Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.     

Pharmacological Inhibition of PERK Attenuates Early Brain Injury After Subarachnoid Hemorrhage in Rats Through the Activation of Akt

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Endoplasmic reticulum (ER) stress was reported to have a vital role in the pathophysiology of neuronal apoptosis in the brain. The present study was designed to investigate the potential effects of ER stress and its downstream signals in early brain injury after SAH. One hundred thirty-four rats were subjected to an endovascular perforation model of SAH. The RNA-activated protein kinase-like ER kinase (PERK) inhibitor GSK2606414 and the Akt inhibitor MK2206 were injected intracerebroventricularly. SAH grade, neurologic scores, and brain water content were measured 72 h after subarachnoid hemorrhage. Expression of PERK and its downstream signals, Akt, Bcl-2, Bax, and cleaved caspase-3, were examined using Western blot analysis. Specific cell types that expressed PERK were detected with double immunofluorescence staining. Neuronal cell death was demonstrated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Our results showed that the expression of p-PERK and its downstream targets, p-eIF2α and ATF4, increased after SAH and peaked at 72 h after SAH. PERK was expressed mostly in neurons. The inhibition of PERK with GSK2606414 reduced p-PERK, p-eIF2α, and ATF4 expression. Furthermore, GSK2606414 treatment increased p-Akt levels and the Bcl-2/Bax ratio as well as decreased cleaved caspase-3 expression and neuronal death, thereby improving neurological deficits at 72 h after SAH. The selective Akt inhibitor MK2206 abolished the beneficial effects of GSK2606414. PERK, the major transducer of ER stress, is involved in neuronal apoptosis after SAH. The inhibition of PERK reduces early brain injury via Akt-related anti-apoptosis pathways. PERK may serve as a promising target for future therapeutic intervention.     

Expression and Cell Distribution of Neuroglobin in the Brain Tissue After Experimental Subarachnoid Hemorrhage in Rats: A Pilot Study

Neuroglobin (Ngb) is a member of the globin superfamily expressed mainly in the nervous system and retina of vertebrates. Accumulated evidence has clearly demonstrated that Ngb has a neuro-protective role enhancing cell viability under hypoxia and other types of oxidative stress. It was suggested that oxidant stress could play an important role in neuronal injury after subarachnoid hemorrhage (SAH). The present study aims to examine the expression of Ngb in the temporal cortex and its cellular localization after SAH. We used a prechiasmatic cistern model of SAH. Ngb expression was examined at 3, 6, 12, 24, 48, and 72 h after SAH by western blot analysis and real-time polymerase chain reaction (PCR). Immunohistochemistry and immunofluorescence were performed to detect the localization of Ngb. Real-time PCR demonstrated that Ngb mRNA levels increased from 3 h after SAH, peaked at 6 h. Western blot showed Ngb protein levels were significantly increased in SAH groups in the temporal cortex and reached the peak at 24 h after SAH. The immunohistochemical staining demonstrated that Ngb was weakly expressed in the cortex in the control group while the enhanced expression of Ngb could be detected in the SAH groups. In addition, immunofluorescence results revealed that the over-expressed Ngb was located in the neuronal and microglia cell cytoplasm. These findings indicated that Ngb might play an important neuro-protective effect after SAH.     

Pentoxifylline Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats: Possibly via Inhibiting TLR 4/NF-κB Signaling Pathway

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) generally causes significant and lasting damage. Pentoxifylline (PTX), a nonselective phosphodiesterase inhibitor, has shown anti-inflammatory and neuroprotective properties in several brain injury models, but the role of PTX with respect to EBI following SAH remains uncertain. The purpose of this study was to investigate the effects of PTX on EBI after SAH in rats. Adult male Sprauge–Dawley rats were randomly assigned to the sham and SAH groups. PTX (30 or 60 mg/kg) or an equal volume of the administration vehicle (normal saline) was administrated at 30 min intervals following SAH. Neurological scores, brain edema, and neural cell apoptosis were evaluated. In order to explore other mechanisms, changes in the toll-like receptor 4 (TLR4) and the nuclear factor-κB (NF-κB) signaling pathway, in terms of the levels of apoptosis-associated proteins, were also investigated. We found that administration of PTX (60 mg/kg) notably improved neurological function and decreased brain edema at both 24 and 72 h following SAH. Treatment with PTX (60 mg/kg) significantly inhibited the protein expressions of TLR4, NF-κB, MyD88 and the downstream pro-inflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). PTX also significantly reduced neural cell death and BBB permeability. Our observations may be the first time that PTX has been shown to play a neuroprotective role in EBI after SAH, potentially by suppressing the TLR4/NF-κB inflammation-related pathway in the rat brain.     

