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Background. Subarachnoid hemorrhage (SAH) is an important subcategory of stroke due to its high mortality rate as well as severe complications such as neurological deficit. It has been suggested that cerebral inflammation is a major factor in advanced brain injury after SAH. Microglia and astrocytes are known supporting cells in the development and maintenance of inflammation in central nervous system. However, the role of microglia and astrocytes in the development of inflammation and neuronal cell apoptosis during the early phase after SAH has not been thoroughly investigated. Materials and Methods. Sprague-Dawley rats were divided into 4 groups ( n = 6 /group): sham group, animals subjected to SAH without treatment, SAH animals pretreated with the microglia inhibitor minocycline (50 mg/kg, ip), and SAH animals pretreated with the astrocyte inhibitor fluorocitrate (50 mg/kg, ip). SAH was induced by injecting autologous blood (1 ml/kg) into the cistern magna on day 0. Pretreatment with minocycline or fluorocitrate was given three days prior to the induction of SAH. Rats were sacrificed 6 hr after SAH, and their cerebral spinal fluids were used to measure protein levels of neuroinflammatory cytokines IL-1β, IL-6, and TNF-α by ELISA. In addition, the cerebral cortex was utilized to determine the levels of caspase-3 by western blot and to evaluate neuronal cell apoptosis by immunohistochemistry staining and detect microglia and astrocyte by immunofluorescence staining for Iba-1 and GFAP. In this study, all SAH animals were given an injection of autologous blood and SAH rats treated with minocycline or fluorocitrate received ip injections on day 1, 2, and 3 before inducing SAH. Neurological outcome was assessed by ambulation and placing/stepping reflex responses on day 7. Results. Immunofluorescence staining showed that SAH induced proliferation of microglia and astrocyte and minocycline inhibited the proliferation of both microglia and astrocyte. However, fluorocitrate inhibited only the proliferation of astrocyte. ELISA analysis showed that SAH upregulated TNF-α and IL-1β, but not IL-6 at 6 hr after SAH. Minocycline, but not fluorocitrate, attenuated the upregulation of TNF-α and IL-1β. Western blot analysis and immunohistochemistry staining showed that SAH induced neuronal cell apoptosis. Pretreatment with minocycline, but not fluorocitrate, decreased SAH-induced neuronal death and cerebral vasospasm. Furthermore, significant improvements in neurobehavioral outcome were seen in the minocycline treatment group, but not in animals treated with fluorocitrate. Conclusions. Microglia may play an important role to regulate neuronal cell apoptosis and cerebral vasospasm through inhibiting inflammation at an early phase after SAH in the rat.

Subarachnoid hemorrhage (SAH) is an important subcategory of stroke due to its unacceptably high mortality rate as well as the severe complications it causes, such as cerebral vasospasm, neurological deficits, and cardiopulmonary abnormality. Hepatoma-derived growth factor (HDGF) is a growth factor related to normal development and is involved in liver development and regeneration. This study explored the relationship between SAH and HDGF. Sixty rats were divided into five groups (n = 12/group): (A) control group; (B) rHDGF ab only group [normal animals treated with 50 µM recombinant HDGF antibodies (rHDGF ab)]; (C) SAH group; (D) SAH + pre-rHDGF ab group (SAH animals pre-treated with 50 µM rHDGF ab into the subarachnoid space within 24 h before SAH); and (E) SAH + post-rHDGF ab group (SAH animals post-treated with 50 µM rHDGF ab into the subarachnoid space within 24 h after SAH). At 48 h after SAH, serum and cerebrospinal fluid (CSF) samples were collected to measure the levels of pro-inflammatory factors by ELISA, and rat cortex tissues were used to measure protein levels by western blot analysis. Immunofluorescence staining for Iba-1, GFAP, TUNEL, and NeuN was detected proliferation of microglia and astrocyte and apoptosis of neuron cells. Neurological outcome was assessed by ambulation and placing/stepping reflex responses. Morphology assay showed that pre-treatment and post-treatment with rHDGF ab attenuated vasospasm after SAH. SAH up-regulated the levels of TNF-α, IL-1β, and IL-6 in both the CSF and serum samples, and both pre- and post-treatment with rHDGF ab inhibited the up-regulation of these pro-inflammatory factors, except for the serum IL-6 levels. Western blot analysis demonstrated that SAH up-regulated pro-BDNF and NFκB protein levels, and both pre- and post-treatment with rHDGF ab significantly reduced the up-regulation. The result from immunofluorescence staining showed that SAH induced proliferation of microglia and astrocyte and apoptosis of neuron cells. Both pre- and post-treatment with rHDGF ab significantly attenuated proliferation of microglia and astrocyte and inhibited apoptosis of neuron cells. Furthermore, treatment with rHDGF ab significantly improved neurological outcome. Blocking HDGF attenuates neuron cell apoptosis and vasospasm through inhibiting inflammation in brain tissue at early phase after SAH.

