Study On The Changes Of NF-κBp65, I-κBαand INOS Following Vertebrobasilar Artery Ischemia Reperfusion In Guinea Pigs And The Protective Effects Of Puerarin

PartⅠThe changes of auditory function and histomorphology of the cochlea following the vertebrobasilar artery ischemia reperfusion in guinea pigsObjective The purpose of this study was to establish an animal model of cochlea ischemia-reperfusion injury through vertebrobasilar artery ischemia reperfusion and to investigate the changes of auditory function and histomorphology of cochlea during the reperfusion of vertebrobasilar artery in guinea pigs.Methods Sixty-eight healthy guinea pigs with normal Preyer's reflex weighed from 200 to 250g were used in this study. All of guinea pigs were obtained from the animal care facilities of Central South University and housed at the same place. They were randomly divided into 6 groups: the normal control group,the ischemia 1 hour group and the reperfusion groups(12, 24, 48hours, 7days). The models of vertebrobasilar artery ischemia reperfusion were established via the skull base approach. Under a surgical microscope, the clivus was gently drilled with a diamond bar. The dura was exposed and cut, the arachnoid membrane covering the basilar artery was removed. After the basilar artery was exposed, then the artery was occluded for 1 hour with a microvascular clamp, the clamp was removed and the blood flow recirculated at the time point of 12 hours, 24 hours, 48 hours and 7 days, respectively. The auditory function was evaluated by evoked auditory brainstem responses(ABR). Each animal was measured before the operation and after ischemia or ischemia-reperfusion, respectively. Some of the animals were observed the changes of ABR during ischemia. The slice of temporal bone by means of the Hematoxylin and Eeosin(H&E) staining and surface preparation of baseal membrane stained by silver nitrate were used for cochlea histopathology observation.Results(1) Auditory function evaluation: After 10-20 minutes the evoked auditory brainstem responses threshold began to increase steadily during verebrobasilar artery ischemia. The changes of ABR during the vertebrobasilar artery ischemia or ischemia-reperfusion consisted of a prolonged in all wave latencies and interpeak latenciesⅠ-Ⅲ, an increase of the auditory threshold, especially 24 hours after ischemia. A statistically significant difference of the auditory threshold shift, interpeak latenciesⅠ-Ⅲand wave latencies was seen between the groups of ischemia and ischemia-reperfusion. A statistically significant difference was seen between the normal control group and the ischemia and ischemia-reperfusion groups, too.(2) The changes of histopathology: After 1 hour ischemia, outer hair cells(OHCs) were swollen, the vertebrobasilar artery reperfusion could cause more severe lesions of the Corti, spiral ganglion cells(SGCs) and stria vascularis, especially 24,48 hours after ischemia. These studies showed that the disapperance of outer hair cells and the degeneration of spiral ganglion cells. 24,48 hours after reperfusion, the loss of OHCs and the degeneration of SGCs became apparent. 24hours after ischemia, the mean percentage of cell loss of OHCs was 10.5％in the basal turn, 7.0％in the second turn, and 3.3％in the apical turn.ConclusionsWe had successfully established an animal model of the vertebrobasilar artery ischemia reperfusion in guinea pigs. Vertebrobasilar artery ischemia reperfusion in guinea pigs could cause the lesions of the Corti, spiral ganglion cells(SGCs) and stria vascularis which were more severe in reperfusion injury. Vertebrobasilar artery ischemia reperfusion could delay the latencies of ABR waves, interpeak latenciesⅠ-Ⅲand increase the threshold. PartⅡStudy on the changes of NF-κBp65, I-κBαand iNOS following vertebrobasilar artery ischemia reperfusion in guinea pigsObjective The aim of this study was to determine the molecular mechanisms of the cochlea injuries on the vertebrobasilar artery ischemia reperfusion in guinea pigs. In this study, activation of the nuclear factor-kappaBp65/I-κBα-iNOS pathway was investigated in the cochlea of guinea pigs induced by vertebrobasilar artery ischemia reperfusion. To study the activation of the nuclear factor-kappaBp65 subunit and nuclear translocation leading to trigger induction of inducible nitric oxide synthase(iNOS) expression. To investigate the degradation of I-κBαin cochlea tissue and to elucidate the spatial distribution and time course of expression of NF-κBp65, I-κBαand iNOS following verebrobasilar artery ischemia reperfusion. This study could provide the foundation for the therapy of ischemia reperfusion injury of cochlea.Methods The spatial and temporal features of NF-κBp65, I-κBαand iNOS induction following ischemia reperfusion were studied by paraffin embedded immunohistochemistry. The nuclear and cytoplasm protein were extracted from the cochlea tissue, respectively. NF-κBp65 and I-κBαprotein levels in cochlea following ischemia reperfusion were detected by the Western blot assay. Electrophoretic mobility shift assay(EMSA) were used to detect the nuclear protein DNA binding activity of NF-κB.Results(1) The negative reaction for NF-κBp65 staining was found in the cochlea in normol control group, The positive reaction was found in the cytoplasm and/or nucleus of the Corti, spiral ganglion cells(SGCs), spiral ligament and stria vasucularis in the ischemia group and all ischemia reperfusion groups. The expression of NF-κBp65 immunoreactivity is prominent in the nucleus after reperfusion. The peaking time of expression was at 24 hours after ischemia, the expression was gradually decreased after 48hours and 7 days reperfusion. Image analysis showed that the expression between normol control group and ischemia or reperfusion groups had a statistically significant difference(P＜0.01). Compared with ischemia group, the NF-κBp65 expression increased in the reperfusion groups also had a statistically significant difference(P＜0.01).