Adiponectin protects hippocampal neurons against kainic acid-induced excitotoxicity

Neuronal damage after seizure is correlated with blood–brain barrier (BBB) leakage. Adiponectin (Ad) has shown protective effects on endothelial function. In this study, we investigated the effects of Ad on cell survival and BBB integrity in the mouse hippocampus after kainic acid (KA) treatment. Twenty-four hours after intracerebroventricular injection of recombinant Ad, mice were treated with KA, and then sacrificed 48 h later. Decreased serum Ad and increased hippocampal Ad receptor 1 in the hippocampus of KA-treated mice were prevented by Ad pretreatment. Using cresyl violet staining, TUNEL analysis, and immunostaining for caspase-3, histological evaluation revealed that the marked cell death noted in the hippocampus of KA-treated mice was not observed in KA-treated mice pretreated with Ad. Impairment of the BBB, which was demonstrated by the presence of IgG, was inhibited by Ad pretreatment. Immunohistochemical analysis indicated that KA caused up-regulation of hippocampal VEGF, eNOS, and NF-κB levels, all of which were reduced in animals that received Ad pretreatment. These data indicate that Ad preserves the integrity of the BBB and has neuroprotective effects in an animal model of seizures.     

Decreased junctional adhesion molecule-A expression during blood–brain barrier breakdown

Tight junctions between brain endothelial cells are one of the specialized structural components of the blood-brain barrier (BBB) to proteins. Research in the last decade has demonstrated that the integral membrane proteins of cerebral endothelial tight junctions are claudin, occludin, and junctional adhesion molecule (JAM). Altered expression of these tight junction proteins could cause BBB breakdown following brain injury leading to edema. In this study, expression of JAM-A, was analyzed by immunostaining and immunoblotting in the rat cortical cold injury model, a well-characterized in vivo model of BBB breakdown. Temporal and spatial expression of JAM-A was examined at 12 hours, days 2, 4, and 6 post-lesion in cold-injured and control rats. Control rats showed punctate JAM-A immunoreactivity at intervals along the circumference of the endothelial layer at tight junctions where JAM-A colocalized with occludin. A significant decrease in JAM-A expression was noted at the lesion site by immunoblotting at 12 h only. At this time period, lesion vessels showed loss of endothelial JAM-A immunostaining while day 2 onwards, there was recovery of endothelial JAM-A immunoreactivity. Dual labelling for JAM-A and fibronectin showed that only lesion vessels with BBB breakdown to fibronectin at 12 h also showed lack of endothelial JAM-A immunoreactivity supporting the evidence that JAM-A contributes to tight junction integrity.     

Relationship of calpain-mediated proteolysis to the expression of axonal and synaptic plasticity markers following traumatic brain injury in mice

The role of neuronal plasticity and repair on the final functional outcome following traumatic brain injury (TBI) remains poorly understood. Moreover, the relationship of the magnitude of post-traumatic secondary injury and neurodegeneration to the potential for neuronal repair has not been explored. To address these questions, we employed Western immunoblotting techniques to examine how injury severity affects the spatial and temporal expression of markers of axonal growth (growth-associated protein GAP-43) and synaptogenesis (pre-synaptic vesicular protein synaptophysin) following either moderate (0.5 mm, 3.5 M/s) or severe (1.0 mm, 3.5 M/s) lateral controlled cortical impact traumatic brain injury (CCI-TBI) in young adult male CF-1 mice. Moderate CCI increased GAP-43 levels at 24 and 48 h post-insult in the ipsilateral hippocampus relative to sham, non-injured animals. This increase in axonal plasticity occurred prior to maximal hippocampal neurodegeneration, as revealed by de Olmos silver staining, at 72 h. However, moderate CCI-TBI did not elevate GAP-43 expression in the ipsilateral cortex where neurodegeneration was extensive by 6 h post-TBI. In contrast to moderate injury, severe CCI-TBI failed to increase hippocampal GAP-43 levels and instead resulted in depressed GAP-43 expression in the ipsilateral hippocampus and cortex at 48 h post-insult. In regards to injury-induced changes in synaptogenesis, we found that moderate CCI-TBI elevated synaptophysin levels in the ipsilateral hippocampus at 24, 48, 72 h and 21 days, but this effect was not present after severe injury. Together, these data highlights the adult brain's ability for axonal and synaptic plasticity following a focal cortical injury, but that severe injuries may diminish these endogenous repair mechanisms. The differential effects of moderate versus severe TBI on the post-traumatic plasticity response may be related to the calpain-mediated proteolytic activity occurring after a severe injury preventing increased expression of proteins required for plasticity. Supporting this hypothesis is the fact that GAP-43 is a substrate for calpain along with our data demonstrating that calpain-mediated degradation of the cytoskeletal protein, alpha-spectrin, is approximately 10 times greater in ipsilateral hippocampal tissue following severe compared to moderate CCI-TBI. Thus, TBI severity has a differential effect on the injury-induced neurorestorative response with calpain activation being one putative factor contributing to neuroregenerative failure following severe CCI-TBI. If true, then calpain inhibition may lead to both neuroprotective effects and an enhancement of neuronal plasticity/repair mechanisms post-TBI.     

