The Fundamental And Imaging Study On Cell Apoptosis Related With Hypoxic-ischemic Brain Damage In Neonatal Rabbits

PREFACENeonatal hypoxic-ischemic brain damage (HIBD) is due to neonatal asphyxia andcan cause whole brain damage with the results of disturbance of blood supply and gasexchange, which is a serious disease for the health of newborn and has a highermorbidity and mortality, and it is one of the reason that bringing mentally disabled andcerebral palsy. The pathological reason for HIBD is very complicated, more and morepoof indicated that neuron apoptosis play a very important role in HIBD, and it has aunique function. Occurrence of neuron apoptosis has a close relation with outcome ofpatients.It is necessary to understand the dynamic morphologic changes of neurons indifferent area of the brain in the process of neonatal HBID, so we can learn the serialprocess of neuron apoptosis in the level of patho-antomy and ultrastructure, whichbenefits to understand the happen and development in neonatal HIBD. In molecularlevel, the process of apoptosis are mediated by multiple gene and cytokine, and thefamily of Bcl-2 are the main focus of study, and the relative ratio of Bax and Bcl-2plays an important role in cell apoptosis. The developments of molecular biology,radioactive chemistry and multiple kinds of diagnostic equipments establish thefundaments of the technique of molecular cell apoptosis imaging and make themstrong. One of the typical examples is annexin V imaging which is belonging toradionuclide apoptosis imaging. The diagnostic model of mainly reflecting thefunctional molecular information of tissues and organs are being gained the focus byclinic. By establishing the neonatal rabbits model of hypoxic-ischemic brain damage, The purposes of our experiment are to observe dynamic changes by the way of thelight microscope, electron microscope, and to observe the regular patterns ofapoptostic cells and its mediating protein by TUNEL dying and immnohistochemistry,and further more, and perform radionuclide apoptostic imaging in vivo with theradiopharmaceuticals of ^99mTc-HYNIC-AnnexinV, to discuss the morphologic changeof apoptostic neuron and the regular expression of apoptostic mediating protein, andfinally discuss the possibilities of apoptostic imaging in vivo. All data from theexperiment could hopefully provide the experimental proof for the study on neonatalhypoxic-ischemic brain damage.MATERIALS1. Experimental animals: neonatal Japanese big ear rabbits after 8 to 10 days ofnewborn, weighted from 100 to 120g, were provided from the Dept. of experimentalanimal in the 2nd affiliated hospital of China Medical University.2. Experimental agents: 5% lidocaine hydrochloride;20% ethyl carbamate; aether;pure oxgen and nitrogen for medical purpose; 4% paraform;0.1M PBS; TTC;Bcl-2 andBax immunohistochemistry; SABC kit; ^99mTcO₄^-, Annenxin V, Sn²⁺-Tricine Kit andHYNIC-Annexin V Kit.3. Experimental equipments: hypoxic cabinet; paraffin section;electronic balance;Olympus optical microscope; JEM-1200EX electron microscope; CY-100 digitalmeasurement meter of oxgen; GE Infinia ^VC Hawkeye double detector SPECT/CT,Philip 3.0 T MR.METHODSNeonatal HIBD models were made according Rice method, new-born rabbits wereanesthetized by aether inspiration, and their right common carotid arteries were ligated.Experimental animals were put into the hypoxic champer (8%FIO2) for two and anhalf hours. Control groups included five ligated animal which didn't undergo hypoxicmanagement.Paper one: neonatal hypoxic-ischemic brain damage in rabbits were made according the above method, experimental animals were divided into six groupsaccording the time after hypoxic-ischemic management.(1) Control group ofimitational operation (n=7);(2) HIBD 1h group (n=5);(3) HIBD 4h group (n=5);(4)HIBD 12h group (n=5);(5) HIBD 24h group (n=5);(6) HIBD 48h group (n=7).The behavior changes of the animals were recorded during the process of makinganimal model. The appearances of brain tissue were also recorded. Every two animalbrains in control group and HIBD 48h group were selected to be stained with TTC.Three animal brains in each group were stained with HE in order to observe themorphological changes of right hemisphere. Two animal brains in each group wereused to make slices of electronic microscope to observe micromorphological changes.Paper Two: Prepare the animal model in rabbits; all animals were divided into sixgroups:(1) Control group(n=5);(2) HIBD 1h group(n=5);(3) HIBD 4h group(n=5);(4)HIBD 12h group(n=5);(5) HIBD 24h group(n=5);(6) HIBD 48h group(n=5). Paraffinimbedding slices were made, which could be used to be stained with TUNEL, Bcl-2and Bax immunohistochemisty. The dynamic distributions of apoptositc cell, proteinexpressions of Bcl-2 and Bax were observed.Paper Three: Prepare the animal model in rabbits; all animals were divided intosix groups:(1) Control group(n=5);(2) HIBD 1h group(n=5);(3) HIBD 4hgroup(n=5);(4) HIBD 12h group(n=5);(5) HIBD 24h group(n=5);(6) HIBD 48hgroup(n=5).Prepare the imaging agents of ^99mTc-HYNIC-Annexin V. All animals ineach group were performed planar brain imaging in vivo with SPECT after injection ofthe agent. The animals in HIBD 48 group were also performed dynamic imaging at5min, 30min, 60min and 120min after injection. For the control group, all animalswere undergone whole body imaging about 1h after injection, and calculated the countratio of target to whole body, including brain, heart, lungs, liver and kidneys. Twoanimal brains in HIBD 48h group were selected to be imaged in vitro. By drawing ROIin the left and right hemisphere, and the average counts in pixel were calculated.The animals in control sacrificed after radionuclide imaging, and the radioactivity in different tissues and organs were recorded in the body of animals. Every twoanimals in control and HIBD 4h groups were selected to be imaged with MRI after thecompletion of radionuclide imaging, so