Genetically modified pigs:Emerging animal models for hereditary hearing loss

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for society.To investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are necessary.Pigs have been extensively used as valuable large animal models in biomedical research.In this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss.Additionally,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL research.Overall,this review provides insights and perspectives for future studies on HHL using porcine models.

Improved methodology for efficient establishment of the myocardial ischemia-reperfusion model in pigs through the median thoracic incision

To investigate the feasibility and effectiveness of establishing porcine ischemia-reperfusion models by ligating the left anterior descending(LAD)coronary artery,we first randomly divided 16 male Bama pigs into a sham group and a model group.After anesthesia,we separated the arteries and veins.Subsequently,we rapidly located the LAD coronary artery at the beginning of its first diagonal branch through a mid-chest incision.Then,we loosened and released the ligation line after five minutes of pre-occlusion.Finally,we ligated the LAD coronary artery in situ two minutes later and loosened the ligature 60 min after ischemia.Compared with the sham group,electrocardiogram showed multiple continuous lead ST-segment elevations,and ultrasound cardiogram showed significantly lower ejection fraction and left ventricular fractional shortening at one hour and seven days post-operation in the model group.Twenty-four hours after the operation,cardiac troponin T and creatine kinase-MB isoenzyme levels significantly increased in the model group,compared with the sham group.Hematoxylin and eosin staining showed the presence of many inflammatory cells infiltrating the interstitium of the myocardium in the model group but not in the sham group.Masson staining revealed a significant increase in infarct size in the ischemia/reperfusion group.All eight pigs in the model group recovered with normal sinus heart rates,and the survival rate was 100%.In conclusion,the method can provide an accurate and stable large animal model for preclinical research on ischemia/reperfusion with a high success rate and homogeneity of the myocardial infarction area.

Ethical inspection about laboratory animals

Laboratory animals and animal experiments are foundations and important support conditions for life sciences, especially for medical research. The animal experiments have drawn extensive attention from the society because of the ethical issue. This paper takes Wenzhou Medical University as an example to give a brief introduction to the ethical review about laboratory animals in the university so as to further draw attention and concerns from the public about the ethical issue of laboratory animals. We successively introduce its scientific projects, nurturing environment and ethical review of laboratory animals.

Bronchoalveolar Lavage and Histologic Characterization of Inhaled Heparin in Asthmatic Guinea Pigs

Objective: To survey the effects of inhaled heparin on airway inflammation inguinea pigs with asthma and investigate the possible mechanism of inhaled heparin in the treatmentof asthma. Methods: The asthma in guinea pigs induced by ovalbumin was treated with inhaled heparin.The changes of cellularities in bronchoalveolar lavage (BAL) fluid and the airway walls wereexamined. Histologic examinations were also done in the guinea pig controls. Results: The number ofeosinophils, lymphocytes, and ciliated epithelial cells in the BAL fluid from the group treated withheparin was significantly lower than that of the group of asthma controls (P<0.01). Within theairway watts of the heparin treated group, the eosinophil infiltration was less prominent than thatof the group of asthma controls (P<0.001) and the number of mast cell was significantly higher thanthat of the group of asthma controls (P<0.01). Histologic examination showed that airway damages inthe heparin treated group were mild. Conclusion: Heparin can inhibit airway inflammation andalleviate airway damage in guinea pigs with asthma.

AN EXPERIMENTAL STUDY OF ELECTROACUPUNCTURE ON AUDITORY IMPAIRMENT CAUSED BY KANAMYCIN IN GUINEA PIGS

Frequency following response(FFR)and auditory brain stem evoked potential response(ABR)were used to determine the auditory acuity in evaluating the effect of electro-acupuncture treatment of kanamycin-induced auditory impairment in guinea pigs.Thesuccinate dehydrogenase(SDH)activity and morphological changes of the inner earreceptors were examined under the light and scanning electron microscope in cochlearspread preparations.The results showed that 1)electro-acupuncture was effective but nosignificant differences were found among the stimulating wave forms;2)Tinggong(SI19),Yifeng(SJ 17),Shenshu(UB 23),Sanyinjiao(Sp 6),Zhubin(K 9)and Waiguan(SJ 5)are all effective acupoints,especially the combination of Tinggong(SI 19),Sanyinjiao(Sp6)and Zhubin(K 9)acupoints;3)improvement in the cochlear function and excitabilityof the cortical and lower auditory center and increase of the mitochondrial SDH activityand energy supply in hair cells might contribute to the mechanism of the treatment.