Nicotinamide reduces hypoxic ischemic brain injury in the newborn rat

Nicotinamide reduces ischemic brain injury in adult rats. Can similar brain protection be seen in newborn animals? Seven-day-old rat pups had the right carotid artery permanently ligated followed by 2.5 h of 8% oxygen. Nicotinamide 250 or 500 mg/kg was administered i.p. 5 min after reoxygenation, with a second dose given at 6 h after the first. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia. Nicotinamide 500 mg/kg reduced brain weight loss from 24.6 +/- 3.6% in vehicle pups (n = 28) to 11.9 +/- 2.6% in the treated pups (n = 29, P < 0.01), but treatment with 250 mg/kg did not affect brain weight. Nicotinamide 500 mg/kg also improved behavior in rotarod performance. Levels of 8-isoprostaglandin F2alpha measured in the cortex by enzyme immune assay 16 h after reoxygenation was 115 +/- 7 pg/g in the shams (n = 6), 175 +/- 17 pg/g in the 500 mg/kg nicotinamide treated (n = 7), and 320 +/- 79 pg/g in the vehicle treated pups (n = 7, P < 0.05 versus sham, P < 0.05 versus nicotinamide). Nicotinamide reduced the increase in caspase-3 activity caused by hypoxic ischemia (P < 0.01). Nicotinamide reduces brain injury in the neonatal rat, possibly by reducing oxidative stress and caspase-3 activity.     

Impact of experimental acute hyponatremia on severe traumatic brain injury in rats: influences on injuries,permeability of blood-brain barrier,ultrastructural features,and aquaporin-4 expression

The effects of acute hyponatremia on severe traumatic brain injury (TBI) in 35 adult male Sprague-Dawley rats were studied in a replicated focal and diffuse injury rat model. Such effects were assessed by the cerebral contusion volume and axonal injury (AI) densities, determined by quantitative immunoreactivity of beta-amyloid precursor protein, by blood-brain barrier (BBB) permeability based on endogenous IgG immunostaining, and by ultrastructural features. Significant increase of contusion volume (P < 0.05) and of AI in the segment of corpus callosum beneath the contusion (P < 0.05) and ipsilateral thalamus (P < 0.05) were observed at 4 h postinjury during the hyponatremic phase. No change in BBB permeability was observed in the hyponatremia + TBI (HT) groups. Significant swelling of perivascular astrocytic foot processes in the HT groups was seen at 4 h (P < 0.01) and 1 day postinjury (P < 0.01) by quantitative image analysis of ultrastructures. However, attenuated swelling of perivascular astrocytic foot processes in severely edematous medulla oblongata with simultaneous swelling of perikaryal astrocytic processes was observed in the HT 1-day group. The ultrastructural features were also correlated with the down-regulation of aquaporin-4 (AQP4) mRNA expression (P < 0.05). Results suggest that acute hyponatremia acts as one of the secondary insults following severe TBI. Such exacerbation may not be attributable to further disruption of BBB permeability, but rather to the ischemia resulting from the swelling of perivascular astrocytic foot processes impeding microcirculation. Down-regulated AQP4 mRNA expression may be one of the molecular mechanisms maintaining water homeostasis in diffusely injured brain exposed to acute hyponatremia.     

