| ObjectiveTo study the changes and trends of nerve growth factor(NGF),and vascular endothelium growth factor(VEGF),of cerebral tissue around the hematoma of rats with intracerebral hemorrhage(ICH);to study the changes and trends of NGF and VEGF affected by electro-acupuncture treatment with different frequency waves.MethodsFor the research,We used totally 232 Sprague-Dawley rats(SD rats).They were all 3 months old male rats,with body weight within the range of 250 to 300 grams,and all were generally healthy.According to random table,we divided these 232 SD rats into 5 groups,i.e.,the control group,the sham -operation group,the operative ICH model group, the high frequency waves electro-acupuncture group and the low frequency waves electro-acupuncture group.The final two groups were experimental groups.8 rats were assigned to be the control group.Comparing with the control group,the other 4 groups of SD rats were further divided each group into 7 sub-groups,i.e,the 6 hours group,the 24 hours group,the 72 hours group,the one week group,the two weeks group,the three weeks group,and the four weeks group.We also took 8 SD rats for eash subgroup.For making an artificial simulative model of intracerebral hemorrhage(ICH)in SD rats,using stereotaxis instrument,0.6U collagenaseⅦ,in 3 microliter(concentrated as 0.2U per microliter)was injected into the caudate nuclus of Rt side brain of ICH-model SD rats,to establish a simulative model of intracerebral hemorrhage.For control,the sham operated SD rats were injected with 3 microliters of physiological saline,into the same area of brain.No any operation or injection was made for the control group,No any treatment was introduced for the sham operated group and the ICH-model group.For those two experimental groups of electro-acupuncture treatment,the acupuncture-points were Dazhui,Fengfu and Shuigou.The Dazhui and Fengfu were connected with electro-acupuncture apparatus,(Brand Huatuo,Model SDZ-IV Electronic Acupuncture Treatment Instrument),with high or low frequency wave stimulation.As to the low frequency waves of electro-acupuncture group,we used 0.175 millisecond wave width,2 hertz(HZ)frequency and 1 milliampere and 1 volt(Ⅴ)electric current.As to the high frequency waves of electro-acupuncture group,we used 0.175 millisecond wave width,100 hertz(HZ)frequency and 1.0 milliampere and 1 volt(Ⅴ) electric current.Each time for electro-acupuncture was 30 minutes and the experimental groups were treated once a day.Principles and methods of ethology,immunohistochemistry and pathology were applied.We observed the variation trend of the neurological deficit score(NDC)every day,NGF and VEGF in different time points of all ICH group & control group of SD rats and studied the effects on those indexes treating with different frequency waves of electro-acupuncture.ResultsFirstly,the affection on the neurological deficit score of the ICH rats treated with different frequency waves of electro-acupuncture resulted as follows:In the model groups,the neurological impairment could be observed in the 6 hours group.Then it appeared its peak after 24 hours.The neurological function began to recover after 72 hours.The neurological impairment was significantly improved after 1 week,and was almost recovered to normal level after 2 weeks and the score was zero after 4 weeks. Statistic data-analysis showed that the neurological deficit score in the 24 hours was significantly higher than that in the 72 hours,1 week,2 weeks,3 weeks and 4 weeks groups (P＜0.01).This proved that the neurological impairment in the 24 hours model group was the most serious.Comparing with both the low frequency and high frequency waves of electro-acupuncture group,the 6 hours groups showed the most serious neurological impairment. The neurological function began to recover in the 24 hours both the low and high frequency waves of electro-acupuncture groups,this was earlier than that in the model group.Statistic analysis showed that the neurological impairment in the 24 hours,72 hours and 1 week subgroups of both the low and high frequency waves of electro-acupuncture groups was significantly less serious than that in the corresponding time points of the model groups(P＜0.01).This proved that the neurological impairment in both the low and high frequency waves of electro-acupuncture groups was less serious than that in the model groups and the neurological function recovered earlier. The rat fatality in 3 groups said above,was 21.43%in the model group,10.71%in the low frequency waves of electro-acupuncture group and 12.50%in the