The Study On Pathologic Alters And NGF Therapeutic Effects In The Region Of Perihematoma By Fundamental Medcine And MR Image After Experimental Intracerebral Hemorrhage

Part â…  : The Setting Up of Intracerebral Hemorrhage Model on Dog in The Frontal Lobes and Evaluation of Image FeaturesObjective: To establish a stable, dominated and reduplicated model of intracerebral heamorrhage in the frontal lobes of dog,which was suitable for the study of medical imaging(SCT/MR) especially.Methods: 20 healthy dogs were chosen to set up ICH models which were made by injecting self-blood into the frontal lobes of the dogs with stereoscopic frame.The region of injection was in the frontal lobes.The volume of blood was 3ml.The time of injection was 5min.The pressure of injection was less than 1 00mmHg.The pattern of injection was at one time.The needle was paid more attention to retain 10min.After ICH ,the examinations were carried out at 3h as follows: (1) CT scanning: The region, volume and shape of hematoma were observed after ICH. (2) MRI scanning: The changes of signal intension in the region of hematoma and perihematoma were observed on T₁WI, T₂WI, T₂*WI, FLAIR, DWI and PWI. (3) Purdy neurological deficit score: The damaged degree of neurological function was assessed objectively after ICH.The ICH models were evaluated generally by CT, MR and Purdy score.Results: (1) CT findings: The shadow of high density was shown in the frontal lobes on dogs after ICH models. The average volume of hemetoma was 2.78±0.42ml.The shapes of hemetoma seemed to be ellipse or round. (2) MRI results: The characteristic change of singal intension were occurred clearly on T1WI, T2WI, FLAIR, T2*WI, DWI and PWI. (3) Purdy score: Purdy neurological deficit score was 8.12±1.37, 5.64±1.53 and 3.32± 1.46 at 1 d, 3d and 1 0d after ICH.It indicated that the degree of damagewas middle. The most impotant symptom of ICH model was that the contralateral limb paralyzed ,especialy posterior limb.Conclusion: That this method can be adopted to set up a stable, reliable model of ICH.It laid a foundation to further study the pathologic changes of ICH by fundamental medcine and advanced MR technology.Part II :The Dynamic Characteristic Study of Experiment Intracerebral Hemorrhage on A Set of MRI Sequences in Contrast With the Changes of PathologyObjective: The signal change of ICH was complicated on MR image,It was influenced by many factors,such as the state of Hb,membranous intactness of RBC, intensity parameter and sequences of MR.So MR had been markedly restricted in the diagnosis and research of ICH for a long time.With the technological development of fMRI, EPI and innovation of instrument, MR was applied in more and more fields . The aim of this article was to study the dynamic characteristics of intracerebral hemorrhage on a set of MRI Sequences through contrasting MR expression to pathology results.Methods: 24 healthy dogs were chosen to be ICH models by injecting self-blood into the frontal lobes. At different points (0.5h, 3h, 6h, 12h, 24h, 48h, 72h, 7d and 15d, the following examinations were carried out: (1) MR scanning: The changes of signal intension in the region of hematoma and perihematoma were observed by Ti WI, T2WI, T2*WI, FLAIR, DWI and PWI. (2) HE staining: The paraffin sectioning and the routine HE staining were done to observe the degeneration, necrosis, edema of the neurons and gliacytes as well as the change of Hb and RBC. (3) The observation of transmission electron microscopy(TEM): The ultrastructure of the neurons and gliacytes were observed under TEM.Results: (1) In the regions of hematoma: T2*WI, DWI and ADC images showed that the centers of hematomas were markedly hypointense at hyperacute stage(0.5³h).At 3⁴8h, T2WI, FLAIR revealed a characteristic thin hypointense rims surrounding the hematoma. At 72h, both T1WI andT2WI were hyperintense in hematoma, which were characteristic features of intracerebral hemorrhage on routine MRI. ( 2 ) In the region of perihematoma:On T2WI and FLAIR, the hyperintense surrounding the