Cerebrospinal fluid from rats given hypoxic preconditioning protects neurons from oxygen-glucose deprivation-induced injury.

Hypoxic preconditioning activates endogenous mechanisms that protect against cerebral ischemic and hypoxic injury. To better understand these protective mechanisms, adult rats were housed in a hypoxic environment (8% O2/92% N2) for 3 hours, and then in a normal oxygen environment for 12 hours. Their cerebrospinal fluid was obtained to culture cortical neurons from newborn rats for 1 day, and then the neurons were exposed to oxygen-glucose deprivation for 1.5 hours. The cerebrospinal fluid from rats subjected to hypoxic preconditioning reduced oxygen-glucose deprivation-induced injury, increased survival rate, upregulated Bcl-2 expression and downregulated Bax expression in the cultured cortical neurons, compared with control. These results indicate that cerebrospinal fluid from rats given hypoxic preconditioning protects against oxygen-glucose deprivation-induced injury by affecting apoptosis-related protein expression in neurons from newborn rats.

Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6-S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our results suggest a novel mechanism of gabapentin-mediated analgesia for visceral inflammatory pain through a PKC-ERK1/2 signaling pathway that may be a future therapeutic target for the treatment of visceral inflammatory pain.

[Protective effect of pretreatment of Salvia miltiorrhiza Bunge. f. alba plasma against oxygen-glucose deprivation-induced injury of cultured rat hippocampal neurons by inhibiting apoptosis].

The present study was to investigate the effect of Salvia miltiorrhiza Bunge. f. alba (SMA) pharmacological pretreatment on apoptosis of cultured hippocampal neurons from neonate rats under oxygen-glucose deprivation (OGD). Cultured hippocampal neurons were randomly divided into five groups (n = 6): normal plasma group, low dose SMA plasma (2.5%) group, middle dose SMA plasma (5%) group, high dose SMA plasma (10%) group and control group. The hippocampal neurons were cultured and treated with plasma from adult Wistar rats intragastrically administered with saline or aqueous extract of SMA. The apoptosis of neurons was induced by glucose-free Earle's solution containing 1 mmol/L Na2S2O4 and labeled by MTT and Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in control group, whereas the number of apoptotic cells was greatly increased in normal plasma group and low dose SMA plasma group. Both middle and high dose SMA plasma could protect cultured hippocampal neurons from apoptosis induced by OGD (P < 0.05). The protective effect of high dose SMA plasma was stronger than that of middle one (P < 0.05). Compared to control, normal plasma and low dose SMA plasma groups, middle and high dose SMA plasma groups both showed significantly higher levels of Bcl-2 (P < 0.05 or 0.01), whereas expressions of Bax was opposite. There were no significant differences of Bcl-2 and Bax expressions between middle and high dose SMA plasma groups. Number of Bcl-2- and Bax-positive cells had similar tendency. Bcl-2/Bax (number of positive cells) ratio was higher in high dose SMA plasma group than those of all the other groups (P < 0.05 or 0.01). These results suggest that pharmacological pretreatment of blood plasma containing middle and high dose SMA could raise viability and inhibit apoptosis of OGD-injured hippocampal neurons by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.

[Lessening effect of hypoxia-preconditioned rat cerebrospinal fluid on oxygen-glucose deprivation-induced injury of cultured hippocampal neurons in neonate rats and possible mechanism].

The present study was to investigate the effect of cerebrospinal fluid (CSF) from the rats with hypoxic preconditioning (HPC) on apoptosis of cultured hippocampal neurons in neonate rats under oxygen glucose deprivation (OGD). Adult Wistar rats were exposed to 3 h of hypoxia for HPC, and then their CSF was taken out. Cultured hippocampal neurons from the neonate rats were randomly divided into four groups (n = 6): normal control group, OGD group, normal CSF group and HPC CSF group. OGD group received 1.5 h of incubation in glucose-free Earle's solution containing 1 mmol/L Na2S2O4, and normal and HPC CSF groups were subjected to 1 d of corresponding CSF treatments followed by 1.5 h OGD. The apoptosis of neurons was analyzed by confocal laser scanning microscope and flow cytometry using Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in normal control group, whereas the number of apoptotic cells was greatly increased in OGD group. Both normal and HPC CSF could decrease the apoptosis of cultured hippocampal neurons injured by OGD (P < 0.01). Notably, the protective effect of HPC CSF was stronger than that of normal one (P < 0.01). Compared to OGD group, normal and HPC CSF groups both showed significantly higher levels of Bcl-2 (P < 0.01), and Bcl-2 expression level in HPC CSF group was even higher than that in normal CSF group (P < 0.01). Whereas the expressions of Bax in normal and HPC CSF groups were significantly lower than that in OGD group (P < 0.01), and the Bax expression in HPC CSF group was even lower than that in normal CSF group (P < 0.01). These results suggest that CSF from hypoxic-preconditioned rats could degrade apoptotic rate of OGD-injured hippocampal neurons by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

[Changes in Bcl-2 and Caspase-3 expressions in cortex of hypoxic preconditioning mice].

