| Radiation injuries of brain (RIB) is a serious complication after neural and cervicaltumor radiation remedy. Radiation remedy might be the only effective treatment for thesetumors because of the restriction of the position and the non obvious effect of surgical. Inrecent years, with the increasing incidence of such tumors and extending of living time inthese patients, sideeffect of radiation injuries got increasing gradually. Currently, hormoneand dehydrator were used in treating RIB, but the treatment effect is poor. So, it seemsnecessary to look for an effective drug. In previous studies, we had built up a rat model ofbrain radiation injuries and find out that study and memory capability of the rat decreasedafter RIB. In this experiment, we treat rats of RIB with dl-3n-butylphthalide, hoping to getproofs of the theoretical basis for treatment of RIB through the observation of the variationof rat’s behaviouristics and histopathology.The capability of learns and remember is the advanced brain neurophysiologicalactivities. Learning is the acquisition of experiences and skills. Memory is the preservingand reproducing of experiences and skills. Learn and remember are two differentneuro-biological processes. Only if they are both interrelated and cooperated with eachother could make the brain accomplish serious of complicated activities. Memory includestransient memory, short-term memory, long-term memory and permanent memory. Manystudies show that the structure of the hippocampus plays an important role in learning,formatting and maintaining in memory, especially short-term memory and permanentmemory. Researchers find that the CA3region is closely related to learn and memoryabilities of space identity. As the knowledge of anatomy, long-term memory is closelyrelated to form and function of the hippocampal synapses, the establishment of the synapticcontact and synaptic plasticity changes. Capabilities of study and memory have a greatrelationship with cholinergic system, monoaminergic system and amino acid system. Therefore, we chose the hippocampus region of rat to do the histopathologic study in thisexperiment.Butylphthalide (dl-3n-butylphthalide, NBP), developed by the Chinese Academy ofMedical Sciences and the National Drug Research Institute of Peking Union MedicalUniversity, is the first drug of national grade have independent intellectual property rights.NBP was first separated, purificated and racemic synthesised from natural plant celery seedand it’s yellow oily liquid, used in the treatment of ischemic stroke. NBP play itspharmacodynamics by reducing cerebral edema, improving cerebral energy metabolism,improving micro-circulation ischemic brain areas, inhibiting neuronal apoptosis,anti-thrombosis, anti-platelet aggregation, inhibiting of glutamate release, decreasing theintracellular calcium concentration, inhibiting free radical and improving antioxidantenzyme activities. Current studies about Butylphthalide are focused on cerebral ischemiafield, lacking of relevant studies on RIB.MMPs is a neutral protease rely on calcium and zinc ions, its main function is todegrade extracellular matrix, it is involved in the pathological process of secondary braininjury. Research has shown that, concentration of MMP-9was increased in the blood ofpatients who had a stroke. It is closely related to the size of the infarct. Has tip role inshort-term prognosis. According to this, we conjectured the concentration of MMP-9wasgoing to increase after RIB. In this experiment, we treat rats of RIB withdl-3n-butylphthalide, hoping to get proofs of the theoretical basis for treatment of RIBthrough the observation of the variation of rat’s behaviouristics and histopathology.ObjectiveTo investigate the study and memory abilities and hippocampal MMP-9expressioneffect of rats with RIB treatment with NBP, discuss the therapeutic mechanism of NBP toRIB.Methods120healthy male Wister rats aged of12-16weeks, weighing about250-300g, weredivided randomly into sham radiation group (n=40), model group (n=40), high-dosebutylphthalide group (n=40)(a dose of6mg/kg) and low dose butylphthalide group (n=40)(a dose of2mg/kg). RIB model group,high-dose butylphthalide group and low dosebutylphthalide group rats receive radiation. Large dose butylphthalide group and low dosebutylphthalide group is given the appropriate dose of butylphthalide intraperitionealinjection from the date of radiation, the RIB model group was given the same amount ofsaline and sham radiation group was not administered. After administration for1d,3d,5d, 7d,14d and30d, at each time point three rats in each group were randomly selected, hadtheir brains removed after cardiac perfusion by normal saline and then paraformaldehyde.Then, the brains were coronal cut after the optic chiasm at1mm and4mm to remove thecerebellum and brain stem, taking the middle of the hippocampus, then fixation,dehydration and embedded into