Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint(TMJ) osteoarthritis(OA);however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4(Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore,Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

Mouse models in liver cancer research:A review of current literature

Primary liver cancer remains one of the most lethal malignancies worldwide. Due to differences in prevalence of etiological factors the incidence of primary liver can-cer varies among the world, with a peak in East-Asia. As this disease is still lethal in most of the cases, research has to be done to improve our understanding of the disease, offering insights for possible treatment options. For this purpose, animal models are widely used, especially mouse models. In this review, we describe the different types of mouse models used in liver cancer research, with emphasis on genetically engineered mice used in this field. We focus on hepatocellular carcinoma (HCC), as this is by far the most common type of primary liver cancer, accounting for 70%-85% of cases.

Establishment of Embryonic Stem Cell Lines Derived from Outbred Mouse Embryos and Production of Chimeras

The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,designated KE1, KE2, and KE5,were isolated from 5 Kunming albino blastocysts. Normal diploid composition of these cell lines was above 70%. By using C57BL/6J and 615 blastocysts as host embryos, one chimera was obtained in living pups. It was shown for the first time that chimeras can be produced by using outhred ES cells. This work implies that to establish ES cell lines from other animal embryos is possible. More interestingly, white color dots from ES cells on the coat of this chimeric mouse enlarged a lot after half a year, indicated that ES cells were inhibited by cells from outbred mouse or the cells of outbred mouse grew vigiously.

SOCS3 Expression Correlates with Severity of Inflammation in Mouse Hepatitis Virus Strain 3-induced Acute Liver Failure and HBV-ACLF

Summary： Recently, suppressor of cytokine signaling-3 （SOCS3） has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure （HBV-ACLF） have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 （MHV-3）-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells （PBMCs） of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immtmohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B （CHB）. Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin （IL）-1 β, IL-6, and tumor necrosis factor （TNF）-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.

Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice

AIM: To optimize the experimental protocols for a simple, sensitive and accurate bleeding assay.METHODS: Bleeding assay was performed in mice by tail tip amputation, immersing the tail in saline at 37 ℃, continuously monitoring bleeding patterns and measuring bleeding volume from changes in the body weight. Sensitivity and extent of variation of bleeding time and bleeding volume were compared in mice treated with the P2 Y receptor inhibitor prasugrel at various doses or in mice deficient of Fc Rγ, a signaling protein of the glycoprotein VI receptor.RESULTS: We described details of the bleeding assay with the aim of standardizing this commonly used assay. The bleeding assay detailed here was simple to operate and permitted continuous monitoring of bleedingpattern and detection of re-bleeding. We also reported a simple and accurate way of quantifying bleeding volume from changes in the body weight, which correlated well with chemical assay of hemoglobin levels(r2 = 0.990, P < 0.0001). We determined by tail bleeding assay the dose-effect relation of the anti-platelet drug prasugrel from 0.015 to 5 mg/kg. Our results showed that the correlation of bleeding time and volume was unsatisfactory and that compared with the bleeding time, bleeding volume was more sensitive in detecting a partial inhibition of platelet's haemostatic activity(P < 0.01). Similarly, in mice with genetic disruption of Fc Rγ as a signaling molecule of P-selectin glycoprotein ligand-1 leading to platelet dysfunction, both increased bleeding volume and repeated bleeding pattern defined the phenotype of the knockout mice better than that of a prolonged bleeding time.CONCLUSION: Determination of bleeding pattern and bleeding volume, in addition to bleeding time, improved the sensitivity and accuracy of this assay, particularly when platelet function is partially inhibited.

A murine model of dengue virus infection in suckling C57BL/6 and BALB/c mice

Dengue is a significant public health concern across tropical and subtropical regions worldwide,principally causing disease in children.Very young children are at increased risk of severe manifestations of dengue infection.The mechanism of dengue disease in this population is not fully understood.In this study,we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis.Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice,showing increased liver enzymes,extended viremia,dissemination to organs and histological alterations in liver and small intestine.Furthermore,the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice.These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study agerelated mechanisms of dengue pathogenesis.