high frequency waves of electro-acupuncture group.There was no significantly difference of fatality among these 4 groups(P＞0.05).Secondly,the pathomorphologyical changes of the brain tissue in rats of the model group were as follows:Under microscope,widespread bleeding,a large area of damage of nerve cell, histolysis and inequality of size of vacuole in intercellular space could be seen in the 6 hours,model group.Red cell lysis in the hematoma,lytic nucleus and neuron with eosinophilous endochylema and considerable swollen cells,obvious edema outside the cells and around the blood vessels,infiltration of generous neutrophil and abscess-formation at the edge of hematoma could be seen in the 24 hours model group.In the 72 hours model group,the red blood cell began to absorb in the centre of hematoma and obvious edema and vacuole in intercellular space could be seen.At the same time,there was foam cell, proliferation of gliocyte and widespread gliocyte around the hematoma.In the 7 days model group,the hematoma was obviously absorbed,the edema was extinctive,cellular swelling was lighten.At the same time,there was a large quantity of foam cell,appeared chronic inflammation and desmoplasia around the hematoma.As to the 2 weeks model group,the dropsy was obviously resolved and there was chronic inflammation and desmoplasia around the hematoma.The shape and alignment of brain cells in both the low and high frequency waves of electro-acupuncture groups were more regular than that in the model group.The degree of edema and the damage degree of nerve cell in the 72 hours of both the low and high frequency waves groups were lighter than that in the model group.The cerebral edema in both the low and high frequency waves groups was fundamentally vanished.Thirdly,the affection on NGF of brain tissue around the hematoma of the ICH rats treated with different frequency waves of electro-acupuncture was as follows:Our study used immunohistoehemical method to mark NGF.The result showed that there was light amber-like color expression of NGF positive cell in the control group.The expression of NGF was low in the 6 hours,24 hours,72 hours and 1 week model groups, just could find a small quantity of hypochromatic expression of NGF positive cell.The expression of NGF began to increase after 2 weeks in the model group.The yellow trachychromatic NGF positive nerve cell around the hematoma could be seen in this group. The high expression of NGF positive nerve cell was lasted till to 4 weeks.Statistic analysis showed that the expression of NGF was fairly low in the 6 hours,24 hours,72 hours and 1 week model groups.It began to increase after 2 weeks and the high expression kept till to 4 weeks.The expression of NGF in the 6 hours,24 hours,72 hours and 1 week model group was significantly lower than that in the 2 weeks,3 weeks and 4 weeks model groups(P＜0.01).The expression of NGF in the 2 weeks model group was significantly lower than that in the 4 weeks model group.This hints that,with the recovery of intracerebral hemorrhage,the expression of NGF was increasing and the increasing expression of NGF in the 2 week model group was more obvious.The expression tendency of NGF of the sham operation group,the low frequency waves of electro-acupuncture group and high frequency waves of electro-acupuncture group was just identical to that of the model group,but significantly lower level than the acupuncture treatment groups.Group comparison showed that the expression of NGF in the 6 hours,24 hours and 72 hours of sham operated groups,the 6 hours,24 hours and 1 week of model group,the 24 hours,72 hours and 1 week of both low and high frequency waves of electro-acupuncture group was significantly lower than that in the control group(P＜0.05 or P＜0.01).But the expression of NGF in the 3 weeks and 4 weeks of sham operated group,the 2 weeks,3 weeks and 4 weeks of model group,the 2 weeks,3 weeks and 4 weeks of both low and high frequency waves of electro-acupuncture group was significantly higher than that in the control group(P＜0.05 or P＜0.01).The expression of NGF in the 2 weeks model group,2 weeks,3 weeks and 4 weeks of both low and high frequency waves of electro-acupuncture group was significantly higher than that in the corresponding time points of the sham operated group(P＜0.01).The expression of NGF in the 6 hours,3 weeks and 4 weeks of both low and high frequency waves of electro-acupuncture group was significantly higher than that in the corresponding time points of the mode/group(P＜0.01).The expression of NGF in the 2 weeks and 4 weeks of high