Application of Gastric Antrum Ultrasound in Assessing the Aspiration Risk during Pediatric Emergency Surgery
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摘要:
目的 探讨即时胃窦部超声检查在小儿急诊手术误吸风险评估中的应用。 方法 选择湖北省荆州市中心医院2020年12月至2021年12月的急诊小儿手术120例,年龄1~12岁,ASA分级I~III级,拟行外科手术治疗,首先根据是否符合美国麻醉医师协会2017年发布的禁饮禁食指南,对患儿进行临床胃排空评估,将患儿分为临床空腹(CE组)和临床饱胃(CF组),然后利用迈瑞M7超声,对小儿胃窦部进行胃内容物的定性和定量评估,将患儿分为超声空腹(UE组)和超声饱胃(UF组),记录临床和超声2种评估方法的一致性。检测右侧卧位下小儿胃管吸引前和吸引后胃窦部横截面积(cross-sectional area,CSA),记录胃管吸引前后CSA的变化,术前小儿的进食种类和进食量,进食与超声检查之间的时间间隔。 结果 最终纳入108例小儿进行了统计分析,临床和超声判断胃排空结果一致性较差,超声和临床均判断为空腹92例(85.2%),超声判断空腹,临床判断饱胃4例(3.7%),超声判断饱胃,临床判断空腹9例(8.3%),超声和临床均判断为饱胃3例(2.8%),(kappa=0.255,P=0.006)。患儿胃管吸引前的进食量与胃窦部CSA存在较好相关性(r =0.840,P<0.05)。吸引前UF组患儿胃窦部CSA明显大于UE组(P<0.05),吸引后UF组患儿胃窦部CSA与UE组无明显差异(P=0.324)。UF组患儿进食以配方奶和高脂肪食物居多,且胃管吸引量,进食量多于UE组(P<0.05),UF组患儿超声检查间隔时间短于UE组(P<0.05)。2组患儿术后呕吐的发生率分别为8.3%和5.2%,差异无统计学意义(P=0.674)。 结论 急诊手术患儿术前行即时胃窦部超声检查结合患儿的进食种类和进食量,可以有效帮助麻醉医师在紧急情况下做出是否饱胃的判断,以便及时调整麻醉策略,选择合适的气道管理方式。 Abstract:Objective To investigate the application of point-of-care gastric antrum ultrasonography in the risk assessment of aspiration in children undergoing emergency surgery. Methods A total of 120 cases of pediatric emergency surgery in Jingzhou Central Hospital of Hubei Province from December 2020 to December 2021, aged 1-12 years, American Society of Anesthesiologists physical status I-III, scheduled for surgical treatment, were selected. First, according to whether it met the fasting guidelines issued by the American Society of Anesthesiologists in 2017, the clinical gastric emptying of the children were evaluated. The children were divided into clinical fasting group(CE group) and clinical full group(CF group), and then Mindray M7 ultrasound was used to qualitatively and quantitatively evaluate the gastric contents in the gastric antrum. The children were divided into ultrasound empty group(UE group) and ultrasound full group(UF group), and the consistency of clinical and ultrasound evaluation methods was recorded. The cross-sectional area(CSA) of the gastric antrum was measured before and after gastric tube suction in children in the right lateral position. The changes of CSA before and after gastric tube suction, the type and amount of food eaten before surgery, and the time interval between eating and ultrasound examination were recorded. Results A total of 108 children were finally included for statistical analysis. The consistency between clinical and ultrasound examination was poor, 92 cases(85.2%) were judged as fasting by both ultrasound and clinical. 