鼻咽腔降溫對(duì)大鼠全腦缺血再灌注損傷的保護(hù)作用

1、鼻咽腔降溫對(duì)大鼠全腦缺血再灌注損傷的保護(hù)作用    鼻咽腔降溫對(duì)大鼠全腦缺血再灌注損傷的保護(hù)作用 The Protection of Nasopharyngeal Cooling Against Globle Cerebral Ischemia-reperfusion in Rats【中文摘要】 目的:采用Pulsinelli四血管阻斷法建立大鼠全腦缺血-再灌注損傷模型,比較全身降溫和鼻咽腔降溫對(duì)降低海馬溫度速度的影響,通過觀察缺血及再灌注期間海馬CA1區(qū)細(xì)胞外液谷氨酸(Glutamic acid,Glu)濃度(Glue)的變化,以及相關(guān)凋亡基因的表達(dá),

2、探討鼻咽腔降溫法對(duì)大鼠全腦缺血再灌注損傷的保護(hù)作用。方法:1實(shí)驗(yàn)分組及動(dòng)物模型制備健康雄性Wistar大鼠18只,體重250±20g(河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供)。隨機(jī)分為3組(每組6只),常溫組(A組),全身降溫組(B組)和鼻咽腔降溫組(C組)。大鼠腹腔注射10%水合氯醛(0.3ml/100g)麻醉后將動(dòng)物俯臥位固定于實(shí)驗(yàn)臺(tái)上,于頸后正中第一、二頸椎處切開皮膚,剝離顯露雙側(cè)翼小孔,燒灼兩側(cè)的椎動(dòng)脈使其閉塞, 24h后再用同法麻醉動(dòng)物,氣管插管吸氧自主呼吸。股靜脈切開置管以1ml/h的速度輸入乳酸鈉林格氏液。作頸前正中切開皮膚,分離出雙側(cè)頸總動(dòng)脈,用4-0絲線穿過頸總動(dòng)脈備用,部分

3、縫合頸部傷口。動(dòng)物立體固定后,頭皮刺入電極,持續(xù)監(jiān)測(cè)腦電圖(EEG),四肢皮下刺入電極,持續(xù)監(jiān)測(cè)心電圖(ECG)。切開頭皮,用微型顱鉆分別在左右海馬CA1區(qū)(前囟bregma3.6mm和中線左、右旁開3mm)打開一直徑約2mm的圓孔,將微透析探針刺入左側(cè)海馬CA1區(qū)2mm,用乳酸鈉林格氏液以2.5l/min的速度持續(xù)灌注,穩(wěn)定60min待腦電圖波形穩(wěn)定后收集腦組織透析液,間隔時(shí)間為10min,直至再灌注后2h。右側(cè)海馬CA1區(qū)置入微型溫度探頭監(jiān)測(cè)海馬溫度。室溫保持在25。之后按照實(shí)驗(yàn)分組給予不同的處理。常溫組(A組):整個(gè)實(shí)驗(yàn)過程中保持大鼠直腸和海馬溫度均為37±0.5,用動(dòng)脈夾夾閉

4、雙側(cè)頸總動(dòng)脈20min。全身降溫組(B組):應(yīng)用電扇、冰袋和酒精等使海馬溫度降至33后再夾閉雙側(cè)頸總動(dòng)脈20min。鼻咽腔降溫組(C組):將氣管插管的大鼠咽喉下部置一棉團(tuán)和吸引設(shè)備避免誤吸。兩側(cè)鼻腔插入自制硅膠管,深度5mm,持續(xù)注入5生理鹽水,速度為100ml·min-1·kg-1 ,直至海馬溫度降至靶溫度33,夾閉雙側(cè)頸總動(dòng)脈20min。B、C兩組低溫維持1h后應(yīng)用變溫毯和白熾燈復(fù)溫至37.0±0.5。以上各組均同時(shí)監(jiān)測(cè)直腸溫度并維持在37±0.5。2標(biāo)本的采集及檢測(cè)方法2.1微透析液的收集及測(cè)定微透析液收集后保存在-70。利用高效毛細(xì)管電泳法測(cè)定出