hematoma standed for vasogenic edema after 3h. The periods of edema peak were at 48⁷2h. The signal changes of perihematoma on DWI and ADC were more complicated.On DWI, ADC values increased in the focal hyperintensive regions,which reflected vasogenic edema.,whereas ADC values decreased in the focal hypointensive regions,which reflected ischmia and cytotoxic edema. In the regions of perihematoma, rCBF was in the state of hypo-perfusion on PWI at 0.5⁶h. As the time went on, rCBF in the perihematoma region recovered and approached to the contralateral region after 24h.Conclusion: A set of MR imaging sequences had important applied values in diagnosis of early hematoma,assessment of pathological injury and estimation of perfusion after intracerebral hemorrhage.PartHI: The Study of Apoptosis and Bcl-2/Bax Gene in the Region of Perihematoma after Experimental Intracerebral HemorrhageObjective: Many researches indicated that there were two forms of cell death: neuronecrosis and apoptosis.Gegerally speaking,acute and serious ischemia resulted in neuronecrosis,whereas chronic and light ischemia resulted in apoptosis.lt was a hot topic whether rCBF drop in perihematoma would cause apoptosic damage or not. The aim of this article was to investigate the space distribution,dynamic change and correlated relationship of apoptosis and expressions of Bcl-2/Bax gene in the region of perihematoma after ICH.Methods: 27 healthy dogs were divided into Control group(n=3) and ICH group(n=24) at random. ICH models were made by injecting self-blood into the frontal lobes.After 4% formaldehydum polymerisatum perfusion and fixation through heart, the brain blocks of ICH was cut into paraffin coronal sections.The following examinations were carried out at 3h> 6h> 121k 24h> 48h> 72h> 7d and 15d after ICH. (1)Determination of TUNEL positive cells:The labeling of the terminal deoxynucleotidyl transferase-mediated dUTP nick end(TUNEL) method was used. TUNEL positive cells were stained brown-yellow colour in the nucleus. (2) Detection of Bcl-2/Bax Protein: immunohistochemistry SP method was adopted. Bcl-2/Bax immune positive cells were stained brown-yellow colour in the cytoplasm. ( 3 ) The examination of cell apoptosis rate: Flow cytometer method(FCM) was applied to observe apoptosis peak of DNA map and calculate apoptosis ratio .Results: (1 )Space distribution of apoptosis cells and genes: TUNEL >< Bcl-2/Bax positive cells mainly existed in perihematoma,but rarely existed in isolateral basal ganglia and contralateral mirror region.(2 )Dynamine changes of apoptosis cells and gene: TUNEL positive cells presented at 12h, reached the peak at 72h, and then decreased gradually at 7d. Bcl-2 cells presented at 6h, reached the peak at 12h and decreased at 72h. Bax cells presented at 12h, reached the peak at 72h, and decreased at 7d. (3) The relationship between TUNEL positive cells and expression of Bcl-2/Bax gene: The expression of Bcl-2 protein was negatively correlated with the number of TUNEL positive cells(r=-0.821,P<0.05),and the expression of Bax protein was positively correlated with the number of TUNEL positive cells (r=0.793,P<0.05). Apoptosis ratio was positively correlated with the number of TUNEL positive cells (r=0. 936,P<0.01).Conclusions: Apoptosis existed in perihematomal regions after ICH.The time window of apoptosis was 7d. Bcl-2/Bax Gene played an important role in the period of apoptosis.This study would provide basis for treatment ICH with anti-apoptosis therapy .PartIV: The Study of Relationship Between the Changes of Blood Flow Perfusion by MRI and NF-kB/p65 Expression in the Region of Perihematoma After Experimental Intracerebral Hemorrhage.Objective: One of the major reasons was that the inflammatory and immune reaction participated in the pathological courses of cerebral ischemia and reperfusion injury. NF-kB/p65 had close relationship withinflammatory reaction,which could induce expression of many inflammatory genes,for example IL-1, IL-8, IL-6, TNF-1, VCAM-1 and ICAM-1. Therefore NF-kB/p65 involved in the