Novel spontaneous myelodysplastic syndrome mouse model

Background:Myelodysplastic syndrome(MDS)is a group of disorders involving he-mopoietic dysfunction leading to leukemia.Although recently progress has been made in identifying underlying genetic mutations,many questions still remain.Animal models of MDS have been produced by introduction of specific mutations.However,there is no spontaneous mouse model of MDS,and an animal model to simulate natu-ral MDS pathogenesis is urgently needed.Methods:In characterizing the genetically diverse mouse strains of the Collaborative Cross(CC)we observed that one,designated JUN,had abnormal hematological traits.This strain was thus further analyzed for phenotypic and pathological iden-tification,comparing the changes in each cell population in peripheral blood and in bone marrow.Results:In a specific-pathogen free environment,mice of the JUN strain are rela-tively thin,with healthy appearance.However,in a conventional environment,they become lethargic,develop wrinkled yellow hair,have loose and light stools,and are prone to infections.We found that the mice were cytopenic,which was due to abnor-mal differentiation of multipotent bone marrow progenitor cells.These are common characteristics of MDS.Conclusions:A mouse strain,JUN,was found displaying spontaneous myelodysplas-tic syndrome.This strain has the advantage over existing models in that it develops MDS spontaneously and is more similar to human MDS than genetically modified mouse models.JUN mice will be an important tool for pathogenesis research of MDS and for evaluation of new drugs and treatments.

The Schlager mouse as a model of altered retinal phenotype

Hypertension is a risk factor for a large number of vision-threatening eye disorders.In this study,we investigated for the first time the retinal neural structure of the hypertensive BPH/2J mouse(Schlager mouse)and compared it to its control counterpart,the normotensive BPN/3J strain.The BPH/2J mouse is a selectively inbred mouse strain that develops chronic hypertension due to elevated sympathetic nervous system activity.When compared to the BPN/3J strain,the hypertensive BPH/2J mice showed a complete loss of outer layers of the neural retina at 21 weeks of age,which was indicative of a severe vision-threatening disease potentially caused by hypertension.To elucidate whether the retinal neural phenotype in the BPH/2J strain was attributed to increased BP,we investigated the neural retina of both BPN/3J and BPH/2J mice at 4 weeks of age.Our preliminary results showed for the first time that the BPH/2J strain develops severe retinal neural damage at a young age.Our findings suggest that the retinal phenotype in the BPH/2J mouse is possibly due to elevated blood pressure and may be contributed by an early onset spontaneous mutation which is yet to be identified or a congenital defect occurring in this strain.Further characterization of the BPH/2J mouse strain is likely to i)elucidate gene defects underlying retinal disease;ii)understand mechanisms leading to neural retinal disease and iii)permit testing of molecules for translational research to interfere with the progression of retinal disease.The animal experiments were performed with the approval of the Royal Perth Hospital Animal Ethics Committee(R535/17-18)on June 1,2017.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

茯苓酸对小鼠黑色素瘤细胞增殖、迁移和侵袭的影响及机制研究

目的:研究茯苓酸(pachymic acid,PA)对小鼠黑色素瘤B16细胞增殖、迁移和侵袭的影响及其可能机制。方法:采用CCK8法和细胞克隆形成实验检测不同浓度(20、40、80μmol/L)PA对B16细胞增殖的影响;细胞划痕和Transwell小室实验观察PA对B16细胞迁移和侵袭能力的影响;Western Blot法和免疫荧光法检测PA对B16细胞的上皮-间质转化相关蛋白表达的变化。结果:PA可以明显抑制B16细胞增殖,并呈一定的浓度依赖性关系(P<0.01);PA干预24 h后可以显著抑制B16细胞的迁移和侵袭能力(P<0.01);40μmol/L的PA能上调B16细胞的E-cadherin蛋白表达(P<0.05)并下调Vimentin蛋白的表达(P<0.05);免疫荧光法检测显示40μmol/L的PA可明显降低B16细胞Vimentin蛋白的表达。结论:PA可以抑制B16细胞的增殖、迁移和侵袭,其机制可能与上调B16细胞的E-cadherin蛋白表达,降低Vimentin蛋白表达,从而抑制B16细胞的上皮-间质转化有关。

