Feng Jia

Activations of GABAergic signaling, HSP70 and MAPK cascades are involved in baicalin's neuroprotection against gerbil global ischemia/reperfusion injury

Baicalin, a flavonoid compound isolated from the plant Scutellaria baicalensis Georgi, is known as a protective agent against delayed neuronal cell death after ischemia/reperfusion. To investigate the neuroprotective mechanism of baicalin, the present study was conducted to explore whether the alterations of GABAergic signaling, heat shock protein 70 (HSP70) and mitogen-activated protein kinases (MAPKs) were involved in its neuroprotection on gerbils global ischemia. The bilateral carotid arteries were occluded by 5 min and baicalin at the dose of 200 mg/kg was intraperitoneally injected into the gerbils immediately after cerebral ischemia. Seven days after reperfusion, neurological deficit was scored and changes in hippocampal neuronal cell death were assessed by Nissl staining as well as NeuN immunohistochemistry. The mRNA and protein expressions of GABAergic signal molecules (GABA(A)R α1, GABA(A)R γ2, KCC2 and NKCC1) were determined in ischemic hippocampus by real-time RT-PCR and Western blot, respectively. In addition, HSP70 and MAPKs cascades (ERK, JNK and p38) were also detected using western blot assay. Our results illustrated that baicalin treatment significantly facilitated neurological function, suppressed the ischemia-induced neuronal damage. Besides, administration of baicalin also caused a striking increase of GABA(A)R α1, GABA(A)R γ2 and KCC2 together with the decrease of NKCC1 at mRNA and protein levels in gerbils hippocampus following an ischemic insult. Furthermore, the protein expressions of HSP70 and phosphorylated ERK (p-ERK) were evidently augmented while the phosphorylated JNK (p-JNK) and phosphorylated p38 (p-p38) were strikingly diminished in ischemic gerbils with baicalin treatment. These findings suggest that baicalin activates GABAergic signaling, HSP70 and MAPKs cascades in global ischemia, which may be a mechanism underlying the baicalins neuroprotection.

Matrix Metalloproteinase-9 Expression and Protein Levels after Fluid Percussion Injury in Rats: The Effect of Injury Severity and Brain Temperature

The temporal and regional expression profiles of matrix metalloproteinase-9 (MMP-9), after moderate or severe traumatic brain injury (TBI) were measured to investigate the effects of post-traumatic hypothermia (33 degrees C) or hyperthermia (39 degrees C). In the first phase of this study, adult male Sprague-Dawley rats were randomly assigned to groups of moderate TBI (1.8-2.2 atm), severe TBI (2.4-2.7 atm), and sham-injured control. The rats were killed at 4, 6, 12, 24, 48, and 72 h, or 1 week after TBI, for mRNA and protein analysis. In the second phase, rats underwent moderate fluid percussion brain injury, followed immediately by 4 h of post-traumatic normothermia (37 degrees C), hyperthermia (39 degrees C), or hypothermia (32 degrees C). The rats were killed at 12 and 48 h after TBI for mRNA expression analyses, or killed at 24 and 72 h after TBI for protein expression analyses. Brain samples, including the cerebral cortex and hippocampus (both ipsilateral and contralateral hemispheres of each group), were assayed using RT-PCR and Western blot techniques. MMP-9 levels in both the ipsilateral and contralateral hemispheres were significantly increased after TBI compared with those of sham injured animals (p < 0.01). Two expression peaks of MMP-9 were observed in the ipsilateral cortex and hippocampus. An increase in injury severity was associated with an increase in mRNA (12 and 48 h), and protein (24 and 72 h) levels of MMP-9. Post-traumatic hypothermia attenuated the increase in both the mRNA and protein levels of MMP-9, compared with normothermia and hyperthermia (p < 0.01). In contrast, hyperthermia had no significant effect on mRNA (at 12 h) and protein levels (at 24 h) of MMP-9, compared with normothermic values (p > 0.05), but resulted in a significant increase in the levels of MMP-9 mRNA and protein at 24 and 72 h, respectively (p < 0.01). Increases in MMP-9 mRNA and protein after TBI were proportional to injury severity in this model. The effects of post-traumatic hypothermia on the expression of MMP-9 may partially explain the observed effects of post-traumatic temperature on secondary injury after TBI.