cerebral ischemia and reperflision injury. The aim of this article was to study the relatinonship between the rCBF and NF-kB/p65 expression in the region of perihematoma after ICH.Methods: 27 healthy dogs were divided into Control group (n=3) and ICH group(n=24) at random. ICH models were made by injecting self-blood into the frontal lobes. At different points (3h,6h,12h,24h,48h,72h,7d and 15d),the following examinations were carried out: (1) Perfusion weighted imaging(PWI)scanning: The changes of rCBF in the region of perihematoma were observed on PWI. (2) CT Perfusion (CTP)scanning: The changes of rCBF in the region of perihematoma were observed on CTP. (3) Detection of NF-kB/p65: After 4% formaldehydum polymerisatum perfusion and fixation through heart,the brain blocks of ICH were cut into paraffin coronal sections.Immunohistochemistry SP method was used to detect expressions of NF-kB/p65. Immune positive cells were stained brown-yellow colour in the cytoplasm and/or nucleus.The marker of activated NF-kB/p65 was stained particulars transformed from the cytoplasm to the nucleus.Results: (1) MR results: In the region of perihematoma, PWI showed hypo-perfusion at less than 12h.,reperfiision or hyper-perfusion at 12²4h,and slightly hypo-perfution at more than 48h. (2) SCT findings: In the region of perihematoma, CTP showed hypo-perfusion at less than 12h.,reperfusion at 12²4h,.and near to contralateral region after 48h. (3) Expression of NF-kB/p65: NF-kB/p65 positive cells presented at 6h, reached the peak at 12⁴8h ,decreased at 72h⁷d.nearly disappeared at 15d.. NF-kB/p65 mainly existed in the region of perihematoma.In the period of peak,NF-kB/p65 were markedly expressed in quantities of neurons,gliacytes and vascular endothelial cells.The activated NF-kB/p65 was clearly shown at the same time.Conclusions:Cerebral ischemia and reperfusion in the region of perihematoma could induce expression of NF-kB/p65, which was involved in the inflammatory and immune reaction and resulted in cerebral ischemia andreperfusion ingury.The result would benefit for neurologists to pay more attention to ischemia and reperfusion ingury when they treated ischemia damage in periheamtoma by means of improving microcirculation.PartVrThe Effect of NGF Gene Therapy on Neural Functional Recovery in the Region of Perihematoma after Experimental Intracerebral Heamorrhageon and Evaluation of NGF by MR ImageObjective: There are two strategies to treat cerebral vascular disease: the endogenous NSC activition and the exogenous NSC transplantation.The suitable microenvironment must be provided for endogenous NSC.The exteral experiment on animals showed that NGF had two biological functions: axon growth and neuron regeneration.The aim of this article was to observe the influence of NGF on neural functional recovery in the region of perihematoma after ICH,and effectiveness of NGF was evaluated by advanced MR image technology.Methods: 21 healthy dogs were divided into three groups at random. NGF group (n=9): NGF(2000AU) were given in the region of perihematoma at 0.5h after ICH; ICH group (n=6): only blood was injected .Control group (n=3): only needle was injected. BrdU were infused by vein at the first three days. The following examinations were carried out at 3d,10d,28d after ICH: (1 )MR scanning: Volume of hematoma and edema was observed by T2WI. ADC was caculated by DWI and rCBF was acquired by PWI. (2) Purdy neural functional deficit score: The leisoned degree of neurological function was assessed by Purdy score. (3) NGF immunohistochemistry staining: Immunohistochemistry SP method was adopted to detect whether or not exogenous NGF could be effectively expressed in the region of perihematoma. NGF immune positive cells were stained brown-yellow colour in the cytoplasm. (4) Detection of confocal laser scanning microscopy (LSCM) scanning: The brain blocks of ICH were cut into frozen sections by freezing microtome. Immunofluorescence labled BrdU,BrdU-NSE and BrdU-GFAP. LSCM detected the number and distribution of BrdU,BrdU-NSE and...