Background Hyperglycemia, a derangement after subarachnoid hemorrhage (SAH), is known to be associated with unfavorable outcomes. Whether the connection between hyperglycemia and poor prognosis results from severe neuronal apoptosis is unknown, and we aim at investigating their relationship. Material and methods Streptozotocin (STZ) was administrated to trigger hyperglycemia before SAH induction in Sprague–Dawley rats that were assigned to one of four groups: control, SAH only, hyperglycemia only, and SAH with hyperglycemia. The severity of neuronal apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nickend labelling (TUNEL) staining of cerebral cortex. Results When subjected to SAH, hyperglycemic animals had worse neurobehavioral functions than normoglycemic ones. Hyperglycemia-exacerbated apoptosis was evident by greater increases in cleaved caspase-3 expression and TUNEL-positive cell density in the SAH with hyperglycemia group than those in the SAH only group, whereas there was no significant difference in cleaved caspase-9 expression and Bax/Bcl-2 ratio between the two groups. Furthermore, there was a remarkable decrease in the ratio of phosphorylated extracellular regulated kinase (ERK)/total ERK in the hyperglycemic rats after SAH. Conclusion Hyperglycemia aggravated neuronal apoptosis after SAH and was associated with impaired neurological outcomes. Activation of the extrinsic caspase cascade through the ERK signal pathway may contribute to hyperglycemia-mediated apoptosis.

Background: Neurological deficits following subarachnoid hemorrhage (SAH) are caused by early or delayed brain injuries. Our previous studies have demonstrated that hyperglycemia induces profound neuronal apoptosis of the cerebral cortex. Morphologically, we found that hyperglycemia exacerbated late vasospasm following SAH. Thus, our previous studies strongly suggest that post-SAH hyperglycemia is not only a response to primary insult, but also an aggravating factor for brain injuries. In addition, mitochondrial fusion and fission are vital to maintaining cellular functions. Current evidence also shows that the suppression of mitochondrial fission alleviates brain injuries after experimental SAH. Hence, this study aimed to determine the effects of mitochondrial dynamic modulation in hyperglycemia-related worse SAH neurological prognosis. Materials and methods: In vitro, we employed an enzyme-linked immunosorbent assay (ELISA) to detect the effect of mitochondrial division inhibitor-1 (Mdivi-1) on lipopolysaccharide (LPS)-induced BV-2 cells releasing inflammatory factors. In vivo, we produced hyperglycemic rats via intraperitoneal streptozotocin (STZ) injections. Hyperglycemia was confirmed using blood-glucose measurements (>300 mg/dL) 7 days after the STZ injection. The rodent model of SAH, in which fresh blood was instilled into the craniocervical junction, was used 7 days after STZ administration. We investigated the mechanism and effect of Mdivi-1, a selective inhibitor of dynamin-related protein (Drp1) to downregulate mitochondrial fission, on SAH-induced apoptosis in a hyperglycemic state, and evaluated the results in a dose–response manner. The rats were divided into the following five groups: (1) control, (2) SAH only, (3) Diabetes mellitus (DM) + SAH, (4) Mdivi-1 (0.24 mg/kg) + DM + SAH, and (5) Mdivi-1 (1.2 mg/kg) + DM + SAH. Results: In vitro, ELISA revealed that Mdivi-1 inhibited microglia from releasing inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. In vivo, neurological outcomes in the high-dose (1.2 mg/kg) Mdivi-1 treatment group were significantly reduced compared with the SAH and DM + SAH groups. Furthermore, immunofluorescence staining and ELISA revealed that a high dose of Mdivi-1 had attenuated inflammation and neuron cell apoptosis by inhibiting Hyperglycemia-aggravated activation, as well as microglia and astrocyte proliferation, following SAH. Conclusion: Mdivi-1, a Drp-1 inhibitor, attenuates cerebral vasospasm, poor neurological outcomes, inflammation, and neuron cell apoptosis following SAH + hyperglycemia.

