| Objective: To observe and compare the protective effects of electro- acupuncture(EA) pretreatment with 3 types of waveforms(continuous wave, dense-disperse wave and intermittent wave) on cerebral injury in sepsis rats, and explore the mechanism of cerebral protection. Furthermore, to evaluate and choose an optimal EA waveform for clinical prevention and treatment of cerebral injury due to sepsis.Method: 60 healthy male SD rats, weighing 180-200 g, were randomly divided into 5 groups(n=12) as sham group(S group), model group(M group), continuous waveform group(CW group), dense-disperse waveform group(DW group)and intermittent waveform group(IW group). The septic rat model was made by means of cecal ligation and puncture(CLP). The EA pretreatment was conducted 30 minutes prior to the CLP event, using 3 different waveforms and corresponding frequency, in same intensity to stimulate the acupoints Baihui and Zusanli. The cognitive function and change were assessed with Morris water maze test 5 days before and 24 hours after operation. Blood sample was withdrawn by puncture of apex immediately after post-operative Morris water maze test. The fresh tissue of hippocampus was harvested from 6 rats in each group immediately after blood sampling and decollation. Then,perfusion and brain-taking were done to remained 6 rats in each group. The content of TNF-α, IL-6, the activity of SOD and the content of MDA in serum and fresh tissue of hippocampus were measured. The pathological changes in hippocampus and cortex were observed by HE staining. The expression of TLR-4 and NF-κB in tissue of hippocampus and cortex were detected with immunohistochemical method. The neuronal apoptosis was detected by TUNEL method, and apoptosis index was been calculated.Results:(1) Morris water maze test: The escape latency, the times of crossing the platform and the swimming time in target quadrant were not significantly different among five groups compared those before operation(p>0.05). In S group, postoperative result of the test was not significantly different compared with preoperative result(p>0.05). Compare with S group, the escape latency prolonged, the times of crossing the platform reduced and the swimming time in target quadrant shortened in M group(p<0.01). In EA groups, the escape latency shortened, the times of crossing the platform increased and the swimming time in target quadrant prolonged, compare with M group(p<0.05, p<0.01). In 3 EA groups, the criterion of maze test in DW group were better than that in IW group, and much better than that in CW group(p<0.05, p<0.01).(2) The content of TNF-α, IL-6 in serum and hippocampus: Compare with S group, The content of TNF-α, IL-6 in M group and 3 EA groups were increased significantly(p<0.01). However, In 3 EA groups, the contents of TNF-α, IL-6 were less than M group(p<0.01). The content of TNF-α, IL-6 in DW group was less than IW group and much less than in CW group(p<0.01).(3) The content of MDA and the activity of SOD in serum and hippocampus: Compare with S group, the content of MDA increased and the activity of SOD reduced in M group with significant difference(p<0.01). Compare with M group, the content of MDA reduced and the activity of SOD increased in the EA groups(p<0.05, p<0.01). In DW group the content of MDA reduced and the activity of SOD increased to more extent than in IW group and to much more extent than in CW group(p<0.05, p<0.01).(4)The pathological changes in hippocampus and cortex(HE staining): In S group, structures of hippocampus and cortex tissue were normal, neurons arranged densely and neatly, interstitial densely without edema, brain cells were morphologically normal characterized as clear nucleolus and homogenous nucleoplasm with no nuclear condensation and dissolution. In M group, most neurons in hippocampus and cortex demonstrated serious damages such as degeneration and necrosis, morphological abnormality, pyramidal cells swelling, irregular arrangement, serious nuclear condensation and strengthened basophilic, hyperchromatic cytoplasm and unclear structure, and large cavitation area due to nerve cell deletion with few normal neurons. Compared with M group, severity of degeneration and necrosis in hippocampus and cortex was less to some extent, and normal nerve cells and degenerative or necrotic cells could be seen in EA groups. In CW group, neurons arranged regularly with alleviated edema, cell loss, morphological irregularity, nuclear condensation, less hyperchromatic nerve cells, more normal nerve cells and more homogeneous nucleoplasm. In IW group, neural cells arranged densely but less regularly with slighter edema, cell loss, nuclear condensation, hyperchromatic cells and more normal nerve cells. In DW group, neurons arranged densely and neatly with a few degeneration and necrosis cells, clear nucleolus and homogeneous nucleoplasm.(5) The expressions of TLR-4 and NF-κB in hippocampus and cortex: Compare with S group, the expressions of TLR-4 and NF-κB in M group increased significantly(p<0.01). In EA groups the expressions of TLR-4 and NF-κB reduced markedly, compared with M group(p<0.05, p<0.01). The expression levels of TLR-4 and NF-κB in DW group were lower than in IW group and much lower than in CW group(p<0.01).(6) The result of cell apoptosis: In S group very little apoptotic cells could be seen. In M group, a large number of apoptotic cells could be seen with higher apoptosis index, compared with S group(p<0.05, p<0.01). Compared with M group, the apoptosis index in EA groups reduced remarkablely(p<0.05, p<0.01). In DW group, number of apoptotic cells and the apoptosis index were less and lower than in IW group and much less and lower than in CW group(p<0.05, p<0.01).Conclusion:(1) EA pretreatment at acupoints ‘Baihui’ and ‘Zusanli’with 3 types of waveform can protect brain of rat from injury due to sepsis, improve cognitive dysfunction.(2) EA pretreatment with dense-disperse wave is superior to intermittent wave and continuous wave for brain protection in rat model of sepsis.(3) The mechanism of cerebral protection may be related with attenuated inflammatory response and oxidative stress, relieved cell apoptosis in cortex and hippocampus. |
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