Characterization of the microvascular glycocalyx in normal and injured spinal cord in the rat.

The glycocalyx of microvasculature in normal and injured spinal cord was characterized by using cationized ferritin to define anionic sites and the lectins concanavalin agglutinin (Con A) and Ricinus communis agglutinin I (RCA) to delineate carbohydrate moities. Binding of cationized ferritin was evaluated at the ultrastructural level in control animals and at 3 hours after spinal cord injury. Horseradish peroxidase (HRP) was administered intravenously before euthanasia. In control spinal cord, there was continuous even binding of cationized ferritin along the luminal front of microvasculature and no evidence of barrier permeability to HRP. After spinal cord injury, there was a reduction in binding of cationized ferritin in those regions of spinal cord that exhibited barrier breakdown to HRP. Lectin binding in the spinal cord was evaluated at 3 hours and 3 days postinjury. At the light microscopic level, there appeared to be increased binding of Con A and RCA in microvessels by 3 days postinjury as compared with the control spinal cord. At the ultrastructural level, a significant increase in RCA binding was noted along luminal fronts in the injured spinal cord. This increased binding coincided with a significant elaboration of the endothelial glycocalyx. These findings demonstrate that the charge, structure, and carbohydrate composition of the endothelial glycocalyx in microvessels in the spinal cord may be dramatically altered after spinal cord injury. Furthermore, there is an association between the loss of charge and disruption of the barrier, suggesting that anionic sites may contribute to maintenance of the blood-spinal cord barrier.

Changes in regional energy metabolism after closed head injury in the rat.

We examined in the present investigation regional ATP, glucose, and lactate content in the cortical and subcortical structures, in a rat model of closed head injury (CHI). In serial tissue sections bioluminescence imaging of ATP, glucose, and lactate was performed at 4 h, 12 h and 24 h (n = 4/5 per time point with) after the induction of CHI or sham surgery. Bioluminescence images were analyzed by computer-assisted densitometry, at the lesion site, in remote cortical areas, and in the subcortical structures (thalamus and caudate nucleus). ATP content was significantly decreased at the lesion site after 4 h and in the remote cortex at 12 h post-injury. At 12 h, the ATP content reached baseline levels on the ipsilateral side and at 24 h also at remote lateral parietal sites. In the contralateral cortex, ATP increased transiently above the baseline at 12 h. No significant changes in ATP were found in the thalamus and caudate nucleus. Cortical glucose and lactate contents could not be discerned over time. Following CHI there is an acute and progressive, yet transient, ischemic cortical profile, which is not reflected in subcortical areas.

Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat.

HSP70 and heme oxygenase-1 (HO-1) are thought to be markers of cell injury and oxidative stress, respectively. We have immunolocalized these proteins in the spinal cord at 1-14 days after contusion. HSP70 and HO-1 were co-induced in glia and macrophages within the injured segment at all time points. This co-induction may reflect complementary functions that serve to protect these cells as they respond to the postcontusional environment.

Endothelin-mediated induction of heme oxygenase-1 in the spinal cord is attenuated in transgenic mice overexpressing superoxide dismutase.

Spinal cord blood flow and the induction of heme oxygenase-1 (HO-1), an indicator of oxidative stress, were studied in the spinal cords of adult wild-type and transgenic mice overexpressing the antioxidant copper, zinc superoxide dismutase (CuZn SOD) after intrathecal administration of the potent vasoactive peptide endothelin-1 (ET-1). Gelfoam, saturated with ET-1 (40, 80, or 400 micromol/L), was positioned in the intrathecal space at the midthoracic level in anesthetized animals. Blood flow was continuously monitored by laser Doppler for 10 min after the intrathecal application of ET-1. There was a significant reduction in spinal cord blood flow to approximately 40% of control values by 10 min after the intrathecal application of the peptide in both wild-type and transgenic mice. Moreover, SB209670, a nonselective endothelin receptor antagonist, blocked this reduction in flow. Each animal was euthanized 24 h after the intrathecal administration of ET-1, and the spinal cord was prepared for quantitative immunocytochemistry. HO-1 was primarily induced in astrocytes near the dorsal surface of the spinal cord in wild-type mice. This induction was attenuated in both wild-type, treated with SB209670, and untreated transgenic mice. Together, these findings suggest that ET-1 mediates oxidative stress in the spinal cord through the modulation of spinal cord blood flow.

Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat.

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress.

Cellular response in the cerebellum after midline traumatic brain injury in the rat.