NP-2 GSK-3βInhibitor Reduced Neonatal Hypoxic-Ischemic Brain Injury in Mice

Hypoxia-ischemia is an important cause of brain injury and neurological morbidity in the newborn infants.Glycogen synthase kinase 3β(GSK-3β)is activated and upregulated following hypoxic-ischemic(HI)brain injury.We have investigated the effects of GSK-3βinhibitors,Tideglusib and TDZD-8,on HI brain injury in neonatal mice.Specifically,postnatal day 7(P7)mouse pups subjected to unilateral common carotid artery ligation followed by 1 h of hypoxia.HI experimental or sham control groups were administered either the GSK-3βinhibitor or vehicle intraperitoneally 20 min prior to the onset of ischemia.The brain infarct volume and whole brain images were used in conjunction with Nissl staining to evaluate the protective effects of GSK-3βinhibitors.Either tideglusib or TDZD-8 significantly reduced cerebral infarct volume and improved neurobehavioral outcomes following HI injury.Western blotting showed that either drug increased phosphorylated GSK-3βand Akt,and reduced the expression of GFAP and p-STAT3.Pretreatment with tideglusib also enhanced the protein level of Notch1,and reduced the cleavage of pro-apoptotic signal caspase proteins,including caspase 3 and caspase 9 following HI.These results indicate that these GSK-3βinhibitors show neuroprotection against hypoxicischemic brain injury in neonatal mice.Either Tideglusib or TDZD-8 is a potential compound for the prevention or treatment of hypoxic-ischemic brain injury in neonates.

Regulatory effects of evodiamine on glucose metabolism-related factors in CT26 colorectal carcinoma-bearing mice

Objective:To investigate the therapeutic effect of evodiamine(EVO)on the expression of hexokinase(HK),lactate dehydrogenase A(LDHA),and pyruvate kinase M(PKM),key enzymes of glycolysis,in the tumor-bearing mice after modeling mouse colon cancer cells(CT26).Methods:A tumor-bearing mouse model was generated by administering axillary injection of CT26 and intraperitoneally injecting different doses of EVO.The therapeutic effects of EVO on CT26 tumor-bearing mice were evaluated by measuring the thymus and spleen indices,tumor volume,tumor suppression rate,and other related indicators in the tumor tissues of mice in each group after the administration of EVO,in addition,histopathological changes in the tumor tissues of the mice in the groups were studied by hematoxylin and eosin staining.The expression levels of HK,LDHA,and PKM in the tumor tissues of each group of mice were measured by performing Western blot to investigate the mechanism of EVO treatment in CT26 tumor-bearing mice.Results:EVO inhibited the growth of tumors in CT26-bearing mice and enhanced their splenic and thymic indices.Western blot results showed that EVO reduced the expression levels of HK,LDHA,and PKM proteins in the tumor tissues of CT26 tumor-bearing mice.Conclusion:EVO has a therapeutic effect on CT26 tumor-bearing mice,and its mechanism of action may be related to the low expression of key enzymes HK,LDHA and PKM of glycolysis in tumor tissues.

基于网络药理学结合UHPLC-MS/MS探讨清感童饮的抗炎作用

目的 基于血清药理学和网络药理学探究清感童饮的体外抗炎作用。方法 采用血清药理学评价方法,验证清感童饮含药血清对脂多糖(LPS)诱导的小鼠巨噬细胞(RAW264.7)释放一氧化氮(NO)、细胞坏死因子α(TNF-α)、白细胞介素6(IL-6)的影响。利用液质联用(UHPLC-MS/MS)技术对清感童饮指标性成分进行含量测定,并根据这些成分进行网络药理学分析,对清感童饮有效成分发挥抗炎作用的潜在靶点及作用通路进行预测。结果 细胞实验证明清感童饮可明显降低NO、TNF-α、IL-6水平(P<0.05,P<0.01,P<0.001)。清感童饮中含量较高的成分为连翘酯苷A、牛蒡苷、绿原酸、野黄芩苷、没食子酸、迷迭香酸、芍药内酯苷、连翘苷。清感童饮中17个活性成分与炎症的交集靶点共215个,主要涉及ALB、VEGFA、IL-6、TNF-α等31个核心靶点,调节AGE-RAGE、PI3K-Akt、MAPK信号通路等多种通路发挥抗炎作用。结论 清感童饮具有多成分-多靶点发挥抗炎作用的特点。