GPR18 Agonist Resolvin D2 Reduces Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage by Multiple Protective Mechanisms

Early brain injury (EBI) is the early phase of secondary complications arising from subarachnoid hemorrhage (SAH). G protein-coupled receptor 18 (GPR18) can exert neuroprotective effects during ischemia. In this study, we investigated the roles of GPR18 in different brain regions during EBI using a GPR18 agonist, resolvin D2 (RvD2). Location and dynamics of GPR18 expression were assessed by immunohistochemistry and western blotting in a rat model of SAH based on endovascular perforation. RvD2 was given intranasally at 1 h after SAH, and SAH grade, brain water content and behavior were assayed before sacrifice. TUNEL and dihydroethidium staining of the cortex were performed at 24 h after SAH. Selected brain regions were also examined for pathway related proteins using immunofluorescence and Western blotting. We found that GPR18 was expressed in meninges, hypothalamus, cortex and white matter before EBI. After SAH, GPR18 expression was increased in meninges and hypothalamus but decreased in cortex and white matter. RvD2 improved neurological scores and brain edema after SAH. RvD2 attenuated mast cell degranulation and reduced expression of chymase and tryptase expression in the meninges. In the hypothalamus, RvD2 attenuated inflammation, increased expression of proopiomelanocortin and interleukin-10, as well as decreased expression of nerve peptide Y and tumor necrosis factor-α. In cortex, RvD2 alleviated oxidative stress and apoptosis, and protected the blood–brain barrier. RvD2 also ameliorated white matter injury by elevating myelin basic protein and suppressing amyloid precursor protein. Our results suggest that GPR18 may help protect multiple brain regions during EBI, particularly in the cortex and hypothalamus. Upregulating GPR18 by RvD2 may improve neurological functions in different brain regions via multiple mechanisms.

     

HLRP 蛋白在大鼠脑中的定位及LPS 对其表达的影响

目的:研究HLRP分子在大鼠脑中的细胞定位和表达特点,观察LPS刺激对动物脑HLRP表达的影响。方法:对原代培养的大鼠神经元、星形胶质细胞、小胶质细胞和大鼠脑组织冰冻切片分别进行免疫荧光染色,观察HLRP的细胞定位和表达特点;给大鼠侧脑室注射LPS,提脑组织蛋白,进行Western blot检测,半定量分析LPS刺激后,大鼠脑HLRP的表达变化。结果:①HLRP选择性表达于部分神经元的细胞核中,正常的星形胶质细胞和小胶质细胞不表达HLRP。②从嗅脑到脑干各节段,HLRP在大鼠脑组织中均匀分布,未发现HLRP阳性神经元聚集的现象。③侧脑室注射LPS 1天以后,HLRP表达明显升高(P<0.05)。结论:大鼠脑中正常表达HLRP,侧脑室注射LPS能刺激HLRP表达。     

Regulated expression of pancreatic triglyceride lipase after rat traumatic brain injury

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT–PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.     

Effects of Tenascin-C Knockout on Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage in Mice

A matricellular protein tenascin-C (TNC) has been suggested to play a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the direct evidence remains lacking. In this study, we examined effects of TNC knockout (TNKO) on cerebral vasospasm after experimental SAH in mice. C57BL/6 wild-type (WT) or TNKO mice were subjected to SAH by endovascular puncture. Ten WT and ten TNKO mice were randomized to WT sham (n = 4), TNKO sham (n = 4), WT SAH (n = 6), and TNKO SAH (n = 6) groups. In addition to neurobehavioral impairments and severity of SAH, cerebral vasospasm was assessed by morphometric measurements of the left internal carotid artery (ICA). Infiltration of inflammatory cells in the subarachnoid periarterial space was also assessed, and expressions of TNC and mitogen-activated protein kinases (MAPKs) in the ICA were immunohistochemically evaluated at 24 h post-surgery. TNC was induced in the smooth muscle cell layers and the adventitia in the spastic ICAs as well as the periarterial inflammatory cells in WT SAH mice. Compared with WT SAH mice, TNKO SAH mice showed better neurological scores and less severe cerebral vasospasm, as well as fewer inflammatory cell infiltration in the periarterial space. Post-SAH activation of MAPKs in the smooth muscle cell layers of the ICAs was also prevented in TNKO SAH mice. The findings in the present study suggest that TNC causes the development of cerebral vasospasm via pro-inflammatory effects and activation of MAPKs.     