We evaluated the response of microglia and Purkinje cells in the cerebellum at 1, 2, 3, 7 and 10 days after a midline fluid percussion brain injury. There was marked activation of microglia and a significant loss of Purkinje cells in the vermis by 7 days postinjury. These findings emphasize the vulnerability of the cerebellum to midline traumatic brain injury.

Attenuated induction of heme oxygenase after intrathecal exposure to lysed blood in mice overexpressing superoxide dismutase.

Induction of heme oxygenase-1 (HO-1) in the spinal cord was studied in adult wildtype and transgenic mice overexpressing the antioxidant copper, zinc superoxide dismutase (CuZn SOD) 24 h after intrathecal infusion of heterologous lysed blood. Double immunolabeling techniques were used to determine the extent to which HO-1 was induced in astrocytes and microglia/macrophages. HO-1 was induced in both astrocytes and microglia/macrophages in the dorsal horns near the site of infusion of lysed blood in all mice. However, the number of HO-1 labeled cells was significantly less in the transgenic as compared to the wildtype animals. Together, these findings suggest that lysed blood preferentially induces HO-1 in glia and macrophages through the generation of oxidative stress.

Changes in ornithine decarboxylase activity and putrescine concentrations after spinal cord compression injury in the rat.

Traumatic spinal cord injury results in direct physical damage to structures and the generation of local factors contributing to secondary pathogenesis. In the present study, we investigated changes in polyamine metabolism after spinal cord compression injury in the rat. This is a stress induced metabolic pathway, of which an activation may indicate both, secondary pathogenesis or induction of neuroprotective response. Ornithine decarboxylase (ODC) activity, the rate limiting step of polyamine synthesis, and levels of the diamine putrescine, the product of ornithine decarboxylase reaction, were analyzed in control (non-laminectomized) animals and at 2 and 4 h after laminectomy or compression injury at the L4 segmental level. ODC activity was significantly increased 4 h after laminectomy in L4 and in adjacent L3 and L5 segments and compression to L4 produced a further increase 4 h after injury as compared with the intact control group. Putrescine levels were likewise significantly elevated to the same extend in the laminectomized and injured cord as compared with the intact control group. These findings demonstrate increased ODC and putrescine levels in the laminectomized and traumatized spinal cord and suggest that laminectomy may be an important 'priming event' that contributes to secondary injury after spinal cord compression injury.

Vascular events after spinal cord injury: contribution to secondary pathogenesis.

Traumatic spinal cord injury results in the disruption of neural and vascular structures (primary injury) and is characterized by an evolution of secondary pathogenic events that collectively define the extent of functional recovery. This article reviews the vascular responses to spinal cord injury, focusing on both early and delayed events, including intraparenchymal hemorrhage, inflammation, disruption of the blood-spinal cord barrier, and angiogenesis. These vascular-related events not only influence the evolution of secondary tissue damage but also define an environment that fosters neural plasticity in the chronically injured spinal cord.

Sustained induction of heme oxygenase-1 in the traumatized spinal cord.

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.

Alterations of norepinephrine levels in plasma and CSF of patients after traumatic brain injury in relation to disruption of the blood-brain barrier.

BACKGROUND: In injured brain tissue with a disrupted blood-brain barrier (BBB) catecholamines such as norepinephrine (NE) are known to enhance glucose consumption and cerebral blood flow but may lead to an energy depletion increasing the risk of ischemia. Therefore it is of great interest whether the exogenous administration of NE used mainly to maintain an adequate cerebral perfusion pressure influences CSF NE levels or not, and whether elevated plasma or CSF levels of NE can influence the actual clinical condition. We addressed this issue by measuring the levels of NE in CSF and plasma and correlating them with the actual clinical condition of the patients. METHODS: In 29 patients with severe TBI (< 8 points on the Glasgow Coma Scale, GCS) NE levels were analysed by high performance liquid chromatography (HPLC) in paired blood and CSF specimens which were collected from days 1 to 14 after severe TBI (total number of pairs = 121). The integrity of the BBB was evaluated by determining the CSF/serum albumin ratio. The clinical condition of the patients was assessed by GCS. RESULTS: Elevated plasma and CSF NE levels were observed in 50% of all samples, most consistently in patients treated with NE. NE elevation in CSF was independent of whether or not the BBB remained intact. There was no correlation between GCS and the levls of NE in CSF or plasma either in samples from the treated or the untreated group. INTERPRETATION: Exogenous administration of NE seems to increase NE levels in plasma and CSF. However, in this group of patients with severe TBI there was no clinical evidence that exogenous administration of NE was detrimental to the traumatized patients.