frequency waves of electro-acupuncture group was significantly higher than that in the corresponding time points of the low frequency waves of electro-acupuncture group(P＜0.05 or P＜0.01).The results showed that the expression of NGF was gradually increased with a temporal lapse after intracerebral hemorrhage.Both types of treatment method with low and high frequency waves of electro-acupuncture could increase the expression of NGF around the hematoma.The expression of NGF after 2 weeks was obviously increased. Statistic analysis showed that the high frequency waves of electro-acupuncture get more dominant than the low frequency waves of electro-acupuncture in this aspect.Fourthly,the affection on VEGF of brain tissue around the hematoma of the ICH rats treated with different frequency waves of electro-acupuncture was as follows: In this study,we used,immunohistochemical method to mark VEGF.The results showed that there was light amber-like color expression of VEGF positive cells in the brain tissues of control group.We could find a large quantity of yellow trachychromatic expression of VEGF positive nerve cell around the hematoma in the 24 hours,72 hours,1 week,2 weeks,3 weeks and 4 weeks model groups,this could be seen in the graph 60-89.The expression of VEGF positive nerve cells began to increased in the 24 hours model group and it was gradually increased with a temporal prolongation till after 4 weeks. Group comparison showed the expression of VEGF in the 6 hours model group was significantly lower than that in the 72 hours,1 week,2 weeks,3 weeks and 4 weeks control groups(P＜0.05 or P＜0.01).It indicated that the expression of VEGF was gradually increased with a temporal prolongation.The expression tendency of the sham operated group,the low frequency waves of elector-acupuncture group and the high frequency waves of elector-acupuncture group was identical to that of the model group.Group comparison showed that the expression of VEGF in the sham operated group, the 24 hours,72 hours,1 week,2 weeks,3 weeks and 4 weeks both in the low and high frequency waves of elector-acupuncture groups,the 72 hours,1 week,2 weeks,3 weeks and 4 weeks model groups was significantly higher than that in the control group(P＜0.05 or P＜0.01).The expression of VEGF in the 2 weeks,3 weeks and 4 weeks model groups, 72 hours,1 week,2 weeks and 3 weeks low frequency waves of electro-acupuncture groups, the 1 week,2 weeks and 3 weeks high frequency waves of electro-acupuncture groups was significantly higher than that in the corresponding time points of the sham operated group (P＜0.05 or P＜0.01).The expression of VEGF in the 24 hours and 72 hours low frequency waves of electro-acupuncture groups was significantly higher than that in the corresponding time points of the model group(P＜0.05 or P＜0.01).As to the expression of VEGF,there was no significant difference between the high frequency waves of electro-acupuncture group and the model group.There was no significant difference between the low frequency waves of electro-acupuncture group and the high frequency waves of elector-acupuncture group (P＞0.05).The results suggested that the expression of VEGF was gradually increased with a temporal lapse after intracerebral hemorrhage.Treatment with low frequency waves of electro-acupuncture could increase the expression of NGF around the hematoma at the acute stage of intracerebral hemorrhage.But there was no significant difference between the high frequency waves of electro-acupuncture group and the model group in this aspect. The results of the increased expression of VEGF by low frequency waves of electro-acupuncture were as follows.Firstly,it might increase the vasopermeability,damage blood-brain barrier;promote cerebral edema so as to aggravate brain injuring.Secondly,it might directly protect the nerve system before the neovascularization by neurotrophy action. This might lengthen the survival time of cells through avoiding the hypoxia and sugar deletion of cortical neuron.Fifthly,the affection on VEGF mRNA of brain tissue around the hematoma of the ICH rats treated with the different frequency waves of electro-acupuncture was as follows:In this study,we used Insitu-Hybride immunohistochemical method to mark VEGF mRNA.The result showed that there was yellowish color expression of VEGF rnRNA positive cells in the brain tissues of the control group.We could find yellowish color expression ofVEGF mRNA positive nerve cells around the hematoma in the 6 hours model group,the low frequency waves of electro-acupuncture group and the high frequency waves of electro-acupuncture group.The expression of VEGF mRNA at the other time points of the model groups was decreased and light stained.Statistic analysis show there was a quite amount of expression of VEGF mRNA in the control group.In all time points of the sham operated group,we also could see a quite amount of expression of VEGF mRNA.There was no significant difference among all these time points of the sham operated group in this aspect(P＞0.05)。