Expression of Nemo-Like Kinase (NLK) in the Brain in a Rat Experimental Subarachnoid Hemorrhage Model

This study aimed to investigate the expression of the Nemo-like kinase (NLK) in the brain after experimental subarachnoid hemorrhage (SAH) in rats. A total of 90 rats were randomly divided into six groups: control group, day 1, day 3, day 5, day 7, and day 14. Day 1, day 3, day 5, day 7, and day 14 groups were all SAH groups in which the rats were killed on days 1, 3, 5, 7, and 14, respectively. In SAH groups, autologous arterial blood was injected into cisterna magna once on day 0. Cross-sectional area of basilar artery was measured by H&E staining. Immunostaining and immunoblotting experiments were performed to detect the expression of NLK protein. Real-time polymerase chain reaction was used to analyze the presence and quantity of NLK mRNA. The level of oxidative stress in the artery was also measured. The basilar arteries exhibited vasospasm after SAH and became the most severe on day 3. The expressions of NLK protein and mRNA were decreased remarkably in SAH groups compared with the control group. The down-regulated expression of NLK was detected after SAH and the low ebb was on day 3, which was oppositely the peak time of oxidative stress. The expression of NLK was present mainly in the neurons in the brain and smooth muscle cells in the basilar artery. NLK is decreasingly expressed in an opposite time-course to the development of cerebral vasospasm (CVS) and SAH-induced brain injury in this rat experimental model of SAH and these findings might have important implications during the administration of specific NLK agonist to prevent or reduce CVS or neuronal apoptosis caused by SAH.     

Folic Acid Acts Through DNA Methyltransferases to Induce the Differentiation of Neural Stem Cells into Neurons

The present study investigated the roles of folic acid and DNA methyltransferases (DNMTs) in the differentiation of neural stem cells (NSCs). Neonatal rat NSCs were grown in suspended neurosphere cultures and identified by their expression of SOX2 protein and capacity for self-renewal. Then NSCs were assigned to five treatment groups for cell differentiation: control (folic acid-free differentiation medium), low folic acid (8 μg/mL), high folic acid (32 μg/mL), low folic acid and DNMT inhibitor zebularine (8 μg/mL folic acid and 150 nmol/mL zebularine), and high folic acid and zebularine (32 μg/mL folic acid and 150 nmol/mL zebularine). After 6 days of cell differentiation, immunocytochemistry and western blot analyses were performed to identify neurons by β-tubulin III protein expression and astrocytes by GFAP expression. We observed that folic acid increased DNMT activity which may be regulated by the cellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and the abundance of neurons but decreased the number of astrocytes. Zebularine blocked these effects of folic acid. In conclusion, folic acid acts through elevation of DNMT activity to increase neuronal differentiation and decrease astrocytic differentiation in NSCs.     

Role of autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) occurred after aneurismal subarachnoid hemorrhage (SAH) strongly determined the patients’ prognosis. Autophagy was activated in neurons in the acute phase after SAH, while its role in EBI has not been examined. This study was designed to explore the effects of autophagy on EBI post-SAH in rats. A modified endovascular perforating SAH model was established under monitoring of intracranial pressure. Extent of autophagy was regulated by injecting autophagy-regulating drugs (3-methyladenine, wortmannin and rapamycin) 30 min pre-SAH intraventricularly. Simvastatin (20 mg/kg) was prophylactically orally given 14 days before SAH induction. Mortality, neurological scores, brain water content and blood–brain barrier (BBB) permeability were evaluated at 24 h post-SAH. Microtubule-associated protein light chain-3 (LC3 II/I) and beclin-1 were detected for monitoring of autophagy flux. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, expression of cleaved caspase-3 and cytoplasmic histone-associated DNA fragments were used to detect apoptosis. The results showed that mortality was reduced in rapamycin and simvastatin treated animals. When autophagy was inhibited by 3-methyladenine and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated and neuronal apoptosis was increased when compared with the SAH animals. Autophagy was further activated by rapamycin and simvastatin, and apoptosis was inhibited and EBI was ameliorated. The present results indicated that activation of autophagy decreased neuronal apoptosis and ameliorated EBI after SAH. Aiming at autophagy may be a potential effective target for preventing EBI after SAH.     