T1W, T2W, DWI and ADC were obtained.RESULTSPaper One: Animals in experimental groups appeared dyphoria 5min afterhypoxia, and breaths became deep and fast 20min after hypoxia, and got drowsiness40-90min after hypoxia. The phenomena of drowsiness became more manifest 90 to2.5h after hypoxia, some animals appeared transit spasm. The behavior of animals incontrol groups appeared normal. The appearances in the brain and cerebellum werenormal in control, HIBD 1h and HIBD 4h groups. Right hemisphere appeared pale andmild swelling compared with left hemisphere, and hyporrhea could be noted undercerebral pia mater. Right hemisphere became obviously swelling and pale, the sign ofhemorrhage under cerebral pia mater is manifest, and the other side of the brain isnormal. The colors of whole brains were stained into rose pink with TTC. The resultsof HE examination was as following: No abnormal changes were found in the animalsof control group; The typical apoptostic cells in the lesion of cortex, hippocamp andcerebellum were respectively found in HIBD 12h,HIBD 24h and HIBD 1h groups andwere largely found in HIBD 48h group for the different part of the right brain.Morphological changes obtained by electronic microscope is an following: Apoptosticcells were found in cerebellum in HIBD 1h, and micro morphological changesappeared obviously in cerebral cortex in HIBD 12h,and also in CA1 area of hippocampin HIBD 24h.Paper Two: The dynamic changes of apoptosis in experimental groups could beobserved by TUNEL method. The results are as following: No obvious abnormalchanges were found in the control group; The positive cells labeled by TUNEL incortex, hipocamp and cerebellum could be largely found, respectively in HIBD12h, HIBD 24h and HIBD 4h groups, and the numbers of apoptostic cells in differentpart of the right brain the maximum in HIBD 48h group. The dynamic expression of Bcl-2 protein in every group was observed withimmunohistochemistry. The results were as following: No abnormal cells were foundin the control group; The positive cells in the cortex, hippocamp and cerebellum whichexpressed Bcl-2 protein were largely found, and respectively in HIBD 4h group(88.4±1.9), HIBD 4h (73.6±3.2) and HIBD 1h groups (93.6±5.08), but with the timegoing on, the number of positive cells in different part of the fight brain decreased.The results of Bax protein were as following: No abnormal cells were found in thecontrol group, and the positive cells which could be largely found in cortex,hippocamp and cerebellum were respectively in HIBD 12h (65.6±14.3), HIBD 24h(76.4±12.0) and HIBD 4h groups (53±6.7).With the time going on, the number of thepositive cells reached the maximum.Paper There: There were no radioactive accumulations in both hemispheres on theimages of ^99mTc-HYNIC-AnnexinV imaging in vivo in control group. There wereslightly abnormal accumulations of imaging agent in fight hemisphere in HIBD 4hgroup, but no concentration of the agent in the area on the counterpart. Theconcentration of imaging agent in the right hemisphere became obvious in HIBD 12h,HIBD 24h and HIBD 48h groups, and also obvious concentration of imaging agent inthe right nuke brain in HIBD 48h, no uptake of the agent was seen in the lefthemisphere. The level of concentration was more obvious than that in the counterpartat 5min after injection of the imaging agent in HIBD 48h group, so the distribution ofthe imaging agent didn't change much with the time going on after injection.For the control group, the radioactive counts in the liver were the highest, and thearea of the brain had the lowest counts among the target organs.The results of MR imaging was as following: There were no abnormal signals inthe brains in control and HIBD 4h groups, and DWI and ADC map were also normal inboth groups.CONCLUSIONSPaper One: 1. Neonatal hypoxic-ischemic brain damage model can be made by performinghypoxia after one side of the common artery of neonatal rabbit is ligated.2. The damage models in this study are belonging to mild level of damage for thebrain, no obvious area of infarction can been in HIBD 48h group.3. The results of injured side of the brain observed by pathological lightmicroscope are found that the liagted side of cerebellum is more sensitive to hypoxiaand ischemia, the next is the gray matter and the area of hippocampus, and alsoapoptostic cells can be observed in above areas, and the level of injury becomesserious with time going on.4. The ligated side of cerebellum is found to have microscopic morphologicalchanges of apoptostic cells at early phase by dynamic observing with electronmicroscope; the next is grey matter and hippocampus. The morphology of cell nucleusand cell organ is clearly seen by electron microscope.Paper Two:1. The apopostic cells can be found in the cerebellum at very early phase after theinsults in the animal HIBD model of neonatal rabbit, and the second is in the cortex,the third is in the area of hippocamp. The number of apopostic cells increases with thetime going on.2. The expressions of Bcl-2 in cortex, hippocamp and cerebellum become obviousin the early time of the insults in the HIBD model of neonatal rabbit, but decreasedwith the time going on and the expressions of Bax increased largely, especiallybecome obvious at early phase in cerebellum。Paper Three:1. Abnormal accumulation of ^99mTc-HYNIC-Annexin V can be seen in the ligatedside of the brain in HIBD 4h group, which indicates that neuron apoptosis appears atearly phase in neonatal HIBD, but no abnormal signals can be found on the images ofMRI and DWI in HIBD 48h group.2. Cell apoptosis can be dynamically, semi-quantitively recorded by radionuclide imaging with ^99mTc-HYNIC-Annexin V.3. There are no distinct differences for the distribution of imaging agent in thebrain between 5min imaging and 2h imaging after injection, which indicate thatapopostic imaging with ^99mTc-HYNIC-Annexin V can be performed at early phaseafter injection.