Background Hyperglycemia is common and showed to be risky for poor prognosis in patients with subarachnoid hemorrhage (SAH). However, the causality and mechanism underlying this observation are not well established. Objective To investigate the relationship between hyperglycemia and cerebral vasospasm with its pathogenesis in a rat model of SAH. Methods One-shot SAH model was employed in male Sprague-Dawley rats. Hyperglycemia was triggered by intraperitoneal streptozotocin administration (50 mg/kg) 7 days before SAH induction. The severity of cerebral vasospasm was determined by the cross-sectional area of basilar artery (BA) in male rats randomly assigned to 1 of 4 groups: control, hyperglycemia only, SAH only, and SAH with hyperglycemia. The expression of endothelial nitric oxide synthase (eNOS) and induced nitric oxide synthase (iNOS) in the BA were analyzed by immunohistochemistry. Results The mean (standard deviation) blood glucose level was 433.0 (98.3) and 156.5 (31.7) mg/dL in streptozotocin -treated and untreated rats, respectively. Hyperglycemic rats exhibited poorer neurobehavioral performance than normoglycemic rats when subjected to SAH. Hyperglycemia-mediated exacerbation of vasospasm was evident by the greater decrease in the BA cross-sectional area in the hyperglycemic SAH group than in the SAH only group. Furthermore, there was more decreased expression of eNOS and increased expression of iNOS within the vessels of the hyperglycemic SAH rats. Conclusion Hyperglycemia exacerbated cerebral vasospasm and was associated with poorer neurological outcomes following SAH. Our findings also suggested the nitric oxide pathway as a potential underlying mechanism via the dysregulation of eNOS and iNOS.

Glioblastoma multiforme is one of the most serious malignant brain tumors and is characterized by resistance to chemotherapy and radiation therapy. Recent studies suggest that autophagy may play an important role not only in the regulation of cancer development and progression but also in determining the response of cancer cells to anticancer therapy. The purpose of the present study was to assess the relationship between protein expressions of two autophagy markers, LC3B and Beclin-1, with clinical parameters in astrocytoma patients. Furthermore, the expression of CD133, a marker of the cancer stem-like cells, in astrocytoma patients was also investigated. A total of 106 thin-section slides were retrospectively collected from astrocytoma patients. LC3B, but not Beclin-1, protein expression was found to significantly correlate with resistance to radiation- or chemotherapy. In addition, high intensity of LC3B staining was predictive of poor prognosis. Furthermore, survival time of patients with high-level expression in both CD133 and LC3B was significantly shorter than those with weak expression in both CD133 and LC3B. These results suggest that astrocytoma cancer stem-like cells together with enhanced autophagy may cause resistance to radiation therapy/chemotherapy and that targeting the cancer stem-like cell in astrocytoma may offer a viable therapeutic approach.

Cerebral vasospasm is the leading cause of mortality and morbidity in patients after aneurysmal subarachnoid hemorrhage (SAH). However, the mechanism and adequate treatment of vasospasm are still elusive. In the present study, we evaluate the effect and possible mechanism of progesterone on SAH-induced vasospasm in a two-hemorrhage rodent model of SAH. Progesterone (8 mg/kg) was subcutaneously injected in ovariectomized female Sprague-Dawley rats one hour after SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Expressions of endothelial nitric oxide synthase (eNOS) and phosphorylated Akt (phospho-Akt) in basilar arteries were evaluated. Prior to perfusion fixation, there were no significant differences among the control and treated groups in physiological parameters recorded. Progesterone treatment significantly(P<0.01)attenuated SAH-induced vasospasm. The SAH-induced suppression of eNOS protein and phospho-Akt were relieved by progesterone treatment. This result further confirmed that progesterone is effective in preventing SAH-induced vasospasm. The beneficial effect of progesterone might be in part related to upregulation of expression of eNOS via Akt signaling pathway after SAH. Progesterone holds therapeutic promise in the treatment of cerebral vasospasm following SAH.

