Neuronal damage and functional deficits are ameliorated by inhibition of aquaporin and HIF1α after traumatic brain injury (TBI).

The present study, using a rodent model of closed-head diffuse traumatic brain injury (TBI), investigated the role of dysregulated aquaporins (AQP) 4 and 9, as well as hypoxia inducible factor -1α(HIF-1α) on brain edema formation, neuronal injury, and functional deficits. TBI was induced in adult (400-425 g), male Sprague-Dawley rats using a modified Marmarou's head impact-acceleration device (450 g weight dropped from 2m height). Animals in each treatment group were administered intravenous anti-AQP4 or -AQP9 antibodies or 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α) 30 min after injury. At 24h post-TBI, animals (n=6 each group) were sacrificed to examine the extent of brain edema by water content, as well as protein expression of AQP and HIF-1α by Western immune-blotting. At 48-hours post-TBI, neuronal injury (n=8 each group) was assessed by FluoroJade (FJ) histochemistry. Spatial learning and memory deficits were evaluated by radial arm maze (n=8 each group) up to 21 days post-TBI. Compared to non-injured controls, significant (p<0.05) increases in the expression of AQP4 and -9 were detected in the brains of injured animals. In addition, significant (p<0.05) brain edema after TBI was associated with increases (p <0.05) both in neuronal injury (FJ labeling) and neurobehavioral deficits. Selective inhibition of either AQP4 or -9, or HIF-1α significantly (p<0.05) decreased the expression of the proteins. In addition, inhibition of the AQPs and HIF-1α significantly (p<0.05) ameliorated brain edema, as well as the number of injured neurons in cortical layers II/III and V/VI, striatum and hippocampal regions CA1/CA3. Finally, compared to the non-treated TBI animals, AQP or HIF-1α inhibition significantly (p<0.01) improved neurobehavioral outcomes after TBI. Taken together, the present data supports a causal relation between HIF-AQP mediated cerebral edema, secondary neuronal injury, and tertiary behavioral deficits post-TBI. The data further suggests that upstream modulation of the molecular patho-trajectory effectively ameliorates both neuronal injury and behavioral deficits post-TBI.

Glycerol accumulation in edema formation following diffuse traumatic brain injury.

Traumatic brain injury (TBI) induces brain edema via water and glycerol transport channels, called aquaporins (AQPs). The passage of glycerol across brain cellular compartments has been shown during edema. Using a modified impact/head acceleration rodent model of diffuse TBI, we assessed the role of hypoxia inducible factor (HIF)-1alpha in regulating AQP9 expression and glycerol accumulation during the edema formation. Adult (400-425 g) male Sprague-Dawley rats received a closed head injury with a weight drop (450 g, 2-m height) and were allowed to survive up to 48 hours. Some rat groups were administered 2-methoxyestradiol (2ME2, a HIF-1alpha inhibitor) 30 minutes after injury and were euthanized at 4 and 24 hours after injury. Brain edema was measured directly by water content, and glycerol concentration was determined by the Cayman Glycerol Assay. HIF-1alpha and AQP9 protein levels were assessed by Western immunoblotting. This study demonstrated a significant (P<0·05) increase in brain water content at 4-48 hours following impact. Cerebral glycerol was significantly (P<0.05) up-regulated at as early as 1 hour and remained at high levels for up to 48 hours. Similarly, significant (P<0.05) increases in HIF-1alpha and AQP9 protein levels were found at 1 hour and up to 48 hours after injury. Compared to untreated but injured rats, inhibition of HIF-1alpha by 2ME2 significantly (P<0.05) reduced the TBI-induced AQP9 up-regulation. This reduction was temporally associated with significant (P<0.05) decreases in both edema and glycerol accumulation. The data suggested an associated induction of HIF-1alpha, AQP9, and extracellular glycerol accumulation in edema formation following diffuse TBI. The implication of HIF-1alpha and AQP9 underlying TBI-induced edema formation offers possibilities for novel TBI therapies.

A mouse model of human repetitive mild traumatic brain injury.