5、各種濃度的谷氨酸標(biāo)準(zhǔn)品的峰面積,并繪制標(biāo)準(zhǔn)曲線、求得回歸方程。利用該方法測(cè)出每個(gè)樣品中谷氨酸的峰面積,根據(jù)回歸方程求得谷氨酸的濃度。2.2 Bcl-2、Bax表達(dá)的檢測(cè)大鼠腦缺血再灌注8h后深麻醉沿肋緣剪開胸廓暴露升主動(dòng)脈和心臟,提起心尖剪開左心室,插灌注針至升主動(dòng)脈并固定,接灌流固定液(50ml肝素鹽水及50ml4%多聚甲醛)同時(shí)剪開右心耳并推注固定液將全身固定。固定完畢后斷頭,剝開顱骨取出全腦,取海馬所在的節(jié)段區(qū)放入4%多聚甲醛溶液中固定。Bcl-2、Bax免疫組化測(cè)定采用SP法,按照試劑盒的要求步驟依次進(jìn)行。結(jié)果:1各組間大鼠的體重比較無統(tǒng)計(jì)學(xué)差異。2降溫速度比較B組海馬溫度從37降到3

6、3所需時(shí)間為31±2.7min,而C組為6.5±0.7min,前者是后者的4.8倍。3缺血前各組間海馬CA1區(qū)Glue比較無統(tǒng)計(jì)學(xué)差異(P>0.05)。4缺血中各組間海馬CA1區(qū)Glue比較缺血10min和20min時(shí),與A組比較,B組、C組Glue明顯降低(P<0.05);B組、C組兩組之間比較無統(tǒng)計(jì)學(xué)差異(P>0.05)。5再灌注后10min、20min、30min時(shí), B組、C組Glue比A組明顯降低(P<0.05);B組、C組之間比較無統(tǒng)計(jì)學(xué)差異(P>0.05)。其余時(shí)間點(diǎn)兩兩比較無統(tǒng)計(jì)學(xué)差異。6各組內(nèi)海馬CA1區(qū)Glue恢復(fù)至缺血前水

7、平的時(shí)間: A組、B組、C組海馬CA1區(qū)Glue恢復(fù)至缺血前水平所需時(shí)間分別為40min、20min、20min。7各組海馬CA1區(qū)Bax表達(dá)的比較與A組比較,B組與C組的Bax免疫陽性細(xì)胞數(shù)及灰度值顯著減少減小(p<0.05),B組與C組比較無統(tǒng)計(jì)學(xué)差異。8各組海馬CA1區(qū)Bcl-2表達(dá)的比較與A組比較,B組與C組的Bcl-2免疫陽性細(xì)胞數(shù)及灰度值顯著增多增大(p<0.05),B組與C組比較無統(tǒng)計(jì)學(xué)差異。結(jié)論:1鼻咽腔降溫與全身降溫同樣具有腦保護(hù)作用,均能抑制缺血再灌注時(shí)腦細(xì)胞外谷氨酸的升高,減弱Bax的表達(dá),增強(qiáng)Bcl-2的表達(dá)。2鼻咽腔降溫法降低海馬溫度的速度明顯快于全身降法

8、,前者約為后者的5倍。【英文摘要】 Objective:By using the method of modified Pulsinellis four-vessel occlusion to establish the model of global cerebral ischemia-reperfusion injury in rats, to observe the speed of the whole body cooling and nasopharyngeal cooling, to detect the concentration of extracellular glutama

9、te (Glue)in hippocampal CA1 field and the expression of Bcl-2 and Bax during global cerebral ischemia-reperfusion injury.Method:Eighteen male Wistar rats weighing 200±50g were randomly divided into 3 groups(n=6): Normal temperature group/group A; Whole body cooling group/group B; Nasopharyngeal