The WW domain-containing oxidoreductase (WWOX) functions as a tumor suppressor by interacting with various proteins in numerous important signaling pathways. WWOX silencing via homozygous deletion of its locus and/or promoter hypermethylation has been observed in various human cancers. However, the relationship between WWOX and tumors in the central nervous system has not been fully explored. In this study, the expression levels of WWOX protein in astrocytomas from 38 patients with different tumor grades were retrospectively analyzed by immunohistochemical staining. The results showed that 19 (50.0%) samples had highly reduced WWOX protein expression when compared with normal controls, while 14 (36.8%) and five (13.2%) cases exhibited moderate and mild decreases in WWOX expression, respectively. Reduction of the expression of WWOX protein correlated with patient age, supra-tentorial localization of the tumor and severity of the symptoms. Furthermore, loss of WWOX expression inversely correlated with survival time. No significant correlation was observed between the loss of WWOX expression and the gender of patients or the difference in pre-operative and post-operative karnofsky performance status scores. Surprisingly, there was no significant correlation between the loss of WWOX protein expression and overall tumor grades. Nevertheless, it was found that 63.6% (7/11) of the grade II astrocytomas had highly reduced WWOX expression and 36.4% (4/11) showed moderately reduced WWOX expression, while none of the samples exhibited mild reductions. Similar results were also found in grade III astrocytomas. The results from this small-size sample pilot study suggest that the loss of WWOX expression may be an early event in the pathogenesis of human astrocytoma.

Axonal regeneration in peripheral nerves after injury is a complicated process. Numerous cytokines, growth factors, channels, kinases, and receptors are involved, and matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis subsequent to nerve injury. In this study, the effect of KMUP-1, an activator of large-conductance Ca(2+)-activated potassium channel, on functional recovery, myelinated axon growth, and immunoreactivity of MMP-9 was evaluated in rats subjected to sciatic nerve crush injury.A total of 144 male Sprague-Dawley rats were divided into the following six groups (n = 24/group): group 1, sham-operated; group 2, sciatic nerve injury without treatment; group 3, injured and vehicle-treated; group 4, injured and treated with 1 mM KMUP-1 by topical application; group 5, injured and treated with 10 mM KMUP-1; group 6, injured and treated with 50 mM KMUP-1. Functional recovery was evaluated using walking track analysis at 1, 2, 3, and 4 weeks (n = 6/group at each time point) after injury. In addition, the number of myelinated axons and MMP-9 in the nerve was also examined.Animals subjected to sciatic nerve crush injury had decreased motor function, a reduced number of myelinated axons, and increased MMP-9 in the nerve. Treatment with KMUP-1 concentration-dependently improved functional recovery, increased the number of myelinated axons, and decreased MMP-9.These results suggest that KMUP-1 may be a novel agent for assisting peripheral nerve recovery after injury. The beneficial effect is probably due to known ability of the compound in activating the nitric oxide/cGMP/protein kinase G pathway.

Patients who have liver cirrhosis are at increased risk of bacterial infections, such as bacteremia, meningitis, pneumonia, urinary tract infections, and spontaneous bacterial peritonitis, due to immunodeficiency associated with the severity of the cirrhosis. Although bacterial infections are frequent in cirrhotic patients, only isolated cases of brain abscess have been reported. In these cirrhotic patients, the initial presentation of brain abscess may not be fever or leukocytosis, but focal neurologic deficits. In addition, for consideration of blood-brain barrier penetration, the anti-biotic choice postoperatively is also quite different from other infections outside the central nervous system. We will discuss two cases of brain abscess in cirrhotic patients with special emphasis on the clinical presentation, magnetic resonance spectroscopic findings, organism encountered, therapeutic strategy, and prognosis.

