Effects Of Gelatinase And Its Inhibitor On Focal Cerebral Ischemia-reperfusion Injury In Rats

Partâ… MRI study in an experimental model of focal cerebral ischemia/reperfusion injuryObjective: To explore time characteristics and dynamic changes of pathology in a rat model of focal cerebral ischemia-reperfusion at different time points with MRI techniques.Methods: Fourteen male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion (MCAO) was induced by an intraluminal suture method. Perfusion-weighted imaging was performed to confirm complete occlusion and reperfusion. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. T₂*-weighted imaging was used at 24h and 5d after reperfusion. MRI lesion volume and signal intensity on postcontrast T₁WI were evaluated at different time point, and the results were expressed in forms of rV-MRI (lesion volume / volume of the ipsilateral hemisphere×100 %) and rSI-MRI (mean grey value of ROIs in the ipsilateral hemisphere/ that of the contralateral hemisphere×100 %).Results: signal attenuation during the first passage of a bolus-contrast indicated a complete occlusion of the artery. On the contrary, a failed occlusion was lack of such a signal change. In the ischemic group hyper-intensity on DWI was first detectable at 1.5h after reperfusion, and the lesion volume on DWI increased with the time of reperfusion (P<0.05). Hyper-intensity on T₂WI and postcontrast T₁WI was detected at 3.5h after reperfusion in the ischemic group.The lesion volume increased at 24h after reperfusion and declined at 5d after reperfusion (P<0.05), but the signal intensity increased all the time (P<0.05). No abnormal signal was found on T₂*WI at any time point. The rats from sham-operated group showed no changes on MRI during the experiment.Conclusions: The apply of MRI in focal cerebral ischemia-reperfusion at different time points can afford dynamic and individual neuroimaging informations of BBB disruption, cerebral edema and infarct size following cerebral ischemia. MRI techniques have shown latent potency in elucidating time characteristics in reperfusion injury and provided a foundation of diagnosis and treatment for cerebral ischemic injury.Partâ…¡Correlation analysis of BBB permeability and activity of gelatinases following transient focal cerebral ischemiaObjective: To explore the relationship between gelatinases activity and disruption of BBB in different phases following cerebral ischemia and reperfusion, and confirm the effects of gelatinases on microvascular damage after transient focal cerebral ischemia in rats.Methods: Thirty-four male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion (MCAO) was induced by an intraluminal suture method. Gd-DTPA enhanced T₁WI was carried out at 3.5h, 24h and 5d after reperfusion. Brain and serum gelatinases activities were analyzed by gelatin zymography at 24h and 5d after reperfusion, respectively. Correlation between MRI findings and gelatinases activities was analyzed. Immunehistochemistry was used to confirm the distribution of MMP-9 expression in the ischemic cerebral tissues.Results: In ischemic group activities of brain MMP-2 and MMP-9 were increased at 24h after reperfusion (P<0.05). MMP-9 was no longer visible on gelatin zymograms after 5d of reperfusion, while MMP-2 activity remained increased at 5d after reperfusion (P<0.05). In sham-operated group, activity of brain MMP-2 remained low all the time, and no MMP-9 was detectable at any time point. Activities of serum MMP-2 and MMP-9 were increased correspondingly at 24h after reperfusion (P<0.05), and kept increased at 5d after reperfusion (P<0.05). In sham-operated group, activities of serum MMP-2 and MMP-9 changed little during the experiment (P>0.05). A significant correlation was observed between activity of brain MMP-9 and rV-T₁WI at 24h of reperfusion (r=0.96, p<0.001). Furthermore, immunehistochemistry results showed distribution of MMP-9 expression corresponded to the area of enhancement on postcontrast T₁WI.Conclusions: Enhanced activities of brain gelatinases play an important role in the disruption of BBB and formation of edema after cerebral ischemia and reperfusion. Activity of brain MMP-9 shows remarkable correlation with reperfusion induced neurovascular damage in cerebral ischemia.Partâ…¢Study on expression of MMPs and TIMPs in rat brain following transient focal cerebral ischemiaObjective: To study the mRNA and protein expression of MMPs and TIMPs in different phases of cerebral ischemia and reperfusion.Methods: Fifty-six male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion (MCAO) was induced by an intraluminal suture method. After 24h and 5d of reperfusion mRNA expression of MMP-2 and MMP-9 was observed by RT-PCR, and the protein expression of MMP-2/TIMP-2 and MMP-9/TIMP-1 was also defined by western blot.Results: mRNA expression of MMP-2 and MMP-9 at 24h and 5d after reperfusion was elevated in the ischemic group(P<0.05), and mRNA level of MMP-9 declined