Effect of Post-Traumatic Mild Hypothermia on Hippocampal Cell Death after Traumatic Brain Injury in Rats

In this investigation, we evaluated the effect of post-traumatic mild hypothermia on cell death in the hippocampus after fluid percussion traumatic brain injury (TBI) in rats. Adult male Sprague-Dawley rats were randomly divided into three groups (n = 40/group): TBI with hypothermia treatment (32 degrees C), TBI with normothermia (37 degrees C), and sham injury. The TBI model was induced by a fluid percussion TBI device. Mild hypothermia (32 degrees C) was achieved by partial immersion in a water bath (0 degrees C) under general anesthesia for 4h. All rats were killed at 24 or 72h after TBI. The ipsilateral hippocampal CA1 in all rats were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick end labeling (TUNEL), and 4,6-diamidino-2-phenylindole (DAPI) staining for determining cell death. Caspase-3 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. At 24h, based on TUNEL and DAPI results, the cell death index was 28.80 +/- 2.60% and 32.10 +/- 1.40% in the normothermia TBI group, while reaching only 14.30 +/- 2.70% and 18.40 +/- 2.10% in the hypothermic TBI group (p < 0.01). Based on RT-PCR and Western blotting results, the expression of caspase-3 was 210.20 +/- 5.30% and 170.30 +/- 4.80% in the normothermic TBI group, while reaching only 165.10 +/- 3.70% and 130.60 +/- 4.10% in the hypothermic TBI group (p < 0.05). At 72h, based on TUNEL and DAPI results, the cell death index was 20.80 +/- 2.50% and 25.50 +/- 1.80% in the normothermic TBI group, while reaching only 10.20 +/- 2.60% and 15.50 +/- 2.10% in the hypothermic TBI group (p < 0.01). Based on RT-PCR and Western blotting results, the expression of caspase-3 was 186.20 +/- 6.20% and 142.30 +/- 5.10% in the normothermic TBI group, versus only 152.10 +/- 3.60% and 120.60 +/- 3.90% in the hypothermic TBI group (p < 0.05). Based on our findings, we conclude that post-traumatic hypothermia significantly attenuates cell death within the hippocampus following fluid percussion injury. Taken together with other studies, these observations support the premise that post-traumatic mild hypothermia can provide cerebral protection for patients with TBI.

Blood–brain barrier permeability is positively correlated with cerebral microvascular perfusion in the early fluid percussion-injured brain of the rat

The blood–brain barrier (BBB) opening following traumatic brain injury (TBI) provides a chance for therapeutic agents to cross the barrier, yet the reduction of the cerebral microvascular perfusion after TBI may limit the intervention. Meanwhile, optimizing the cerebral capillary perfusion by the strategies such as fluid administration may cause brain edema due to the BBB opening post trauma. To guide the TBI therapy, we characterized the relationship between the changes in the cerebral capillary perfusion and BBB permeability after TBI. First, we observed the changes of the cerebral capillary perfusion by the intracardiac perfusion of Evans Blue and the BBB disruption with magnetic resonance imaging (MRI) in the rat subjected to lateral fluid percussion (FP) brain injury. The correlation between two variables was next evaluated with the correlation analysis. Since related to BBB breakdown, matrix metalloproteinase-9 (MMP-9) activity was finally detected by gelatin zymography. We found that the ratios of the perfused microvessel numbers in the lesioned cortices were significantly reduced at 0 and 1u2009h post trauma compared with that in the normal cortex, which then dramatically recovered at 4 and 24u2009h after injury, and that the BBB permeability was greatly augmented in the ipsilateral parts at 4, 12, and 24u2009h, and in the contralateral area at 24u2009h after injury compared with that in the uninjured brain. The correlation analysis showed that the BBB permeability increase was related to the restoration of the cerebral capillary perfusion over a 24-h period post trauma. Moreover, the gelatin zymography analysis indicated that the MMP-9 activity in the injured brain increased at 4u2009h and significantly elevated at 12 and 24u2009h as compared to that at 0 or 1u2009h after TBI. Our findings demonstrate that the 4u2009h post trauma is a critical turning point during the development of TBI, and, importantly, the correlation analysis may guide us how to treat TBI.

MMP-9 Inhibitor SB-3CT Attenuates Behavioral Impairments and Hippocampal Loss after Traumatic Brain Injury in Rat

The aim of this study was to evaluate the potential efficacy of SB-3CT, a matrix metallopeptidase 9 inhibitor, on behavioral and histological outcomes after traumatic brain injury (TBI) in rats. Adult male Sprague-Dawley rats were randomly divided into three groups (n=15/group): TBI with SB-3CT treatment, TBI with saline, and sham injury. The TBI model was induced by a fluid percussion TBI device. SB-3CT (50u2009mg/kg in 10% dimethyl sulfoxide) was administered intraperitoneally at 30u2009min, 6u2009h, and 12u2009h after the TBI. Motor function (beam-balance/beam-walk tests) and spatial learning/memory (Morris water maze) were assessed on post-operative Days 1-5 and 11-15, respectively. Fluoro-Jade staining, immunofluorescence, and cresyl violet-staining were carried out for histopathological evaluation at 24u2009h, 72u2009h, and 15 days after TBI, respectively. It was shown that TBI can result in significant behavioral deficit induced by acute neurodegeneration, increased expression of cleaved caspase-3, and long-term neuronal loss. SB-3CT intervention via the current regime provides robust behavioral protection and hippocampal neurons preservation from the deleterious effects of TBI. Hence, the efficacy of SB-3CT on TBI prognosis could be ascertained. It is believed that the current study adds to the growing literature in identifying SB-3CT as a potential therapy for human brain injury.

