Empirical Study Of Encouragement That Osmotic Opening Of The Blood Brain Barrier Bring About Nerve Growth Factor's Pass

Background and Objective:Hypoxic-ischemic brain damage (HIBD) is a frequent diffuse damage of central nervous system in neonatus. Dysbolism of energy, intracellular calcium overload, neurotoxicity of excitatory amino acids, nitric oxide and oxygen free radical oxyradical's damage and apoptosis co- participate the patho-course of HIBD. Nerve growth factor (NGF) has double-acting on nerve cells of nurturing them and encouraging the ecphymas to grow. NGF has been producing a marked effect on survival , differentiation, regeneration as well as expressing function and characteristics of both central and peripheral nerve cells. NGF is an important factor which participates regeneration and function's renovation of damaged nerves. It has been indicated by animal experiments, that growth associated protein-43's (GAP-43) activity of immune reaction enhanced in rats' some encephalic regions after they were injected NGF in cerebral ventricles. GAP-43, a glucoprotein which is endemic in nerve tissue, has been producing a marked effect on growth development regeneration retaining synapse's function and releasing transmitters of nerves. It has been indicated by generous researches, that there were considerable GAP-43 in endings of neurofibrils which were growing or differentiating. So this certify that GAP-43 has close relation with growth, regeneration and differentiation of nerves.However, NGF is a macromolecule which can't pass blood brain barrier (BBB) customarily. Now it is need to find a available and reasonable way for NGF's central administer. The most important element constitute of blood brain barrier are brain micrangium endothelial cells(BMECs), and their tight junction can be reversibly opened under the hyperosmosis, so that some hydrosoluble drug protein and some other solvents can enter brain. It has been confirmed by animal experiment that perfusion of carotid artery of hyperosmotic solution can open blood brain barrier. Experiments have also proved that blood brain barrier can be osmotic opened pro tempore by intravenous injection of Mannitol. In experiments and clinic, the osmotic method has been used to treat brain tumor well. This research is animal experiment ,and we are intend to study the effect which NGF make on expressing of GAP-43 in rats' brains, then initially approach quantitative different of NGF that pass blood brain barrier between before and after intravenous injection of Mannitol, so to guide clinical treatment.Material and method1. Preparing the rat models of HIBD and divide into groupsFirstly 7-days rats were divided into two parts. One part was divided into three groups-treatment group( I ) control group (II )and sham operated group(III),there were 20 in every group. Rats in I and II were hold heads and extremities to keep a dorsal position, after they were marked weigh and anesthetized by diethyl ether. Then their skin were degermed, the median incision of cervicum were choosen. The left common carotid artery was freed and ligatured then, sew up the cut and rest them for 1 hour. Subsequently them were put in a oxygen deficiency box (inhaling 8 % O₂ and 92 % N₂)for 2 hours. In the end, those were the rat models of HIBD. Rats in III were just freed the left common carotid artery ,neither ligation nor hypoxia, the way of breed were same to the former two groups. The other part were divided into four-both Mannitol and NGF treated group( i ) just NGF treated group( ii) control group(iii) and sham operated group(iv),there were 10 in every group. Treated rats in i ii and iii as those in I and II of first part,and treated those in iv as those in III of first part. 2. Drug application and type collectionThe treat group( I )in first part were treated with intraperitoneal injection of 100U NGF(5000-7000U/kg) instantly after models were prepared, and once a day for consecutive 7 days. The control group (II )in this part were treated similarly with intraperitoneal injection of isotonic Na chloride. Sham operated group(III) received no treat. Ten rats were randomly selected from every group in this part and them were killed at the eighth day. Dissect the 30 rats and free the brains quickly after infusing heart, fix the brains, took the center parts to be anhydrated and paraffin imbed, then made histological sections form them; Then the other part, 24 hours after models were prepared, inject 20% Mannitol 1.5g/kg from sinuses venosus to both Mannitol and NGF treated group( i ) quickly, and inject NGF 100U the same way 10 min later. Just inject NGF 100U from sinuses venosus to just NGF treat group( ii ). All rats were killed in this two groups 30 min after injection, and then took their brain to make homogenate. Those in last two groups without injection were killed and make homogenate.3. ObservationHistological sections were HE stained or immunohistochemistry stained to observe pathological change or expression of GAP-43.The last rats in first part were tested at the 28th day by maze test, so that the message of their study and memory ability were observed. Brain tissue homogenate were tested by enzyme linked immunosorbent assay(ELISA)to determin the content of NGF.4. Statistics analysisDepend on SPSS11.0 software, measurement datas were expressed as mean±standard deviation (x|-±s), and group comparison were done by one-way analysis of variance. Statistical significance was signed by P<0. 05.Result1. General dataDiscrepancy of rats' weight, time of anaesthesia and operation between every groups had no significance (P>0.05 ) . 2. Pathological changeObserved through microscope, chiefly at cerebral cortex corpora striata cornu ammonis and cerebral ganglion. Apomorphosis and cellular necrosis of cellula nervosa could be observed in control group (II),while pathological changes of treat group( I ) were slight.3. Expression of GAP-43The average gray scale value of GAP-43 expressing spot in sham operated group(III) were significantly higher than that in control group (II )(P<0. 05); The average gray scale value in treat group( I) were significantly higher than that in control group (II) (P<0.05) and sham operated group(III) (P<0.05) .4. Effect of NGF on rats' study and memory abilityRats in control group (II )need significantly more times to study than those in treated group( I ) (P<0.05) and sham operated group(III) (P<0.05) at the first and second day, and the right reaction rate were significantly lower than those two groups; However, rats in treated group( I )need significantly more times to study than those in sham operated group(III) (P<0.05 ) at the first and second day, and the right reaction rate were significantly lower than that of sham operated group(III).5. Content of NGF in Brain tissue homogenateContent of NGF in both Mannitol and NGF treated group( i )were significantly more than that in just NGF treat group(ii) (P<0.05) , control group(iii) (P<0.05) . and sham operated group(iv) (P<0.05) ;Just NGF treated group( ii) were significantly more than control group(iii) (P<0.05) and sham operated group(iv) (P<0.05) ;However, content of NGF in control group(iii) and sham operated group(iv) have no significant difference.Conclusion1. NGF can protect rats' nerve cell from apomorphosis and necrosis after HIBD.2. NGF can increase expression of GAP-43 in brain tissue, and HIBD rats' study and memory ability can be improved at the same time.3. Osmotic opening blood brain barrier (BBB) by exogenous Mannitol from vein can significantly augment deal of NGF that passed the BBB of rats.