Peripheral thermal injury causes blood-brain barrier dysfunction and matrix metalloproteinase (MMP) expression in rat

Mortality after serious systemic thermal injury may be linked to significant increases in cerebral vascular permeability and edema due to blood-brain barrier (BBB) breakdown. This BBB disruption is thought to be mediated by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs). The gelatinases, MMP-2 and MMP-9, digest the endothelial basal lamina of the BBB, which is essential for maintaining BBB integrity. The current study investigated whether disruption of microvascular integrity in a rat thermal injury model is associated with gelatinase expression and activity. Seventy-two adult Sprague-Dawley rats were anesthetized and submerged horizontally, in the supine position, in 100 degrees C (37 degrees C for controls) water for 6 s producing a third-degree burn affecting 60-70% of the total body surface area. Brain edema was detected by calculating water content. Real time PCR, Western blot, and zymography were used to quantify MMP mRNA, protein, and enzyme activity levels. Each group was quantified at 3, 7, 24, and 72 h post thermal injury. Brain water content was significantly increased 7 through 72 h after burn. Expression of brain MMP-9 mRNA was significantly increased as early as 3 h after thermal injury compared to controls, remained at 7 h (p<0.01), and returned to control levels by 24 h. MMP-9 protein levels and enzyme activity began to increase at 7 h and reached significant levels between 7 and 24 h after thermal injury. While MMP-9 protein levels continued to increase significantly through 72 h, enzyme activity returned to control level. The increase in MMP-9 expression and activity, associated with increased BBB permeability following thermal injury, indicates that MMP-9 may contribute to observed cerebral edema in peripheral thermal injury.     

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 microg/kg) or CEPO (40 microg/kg). Results indicated that 3 days post-injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase-mediated dUTP nick-end labelling (TUNEL)-labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker beta-amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the beta common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.     

Rapid loss and partial recovery of neurofilament immunostaining following focal brain injury in mice

Neurofilaments (NF), the intermediate filaments of the neuronal cytoskeleton, provide mechanical stability to the cell. High-molecular-weight NF (NFH) comprises a heavily phosphorylated carboxyl terminal ("sidearm") domain which helps determine interfilament spacing distances. Experimental evidence suggests that dephosphorylation greatly increases the rate and extent of proteolysis of NFH. Because NF proteolysis has been implicated as one pathogenic mechanism underlying cell death following traumatic brain injury (TBI), we analyzed the patterns of acute NFH damage in relation to phosphorylation state following focal, concussive, controlled cortical impact (CCI) brain injury in mice. Brains from C57BL/6 male mice (n = 4 injured and n = 1 sham per time point) were evaluated 5 min, 15 min, 90 min, 4 h, and 24 h following CCI injury (1 mm depth, 5 m/s). Immunohistochemistry was performed using antibodies that recognize epitopes on either dephosphorylated (d-NFH) or phosphorylated (p-NFH) sidearms or on the core (c-NFH) domain. As early as 5-15 min postinjury, immunoreactivity for d-, p-, and c-NFH decreased in the ipsilateral cortex, and hippocampal CA3, CA1, and dentate areas. This marked decrease of NFH labeling occurred in the absence of notable cell loss. Furthermore, partial recovery of NFH labeling was observed as early as 90 min postinjury in the cortex and by 24 h postinjury in hippocampal CA3 and dentate. The results of this study suggest that both phosphorylated and dephosphorylated NFH are vulnerable almost immediately following focal brain injury in mice, but that injured neurons may have an adaptive capability to partially restore this important cytoskeletal protein.     

Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex.

Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain-specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24-72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI.     

Broad neuroprotective profile of nicotinamide in different mouse models of MPTP-induced parkinsonism

The factors contributing to substantia nigra pars compacta (SNc) dopamine (DA) neuron death and striatal DA depletion in Parkinson's disease (PD) are still poorly understood. However, mitochondrial dysfunction, cellular energy depletion and oxidative stress appear to play important roles in the pathogenesis of PD. In view of this, the current study examined the potential of nicotinamide, a form of the B-complex vitamin niacin, to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced SNc cell loss and striatal DA depletion in two mouse MPTP models that respond differently to putative neuroprotective agents. Adult male C57Bl/6 mice received nicotinamide (125, 250 or 500 mg/kg i.p.) prior to either acute (four injections in 1 day at 2-h intervals) or sub-acute (two injections per day at 4-h intervals for 5 days) MPTP administration. Striatal DA levels, changes in numbers of tyrosine hydroxylase (TH)- and cresyl violet-stained cells in the SNc at 2 and 6 weeks following the last MPTP exposure were analyzed. Nicotinamide administration resulted in a dose-dependent sparing of striatal DA levels and SNc neurons in acute MPTP-treated animals. Only the highest dose of nicotinamide had similar effects in sub-acute MPTP-treated animals. At 6 weeks after MPTP exposure, there was some spontaneous recovery of striatal DA levels in both models: neuroprotective effects were still apparent in acute but not sub-acute MPTP-treated animals. These results show neuroprotective effects of nicotinamide in different mouse Parkinson models associated with different forms of cell death and suggest that nicotinamide may have broad neuroprotective potential in PD.     