Improvement of Hypoxia–Ischemia-Induced White Matter Injury in Immature Rat Brain by Ethyl Pyruvate

Ethyl pyruvate (EP) has been reported to be neuroprotective in several models of brain injury, yet its influence on periventricular leukomalacia still remains elusive. Here we investigated whether repeated administration of EP could protect against white matter injury after hypoxia–ischemia (HI) (right common carotid artery ligation and 6 % O₂ for 60 min) in post-natal 3 day rat pups. EP was injected (50 mg/kg, intraperitoneally) 10 min, 1 and 24 h after HI insult. Treatment with EP significantly reduced HI-induced ventricular enlargement, loss of developing oligodendrocytes, and hypomyelination. We further demonstrated a marked inhibitory effect of EP on inflammatory responses, as indicated by the decreased number of activated microglia and astrocytes and the reduced release of proinflammatory cytokines. Moreover, EP down-regulated the expression of cleaved caspase-3 and Bax, and up-regulated Bcl-2 expression after HI exposure. In conclusion, our results demonstrated that EP was able to provide potent protection on white matter injury through blocking the cerebral inflammatory responses and modulating the apoptotic death program of oligodendrocytes, indicating a potential neuroprotective agent in neonatal brain injury.     

The expression pattern of ADP-ribosyltransferase 3 in rat traumatic brain injury

Mammalian ecto ADP-ribosyltransferases (ARTs) can regulate the biological functions of various types of cells by catalyzing the transfer of single ADP-ribose moiety from NAD⁺ to a specific amino acid in a target protein. ART3 is a member of the known ART family which is involved in cell division, DNA-repair and the regulation of the inflammatory response. To elucidate the expression, cellular localization and possible functions of ART3 in central nervous system (CNS) lesion and repair, we performed an acute traumatic brain injury model in adult rats. Western blot analysis showed that the expression of ART3 in ipsilateral brain cortex increased, then reached a peak at day 3 after traumatic brain injury (TBI), and gradually declined during the following days. But in the contralateral brain cortex, no obvious alterations were observed. Immunohistochemistry revealed the highly significant accumulation of ART3 at the ipsilateral brain in comparison to contralateral cerebral cortex. Double immunofluorescence labeling suggested that ART3 was localized mainly in the plasmalemma of neurons, but not in astrocytes or microglias within 3 mm from the lesion site at day 3 post-injury. In addition, we detected the expression profiles of caspase-3 and growth associated protein 43 (GAP-43) whose changes were correlated with the expression profiles of ART3 in this TBI model. Besides, co-localization of ART3/active caspase-3 and ART3/GAP43 were detected in NeuN-positive cells, respectively. Moreover, Pheochromocytoma (PC12) cells were treated with H₂O₂ to establish an apoptosis model. The results showed that the expression of ART3 was increased in the concentration and time dependence way. To further examine the involvement of ART3 in apoptosis of PC12, 3-Methoxybenzamide was used in flow cytometry analysis of apoptotic cells stained with Annexin V and PI. The experimental group in which 3-Methoxybenzamide used had a relative low level of apoptotic index compared with the untreated group. Together with previous reports, we hypothesize that ART3 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.     

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.     

Docosahexaenoic Acid Alleviates Oxidative Stress-Based Apoptosis Via Improving Mitochondrial Dynamics in Early Brain Injury After Subarachnoid Hemorrhage

