通过结合70%部分切除术和对乙酰氨基酚，生成急性肝衰竭大鼠模型
Summary
本研究开发的急性肝衰竭动物模型为研究潜在疗法提供了可行的替代方法。目前的模型采用身体和药物引起的肝损伤的综合效应，为研究新疗法的潜力提供了一个合适的时间窗口。
Abstract
急性肝衰竭（ALF）是由各种病因引起的临床疾病，导致肝脏代谢、生化、合成和排毒功能的丧失。在大多数不可逆转的肝损伤病例中，正交肝脏移植（OLT）仍然是唯一可用的治疗方法。为了研究ALF治疗的治疗潜力，它事先在ALF的动物模型中进行测试是至关重要的。在目前的研究中，通过结合70%的局部肝切除术（PHx）和注射对乙酰氨基酚（APAP），在大鼠中开发了ALF模型，这种注射提供了48小时的治疗窗口。肝脏的中位和左侧叶被切除，以切除70%的肝质量，APAP在手术后给予24小时2天。发现ALF诱导动物的存活率严重下降。ALF的发展通过酶丙氨酸氨基转移酶（ALT）、阿斯巴酸氨基转移酶（AST）、碱性磷酸酶（ALP）的血清水平改变得到确认;孕激素时间的变化（PT）;以及国际标准化比率（INR）的评估。qPCR对基因表达特征的研究表明，参与凋亡、炎症和肝损伤进展的基因表达水平增加。通过组织学评价，观察到肝细胞扩散变性和免疫细胞渗透。ALF的可逆性通过恢复ALT、AST和ALP的存活和血清水平，在合成健康大鼠肝细胞的静脉内移植后得到确认。该模型为研究ALF的病理生理学以及评估ALF新疗法的潜力提供了一种可靠的替代方法。使用两种不同的方法也使得研究身体和药物引起的肝损伤的综合作用成为可能。现有程序的可重复性和可行性是该模型的附加优势。
Introduction
急性肝功能衰竭（ALF）被美国肝病研究协会定义为急性肝损伤的迅速发展，没有任何损害前的迹象，其特点是肝脏的合成、代谢和排毒功能严重受损。ALF不同于慢性肝衰竭，其中心力衰竭是由于长期引起的肝损伤和急性慢性肝衰竭（ACLF），其中突然肝损伤发生慢性肝病2，3，4。ALF 的唯一可用治疗方法是正交肝脏移植 （OLT），否则可能发生死亡。由于肝捐赠者短缺，ALF患者的死亡率非常高。
为了研究替代治疗方法的潜力，并更好地了解ALF的病理生理学，需要能够反映人类ALF发生的动物模型。许多已经可用的ALF动物模型有几个缺点。对乙酰氨基酚 （APAP） 效应难以重现，但在时间、临床、生化和病理参数方面有最接近的相似性。APAP诱导的动物模型经常遇到问题，由于由APAP及其中间体5，6，7氧化引起的血红蛋白血症的存在。另一个问题是缺乏可重复性，这反映在不可预测的剂量反应和死亡时间上。使用四氯化碳（CCl4）生产的ALF动物模型具有较差的可重复性8，9，10，11。Concavalin A（Con A）和利波糖（LPS）诱导的ALF动物模型并不反映人类疾病的临床模式，尽管在研究自身免疫性肝病的细胞机制和脓毒症研究中分别具有优势12、13、14、15。同样，硫乙酰胺（TAA）也需要生物转化到活性代谢物硫乙酰胺，并显示物种变异16，17，18，19。D-加曲霉素（D-Gal）产生一些生物化学、代谢和生理变化，类似于ALF，但无法反映整个ALF病理状况20、21、22、23。很少有尝试结合两个或两个以上这些方法，以开发ALF模型，能够更好地反映ALF综合征13。因此，需要进一步的研究，以开发一个模型，能够反映疾病参数，具有更好的可重复性，并提供足够的时间来研究治疗干预的效果。
在目前的研究中，结合部分肝切除术（PHx）和低剂量的肝毒试剂，在大鼠中创建了一种替代ALF模型。APAP在造成肝损伤5，24，25方面具有公认的作用。它是一种广泛使用的镇痛剂，通过形成有毒的代谢物，在超治疗剂量下对肝脏有毒。在发达国家，APAP是许多死亡的原因。部分肝切除术引起的身体损伤启动炎症和肝脏再生过程中的各种过程的激活。注射肝毒剂APAP在肝脏中造成恶劣的环境，防止肝细胞的增殖。这减少了动物的压力期，当与小剂量的肝毒素结合，导致更好的重复程序。因此，利用该模型，研究了两种肝损伤的组合效应。为了描述已开发的ALF动物模型，研究了生理和生化参数。通过联合体健康大鼠肝细胞移植，证实了ALF的成功可逆性。
Protocol
下文所述的程序已获得新德里国家免疫学研究所机构动物伦理委员会的批准。批准的序列号为 IAEC#355/14。 1. 准备 准备手术，如Das B等人26所述。 使用6~8周大、体重为200~250克的近亲繁殖的威斯塔大鼠。 将动物放在标准动物护理条件下，在手术前后用大鼠粪便和利他来喂食。 当执行 70% PHx 时，使用盐酸氯胺酮（100 毫克/千克体重?…
Representative Results
ALF动物模型中的存活率APAP的最佳剂量导致ALF与70%PHx结合被标准化为750毫克/千克体重。治疗方案在70%PHx后24小时开始，当动物从手术中完全恢复时，由24小时间隔的两个APAP剂量组成。第二剂APAP（手术后48小时）施用后，死亡率观察到80%。通过卡普兰-迈尔方法分析和绘制生存百分比（图1）。该模型提供的死亡时间和时间段的可重复性使其成为研究针对ALF的治疗?…
Discussion
为ALF开发适当的动物模型对于更好地了解ALF的发病机制和进展至关重要。具有良好特征的ALF动物模型也为针对ALF的新治疗方法的开发和试验提供了机会。已多次尝试开发与临床相关的ALF6、12、21、23、46、47、48的临床模型。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了印度政府生物技术部向新德里国家免疫学研究所提供的核心赠款的支持。
Materials
| Acetaminophen (Biocetamol) | EG Pharmaceuticals | No specific Catalog Number (Local Procurement) | |
| Alkaline Phosphatase Kit (DEA) | Coral Clinical System, India | No specific Catalog Number (Local Procurement) | |
| Automated analyser | Tulip, Alto Santracruz, India | Screen Maaster 3000 | Biochemical analyser for liver functional test |
| Betadine (Povidon-Iodine Solution) | Win-Medicare; India | No specific Catalog Number (Local Procurement) | |
| Biological safety cabinet (Class I) | Kartos international; India | No specific Catalog Number (Local Procurement) | |
| Bright Field Microscope | Olympus, Japan | LX51 | |
| Cefotaxime (Taxim®) | AlKem; India | cefotaxime sodium injection, No specific Catalog Number (Local Procurement) | |
| Cell Strainer | Sigma; US | CLS431752 | |
| Collagenase Type I | Gibco by Life Technologies | 17100-017 | |
