Christian W. Kreipke

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.

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.

Neovascularization following traumatic brain injury: possible evidence for both angiogenesis and vasculogenesis

Abstract Objective: Our goal was to characterize the angiogenic response following traumatic brain injury (TBI). Methods: Western analysis for vascular endothelial growth factor (VEGF) expression, double immunofluorescence labeling of endothelium and vascular endothelial growth factor receptor 2 (VEGFR2), bromodioxyuridine (BrdU) incorporation and measurement of capillary density, were all used to determine the temporal angiogenic response following TBI. Results: The angiogenic factors, VEGF and VEGFR2, increase following trauma. Capillary density increases and BrdU incorporation confirm the presence of newly formed vessels up to 48 hours post-injury. Discussion: Our results indicated that following TBI, there is a substantial increase in angiogenesis and based on morphologic characterization of BrdU-positive nuclei within the endothelium, we provide evidence for vasculogenesis following injury.

Hypoxia-Inducible Factor-1α 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-1α 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 Marmarous 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.

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.

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.

Receptor for advanced glycation end products and neuronal deficit in the fatal brain edema of diabetic ketoacidosis.

Radiologic and neuropsychologic studies suggest that diabetes mellitus causes structural changes in the brain and adversely effects cognitive development. Experimental animal models of type 1 diabetes mellitus (T1DM) have advanced these findings by demonstrating duration-related neuronal and cognitive deficits in T1DM BB/Wor rats. We studied the expression of receptor for advanced glycation end products (RAGE) and neuronal densities in the brains of two patients who died as the result of clinical brain edema(BE)that developed during the treatment of severe diabetic ketoacidosis (DKA). RAGE was markedly and diffusely expressed in blood vessels, neurons, and the choroid plexus and co-localized with glial fibrillary acidic protein (GFAP) in astrocytes. Significant neuronal loss was seen in the hippocampus and frontal cortex. Astrocytosis was present and white matter was atrophied in both cases when compared to age-matched controls. Our data supports that a neuroinflammatory response occurs in the BE associated with DKA, and that even after a relatively short duration of poorly controlled T1DM, the pathogenesis of primary diabetic encephalopathy can be initiated.

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 Marmarous 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.

Differential expression of capillary VEGF isoforms following traumatic brain injury

Abstract Objectives: While it is known that angiogenesis occurs after trauma, we sought to characterize the expression of vascular endothelial growth factor (VEGF) subtypes, vascular endothelial growth factor receptor 2 (VEGFR2) and angiopoietin within capillaries of animals subjected to traumatic brain injury (TBI). Further, we sought to characterize pericyte cell death in isolated capillaries. Methods: We used Marmarous acceleration impact model to induce head trauma and measured VEGF, VEGFR2 and angiopoietin levels in isolated capillaries. TUNEL was used to determine pericyte cell death. Results: The VEGF response was restricted to the VEGF120 isoform. No increase in transcripts for VEGF164 and VEGF188 was observed. VEGFR2 was marginally increased and angiopoietin was increased. A subset of pericytes were TUNEL-positive. Discussion: These results show a distinct expression pattern of angiogenic factors following injury and suggest that pericyte involvement in adaptive angiogenesis may be altered following TBI.