Bradford T. Stokes

Cellular inflammatory response after spinal cord injury in sprague-dawley and lewis rats

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.

Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury

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.

Cytokine mRNA Profiles in Contused Spinal Cord and Axotomized Facial Nucleus Suggest a Beneficial Role for Inflammation and Gliosis

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.

CONCEPT OF AUTOIMMUNITY FOLLOWING SPINAL CORD INJURY: POSSIBLE ROLES FOR T LYMPHOCYTES IN THE TRAUMATIZED CENTRAL NERVOUS SYSTEM

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.

Brain-Derived Neurotrophic Factor Stimulates Hindlimb Stepping and Sprouting of Cholinergic Fibers after Spinal Cord Injury

Neurotrophic factors have been proposed as a therapeutic treatment for traumatic brain and spinal cord injury. The present study determined whether exogenous administration of one such factor, brain-derived neurotrophic factor (BDNF), could effect behavioral recovery and/or histopathological changes after spinal cord injury. Adult rats received a mild or moderate contusion injury or complete transection of the mid-thoracic spinal cord. Immediately thereafter, they were infused intrathecally with vehicle or BDNF for 28 days. Behavioral recovery was evaluated for 6 weeks after injury, at which time the rats were sacrificed and the spinal cord tissue was examined histologically. The infusion of BDNF resulted in acute stimulation of hindlimb activity. These effects included activation of alternating airstepping in injured rats when the hindlimbs were unloaded as well as slight improvements in the rate of recovery in open field locomotion score. BDNF infusion was also associated with enhanced growth of cholinergic fibers at the injury epicenter, but did not affect white matter sparing or density of serotonergic axons at or below the injury site. Based on immunohistochemical detection of BDNF protein distribution, these described effects are likely to be mediated by the activation of cells and axons within the central injury region and the along the peripheral rim of the spinal cord. Together, these findings demonstrate that the exogenous infusion of BDNF after spinal trauma can influence postinjury outcome through mechanisms that include acute stimulation of hindlimb activity and neuritogenesis at the injury site.

A quantitative spatial analysis of the blood-spinal cord barrier. I. Permeability changes after experimental spinal contusion injury

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.

Extracellular calcium activity in the injured spinal cord

The extracellular concentration of calcium ion was measured in canine spinal cord subsequent to spinal injury. In the control animal, we found that calcium activities changed little independent of electrode placement in the spinal cord, were stable during the 3 h necessary to make injury measurements, and were comparable to other estimates of calcium in the interstitial space. After injury, calcium activities decreased to micromolar levels that were incompatible with neural function. An incomplete recovery of extracellular calcium occurred during the next 3 h to about one-third (0.44 +/- 0.01 mM) of the normal value (1.1 +/- 0.08 mM). Such a pattern of changes in extracellular calcium was specific for the injury site itself and did not occur at nearby anatomic loci. These results are interpreted as having both short- and long-term effects on neuronal function and subsequent reorganization of spinal pathways.

Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury

Following traumatic injury to the spinal cord, hematogenous inflammatory cells including neutrophils, monocytes, and lymphocytes infiltrate the lesion in a distinct temporal sequence. To examine potential mechanisms for their recruitment, we measured chemokine mRNAs in the contused rat spinal cord, using specific and sensitive reverse transcriptase polymerase chain reaction (RT‐PCR) dot‐blot hybridization assays. The neutrophil chemoattractant GRO‐α was 30‐fold higher than control values at 6 hr postinjury and decayed rapidly thereafter. LIX, a highly related α‐chemokine, also was elevated early postinjury. Monocyte chemoattractant peptide (MCP)‐1 and MCP‐5 mRNAs, potent chemoattractants for monocytes, were significantly elevated at the lesion epicenter at 12 and 24 hr postinjury and declined thereafter. Interferon‐γ‐inducible protein, 10 kDa (IP‐10), chemoattractant towards activated T‐lymphocytes, was significantly elevated at 6 and 12 hr postinjury. The dendritic cell chemoattractant MIP‐3α also was increased, perhaps contributing to the development of T‐cell autoreactivity to neural components after spinal cord injury (SCI) in rats. Other β‐chemokines, including MIP‐1α and RANTES (regulated on expression normal T‐cell expressed and secreted), were minimally affected by SCI. Expression of chemokines, therefore, directly precedes the influx of target neutrophils, monocytes, and T‐cells into the spinal cord postinjury, as noted previously. Thus, selective chemokine expression may be integral to inflammatory processes within the injured spinal cord as a mechanism of recruitment for circulating leukocytes. J. Neurosci. Res. 53:368–376, 1998.

Behavioral and Histological Outcomes Following Graded Spinal Cord Contusion Injury in the C57Bl/6 Mouse

A computer-controlled electromagnetic spinal cord injury device (ESCID) has been adapted to develop a mouse model of spinal cord contusion injury. In the present study, we have extended this model in C57Bl/6 mice with behavioral and histopathological outcome assessment. Three groups of mice received a laminectomy at the T(9) vertebral level followed by a contusion injury from a predetermined starting load of 1500 dynes. Contusion was produced by rapid displacement of the spinal cord to a peak distance of 0.3, 0.5, or 0.8 mm, with the entire injury and retraction procedure completed over a 23-ms epoch. Control groups received laminectomy alone or complete transection. Functional recovery was examined for 9 weeks after injury using the BBB locomotor rating scale, grid walking, and footprint analysis. Distinct patterns of locomotor recovery were evident across the five groups. Measurements of spared white matter at the epicenter, lesion length, and cross-sectional area of fibronectin-immunopositive scar tissue were also significantly different between injury groups. The severity of injury corresponded with the biomechanical measures recorded at the time of impact as well as with behavioral and histological parameters. The results demonstrate that graded contusion injuries can be produced reliably in mice using the ESCID. The data provide a thorough and quantitative analysis of the effects of contusion injury on long-term behavioral and histological outcome measures in this strain and species.

Fetal grafts alter chronic behavioral outcome after contusion damage to the adult rat spinal cord.

In the present experiments, we have examined the capacity of intraspinal transplants to effect alterations in certain locomotor behaviors after spinal contusion injuries. An electromechanical impactor that was sensitive to tissue biomechanical characteristics was used to produce rapid (20 ms) compression injuries to the thoracic spinal cord (T8). Suspensions of fetal spinal tissue (14-day) were placed at 10 days postinjury into the intraspinal cavity created by these reproducible spinal injuries. In the pre- and postinjury period, a number of general and sensitive motor behaviors were used to characterize the immediate and long-term progress of hindlimb behavioral recovery over an extended period of time (73 days). Our data reveal that a lasting alteration in some motor behaviors can be achieved by suspension grafts. While little improvement in some generalized motor tasks (inclined plane analysis, grid walking) takes place, fetal transplants precipitate a rapid and enduring change in certain motivated fine motor behaviors (gait analysis). The base of support and stride length of the hindlimbs were improved by 7 days post-transplantation and the effect was stable over time. The angle of rotation was, however, not altered. The lasting effect in two gait parameters noted was accompanied by the presence of well-developed spinal grafts that often fused with the host spinal parenchyma. These results provide the first documentation of an influence of fetal transplants on motivated locomotor capacity in a well-characterized spinal injury model that mimics lesions seen in the contused adult human spinal cord.