The Up-Regulation of Voltage-Gated Sodium Channel NaV1.6 Expression Following Fluid Percussion Traumatic Brain Injury in Rats

BACKGROUNDThe influx of Na+ and the depolarization mediated by voltage-gated sodium channels (VGSCs) is an early event in traumatic brain injury (TBI) induced cellular abnormalities and is therefore well positioned as an upstream target for pharmacologic modulation of the pathological responses to TBI. Alteration in the expression of the VGSC α-subunit has occurred in a variety of neuropathological states including focal cerebral ischemia, spinal injury, and epilepsy. OBJECTIVEIn this study, changes in Nav1.6 mRNA and protein expression were investigated in rat hippocampus after TBI. METHODSForty-eight adult male Sprague Dawley rats were randomly assigned to control or TBI groups. TBI was induced with a lateral fluid percussion device. Expression of mRNA and protein for Nav1.6 in the bilateral hippocampus was examined at 2, 12, 24, and 72 hours after injury by real-time quantitative polymerase chain reaction and Western blot. Immunofluorescence was performed to localize the expression of Nav1.6 protein in the hippocampus. RESULTSExpression of >Nav1.6 mRNA was significantly up-regulated in the bilateral hippocampus at 2 and 12 hours post-TBI. Significant up-regulation of Nav1.6 protein was identified in the ipsilateral hippocampus from 2 to 72 hours post-TBI and in the contralateral hippocampus from 2 to 24 hours post-TBI. Expression of Nav1.6 occurred predominantly in neurons in the hippocampus. CONCLUSIONResults of the study showed significant up-regulation of mRNA and protein for Nav1.6 in rat hippocampal neurons after TBI.

The Effect of Hypothermia on the Expression of TIMP-3 after Traumatic Brain Injury in Rats

Here we investigate the effect of hypothermia on the expression of apoptosis-regulating protein TIMP-3 after fluid percussion traumatic brain injury (TBI) in rats. We began with 210 adult male Sprague-Dawley rats and randomly assigned them to three groups: TBI with hypothermia treatment (32°C), TBI with normothermia (37°C), and sham-injured controls. TBI was induced by a fluid percussion TBI device. Mild hypothermia (32°C) was achieved by partial immersion in a water bath (0°C) under general anesthesia for 4u2009h. The rats were killed at 4, 6, 12, 24, 48, and 72u2009h and 1 week after TBI. The mRNA and protein level of TIMP-3 in both the injured and uninjured hemispheres of the brains from each group were measured using RT-PCR and Western blotting. In the normothermic group, TIMP-3 levels in both the injured and uninjured hemispheres were significantly increased after TBI compared with those of sham-injured animals (pu2009<u20090.01). In contrast, post-traumatic hypothermia significantly attenuated this increase. According to the RT-PCR and Western blot analyses, the maximum mRNA levels of TIMP-3 were reduced to 60.60u2009±u20092.30%, 55.83u2009±u20091.80%, 66.03u2009±u20092.10%, and 64.51u2009±u20091.50%, respectively, of the corresponding values in the normothermic group in the injured and uninjured hemispheres (cortex and hippocampus) of the hypothermia group (pu2009<u20090.01), while the respective maximum protein levels of TIMP-3 were reduced to 57.50u2009±u20091.50, 52.67u2009±u20092.20, 60.31u2009±u20092.50 and 54.76u2009±u20091.40 (pu2009<u20090.01). Our data suggest that moderate fluid percussion brain injury significantly upregulates TIMP-3 expression, and that this increase may be suppressed by hypothermia treatment.