Guinea pigs as an animal model for sciatic nerve injury

The overwhelming use of rat models in nerve regeneration studies is likely to induce skewness in treatment outcomes.To address the problem,this study was conducted in 8 adult guinea pigs of either sex to investigate the suitability of guinea pig as an alternative model for nerve regeneration studies.A crush injury was inflicted to the sciatic nerve of the left limb,which led to significant decrease in the pain perception and neurorecovery up to the 4th weak.Lengthening of foot print and shortening of toe spread were observed in the paw after nerve injury.A 3.49 ± 0.35 fold increase in expression of neuropilin 1（NRP1） gene and 2.09 ± 0.51 fold increase in neuropilin 2（NRP2） gene were recorded 1 week after nerve injury as compared to the normal nerve.Ratios of gastrocnemius muscle weight and volume of the experimental limb to control limb showed more than 50% decrease on the 30 th day.Histopathologically,vacuolated appearance of the nerve was observed with presence of degenerated myelin debris in digestion chambers.Gastrocnemius muscle also showed degenerative changes.Scanning electron microscopy revealed loose and rough arrangement of connective tissue fibrils and presence of large spherical globules in crushed sciatic nerve.The findings suggest that guinea pigs could be used as an alternative animal model for nerve regeneration studies and might be preferred over rats due to their cooperative nature while recording different parameters.

Myopia induced by flickering light in guinea pigs: a detailed assessment on susceptibility of different frequencies

AIM: To investigate the effectiveness and feasibility of inducing myopia in guinea pigs by flickering light (FL) stimulation with different frequencies. METHODS: Seventy 2 -week-old guinea pigs were randomly assigned to six groups: five FL groups and a control group (n =12 for each). Animals in the five FL groups were raised under 500lx illumination with a duty diurnal cycle of 50% at a flash rate of 5, 1, 0.5, 0.25 and 0.1Hz respectively. Those in the control group were reared under steady 250lx illumination. Refraction, axial length, and radius of curvature were measured before and at 2, 4, 6, 8, 10 and 12 weeks after treatment. At week 12, the eyeballs were taken out and three ocular dimensions and dry weight of sclera were measured. RESULTS: A myopic shift and axial eye length increase developed in the five FL groups. Stimulation at 0.5Hz caused greater changes in myopic shift, axial elongation, eyeball dimension, and dry weight of sclera than stimulation at other frequencies. Compared with controls, eyes in 0.5Hz group were approximately -5.5 ±1.5D more myopic with increase in horizontal, vertical, axial dimensions by 0.89 ±0.3mm, 0.69 ±0.2mm, 1.12 ±0.2mm respectively and with increase in dry weight of sclera by 0.44mg. CONCLUSION: Chronic exposure to periodic illumination at temporal frequency is attended by development of excessive ocular enlargement and myopic refractive error. Emmetropization could bedisrupted differently by frequency alteration.

Comprehensive evaluation of the metabolic effects of insect meal from Tenebrio molitor L.in growing pigs by transcriptomics, metabolomics and lipidomics

Background:The hypothesis was tested that insect meal(IM)as protein source influences intermediary metabolism of growing pigs.To test this,30 male,5-week-old crossbred pigs were randomly assigned to 3 groups of 10 pigs each with similar body weights(BW)and fed isonitrogenous diets either without(CON)or with 5%IM(IM5)or 10%IM(IM10)from Tenebrio molitor L.for 4 weeks and key metabolic tissues(liver,muscle,plasma)were analyzed using omics-techniques.Results:Most performance parameters did not differ across the groups,whereas ileal digestibilities of most amino acids were 6.7 to 15.6%-units lower in IM10 than in CON(P<0.05).Transcriptomics of liver and skeletal muscle revealed a total of 166 and 198,respectively,transcripts differentially expressed between IM10 and CON(P<0.05).Plasma metabolomics revealed higher concentrations of alanine,citrulline,glutamate,proline,serine,tyrosine and valine and a lower concentration of asparagine in IM10 than in CON(P<0.05).Only one out of fourteen quantifiable amino acid metabolites,namely methionine sulfoxide(MetS),in plasma was elevated by 45%and 71%in IM5 and IM10,respectively,compared to CON(P<0.05).Plasma concentrations of both,major carnitine/acylcarnitine species and bile acids were not different across groups.Lipidomics of liver and plasma demonstrated no differences in the concentrations of triacylglycerols,cholesterol and the main phospholipids,lysophospholipids and sphingolipids between groups.The percentages of all individual phosphatidylcholine(PC)and phosphatidylethanolamine(PE)species in the liver showed no differences between groups,except those with 6 double bonds(PC 38:6,PC 40:6,PE 38:6,PE 40:6),which were markedly lower in IM10 than in CON(P<0.05).In line with this,the percentage of C22:6n-3 in hepatic total lipids was lower in IM10 than in the other groups(P<0.05).Conclusions:Comprehensive analyzes of the transcriptome,lipidome and metabolome of key metabolic tissues indicate that partial or complete replacement of a conventional protein source by IM in the diet has only a weak impact on the intermediary metabolism of growing pigs.Thus,it is concluded that IM from Tenebrio molitor L.can be used as a dietary source of protein in pigs without causing adverse effects on metabolism.