The purpose of the present study was to explore the roles of Bcl-2 and Caspase-3 in mouse cortex in hypoxic preconditioning. Blb/c mice were randomly divided into three groups: control group, hypoxic group and hypoxic preconditioning group. Fluorescence intensity of Bcl-2 and Caspase-3 was observed and number of positive cells was counted in parietal cortex by immunofluorescence and confocal laser scanning microscope. Fluorescence intensity of Bcl-2 in the normal group, hypoxic group and hypoxic preconditioning group was 6.2±1.7, 68.5±13.1, 180.6±34.8, respectively, and number of Bcl-2-positive cells was 18.5±4.9, 52.3±10.5, 150.8±24.7, respectively. Fluorescence intensity of Caspase-3 in the control group, hypoxic group and hypoxic preconditioning group was 8.6±2.0, 40.2±8.2, 26.4±6.1, respectively, and number of Caspase-3-positive cells of was 4.3±1.2, 63.6±12.5, 45.7±9.8, respectively. The results showed that the expressions of Bcl-2 in both hypoxic group and hypoxic preconditioning group were significantly higher than that in the control group; and the expression of Bcl-2 in hypoxic preconditioning group was even higher than that in hypoxic group. The expressions of Caspase-3 in hypoxic group and hypoxic preconditioning group were also significantly higher than that in the control group; whereas the expression of Caspase-3 in hypoxic preconditioning group was significantly lower than that in hypoxic group. These results suggest that cortex cells are resistant to apoptosis via increased expression of Bcl-2 and lowered expression of Caspase-3 in the cortex and brain cells are thereby protected during hypoxic preconditioning.

Expression of the receptors of VEGF and the influence of extract of Ginkgo biloba after cisternal injection of autologus arterial hemolysate in rats.

The study was aimed to investigate the alterations of vascular endothelial growth factor (VEGF) receptors and the influence of extract of Ginkgo biloba (EGb) after subarachnoid hemorrhage (SAH). Wistar rats were divided into non-SAH, SAH, vehicle, EGb1 (lower dose), and EGb2 (higher dose) groups. Autologus arterial hemolysate was injected into cisterna magna to induce SAH. The non-SAH rats received cisternal injection of saline instead. Rats underwent RT-PCR determination of one of the VEGF receptors flt-1mRNA, and immunohistochemistry for VEGF receptors Flt-1 and Flk-1. The results revealed that there was only slight expression of flt-1mRNA in the brain tissue in non-SAH rats. The expression in SAH group was enhanced 24 hours and 72 hours after cisternal injection. No Flt-1 and Flk-1 positive cell was observed in the brain in non-SAH group. A good few Flt-1 and Flk-1 positive cells were found in cortex and other regions of the brain in SAH group. The expression of flt-1mRNA, Flt-1 and Flk-1 proteins were increased by the use of two doses of EGb. It was concluded that the up-regulated expression of the two kinds of VEGF receptors may be an intrinsic protective mechanism in the process of SAH, which can be enhanced by EGb.

[Dephosphorelation of Bad and upregulation of Bcl-2 in hippocampus of rats following limbic seizure induced by kainic acid injection into amygdaloid nucleus].

The purpose of the present study was to explore the seizure-induced changes in Bad (Bcl-2-associated death protein), 14-3-3, phosphoBad, Bcl-2 and Bcl-XL expression in the rat model of focal limbic seizure. Unilateral intra-amygdaloid injection of kainic acid (KA) was made to induce seizure. Electroencephalogram (EEG) and regional cerebral flow (r-CBF) were monitored continuously. Diazepam (30 mg/kg) was administered to terminate the seizure. The apoptotic and surviving neurons in the hippocampus were observed by terminal deoxynucleotidyl transferrase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Bad, 14-3-3, phosphoBad, Bcl-2 and Bcl-XL were detected with immunofluorescence, Western blot and immunoprecipitation. The results showed that TUNEL-positive neurons appeared at 8 h and reached maximum at 24 h following seizure cessation within the ipsilateral CA3 subfield of the hippocampus. Seizure induced the dephosphorylation of Bad and the dissociation of Bad from its chaperone protein 14-3-3 and subsequent dimerization of Bad with Bcl-XL. The expression of phosphoBad decreased and Bcl-2 increased. There was little change in r-CBF after the seizure. These results suggest that seizure leads to a dephosphorylation of Bad and an upregulation of Bcl-2. Dephosphorylation of Bad may be injurious while the upregulation of Bcl-2 may be protective to the brain damage induced by seizures, but not related with r-CBF.