A novel method for the study of repetitive mild traumatic brain injury (rmTBI) that models the most common form of head injury in humans is presented. Existing animal models of TBI impart focal, severe damage unlike that seen in repeated and mild concussive injuries, and few are configured for repetitive application. Our model is a modification of the Marmarou weight drop method and allows repeated head impacts to lightly anesthetized mice. A key facet of this method is the delivery of an impact to the cranium of an unrestrained subject allowing rapid acceleration of the free-moving head and torso, an essential characteristic known to be important for concussive injury in humans, and a factor that is missing from existing animal models of TBI. Our method does not require scalp incision, emplacement of protective skull helmets or surgery and the procedure can be completed in 1-2 min. Mice spontaneously recover the righting reflex and show no evidence of seizures, paralysis or impaired behavior. Skull fractures and intracranial bleeding are very rare. Minor deficits in motor coordination and locomotor hyperactivity recover over time. Histological analyses reveal mild astrocytic reactivity (increased expression of GFAP) and increased phospho-tau but a lack of blood-brain-barrier disruption, edema and microglial activation. This new animal model is simple and cost-effective and will facilitate characterization of the neurobiological and behavioral consequences of rmTBI. It is also ideal for high throughput screening of potential new therapies for mild concussive injuries as experienced by athletes and military personnel.

Endothelin-1 is upregulated after traumatic brain injury: a cross-species, cross-model analysis.

OBJECTIVES: This work was designed to compare levels of endothelin-1 following brain injury in both rat and porcine models of head injury. In a broader sense, this work also determines the feasibility of testing traumatic brain injury-related phenomenology across species and models. METHODS: Male Sprague-Dawley rats (400-450 g) were subjected to traumatic brain injury using a weight acceleration impact injury device (n = 5 per group). Following impact, cerebrospinal fluid was collected for enzyme-linked immunosorbent assay analysis of endothelin-1 concentration using a standard endothelin-1 detection kit at 4 hours, 24 hours, 48 hours, and 7 days post-traumatic brain injury. Sham operated animals (n = 5) were used as controls. In another set of experiments, traumatic brain injury was induced in newborn and juvenile pigs (n = 6 per group) using a lateral fluid percussion model of brain injury. Cerebrospinal fluid was collected at 4 hours, 8 hours, 72 hours, and 7 days post-injury and endothelin-1 levels were measured using a radiolabeled kit. RESULTS: Endothelin-1 levels rapidly increased from ∼35 in sham operated animals to over 200 pg/g tissue 4 hours post-impact in both rat cortex and hippocampus. This elevation was sustained through 48 hours post-impact. By 7 days post-injury, endothelin-1 levels returned to normal, control concentrations. This trend was consistent with the porcine model, being more pronounced in newborn versus juvenile pigs. CONCLUSION: These results show that endothelin-1 peptide concentration elevation is a consistent finding between rat and pig and between weight acceleration and fluid percussion models of traumatic brain injury. This suggests that endothelin-1 elevation is not only a conserved phenomenon in different models of traumatic brain injury, but that it is a likely target for understanding the observed enhanced vascular response to traumatic brain injury and ultimately developing strategies to improve outcome following traumatic brain injury.

Endothelin receptors A and B are expressed in distinct cellular compartments of rat hippocampus following global ischemia: an immunocytochemical study.

