Role of protein kinase C in the mechanism of central sensitization of migraine / 中华神经医学杂志

Objective To explore the role of protein kinase C (PKC) in the mechanism of central sensitization of migraine.Methods Sixty healthy adult male SD rats,weighting from 200 to 250 g,were randomly divided into five groups:normal group,sham-operated group,migraine model group,chloroform treatment group and H-7 (the inhibitor of PKC) treatment group (n=12).Dural blood flow monitor was performed by laser Doppler blood flow imager and extracellular discharge frequency in the spinal trigeminal nucleus was observed by multi-conductive polygraph; the dural blood flow and discharge frequency changes were analyzed and compared.Results Two hours after the success of model making,the dural blood flow in the migraine model group increased obviously as compared with that in the sham-operated group (P＜0.05); as compared with that in the migraine model group,the dural blood flow in the H-7 treatment group decreased obviously (P＜0.05); as compared with sham operation group,blood flow decreased obviously in H-7 group (P＜0.05).Extracellular discharge frequency in the spinal trigeminal nucleus increased 2 h after the success of model making; 2 hours after model making,the extracellular discharge frequency was (323.82±11.00) ％ of baseline level; as compared with that in the migraine model group,discharge frequency in the H-7 treatment group decreased obviously (P＜0.05); as compared with that in the sham-operated group,discharge frequency in the H-7 treatment group had no obvious changes (P＞0.05).Conclusion PKC may play an important role in the mechanism of central sensitization of migraine.

Cerebral lymphatic blockage aggravates apoptosis of cortical neurons after subarachnoid hemorrhage in rats in vivo / 生理学报

This present study was performed to investigate the influence of cerebral lymphatic blockage (CLB) on apoptosis of cortical neurons after subarachnoid hemorrhage (SAH) in rats in vivo. Healthy adult Wistar rats were randomly divided into normal control group, SAH group and SAH+CLB group. SAH model was made by double injection of autologous blood into the cisterna magna. On the third day after the second cisternal injection, morphological changes of cortical cells were observed by hematoxylin-eosin (HE) combined with propidium iodide (PI) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was applied to determine in situ apoptosis in the cerebral cortex. Immunohistochemistry was conducted to detect the expression of Caspase-3 and Bcl-2 in cortical neurons. HE and PI staining showed that cortical neurons of SAH rats were partly shrinkable; the nuclei showed wavy, folded or wrinkled appearance, and some nuclei had the shape of crescent. The cortical neurons in SAH+CLB group distributed sparsely and the nuclear fragmentation, apoptotic bodies were found, surrounded by the formation of vacuoles. The numbers of TUNEL-positive cells in SAH group and SAH+CLB group were higher than that in the normal control group, while the number in SAH+CLB group was significantly higher than that in the SAH group. Caspase-3 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in SAH+CLB group was significantly higher than that in the SAH group. Bcl-2 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in the SAH+CLB group was significantly lower than that in SAH group. The results obtained suggest that CLB exacerbates the apoptosis of cortical neurons in rats after SAH by up-regulating Caspase-3 expression and down-regulating Bcl-2 expression.

Protective effect of hypoxic preconditioning against cerebral ischemic injury induced by acute cerebral infarction in mice / 中华神经医学杂志

Objective To observe the protective effect of hypoxic preconditioning against cerebral ischemic injury induced by acute cerebral infarction in mice. Methods Blb/c mice were randomized into normal control, sham-operated, acute cerebral infarction (CI), and hypoxic preconditioning with CI (HP+CI) groups. In the latter two groups, acute cerebral cortical infarction was induced photochemically by cold light exposure of the brain tissue following intravenous Rose Bengal injection. The mice in the sham-operated group received only cold light exposure without Rose Bengal injection. Behavioral test, immunofluorescence assay and confocal laser scanning microscopy were performed to evaluate the neurological deficits of the mice and assess the cell apoptosis, and the cerebral infarction volume was measured. Results No infarct was found in the normal control and sham-operated groups, whereas obvious isehemic infarction foci were found in CI and HP+CI groups, but the infarction volume was significantly smaller in HP+CI group (P<0.05). The mice in the normal control and sham-operated groups presented with no neurological impairment, but in CI and HP+CI groups, obvious symptoms of neurological impairment were observed with lowered neurological scores. The neurological scores were significantly higher in HP+CI group than in CI group (P<0.05). Occasional apoptotic cells were found in the normal control and sham-operated groups, while in the other two groups, the TUNEL-positive cell number increased significantly. The apoptotic cell number was significantly smaller in HP+CI group than in CI group (P<0.05). Conclusion Hypoxic preconditioning may protect against cerebral ischemic injury induced by acute cerebral infarction in mice possibly by reducing the cerebral infarction volume and cell apoptosis.

