Моделирование ишемически-реперфузионного повреждения печени и почек в эксперименте: отработка методики

Введение. Ощутимый дефицит качественных донорских органов по-прежнему остаётся одним из наиболее острых вызовов, особенно если речь идёт о донорах с расширенными критериями или об асистолических донорах. Решение этой проблемы возникает на стыке хирургического мастерства, продвинутых биомедицинских технологий и глубокого понимания механизмов ишемически-реперфузионного повреждения (ИРП).
Цель. Данное исследование выполнено с целью обоснования и отработки методики экстракорпоральной ex-vivo перфузии трансплантата печени на животной модели при помощи аппарата перфузионного для экстракорпоральной оксигенации “Ex-Stream” по ТУ 32.50.21-002-75538036-2020 (держатель РУ ООО «Трансбиотек», Санкт-Петербург, Россия, производитель ООО «Биософт-М», Москва, Россия).
Материалы и методы. Исследование проводилось на мужских особях свиней массой 15–30 кг (n = 5). В основу исследования включён анализ результатов гипотермической оксигенированной перфузии трансплантата печени в условиях вивария с использованием аппарата искусственного кровообращения. Исследование проводилось по следующему протоколу: у животного проводились изъятие донорской печени с формированием временного вено-венозного обхода, фармако-холодовая консервация органа при помощи аппарата «Ex-Stream» и её последующая реплантация.
Результаты. В серии из 5 наблюдений были получены следующие результаты. Маркеры повреждения тканей (АСТ, АЛТ, ЛДГ, ГГТП) показали постепенное увеличение их уровня в перфузате с течением времени ишемии. Средние значения АСТ и АЛТ возросли в 2–3 раза, ЛДГ – в 1,5–2 раза, а ГГТП – в 1,2–1,5 раза по сравнению с исходными значениями. Уровень малонового диальдегида, отражающий окислительный стресс, повысился в среднем на 30–40% к концу эксперимента, в то время как уровень глутатиона снизился на 20–25 %. Концентрации провоспалительных цитокинов (TNF-α, IL-6, IL-1β) в перфузате увеличились в 2–4 раза по сравнению с базовыми значениями, указывая на развитие воспалительного ответа. Микроскопическое исследование в окраске гематоксилин-эозин выявило признаки ишемического повреждения гепатоцитов, такие как вакуолизация цитоплазмы, пикноз ядер и нарушение балочной структуры. Степень повреждения нарастала с увеличением времени ишемии. Окраска по Масону показала умеренное увеличение соединительной ткани в портальных трактах и перицентральных зонах, свидетельствующее о начальных фибротических изменениях. Ультрамикроскопическое исследование (просвечивающая электронная микроскопия) выявило набухание митохондрий, нарушение целостности их крист, расширение эндоплазматического ретикулума и образование аутофагосом в гепатоцитах. Потребление кислорода тканью печени постепенно снижалось в ходе эксперимента, достигая 60–70 % от исходного уровня к концу наблюдения. Продукция углекислого газа также уменьшалась, но в меньшей степени, составляя 75–85 % от базовых значений. Анализ перфузата с помощью потенциостата-гальваностата ИПС показал постепенное снижение окислительно-восстановительного потенциала, указывающее на нарастание гипоксии и истощение антиоксидантных резервов. Активность супероксиддисмутазы и каталазы, ключевых антиоксидантных ферментов, снизилась на 30–40 % и 20–30 % соответственно по сравнению с исходными значениями, свидетельствуя об ослаблении антиоксидантной защиты.
Заключение. Полученные результаты свидетельствуют о том, что разработанная модель с использованием аппарата Ex-Stream является воспроизводимой и позволяет эффективно изучать состояние ишемически-реперфузионного повреждения. Это открывает возможности для проведения более масштабной и всесторонней серии экспериментов, результаты которой станут предметом наших дальнейших исследований.

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