| Cotton Buds | Pure Swabs Pvt Ltd; India | No specific Catalog Number (Local Procurement) | |
| Drape Sheet | JSD Surgicals, Delhi, India | No specific Catalog Number (Local Procurement) | |
| DPX Mountant | Sigma; US | 6522 | |
| Eosin Y solution, alcoholic | Sigma; US | HT110132 | |
| Forceps | Major Surgicals; India | No specific Catalog Number (Local Procurement) | |
| Gas Anesthesia System | Ugo Basile; Italy | 211000 | |
| Glucose | Himedia, India | GRM077 | |
| Hair removing cream (Veet®) | Reckitt Benckiser, India | No specific Catalog Number (Local Procurement) | |
| Hematoxylin Solution, Mayer's | Sigma; US | MHS16 | |
| Heparin sodium salt | Himedia; India | RM554 | |
| Hyaluronidase From Sheep Testes | Sigma; US | H6254 | |
| I.V. Cannula (Plusflon) | Mediplus, India | Ref 1732411420 | |
| Insulin Syringes | BD; US | REF 303060 | |
| Isoflurane (Forane®) | Asecia Queenborough | No B506 | Inhalation Anaesthetic |
| Ketamine (Ketamax®) | Troikaa Pharmaceuticals Ltd. | Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement) | |
| Meloxicam (Melonex®) | Intas Pharmaceuticals Ltd; India | No specific Catalog Number (Local Procurement) | |
| Micro needle holders straight & curved |
Mercian; England | BS-13-8 | |
| Micro needle holders straight & curved |
Mercian; England | BS-13-8 | |
| Microtome | Histo-Line Laboratories, Italy | MRS3500 | |
| Nylon Thread | Mighty; India | No specific Catalog Number (Local Procurement) | |
| Paraformaldehyde | Himedia; India | GRM 3660 | |
| Percoll® | GE Healthcare | 17-0891-01 | |
| Refresh Tears/Eyemist Gel | Allergan India Private Limited/Sun Pharma, India | P3060 | No specific Catalog Number |
| RPMI | Himedia; India | No specific Catalog Number (Local Procurement) | |
| Scalpel | Major Surgicals; India | No specific Catalog Number (Local Procurement) | |
| Scissors | Major Surgicals; India | No specific Catalog Number (Local Procurement) | |
| SGOT (ASAT) KIT | Coral Clinical System, India | No specific Catalog Number (Local Procurement) | |
| SGPT (ALAT) KIT | Coral Clinical System, India | No specific Catalog Number (Local Procurement) | |
| Shandon Cryotome E Cryostat | Thermo Electron Corporation; US | No specific Catalog Number | |
| Sucrose | Sigma; US | S0389 | |
| Surgical Blade No. 22 | La Medcare, India | No specific Catalog Number (Local Procurement) | |
| Surgical Board | Locally made | No specific Catalog Number (Local Procurement) | |
| Surgical White Tape | 3M India; India | 1530-1 | Micropore Surgical Tape |
| Sutures | Ethicon, Johnson & Johnson, India | NW 5047 | |
| Syringes (1ml, 26 G) | Dispo Van; India | No specific Catalog Number (Local Procurement) | |
| Trimmer (Clipper) | Philips | NL9206AD-4 DRACHTEN QT9005 | |
| Weighing Machine | Braun | No specific Catalog Number (Local Procurement) | |
| William's E Media | Himedia; India | AT125 | |
| Xylazine (Xylaxin®) | Indian Immunologicals Limited | Sedative, Pre-Anaesthetic, Analgesic and muscle relaxant |
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Cite This Article
Sahay, P., Jain, K., Sinha, P., Das, B., Mishra, A., Kesarwani, A., Sahu, P., Mohan, K. V., Kumar, M. M., Nagarajan, P., Upadhyay, P. Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen. J. Vis. Exp. (153), e60146, doi:10.3791/60146 (2019).