Effect of Transport Times on Welfare of Pigs

The aim of the study was to investigate effect of transport times of up to 12 h on pigs＇ welfare. An observation box was located on the 3rd floor of the vehicle. Device to measure temperature, humidity and video camera to monitor pigs＇ behaviors were fitted in the box. Eighteen measurements were performed during two seasons for 4, 8, and 12 h of transport time with three replications. Meat samples were taken from longissimus dorsi （LD） and the carcases were chilled for 24 h at ＋4 ~C for pH determination. To determine cortisol, glucose, lactate, and creatine kinase concentration levels, blood samples were collected before and after transport from 90 pigs and from 20 controls that were not transported for control purposes. To evaluate behavioural alterations, frequency of events and durations were considered. Highest pH24 of 5.99 ＋ 0.29 occurred during summer at 12 h transport time. Cortisol concentrations elevated during short and decreased with an increase of transport time （P 〈 0.001）. Highest and lowest glucose concentrations for winter and summer were at 8 and 12 h transport time, respectively （P 〈 0.01）. Concentrations of lactate and creatine kinase positively correlated with transport time （P 〈 0.002）. Lying, sitting, rooting and vocalization behaviours correlated with transport time （P 〈 0.009）. Certain THI values during summer surpassed normal （〉 74） level during loading. The pH24 value correlated with transport time during summer. Lactate and creatine kinase concentrations increased with an increase in transport time but for Glucose, highest value was at 8 h transport time.

Large animal models for Huntington's disease research

Huntington'sdisease(HD)isahereditary neurodegenerative disorder for which there is currently no effectivetreatmentavailable.Consequently,the development of appropriate disease models is critical to thoroughly investigate disease progression.The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin(HTT)gene,leading to the expansion of a polyglutamine repeat in the HTT protein.Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain,which precipitate selective neuronal loss in specific brain regions.Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets.Due to the marked species differences between rodents and larger animals,substantial efforts have been directed toward establishing large animal models for HD research.These models are pivotal for advancing the discovery of novel therapeutic targets,enhancing effective drug delivery methods,and improving treatment outcomes.We have explored the advantages of utilizing large animal models,particularly pigs,in previous reviews.Since then,however,significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD.In the current review,we provide a comprehensive overview of large animal models of HD,incorporating recent findings regarding the establishment of HD knock-in(KI)pigs and their genetic therapy.We also explore the utilization of large animal models in HD research,with a focus on sheep,non-human primates(NHPs),and pigs.Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

我国实验用猪的健康管理与应用前景

猪在生理生化指标、解剖结构、系统功能、营养代谢以及疾病发生和发展等方面与人类机体相对应的系统和功能相类似。目前人医和动物医学领域各项研究中,越来越多研究者首选使用猪进行科学试验。本文将着重讨论我国当下实验用猪猪群的健康管理,包括实验用猪猪群生物安全体系建立、生产管理、疫病监测机制、药物保健等,以提升实验用猪在饲养繁育和管理过程中的微生物等级与健康管理水平,减少其在实验过程中对科学实验造成的干扰。

2013-2021年实验动物质量检测能力验证情况

目的分析我国实验动物质量检测能力现状,探讨实验动物质量检测能力验证组织实施现状,研究影响能力验证满意率的关键因素。方法通过Excel2010和Powerpoint2010软件进行实验室数量、项次、满意率以及频次的变化分析。结果2013—2021年共实施27个能力验证计划,包括22种检测能力参数,发放92种生物样本。共有70家实验室参加552个项次,整体满意率达到90.22%。结论持续参加能力验证活动可以提升实验室的检测能力。