Expression and Localization of the Orexin-1 Receptor (OX1R) After Traumatic Brain Injury in Mice

Orexins are neuropeptides that have a wide range of physiological effects, and recent studies have suggested that the orexin system may be involved in traumatic brain injury. However, the expression and localization of orexin receptors have not been examined yet under brain injury conditions. In the present study, we used immunohistochemical techniques to investigate the expression of orexin-1 receptor (OX1R) and its time-dependent changes in the mouse brain after controlled cortical impact (CCI) injury. OX1R-like immunoreactivity was first detected 6 h after injury in the surrounding penumbra of the injury. The intensity of this immunoreactivity was increased at 12 h, peaked at day 1, and then decreased from day 2 to day 7. To identify the cellular localization of OX1R, we also performed double-immunohistochemical staining with OX1R and several cell marker antibodies. OX1R-like immunopositive cells were clearly co-localized with immunoreactivity for the neuronal marker NeuN at day 7. It was also expressed on the periphery of cells immunopositive for CD11b, a microglial cell marker, at days 1 and 7. These results suggest that orexin and its receptor may play roles in traumatic brain injury, and that OX1R is induced in neurons and microglial cells after traumatic brain injury.     

PACAP38 Suppresses Cortical Damage in Mice with Traumatic Brain Injury by Enhancing Antioxidant Activity

The production of reactive oxygen species (ROS) and the resulting oxidative stress in mice in response to a controlled cortical impact (CCI) are typical exacerbating factors associated with traumatic brain injury (TBI). Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a multifunctional peptide that has been shown to exhibit neuroprotective effects in response to a diverse range of injuries to neuronal cells. We recently reported that PACAP38 might regulate oxidative stress in mice. The aim of the present study was to determine whether PACAP38 exerts neuroprotective effects by regulating oxidative stress in mice with TBI. Reactive oxidative metabolites (ROMs) and biological antioxidant potential (BAP) were measured in male C57Bl/6 mice before and 3, 4, and 24 h after CCI. PACAP38 was administered intravenously immediately following CCI, and immunostaining for the oxidative stress indicator nitrotyrosine (NT), and for neuronal death as an indicator of the area affected by TBI, was measured 24 h later. Western blot experiments to determine antioxidant activity [as indicated by superoxide dismutase-2 (SOD-2) and glutathione peroxidase 1 (GPx-1)] in the neocortical region were also performed 3 h post-CCI. Results showed that plasma BAP and ROM levels were dramatically increased 3 h after CCI. PACAP38 suppressed the extent of TBI and NT-positive regions 24 h after CCI, and increased SOD-2 and GPx-1 levels in both hemispheres. Taken together, these results suggest that increasing antioxidant might be involving in the neuroprotective effect of PACAP38 in mice subjected to a CCI.     

USP14 inhibition promotes recovery by protecting BBB integrity and attenuating neuroinflammation in MCAO mice

Aim

Blood–brain barrier (BBB) dysfunction is one of the hallmarks of ischemic stroke. USP14 has been reported to play a detrimental role in ischemic brain injury. However, the role of USP14 in BBB dysfunction after ischemic stroke is unclear.

Methods

In this study, we tested the role of USP14 in disrupting BBB integrity after ischemic stroke. The USP14-specific inhibitor IU1 was injected into middle cerebral artery occlusion (MCAO) mice once a day. The Evans blue (EB) assay and IgG staining were used to assess BBB leakage 3 days after MCAO. FITC-detran test was slected to examine the BBB leakage in vitro. Behavior tests were conducted to evaluate recovery from ischemic stroke.