(2) The positive reaction of I-κBαwas found in the cytoplasm of the Corti, spiral ganglion cells(SGCs), spiral ligament and stria vascularis in normol control group. The expression of I-κBαimmunoreactivity was prominent in the cytoplasm. After ischemia and reperfusion, the expression was gradually decreased, but re-upregulated again after 48hours. We could see positive immunoreactivity in the nucleus at 48 hours and 7 days reperfusion groups. Image analysis showed that the expression between normol control group and ischemia or reperfusion groups had a statistically significant difference(P＜0.01).(3) The negative reaction of iNOS staining was found in the cochlea in normol control group, the positive reaction could be found in the cytoplasm of the Corti, spiral ganglion cells(SGCs), and stria vascularis in the ischemia group and ischemia reperfusion groups. The peaking time of expression was at 24 hours after ischemia. Image analysis showed that the expression between normol control group and ischemia or reperfusion groups had a statistically significant difference(P＜0.01). Compared with ischemia group, the iNOS expression increased in the reperfusion groups also had a statistically significant difference(P＜0.01).(4) The NF-κBp65 subunit protein levels in the nucleus was detected by Western blot assay. Semi-quantitative data showed that the upregulation expression of NF-κBp65 began at the 12 hours after ischemia. It reached the peaking expression at the 24 hours after ischemia and began to decline at 48 hours. At the same time, NF-κBp65 subunit protein levels in cytoplasm had an opposition changes. In the normal control group, the I-κBαprotein in cytoplasm had a high levels, it began to significantly decline at 12 hours after ischemia and reached the lowest levels at 24 hours after ischemia.(5) The radiolabeled bound probe differently to nuclear protein extracted from the normal control group and ischemia-reperfusion groups showed that ischemia-reperfusion injury significantly increased the DNA binding activity of NF-κB. The obvious upregulation of NF-κB activation was observed in ischemia-reperfusion cochlea tissue, as compared to the normal control group. It reached the peaking binding activity at the 24 hours after ischemia and began to decline at 48 hours.Conclusions(1) The activation of NF-κBp65, the degradation of I-κBαand iNOS were involved in ischemia-reperfusion injury of the cochlea. NF-κBp65, I-κBα, iNOS had an abnormal expression during the ischemia-reperfusion of cochlea.(2) Western blot assay approved the activation of NF-κBp65 and nuclear translocation, the degradation of IκBαduring the ischemia reperfusion of cochlea.(3) Electrophoretic mobility shift assay demonstrated that ischemia-reperfusion of cochlea could enhance the DNA binding activity of NF-κB. PartⅢThe protective effect of Puerarin on the auditory function and the expression of NF-κBp65, I-κBαand iNOS after vertebrobasilar artery ischemia reperfusion in guinea pigs.Objective To investigate the protective effects of Puerarin on vertebrobasilar artery ischemia reperfusion injury of cochlea in guinea pigs and the mechanism of its protection.Methods Thirty-two healthy guinea pigs with normal Preyer's reflex weighed from 200 to 250g were randomly divided into four groups with eight animals in each group: normal control group, reperfusion 7 days group, Puerarin group and normal saline group. The models of vertebrobasilar artery ischemia reperfusion were established via the skull base approach. In Puerarin group, Puerain 150mg/kg/d was given daily by intraperitoneal injection from the first day of ischemia for six days. In normal saline group, normal saline of the same volume was used as Puerarin group for six days too. The auditory function was evaluated by evoked auditory brainstem responses. The slice of temporal bone by means of the Hematoxylin and Eeosin(H&E) staining was used for cochlea histopathology observation. The effect of Puerarin on expressions of NF-κBp65, I-κBαand iNOS were studied using paraffin embedded immunohistochemistry. ResultsPuerarin could prevent effectively all wave latencies and interpeak latenciesⅠ-Ⅲof ABR from delaying by ischemia reperfusion injury and decreasing the auditory threshold. The activation of NF-κBp65 and the expression of iNOS were found at reperfusion 7 days group and normal saline group, they were obviously higher than normal control group(P＜0.05). The expression of I-κBαat reperfusion 7 days group and normal saline group was significantly decline, they were obviously lower than normal control group(P＜0.05). In Puerarin group, the activation of NF-κBp65 and the expression of iNOS were significantly decreased, and the expression of I-κBαwas increased, they were neared to the normal control group.Conclusions(1) The study showed that Puerarin can protect the cochlea from ischemia reperfusion injury in guinea pigs.(2) The protective effects of Puerarin aganist the cochlea of ischemia reperfusion injury may be related to increase the expression of I-κBαand decrease the activation of NF-κBp65 and the expression of iNOS.