As to the model group,the expression of VEGF mRNA began to decrease after 24 hours.The expression of VEGF mRNA in the 6 hours model group was significantly higher than that in the other time points of the model group(P＜0.05 or P＜0.01).There was no significant difference among the other time points of the model group(P＞0.05).There was no significant difference among all time points of the low frequency waves of electro-acupuncture groups in the expression of VEGF mRNA(P＞0.05).The expression of VEGF mRNA in the 6 hours high frequency waves of electro-acupuncture group was significantly higher than that in the 24 hours,1 week,2 weeks and 3 weeks high frequency waves of electro-acupuncture groups(P＜0.05 or P＜0.01).There was no significant difference among other time points of the high frequency waves of elector-acupuncture groups in the expression of VEGF mRNA(P＞0.05).Group comparison showed that the expression of VEGF mRNA in the 6 hours,1 week, 2 weeks,3 weeks and 4 weeks sham operated groups,24 hours,1 week,2 weeks,3 weeks and 4 weeks model groups,4 hours,1 week,2 weeks,3 weeks high frequency waves of electro-acupuncture group was significantly lower than that in the control groups(P＜0.05 or P＜0.01).There was no significant difference between all time points of the low frequency waves of elector-acupuncture groups and the control group in the expression of VEGF mRNA(P＞0.05).The expression ofVEGF mRNA in the 1 week,2 weeks and 3 weeks low frequency waves of elector-acupuncture groups was significantly higher than that in the corresponding time points of the model groups(P＜0.05 or P＜0.01).There was no significant difference between the low frequency waves of elector-acupuncture group and the high frequency waves of elector-acupuncture group in the expression of VEGF mRNA (P＞0.05).Obviously,the expression of VEGF and the expression of VEGF mRNA were disagreeing.The expression of VEGF was appeared in the 24 hours model group and gradually increased with a temporal lapse,till after 4 weeks.As to the expression of VEGF mRNA in the model group,it began to decrease in the 24 hours group and kept low expression till after 4 weeks.Both the low and high frequency waves of elector-acupuncture could not increase or decrease the expression of VEGF mRNA.We just could see the low frequency waves of electro-acupuncture could suppress the low level expression of VEGF mRNA after intracerebral hemorrhage.The expression of VEGF mRNA in the 1 week,2 weeks and 3 weeks low frequency waves of elector-acupuncture group was significantly higher than that in the corresponding time points of the model group.On the other hand,only the expression of VEGF mRNA in the 3 weeks high frequency waves of elector-acupuncture group was significantly higher than that in the 3 weeks of the model group.ConclusionResults of pathobiology and immunohistochemistry showed that there did have increasing expression of NGF and VEGF with temporal traction after an intracerebral hemorrhage episode.At the same time,the expression of VEGF mRNA was decreasing. The treatment methods of both low and high frequency waves of electro-acupuncture could increase the expression of NGF around the hematoma and this increased expression was more obvious after 2 weeks.The high frequency waves of electro-acupuncture showed more effective than the low frequency waves of electro-acupuncture in this aspect.On the other hand,the method of the low frequency waves of electro-acupuncture could increase the expression of VEGF and keep the expression level of VEGF mRNA.Now there present some argument about the meaning of VEGF in the acute stage of intracerebral hemorrhage.This study may leading to a suggestion that high frequency waves of electro-acupuncture should be the first choice for treating ICH.And the increase of NGF & VEGF in brain tissue paid a significant role in the healing process of ICH,either treated or nontreated.Increase of NGF & VEGF also play a major role in promoting convalenscence from ICH attack with electronic acupuncture treatment,especially using high frequency.
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