Results

Middle cerebral artery occlusion increased endothelial cell USP14 expression in the brain. Furthermore, the EB assay and IgG staining showed that USP14 inhibition through IU1 injection protected against BBB leakage after MCAO. Analysis of protein expression revealed a reduction in the inflammatory response and chemokine release after IU1 treatment. In addition, IU1 treatment was found to rescue neuronal loss resulting from ischemic stroke. Behavior tests showed a positive effect of IU1 in attenuating brain injury and improving motor function recovery. In vitro study showed that IU1 treatment could alleviate endothelial cell leakage induced by OGD in cultured bend.3 cells through modulating ZO-1 expression.

Conclusions

Our results demonstrate a role for USP14 in disrupting the integrity of the BBB and promoting neuroinflammation after MCAO.     

Impairment of the blood-nerve and blood-brain barriers in apolipoprotein e knockout mice

Apolipoprotein E (apoE) is well characterized as a plasma lipoprotein involved in lipid and cholesterol metabolism. Recent studies implicating apoE in Alzheimer's disease and successful recovery from neurological injury have stimulated much interest in the functions of apoE within the brain. To explore the functions of apoE within the nervous system, we examined apoE knockout (KO) mice. Previously, we showed that apoE KO mice have a delayed response to noxious thermal stimuli associated with a loss and abnormal morphology of unmyelinated fibers in the sciatic nerve. From these data, we hypothesized that apoE KO mice could have an impaired blood-nerve barrier (BNB). In this report, we demonstrate functionally impaired blood-nerve and blood-brain barriers (BBB) in apoE KO mice using immunofluorescent detection of serum protein leakage into nervous tissue as a diagnostic for decreased BNB and BBB integrity. Extensive extravasation of serum immunoglobulin G (IgG) is detected in the sciatic nerve, spinal cord, and cerebellum of apoE KO but not WT mice. In a subpopulation of apoE KO mice, IgG also extravasates into discrete cortical and subcortical locations, including hippocampus. Loss of BBB integrity was additionally confirmed by the ability of exogenously supplied Evans blue dye to penetrate the BBB and to colocalize with IgG immunoreactivity in CNS tissue. These observations support a role for apoE in maintaining the integrity of the BNB/BBB and suggest a novel relationship between apoE and neural injury.     

Temporal profile of apoptotic-like changes in neurons and astrocytes following controlled cortical impact injury in the rat.

Apoptotic cell death has been observed in both neurodegenerative diseases and acute neurological traumas such as ischemia, spinal cord injury, and traumatic brain injury (TBI). Recent studies employing different models of TBI have described morphological and biochemical changes characteristic of apoptosis following injury. However, no study has examined the temporal profile of apoptosis following controlled cortical impact (CCI) injury in the rat. In addition, the relative frequency of apoptotic profiles in different cell types (neurons versus glia) following CCI has yet to be investigated. In the present experiments, injured cortex was subjected to DNA electrophoresis, and serial sections from the contusion area were stained with hematoxylin and eosin or Hoechst 33258 or double-labeled with TUNEL and neuronal or glial markers. The results of the present study indicate that CCI produces a substantial amount of DNA damage associated with both apoptotic-like and necrotic-like cell death phenotypes primarily at the site of cortical impact and focal contusion. DNA damage, as measured by TUNEL and DNA electrophoresis, was most apparent 1 day following injury and absent by 14 days post-TBI. However, quantitative analysis showed that the majority of TUNEL-positive cells failed to exhibit apoptotic-like morphology and were probably undergoing necrosis. In addition, apoptotic-like morphology was predominantly observed in neurons compared to astrocytes. The present study provides further evidence that apoptosis is involved in the pathology of TBI and could contribute to some of the ensuing cell death following injury.     

Increase in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury

Traumatic brain injury (TBI) as a consequence of exposure to blast is increasingly prevalent in military populations, with the underlying pathophysiological mechanisms mostly unknown. In the present study, we utilized an air-driven shock tube to investigate the effects of blast exposure (120 kPa) on rat brains. Immediately following exposure to blast, neurological function was reduced. BBB permeability was measured using IgG antibody and evaluating its immunoreactivity in the brain. At 3 and 24 hr postexposure, there was a transient significant increase in IgG staining in the cortex. At 3 days postexposure, IgG immunoreactivity returned to control levels. Quantitative immunostaining was employed to determine the temporal course of brain oxidative stress following exposure to blast. Levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) were significantly increased at 3 hr postexposure and returned to control levels at 24 hr postexposure. The response of microglia to blast exposure was determined by autoradiographic localization of (3) H-PK11195 binding. At 5 days postexposure, increased binding was observed in the contralateral and ipsilateral dentate gyrus. These regions also displayed increased binding at 10 days postexposure; in addition to these regions there was increased binding in the contralateral ventral hippocampus and substantia nigra at this time point. By using antibodies against CD11b/c, microglia morphology characteristic of activated microglia was observed in the hippocampus and substantia nigra of animals exposed to blast. These results indicate that BBB breakdown, oxidative stress, and microglia activation likely play a role in the neuropathology associated with TBI as a result of blast exposure.     