完善实验动物废物处置质量管理体系的思考:以中国食品药品检定研究院为例

科技发展是推动人类社会前进的巨大动力,而实验动物资源是生命科学、医药等领域科技发展的条件保障。但在开展医药研发、检验检测等实验过程中,使用实验动物产生的废物也为城市管理带来了新的考验。废物的分类、回收、有效利用和生物安全危害等问题接踵而至,对法规、标准、规范等的制定提出了与时俱进的要求。质量管理体系建设是检验检测机构管理的地基和框架,具备较强的操作性和检查性,可以不断提升管理水平,加强基础管理的稳定性。随着各层级新的管理办法出台,很多单位现行的质量管理体系内关于实验动物废物管理的相关内容欠妥,存在未明确非医疗机构实验动物废物处置流程、实验动物废物分类不准确、不同分类的废物未能按照不同方式处置等问题。本文针对目前存在的问题,梳理了中国食品药品检定研究院质量管理体系需要增加或者删改的流程,并制订了现行有效的标准作业流程,在如何加强监督管理方面提出了切实可行的措施,并开创性地把6S管理内容纳入质量管理体系中。因此,针对实验动物废物的管理应从完善质量管理体系的角度出发,从源头做好废物分类和管理,有效控制生物危害,最大限度地减少环境污染,为可持续发展创造条件。

基于科研范式变革的实验动物资源创新发展

实验动物是支撑生命科学和生物医药等领域科技创新的重要基础科研条件之一。随着科研范式的变革,准确把握科学前沿和国家重大需求,进一步凝练实验动物资源创制与发展中的难点和疑点,对加快推动实验动物新品种/新品系开发和动物模型创制与发展具有重要意义。本文梳理了我国实验动物资源建设和应用情况,就应对新形势下的科研范式变革中存在的主要问题进行了深入探讨,展望了我国实验动物资源发展新动向,以期为实验动物新资源研究开发提供新视角。

Clinical/Behavioral Monitoring of Rodents and Rabbits Undergoing Scientific Experiments

Background: We currently have international and national guidelines regarding the assessment and monitoring of clinical signs and humane endpoints in animals used in teaching and research, which make the performance of these activities mandatory for any experiment and professional working in this area. Assigning the severity of a research experiment is the result of an analysis of records of observations of the animal’s behavior, and clinical signs. The aim of this study was to describe the importance of carrying out a severity assessment associated with clinical and behavioral monitoring of rodents and rabbits during experimentation to maintain the welfare of these animals undergoing scientific research. Methods: The literature search was carried out using the following terms: “Monitoring”;“Humane endpoints”;“Animal welfare”, “Rodents”;“Rabbits”, and as connectors “and”;“or”, in the following databases: PubMed;LILACS/BIREME and SciELO. Results: A total of 987 articles were identified in the databases, and 20 of these studies were included in this review. Conclusions: Humane endpoint protocols and procedure severity tables are of the utmost importance, both from an ethical point and to refine the results of research conducted on laboratory animals. They should be drawn up jointly by the teams responsible for the project and the maintenance of the animals during the research period, and the data obtained should be published so that the scientific community can have access to it, helping to disseminate these practices, as well as helping to draw up new procedures. Monitoring and evaluating the welfare and clinical condition of animals undergoing scientific research procedures is the responsibility of the professors, researchers, veterinarians, and animal facility coordinators. The Ethics Committee on the Use of Animals must monitor all the activities conducted with the animals, by inspecting the experimental procedures and the physical environment of the laboratory animal facility where the animals are housed.

《广东省实验动物管理条例》制度特点及近十年实施情况分析

《广东省实验动物管理条例》(以下简称《条例》)于2010年10月正式颁布实施,明确授权广东省实验动物监测机构开展实验动物质量监测工作并承担相应法律责任,明确了实验动物生产和使用规范,推动了实验动物福利伦理关注,标志着广东省实验动物管理工作进入了法制化管理的新阶段。通过制定《条例》配套文件,广东省积极推行行政执法工作规范化、标准化以及“放管服”措施,在促进广东省实验动物规范管理和支撑生物医药创新发展方面发挥了积极的作用,在实验动物科技平台建设、标准化工作、特色资源保存研制和人才队伍建设等方面取得了显著成绩。然而,随着科技进步、社会经济发展和立法工作的深入,《条例》在实施过程中也发现了一些需要完善的内容,如实验动物福利内容规定需进一步明确、基因工程动物管理需加强具体指引、从业人员安全福利保障相关规定需完善、“一票否决制”需进一步细化落实、实验动物标准化建设仍需进一步推动、实验资源共享需探索有效机制。本文对《条例》的制度特点、立法后的实施情况和成效,以及实施中存在的问题及对策等方面进行梳理归纳,总结了广东省近10年的实验动物管理实践经验,以期为实验动物立法提供参考。