4 cases(3.7%) were judged as fasting by ultrasound but full stomach by clinical examination. 9 cases(8.3%) were judged as full stomach by ultrasound but fasting by clinical examination. 3 cases(2.8%) were judged as full stomach by both ultrasound and clinical, (kappa=0.255, P=0.006). There was a well correlation between food intake and CSA in gastric antrum before gastric tube aspiration(r =0.840, P<0.05). Before aspiration, the gastric antrum CSA in the UF group was significantly larger than the UE group(P<0.05); After suction, there was no significant difference in gastric antrum CSA between the two groups(P=0.324). The UF group consumed more formula milk and high-fat food, the amount of food suctioned by the gastric tube and the food intake were more than UE group(P<0.05), The ultrasound interval time in the UF group was shorter than the UE group(P<0.05). The incidence of vomiting in the two groups was 8.3% and 5.2% respectively, and there was no significant difference between the two groups(P=0.674). Conclusion Preoperative gastric antral ultrasonography combined with the type and amount of food intake in children undergoing emergency surgery can effectively help anesthesiologists to make an appropriate judgment of whether the stomach is full under emergency circumstances, so as to adjust the anesthesia strategy in time and choose the appropriate airway management. -
Key words:
- Gastric ultrasonography /
- Pediatric emergency surgery /
- Aspiration /
- Fasting
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动脉粥样硬化(atherosclerosis,AS)是一种慢性炎症主导的疾病,其首要驱动因素是巨噬细胞表型失调[1]。激活的巨噬细胞主要被极化成两种表型,经典激活的M1和间接激活的M2巨噬细胞,启动并促进炎症是M1巨噬细胞的主要作用,而M2巨噬细胞的主要作用为抑制炎症[2]。在炎症的早期启动中,由于各种炎症因子的诱导,M1巨噬细胞被大量激活,能够有效清除病原体;随着炎症进展,激活了大量的M2巨噬细胞,炎症反应被抑制[2-3]。Schmitz等[4-5]发现,M2巨噬细胞主要存在于稳定性动脉粥样硬化斑块中,而M1巨噬细胞主要存在于不稳定性动脉粥样硬化斑块中,提示斑块的不稳定可能是由于M1和M2之间失衡。
MicroRNA在单核细胞向巨噬细胞分化过程中起着重要的调控作用。最近研究显示,随着单核细胞分化为M1巨噬细胞,miR-125a-3p和miR-125a-5在此过程中也表达升高。M1巨噬细胞比例的升高,M1/M2巨噬细胞的分化失衡,导致不稳定斑块的形成[6-7]。研究发现MMP-9、VEGF在不稳定斑块中呈高表达[8-9],并且MMP导致巨噬细胞积累,VEGF将巨噬细胞诱导为M1巨噬细胞[10-11],表明MMP-9、VEGF在不稳定斑块中起重要作用。笔者的研究[12]显示miR-125a-3p在动脉粥样硬化斑块的不稳定及破裂中也起着重要的作用。本研究拟进一步在动物体内实验,观察miR-125a-3p抑制剂对动脉粥样硬化斑块形成、M1/M2巨噬细胞以及斑块组织中MMP-9和VEGF表达的影响,以探讨其在动脉粥样硬化斑块的稳定性中的作用,为不稳定斑块的防治提供新的药物靶点。
