Effects Of PACAP On The Expressions Of P-ERK,P-JNK And Apoptosis Of Rats After Ischemia-reperfusion

Background and ObjectiveFor the high incidence, mortality and disability rate, ischemic cerebrovascular disease (ICVD) is seriously endangering man's health. The mechanisms of neuronal injury and death have been being the emphasis of fundamental and clinical medicine study. In recent years, scientists have payed close attention to the injury of ischemia -reperfusion. Neurons are found apoptosis after cerebral ischemia-reperfusion. Apoptosis is the main way of neuron death in ischemic penumbra. The key for therapy of the injury is to save the still alive neurons in penumbra.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP can stimulate cAMP formation . PACAP is widely distributed in the central and peripheral nervous systems. Currently available data indicate that PACAP is a promising neuroprotective peptide. PACAP plays an important role during the development of the nervous system and in regeneration following nervous injuries both in vitro and in vivo. Researches have showed that external PACAP can go through blood brain barria (BBB) and reduce infarction volume after cerebral ischemia-reperfusion. But the mechanism is unclear to date.Extracellular signal-regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) are key proteins in mitogen activated protein kinase (MAPK) signaling pathway which mediates cell stress and injury responses. Both of them are activated in the cytoplasm and translocated into nucleus to activate transcription factors resulting in gene transcription. The ERK signaling pathway is one of the major survival signals, while the JNK is implicated in neuronal apoptosis. The expressions and dynamic balance of them determine the level of injury of cerebral ischemia -reperfusion. But the roles of variation of space-time don't reach a concordance.By creating rat focal cerebral ischemia reperfusion model established with suture emboli method and injecting PACAP-38 through caudal vein, the changes of nerve score and injury of nerve cell under light microscope were measured. By using immunohistochemical techniques and TUNEL detection, the expressions of p-ERK,p-JNK and apoptosis were observed to investigate the mechanism of neuron death after focal cerebral ischemia-reperfusion and the protecting effect and mechanism of PACAP. It can provide experimental and theoretical proof for PACAP curing ICVD.Materials and Methods(1)Seventy-two healthy male Sprague-Dawley rats weighing 280-320 grams were randomly divided into three groups (twenty-four rats each group).Group A were sham-operated. Group B were ischemia-reperfusion. Group C were ischemia -reperfusion plas PACAP treatment. Each group was again randomly divided into four subgroups (six rats each group) according to different time after ischemia- reperfusion. The models were made by occluding left middle cerebral artery(MCA) for 2hr and reperfused for 2,12,24 and 48hr. Animals in the sham operated groups were treated in the same way without MCAO. Horner's sign in left side and paralysis of the right front limbs were observed to judge the success of MCAO model. Group C received an intravenous injection through caudal vein of PACAP-38 1×10^-9mol/kg (dissolved in 1ml saline ) after ischemia-reperfusion within 30min,while Group A and B received 1 ml saline through caudal vein at the same time.(2) Neurological scoring was done at different time after reperfusion . Then the animals were deeply anesthetized and perfused. The brains were removed, cutted and postfixed. The slice was embed in paraffin and made pathological sections. By using HE staining, immunohistochemical SP methods and TUNEL detection, we examined injury of nerve cell under light microscope and detected the number of p-ERK, p-JNK and apoptosis. The concentration of primary antibodies against p-ERK and p-JNK were both 1:300.(3) All data were entered into a computer and analyzed using software SPSS 11.0 for windows. The data were presented as means±SD( x±s ). Unpaired Student's t-tests were used to determine significant differences between two groups, and one-way analysis of variance was used to determine significant differences among three or four groups. The significant testing standard was a =0.05.Results(l)After the operations , all the animals of group B and C behaved low spirit and the myodynamia of right extremities decreased. Quantitative evaluation revealed that the positive effects of PACAP at 2, 12, 24, 48hr on neurological function of rats were statistically significant (P<0.05) compared with group B .(2)The result of pathological section with HE staining in every group: There was no infarction in group A, the shape or structure of the neurons was normal. There was obvious infarction in group B, the neurons in core of ischemic cortex were found swollen, deformed and softened and normal neurons were less. More inflammatory cells infiltrated. Some nerve cells were shrunken and concentrated in cerebral penumbra. The infarction was smaller in group C , the number of injured neurons and inflammatory cells were less than of group B. Few neurons were shrunken and concentrated in cerebral penumbra.(3)Few apoptotic cells were observed in sham operated group. After ischemia -reperfusion caused by MCAO, the apoptotic neurons enhanced in the penumbra of the ischemic cortex in group B and C. The decease of apoptotic neurons were seen in group C at 12 and 48hr compared with group B. Differences were statistically significant (P<0.05).(4) Immunohistochemistry showed a weak immunoreactivity for p-ERK in the cortex of the sham operated rats. After ischemia-reperfusion, the prominent staining mainly appeared in the penumbra of the ischemic cortex in group B and C. P-ERK immunoreactivity seemed to be mainly located in the nucleus of the neurons. The expression of p-ERK reached the peak at 2hr (P<0.05). The experiment again revealed that the positive cells in group C at 2, 12, 24 and 48hr after reperfusion were higher than that in group B. Differences were statistically significant (P<0.05).(5) Immunohistochemistry showed a weak immunoreactivity for p-JNK in the cortex of the sham operated rats. After ischemia-reperfusion, the prominent staining for p-JNK appeared in the penumbra and cores of the ischemic cortex at 2hr in group B and C, While positive cells mainly appeared in the penumbra at other time. P-JNK immunoreactivity seemed to be mainly located in the nucleus of the neurons. The expression of p-JNK reached the peaks at 2 hr and 48 hr after reperfusion in ischemic cortex(P<0.05). The experiment again revealed that the positive cells in group C at all time after reperfusion were lower than group B even though they were still higher than sham operated groups. Differences were statistically significant (P<0.05).Conclusions(1) The roles of expressions of p-ERK and p-JNK are dynamic and space-time dependented. The expressions of p-ERK reach the peak at earlier period and appear in the penumbra of the ischemic cortex. The expressions of p-JNK reach two peaks at earlier period and later period and appear in the penumbra and cores of the ischemic cortex .(2) PACAP-38 could inhibit apoptosis of cortical neurons in ischemic penumbra . The effect might be mediated by up-regulating the expression of p-ERK and down-regulating the expression of p-JNK in ischemic cortex.(3) PACAP-38 injected through caudal vein could attenuate the injury of cerebral ischemia-reperfusion. The neuroprotective effect of PACAP-38 is obvious.