Effect of brain homogenate from hypoxia-preconditioned mice on rat embryonic hippocampal neurons with hypoxia/reoxygenation injury / 中华神经医学杂志

Objective To observe the effect of brain homogenate (BH) extracted from hypoxia-preconditioned mice on the viability and apoptosis of rat embryonic hippocampal neurons with hypoxia/reoxygenation-induced injury. Methods Rat embryonic hippocampal neurons primarily cultured for 8 days in 96 well tissue culture plate were divided into 5 groups, namely the normal control group (treated with PBS), H<,4>R<,48> group (with hypoxia for 4 h followed by reoxygenation for 48 h and PBS treatment), H0 group (with BH from normal mice prior to hypoxia/reoxygenation), H1 group (with BH from acute hypoxia-preconditioned mice and hypoxia/reoxygenation), and H4 group (with BH from hypoxia-preconditioned mice and hypoxia/reoxygenation). The viability and apoptosis of the cells in the 5 groups were observed by MTT assay and flow cytomertry, respectively. Results The cell viability was significantly higher in the normal control group than in H<,4>R<,48> group. In H0, H1, and H4 groups, the cell viability increased significantly as compared with that in H<,4>R<,48> group, and the cells in H4 group showed the highest viability. Apoptotie cells were scarcely observed in the normal control group, but were numerous in H<,4>R<,48> group. Compared with H<,4>R<,48> group, H0, H1, and H4 groups showed obviously reduced apoptotic cells, and the reduction was the most conspicuous in H4 group. Conclusion BH extracted from hypoxia-preconditioned mice may offer protection against hypoxic injury of rat embryonic hippocampal neurons challenged with hypoxia-reoxygenation by promoting the cell viability and decreasing cell apoptosis.

Cerebral lymphatic blockage aggravates apoptosis of hippocampal neurons induced by cerebrospinal fluid from experimental subarachnoid hemorrhage / 生理学报

This work was performed to determine the role of cerebral lymphatic drainage pathway in the development of neural injury following subarachnoid hemorrhage (SAH). SAH and cerebral lymphatic blockage (CLB) models in adult New Zealand rabbits were used. Cerebrospinal fluid (CSF) was obtained from experimental animals 5 d after modeling and was added into cultured rat hippocampal neurons. The neurons were randomly divided into blank control, normal CSF, SAH, and SAH+CLB groups. At different points of time, lactate dehydrogenase (LDH) leakage was detected by colorimetric method. Flow cytometry was used to detect the apoptosis of neurons. Expressions of Bax and heat-shock protein 70 (Hsp70) were determined by immunohistochemical staining. LDH leakage detection revealed that, compared with blank control group, CSF from normal rabbit did not damage the neurons, whereas the leakage of LDH increased in SAH group and SAH+CLB group. The increasing effect was more obvious in SAH+CLB group than that in SAH group. Normal CSF did not induce the apoptosis of neurons, whereas neuron apoptosis was found in SAH group and the apoptosis was even more severe in SAH+CLB group. Bax and Hsp70 protein expressions were found in both SAH and SAH+CLB groups. Expression of Bax protein in SAH+CLB group was stronger than that in SAH group in a time-dependent manner. At 0.5 h and 1 h, the expression of Hsp70 protein in SAH+CLB group was stronger than that in SAH group, whereas the expression became weaker at 2 h and 4 h. These results suggest that blockage of cerebral lymphatic drainage pathway deteriorates the damage of neurons treated with CSF from SAH, indicating this pathway may act as an endogenous protective role in SAH.

Changes in Bcl-2 and Caspase-3 expressions in cortex of hypoxic preconditioning mice / 生理学报

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.

Dephosphorelation of Bad and upregulation of Bcl-2 in hippocampus of rats following limbic seizure induced by kainic acid injection into amygdaloid nucleus / 生理学报

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.

The changes of bcl-2, bax expression and neuron apoptosis in the hippocampus after the blockade of cervical lymphatics of rats / 生理学报

To investigate the changes in bcl-2, bax expression and neuron apoptosis in the hippocampus after the blockade of cervical lymphatics, the model of lymphostatic encephalopathy was established by occluding and removing both the superficial and deep cervical lymph nodes in rats. The animals were sacrificed at 1, 2, 3, 5, 7 and 14 d after operation. H and E staining was used to observe the structure of brain tissues and TUNEL staining was used to detect in situ cell apoptosis in the hippocampus. The expression of bcl-2 and bax in the hippocampus were examined by RT-PCR. The results showed that cerebroedema appeared at day 2 and was most serious at day 5 after the blockade of cervical lymphatics. The number of TUNEL positive cells began to increase at day 2 and reached the maximum at day 5. The expression of bax began to increase at day 1 and reached the maximum at day 2. The expression of bcl-2 began to decrease at day 1 and dropped to the minimum at day 5. The items mentioned above recovered to control level at day 14. These results suggest that lymphostatic encephalopathy following the blockade of cervical lymphatics result in changes in bcl-2 and bax expression in the hippocampus and that apoptosis is the main form of neuron death.