Mitochondrial dysfunction is considered a crucial therapeutic target for early brain injury following subarachnoid hemorrhage (SAH). Emerging evidence indicates that docosahexaenoic acid (DHA), an essential omega-3 fatty acid, protects mitochondria in various chronic diseases. This study aimed to investigate the neuroprotective effects of DHA on mitochondrial dynamic dysfunction after EBI using in vivo and in vitro approaches. For in vivo experiments, the rat endovascular perforation SAH model was performed, whereby DHA was administered intravenously 1 h after induction of SAH. Primary cultured neurons treated with oxyhemoglobin (OxyHb) for 24 h were used to mimic SAH in vitro. Our results demonstrated that DHA improved neurological deficits and reduced brain edema in rats with SAH, and attenuated OxyHb-induced neuronal death in primary cultured cells. DHA reduced the amount of reactive oxygen species-positive cells and improved cell viability when compared to the SAH?+?vehicle group in vitro. DHA attenuated malondialdehyde levels and superoxide dismutase stress, increased Bcl2 and Bcl-xl, and decreased Bax and cleaved caspase-3 in vivo. Additionally, DHA ameliorated mitochondrial dysfunction, upregulated the mitochondrial fusion-related protein Optic Atrophy 1, and downregulated the mitochondrial fission-related protein Dynamin-Related-Protein 1 (Drp1) and Serine 616 phosphorylated Drp1 after SAH both in vitro and in vivo. Taken together, our current study demonstrates that DHA might prevent oxidative stress-based apoptosis after SAH. The characterization of the underlying molecular mechanisms may further improve mitochondrial dynamics-related signaling pathways.     

A mechanistic investigation into non-infarcted brain injury induced by cerebral artery microemboli

To establish a rat brain injury by non-infarction process model induced by cerebral artery microemboli which would be used to further explore the neural injury mechanisms of cerebral artery microemboli. Seventy-two Sprague–Dawley rats were randomly divided into the microemboli group and the sham group; 100 25–50 μm microemboli in 300 μl or the same amount of saline were injected into the left carotid artery, respectively. The severity of neuron damage was assessed 3 and 7 days after the operation, using haematoxylin-eosin (HE) staining and immunohistochemical staining for caspase-3. Immunohistochemical staining for CD11b and GFAP were used to quantitatively analyse hyperplasia and the activation of microglia and astrocytes. TNF-α expression was detected by using ELISA and the NF-κB expression was detected by employing Western blotting. The results of HE staining had shown that ischaemic infarct foci were not detected in either the microemboli group or sham group. Only a few apoptotic cells and a few cells with the positive expression of CD11b and GFAP were detected in the sham group. And compared with that of the sham group, the number of apoptotic cells and the positive expression of CD11b and GFAP in the microemboli group were significantly increased (P < 0.001). These parameters were also significantly increased 7 days after the operation compared to rats 3 days after surgery (P < 0.001). The expressions of TNF-α and NF-κB were significantly increased in the microemboli group (P < 0.001), and the increase of the expression of TNF-α and NF-κB on the 3 days was more significant compared to that of TNF-α and NF-κB on 7 days (P < 0.001). Injection of 25–50 μm microemboli at a dose of 100 microemboli in 300 μl into the carotid artery of rats did not result in cerebral infarction, but led to neuronal apoptosis, hyperplasia and activation of microglia and astrocytes. This leads us to conclude that TNF-α and NF-κB may play important roles in the pathogenesis of neuronal apoptosis induced by microemboli in the cerebral arteries.     

Attenuation of Early Brain Injury and Learning Deficits Following Experimental Subarachnoid Hemorrhage Secondary to Cystatin C: Possible Involvement of the Autophagy Pathway

Cystatin C (CysC) is a cysteine protease inhibitor and previous studies have demonstrated that increasing endogenous CysC expression has therapeutic implications on brain ischemia, Alzheimer’s disease, and other neurodegenerative disorders. Our previous reports have demonstrated that the autophagy pathway was activated in the brain after experimental subarachnoid hemorrhage (SAH), and it may play a beneficial role in early brain injury (EBI). This study investigated the effects of exogenous CysC on EBI, cognitive dysfunction, and the autophagy pathway following experimental SAH. All SAH animals were subjected to injections of 0.3 ml fresh arterial, nonheparinized blood into the prechiasmatic cistern in 20 s. As a result, treatment with CysC with low and medial concentrations significantly ameliorated the degree of EBI when compared with vehicle-treated SAH rats. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosomes, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased in the cortex 48 h after SAH and were further up-regulated after CysC therapy. By ultrastructural observation, there was a marked increase in autophagosomes and autolysosomes in neurons of CysC-treated rats. Learning deficits induced by SAH were markedly alleviated after CysC treatment with medial doses. In conclusion, pre-SAH CysC administration may attenuate EBI and neurobehavioral dysfunction in this SAH model, possibly through activating autophagy pathway.