The Relationship Between Neuropeptide Y In Plasma And Cerebral Vasospasm AfterSubarachnoid Hemorrhage In Rats

Subarachnoid hemorrhage is a syndrome caused by blood into subarach-noidealis of intracalvarium or vertebral canal for some reason. Cerebral vaso-spasm( CVS) , an important complication of SAH, is the leading cause of disability and death. According to literature, angiography shows that 70% of SAH patients may present cerebrovascular spasm, in which 30% may be with a secondary ischaemic damage. At present, the exact mechanism of cerebrovascular spasm is not very clear. In feneral, two mechanisms body fluid regulation and nervous regulation exist simultaneous. When SAH occurs, the distribution of Nerve fiber in cerebral blood vessel and the concentration of neurotransmitter in blood plasma change. The role of such changes in cerebrovascular spasm caused by SAH has been focused on. Molecular biological and immunohistochemistry experiments confirmed that NPY distributes widely in many animal and human organs , even in various part of brain. NPY is found as a strong vascular contracting substance. NPY injected into the internal carotid artery will result in marked reduction of cerebral blood flow in internal carotid artery, and a dose -depending reduction of blood flow in homolateral striate body. All these disclose that NPY is an important vascular contracting substance. However, little has been shown about Whether NPY level of blood plasma varies under condition of CVS. Its clinical significance. If the blood plasma level of NPY is positively correlated with the degree of cerebral spasm, the treatment focusing on NPY level will benefit for the CVS. Here, SAH animal model is constructed and the relationship of NPY and CVS is investigated in SAH. It will provide some data forclinical treatment of CVS in SAH to decrease the morbility and mortality.Materials and methodsReagents and laboratory equipments1. Conventional reagents Polyoxymethylene, pbs, DAB solution, hematoxy-lin, eosin, Glacial Acetic Acid (analytically pure) , distilled water.2. radioimmunological reagents neuropeptides Y radioimmunoassay3. laboratory equipmentsLDF - Laser Doppler Perfusion Monitors ( pf3 , PERIMED) N paraffin slicing machine ? thermostatic device ^ Transferpette ^ High - speed Refrigerated Centrifuge ^Y Radioimmunoassay Counter N instruments ^electronic scales % Dental burMethods1. Animal45 Wistar rats of both sexes weighing 300 - 350g were used in this study ( supplied by the experimental animal centre, China Medical University).2. GroupThey were randomly assigned to control group ^ sham SAH group N SAH group (include post -SAH lh^6h^l2hNldN3dN5dN7d) ,5 rats each group.3. Animal ProceduresThe experimental model of SAH was completed by double injection autoge-neic arterial blood into cistema magna . The rats were anesthetized with intrap-eritoneal 10% Chloral Hydrate (40mg/100g). Under sterile conditions, the posterior scalp was incised in the midline and the occipital bone exposed at the junction of the occipital bone and the arch of Cl. The occipital muscles were carefully dissected off the occipital bone. The atlanto - occipital membrane was then exposed , covered with sterile gauze, a 0. 3ml of blood sample was drawn from femoral artery which had been exposed, after an equivalent volume of cere-brospinal fluid (CSF) was drawn from intracisternal space, the 0. 3ml of hepa-rinized autoblood sample was injected slowly into cistema magna over a period of 1 min. Notable changes in animals'respiration pattern even transient apnea wasobserved by the end of injection. Animals were immediately placed in a head -down position for 30 min to facilitate the diffusion of the autoblood in the basal cisterns. After no significant blood or cerebrospinal fluid leakage from the insertion site was found, the muscles and the incisions were closed with suture and sterilized. All the rats were fed cleanly and antibiotic was administered by mouth for infection prevention. Forty - eight hours afer the initial intracistemal blood injection, the rats in SAH group were reanesthetized and 0. 3ml of autoblood was reinjected into the cisterna magna. The sham SAH group underwent the same basic procedure as the SAH - animals, except that physiological saline was injected into the intracistemal space rather than blood. The control group was untreated.4. Measurement of Cerebral Blood Flow( CBF)The SAH — animals were reanesthetized as described above for the SAH procedure at 1 h to 7 days post - SAH ( control group and sham group at a fixation time) . The posterior scalp was incised in the midline and a hole( D = 3mm) was drilled by a dental drill at the point which was 3 mm away from the midline and the bregma respectively on the left of the skull. The rats cerebral blood flow (rCBF) was measured by a detecting head ( D =2. 5mm) of laser Doppler flowmetry which was placed vertically on the cerebral dura mater.5. Materialsmeasure rcfb of experimental animals, collects 2 ml blood from auriculadextra, then infuse it to the tube containing 40 ul of 10% EDTA and 40 ul of Apro-tinin, mix homogeneously, centrifuge at 4 Tl for lOmin (3000rpm /min) , store at "^20Tl. Puncture at apex, first , infuse 20Chnl of 371 saline , shear auriculadextra at the same time to eject blood of whole body untill seeing clear fluid. Then dissect brain out, cut off the brain stem and That the brain, insure the integrity of basilar arterial vascular , dissect a section of brain stem and put it into Polyoxymethylene 4% for proteopexy.6. H&E staining7. NPY concentration measurements Prior to measurement , the samples should be placed at RT for 30 min, get homogenous mixture, and centrifuge at 4 C for 25 min(3500rpm /min) . Discard the supernatant, and get the cpm valuesby Y Radioimmunoassay Counter .8. StatisticsUse SPSS11. 