frozen sections. HE staining were used to observehippocampal pathology. Immunohistochemical staining were used to observe theexpression of MMP-9. Capability of study and memory of the remaining rats in each groupwas tested by platform avoidance test and Morris’ water maze test. Quantitative data wereexpressed as mean±standard deviation. Statistical comparisons were carried out with aone-way analysis of variance (SPSS11.0). LSD-t test was used for each pairwisecomparison between groups. A value of P<0.05was considered statistically significant.Results1. The general situation of experimental animalsRats of sham radiation group keep good spiritual state, acting agilely, drinking andeating normally, reacting quickly. The RIB model group keeps low spirit, had a negativeaction, response slowly, drunk and eat less. After the model established successfully, therats’ spirit, activity, response and eating of high-dose butylphthalide group and low dosebutylphthalide group were gradually lifting2-3days and back to normal after one week.Rats in high-dose butylphthalide group recovered faster than low dose butylphthalidegroup.2. Platform avoidance test and Morris’ water maze testThe rats were tested by platform avoidance test and Morris water maze test after RIB.Performances of Day31were considered as study performances. Wile Day32was amemory performances. In platform test, we use escape latency period of rats and thenumber of errors as the academic performance, while latency and error frequency as thememory performance. In Morris’ water maze test, we use escape latency period of rats anderror frequency as the academic performance, while the time of staying in the targetquadrant as the memory performance.2.1Platform test results showed that, in the learning capability test, compared to RIBmodel group, high-dose butylphthalide group and low dose butylphthalide group havesignificantly shorter escape latency respectively (85.23±12.71,56.18±11.36,57.96±11.48)and significantly less error frequency, respectively (4.78±0.79,2.08±0.41,2.09±0.45)(P<0.01). In the memory ability test, compared with RIB model group, high-dosebutylphthalide group and low dose butylphthalide group have significantly prolonged latency, respectively (72.84±10.26,152.27±11.70,153.19±11.58) and decreased errorfrequency, respectively (1.86±0.13,0.53±0.05,0.55±0.06)(P<0.01).Swimming speedincrease significantly, respectively (0.52±0.03,0.5±0.02,1.83±0.1)(P<0.01)2.2Morris water maze test results show that: in the learning capability test, comparedto RIB model group, high-dose butylphthalide group and low dose butylphthalide grouphave significantly shorter escape latency, respectively (80.71±8.27,43.25±5.29,44.74±5.82) and significantly less error frequency, respectively (37.53±7.69,14.76±5.01,15.68±5.32)(P<0.01). In the memory ability test, compared with RIB model group,high-dose butylphthalide group and low dose butylphthalide group have significantlylonger former platform quadrant time, respectively (18.67±5.39,32.35±4.27,31.19±4.78).Swimming speed increase significantly, respectively (10.09±3.67,17.36±2.11,18.18±2.09).3. HE staining in hippocampus of ratsSham radiation Group: Neurons hydroncus and becomes deformation and necrosis notobvious, nucleus stained deep and big, cells were arranged orderliness, the boundary wasclear. RIB model group: Neurons hydroncus and becomes deformation and necrosisobviously, nucleus stained pyknosis,even karyolysis. Butylphthalide group: edema,deformation and necrosis of neurons was obviously lightened than sham radiation group,neuronal cell has more plain structure, damaged cell number reduced obviously, comparedwith low dose butylphthalide, cellular edema of high-dose butylphthalide was milder.4. MMP-9immunohistochemical stainingThe rats were respectively narcotized at the1d,5d,15d,30d,60d and90d by10%chloral hydrate, their brains removed after cardiac perfusion by normal saline and thenparaformaldehyde. Then fixation, dehydration and embedded into frozen sections. SPimmunohistochemical staining method is used to observe expression of the MMP-9. Imageacquisition system and image analysis software was used to analyze the images. Comparedwith model group, high-dose butylphthalide group and low dose butylphthalide group hadtheir expression of MMP-2and MMP-9at all time points significantly reduced(P<0.05),the expressing degree of MMP-9in high-dose butylphthalide group was much higher thanthe low-dose group.Conclusion1. Butylphthalide can improve study and memory abilities of RIB rats in platformavoidance test and Morris water maze test.2. Expression of MMP-9was significantly increased in hippocampal neurons of RIB rats.3. Butylphthalide can significantly reduce the expression of MMP-2and MMP-9inhippocampal neurons.4. Butylphthalide reducing expression of MMP-2and MMP-9may be involving forNBP’s treatment of RIB. |
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