藤黄酸对脓毒症大鼠肠道损伤的保护作用

目的探讨藤黄酸(GA)对脓毒症大鼠肠损伤的影响以及对RIP1/RIP3/MLKL信号通路的调节作用。方法取40只SPF级SD大鼠,按随机数字表法分为假手术(Sham)组、脓毒症(Spesis)组、低剂量GA组和高剂量GA组,各10只。除Sham组外,其余3组大鼠均采用盲肠穿孔结扎法(CLP)建立脓毒症大鼠模型。低剂量GA组和高剂量GA组于造模前24 h分别灌胃给予50、100 mg·kg^(-1)GA。造模后12 h麻醉处死,收集外周血和小肠组织。采用酶联免疫吸附(ELISA)法检测血清中肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6)表达水平;苏木素-伊红(HE)染色观察小肠组织病理变化并进行Chui’s评分;蛋白免疫印迹(Western blotting)法检测凋亡相关蛋白(Caspase-3、Bax和Bcl2)、紧密连接蛋白(ZO-1和Occludin)及RIP1/RIP3/MLKL信号通路相关蛋白的表达水平。结果与Sham比较,Spesis组大鼠肠黏膜受损明显,Chui’s评分显著升高(P<0.05);血清中炎性因子TNF-α、IL-1β、和IL-6表达明显增加(P<0.05);小肠组织中Bax、ZO-1和Occludin蛋白表达明显降低(P<0.05),而Caspase-3、Bcl2、MLKL、RIP1以及RIP3蛋白表达明显增加(P<0.05);与Spesis组比较,低剂量GA组和高剂量GA组大鼠肠黏膜损伤均有不同程度改善,Chui’s评分均明显降低(P<0.05);血清中炎性因子TNF-α、IL-1β、和IL-6表达均显著降低(P<0.05);小肠组织中Bax、ZO-1和Occludin蛋白表达明显增加(P<0.05),而Caspase-3、Bcl2、MLKL、RIP1以及RIP3蛋白表达明显降低(P<0.05)。结论GA能够减轻脓毒症诱导的大鼠肠道损伤,这或许与RIP1/RIP3/MLKL信号通路密切相关。

小型猪气管插管时异氟醚半数有效剂量的探索

目的本研究拟探讨小剂量舒泰(1.5 mg/kg)肌注复合异氟烷吸入用于保留自主呼吸的小型猪气管插管时50%和95%有效的异氟烷最小肺泡气浓度(50%and 95%minimum alveolar concentrations effective inhaled,MAC EI50and MAC EI95),为小型猪提供安全的麻醉插管方法。方法44例全身麻醉的巴马小型猪,肌注舒泰镇静后,面罩吸入异氟烷进行麻醉诱导,监测心率、血压、呼吸、体温、氧饱和度、呼末二氧化碳浓度和呼末异氟烷浓度。初始呼末异氟烷浓度设置为2.0%,采用Cooper’s评分评价气管插管满意度,采用Dixon’s改良序贯法测算剂量-效应关系。Probit回归法计算MAC EI50和MAC EI95及95%可信区间(confidence interval,CI)。结果小剂量舒泰复合异氟烷吸入诱导直接喉镜气管插管时,小型猪肺泡气浓度的MAC EI503.10%(95%CI 2.79%~3.56%),MAC EI953.77%(95%CI 3.41%~6.42%);在做好监测和气道管理预案的基础上,肺泡异氟烷浓度3.10%~3.75%,均能保证小型猪生命体征稳定。结论舒泰复合异氟烷吸入用于保留自主呼吸的小型猪气管插管是建立小型猪经口气道较好且安全的麻醉方法,具有推广价值。

黑线姬鼠实验动物化研究进展

自新冠肺炎疫情以来,全球以动物为媒介的传染病不断发生,人们需要更多种类的实验动物作为研究样本。黑线姬鼠(Apodemus agrarius)是一种我国常见的农田害鼠,繁殖期短、适应能力强,在我国分布广泛,是影响我国农业生产的主要害鼠之一。另外,黑线姬鼠也是汉坦病毒(Hantavirus)、莱姆病伯氏疏螺旋体(Borrelia burgdorferi)和刚地弓形虫(Toxoplasma gondii)的天然携带者,是流行病学研究的重要调查样本,同时在构建疾病模型和探究病原体致病机制方面也更具优势,其实验动物化的研究情况应该受到重视。本文总结了黑线姬鼠分类及其分布、繁殖学研究、生理学研究、分子生物学研究、携带病原体概况,并对黑线姬鼠的繁殖研究、多组学应用、病原探究和发展前景进行展望,以期为黑线姬鼠后续实验动物资源开发提供帮助。