彭羅根，趙會(huì)民，張劍鋒

創(chuàng)傷性失血性休克淺低溫動(dòng)物模型的建立

彭羅根，趙會(huì)民，張劍鋒

目的 建立一種簡(jiǎn)單穩(wěn)定的創(chuàng)傷性失血性休克淺低溫動(dòng)物模型。 方法 新西蘭兔20只隨機(jī)分為模型組和對(duì)照組，每組10只。模型組以頸動(dòng)脈放血并股骨骨折法建立創(chuàng)傷性失血性休克(traumatic hemorrhagic shock，T/HS)模型，對(duì)照組實(shí)施假手術(shù)。兩組均經(jīng)歷麻醉，輔以冷液體及外加冰塊等誘導(dǎo)低溫過程，分別檢測(cè)休克前(t0)、休克30 min后(t1)、低溫2 h(t2)及復(fù)溫2 h(t3)的血常規(guī)，記錄體溫(temperature，T)、心率(heart rate，HR)、休克指數(shù)(shock index，SI)、平均動(dòng)脈壓(mean arterial pressure，MAP)、血紅蛋白(hemoglobin，HGB)及呼吸(respiration，R)等相關(guān)生理指標(biāo)，觀察兩組成活率。結(jié)果 兩組動(dòng)物創(chuàng)傷早期(4 h)的存活率100%。與對(duì)照組相比，模型組尿量明顯減少(t=3.3，P<0.05)。在t1時(shí)間點(diǎn)，模型組MAP(t=22.0，P<0.01)、HGB(t=4.0，P<0.01)水平明顯低于對(duì)照組；而R(t=2.9，P<0.01)、HR(t=2.1，P<0.05)及SI(t=4.0，P<0.01)水平則顯著高于對(duì)照組。在t2時(shí)間點(diǎn)，模型組MAP(t=4.5，P<0.01)、HGB(t=6.4，P<0.01)仍舊顯著低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。在t3時(shí)間點(diǎn)，模型組HGB(t=9.8，P<0.01)顯著低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。t2時(shí)間點(diǎn)與t1時(shí)間點(diǎn)相比：模型組MAP(t=20.0，P<0.01)顯著升高，對(duì)照組HGB(t=19.0，P<0.01)顯著升高，差異有統(tǒng)計(jì)學(xué)意義。對(duì)照組MAP、HR、R、SI水平在各時(shí)間點(diǎn)均無明顯變化(P>0.05)。結(jié)論 成功建立兔創(chuàng)傷性失血性休克淺低溫模型，為創(chuàng)傷性失血性休克早期淺低溫研究提供了動(dòng)物模型。

創(chuàng)傷性失血性休克；淺低溫；動(dòng)物模型

隨著社會(huì)進(jìn)步，人類面臨的災(zāi)害已從自然災(zāi)害(如地震、洪水、泥石流等)向人為災(zāi)難(如交通事故、恐怖襲擊等)發(fā)展。復(fù)雜外傷或合并創(chuàng)傷性失血性休克是事發(fā)現(xiàn)場(chǎng)早期最常見的死亡原因。而低體溫、酸中毒及凝血功能障礙被認(rèn)為是嚴(yán)重創(chuàng)傷致死的重要危險(xiǎn)因素，常規(guī)救治主張對(duì)T/HS患者進(jìn)行保暖復(fù)溫，然而低溫復(fù)蘇在卒中患者器官損傷抑制的益處顯而易見，近年來國內(nèi)外學(xué)者試圖探討低溫對(duì)創(chuàng)傷性休克患者早期復(fù)蘇的利弊[1-3]。因此，筆者試圖建立一種簡(jiǎn)單的T/HS淺低溫模型，為T/HS淺低溫研究提供實(shí)驗(yàn)動(dòng)物支持。

1 材料與方法

1.1 材料 健康成年新西蘭兔20只(動(dòng)物許可證號(hào)：scxk桂2009-0002)，體重(2.5±0.4)kg，SPF級(jí)，雌雄不限，按隨機(jī)數(shù)字表法分為模型組(10例)和對(duì)照組(10例)。