1. 材料与方法
1.1 实验动物
15只成年雄性健康日本大耳兔,体重2~2.5 kg,由昆明医科大学实验动物学部提供。本研究经医院伦理委员会审批,实验动物的处理符合实验动物相关要求。
1.2 主要试剂和仪器
牛血清白蛋白(Solarbio)、0.5%胆固醇,l0%蛋黄,5%猪油、miR-125a-3p干扰慢病毒载体(广州复能基因有限公司)、生理盐水、甲醛溶液、70%乙醇、4%中性福尔马林、油红O染液、0.01M的PBST、2%BSA、CD11c抗体(Affinity)、CD206抗体(Affinity)、VEGF抗体(Santa Cruz Biotechmology)、MMP-9抗体(Solarbio)。Axio Lab A1显微镜(蔡司)、Axio Observer A1荧光显微镜(蔡司)。
1.3 实验方法
1.3.1 兔动脉粥样硬化模型的建立及miR-125a-3p抑制剂干预
15只成年雄性健康日本大耳兔,给予普通饲料和水适应性喂养7 d,再将其随机分为3组,对照组、动脉粥样硬化模型组(AS模型组)和miR-125a-3p抑制剂干预组(miR-125a-3p抑制剂组)。对照组给以普通饲料喂养,动脉粥样硬化模型组和miR-125a-3p干预组一次性静脉注射牛血清白蛋白(250 mg/kg),即日起给予(0.5%胆固醇,l0%蛋黄,5%猪油)150 g/d喂养2周,给予动脉粥样硬化模型组和miR-125a-3p干预组第2次注射牛血清白蛋白(250 mg/kg),并继续给予高胆固醇饲料喂养。期间,所有组提供充足干净水,定期打扫和清洗兔笼。分别于动脉粥样硬化建立4周和8周后, miR-125a-3p干预组注射以miR-125a-3p干扰慢病毒载体(5 mL/只),对照组和动脉粥样硬化模型组注射以等量生理盐水,12周时耳缘静脉注射空气处死兔子,立即剖开胸、腹腔,游离主动脉全长,剥离外膜脂肪组织,纵行剖开,行以下述处理。
1.3.2 血管内膜油红“O”染色血管粥样斑块大体标本染色步骤
(1)从甲醛溶液中取出标本,用流水洗15 min,剥去外膜,再用蒸馏水浸洗;(2)取储备液60 mL加入蒸溜水至100 mL,混匀放置10 min后染色,染色时间为2~4 min;(3)用70%乙醇浸泡标本至斑块呈红色,底色呈白色为止,最后用蒸馏水浸泡标本,浸入甲醛溶液中保存。油红O染液的配制:储备液:油红O 0.5 g,98%异丙醇 100 mL。临用前取上液6 mL,加蒸馏水4 mL,静置10 min,过滤即可。
1.3.3 免疫组织化学方法检测斑块组织中MMP-9、VEGF
主动脉经4%中性福尔马林固定后,石蜡切片进行MMP-9、VEGF表达测定(SP法,阴性对照一抗用0.01 mol/L PBS代替)。显微镜下拍照后用“Image Pro Plus 4.5”软件进行图像分析,测定阳性物质表达面积和积分光密度。
1.3.4 免疫荧光法检测M1标志物CD11c、M2
标志物CD206 石蜡切片经脱蜡、酒精脱水后,进行抗原修复,用0.01 M的PBST漂洗5 min,重复3次;以2% BSA于37 ℃湿盒内封闭30 min;标本片上滴加适当稀释的荧光标记抗体,放在湿盒中,37 ℃孵育30 min,0.01 mol/L PBS漂洗5 min,重复3次,不时震荡。缓存甘油封片,镜检。
1.4 统计学处理
SPSS 11.0软件进行统计学分析。计量资料以均数±标准差表示,多组间均数比较应用方差分析,P < 0.05为差异有统计学意义。
2. 结果
2.1 血管内膜油红“O”染色结果
血管内膜油红“O”染色显示,动脉粥样硬化病变处被染成红色。血管内膜油红“O”染色统计结果显示动脉粥样硬化组病变区域远大于miR-125a-3p抑制剂干预组,各组间的差异具有显著性(P < 0.01)(图1、图2)。结果表明:miR-125a-3p抑制剂能减少动脉粥样硬化斑块的形成。
2.2 免疫组织化学方法检测斑块组织中MMP9的结果
MMP-9免疫组化染色结果显示,与对照组比较,AS模型组的MMP-9水平显著升高(P < 0.001),与AS模型组比较,miR-125a-3p抑制剂干预后MMP-9水平显著下降(P < 0.01)(图3、图4)。结果表明:miR-125a-3p抑制剂干预后,降低了斑块组织中MMP-9的表达。
2.3 免疫组织化学方法检测斑块组织中VEGF的结果
VEGF免疫组化染色结果显示,与对照组对比,AS模型组的VEGF水平显著提高(P < 0.01);与AS模型组对比,miR-125a-3p抑制剂干预后VEGF水平下降,(P < 0.01)(图5、图6)。结果表明:miR-125a-3p抑制剂干预后,降低了斑块组织中VEGF的表达。
2.4 免疫荧光染色检测M1标志物CD11c的结果
免疫荧光检测M1标志物CD11c染色结果显示:与对照组比较,AS模型组的CD11c水平有明显升高(P < 0.0001);与AS模型组比较,miR-125a-3p抑制剂干预后CD11c水平显著下降(P < 0.0001)(图7、图8)。结果表明:miR-125a-3p抑制剂减少了斑块组织中M1 巨噬细胞。
2.5 免疫荧光染色检测M2 标志物CD206的结果
免疫荧光检测M2标志物CD206染色结果显示,与对照组对比,AS模型组的CD206水平显著下降(P < 0.0001),miR-125a-3p抑制剂干预后CD206水平明显升高(P < 0.0001)(图9、图10)。结果表明:miR-125a-3p抑制剂增加了斑块组织中M2 巨噬细胞。
