【摘要】：目的：本研究建立7日齡SD大鼠缺氧缺血性腦損傷（hypoxic-ischemic braindamage，HIBD）模型，建模后將血管內(nèi)皮生長因子（vascular endothelial growthfactor，VEGF）分兩個(gè)不同時(shí)間段經(jīng)鼻給予新生大鼠HIBD模型。通過觀察新生大鼠運(yùn)動(dòng)行為學(xué)改變、改良神經(jīng)功能缺失評分變化、腦組織形態(tài)學(xué)改變、腦組織病理改變以及腦細(xì)胞凋亡的變化，探討VEGF在新生大鼠HIBD后發(fā)揮神經(jīng)保護(hù)作用的最佳用藥時(shí)間及安全有效的方法，為臨床安全有效地應(yīng)用外源性VEGF治療新生兒缺氧缺血性腦病提供依據(jù)。 方法：(1)7日齡SD大鼠64只，隨機(jī)分為8組，每組8只：A組為假手術(shù)組，行頸部切口并分離組織后皮膚縫合不做HIBD處理，行假手術(shù)后當(dāng)天處死；B H組均參照改良Rice法制備HIBD模型，B組HIBD模型建模后當(dāng)天處死；C組建模后第6d處死；D組建模后第9d處死；E H組建模后將10mg/L的VEGF分兩個(gè)時(shí)間段經(jīng)鼻給予新生大鼠，其中E組、F組建模后第13d給藥，每天1次，連續(xù)給藥3d，E組建模后第6d處死；F組建模后第9d處死；G組、H組在46d給藥，每天1次，連續(xù)給藥3d，G組在建模后第6d處死；H組在建模后第9d處死。(2)各組新生大鼠處死前觀察新生大鼠運(yùn)動(dòng)行為學(xué)改變、改良神經(jīng)功能缺失評分變化，處死后觀察腦組織病理改變、腦組織大體形態(tài)學(xué)改變、TUNEL法檢測腦細(xì)胞凋亡的變化。 結(jié)果：(1)新生大鼠運(yùn)動(dòng)行為學(xué)改變A組新生大鼠運(yùn)動(dòng)、感覺、反射和平衡能力正常。B H組HIBD模型建模后進(jìn)行連續(xù)觀察2h：所有新生大鼠相繼出現(xiàn)間斷性的抖動(dòng)、皮膚青紫、軀體顫抖、尾部抽搐、翻身困難、尾部及臀部翹起并向左旋轉(zhuǎn)、走路不穩(wěn)等明顯異常的運(yùn)動(dòng)行為學(xué)變化。HIBD后的新生大鼠右側(cè)眼睛正常而左側(cè)眼睛張開遲緩，眼裂小，上瞼下垂。建模后第6d：C組、D組新生大鼠可以行走，，但行動(dòng)遲緩，不能翻身，平衡能力差易向左側(cè)傾倒；E組、F組可以行走，但不能順利翻身，平衡能力較差向左側(cè)偏斜；G組、H組可以行走，部分新生大鼠能較順利翻身，平衡能力較差向左側(cè)偏斜。建模后第9d：D組行走比較穩(wěn)，大部分能翻身，但平衡能力較假手術(shù)組有明顯差異；F組行走穩(wěn)，可順利翻身，平衡能力較HIBD組好；H組9天后行走穩(wěn)，可順利翻身，平衡能力較好。(2)新生大鼠改良神經(jīng)功能缺失評分A組新生大鼠評分0，無神經(jīng)功能缺失；B組14.63±1.41，嚴(yán)重神經(jīng)功能損傷。建模后第6d：C組9.63±1.51，中度損傷；E組6.75±1.39，中度損傷；G組4.00±0.76，輕度損傷。建模后第9d：D組7.63±1.92，中度損傷；F組5.25±1.49，輕度損傷；H組2.75±0.89，輕度損傷。建模后4~6d給予VEGF治療的G、H組評分分別低于1~3d給藥的E、F組，給藥治療的E~H組均低于未給藥的C、D組，且p0.05，差異有統(tǒng)計(jì)學(xué)意義。(3)腦組織大體形態(tài)學(xué)變化A組新生大鼠腦組織大體外觀正常，兩側(cè)半球?qū)ΨQ，無水腫、液化及萎縮；B H組HIBD模型建模后各時(shí)間點(diǎn)處死的新生大鼠腦組織左側(cè)大腦出現(xiàn)水腫、蒼白、液化等不同程度的異常改變。建模后第6d：C組新生大鼠腦組織左側(cè)大腦大片壞死液化區(qū)，經(jīng)VEGF治療的E組、G組腦組織大體病變明顯輕于未治療各組，表現(xiàn)為中度的水腫，無大片液化壞死區(qū)。