1.2 創(chuàng)傷性休克動(dòng)物模型制備及低溫復(fù)溫方法 實(shí)驗(yàn)前適應(yīng)性喂養(yǎng)1周，術(shù)前禁食12 h，禁水4 h。實(shí)驗(yàn)組操作方法參照Capnoe和Park等[2,4]的研究，略有改進(jìn)，具體是：(1)建立頸外靜脈通路作為麻醉用藥及補(bǔ)液通路；以20%烏拉坦(5 ml/kg)靜脈麻醉，切開右大腿皮膚，分離肌群，暴露并鉗斷股骨(以無菌紗塊保護(hù)創(chuàng)面且便于稱重記錄失血量)；分離左側(cè)頸動(dòng)脈并置入以枸櫞酸鈉抗凝的動(dòng)脈留置管，外接三通閥以供放血并記錄失血量及記錄心率、血壓，在15 min內(nèi)將MAP降至45 mmHg(1 mmHg=0.133 kPa)，放血速率2 ml/(kg·min)，本組平均失血量為(38±5.0)ml；低血壓穩(wěn)定持續(xù)30 min視為創(chuàng)傷性休克模型成功。(2)以20℃乳酸林格氏液和羥乙基淀粉40氯化鈉注射液(體積比2∶1)的液體補(bǔ)液維持MAP在目標(biāo)血壓45 mmHg。(3)輔以乙醇冰水擦浴降溫，中心體溫(膀胱)(34±0.5)℃維持2 h，以電熱毯、紅外線照射緩慢復(fù)溫2 h。對(duì)照組僅進(jìn)行假手術(shù)(皮膚切開，不放血不輸液，同樣經(jīng)歷低溫復(fù)溫過程)。

1.3 采集與指標(biāo)測(cè)定 記錄動(dòng)物存活率及總失血量(注射器采血和紗塊稱重法并用，為頸動(dòng)脈失血量和骨折創(chuàng)傷失血量之和)、尿量和補(bǔ)液量；分別于術(shù)前(t0)、術(shù)后30 min(t1)、降溫2 h(t2)及復(fù)溫2 h(t3)采集血樣本，優(yōu)利特全自動(dòng)血細(xì)胞分析儀(3010型)檢測(cè)血常規(guī)；泰盟(BL-420S)生物機(jī)能試驗(yàn)系統(tǒng)實(shí)時(shí)監(jiān)測(cè)MAP、收縮壓(systolic blood pressure，SBP)、R、HR等相關(guān)數(shù)據(jù)；計(jì)算SI(脈搏與收縮壓的比值)；自動(dòng)電子體溫計(jì)每30 s記錄膀胱溫度(T)。實(shí)驗(yàn)完畢常規(guī)處死尸檢。

2 結(jié) 果

2.1 模型組與對(duì)照組MAP、HGB、SI、HR、R在不同時(shí)間點(diǎn)的比較 實(shí)驗(yàn)前模型組與對(duì)照組比較，MAP、HGB、HR、R及SI差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)。在t1時(shí)間點(diǎn)，模型組MAP(t=22.0，P<0.01)、HGB(t=4.0，P<0.01)顯著低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義；模型組HR(t=2.1，P<0.05)、R(t=2.9，P<0.01)及SI(t=4.0，P<0.01)顯著高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。在t2時(shí)間點(diǎn)，模型組MAP(t=4.5，P<0.01)、HGB(t=6.4，P<0.01)仍舊顯著低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。在t3時(shí)間點(diǎn)，模型組HGB(t=9.8，P<0.01)顯著低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。t2時(shí)間點(diǎn)與t1時(shí)間點(diǎn)相比：模型組MAP(t=20.0，P<0.01)顯著升高，對(duì)照組HGB(t=19.0，P<0.01)顯著升高。對(duì)照組MAP、HR、R、SI水平在各時(shí)間點(diǎn)均無變化(P>0.05)。見表1。