OBJECTIVES: The syntheses of endothelin receptors A and B were previously shown to be upregulated in rat dorsal hippocampus after traumatic brain injury. Here we characterize endothelin receptor A and endothelin receptor B cellular distribution in hippocampus after permanent global brain ischemia and their possible association to nerve cell injury. METHODS: Twenty-minute global ischemia was induced using the Pulsinelli's four-vessel occlusion in conjunction with systemic hypovolemia in male rats. Endothelin receptor A and endothelin receptor B immunoreactivities from sham-operated and ischemic rats were assessed qualitatively in dentate gyrus, Cornu Ammonis, and hilus regions of the hippocampus. Quantitative immunoreactivity measurements were also obtained by optical densitometry. RESULTS: In sham-operated control hippocampus, endothelin receptor A immunoreactivity was absent in nerve cell bodies but strongly expressed in the mossy fiber pathway (axons of dentate gyrus granule cells). After ischemia endothelin receptor A immunoreactivity in the same regions was reduced by 40-50% from control. In contrast, endothelin receptor B immunoreactivity in control hippocampus was widely distributed in pyramidal neurons, granule cells and glial cells, this immunoreactivity increasing by approximately 25-30% after ischemia. DISCUSSION: Endothelin receptor A's marked decrease in mossy fibers after ischemia may contribute to glutamate release from mossy fiber terminals, thus enhancing excitotoxic effects on their Cornu Ammonis synaptic targets. Additionally, endothelin receptor B increased expression in neurons and glia could be related to a more generalized activation of survival mechanisms involving elements of the neurovascular unit.

Pericyte-mediated vasoconstriction underlies TBI-induced hypoperfusion.

OBJECTIVES: Endothelin-1 is a 21-amino acid peptide that together with specific receptors, A (ETrA) and B (ETrB) is induced following traumatic brain injury (TBI) and has been closely linked to regulation of cerebral vasospasm, oxidative stress, and hypoperfusion. Specific endothelin receptor antagonists have been shown to ameliorate early evidence of neuronal cell injury, activation of microglial cells, and hypoperfusion following TBI. The exact mechanism involved in TBI-induced hypoperfusion is still unclear; however, it is thought that endothelin-1 engagement of ETrA is primarily responsible for changes in blood flow. In this study we question the role of the microvascular pericyte in endothelin-1-mediated pathophysiology in TBI. METHODS: Pericyte expression of endothelin-1, ETrA, and ETrB was examined in primary culture and in sham and impacted rat brain. Adult male rats were also given intracerebroventricular injections of ETrA (BQ-123) before being subjected to TBI using a closed head acceleration impact model. RESULTS: Primary pericytes express both endothelin-1 and its receptors ETrA and ETrB. Following TBI, the number of alpha-smooth muscle actin (SMA) positive pericytes located in microvessels is significantly increased by 4 hours post-traumatic impact. Increases in pericyte expression of alpha-SMA correlated with evidence of a reduction in both arteriolar and capillary diameter. Capillary endothelin-1, ETrA, and ETrB transcript and protein was also increased. Increased endothelin-1 expression was seen by 2-4 hours post-impact. Upregulation of receptors was observed by 4-8 hours and maximum by 24 hours. ETrA antagonists decreased the number of alpha-SMA(+) pericytes as well as changes in microvascular diameter. CONCLUSION: These results suggest that decreased vasoconstriction following TBI may be due to an endothelin-1-induced pericyte-mediated regulation of microvessel blood flow following TBI. Furthermore, results suggest that ETrA antagonists ameliorate trauma induced hypoperfusion, in part, by inhibiting endothelin-1-mediated upregulation of alpha-SMA in pericytes.

Endothelin receptor A antagonism reduces the extent of diffuse axonal injury in a rodent model of traumatic brain injury.

OBJECTIVES: While endothelin-1 and its receptors have traditionally been associated with mediating vasoreactivity, we have recently shown that the vast majority of endothelin receptor A expression following traumatic brain injury is localized within the neuron. While it has been suggested that endothelin receptor A plays a role in influencing neuronal integrity, the significance of neuronally expressed endothelin receptor A remains unclear. One report suggests that endothelin-1 signaling mediates diffuse axonal injury. Therefore, this work sought to determine whether treatment with BQ-123, a selective endothelin receptor A antagonist, diminishes the extent of diffuse axonal injury following trauma. METHODS: A total of 12 male Sprague-Dawley rats (350-400 g) were used in this study. Two groups (n = 6 per group) were generated as follows: sham operation and traumatic brain injury+1·0 mg/kg BQ-123 delivered intravenously 30 minutes prior to the injury. Trauma was induced using a weight acceleration impact device. Animals were terminated 24 or 48 hours after trauma, and a series of six coronal sections through the entire anterior-posterior extent of the corpus callosum were selected from each brain for quantification of diffuse axonal injury by beta-amyloid precursor protein immunostaining. RESULTS: Our data indicated that animals treated with BQ-123 30 minutes prior to trauma showed a significant reduction in diffuse axonal injury in corpus callosum at both 24 and 48 hours post-injury. CONCLUSION: The results show that endothelin receptor A antagonism reduced the extent of diffuse axonal injury, demonstrating a potential influence of the endothelin system on the intra-axonal cascade of molecular events underlying diffuse axonal injury.