三种小鼠肠道病毒多重PCR检测方法的建立和初步应用

目的建立一种同时检测小鼠细小病毒(MVM)、肝炎病毒(MHV)和诺如病毒(MNV)的多重PCR检测方法。方法分别根据MVM、MHV和MNV基因保守区应用软件设计特异性引物,构建在同一反应体系中可同时检测MVM、MHV和MNV的多重PCR检测方法,验证其特异性、敏感性及重复性,并进行初步检测应用。结果通过优化PCR扩增条件成功建立多重PCR检测方法,特异性实验结果表明,该方法可同时扩增MVM、MHV和MNV的目的片段,片段大小与预期相符,未出现非特异性片段;敏感性测试显示,MVM、MHV和MNV三种病毒的最低检测下限为10^(2)、10^(4)、10^(2) copies/μL;重复性实验显示该方法在不同时间、不同场所应用的重复性稳定。应用该方法对287份小鼠结肠匀浆样品进行检测,与单重PCR检测结果完全一致。结论所建立的MVM、MHV和MNV三种病毒多重PCR检测方法具有特异性高、敏感性强和重复性优的特点,适用于三种病原体感染的临床样品检测和大量样品的流行病学调查。

免疫系统人源化小鼠及其在肿瘤免疫治疗中的应用

肿瘤免疫治疗的有效性多借助于小鼠模型进行评价。但由于物种差异,传统小鼠模型不能完全模拟人体免疫应答的真实情况,导致大部分基于小鼠的研究成果不适用于人体。近年随着免疫缺陷小鼠的出现和品系改良,基于免疫缺陷小鼠构建的免疫系统人源化小鼠有望帮助研究者克服这一难题。本文就免疫缺陷小鼠模型和免疫系统人源化小鼠模型的最新研究进展和存在的问题以及免疫系统人源化小鼠在肿瘤免疫研究中的应用进行总结,为后续人源化小鼠模型的完善及其在肿瘤免疫治疗中的应用提供参考。

小鼠肝炎病毒以不同途径感染4个品系小鼠的比较

用小鼠肝炎病毒A59株以滴鼻、灌胃、腹腔注射3种途径人工感染近交系小鼠(BALB/c小鼠、C57BL小鼠)、免疫缺陷小鼠(NIH-III小鼠、BALB/c-nu/nu小鼠),接种病毒后3、5、7、14、21、28d分别取各鼠的肝脏,提取肝脏总RNA,用RT-PCR方法检测小鼠肝炎病毒的感染情况。结果显示:腹腔注射病毒的小鼠在肝脏中能最早检测出病毒,其次是灌胃,滴鼻则最晚;另外,近交系小鼠及免疫缺陷小鼠感染后3d在肝脏中检测出病毒,而转基因小鼠则在感染后7d在肝脏中检测出病毒。

新生小鼠睾丸组织移植到裸鼠体内不同时期移植物的组织学观察

目的 通过测量不同发育时期移植物的重量及观察其中生精小管结构和生精细胞组成情况，对去势雄性小鼠中移植的新生小鼠睾丸组织生长和发育进行系统观察。方法 将出生1．2d昆明小鼠睾丸移植到7—12周去势雄性免疫缺陷小鼠背部；在移植后不同时间段（分为3d、1—11周和3．6月16个组）取出移植物，计算移植物的回收率，测定移植物重量，观察移植物中生精小管结构及生精细胞的组成。结果 从移植的450个新生小鼠睾丸组织，回收到405个移植物，总回收率为90．0％。重量比移植前增加约40倍。移植物中生精小管的发育及各阶段生精细胞出现时间与在正常小鼠中所见基本相同。移植时间超过8周后，生精上皮的退化现象显著增加。结论 将新生小鼠睾丸组织异位移植到受体背部后的发育进程与在体情况相同，第1次生精波结束后的时间应为获取精子细胞和精子的最佳时间，大约是移植后5．7周。