時(shí)間MAP(mmHg)模型組對(duì)照組HGB(g/L)模型組對(duì)照組HR(次/min)模型組對(duì)照組R(次/min)模型組對(duì)照組SI(次/minmmHg)模型組對(duì)照組t095±392±6114±13125±10347±53327±5251±1047±103.1±0.43.0±0.6t145±6①88±597±11①117±12398±82①334±4663±13①46±104.5±0.9①3.1±0.5t275±8①②86±578±17①113±14②343±73325±4448±1042±93.5±1.03.2±0.6t384±688±474±23①117±9 362±66331±4851±1445±83.6±0.73.2±0.6

注：模型組為頸動(dòng)脈放血并股骨骨折建立創(chuàng)傷性失血性休克模型，對(duì)照組為假手術(shù)組。①與對(duì)照組同時(shí)間點(diǎn)比，P<0.05 ；②與同組t1比，P<0.05

2.2 模型組與對(duì)照組尿量及補(bǔ)液量比較 記錄創(chuàng)傷性休克動(dòng)物模型制備及低溫復(fù)溫過程兩組動(dòng)物的補(bǔ)液量及尿量得出，模型組與對(duì)照組4 h內(nèi)的補(bǔ)液量分別為(73±10)ml、(18±7)ml，兩組比較差異有統(tǒng)計(jì)學(xué)意義(t=14.0，P<0.01)；模型組與對(duì)照組4 h內(nèi)的尿量分別為(12±6)ml、(21±8)ml，兩組比較差異有統(tǒng)計(jì)學(xué)意義(t=3.3，P<0.01)。

3 討 論

目前，低溫療法已被用于心臟驟停、創(chuàng)傷、出血性休克等，其保護(hù)作用也得到廣泛認(rèn)可[4,5]。最近美國匹茲堡大學(xué)把低溫療法應(yīng)用到嚴(yán)重T/HS患者中，為后續(xù)搶救性治療贏得時(shí)間，對(duì)T/HS低溫治療價(jià)值有了新認(rèn)識(shí)。本實(shí)驗(yàn)采用開放性長(zhǎng)骨骨折及控制性失血，輔以冷液體和物理降溫成功復(fù)制了T/HS淺低溫模型。模型組處于重度休克(SI=4.5)，經(jīng)歷淺低溫后，休克程度(SI=3.5)明顯減輕，且平均動(dòng)脈壓較休克時(shí)顯著上升，可能與淺低溫對(duì)T/HS的保護(hù)作用有關(guān)。本方法構(gòu)建的模型具有相對(duì)穩(wěn)定性、實(shí)用性和合理性，為臨床研究T/HS早期(4 h)一線、院前期基礎(chǔ)救治研究及低溫抗休克等提供了多方面的參考依據(jù)，也為T/HS的救護(hù)贏得了時(shí)間。

近年來，國外研究表明低溫可提高失血性休克動(dòng)物早期存活率，延長(zhǎng)救治“黃金時(shí)間”，對(duì)于失血性休克后全身炎性反應(yīng)也有一定抑制作用[6]。若溫度繼續(xù)低于34 ℃，死亡率隨溫度的降低而明顯增加[7]。另外，快速誘導(dǎo)低溫對(duì)心臟驟停、創(chuàng)傷、出血性休克等的保護(hù)作用已得到廣泛認(rèn)可[4,5]。傳統(tǒng)方法使用冰毯、醇浴等物理降溫很難迅速降低至目標(biāo)體溫，反而在降溫過程中增加機(jī)體耗氧等。另外有學(xué)者用“冬眠合劑”加冰毯也能成功快速誘導(dǎo)低溫，但不良反應(yīng)也不可避免，如引起血壓下降、抑制體溫及呼吸中樞等[8]。本實(shí)驗(yàn)以快速輸注低溫液體(乳酸林格氏液和羥乙基淀粉40氯化鈉注射液體積比2∶1)總量少于2倍失血量，降溫至目標(biāo)溫度，輔以乙醇擦浴大血管冰塊降溫維持中心體溫(膀胱)目標(biāo)溫度。本實(shí)驗(yàn)?zāi)繕?biāo)溫度為34 ℃，輸注液體溫度為20 ℃，復(fù)溫以電熱毯、紅外線照射緩慢復(fù)溫，能較好地控制溫度。文獻(xiàn)[9，10]報(bào)道，限制性液體復(fù)蘇能夠降低重度失血性休克早期的死亡率，小劑量補(bǔ)液較快速大量補(bǔ)液更有利于抑制酸中毒進(jìn)展及高鉀血癥。