5 Software to analyze the data. The measured values should be shown with the form of X it S. Analysis of variance should be used to test the significance of NPY between groups ( p <0. 05).Result1. Make SAH rat model Successful, blood clots deposition is found in cis-ternals of rats of SAH Group. Abnormal changes ( vessel wall incrassation and collapsed endodermis) of basilar artery can be shown by HE dyeing.2. The values of Post -SAH rCBF changed obviously before and after, assuming the bidirectional changes.3. The concentration of NPY in plasma of the rats in SAH Group increases lhr after the occurrence and come to get down from then on and to the normal on the 5th day.4. The study also showed a negative correlation between the Cerabral blood flow and the concentration of NPY in plasma.DiscussionIn this research we infused blood into cisternals to build Cerebrovascular Spasm models and observed the morphological changes of basilar artery of rats through optical microscope. After the rats being processed, the head dissection confirmed that there was blood clot deposition in the cisternals.The abnormal changes - - vessel wall incrassation and collapsed endodermis - - have been found at lhr after blood infusion during the morphological observation through Optical microscope, and these changes obviously reduced on the 7th day.At the same time, we used laser Doppler to measure the cerebral blood flows of rats and found that the blood flows obviously reduced when SAH hap-pened and also rCBF assumed the bidirectional - changes. The reasons may be the followings: when SAH the vasoconstriction - dilatation balance was broken, resulting in vasoconstriction. Because of stress reaction and by the stimulation of SAH blood block and the released factors, the negative feedback of neurohumor-al regulation in the body, which ought to keep the balance to recover rCBF, firstly push this dynamic balance to the direction of constriction again and then gradually back to the equilibrium point.Cerebrovascular Spasm ( CVS) , one of most important complications, is a main lethal and disable cause after SAH. At present, the accurate mechanism of Cerebrovascular Spasm is not clear and most scientists agree that there are two kind of mechanisms that can cause CVS, namely, humoral regulation and nervous regulation. Scientists has attached the importance to the functions of NPY on Subarachnoid hemorrhage induced CVS.The NPY is a type of peptide hormone and wide spreads in each region of cerebrum, of which content is maximum.As an accepted neurotransmitter, NPY can directly cause vascular smooth muscle constriction. Radioimmunology is used in this experiment to measure the concentrations of NPY in plasma of 3 groups of rats: SAH Group, Pseudo - surgery Group and Control Group. We found that NPY level of each group was changing at different time after attack. NPY Level of SAH group began to increase lhr later and reached at the top Id later, then came to decrease and recovered 5 days later.The experiment testified that the concentration of NPY in plasma obviously increased but the value of rCBF distinctively fell down. So we think NPY can act on cerebral vessel and cause contradiction resulting in CVS. At present we think that NPY functions by NPY - Y 1 receptor and its mechanism follows below: Coupling with G - protein, NPY - Y 1 receptors can contribute to IP2 hydrolysis by activating PLC, thus IP3 and DAG, which are secondary messengers to modulate the release of Ca2 + , are generated. IP3 induces a large amount of free Ca2 + release from " Calcium Library" to cytoplasm and thus promotes the vasoconstriction. Furthermore, there are lots of NPY - Y2 receptors on the vascular smooth muscles which functions may be against those of NPY - Yl receptors.Also NPY can boost up the abilities of other vasoconstrictive substances and inhibit the functions of vasodilatation of some media.We think that the increase of NPY Level after SAH attack is related to following factors:1. Stress Reaction, which actives sympathetic nerves to release neurotrans-mitters excessively.2. Endangium Damage and Stress Reaction, which active thromboplastids releasing over many NPY.3. Intracranial Hematoma or Hydrocephalus, which leads to high toin-tracranial pressure resulting in autonomic nerve dysfunction and NPY increase.4. Direct Damage to cerebrum by Hematoma, which causes NPY releasing and breaking into blood circulation via destructive blood - brain barrier.High NPY concentration leads to cerebrovascular spasm and NPY can:1. Directly contribute to Vascular smooth muscles constriction.2. Enhance the abilities of other vasoconstrictive substances (for example, 5 - HT and Noradrenalin) .3. Inhibit the functions of vasodilatation of some media.4. Stimulate vascular endothelial cells to release endodermis - derived dias-tolic factors giving a comprehensive effect of vasoconstriction. Based on a body of investigation on the relationship between NPY and Post - SAH CVS of rats, this experiment is intended to provide strong and capable proofs for the research on mechanisms of Post - SAH CVS and the clinical therapeutics of CVS.,.: conclusion ,,:In current SAH rat model, obvious CVS correlates with plasmic NPY increasing tightly, indicating NPY contributing much to the setup and development of CVS.