dramatically after 5d of reperfusion (P<0.05), while expression of MMP-2 mRNA kept increased (P<0.05). Only low level of MMP-2 mRNA was detected in the sham-operated group at any time point without obvious fluctuation (P>0.05). Expression of TIMP-1 and TIMP-2 was significant higher in the ischemic group at 24h after reperfusion (P<0.05). After 5d of reperfusion expression of TIMP-1 declined dramatically (P<0.05) while protein level of TIMP-2 still increased (P<0.05). Low expression of TIMP-1 and TIMP-2 was confirmed in the sham-operated group.Conclusions: Cerebral ischemia/reperfusion induces expression of MMP-2 and MMP-9 in rat brain. In the early stage of reperfusion, expression of gelatinases is charactered with high level of MMP-9, whereas in the late phase of reperfusion, expression of MMP-2 seems to be predominant. Expression of TIMP-1 and TIMP-2 after cerebral ischemia changes correspondingly with expression of gelatinases, which indicates an auto-regulation in vivo.Partâ…£Effects of anti-MMPs therapy on cerebral ischemia/reperfusion induced injury in rat brainObjective: To observe the effects of anti-MMPs therapy on reperfusion induced injury in a rat model of transient focal cerebral ischemia.Methods: Twenty-eight male Wistar Rats were randomized into BB-94 group and the control group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion (MCAO) was induced by an intraluminal suture method. BB-94, an inhibitor of MMPs, was injected intraperitoneally (50mg/kg) at different time point in the BB-94 group, and equal volume of saline was injected intraperitoneally at the same time in the control group. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. All rats received neurological evaluation before sacrifice.Results: After 3.5h and 24h of reperfusion rV-DWI in the BB-94 group was significant lower than that of control (P<0.05). BB-94 reduced rV-T₂WI, rV-T₁WI and rSI-T₁WI at 3.5h, 24h and 5d of reperfusion, respectively (P<0.05). Neurological outcome after 24h and 5d of reperfusion was also improved by BB-94 as compared with the control group (P<0.05).Conclusions: Anti-MMPs therapy shows significant neuroprotective effects by attenuating BBB disruption, reducing infarct volume and improving neurological outcome after transient focal cerebral ischemia in rats. Partâ…¤Study on neuroprotective effects of minocycline in ischemia- reperfusion induced injury and its mechanismObjective: To observe the effects of minocycline on reperfusion induced BBB disruption, infarct size and neurological outcome following transient focal cerebral ischemia in rats, and explore the underlying mechanism.Methods: Fifty-six male Wistar Rats were randomized into minocycline group and the control group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion (MCAO) was induced by an intraluminal suture method. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. All rats received neurological evaluation before sacrifice. At 24h and 5d of reperfusion, activities of brain and serum gelatinases were analysed by zymography. Effects of minocycline on brain mRNA level of MMP-2, MMP-9 and protein expression of MMP-2/TIMP-2, MMP-9/TIMP-1 were defined by RT-PCR and western blot, respectively. Results: After 3.5h and 24h of reperfusion rV-DWI in the minicycline group was significantly lower than that of control (P<0.05). Minocycline reduced rV-T₂WI, rV-T₁WI and rSI-T₁WI at 3.5h, 24h and 5d of reperfusion, respectively (P<0.05). Neurological outcome after 24h and 5d of reperfusion was also improved by minocycline as compared with the control group (P<0.05). Brain gelatinases activities were inhibited by minocycline at 24h and 5d after reperfusion (P<0.05). Serum MMP-9 activity at 24h and serum MMP-2 activity at 5d after reperfusion were also inhibited by minocycline (P<0.05). After 24h of reperfusion brain mRNA and protein level of MMP-2 and MMP-9 were significantly reduced by minocycline as compared with the control group (P<0.05). Expression of brain TIMP-2 was increased in the minocycline group (P<0.05), while expression of TIMP-1 showed no difference between the two groups (P>0.05). After 5d of reperfusion brain mRNA and protein level of MMP-2 remained lower than that of control (P<0.05). There was no difference in the expression of TIMP-1 and TIMP-2 between the two groups any more (P>0.05). Expression of MMP-9 mRNA and protein was invisible in both minocycline and control group at 5d after reperfusion.Conclusions: Minocycline attenuates BBB disruption in both the early and late phases of cerebral ischemia and reperfusion. Treatment with minocycline reduces infarct volume and improves neurological outcome in a rat model of transient focal cerebral ischemia. Inhibiting the activities of brain and serum gelatinases, reducing their expression in the brain, and elevating the level of brain TIMP-2, which is capable of inhibiting the activity of gelatinases, may be responsible for the neurovascular protection of minocycline.