10、 cooling group/group C.Anaesthesia was induced by an intraperitoneal injection of 10% chloral hydrate (0.3ml/100g). The trachea was cannulated via endotracheal intubation. Electroencephalogram (EEG) and electrocardiogram (ECG) were continuously monitored. A cannula was placed through the right femor

11、al vein into the vena cava for local anesthetic and sodium lactated Ringers solution infusion. The global cerebral ischemic model was established by Pulsinellis four-vessel occlusion: The bilateral vertebral arteries were electrocauterized permanent, after 24h, the bilateral common carotid arteries

12、were occlused for 20 min. After placement in a stereotaxic apparatus, A microdialysis probe was implanted in the left hippocampal CA1 region (2 mm to the left and 3.6 mm posterior to the bregma and 2 mm below the cortical surface). The microdialysis probe was perfused with Ringers solution at a rate

13、 of 2.5L/min using an infusion pump, and the dialysates were collected every 10 mins for 2h after onset of reperfusion. The right hippocampal temperature was measured by placing a small thermocouple in the CA1 region.Normal temperature group/group A: both the rectal temperature and the hippocampal t

14、emperature were maintained at 37±0.5by using a heated water blanket and an infrared lamp during the experiment. The whole body cooling group/group B: the hippocampal temperature was reduced to 33and maintained it for one hour by using a fan and a ice bag. Nasopharyngeal cooling group/group C: g

15、el silica pipes were inserted into both nasal cavities, and cold physiologic saline (5) was infused at a rate of 100 mL·min-1·kg-1 until the hippocampal temperature decreased to 33. A cotton ball was placed in the lower pharynx and a suction device was placed for drainage, so as not to cau

16、se aspiration of saline. The rectal temperature was maintained at 37.0±0.5using a heated water blanket and an infrared lamp. Arteria carotis communes were clamped for 20min when the temperature was to 33in group B and group C, and the objective temperature was maintained for 60 mins, and then r

17、ewarming was initiated with the use of heated water blanket and an infrared lamp.After reperfusion for 8h, all rats were anesthetized again, fixed with 4% PFA perfused via ascending aorta. Then, the ratsbrain were carefully sampled and stored in 4. Whats more, specimans of the brain were used in imm

18、unohistochemical test to observe the expression of Bcl-2 and Bax. The dialysates were stored under -70and be used to determine the concentration of glutamate by high performance capillary electrophoresis(HPCE).Result:1. There were no statistical differences of the ratsweight in the three groups.2. T

19、he comparison of the cooling speed of hippocampal temperature from 37to 33It took 31±2.7min in group B and 6.5±0.7min in group C for reducing to 33from 37of hippocampus, this onset latency in hippocampal temperature reaching 33was about 5 times longer than that in the nasopharyngeal coolin

20、g group.3. There were no statistical differences of the Glue before ischemia in the three groups.4. The comparison of Glue in hippocampus CA1 field during ischemiaAt 10min and 20min of ischemia, the Glue was significantly lower in both group B and group C compared with group A (P<0.05). There wer

21、e no statistical differences between group B and group C(P>0.05). 5. At 10min, 20min, 30min after ischemia, the Glue in hippocampus CA1 field was significantly lower in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P&

22、gt;0.05).6. The time of Glue in hippocampal CA1 field returning to the level of control The time of Glue in hippocampal CA1 field returning to the level of control was 40 min、20min、20min in group A、B and C respectively.7. The expression of Bax in hippocampal CA1 field The expression of Bax level in

23、hippocampal CA1 field was significantly lower in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05).8. The expression of Bcl-2 level in hippocampal CA1 field The expression of Bcl-2 level in hippocampal CA1 field w

24、as significantly higher in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05).Conclusion:1. Nasopharyngeal cooling protects brain from global cerebral ischemia-reperfusion same as whole body cooling does. Both of cooling methods reduce Glue, weaken the expression of Bax and strengthen the expression of Bcl-2 in hippocamp