相比于傳統(tǒng)的創(chuàng)傷性休克模型常用方法，如①拍擊肢體法、②轉(zhuǎn)筒法、③鈍器沖擊腹部臟器致創(chuàng)傷(肝、脾、腎等)等[11]，本實(shí)驗(yàn)采用開放性長(zhǎng)骨骨折及控制性失血制作T/HS淺低溫模型，較好地量化了創(chuàng)傷和休克程度[12-15]。前期預(yù)實(shí)驗(yàn)MAP按Park等[16]方法降至40 mmHg左右，再予以創(chuàng)傷和低溫后，在不輸液時(shí)動(dòng)物死亡率非常高(3/4)，給予輸液后勉強(qiáng)能維持在40 mmHg，但輸液量較多(多于失血量的4倍)，這很大程度影響了內(nèi)環(huán)境。若MAP在45 mmHg左右，同樣的操作后不需干預(yù)也能維持平穩(wěn)血壓，又能有典型的休克體征。本實(shí)驗(yàn)主要觀察創(chuàng)傷性失血性休克早期(4 h)淺低溫的病理生理變化及救治，更長(zhǎng)時(shí)間的研究和后續(xù)治療有待于進(jìn)一步研究。

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(2014-12-02收稿 2014-01-07修回)

(責(zé)任編輯 張亞麗)

Establishment of hypothermia animal model of traumatic haemorrhagic shock

PENGLuogen,ZHAOHuimin,andZHANGJianfeng.

DepartmentofEmergencyMedicine,ThefirstAffiliatedHospital,GuangxiMedicalUniversity,GuangxiZhuangAutonomousRegion,Nanning530021,China

ZHAOHuimin,E-maill:hmzhao2006@163.com

Objective To establish animal model of hypothermia of traumatic hemorrhagic shock. Methods 20 rabbits were randomly divided into control group and model group, 10 cases each. Carotid artery bloodletting and femoral fracture were applied to model group to establish traumatic hemorrhagic shock(T/HS) model, while sham operation was used in control group. Both of them experienced anesthesia and low temperature process induced by cold liquid and ice. Then the routine blood, temperature (T), heart rate (HR), shock index (SI), mean arterial pressure (MAP), hemoglobin (HGB), respiration(R)and related physiological indexes were recorded at different time including before shock(t0), 30 minutes after shock(t1), 2 hours after hypothermia(t2) and 2 hours after resuscitation(t3), and survival rates of two groups were also observed. Results Survival rates in both groups were 100%. Compared with control group, urine volume of model group was significantly decreased(t=3.3,P<0.05). 30 minutes after shock, in model group, the level of MAP(t=22.0，P<0.01) and HGB(t=4.0，P<0.01) were significantly lower, while R(t=2.9，P<0.01), HR(t=2.1，P<0.05), and SI(t=4.0，P<0.01) were significantly higher than that of control group. 2 hours after hypothermia, the level of MAP(t=4.5，P<0.01)and HGB(t=6.4，P<0.01) of model group were still lower than those of control group. 2 hours after resuscitation, the level of HGB(t=9.8，P<0.01) of model group were lower than those of control group. The level of MAP(t=20.0，P<0.01) in model group and HGB(t=19.0，P<0.01) in control group 2 hours after hypothermia were increased significantly than that 30 minutes after shock. The level of MAP, HR, R and SI of control group didn’t change significantly in all of the time points. Conclusions The rabbit T/HS model was established by this way successfully, and provide an animal model of hypothermia with T/HS for further researching.

traumatic hemorrhagic shock; hypothermia; animal model

10.13919/j.issn.2095-6274.2015.01.004

廣西青年基金項(xiàng)目(NO.桂科青0832040)

彭羅根，碩士，住院醫(yī)師，E-mail:pengluogen2012@163.com

530021 南寧，廣西壯族自治區(qū)廣西醫(yī)科大學(xué)第一附屬醫(yī)院急診科

趙會(huì)民，E-mail：hmzhao2006@163.com

R361