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.

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.