Differential effects of endothelin receptor A and B antagonism on behavioral outcome following traumatic brain injury.

OBJECTIVES: Previously we have reported that endothelin receptor A and B antagonists elicit differential effects on cerebral blood flow and cellular damage. In summary, endothelin receptor A antagonists restore microcirculation and diminish cellular damage after injury, while endothelin receptor B antagonists had no effect on either parameter. However, what is not known is the effect of either antagonist on behavioral outcome. Therefore, this work was designed to test the effects of endothelin receptor A and B antagonism on behavioral outcome following traumatic brain injury (TBI). METHODS: A total of 48 male Sprague-Dawley rats (400-450 g) were used in this study. Four groups (n = 12 per group) were generated as follows: sham operation, trauma+vehicle (0·9% saline), trauma+40 nmol BQ-123 (a selective endothelin receptor A antagonist) and trauma +20 nmol BQ-788 (a selective endothelin receptor B antagonist). All treatments were delivered via intracerebroventricular injection. Trauma was induced using a weight acceleration impact device. Twenty-four hours post-injection animals were tested for 21 days on a radial arm maze task to determine cognitive outcome. RESULTS: Our data indicated that endothelin receptor A antagonism significantly reduced the extent of behavioral deficits following TBI while endothelin receptor B and vehicle injection had no effect. CONCLUSION: The results suggest that endothelin receptor A, but not endothelin receptor B, antagonism improves behavioral outcome following TBI. Furthermore, these data provide a functional correlate to previously published findings in our laboratory showing that endothelin receptor A antagonism improves both blood flow and cellular outcome following TBI. In a broader sense, this work demonstrates that hypoperfusion following TBI likely contributes to poor outcome following head injury.

Clazosentan, a novel endothelin A antagonist, improves cerebral blood flow and behavior after traumatic brain injury.

OBJECTIVES: The purpose of this study was to test the efficacy of a novel endothelin receptor A antagonist on blood flow and behavioral outcome given 30 minutes following traumatic brain injury. METHODS: Male Sprague-Dawley rats (400-450 g) were used in this study. All animals were scanned for initial blood flow using arterial spin labeling magnetic resonance imaging (n = 72 total). Half were subjected to traumatic brain injury using a weight acceleration impact device (n = 36 total). Sham operated animals were used as control (n = 36 total). Thirty minutes following traumatic brain injury, animals were given one intravenous injection of vehicle (0·9% saline) or 1·0 mg/kg clazosentan, a novel endothelin receptor A antagonist, for a total of four groups. At 4, 24, and 48 hours post-traumatic brain injury, blood flow determination continued. On the second day post-traumatic brain injury/sham operation, behavioral testing commenced using a radial arm maze to assess cognitive function. RESULTS: Our results indicate that 1·0 mg/kg clazosentan was effective in ameliorating hypoperfusion seen after traumatic brain injury. Saline had no effect. Furthermore, clazosentan treatment was effective in significantly improving behavioral outcome following traumatic brain injury. CONCLUSION: Collectively, these results indicate that clazosentan, given at 30 minutes post-traumatic brain injury, is effective in improving outcome following injury.

Polyadenylated mRNA staining reveals distinct neuronal phenotypes following endothelin 1, focal brain ischemia, and global brain ischemia/ reperfusion.