建模后第9d：D組新生大鼠腦組織左側(cè)大腦萎縮明顯，經(jīng)VEGF治療的F組、H組腦組織大體表現(xiàn)為輕中度的水腫。(4)腦組織病理改變（HE染色） A組新生大鼠腦組織結(jié)構(gòu)層次清楚，細(xì)胞輪廓清晰，形態(tài)正常，細(xì)胞核居中。B~H組在建模后左側(cè)大腦皮質(zhì)區(qū)、海馬區(qū)、丘腦出現(xiàn)神經(jīng)細(xì)胞大片水腫、液化、壞死區(qū)。建模后第6d：C組新生大鼠腦組織切片左側(cè)大腦大片壞死液化區(qū)，細(xì)胞數(shù)目明顯減少；E組可見細(xì)胞壞死及水腫，但沒有成大片壞死區(qū)域，細(xì)胞數(shù)目有所減少；G組可見明顯水腫細(xì)胞，細(xì)胞數(shù)目減少，可見膠質(zhì)細(xì)胞增生。建模后第9d：D組新生大鼠腦組織切片細(xì)胞數(shù)目減少更加明顯，有膠質(zhì)細(xì)胞增生；F細(xì)胞數(shù)目有所減少，細(xì)胞水腫減輕，膠質(zhì)細(xì)胞增生；H組可見細(xì)胞數(shù)目減少，細(xì)胞間隙增寬，膠質(zhì)細(xì)胞明顯增生。(5) TUNEL法檢測腦細(xì)胞凋亡A組切片染色后偶可見核棕染的凋亡細(xì)胞，凋亡細(xì)胞數(shù)3.50±0.93個(gè)。B組左側(cè)缺血側(cè)大腦切片可見大量核棕染的凋亡細(xì)胞，凋亡細(xì)胞數(shù)58.13±5.74個(gè)。建模后第6d：C組66.38±8.18個(gè)；E組為53.12±7.92個(gè)；G組為41.38±6.78個(gè)。建模后第9d：D組50.50±5.68個(gè)；F組為35.63±6.05個(gè)，H組為29.25±3.49個(gè)，凋亡細(xì)胞數(shù)明顯低于未給藥治療的C、D組。建模后4~6d給予VEGF治療的G、H組凋亡細(xì)胞數(shù)分別低于1~3d給藥的E、F組，給藥治療的E~H組均低于未給藥的C、D組，且p0.05，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論：(1)經(jīng)鼻將VEGF給予新生大鼠HIBD模型后可以有效改善大鼠神經(jīng)行為功能，減輕腦水腫，改善神經(jīng)細(xì)胞病理變化，減少神經(jīng)細(xì)胞凋亡。提示VEGF可通過這種無創(chuàng)方法用于新生兒HIE的治療。(2)本實(shí)驗(yàn)G組、H組效果優(yōu)于E組、F組即在新生大鼠HIBD建模3d后應(yīng)用效果優(yōu)于起始應(yīng)用。提示VEGF用于新生兒HIE的治療可在腦水腫高峰過后應(yīng)用。
[Abstract]:AIM: To establish a 7-day-old model of hypoxic-ischemic brain damage (HIBD) in SD rats. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) was injected nasally into the model of HIBD in neonatal rats in two different periods after the model was established. To explore the best time and safe and effective method for the use of vascular endothelial growth factor (VEGF) to exert neuroprotective effect after HIBD in neonatal rats, and to provide evidence for the safe and effective use of exogenous VEGF in the treatment of neonatal hypoxic-ischemic encephalopathy.
