The Effect Of Facal Mild Hypothermia On The Excitatory Amino Acid And NMDAR₁ Levels Of Experimental Intracerebral Hemorrhage In Rat

Intracerebral hemorrhage (ICH) is a serious disease that has highmortality ratio without effective treatment. The direct damage to theearly and following neurological disability were mainly induced byfurther expansion of the cerebral hematoma, beside that it owed tosecondary brain injury, such as ischemia surrounding the hematomaand exitotoxicity. Several studies reported that mild hypothermia(MHT) can increase survival ratio and life quality of ICH patients as aadjunctive therapy. But the mechanism of MHT treated ICH is notclear. Recent studies confirmed that excitatory amino acid (EAA) isnot only neurotransmitter, but has the exitotoxicity. The theory ofexitotoxicity has become one of the most important mechanisms aboutapomorphosis and necrosis of neurone. Glumatic acid (Glu), asparticacid (Asp), as two widest distributed EAAs in central nervous system,and their common receptor, N-methyl-D-aspartate receptor(NMDAR)play key roles in ICH pathology. The purpose of this experiment is tostudy the effect of skull Focal mild hypothermia on EAA in peripheralblood and brain tissue homogenate and on NMDAR1 in tissuesurround the hematoma of ICH. The rat model of experimental ICH was made by the method ofinjection of autologous arterial blood into the caudate nucleus. A totalof 144 male Wistar rats were divided into 2 parts at random.Part A(n=72) were used to detect the content of EAA and brain water content;Part B (n=72) were used to observe the pathological change. Each partwas randomly divided into 3 groups, including sham-operated controlgroup (n=24) and normothermia group (n=24) and local mildhypothermia group (n=24). Each group was randomly divided into 4subgroups, which would be killed at 3, 6, 24, 72 hours (the rats forAsp content were killed at 3 hours). After anesthesia, thenormothermia group and the mild hypothermia group received andinjection of 50μl of autologous blood into the basalganglia. 30minutes later,a head cooling therapeutic instrument was introducedinto the mild hypothermia group immediately after the injection. Thecooling pad of the instrument was placed on the operated side of therats' head and immobilized. The tympanic temperature was used torepresent the brain temperature. Maintain the tympanic temperaturebetween 32 and 34 ℃ , and the rectal temperature between 36 and 37 ℃.At 3h group rats were continually cooled 2 hours. At 6h and 24h grouprats were continually cooled 4 hours. At 72h group rats werecontinually cooled 4 hours every day. The sham-operation groupunderwent the same operation but didn't receive injection. Thenormothermia group didn't receive hypothermia. About Part A: at thecorresponding time spot, 1000μl blood specimen was taken from tails,and sufficient brain tissue surrounding the hematoma , admixingisotonic saline solution was homogenated, which were used tomeasure the excitatory amino acid. Some brain tissue was used tomeasure the water content. About part B: Animals were anesthetizedand killed by perfusion with 4% paraformaldehyd at the correspondingtime spot. Fixed cerebral tissues were cut coronally through the needleentry site, and slices including the hematoma site were embedded inparaffin. The coronal slides of cerebral tissue were stained by HE andwith rabbit polyclonals anti-NMDAR1 antibodies by immunohisto-chemical technique. The positive cells were counted for statisticanalysis.The result showed: ① the water content of brain tissuesurrounding the hematoma was increasing from 3h to 72h. It is evidentthat brain tissue edema of 3h ICH group rats isn't different, From 6hto 72h ,brain tissue edema of mild hypothermia group wasreduced ,compared with the normothermia group, having significantdeviation (p<0.05). ② the content of Glu and Asp in blood plasmawas decreasing from 3h to 72h. it was reduced in Mild hypothermiagroup (p<0.05) compared with the normothermia group.③ the contentof Glu and Asp in brain tissue was also reduced in mild hypothermiagroup compared with the normothermia group. ④ The roundhematoma in the area of caudate nucleus was existed by the grossobservation of the normothermia group and the mild hypothermiagroup, and the diameter of the hematoma was about 3mm, whichshowed the experimental ICH model was successful. The operatedside of the brain was swelling, edema of the normothermia group.Herniation could be seen in some samples. In the mild hypothermiagroup, the damage was less than the normothermia group. In thesham-operation group, only the needle hole could be seen. ⑤HE: Thepathological change around the hematoma was not different at 3h, 6h.From 24h to 72h, the changes became significant, included tissuesoftening, edema, swelling and necrosis of neuron and astrocyte, nervefibers rupture and demyelination, inflammatory cells emerging, smallvessels hemorrhage. Of the mild hypothermia group edema andswelling around the hematoma was alleviated, necrotic cells werefewer, a few small vessels hemorrhage and astrocyte reaction can beseen. There were few inflammatory cells near the needle tunnel in thesham-operation group. ⑥ Immunohistochemistry: Immunohistoch-emical labeling indicated that NMDAR1 expresses moderately innormal neurones, but not in glial cells. ICH was accompanied byelevated expression of NMDAR1. From 3h to 72h, the expression sitesof the NMDAR1 focused on the circumambience of the hematoma,cortex, hippocampus, et al, and reach the top at 24h. Positive cellsincluded neurons, glial cells and carrier cell. Compared with thesham-operation group, the quantity of positive cells has significantdeviation. The expressional level of NMDAR1 was also reduced in themild hypothermia group compared with the normothermia group,having significant deviation.According to the result, we can conclude: ①Focal mildhypothermia has the effect of decreasing brain edema afterexperimental ICH in rats. ②The excitatory amino acid levelssignificantly increased not only in blood but also in brain tissue afterhemorrhage. Focal mild hypothermia can significantly depress theincreasing of excitatory amino acid after hemorrhage. ③Theexpression of NMDAR1 surrounding the hematoma significantlyincreased after hemorrhage. Focal mild hypothermia can also depressthe increasing. ④F ocal mild hypothermia can depress the increasingof excitatory amino acid and NMDAR1 after hemorrhage which mightbe one of the mechanisms of it in the treatment of ICH.