OBJECTIVES: Most work on ischemia-induced neuronal death has revolved around the relative contributions of necrosis and apoptosis, but this work has not accounted for the role of ischemia-induced stress responses. An expanded view recognizes a competition between ischemia-induced damage mechanisms and stress responses in the genesis of ischemia-induced neuronal death. An important marker of post-ischemic stress responses is inhibition of neuronal protein synthesis, a morphological correlate of which is the compartmentalization of mRNA away from ribosomes in the form of cytoplasmic mRNA granules. METHODS: Here we assessed the generality of this mRNA granule response following either 10 or 15 minutes global brain ischemia and 1 hour reperfusion, 4 hours focal cerebral ischemia alone, and endothelin 1 intraventricular injection. RESULTS: Both global and focal ischemia led to prominent neuronal cytoplasmic mRNA granule formation in layer II cortical neurons. In addition, we report here new post-ischemic cellular phenotypes characterized by the loss of nuclear polyadenylated mRNA staining in cortical neurons following endothelin 1 treatment and 15 minutes global ischemia. Both mRNA granulation and loss of nuclear mRNAs occurred in non-shrunken post-ischemic neurons. DISCUSSION: Where cytoplasmic mRNA granules generally appear to mark a protective response in surviving cells, loss of nuclear mRNAs may mark cellular damage leading to cell atrophy/death. Hence, staining for total mRNA may reveal facets of the competition between stress responses and damage mechanisms at early stages in post-ischemic neurons.

The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood-brain barrier disruption and brain edema after traumatic brain injury.

OBJECT: The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α), aquaporin-4 (AQP-4), and matrix metalloproteinase-9 (MMP-9) in blood-brain barrier (BBB) permeability alterations and brain edema formation in a rodent traumatic brain injury (TBI) model. METHODS: The brains of adult male Sprague-Dawley rats (400-425 g) were injured using the Marmarou closed-head force impact model. Anti-AQP-4 antibody, minocycline (an inhibitor of MMP-9), or 2-methoxyestradiol (2ME2, an inhibitor of HIF-1α), was administered intravenously 30 minutes after injury. The rats were killed 24 hours after injury and their brains were examined for protein expression, BBB permeability, and brain edema. Expression of HIF-1α, AQP-4, and MMP-9 as well as expression of the vascular basal lamina protein (laminin) and tight junction proteins (zona occludens-1 and occludin) was determined by Western blotting. Blood-brain barrier disruption was assessed by FITC-dextran extravasation, and brain edema was measured by the brain water content. RESULTS: Significant (p < 0.05) edema and BBB extravasations were observed following TBI induction. Compared with sham-operated controls, the injured animals were found to have significantly (p < 0.05) enhanced expression of HIF-1α, AQP-4, and MMP-9, in addition to reduced amounts (p < 0.05) of laminin and tight junction proteins. Edema was significantly (p < 0.01) decreased after inhibition of AQP-4, MMP-9, or HIF-1α. While BBB permeability was significantly (p < 0.01) ameliorated after inhibition of either HIF-1α or MMP-9, it was not affected following inhibition of AQP-4. Inhibition of MMP reversed the loss of laminin (p < 0.01). Finally, while inhibition of HIF-1α significantly (p < 0.05) suppressed the expression of AQP-4 and MMP-9, such inhibition significantly (p < 0.05) increased the expression of laminin and tight junction proteins. CONCLUSIONS: The data support the notion that HIF-1α plays a role in brain edema formation and BBB disruption via a molecular pathway cascade involving AQP-4 and MMP-9. Pharmacological blockade of this pathway in patients with TBI may provide a novel therapeutic strategy.

Soman increases neuronal COX-2 levels: possible link between seizures and protracted neuronal damage.