Background and Purpose. The vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). Previous results showed that CGS 26303, an endothelin converting enzyme (ECE) inhibitor, effectively prevented and reversed arterial narrowing in animal models of SAH. In the present study, we assessed the effect of CGS 26303 on neurological deficits in SAH rats. The involvement of vasoactive pathways downstream of ET-1 signaling in SAH was also investigated.Methods. Sprague-Dawley rats were divided into five groups (n=6/group): (1) normal control, (2) SAH, (3) SAH+vehicle, (4) SAH+CGS 26303 (prevention), and (5) SAH+CGS 26303 (reversal). SAH was induced by injecting autologous blood into cisterna magna. CGS 26303 (10 mg/kg) was injected intravenously at 1 and 24 hr after the initiation of SAH in the prevention and reversal protocols, respectively. Behavioral changes were assessed at 48 hr after SAH. Protein expression was analyzed by Western blots.Results. Deficits in motor function were obvious in the SAH rats, and CGS 26303 significantly improved the rate of paraplegia. Expressions of rho-kinase-II and membrane-bound protein kinase C-δand rhoA were significantly increased, while those of soluble guanylyl cyclaseα1andβ1as well as protein kinase G were significantly decreased in the basilar artery of SAH rats. Treatment with CGS 26303 nearly normalized these effects.Conclusions. These results demonstrate that the rhoA/rho-kinase and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. It also shows that ECE inhibition is an effective strategy for the treatment of this disease.

Background. Central neurocytoma and oligodendroglioma are rare tumors of the central nervous system. However, diagnosis between these two types of tumors is challenging due to their many cytological and histological similarities. Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine protein kinase involved in many apoptosis pathways, and repressed expression of DAPK by promoter hypermethylation has been found in a variety of human cancers. The purpose of this study was to assess DAPK protein expression and promoter hypermethylation in central neurocytoma and oligodendroglioma.Method. Central neurocytoma and oligodendroglioma samples were obtained from age- and sex-matched patients. DAPK protein expression was performed using immunohistochemical assays in formalin-fixed, paraffin-embedded sections. DAPK promoter hypermethylation was carried out using bisulfite-modified genomic DNA in methylation-specific PCR followed by separation in agarose gels.Findings. A statistically significant difference (P=0.021) in DAPK promoter hypermethylation between central neurocytoma (76.9%) and oligodendroglioma (20%) was observed. High levels of DAPK protein expression were generally found in oligodendroglioma (90%), compared with 38.5% in central neurocytoma (P=0.054; not statistically significant). There was an inverse correlation between DAPK protein expression and DAPK promoter hypermethylation in the cohort of 23 patients (P=0.002).Conclusions. The results show that DAPK promoter hypermethylation and repressed expression of DAPK protein were more common in central neurocytoma than in oligodendroglioma. Thus, DAPK promoter hypermethylation could be useful for differential diagnosis between these two types of tumors, whereas DAPK protein expression might be less predictive. The role of DAPK promoter hypermethylation in the pathogenesis of central neurocytoma warrants further study.

The WW domain-containing oxidoreductase (WWOX) functions as a tumor suppressor by interacting with various proteins in numerous important signaling pathways. WWOX silencing via homozygous deletion of its locus and/or promoter hypermethylation has been observed in various human cancers. However, the relationship between WWOX and tumors in the central nervous system has not been fully explored. In this study, the expression levels of WWOX protein in astrocytomas from 38 patients with different tumor grades were retrospectively analyzed by immunohistochemical staining. The results showed that 19 (50.0%) samples had highly reduced WWOX protein expression when compared with normal controls, while 14 (36.8%) and five (13.2%) cases exhibited moderate and mild decreases in WWOX expression, respectively. Reduction of the expression of WWOX protein correlated with patient age, supra-tentorial localization of the tumor and severity of the symptoms. Furthermore, loss of WWOX expression inversely correlated with survival time. No significant correlation was observed between the loss of WWOX expression and the gender of patients or the difference in pre-operative and post-operative karnofsky performance status scores. Surprisingly, there was no significant correlation between the loss of WWOX protein expression and overall tumor grades. Nevertheless, it was found that 63.6% (7/11) of the grade II astrocytomas had highly reduced WWOX expression and 36.4% (4/11) showed moderately reduced WWOX expression, while none of the samples exhibited mild reductions. Similar results were also found in grade III astrocytomas. The results from this small-size sample pilot study suggest that the loss of WWOX expression may be an early event in the pathogenesis of human astrocytoma.