured (3, 7, 14, and 28 days postinjury), laminectomy control, and uninjured control animals. Permeability was quantitated using four separate imaging techniques in gray and white matter throughout the rostro-caudal extents of the forming lesion. Away from the epicenter, gray matter permeability was further differentiated within discrete spinal lamina using computerized templates. Regardless of the type of analysis used, increased AIB permeability (Ki values) was noted at all survival times in all tissue regions with respect to both uninjured and laminectomy control groups. The data indicate a large increase in individual Ki values throughout the dorsoventral axis of the spinal cord at 3 days postinjury (approximately 6-9 ml/kg/min). By 7 days, Ki values were quantitatively smaller (approximately 4-5 ml/kg/min) in all regions compared with 3-day tissues. Despite further attenuation of AIB uptake in the gray matter at 14 and 28 days postinjury, circumferential white matter tracts showed a secondary increase in permeability compared to 7-day tissue. Permeability in the white matter at 14-28 days postinjury (approximately 5-6 ml/kg/min) was comparable to that at 3 days postinjury (6-7 ml/kg/min). Measurements of the axial distribution of AIB permeability indicate increased BSB permeability over several segments rostral and caudal to the lesion epicenter (approximately 3 cm in both directions). Secondary elevations of AIB transfer in the spinal white matter between 14 and 28 days were colocalized with zones of immunohistochemically defined microglial clusters. The known plasticity of this cell type in response to changes in the extracellular microenvironment suggests that the spinal white matter at later survival times (14-28 days postinjury) is an area of dynamic vascular and/or axonal reconstruction. The implications of increased permeability to both tissue injury and neural regeneration are discussed.
Asunto(s)
Barrera Hematoencefálica/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiología , Ácidos Aminoisobutíricos/farmacocinética , Animales , Autorradiografía , Radioisótopos de Carbono , Permeabilidad de la Membrana Celular/fisiología , Contusiones/fisiopatología , Femenino , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Cinética , Microcirculación/fisiología , Regeneración Nerviosa/fisiología , Neuritis/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/irrigación sanguínea , Médula Espinal/químicaRESUMEN
The effect of immunological activation on the neuropathologic sequelae and neurologic outcome from spinal cord injury is unclear. Similar to models of neuroinflammatory disease (e.g., experimental autoimmune encephalomyelitis; EAE), injury to the spinal cord precipitates the activation of resident microglia and the recruitment of circulating inflammatory cells (e.g., macrophages and lymphocytes). In EAE, these cells are known to cause tissue damage and loss of neurological function via autoimmune reactions to myelin proteins. The role these cells play in the pathology of traumatic injury to the spinal cord has not been clarified. In this review, data are presented that indicate that T cells isolated from spinal-injured rats are capable of causing neurologic deficits and histopathologic changes similar to EAE when injected intravenously into naive animals. These data are consistent with the concept of trauma-induced autoimmune reactions. However, disease transfer was only possible when T cells were obtained from animals at 1 week post-injury. Thus, the encephalitogenic T-cell repertoire appears to be rapidly regulated. It is possible that trauma-induced autoimmunity evolves into a mechanism by which the autoreactive repertoire regulates ongoing central nervous system (CNS) immunologic responses. Similar immunoregulatory networks have been proposed in EAE and are discussed here in the context of CNS trauma and neurodegenerative disease.