Observation Of Early Seizure And Hippocampus Change In Rats With Traumatic Brain Injury

Objective: To make model of early seizures after fluid percussion injury in brain of rats ,observe their behavior changes and abnormal EEG under different cracking down; combined with hippocampal glial fibrillary acidic protein(GFAP) expression and hippocampal mossy fibers, and to explore the early post-traumatic mechanism of epilepsy seizures. Methods:1 .Making the animal model of early epileptic seizures in rats after fluid-percussion brain injurySD rats (50, either sex, weighing 300g-350g) were randomly divided into:①control group 10, do not give any treatment.②control group (sham group) 10, experimental rats given the same anesthesia and surgery, without any strike.③operation group 30, according to different crackdown (0.1kPa, 0.2kPa, 0.3kPa) were divided into three sub-groups, each sub-group of 10 rats. After intraperitoneal anesthesia by 10% chloral hydrate, the skin of the rats'longitudinal roof craniotomy were cut . In the left parietal bone (from the sagittal suture, coronal suture of 5mm) a bone window of 5mm in diameter was removed by the dental burr , maintaining dural integrity. The hydraulic device to combat anti-pipe was fixed on bone window, zinc polycarboxylate cement fixed sealing connections. The rats were fixed on animal stereotactic instrument. When conscious partly, we adjusted the striking force by 0.1kPa, 0.2kPa, 0.3kPa, to cause mild, moderate and severe brain damage to rats. Suture wounds and maked the rats eat food and drink water freely after injury. The rats could not eat water had been given nasal feeding .All rats were sacrificed on the 7th days.2. Behavioral observation and EEG monitoring after injury.Behavioral observation after injury: observed the time ,degree of the epileptic seizures in rats under different intensity of the attack. The severity of behavioral seizures was scored according to a slightly modified Racine's scale (Racine,1972):score 0= electrographtly seizure without any detectable motor manifestation;score 1=mouth and face clonus ,head nodding; score 2=clonic jerks of one forelimb; score 3=bilateral forelimb clonus ;score 4=forelimb clonus and rearing;and score 0-2 were considered partial, whereas siezures scored 3-5 were considered secondarily generalized . In our study ,animals with two or more seizures during the entire period of monitoring were considered considered epileptic.At the same time EEG monitoring was executed after injury for 3 days. The laying of measuring electrode was fixed on the injured side of the brain: the shallow electrode on superficial layer of Cortex was located behind the bregma 1.0mm, middle next to the 2.0mm and hippocampal electrode: 3.8mm behind the bregma, 2.0mm next to the middle, depth 3.4mm. EEG monitoring of rats up to 3 days after injury and epileptic seizures was determined when sharp wave, spike wave, sharp (spike) slow complex wave appeared.3 Regional changes in rat hippocampus3.1 Pathological changes in the hippocampus region5 rats in each sub-group randomly were killed on the 7th day after injury. After Left ventricle - ascending aorta cannulation, the rats were injected with normal saline 150 ml irrigation., 4% paraformaldehyde 300ml for 2 hours Slow down after, then fixed in 4% paraformaldehyde 24 hours or more . The brain was taken out and done continuous coronal slices to the hippocampus as the center both sides . A set of slices for conventional dehydration, transparent, Baptist wax, hematoxylin-eosin staining (HE), another for immunohis- tochemical staining to detect the hippocampus GFAP.3.2 Detection of GFAP in hippocampal area15 rats of 3 sub-group were killed after the deep anesthesia. After left ventricle - ascending aorta cannulation, the animals were were injected with 0.4% sodium sulfide 50ml, 4℃, 4% paraformaldehyde 300ml slow down after the first infusion over 1h. The brains were taken out and fixed in 4% paraformaldehyde for 30min-1h, next at 30% sucrose overnight soak in the sink to the bottom, taken out, in the cryostat been made consecutive coronal slices, thickness 30μm.According to Danscher's improved staining method, the slices were added into the preparared Timm Staining in the dark room, adding 17% silver nitrate 0.5ml, beening Incubated in water bath box, for 1.5 hours at room temperature. Then ,after Water washing, dehydration, transparent, sealing, and the slices were observed under microscope, images captured, doing Statistical analysis. The control group were do Timm staining in the same wayResults:1 Behavioral performanceSeizure frequency and intensity increased with the increase of efforts to crack down. There were 7 rats with Severe injury (0.3kPa) caused early generalized epileptic seizures , showed paroxysmal clonic limb or body - convulsive seizures, In severe cases, convulsions or generalized tonic - clonic seizures. 3 rats expressed as partial seizures, behavior showed rigid-like nodding , chewing, facial tics and other behaviors attack. The rats with mild combat injury (0.1kPa) had not any epileptic behavior, only showing Camponotus, beard trembling.2 EEG performanceControl rats'EEG expressed asαandβwaves waves waves as the main basis of rhythm. Three subgroups of rats after injury, both had short-term inhibition of low-voltage, lost.within 1.5 hour. Epileptic-like brain waves in severe blow could be detected at about 90±45min after injury ; moderate injury group within 120±25min after injury .However, Mild combat group of rats had not been observed epileptiform EEG waveforms. Epileptiform EEG waveforms showed high amplitude sharp peaks and short sharp burst of tall waves, spike wave, sharp (spike) slow complex wave3 Changes in the hippocampus3.1 Pathological changes in the hippocampus7 days after injury in the light microscope, hippocampal tissue HE staining showed: hippocampal CA3 area in control group rats whorse number of nerve cells, morphology and distribution were normal, Fluid percussion injury group:7 days after injury, hippocampal injury in the central area of each group can be seen loose organization, nerve cells, deformation, deep dyeing, the cells surrounding vacuolation, nuclear pyknosis seen, glial cell hypertrophy. Severe combat injuries performanced significantly.3.2 Determination of the hippocampus GFAPBlank control group and the sham-operated group hippocampal GFAP integral optical density measurement, statistical analysis no significant difference (P>0.05), GFAP-positive cells in small, sporadic expression of obvious cell body, dendrites and small, dyed light. Three sub-groups of experimental group,and the control group, were significantly different (P <0.05): astrocytes GFAP immunohistochemical staining increased as well as cell body and branch number,cell processes became more thickening.3.3 Hippocampal mossy fiber Timm staining experimentsAfter Timm staining in rat hippocampus, the mossy fiber sprouting of the slices were observed . Control rats hippocampus dentated gyrus and CA3 layers of clear zones, and early seizure group (severe combat trauma group) in hippocampal dentated gyrus of uneven layers of shading and coloring were well aware, showing in the dentated gyrus of molecular layer of granule cells in the upper zone and a continuous, concentrated dye ribbon Timm's staining granules.Conclusions:1. Fluid percussion injury in rat model of early seizures,. simulated the human brain injury early clinical features of epilepsy betterly and produced a similar disease of human physiological changes.So it had good stability and reproducibility2 Early epileptic seizures related to the combat strengtht of brain injury are closely . With the degree of injury increasing, the frequency of seizures significantly increased.3.The proliferation of mossy fiber sprouting in the hippocampus region increased markedly in early brain injury, and closely related with the sensitivity of epileptic seizure.