Genetic disruption of cyclooxygenase-2 does not improve histological or behavioral outcome after traumatic brain injury in mice

Increasing evidence suggests a role for cyclooxygenase-2 (COX-2) in traumatic brain injury (TBI). In the present study, the role of COX-2 in TBI was investigated using COX-2 gene-disrupted (COX-2 null) mice and wild-type (WT) controls that were subjected to the controlled cortical impact (CCI) model of TBI. There was increased expression of COX-2 in ipsilateral hippocampus in WT mice subjected to CCI. CCI resulted in a significant increase in prostaglandin E(2) concentrations in WT compared with COX-2 null hippocampi. There was a significant increase in TUNEL staining of CA1 neurons 24 hr after CCI in WT, but not in COX-2 null mice, compared with sham-operated controls, which is consistent with a protective role for COX-2 in the early phase of injury after TBI. However, there was no difference in lesion volume 21 days after CCI in COX-2 null and WT mice. COX-2 gene disruption did not alter Morris water maze performance. Taken together, these results suggest only a minor role for COX-2 activity in determining outcome after TBI in mouse.     

脑损伤后水通道蛋白4表达与血脑屏障通透性的关系

目的研究脑损伤后，水通道蛋白4（AQP4）的表达变化与血脑屏障（BBB）通透性之间的关系。方法健康成年Wistar大鼠，随机分成创伤性（TBI）组和假手术（SO）组。自由落体硬膜外撞击方法致重度脑创伤模型。于伤后4h、8h、12h、1d、3d、5d、7d取出大鼠脑组织，进行以下实验：①测创伤脑组织中伊文思蓝（EB）外渗的量，以EB外渗的量反应BBB通透性的变化；②免疫组化（IHC）和原位杂交（ISH）检测AQP4的表达变化。结果脑损伤后，BBB通透性增加，其增加有两个高峰，分别在TBI后12h和3d，后者尤为更明显。IHC和ISH显示，脑损伤后AQP4在脑组织中的表达逐渐上调，1d达高峰，持续至3d后下降，7d接近SO组水平。AQP4的表达变化与脑组织伊文思蓝（EB）含量的变化呈正相关（r=0．894，P〈0．05）。结论脑损伤后BBB通透性的增加与脑水肿的形成密切相关。TBI后BBB通透性增加，可能与AQP4表达上调有关，两者的变化影响TBI后脑水肿的发生、发展。     

Acutely increased cyclophilin a expression after brain injury: a role in blood-brain barrier function and tissue preservation

Blood-brain barrier (BBB) compromise is a significant pathologic event that manifests early following traumatic brain injury (TBI). Because many signaling cascades are initiated immediately after the traumatic event, we were interested in examining acute differential protein expression that may be involved in BBB function. At acute time points postinjury, altered protein expression may result from altered translation efficiency or turnover rate rather than from a genomic response. The application of tandem 2-D gel electrophoresis and mass spectrometry analysis is a powerful approach for directly screening differential protein expression following TBI. Using comparative 2-D gel analysis, we selected candidate protein spots with apparent altered expression and identified them by mass spectrometry. Cyclophilin A was selected for further analysis because it has been implicated in endothelial cell activation and inflammation, and studies have suggested cyclosporine A, an inhibitor of all cyclophilin isoforms, might be beneficial after TBI. We examined if altered expression of cyclophilin A in the brain vasculature might play a role in BBB function. We found significantly increased cyclophilin A levels in isolated brain microvessels 30 min following injury. Postinjury administration of cyclosporine A significantly attenuated BBB permeability measured 24 hr postinjury, suggesting cyclophilin activity after TBI may be detrimental. However, direct injection of purified recombinant cyclophilin A attenuated both BBB permeability and tissue damage in a stab wound model of injury. These findings suggest that increased expression of cyclophilin A may play a protective role after TBI, whereas other cyclophilin isoforms may be detrimental.