Methods: (1) Sixty-four seven-day-old SD rats were randomly divided into 8 groups, each group consisting of 8 rats: group A was sham-operated, the neck incision was made and the skin was sutured without HIBD treatment, and the rats were executed on the day after sham operation; group B was made HIBD model according to the modified Rice method, and group B was executed on the day after modeling; group C was executed on the 6th day after modeling; group D was executed on the day after modeling; The rats in group E were given 10 mg/L of vascular endothelial growth factor by nasal administration at the 9th day after the establishment of the model. The rats in group E were given 10 mg/L of vascular endothelial growth factor by nasal administration at the 13th day after the establishment of the model, once a day for 3 days, and the rats in group E were sacrificed at the 6th day after the establishment of the model. (2) The neonatal rats in each group were sacrificed on the 9th day to observe the changes of motor behavior, modified neurological deficit score, pathological changes of brain tissue, gross morphological changes of brain tissue, and apoptosis of brain cells by TUNEL.
Results: (1) Neonatal rats had normal motor, sensory, reflex and balance abilities. The HIBD model of group B H was established and observed continuously for 2 hours. All the neonatal rats had intermittent shaking, blue skin, body trembling, tail twitching, difficulty in turning over, tail and buttock warping up and turning to the left, and no walking. After HIBD, the right eye of neonatal rats was normal, but the left eye was slow to open, the cleft was small, and the upper eyelid was drooping. Group G, group H can walk, part of the newborn rats can roll over smoothly, balance ability is poor to the left deviation. 9 days after modeling: group D walks more stable, most can roll over, but the balance ability is significantly different from the sham operation group; group F walks steadily, can roll over smoothly, balance ability is better than the HIBD group 9 days later; (2) Neonatal rats in group A scored 0, without neurological deficit; group B scored 14.63 + 1.41, with severe neurological impairment. Sixth day after modeling: group C 9.63 + 1.51, moderate injury; group E 6.75 + 1.39, moderate injury; group G 4.00 + 0.76, mild injury. Group D was 7.63 [1.92], moderate injury; Group F was 5.25 [1.49], mild injury; Group H was 2.75 [0.89], mild injury. After modeling, the scores of group G and group H were lower than those of group E and group F, which were given VEGF for 4-6 days, respectively. The scores of group E~H were lower than those of group C and group D, and the differences were statistically significant. (3) Gross morphological changes of brain tissue in group A were statistically significant. The brain tissue was normal in general appearance, bilateral hemisphere symmetry, no edema, liquefaction and atrophy; the left side of the brain tissue of neonatal rats in group B H was edema, paleness, liquefaction and other abnormal changes in varying degrees after the HIBD model was established. In group E, the gross lesion of brain tissue in group G was milder than that in untreated group, showing moderate edema without large areas of liquefied necrosis. In group B~H, there were large areas of edema, liquefaction and necrosis of neurons in the left cerebral cortex, hippocampus and thalamus. Necrosis and edema were observed, but there was no large area of necrosis, and the number of cells was decreased in group G. The number of edematous cells and glial cells were decreased in group G. The number of glial cells in brain slices of neonatal rats in group 9:D was decreased more obviously than that in group G. The number of F cells was decreased and the number of glial cells was edema. The number of apoptotic cells in group A was detected by TUNEL, and the number of apoptotic cells in group B was 3.50 (+ 0.93). A large number of apoptotic cells and apoptotic cells were found in the left ischemic side of the brain. On the 6th day after modeling, there were 66.38+8.18 cells in group C, 53.12+7.92 cells in group E, 41.38+6.78 cells in group G, 50.50+5.68 cells in group D, 35.63+6.05 cells in group F, 29.25+3.49 cells in group H, and the number of apoptotic cells in group H was significantly lower than that in group C and D. The number of apoptotic cells in group G treated with VEGF was lower than that in group 1 on the 4th to 6th day after modeling, respectively. The levels of E, F and E~H were lower than those of C and D, and the difference was statistically significant (p0.05).
Conclusion: (1) After intranasal administration of VEGF to HIBD model of neonatal rats, it can effectively improve neurobehavioral function, reduce brain edema, improve pathological changes of nerve cells and reduce apoptosis of nerve cells. The results showed that the application of VEGF in the treatment of neonatal HIE could be used after the peak of cerebral edema.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R722.1

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