两种实验性糖尿病模型小鼠的比较

目的:通过对链脲佐菌素(STZ)模型鼠和db/db模型鼠的糖尿病模型鼠的特点以及对三类降糖药反应性的比较,客观评价两种糖尿病模型鼠在药物研究中的应用价值。方法:离乳、雄性BALB/c小鼠高脂饲料喂养4周后,空腹12h STZ(45mg/kg)腹腔注射,连续3次,一周后检测糖耐量受损、空腹血糖大于8.0mmol/L的为STZ模型鼠。将STZ模型鼠和8周龄空腹血糖大于8.0mmol/L的db/db模型鼠各随机分组,每组8只分别给予罗格列酮(12mg/kg)、格列苯脲(45mg/kg)、胰岛素(1U/kg)和生理盐水(0.1mL/10g),每天1次,连续给药4周后检测两模型鼠及其对照组糖脂代谢相关的生化指标及激素水平。结果:STZ模型鼠和db/db模型鼠分别与正常BALB/c鼠、C57BL鼠对比,STZ模型鼠和db/db模型鼠均口服葡萄糖耐量试验(OGTT)异常,空腹血糖、血TG和TC显著升高。但STZ模型鼠的胰岛素与C肽水平低于正常;而db/db鼠血C肽、胰岛素浓度显著升高;3种降糖药给药4周后STZ模型鼠的空腹血糖均明显降低,OGTT显著改善;罗格列酮对db/db模型鼠也可显著降低空腹血糖并改善糖耐量,但相同剂量的格列苯脲和胰岛素对db/db模型鼠的空腹血糖和糖耐量无影响。给予3倍剂量的胰岛素,db/db模型鼠才表现出空腹血糖的显著降低。结论:STZ模型鼠胰岛功能受损并有一定程度的胰岛素抵抗,对3种降糖药均敏感。db/db模型鼠表现出高度的胰岛素耐受,对内源性和外源性的胰岛素都不敏感,对罗格列酮的敏感性与STZ模型鼠相当。

诱发性2型糖尿病小鼠模型与自发性db/db小鼠特性的比较

目的建立诱发性2型糖尿病小鼠模型,并将其与自发性2型糖尿病小鼠db/db进行比较分析。客观评价两种2型糖尿病小鼠模型,为糖尿病研究中动物模型的选择与实际应用提供实验依据。方法高脂饲料喂养C57BL/6J小鼠4周,腹腔连续3次注射STZ,建立诱发性2型糖尿病小鼠模型。感染后4周,大体肉眼观察小鼠的肝脏、肾脏,测定糖耐量,血清生化指标及血清细胞因子IL-2、IL-4、IL-6、IFN-γ、TNF-α、IL-17、IL-10表达量,将其与同龄的自发性2型糖尿病小鼠db/db进行比较分析。结果肉眼观察发现,两组模型小鼠的肝脏、肾脏与对照组均具有明显差异。糖耐量分析中,两组模型小鼠与对照组小鼠各时间点的血糖值均具有统计学差异(P<0.05),耐糖功能低下,两组模型小鼠间血糖值无统计学差异。血液生化指标中,与对照组小鼠相比,两组模型小鼠GLU、CHOL、LDLC明显升高(P<0.05);两组模型小鼠相互比较,诱发性2型糖尿病小鼠血脂水平较高(P<0.05)。免疫指标比较显示:除IL-2外,两组模型小鼠血清中细胞因子水平均较对照组小鼠明显升高(P<0.05),而db/db小鼠血清中细胞因子表达较诱发性糖尿病小鼠高,其中IL-6、IFN-γ、TNF-α具有显著性差异(P<0.05)。结论两组2型糖尿病模型小鼠均在一定程度上模拟了人类糖尿病患者症状,但由于糖尿病产生的原因不同而存在着一定的差异,研究者可根据实际需要参照相关数据进行选择。

白芍解郁颗粒对小鼠抑郁模型的影响

目的 研究白芍解郁颗粒对小鼠抑郁模型的影响。方法 选择小鼠强迫游泳、悬尾应激及利血平所致眼睑下垂小鼠抑郁模型 ,用白芍解郁颗粒对其进行治疗观察。结果 白芍解郁颗粒可以对抗小鼠因强迫悬尾及强迫游泳造成的抑郁症状 ,并可对抗因利血平所致小鼠眼睑下垂和体温下降。