| Background and ObjectiveImmunological reaction play an important role in cerebral ischemia-reperfusion . As the propounding study on the inflammation mechanism of cerebral ischemia , more and more attention was payed to the inflammatory factors . The up-regulation of expression of focal inflammatory factors ,chemotactic factors , and adhesion molecule make the foundation of the transition from cerebral ischemia to cerebral inflammatory damage. Then the aggregation and infiltration of leucocyte into the ischemia area can induce the next neuron damage and the expansion of infarction.NF-κB is one of the important transcription factor in inflammation reaction ,and is the critical point of the inflammation reaction in cerebral ischemia focal . TNF-αis also an important inflammatory factor ,which play the role of cascade amplification in inflammation reaction of cerebral ischemia-reperfusion. adhesion molecule play an important role in the development of inflammation reaction .ICAM-1 is one kind of adhesion molecule,Which can reduce the leucocyte and the cerebral micro- vascular endothelial cell(MVEC); aggregation and infiltration into the cerebral ischemia area , and promote the inflammation reaction after ischemia .Then bring the ischemia-inflammation- ischemia infernal circle.PACAP is a new kind of hypothalamus hypophysiotropic hormone ,which is deposited generally in central nervous system; and its effect of neurotrophy was paid more and more attention . In normal condition , PACAP can permeation the blood brain barrier ,work through its combination to PACAP receptor . It is founded by abroad animal experiment that the PACAP can deflate the area of infarction in both focal and global cerebral ischemia ,reduce the neuron damage after ischemia-reperfusion . But the mechanism of its cerebral protection is still unknown . we made the rat ischemia-reperfusion model by thread embolism , observe the effect of PACAP on the expression of NF-κB, TNF-α,ICAM-1 after ischemia-reperfusion . Approach the mechanism of its neuroprotective function. The experiment can be served as theoretical testimony for PACAP curing ischemic cerebrovascular diseases in clinic.Studies have shown PACAP can decreased the infarct volume and neuron injure of focal and globe cerebral ischemia. But the molecular mechanism of PACAP-induced protection has not been known at present. This experiment aimed to explore the mechanism of PACAP-induced protection by means of detection the expression of NF-κB, TNF-αand ICAM-1 in the cortex of rats with focal cerebral ischemia-reperfusion injury. All of this will provide some experimental proofs for PACAP preventing and curing ICVD.Materials and methods1. The model of middle cerebral artery occlusion(MCAO) by suture embolus was established according to Koizumi's report. The suture embolus was inserted in left MCA abort 17.5±1.0mm from the origination of internal carotid artery to occlude the blood supply of MCA. When the suture was put out of the ECA, 12h, 24h reperfusion occurred. Sham-operation group received the same operation except the suture embolus was inserted about 10mm. The standards of successful model included Homer's sign in left side, Paralysis in right limbs and Longa's scores.2. 72 male Sprague-Dawley rats each weighing 300±20g were randomly divided into Sham-operation group,ischemia-reperfusion group and PACAP group. PACAP group received tail vein injection of PACAP(1×10-9 mol/kg) daily just at half past very outset of ischemia, while Sham-operation group received the same doses of saline. After ischemia for 2 hours and reperfusion for 12h, 24h, the brains were removed. Some of the brains were prepared for paraffin sections, some were prepared for in Western-blot.3. Sections of every rat were processed with HE staining, and NF-κB, TNF-α, ICAM-1 immunohistochemistry staining. Some rats' brains were processed Western-blot.4. The dates were expressed with x|-±s and analyzed with one-way analysis of variance among groups and t-test between two groups. Longa's scores were analyzed with q-test. The significant testing standard was a =0.05.Results1. Longa's scores in Sham-operation group were 0, while I/R group rats were 2.00±0.10 at 12h reperfusion, 2.36±0.76 at 24h reperfusion. Comparing with sham-operation group, there is significant difference(P O.05). The scores of rats with PACAP administration are 1.12±0.46 at 12h reperfusion, 1.01±0.22(24h). Compare all the PACAP groups and ischemia-reperfusion's groups, there are significant difference(P<0.01).2. The Pathological changes: The rat cerebral section of Sham-operation group hadn't obvious abnormal under light microscope. The cerebral section of ischemia reperfusion group showed normal neuron was less, deformation of neuron bodies and concentration of nucleus and bubble in cytoplasm were more, more inflammatory cells infiltrated. The section of PACAP group showed the number of injured neuron and inflammatory cells less then that of ischemia-reperfusion group.3. The expression of NF-κB : 12 to 24 hours after the reperfusion . NF-κB was significantly translocated from the cytoplasm into the nucleus; however, NF-kappa B remained in the cytoplasm of bilateral cortexes in the sham operation groups, and the nonischemic cortexes in the I/R and PACAP groups. The translocation of NF-κB from cytoplasm into nucleus was significantly inhibited in the ischemic cortex of the PACAP group. The expression values of NF-κB in the nuclei of ischemic cortexes in the I/R group 12 to 24 hours after reperfusion were significantly higher than those in the sham operation group and the nonischemic cortexes of the I/R and PACAP groups (all P < 0.01). The expression values of NF-κB in the ischemic cortex of the PACAP group 12 to 24 hours after reperfusion was significantly lower than that of the I/R group (all P < 0.01).4. The expression of TNF-α: Sham-operation group:2.83±1.47(12h),2.96±0.68(24h), ischemia-reperfusion group:40.82±3.71(12h), 60.67±3.49(24h). Comparing with sham-operation group, there is significant difference (P <0.01). PACAP group: 20.01±1.33(12h),30.71±2.48(24h). Comparing with I/R group, there is significant difference(P <0.01).5. The expression of ICAM-1: Sham-operation group:0.56±0.16(12h), o.64±1.49(24h), ischemia-reperfusion group:6.00±0.66(12h), 12.02±0.51(24h). Comparing with sham-operation group, there is significant difference(P <0.01). PACAP group: 1.68±1.33(12h),4.02±0.50(24h). Comparing with I/R group, there is significant difference(P <0.01).Conclusion1. Middle cerebral artery occlusion method was adopted to make focal ischemic cerebrovascular model in this research. The model was similar to clinical cerebral ischemia. It can control accurately the time of ischemia-reperfusion. It had the high rate of repeating.2. The administration of PACAP could lessen brain tissue pathological changes and the score of rats after cerebral ischemia-reperfusion injury in rats.3. The administration of PACAP could decrease the expression of NF-κB, TNF-α, ICAM-1 after cerebral ischemia-reperfusion injury in rats.4. The experiment can be served as theoretical testimony for PACAP curing ischemic cerebrovascular diseases in clinic.
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