Antagonism of R-Type Calcium Channels Significantly Improves Cerebral Blood Flow after Subarachnoid Hemorrhage in Rats

The effects of R-type calcium channels on cerebral blood flow (CBF) and vasospasm pathways following subarachnoid hemorrhage (SAH) have not been well studied. The aim of this study was to investigate the role of R-type calcium channels in vasospasm development and treatment. Sixty-five rats were randomly divided into four groups: sham (nu2009=u200914), SAH (nu2009=u200917), SAH + nimodipine (nu2009=u200917), and SAH + SNX-482 (nu2009=u200917). A prechiasmatic SAH model was constructed on day 0. Then 5u2009μg of nimodipine (an L-type calcium channel antagonist) or 0.1u2009μg of SNX-482 (an R-type calcium channel antagonist) was infused intracisternally on days 1 and 2. On day 3, neurological status was evaluated and CBF was determined using fluorescent microspheres. The extent of myosin light chain-2 (MLC2) phosphorylation was determined by urea-glycerol polyacrylamide gel electrophoresis, followed by immunoblotting. The relative presence of R-type calcium channels and calponin was determined by SDS polyacrylamide gel electrophoresis, followed by immunoblotting. Numbers of R-type calcium channels increased following SAH, and neurological deficit, CBF reduction, and enhancement of MLC2 phosphorylation as well as calponin degradation were all found to be present. There were no statistically significant differences in neurological scores among the SAH, SAH + nimodipine, and SAH + SNX-482 groups. Nimodipine had no significant effect on CBF reduction compared to the SAH group (pu2009>u20090.008), whereas SNX-482 significantly inhibited CBF reduction (pu2009<u20090.008). Both MLC2 phosphorylation and calponin degradation appeared to be inhibited by SNX-482, whereas the effects of nimodipine were relatively blunted. We concluded that an R-type calcium channel antagonist may improve CBF following SAH by partially inhibiting MLC2 phosphorylation and calponin degradation, and may exceed the potential of an L-type calcium channel antagonist, which suggests a more crucial role for R-type calcium channels in the development of SAH vasospasm and its treatment.

Migration of neural stem cells to ischemic brain regions in ischemic stroke in rats

An established rat model of ischemic stroke, produced by temporary middle cerebral artery occlusion and reperfusion (MCAO/R), was used in the evaluation of organ migration of intra-arterial (IA) transplantation of neural stem cells (NSCs). Immediately after transplantation, ischemic rats (n=8) transplanted with either NSCs (MCAO/R+NSC group) or NSC growth medium (MCAO/R+medium group) exhibited neurological dysfunction but rats in a sham+NSCs group (n=5) did not. During the post-operative period, neurological function improved to a similar extent in both MCAO/R groups. At 10 and 14 days post-transplantation, neurological function in the MCAO/R+NSC group was superior to that in the MCAO/R+medium group (p<0.001). Hematoxylin-eosin staining showed neuronal degeneration and necrosis in ischemic rats. Immunofluorescence staining revealed that NSCs had migrated to the frontal and parietal lobes, caudate, and putamen. Some cells had begun differentiating into neurons and astrocytes. Rat NSCs can migrate into the ischemic region, survive, and differentiate into astrocytes and neurons, and thereby potentially improve neurologic function after cerebral ischemia.

Blockage of the Upregulation of Voltage-Gated Sodium Channel Nav1.3 Improves Outcomes after Experimental Traumatic Brain Injury

Excessive active voltage-gated sodium channels are responsible for the cellular abnormalities associated with secondary brain injury following traumatic brain injury (TBI). We previously presented evidence that significant upregulation of Nav1.3 expression occurs in the rat cortex at 2u2009h and 12u2009h post-TBI and is correlated with TBI severity. In our current study, we tested the hypothesis that blocking upregulation of Nav1.3 expression in vivo in the acute stage post-TBI attenuates the secondary brain injury associated with TBI. We administered either antisense oligodeoxynucleotides (ODN) targeting Nav1.3 or artificial cerebrospinal fluid (aCSF) at 2u2009h, 4u2009h, 6u2009h, and 8u2009h following TBI. Control sham animals received aCSF administration at the same time points. At 12u2009h post-TBI, Nav1.3 messenger ribonucleic acid (mRNA) levels in bilateral hippocampi of the aCSF group were significantly elevated, compared with the sham and ODN groups (p<0.01). However, the Nav1.3 mRNA levels in the uninjured contralateral hippocampus of the ODN group were significantly lowered, compared with the sham group (p<0.01). Treatment with antisense ODN significantly decreased the number of degenerating neurons in the ipsilateral hippocampal CA3 and hilar region (p<0.01). A set of left-to-right ratio value analyzed by magnetic resonance imaging T2 image on one day, three days, and seven days post-TBI showed marked edema in the ipsilateral hemisphere of the aCSF group, compared with that of the ODN group (p<0.05). The Morris water maze memory retention test showed that both the aCSF and ODN groups took longer to find a hidden platform, compared with the sham group (p<0.01). However, latency in the aCSF group was significantly higher than in the ODN group (p<0.05). Our in vivo Nav1.3 inhibition studies suggest that therapeutic strategies to block upregulation of Nav1.3 expression in the brain may improve outcomes following TBI.