Nerve agent-induced seizures cause neuronal damage in brain limbic and cortical circuits leading to persistent behavioral and cognitive deficits. Without aggressive anticholinergic and benzodiazepine therapy, seizures can be prolonged and neuronal damage progresses for extended periods of time. The objective of this study was to determine the effects of the nerve agent soman on expression of cyclooxygenase-2 (COX-2), the initial enzyme in the biosynthetic pathway of the proinflammatory prostaglandins and a factor that has been implicated in seizure initiation and propagation. Rats were exposed to a toxic dose of soman and scored behaviorally for seizure intensity. Expression of COX-2 was determined throughout brain from 4h to 7 days after exposure by immunohistochemistry and immunoblotting. Microglial activation and astrogliosis were assessed microscopically over the same time-course. Soman increased COX-2 expression in brain regions known to be damaged by nerve agents (e.g., hippocampus, amygdala, piriform cortex and thalamus). COX-2 expression was induced in neurons, and not in microglia or astrocytes, and remained elevated through 7 days. The magnitude of COX-2 induction was correlated with seizure intensity. COX-1 expression was not changed by soman. Increased expression of neuronal COX-2 by soman is a late-developing response relative to other signs of acute physiological distress caused by nerve agents. COX-2-mediated production of prostaglandins is a consequence of the seizure-induced neuronal damage, even after survival of the initial cholinergic crisis is assured. COX-2 inhibitors should be considered as adjunct therapy in nerve agent poisoning to minimize nerve agent-induced seizure activity.

Differential effects of endothelin receptor A and B antagonism on cerebral hypoperfusion following traumatic brain injury.

OBJECTIVES: Our laboratory has previously shown that endothelin 1 (ET-1), a powerful vasoconstrictor, and its receptors, A (ETrA) and B (ETrB), are up-regulated following trauma. This up-regulation coincides temporally with enhanced vasoreactivity in cerebral cortical microvessels, which leads to a state of chronic hypoperfusion for up to 48 hours following traumatic brain injury (TBI). However, the direct contribution of either receptor up-regulation to decreased cerebral blood flow (CBF) after closed head trauma has not been determined. Furthermore, how ET-1 blockade may affect histological outcome following TBI has not been explored. Therefore, the effects of ETrA and B antagonism on TBI induced hypoperfusion of CBF and cell injury in sensorimotor cortex (smCx) and hippocampus (Hipp) were assessed by arterial spin labeling magnetic resonance imaging and Fluoro-Jade staining, respectively. METHODS: Adult male rats were given intracerebroventricular injections of ETrA (BQ123) or ETrB antagonist (BQ788) before being subjected to TBI using a closed head acceleration impact model. Following TBI, CBF was measured and histological examination of cell integrity was carried out. RESULTS: ETrA blockade ameliorated TBI induced hypoperfusion in smCx and Hipp at 4 and 24 hours after TBI and caused a mild hyperemia in both centers by 48 hours after injury. Furthermore, ETrA antagonism reduced the extent of Fluoro-Jade labeled cells within smCx and Hipp as compared with TBI only. ETrB blockade had little effect on TBI induced hypoperfusion and did not change the extent of cell injury following TBI. DISCUSSION: These results suggest that decreased CBF following TBI may be caused by ETrA, but not ETrB, up-regulation. Furthermore, these results suggest that TBI induced hypoperfusion may contribute to poor neurologic outcome following TBI. In this work, we provide a rationale for studying the clinical relevancy of use of ETrA antagonists following TBI.

Sequential abnormalities in type 1 diabetic encephalopathy and the effects of C-Peptide.

Diabetic encephalopathy is a recently recognized complication in type 1 diabetes. In this review, we summarize a series of experimental results obtained longitudinally in the spontaneously type 1 diabetic BB/Wor-rat, and bringing out the beneficial effects of C-peptide replacement. It is increasingly clear that lack of insulin and C-peptide, and perturbations of their signaling cascades in type 1 diabetes are detrimental to the regulation of neurotrophic factors and their receptors. Other consequences of such deficits and perturbations are innate inflammatory responses with effects on synaptogenesis, neurite degeneration, and early behavioral abnormalities. Replacement of C-peptide, which does not effect hyperglycemia, has beneficial effects on a variety of pro-apoptotic stressors, oxidative stressors, and finally on apoptosis. Eventually, this cascade of events leads to neuronal loss and decreased densities of white matter myelinating cells, with more profound deficits in behavioral and cognitive function. Such changes are likely to underlie gray and white matter atrophy in type 1 diabetes, and are significantly prevented by full C-peptide replacement. Present data demonstrate that C-peptide replacement has beneficial effects on numerous sequential and partly interrelated pathogenetic mechanisms, resulting in prevention of neuronal and oligodendroglial cell loss, with significant prevention of neurobehavioral and cognitive functions.