3. 讨论
AS是一种慢性炎症性疾病,严重威胁人体健康。研究表明,不稳定AS斑块容易导致不良心血管事件的发生[13-14]。巨噬细胞是AS病变中主要的免疫细胞,在AS整个病理生理过程中都发挥着重要作用[5]。在动脉粥样硬化中,当循环中的单核细胞进入内膜后,单核细胞会分化为巨噬细胞[15]。巨噬细胞和单细胞暴露在炎症细胞因子、氧化脂质、胆固醇晶体和其他因素中。所有这些刺激不仅诱导特定的转录反应,而且还广泛相互作用,导致动脉粥样硬化斑块中巨噬细胞的转录[7]。基于不同的激活方式,巨噬细胞主要分为两种表型,即经典激活的M1和替代激活的M2巨噬细胞[2-3]。M1巨噬细胞能够开始并维持炎症反应,分泌促炎细胞因子,激活内皮细胞,并诱导其他免疫细胞募集到发炎组织中;另一方面,M2巨噬细胞促进炎症的消退,吞噬糖凋亡细胞,驱动胶原蛋白沉积,协调组织完整性,释放抗炎介质,主要参与组织修复,具有吞噬、促血管生成和促纤维化能力。M1巨噬细胞在促进斑块不稳定方面发挥了重要作用,而M2巨噬细胞则维持斑块的稳定,提示斑块的不稳定可能是M1和M2亚型之间失衡的结果[6, 16]。
miRNA是一种小型、进化保守的非编码RNA,长度为18~25个核苷酸,在基因调控中具有重要作用,转录后调控基因表达[17-18]。miRNA可以抑制数千个目标基因并协调正常过程,包括细胞增殖、分化和凋亡[19]。同时,miRNA对巨噬细胞的激活起到重要调节作用[7],例如Lin-Li Lv等的研究表明[20],外体miR-19b-3p调节的TEC和巨噬细胞之间的通路,导致M1巨噬细胞激活。由于M1巨噬细胞加剧斑块的不稳定,因此,笔者可以推测miRNA对不稳定斑块的发生起到了一定的作用。笔者的研究[12]也提示miR-125a-3p在动脉粥样硬化斑块的不稳定及破裂中起着重要的作用。
VEGF是一种有效的内皮细胞生长因子和血管生成诱导因子,对血管的完整性和血管功能具有重要意义。研究发现,VEGF在不稳定斑块中发挥着重要作用,能在一定程度上加速动脉粥样硬化斑块的进展和不稳定性。其机制可能是VEGF增加血管通透性,引起红细胞外渗,进而导致斑块内出血,加速动脉粥样硬化[21]。基质金属蛋白酶-9(MMP-9)属于MMP家族,并已被广泛研究。随着MMP-9活性的增加,MMP-9可导致纤维帽变薄,从而导致斑块的不稳定[22]。
本研究结果显示,抑制miR-125a-3p可使动脉粥样硬化斑块病变区域面积减少,斑块组织中M1 巨噬细胞、MMP-9,VEGF表达减少,M2 巨噬细胞增加。提示miR-125a-3p抑制可以减轻动脉粥样硬化斑块形成,平衡M1/M2巨噬细胞,减少MMP-9,VEGF表达,促进斑块稳定,miR-125a-3p可能是治疗不稳定动脉粥样硬化斑块的新靶点。
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表 1 2组患儿一般资料比较(
$ \bar{x}\pm s $ )Table 1. Comparison of general data between two groups of patients(
$ \bar{x}\pm s $ )指标 UE组
(n = 96)UF组
(n = 12)t/χ2 P 男/女(n) 68/28 7/5 0.307 0.580 年龄(岁) 7.39 ± 2.18 8.00 ± 1.86 −0.933 0.353 BMI(kg/m2) 16.55 ± 2.58 16.37 ± 1.95 0.376 0.708 手术时间(min) 48.33 ± 9.37 45.00 ± 6.74 1.193 0.236 表 2 临床和超声判断胃容量结果一致性评价[n(%)]
Table 2. Consistency of gastric volume measured by clinical and ultrasound[n(%)]
临床判断 超声判断 kappa P UE组 UF组 CE组 92(85.2) 9(8.3) 0.255 0.006* CF组 4(3.7) 3(2.8) *P<0.05。 表 3 超声检查空腹和饱胃相关指标比较[M(Q1,Q3)/n(%)]
Table 3. Comparison of related parameters between UE and UF[M(Q1,Q3)/n(%)]
指标 UE组
(n = 96)UF组
(n = 12)Z/t/χ2 P BCSA(cm2) 2.10(1.80,2.80) 3.90(3.80,4.20) 15.088 <0.001* ACSA(cm2) 1.80(1.70,1.90) 1.85(1.80,2.00) 0.973 0.324 胃管吸引量(mL/kg) 0.3(0.2,0.6) 1.2(1.0,1.3) 23.160 <0.001* 进食种类(1/2/3/4)(n) (64/30/2/0) (1/3/8/0) 34.900 <0.001* 进食至超声检查间隔(h) 5.92 ± 1.06 2.42 ± 0.52 11.208 <0.001* 进食量(g) 120(100,150) 225(203,238) 16.255 <0.001* 呕吐[n(%)] 5(5.2) 1(8.3) 0.177 0.674 BCSA:胃管吸引前CSA,ACSA:胃管吸引后CSA,胃管吸引量和进食量均采用[M(Q1,Q3)]表示;*P < 0.05。 -
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