Inflammation in Diabetic Encephalopathy is Prevented by C-Peptide.

Encephalopathy is an increasingly recognized complication of type 1 diabetes. The underlying mechanisms are not well understood, although insulin deficiency has been implicated. The spontaneously diabetic BB/Wor-rat develops neuro-behavioral deficits and neuronal cell death in hippocampus and frontal cortex, which can be prevented by insulinomimetic C-peptide. Here we examined whether contributing factors such as activation of innate immune mediators are responsive to C-peptide replacement. Seven-month diabetic BB/Wor-rats and those treated with full C-peptide replacement were compared to age-matched control rats. Hippocampi of diabetic rats showed upregulation of RAGE and NF-kappaB, the former being localized to proliferating astrocytes. These changes were associated with increased expression of TNF-alpha, IL-1beta, IL-2 and IL-6 in hippocampi of diabetic rats. Full C-peptide replacement, which did not induce hyperglycemia, resulted in significant prevention of upregulation of RAGE expression, activation of NF-kappaB and activation of pro-inflammatory factors. In conclusion, impaired insulin activity is associated with upregulation of RAGE and pro-inflammatory factors, and these are likely to contribute to previously described oxidative and apoptotic neuronal cell death. Replacement of insulinomimetic C-peptide significantly prevents this cascade of events.

Hypoxia-inducible factor-1alpha signaling in aquaporin upregulation after traumatic brain injury.

Previous studies have demonstrated that traumatic brain injury (TBI) causes brain edema via aquaporins (AQPs), the water-transporting proteins. In the present study, we determined the role of hypoxia inducible factor-1alpha (HIF-1alpha), which is a transcription factor in response to physiological hypoxia, in regulating expression of AQP4 and AQP9. Adult male Sprague-Dawley rats (400-425g) received a closed head injury using the Marmarou weight drop model with a 450g weight and survived for 1, 4, 24 and 48h. Some animals were administered 30min after injury with 2-methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol which is known to post-transcriptionally down-regulate HIF-1alpha expression, and sacrificed 4h after injury. Real-time PCR and Western blot were used, respectively, to detect gene and protein expressions of manganese superoxide dismutase (MnSOD, showing hypoxic stress), HIF-1alpha, AQP4, and AQP9. ANOVA analysis demonstrated a significant (p<0.05) increase in gene expression of MnSOD, HIF-1alpha, AQP4, and AQP9, starting at 1h after injury through 48h. Western blot analysis further indicated a significant (p<0.05) increase in protein expression of these molecules at the same time points. Pharmacological inhibition of HIF-1alpha by 2ME2 reduced the up-regulated levels of AQP4 and AQP9 after TBI. The present study suggests that hypoxic conditions determined by MnSOD expression after closed head injury contribute to HIF-1alpha expression. HIF-1alpha, in turn, up-regulates expression of AQP4 and AQP9. These results characterize the pathophysiological mechanisms, and suggest possible therapeutic targets for TBI patients.

Synapse loss regulated by matrix metalloproteinases in traumatic brain injury is associated with hypoxia inducible factor-1alpha expression.

The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor up-regulated during hypoxia, in the regulation of MMP-2 and -9 expression post-TBI. Adult male Sprague-Dawley rats (n=6 per group, 400 g-425 g) were injured using Marmarou's closed-head acceleration impact model and allowed to survive for 1, 4, 24 and 48 h. In another set of experiments, 30 min after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1alpha) and sacrificed at 4 h after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1alpha, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 h through 48 h after TBI. A significant increase in gene and protein expressions of HIF-1alpha, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1alpha significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1alpha reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1alpha in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals.