Долгосрочные последствия нарушения развития мозга крыс, вызванного пренатальной гипоксией тема диссертации и автореферата по ВАК РФ 00.00.00, доктор наук Ветровой Олег Васильевич
Ветровой Олег Васильевич
доктор наук
2022
- Специальность ВАК РФ00.00.00
- Количество страниц 236
Оглавление диссертации доктор наук Ветровой Олег Васильевич
Введение
Глава 1. Эпигенетические модификации в мозге крыс, переживших
пренатальную гипоксию
Глава 2. Роль индуцируемого гипоксией фактора Н^1а в патогенезе постгипоксической реоксигенации гиппокампа взрослых крыс и особенности его экспрессии у крыс, переживших пренатальную
гипоксию
Глава 3. Центральные и периферические нарушения глюкокортикоидной системы крыс, переживших пренатальную
гипоксию
Глава 4. Особенности функционирования глутаматэргической системы
гиппокампа крыс, переживших пренатальную гипоксию
Заключение
Выводы
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Введение диссертации (часть автореферата) на тему «Долгосрочные последствия нарушения развития мозга крыс, вызванного пренатальной гипоксией»
ВВЕДЕНИЕ
Актуальность темы исследования. Значительная часть распространенных заболеваний, в том числе неврологических и нервно-психических, возникает в результате повреждающего действия на мозг различных неблагоприятных факторов, среди которых большая роль отводится гипоксии/ишемии (Graham et al., 2008; Li et al., 2012; Gonzalez-Rodriguez et al., 2014).
В первую очередь к гипоксическим патологиям относятся ассоциированные с возрастом заболевания, такие как инфаркт миокарда и ишемический инсульт. Однако, крайне опасно действие гипоксии и в раннем онтогенезе - в период пренатального и раннего постнатального развития (Rice, Barone, 2004; Golan, Huleihel, 2006). Пренатальная гипоксия хорошо известна как фактор риска расстройств аутистического спектра, шизофрении и ненаследственных форм нейродегенеративных заболеваний. Проявляясь в младенчестве в нарушении физического, эмоционального и умственного развития ребенка, последствия пренатальной гипоксии сохраняются у взрослых и усугубляются с возрастом, ведя к преждевременному старению и ранней смертности (Langley-Evans, McMullen, 2010; Warner, Ozanne, 2010; Dudley et al., 2011; Xiong, Zhang, 2013).
К формированию патологий развития ведут нарушения настройки порогов чувствительности сигнальных систем организма плода,
опосредованные не только самой гипоксией, но и выбросом стрессорных гормонов организмом матери (Li et al., 2012; Tomalski, Johnson, 2010).
Настоящая работа посвящена исследованию механизмов нарушения развития и дальнейшего функционирования мозга крыс вследствие гипоксического стресса и стрессорного ответа матери на гипоксию, перенесенных во время внутриутробного развития.
При изучении последствий пренатальной гипоксии на развитие
/»" и и
мозга особый интерес представляет гиппокамп - важнейшая экстрагипоталамическая структура мозга, контролирующая глюкокортикоидную отрицательную обратную связь, а также обеспечивающая формирование эмоций и пространственную память (Fanselow, Dong, 2010). Известно, что 14-16-е сутки эмбрионального развития головного мозга крыс соответствуют 5-7-ой неделе беременности человека. В этот период начинается формирование гиппокампа (Golan, Huleihel, 2006). Воздействие гипоксии и стрессорный ответ матери в период его развития могут привести к устойчивым нарушениям функционирования этой структуры на молекулярно-клеточном уровне. В фокусе внимания нашего исследования были особенности паттернов эпигенетических модификаций хроматина в мозге, модификация активности индуцируемого гипоксией транскрипционного фактора HIF1, периферические и центральные компоненты глюкокортикоидной нейроэндокринной системы на протяжении всей жизни крыс, подвергавшихся пренатальной гипоксии, а также возраст-ассоциированные изменения пространственной памяти и
функционирования глутаматэргической медиаторной системы гиппокампа.
Степень разработанности темы исследования. Клинические и экспериментальные исследования показывают, что внутриутробный стресс влияет на развитие плода, что приводит к задержкам в развитии в период раннего постнатального онтогенеза и потенциальному риску дальнейшего развития нейродегенеративных заболеваний (Dudley et al., 2011; Xiong, Zhang, 2013). По наблюдениям клиницистов, особое место среди факторов риска нарушений развития в пренатальном онтогенезе занимает гипоксия, являющаяся одним из самых часто встречающихся осложнений при беременности и родах. Внутриутробная гипоксия плода и асфиксия новорожденного, во многом определяющие уровень мертворождаемости, неонатальной и младенческой смертности, являются одной из наиболее актуальных проблем акушерства и перинатологии. Значимость данной проблемы обусловлена не только существенными перинатальными потерями, но и тем, что и у выживших детей, перенесших воздействие перинатальной гипоксии, в дальнейшем нередко развиваются различные нарушения здоровья, приводящие к формированию инвалидности и снижению качества жизни в целом. В структуре заболеваемости новорожденных, перенесших перинатальную гипоксию, одно из важных мест занимает перинатальное поражение мозга, что связано с высокой распространенностью данной патологии (Li et al., 2012; Gonzalez-Rodriguez et al., 2014). По данным Комитета
экспертов Всемирной Организации Здравоохранения у 10% детей регистрируются нервно-психические заболевания, 70-80% которых связаны с перинатальными поражениями мозга. От 46% до 60% случаев перинатальных поражений центральной нервной системы у детей имеют гипоксическую природу.
Эффекты пренатальной гипоксии не всегда соотносятся со степенью ее тяжести. Первостепенное значение имеют сроки онтогенеза, в которые произошло воздействие. В течение пренатального и раннего постнатального онтогенеза выделяют несколько критических периодов, когда организм и, в особенности, мозг становятся особенно восприимчивыми к неблагоприятным воздействиям, а негативные последствия таких воздействий наиболее значимы (Rice, Barone, 2000; Golan, Huleihel, 2006). Нарушения, обусловленные действием повреждающих факторов в эти критические периоды раннего онтогенеза, могут приводить к возникновению не только грубых дефектов развития (врожденные аномалии - уродства), но и многочисленных функциональных расстройств в деятельности клеток, органов и систем всего организма. Если воздействия происходят в период раннего пренатального онтогенеза (активного органогенеза), то оно может приводить к увеличению частоты хромосомных аберраций в клетках тканей организма, а также появлению таких пороков развития, как аненцефалия, ацефалия, анофтольмия, заячья губа, фокомелия, амелия, атрезия ротового отверстия (Dunaeva et al., 2008). В тоже время, появление функциональных нарушений связывают с неблагоприятными
воздействиями на более поздних сроках пренатального онтогенеза, период активного нейрогенеза в различных областях развивающегося мозга, включая неокортекс и гиппокамп (7Иигэу1п е1 а1., 2004; Уа1аеуа е1 а1., 2004, 2005, 2007; ЭиЬгоУБкауа, 7Иигау1п, 2010; Тюлькова и др., 2015). Несмотря на то, что к настоящему времени накоплен достаточно большой массив данных по эффектам и механизмам гипоксических воздействий в раннем онтогенезе, концептуальное осмысление этих сведений, зачастую противоречивых, затруднено в связи с тем, что эти данные получены в разнообразных моделях ишемии и гипоксии, применяемых в различные периоды пре- и перинатального онтогенеза. В основном исследуются влияния ишемической и/или нормобарической гипоксии в течение всего периода беременности или тестируются отдельные (точечные) сроки, и, как уже отмечалось, полученные в этих работах данные не всегда согласуются. Требуется последовательное и комплексное изучение эффектов и механизмов пренатальной гипоксии в одной модели на всех уровнях, включая нарушения поведения, нейроэндокринную регуляцию и особенности функционирования медиаторных систем. В качестве
/»" и и /»" и
удобной экспериментальной модели для подобного рода исследований служит гипобарическая (высотная) гипоксия, создаваемая в барокамере, поскольку она является физиологически адекватным воздействием (встречается в естественных условиях при подъеме на высоту), легко контролируется и дозируется, что создает возможности для ее применения в различных режимах.
Гипоксия опосредует свое влияние на плод через организм матери и плаценту. При остром недостатке кислорода запускается целый каскад эффектов, включающих выброс стрессовых гормонов в кровь матери и структурно-функциональные изменения в материнской и фетальной частях плаценты. В какой степени наблюдающиеся при повреждающих гипоксических/ишемических воздействиях во время беременности изменения обусловлены собственно гипоксическим фактором, а в какой -неспецифической стрессовой реакцией матери, до сих пор остается неясным. Неспецифичным элементом реакции на стрессорные воздействия является увеличение концентрации гормонов гипоталамо-гипофизарно-адренокортикальной системы (ГГАС): кортикостероидов (глюко- и минералокортикоидов) и адренокортикотропного гормона гипофиза. Именно эти гормоны определяют увеличение адаптационных возможностей организма, вызывая перестройку метаболических процессов. Глюкокортикоиды не только обеспечивают адаптивные реакции организма на стресс, но и регулируют широкий спектр биологических процессов у взрослых животных, а также играют важную роль в развитии плода. Недостаток глюкокортикоидов, так же, как и их избыток может играть фатальную роль в развитии организма. Концентрация глюкокортикоидов в сыворотке крови плода низка на протяжении большей части беременности, однако она резко увеличивается на 36 неделе гестации у человека и после 15 дня эмбрионального развития у крыс и мышей. Регуляция образования этих гормонов у плода намного сложнее, чем у взрослых, поскольку
материнские глюкокортикоиды могут проникать через плаценту. Их синтез лишь частично регулируется гипоталамо-гипофизарно-надпочечниковой системой и в основном зависит от экспрессии ферментов синтеза глюкокортикоидов (Busada, Cidlowski, 2017). Несмотря на то, что наиболее известной функцией глюкокортикоидов является их участие в стимуляции дифференциации и функционального становления легких, они также играют решающую роль в развитии других органов, в частности, головного мозга. В последние годы идентифицировано множество генов, регулируемых глюкокортикоидами, в том числе порядка 80-90 генов в головном мозге. Это гены различных факторов роста, факторов транскрипции, белков сигнальных путей, ферментов метаболизма (в частности глюкозы) и пр. (Juszczak, Stankiewicz, 2018). Показано, что глюкокортикоиды выполняют нейротрофическую и нейропротекторную роль в ЦНС, необходимы для созревания и поддержания жизнедеятельности различных клеток, включая нейрональные клетки гиппокампа, мозжечка (клетки Пуркинье и гранулярные клетки), коры, а также олигодендроцитов (Malaeb, Stonestreet, 2014) и определяют сбалансированность процессов синтеза и деградации медиаторных молекул как напрямую, так и посредством регуляции поступления глюкозы в мозг, с одной стороны являющейся энергетическим субстратом, а с другой представляющей собой предшественник синтеза таких медиаторов как гамма-аминомасляная кислота и глутамат. В то же время избыток глюкокортикоидов (хронический стресс матери или введение синтетических гормонов)
может оказывать негативные эффекты и приводить к развитию различного рода заболеваний уже в постнатальном периоде. При этом глюкокортикоиды широко используются в перинатальной и неонатальной медицине. Их назначают беременным женщинам для предотвращения преждевременных родов, как противовоспалительные и противоаллергические препараты, а также новорожденным детям с нарушением дыхательной функции. В настоящее время, в связи с накоплением данных о долгосрочном влиянии введения глюкокортикоидов при беременности, в особенности на нервную систему, их применение в этот период вызывает много вопросов (Moors e.al., 2012, Khalife et al., 2013, Sorrells et al., 2014). Стоит также отметить, что влияние глюкокортикоидов на развивающийся мозг во многом зависит как от стадии развития во время воздействия, так и продолжительности воздействия. Однако молекулярные механизмы, ответственные за долгосрочные эффекты глюкокортикоидов, по-прежнему изучены недостаточно. Известно, что действие глюкокортикоидов опосредуется двумя типами рецепторов: минералокортикоидными и глюкокортикоидными, которые в комплексе с гормонами функционируют как транскрипционные факторы (de Kloet, 2013). При этом глюкокортикоиды могут как стимулировать, так и подавлять транскрипцию (Cohen, Steger, 2017). Важно отметить, что как эндогенные, так и синтетические глюкокортикоиды могут вызывать эпигенетические изменения, которые оказывают влияние на развитие плода (Concepcion, Zhang, 2018). Кроме того, глюкокортикоиды могут
оказывать быстрые негеномные эффекты, влияющие, например, на возбудимость нейронов или вызывающие ингибирование пролиферации нейронов, сопровождающееся неврологическими дефектами, наблюдаемыми у младенцев после хронического стресса у матерей или после лечения синтетическими глюкокортикоидами (Виsada, Cidlowski, 2017). В настоящее время совершенно очевидно вовлечение устойчивых отклонений чувствительности глюкокортикоидной системы в экстрагипоталамических структурах мозга, участвующих в обеспечении
и /"" и
глюкокортикоидной обратной связи, таких как гиппокамп, в развитии ассоциированных с возрастом эндокринных и неврологических расстройств. Исследованию конкретных молекулярных механизмов подобных нарушений, закладываемых в пренатальном онтогенезе и определяющих качество всей дальнейшей жизни и была посвящена настоящая работа.
Цель исследования. Изучить влияние пренатальной гипоксии на некоторые аспекты функционирования мозга крыс на протяжении постнатального онтогенеза.
Задачи. Для достижения указанной цели были поставлены следующие задачи:
1. Исследовать влияние пренатальной гипоксии на паттерны эпигенетических модификаций мозга крыс на протяжении постнатального онтогенеза.
2. Оценить особенности функционирования индуцируемого гипоксией транскрипционного фактора HIF1a в гиппокампе на протяжении жизни крыс, переживших пренатальную гипоксию.
3. Сравнить эффекты стрессорного ответа матери на гипоксию и введения синтетического глюкокортикоида дексаметазона на экспрессию и транскрипционную активность глюкокортикоидных рецепторов в гиппокампе потомства.
4. Провести комплексный анализ особенностей функционирования периферических компонентов глюкокортикоидной нейроэндокринной системы на протяжении жизни крыс, переживших пренатальную гипоксию.
5. Проанализировать особенности функционирования глутаматэргической системы гиппокампа крыс, переживших пренатальную гипоксию.
6. Изучить влияние пренатальной гипоксии на особенности пространственной памяти и ассоциированную с возрастом потерю нейронов в гиппокампе крыс.
Материалы. Работа выполнена на взрослых самках крыс линии Wistаr весом 350г и их потомстве. Животные были получены из Биоколлекции Института физиологии им. И.П. Павлова РАН. Все измерения проведены с использованием материалов минимум из двух независимых экспериментов. В каждой экспериментальной группе (контрольной или
и \ и и /-
опытной) на каждой временной точке использовано не менее 6
животных.
Методы. В работе использована тяжелая гипобарическая гипоксия (ТГ) (180 мм.рт.ст, 5 % O2, 3 сеанса по 3 часа с интервалами в 24 часа), которой подвергали беременных самок крыс на 14, 15 и 16 сутки беременности (пренатальная гипоксия, ПГ). В этот период у плода происходит закладка гиппокампа, важнейшей экстрагипоталамической структуры, контролирующей глюкокортикоидную отрицательную обратную связь, а также отвечающей за пространственную память и обучение. Кроме того, стрессорный ответ матери был имитирован посредством введения синтетического глюкокортикоида дексаметазона (0,8 мг/кг) на 14, 15 и 16 сутки беременности. В дополнительной серии экспериментов беременным самкам крыс перед сеансами гипоксии или без них на 14, 15 и 16 сутки беременности вводили блокатор синтеза глюкокортикоидов метирапон (группы Мет и ПГ+Мет).
Количество кортикостерона, основного глюкокортикоидного гормона крыс, определяли в плазме крови беременных самок и у самцов потомков в возрасте 1 день (новорожденные), 2 недели (ювенильные), 3 месяца (взрослые) и 18 месяцев (стареющие) методом конкурентного иммуноферментного анализа.
Методом вестерн блот изучали распределение транскрипционных факторов HIF1a и глюкокортикоидных рецепторов (GR) между ядерной и цитозольной фракциями гиппокампа 1-дневных крысят (цитозольный
референсный белок Актин-0, ядерный - NeuN), а также проводили количественный анализ метаботропных глутаматных рецепторов 1го типа (mGluR1) в тотальной фракции гиппокампа 18-месячных крыс.
Количество и локализацию HIF1a, GR, а также посттрансляционных эпигенетических модификаций хроматина в мозге и печени 1-дневных, 2-недельных, 3- и 18-месячных самцов крыс определяли иммуногистохимическим методом. Кроме того,
иммуногистохимическим методом также исследовали возрастные изменения количества нейронов, астроцитов, mGluR1 и рецепторов инозитол-3-фосфата (IP3R1) в гиппокампе.
Изменение количества инозитол-3-фосфата (^3) на переживающих срезах в ответ на аппликацию агониста mGluR1 DHPG определяли хроматографически.
Возрастные изменения количества глутамата в гиппокампе крыс изучали энзиматическим колориметрическим методом.
Функциональное состояние системы рецепции глутамата определяли на взрослых крысах с использованием модели ТГ в качестве эксайтотоксического теста. Эффективность рецепции глутамата измеряли по изменению продуктов перекисного окисления липидов (ПОЛ) спектрофотометрическими и флуориметрическими методами.
Эффективность периферической рецепции глюкокортикоидов определяли по активности глюкозо-6-фосфатазы (энзиматический
колориметрический метод), количеству гликогена (РЛБ-реакция) в
печени и количеству глюкозы в капиллярной и артериальной крови.
Количество мРНК HIF1-зависимых, глюкокортикоид-зависимых генов, а также генов ферментов метаболизма глутамата, глутаматных транспортеров в гиппокампе определяли методом количественной полимеразной цепной реакции в реальном времени (РВ ПЦР).
Пространственную память и способность к обучению оценивали с использованием водного лабиринта Морриса у взрослых (3 месяца) и стареющих (18 месяцев) контрольных крыс и крыс, переживших ПГ.
Для оценки достоверности различий между группами использовали однофакторный дисперсионный анализ (ANOVA) с последующим тестом Тьюки. Статистическая значимость была установлена на уровне р <0,05.
Положения, выносимые на защиту:
1. Пренатальная гипоксия вызывает устойчивые изменения паттернов эпигенетических модификаций хроматина, ассоциированные со снижением его активности, в гиппокампе и неокортексе крыс на протяжении всей жизни.
2. Увеличение содержания и транскрипционной активности индуцируемого гипоксией транскрипционного фактора HIF1a в гиппокампе крыс, переживших пренатальную гипоксию, сохраняется после рождения, стабилизируясь к половозрелому возрасту. При этом в процессе старения вновь происходит увеличение количества этого белка, что может отражать возрастные нарушения
энергетического метаболизма мозга или вовлекаться в их формирование.
3. Стрессорный ответ матери на гипоксию, так же, как и введение синтетического глюкокортикоида дексаметазона во время беременности, вызывают снижение чувствительности гиппокампа потомства к глюкокортикоидам в результате снижения экспрессии глюкокортикоидных рецепторов и эффективности глюкокортикоид-зависимой транскрипции. Снижение чувствительности к глюкокортикоидам гиппокампа крыс, переживших пренатальную гипоксию, стабильно на протяжении всей жизни. При этом использование ингибитора синтеза кортикостерона предотвращает уменьшение количества глюкокортикоидных рецепторов в гиппокампе после пренатальной гипоксии. Это свидетельствует о центральной роли стрессорного ответа матери на гипоксию в стабильном нарушении чувствительности гиппокампа потомства к глюкокортикоидам, что в дальнейшем определяет центральные и периферические нарушения функционирования глюкокортикоидной системы.
4. Следствием нарушения чувствительности гиппокампа крыс, переживших пренатальную гипоксию, к глюкокортикоидам является стабильная гиперактивация гипоталамо-гипофиз-адренокортикальной оси. Устойчивое увеличение базального уровня глюкокортикоидов сопровождается возрастными
нарушениями их периферической рецепции и, как следствие, реализации глюкокортикоид-зависимых функций.
5. Пренатальная гипоксия вызывает возраст-ассоциированное нарушение синтеза и увеличение деградации глутамата, сопровождающееся неэффективной гиперактивацией рецепторного аппарата глутаматэргической системы в гиппокампе крыс.
6. Долгосрочные последствия пренатальной гипоксии выражаются в развитии состояния «ранней старости», проявляющегося в преждевременной потере нейронов и, как следствие, прогрессирующем с возрастом когнитивном дефиците.
Научная новизна. Ранее нами и другими исследователями было установлено, что пренатальная гипоксия приводит к устойчивым нарушениям двигательного, эмоционального, исследовательского поведения и способности к обучению. Показано, что эти нарушения наиболее выражены, если гипоксическое воздействие приходится на начало третьей недели гестации - период активного нейрогенеза в различных областях развивающегося мозга, в первую очередь гиппокампа.
Результаты многолетних исследований, проводимых в нашей лаборатории, позволили выдвинуть гипотезу о механизмах формирования отдаленных негативных последствий пренатальной гипоксии. Согласно этой гипотезе патологии развития мозга, вызванные пренатальной гипоксией, помимо нарушения кислородного снабжения
определяются неадекватным уровнем глюкокортикоидной стимуляции плода, которая ведет к снижению чувствительности гиппокампа к глюкокортикоидам и последующему проявляющемуся с возрастом ослаблению периферической регуляции глюкокортикоид-зависимых процессов. Изменения функциональной активности гиппокампа также проявляются в нарушении работы индуцируемого гипоксией сигналинга и дисфункции глутаматэргической медиаторной системы гиппокампа, приводящим к дефициту пространственной памяти и ранней потере нейронов. Впервые показано, что эти изменения связаны с устойчивыми изменениями эпигенетического кода. Полученные данные о механизмах действия гипоксии и глюкокортикоидов матери на развивающийся мозг плода, а также о последствиях нарушения функционирования гиппокампа открывают широкие возможности для ранней диагностики и эффективной коррекции широкого спектра возрастных неврологических заболеваний спорадического характера.
Теоретическая и практическая значимость работы. Результаты работы расширяют современные представления о молекулярно-биологических процессах, приводящих к развитию патологий постнатального развития вследствие пренатальной гипоксии. Результаты данного фундаментального исследования имеют не только теоретическую, но и прикладную ценность. Высокая практическая значимость работы определяется клинической необходимостью разработки новых способов ранней диагностики последствий пренатальных патологий и поиска
эффективных путей повышения общей резистентности организма. Полученные результаты на молекулярно-клеточном уровне могут быть использованы при создании нового поколения эффективных фармакологических препаратов, направленных на предотвращение последствий пренатального стресса.
Степень достоверности и апробации результатов. Результаты получены с помощью современных биохимических, молекулярно-биологических и гистохимических и поведенческих методов. Объем выборок и число независимых экспериментов позволили оценить значимость результатов после обработки с помощью адекватных методов статистического анализа. Результаты работы были представлены и обсуждены на 45 всероссийских и международных конференциях, наиболее значимые из которых: ISN-ESN Meeting (Paris, France, 2024.08.2017), FENS Forum of Neuroscience (Berlin, Germany, 7-11.07.2018), ISN-ASN Meeting (Montreal, Canada, 4-8.08.2019), ESN Conference on Molecular Mechanisms of Regulation in the Nervous System (Milan, Italy, 14.09.2019), online FENS Forum of Neuroscience (Glasgow, Scotland, 1115.07.2020), 1st ESN Virtual Conference "Future perspectives for European neurochemistry - a young scientists conference" (25-26.05.2021).
Публикации. По теме диссертации опубликована 81 работа, из них 21 статья в рецензируемых журналах, рекомендованных ВАК РФ, 60 тезисов конференций.
Личный вклад соискателя. Личный вклад диссертанта состоял в анализе литературы по проблеме исследования, разработке гипотезы, планировании экспериментов и их выполнении, статистической обработке полученных результатов, обсуждении результатов, подготовке публикаций по теме диссертации.
Структура диссертации. Диссертация включает введение, четыре главы, заключение и выводы; изложена на 121 странице текста, содержит 28 иллюстраций и 148 ссылок на использованные источники литературы.
Финансовая поддержка. Работа выполнена при поддержке грантов РФФИ № 16-04-00872, 16-34-00027, 17-04-01118.
ГЛАВА 1. Эпигенетические модификации в мозге крыс, переживших
пренатальную гипоксию
В рамках работ, направленных на исследование влияния ПГ и стрессорного ответа матери во время беременности на паттерны эпигенетических модификаций в мозге крыс на протяжении постнатального онтогенеза с использованием количественного иммуногистохимического метода нами были изучены посттрансляционные модификации гистона Н3 - ацетилирование по лизину 24 (асН3К24) (Тюлькова и др., 2017, 2019), метилирование по лизинам 4 и 9 (теН3К4 и теН3К9), а также общее метилирование ДНК (meDNA) в клетках пятого слоя неокортекса (НеоУ) и СА1 поля гиппокампа крыс (Тюлькова и др., 2020) (Рис. 1).
Эпигенетические модификации вследствие воздействия пренатальной гипоксии
Активационные модификации хроматина Тормозные модификации хроматина
acH3k24 шеНЗк4 meH3k9 meDNA
3 месяца 2 недели 3 месяца 18 месяцев 2 недели 3 месяца 18 месяцев 2 недели 3 месяца 18 месяцев
Д N N ti и fi- fi t
N 4 N N t N fí fi N
Рисунок 1. Результаты иммуногистохимического исследования эпигенетических модификаций в гиппокампе (СА1 поле) и неокортексе (НеоУ) крыс, переживших ПГ. асН3К24, ацетилирование по лизину 24 гистона Н3; теН3К4 и теН3К9, метилирование по лизинам 4 и 9 гистона Н3, соответственно, meDNA, метилирование ДНК.
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БЛАГОДАРНОСТИ
В первую очередь автор выражает свою благодарность своим научным наставникам (Тюлькова Е.И., Ещенко Н.Д., Рыбникова Е.А.) за неоценимый вклад в осуществление данной работы, конструктивные дискуссии и концептуальный анализ полученных результатов, а также за бесценные советы по улучшению диссертации.
Также хотелось бы поблагодарить сотрудников лаборатории регуляции функций нейронов мозга Института Физиологии им. И.П. Павлова РАН (Аксенова Е.В., Баранова К.А., Сариева К.В., Стратилов В.А.), сотрудников кафедры биохимии и ресурсного центра «Обсерватория экологической безопасности» Санкт-Петербургского Государственного Университета (Галкина О.В., Лянгузов А.Ю.), Института Цитологии РАН (Ломерт Е.В.) и Института регенеративной медицины Московского Государственного Университета им. М.В. Ломоносова (Макаревич П.И., Нимирицкий П.П.), совместно с которыми данная работа была осуществлена. Работа выполнена при поддержке грантов РФФИ № 16-04-00872, 16-3400027, 17-04-01118.
Pavlov Institute of Physiology Russian Academy of Sciences
Manuscript copy
Oleg V. Vetrovoy
LONG-TERM EFFECTS OF PRENATAL HYPOXIA-INDUCED IMPAIRMENT OF
RAT BRAIN DEVELOPMENT
1.5.4. Biochemistry
THE DISSERTATION submitted for the degree of Doctor of Biological Sciences
Translation from Russian
Scientific Advisors: Doctor of Biological Sciences, Ekaterina I. Tyulkova Doctor of Biological Sciences, Professor, Natalia D. Eschenko
Saint Petersburg, 2022
123
CONTENT
INTRODUCTION....................................................................................................124
Chapter 1. Epigenetic modifications in the brain of rats survived prenatal
hypoxia..........................................................................................................139
Chapter 2. The role of hypoxia-inducible factor HIF1a in the pathogenesis of post-hypoxic reoxygenation in hippocampus of adult rats and features of its
expression in rats survived prenatal hypoxia.................................................145
Chapter 3. Central and peripheral disturbances of the glucocorticoid system in
rats survived prenatal hypoxia.............................................................................160
Chapter 4. Prenatal hypoxia induces impaired function of the glutamate
system in rat hippocampus............................................................................177
SUMMARY.....................................................................................................198
CONCLUSIONS...............................................................................................204
REFERENCES..................
ACKNOWLEDGEMENTS
236
124
INTRODUCTION
Relevance of the research topic. A significant part of common diseases, including neurological and neuropsychiatric ones, arise from damaging effect on the brain of various adverse factors, among which the leading role is played by hypoxia / ischemia. (Graham et al., 2008; Li et alv 2012; Gonzalez-Rodriguez et al., 2014).
First of all, age-associated diseases such as myocardial infarction and ischemic stroke are related to hypoxic pathologies. However, hypoxia has an extremely dangerous effect within early ontogenesis - during prenatal and early postnatal development. (Rice, Barone, 2004; Golan, Huleihel, 2006). Prenatal hypoxia is well known as a risk factor for autism spectrum disorders, schizophrenia, and non-hereditary forms of neurodegenerative diseases. In infancy manifested as violation of the physical, emotional, and mental development of the child, the consequences of prenatal hypoxia persist in adults and then worsen with age, leading to premature aging and early mortality (Langley-Evans, McMullen, 2010; Warner, Ozanne, 2010; Dudley et al., 2011; Xiong, Zhang, 2013).
Violations in setting the sensitivity thresholds of fetal signaling systems are mediated not only by hypoxia itself, but also by the release of stress hormones of maternal organism, eventually lead to formation of developmental pathologies (Tomalski, Johnson, 2010; Li et al., 2012).
This work is devoted to the study of the mechanisms of impairment of the development and further functioning of the rat brain due to hypoxic stress
and maternal stress response to hypoxia, which has its effect during intrauterine development.
The hippocampus is of particular interest for studying the consequences of prenatal hypoxia on brain development, since it is the most important extrahypothalamic structure of the brain, which controls glucocorticoid negative feedback, and provides the emotional processing and spatial memory formation (Fanselow, Dong, 2010). It is known that the 14-16th day of embryonic development of the rat brain corresponds to the 5-7th week of human pregnancy. Hippocampal development begins during this period (Golan, Huleihel, 2006). Exposure to hypoxia and the maternal stress response during its covering can lead to permanent disruptions in the functioning of this structure at the molecular-cellular level. Our study focused on the features of the patterns of epigenetic modifications of chromatin in the brain, alterations in the activity of the hypoxia-induced transcription factor HIF1, peripheral and central components of the glucocorticoid neuroendocrine system throughout the life of rats subjected to prenatal hypoxia, as well as age-associated changes in spatial memory and functioning of hippocampal glutamate system.
Elaboration of the research topic. Clinical and preclinical studies show that intrauterine stress affects fetal development, it leads to developmental delays during early postnatal ontogenesis and risk of neurodegenerative pathologies in later life (Dudley et al., 2011; Xiong, Zhang, 2013). According to the observations of clinicians, hypoxia occupies a special place among the risk
factors for developmental disorders in prenatal ontogenesis, which is one of the most common complications of pregnancy and childbirth. Fetal hypoxia and asphyxia of the newborn in many respects determining the level of stillbirth, neonatal and infant mortality, is one of the most pressing problems of obstetrics and perinatology. The significance of this problem is not only an effect of significant perinatal losses but also is that children who have survived perinatal hypoxia often develop various health problems later on, leading to the formation of disability and reduced quality of life in general.
In the structure of the incidence of newborns with perinatal hypoxia, one of the important places is occupied by perinatal brain damage, which is associated with a high prevalence of this pathology and increased importance for the subsequent neuropsychological development of a child (Li et al., 2012; Gonzalez-Rodriguez et al., 2014). According to the World Health Organization Expert Committee, neuropsychiatric diseases are registered in 10% of children, and 70-80% of them are associated with perinatal brain lesions. From 46% to 60% of cases of perinatal lesions of the central nervous system in children are hypoxic.
The effects of prenatal hypoxia do not always correlate with its severity. The period of the ontogenesis at which the exposure occurred is crucial. During prenatal and early postnatal ontogenesis, there are several critical periods when the body and, in particular, the brain become especially susceptible to adverse effects, and the negative consequences of such effects are most significant (Rice, Barone, 2000; Golan, Huleihel, 2006) Violations caused by the action of damaging factors during these critical periods of early
ontogenesis can lead to the appearance of not only serious developmental defects (congenital anomalies and malformations), but also numerous functional disorders in the activity of cells, organs, and systems of the whole organism. If the effects occur during the period of early prenatal ontogenesis (active organogenesis), then it can lead to an increase in the frequency of chromosomal aberrations in the cells of body, as well as the appearance of such malformations as anencephaly, acephaly, anophthalmia, cleft palate, phocomelia, amelia, and oral atresia. (Dunaeva et al., 2008). Meanwhile, appearance of functional disorders is associated with adverse effects at a later stage of prenatal ontogenesis, which is a period of active neurogenesis in various areas of the developing brain, including the neocortex and hippocampus (Zhuravin et al., 2004; Vataeva et al., 2004; 2005; 2007; Dubrovskaya, Zhuravin, 2010; Tyulkova et al., 2015). Despite the fact that a large body of data on the effects and mechanisms of hypoxic effects in early ontogenesis has been accumulated to date, the conceptual understanding of this often-contradictory information is difficult due to these data are obtained in various models of ischemia and hypoxia used in various periods of pre- and perinatal ontogenesis. Basically, the effects of ischemic and/or normobaric hypoxia are studied during the entire or individual period of pregnancy and as already noted, the data obtained in these works are not always consistent. As a convenient experimental model for this kind of research hypobaric hypoxia created in a pressure chamber is used, since it is a physiologically adequate effect (found in natural conditions when climbing to a height), it is easily
controlled and dosed, which makes it possible to use it in the various regimens.
Hypoxia mediates its effect on the fetus through the mother's body and placenta. In acute oxygen deficiency, a whole cascade of effects is triggered, including the release of stress hormones into the mother's blood and structural and functional changes in the maternal and fetal parts of the placenta. It is still not clear whether what extent the changes observed during damaging hypoxic/ischemic effects during pregnancy are due to the hypoxic factor itself, and whether what extent the nonspecific stress response of the mother. A non-specific element of the stress reaction is an increase in the concentration of the hormones of the hypothalamic-pituitary-adrenocortical system: corticosteroids (gluco- and mineralocorticoids) and adrenocorticotropic hormone of the pituitary gland. they are these hormones determine the increase in the adaptive capacity of the body, causing a restructuring of metabolic processes. Glucocorticoids not only provide the body's adaptive responses to stress, but also regulates a wide range of biological processes in adult animals, and also play an important role in the development of the fetus. Lack of glucocorticoids, as well as their excess, can play a fatal role in the development of the organism. The concentration of glucocorticoids in the serum of the fetus is low throughout most of the pregnancy, but it increases dramatically at 36 weeks of gestation in humans and after 15 days of fetal development in rats and mice. Regulation of the formation of these hormones in the fetus is much more difficult than in adults since maternal glucocorticoids can penetrate through the placenta. Their
synthesis is only partially regulated by the hypothalamic-pituitary-adrenal system and mainly depends on the expression of glucocorticoid synthesis enzymes (Busada, Cidlowski, 2017). Despite the fact that the most well-known function of glucocorticoids is their participation in stimulating the differentiation and functional formation of the lungs, they also play a crucial role in the development of other organs, including the brain. In recent years, many glucocorticoid-regulated genes have been identified, including about 80-90 genes in the brain. These are genes of various growth factors, transcription factors, proteins of signaling pathways, metabolism enzymes (in particular, glucose), etc. (Juszczak, Stankiewicz, 2018). In addition, glucocorticoids determine the balance of the processes of synthesis and degradation of mediator molecules, both directly and by regulating the flow of glucose into the brain, on the one hand being an energy substrate, and on the other representing a precursor to the synthesis of mediators such as gamma-aminobutyric acid and glutamate. It is shown that glucocorticoids play a neurotrophic and neuroprotective role in the central nervous system and are necessary for the maturation and maintenance of various cells, including neuronal cells of the hippocampus and cerebellum (Purkinje cells and granular cells), cortex, and oligodendrocytes (Malaeb, Stonestreet, 2014). On the other hand, an excess of glucocorticoids (chronic maternal stress or the introduction of synthetic hormones) can have negative effects and lead to the development of various diseases already in the postnatal period. Currently, glucocorticoids are widely used in perinatal and neonatal medicine. They are prescribed to pregnant women to prevent preterm birth, as an anti-
inflammatory and antiallergic drugs, as to newborns with impaired respiratory function. At present, due to the accumulation of data on the long-term effect of glucocorticoid administration during pregnancy, especially on the nervous system, their using within this period raises many questions (Moors et al., 2012, Khalife et al., 2013, Sorrells et al., 2014). It is also worth noting that the effect of glucocorticoids on the developing brain depends largely on both the stage of development during exposure and the duration of exposure. However, the molecular mechanisms responsible for the long-term effects of glucocorticoids are still not well understood. It is known that glucocorticoid action is mediated by two types of receptors: mineralocorticoid and glucocorticoid, which function as transcription factors (de Kloet, 2013). In addition, glucocorticoids can both stimulate and suppress transcription (Cohen, Steger, 2017). It is important to note that both endogenous and synthetic glucocorticoids can cause epigenetic changes that affect the development of the fetus (Concepcion, Zhang, 2018). In addition, glucocorticoids can have fast non-genomic effects, for example, affecting neuronal excitability or causing inhibition of neuronal proliferation, accompanied by neurological defects observed in infants after chronic stress in mothers or after treatment with synthetic glucocorticoids (Busada, Cidlowski, 2017).
At present, the involvement of persistent deviations in the sensitivity of the glucocorticoid system in the extrahypothalamic brain structures involved in providing glucocorticoid feedback, such as the hippocampus, in the development of age-related endocrine and neurological disorders is quite
obvious. The present project is devoted to the study of the specific molecular mechanisms of such disorders, which take their origin in prenatal ontogenesis and determine the quality of the rest of life, the identification of early markers, as well as the attempt to timely correction.
Purpose of the study is to investigate the effect of prenatal hypoxia on the rat brain functioning during postnatal ontogenesis.
Tasks. To achieve this goal, the following tasks were set:
1. To study the effect of prenatal hypoxia on the patterns of epigenetic modifications of the rat brain during postnatal ontogenesis.
2. To assess the features of the functioning of the hypoxia-inducible transcription factor HIF1a in the hippocampus during lifespan of rats that survived prenatal hypoxia.
3. To compare the effects of the maternal stress response to hypoxia and the synthetic glucocorticoid dexamethasone injection on the expression and transcriptional activity of glucocorticoid receptors in the hippocampus of the offspring.
4. To carry out a comprehensive analysis of the functional features of the peripheral components of the glucocorticoid neuroendocrine system during lifespan of rats that survived prenatal hypoxia.
5. To analyze the features of the functional features of hippocampal glutamate system in rats that survived prenatal hypoxia.
6. To study the effect of prenatal hypoxia on the features of spatial memory and the age-associated neuronal loss in the rat hippocampus.
Materials. The work was performed on adult female Wistar rats weighs 350 g and their offspring. The animals were obtained from the Biocollection of the Pavlov Institute of Physiology Russian Academy of Sciences. All measurements were made using materials from at least two independent experiments. In each experimental group (control or experimental), at least 6 animals were used at each time point.
Methods. Pregnant female rats were exposed to severe hypobaric hypoxia (SH) (180 mm Hg, 5% O2, 3 sessions of 3 hours at intervals of 24 hours) on the 14th, 15th and 16th days of pregnancy to model prenatal severe hypoxia (PSH). The fetal hippocampus begins to develop during this period. It is one the most important extrahypothalamic structure of mature brain, which controls glucocorticoid negative feedback and is also responsible for spatial memory and learning. In addition, the maternal stress response was mimicked by administering the synthetic glucocorticoid dexamethasone (0.8 mg / kg) on days 14, 15 and 16 of pregnancy. In an additional series of experiments, a inhibitor of glucocorticoid synthesis metyrapone (Met and PSH + Met groups) was administered to pregnant female rats before or without hypoxia sessions on days 14, 15, and 16 of pregnancy.
The amount of corticosterone, the main glucocorticoid hormone in rats, was determined in the blood plasma of pregnant females and in male offspring at the age of 1 day (newborns), 2 weeks (juvenile), 3 months (adults) and 18 months (aging) by the method competitive enzyme immunoassay.
The distribution of transcription factors HIF1a and glucocorticoid receptors (GR) between the nuclear and cytosolic fractions of the hippocampal cells of 1-day-old rat pups (cytosolic reference protein Actin-0, nuclear - NeuN) was studied using the Western blot method, and quantitative analysis of type 1 glutamate receptors (mGluR1) was performed in the total fraction of the hippocampus of 18-month-old rats.
The number and localization of HIF1a, GR, as well as post-translational epigenetic modifications of chromatin in the brain and liver of 1-day-old, 2-week-old, 3-, and 18-month-old male rats were determined by immunohistochemical method. In addition, age-related changes in the number of neurons, astrocytes, mGluR1 and inositol-3-phosphate receptor type 1 (IP3R1) in the hippocampus were also studied using the immunohistochemical method.
The change in the amount of inositol-3-phosphate (IP3) in acute brain slices in response to the application of the mGluR1 agonist DHPG was determined chromatographically.
Age-related changes in the amount of glutamate in the rat hippocampus were studied using the enzymatic colorimetric method.
The functional state of the glutamate reception was determined in adult rats using the SH model as an excitotoxic test. The efficiency of glutamate reception was measured by the change in lipid peroxidation (LPO) products by spectrophotometric and fluorimetric methods.
The efficiency of peripheral glucocorticoid reception was determined by the activity of glucose-6-phosphatase (enzymatic colorimetric method), the
amount of glycogen (PAS reaction) in the liver, and the amount of glucose in capillary and arterial blood.
The number of mRNA of HIF1-dependent, glucocorticoid-dependent genes, as well as genes of enzymes of glutamate metabolism, glutamate transporters in the hippocampus was determined by the quantitative polymerase chain reaction in real time (RT PCR).
Spatial memory and learning ability were assessed using the Morris water maze in adult (3 months) and aged (18 months) control and PSH rats.
One way analysis of variance (ANOVA) followed by Tukey's test were used to assess the significance of differences between groups. Statistical significance was set at p <0.05.
The following statements are presented for the consideration:
1. Prenatal hypoxia induces persistent changes in the patterns of epigenetic modification of chromatin associated with decreased hippocampal and neocortical activity in rats during lifespan.
2. An increase in the content and transcriptional activity of the hypoxia-inducible transcription factor HIF1a in the hippocampus of rats which survived prenatal hypoxia persists after birth and stabilize to adulthood. Meanwhile, in the process of aging, the amount of this protein increases, which might reflect age-related disorders of the energy metabolism of the brain or be involved in their development.
3. The maternal stress response to hypoxia, as well as the administration of the synthetic glucocorticoid dexamethasone during pregnancy, causes a
decrease in the sensitivity of the offspring's hippocampus to glucocorticoids because of a decrease in the expression of glucocorticoid receptors and the efficiency of glucocorticoid-dependent transcription. The decrease in sensitivity to glucocorticoids in the hippocampus of rats survived prenatal hypoxia is stable throughout their lives. At the same time, the using of an inhibitor of corticosterone synthesis prevents a decrease in the number of glucocorticoid receptors in the hippocampus after prenatal hypoxia. This indicates the central role of the maternal stress response to hypoxia in the stable disturbance of the sensitivity of the offspring hippocampus to glucocorticoids, which further determines the central and peripheral disturbances in the functioning of the glucocorticoid system.
4. The consequence of impaired sensitivity of the hippocampus of rats that survived prenatal hypoxia to glucocorticoids is stable hyperactivation of the hypothalamic-pituitary-adrenocortical axis. A steady increase in the basal level of glucocorticoids is accompanied by age-related disorders of their peripheral reception and, as a consequence, the implementation of glucocorticoid-dependent functions.
5. Prenatal hypoxia causes age-associated impairment of synthesis and increased degradation of glutamate, accompanied by ineffective hyperactivation of the receptor apparatus of the glutamate system in the hippocampus of rats.
6. The long-term consequences of prenatal hypoxia are expressed in the development of "early old age" state, which manifests itself in
premature loss of neurons and, consequently, cognitive deficit that
progresses with age.
Scientific novelty. Previously, we and other researchers found that prenatal hypoxia leads to persistent disorders of motor, emotional, exploratory behavior and learning ability. It has been shown that these disorders are most expressed if the hypoxic effect occurs at the beginning of the third week of gestation - the period of active neurogenesis in various areas of the developing brain, primarily the hippocampus.
Many years of research carried out in our laboratory allowed us to put forward a hypothesis on the mechanisms of the formation of long-term negative consequences of prenatal hypoxia. According to this hypothesis, pathologies of brain development caused by prenatal hypoxia, in addition to impaired oxygen supply, are determined by an inadequate level of glucocorticoid stimulation of the fetus, which leads to a decrease in the sensitivity of the hippocampus to glucocorticoids and a subsequent weakening of peripheral regulation of glucocorticoid-dependent processes that manifests itself with age. Changes in the functional activity of the hippocampus are also manifested in the disruption of the hypoxia-induced signaling and dysfunction of the hippocampal glutamate system, leading to a deficit in spatial memory and early neuronal loss. We are the first ones to show that these changes are associated with persistent changes in the epigenetic code. The obtained data on the mechanisms of action of maternal hypoxia and glucocorticoids on the developing fetal brain, as well as on the
consequences of dysfunction of the hippocampus, open up wide opportunities for early diagnosis and effective correction of a wide range of age-related neurological diseases of a sporadic nature.
Theoretical and practical significance of the research. The results of the work expand the modern understanding of molecular biological processes leading to the development of pathologies of postnatal development due to prenatal hypoxia. The results of this fundamental research have not only theoretical but also practical value. The high practical significance of the work is determined by the clinical need to develop new methods for early diagnosis of the consequences of prenatal pathologies and the search for effective ways to increase the overall resistance of the organism. The results obtained at the molecular-cellular level can be used to create a new generation of effective pharmacological drugs aimed at preventing the consequences of prenatal stress.
The reliability and approval of the results. The results were obtained using modern biochemical, molecular-biological, histochemical, and behavioral methods. The sample size and the number of independent experiments made it possible to assess the significance of the results after processing with adequate methods of statistical analysis. The results of the work were presented and discussed at 45 all-Russian and international conferences, the most significant of which are: ISN-ESN Meeting (Paris, France, 20-24.08.2017), FENS Forum of Neuroscience (Berlin, Germany, 7-
11.07.2018), ISN-ASN Meeting (Montreal, Canada, 4-8.08.2019), ESN Conference on Molecular Mechanisms of Regulation in the Nervous System (Milan, Italy, 1-4.09.2019), online FENS Forum of Neuroscience (Glasgow, Scotland, 11-15.07.2020), 1st ESN Virtual Conference "Future perspectives for European neurochemistry - a young scientists conference" (25-26.05.2021).
Publications. 81 papers have been published on the topic of the dissertation, part of them are 21 articles in peer-reviewed journals recommended by the Higher Attestation Commission of the Russian Federation, the others are 60 conference abstracts.
Applicant's personal contribution. The personal contribution of the dissertation candidate consisted in the analysis of the literature on the research problem, the development of a hypothesis, the planning of experiments and their implementation, the statistical processing of the results obtained, the discussion of the results, and the preparation of publications on the topic of the dissertation.
Dissertation structure. The dissertation includes an introduction, four chapters, summary, and conclusions; set out on 115 pages of text, contains 28 illustrations and 148 references to used literature sources.
Financial support. This work was supported by RFBR grants No. 16-0400872, 16-34-00027, 17-04-01118.
Chapter 1. Epigenetic modifications in the brain of rats survived prenatal
hypoxia
We studied post-translational modifications of histone H3 - acetylation at lysine 24 (acH3K24) (Tyulkova et al., 2017, 2019), methylation for lysines 4 and 9 (meH3K4 and meH3K9), as well as total DNA methylation (meDNA) in cells of the fifth layer of the neocortex (NeoV) and CA1 of the rat hippocampus (Tyulkova et al., 2020) within the work aimed to study the effect of PSH and the maternal stress response during pregnancy on the patterns of epigenetic modifications in the postnatal rat brain by quantitative immunohistochemical method (Fig. 1).
Epigenetic modifications due to exposure to prenatal hypoxia
Activation chromatin modifications Inhibitory chromatin modifications
acH3k24 meH3k4 meH3k9 meDNA
3 months 2 weeks 3 months IS months 2 weeks 3 months 18 months 2 months 3 months 18 months
I N G N t tr t t t
N 4 N I N tr N t a N
Figure 1. Results of immunohistochemical studies of epigenetic modifications in the hippocampus (CA1 field) and neocortex (NeoV) of PSH rats. acH3K24, acetylation of histone H3 at lysine 24; meH3K4 and meH3K9, methylation at lysines 4 and 9 of histone H3, respectively, meDNA, DNA methylation.
Age-related changes in the number of inhibitory chromatin modifications -meDNA and meH3K9 (but not activation modification meH3K4) were found in
control animals, namely, a decrease in the level of meDNA and meH3K9 in aged 18-month-old rats compared to adult 3-month-old rats (Tyulkova et al., 2020). Age-related changes in the level of meDNA and meH3K9, modifications that contribute to the inhibition of gene expression, may be involved in the discovered disorders of the learning ability of rats in the Open Field and the Morris maze during natural aging (Tyulkova et al., 2015; Stratilov et al. 2021; Vetrovoy et al., 2021c).
Exposure to hypoxic stress in the prenatal period is an example of "early-stage programming of brain development " - the phenomenon that occurs during critical periods of development and caused by the action of stress factors, leads to persistent changes in physiological functions and behavior in later life (Barker, 1995; Nyirenda, Seckl, 1998). Reprogramming of the epigenome during the early development of the organism is a very complex and well-organized process, which involves the interaction of molecular changes in DNA and histone proteins (Dasgupta et al., 2012; Liu et al., 2016). It determines the balance of expression of genes involved in maintaining cell plasticity during adaptation to changing environmental conditions. In addition, changes in the balance of DNA methylation / demethylation and histone modification during embryonic development play an essential role in the regulation of progenitor cell differentiation between neurogenesis and astrogliogenesis (Takizawa et al., 2001; Sauvageot, 2002).
PSH on the 14-16th day of embryonic development stably increases the meDNA level in all studied brain structures, regardless of the age of the rats (Tyulkova et al., 2020). Administration of dexamethasone at the same time of
pregnancy in order to simulate the maternal stress response also increases the degree of meDNA, and this is mostly expressed in juvenile animals (Tyulkova et al., 2020).
DNA methylation is a fundamental epigenetic mechanism for controlling a mammalian gene expression, associated mainly with the repression of transcription, which consists in the attachment of a methyl group to the carbon at the 5th position of the cytosine molecule to form 5-methylcytosine. Enhanced DNA methylation suppresses the expression of hypoxia-inducible transcription factor HIFla (Koslowski et al., 2001; Walczak-Drzewiecka et al., 2010), the main mediator of transcriptional responses to hypoxia, which contributes to the formation of a hypoxic-related phenotype (Watson et al., 2010). Meanwhile, an inverse relationship was shown between the baseline level of HIF1a in neocortex neurons and the genetically programmed tolerance of the organism to hypoxia (Kirova, 2012). The increase in meDNA is consistent with a decrease in baseline HIF1a expression in the neocortex of these animals.
When analyzing the results on the modification of histone H3 methylation by lysines 4 and 9, it turned out that the most pronounced changes are manifested mainly in the long term after exposure, namely, in the brain structures of 18-month-old rats. This increases the level of meH3K4, which promotes the activation of transcription, and decreases meH3K9, which, like meDNA, is associated with gene repression (Tyulkova et al., 2020).
The observed decrease in the degree of histone H3 methylation at lysine 4 in the studied brain structures of old rats exposed to severe hypobaric
hypoxia on the 14-16th day of prenatal ontogenesis is consistent with the modifications shown by us (Tyulkova et al., 2017) of histone H3 acetylation at 24 lysine the hippocampus and neocortex of adult rats after the same exposure. acH3K24 also promotes the activation of genes and correlates with the revealed abnormalities in the expression of gluco- and mineralocorticoid receptors that regulate the expression of target genes, including HIFla transcription factors, neurohormones corticoliberin and vasopressin, and antioxidants (Kodama et al., 2003). Delayed changes in the degree of histone H3 methylation can cause deterioration of the functional activity of the brain that increases with age, leading to a weakening of memory and learning ability in animals exposed to severe hypobaric hypoxia.
It is interesting to note that in experiments on adult animals sessions of damaging hypoxia caused patterns of epigenetic modifications of chromatin similar to those observed in animals subjected to prenatal hypoxia. However, already fourth day after exposure, the levels of meDNA as well as histone H3 methylation and acetylation returned to control values (Samoilov et al., 2016, Vetrovoy et al., 2019a, 2020a).
It could be assumed that shown long-term changes in behavior and learning ability, which are caused by changes in the activity of the main intracellular regulatory systems (calcium and phosphoinositide), and also disorders of glutamate signal transduction, the ratio of pro- and antioxidant systems in the brain of rats subjected to severe hypobaric hypoxia during prenatal development , are affected by modifications of the epigenetic status (Tyulkova et al., 2010a, 2015; Vataeva et al., 2018). The profile of histone
acetylation, modifications of histone and DNA methylation caused by prenatal administration of increased doses of glucocorticoid hormones (dexamethasone) differs in severity from the action of prenatal hypoxia (Tyulkova et al., 2017, 2019, 2020), which may underlie the ambiguous manifestations of these effects on molecular, cellular and behavioral levels (Tyulkova et al., 2015; Vataeva et al., 2018).
The results of this block of studies are published in the articles:
1) M. Samoilov, A. Churilova, T. Gluschenko, O. Vetrovoy, N. Dyuzhikova, E. Rybnikova. Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance. Acta Histochemica, vol. 118, 2016, pp. 80-89
2) E.I. Tyulkova, O.V. Vetrovoy, K.V. Sarieva, L.A. Vataeva, and T.S. Glushchenko. The characteristics of acetylation of histone H3 at Lys24 in the hippocampus and neocortex of rats that were exposed to hypoxic stress at different stages of prenatal development. Neurochemical Journal, vol. 11, 2017, pp. 309-314
3) O.V. Vetrovoy, T.S. Glushchenko, K.V. Sariyeva, Ye.I. Tyulkova, and Ye.A. Rybnikova. Changes in histone H3 acetylation in the rat hippocampus due to severe hypoxia and the role of hypoxic postconditioning. Neuroscience and Behavioral Physiology, vol. 49, 2019, pp. 1022-1026
4) E.I. Tyulkova, L.A. Vataeva, O.V. Vetrovoi, K.V. Sarieva, V.A. Stratilov. Prenatal administration of dexamethasone leads to decreased Lysine 24 acetylation of histone H3 in the neocortex and hippocampus of adult rats. Cell and Tissue Biology, vol. 13, 2019, pp. 305-311
5) O.V. Vetrovoy, E.I. Tyulkova, V.A. Stratilov, K.A. Baranova, M.O. Samoilov. The pattern of DNA and histone H3 methylation in rat brain in response to severe hypobaric hypoxia and hypoxic postconditioning. Cell and Tissue Biology, vol. 14, 2020, pp. 36-42
DOI: 10.1134/S1990519X20010101
6) E.I. Tyulkova, L.A. Vataeva, V.A. Stratilov, V.S. Barysheva, O.V. Vetrovoy. Peculiarities of DNA and histone H3 methylation in the hippocampus and
neocortex of rats subjected to pathological treatments during the prenatal period. Neurochemical Journal, vol. 14, 2020, 64-72
Chapter 2. The role of hypoxia-inducible factor HIFla in the pathogenesis of post-hypoxic reoxygenation in hippocampus of adult rats and features of its expression in rats survived prenatal hypoxia
The fundamental importance of oxygen for multicellular organisms is undeniable. Animals need oxygen for the efficient flow of energy metabolism, and deviations of its concentration from the norm can threaten normal life and cause death. Considering the extreme importance of maintaining oxygen homeostasis in aerobic organisms, it was simply inevitable that a mechanism for providing adaptive responses to changes in oxygen supply had to be formed. Signaling provided by a hypoxia-inducible factor (HIF1) is recognized as such a very widespread mechanism among animals (Semenza et al., 1991; Iliopoulos et al., 1995, 1996; Wang et al., 1995; Maxwell et al., 1999; Jakkola et al., 2001; Mircea et al., 2001). HIF1 is a transcription factor that is a heterodimer of the HIF1a and HIF10 proteins. HIF10 is expressed constitutively, while HIF1a, being a regulated subunit, accumulates under hypoxic conditions. This leads to its dimerization with HIF10, translocation into the nucleus and the start of transcription of target genes (O'Rourke et al., 1999).
Inhibition of the pentose phosphate pathway by the transcription factor HIF1 as a pathogenetic mechanism of posthypoxic reoxygenation. Among the more than a hundred transcriptional targets of HIF1, it is worth highlighting the vascular endothelial growth factor (VEGF), neuronal and glial
glucose transporters, glycolysis enzymes, lactate dehydrogenase, the cytokine erythropoietin and many other genes critical for the functioning of cells under conditions of chronic hypoxia (Dengler et al., 2014).
However, the pathogenesis of severe forms of hypoxia / ischemia is primarily realized during the period of reoxygenation, when uncontrolled activation of this transcription factor is potentially capable of exerting a maladaptive effect (Vetrovoy, Rybnikova, 2019). We made an assumption about the role of HIF1 in the implementation of posthypoxic pathology through the negative regulation of the pentose phosphate pathway (PPP), which is necessary for providing antioxidant protection and maintain the integrity of cell membranes (Galkina et al., 2013, 2021). To test this hypothesis, we evaluated the role of HIF1 in realization of the effects of pathological hypoxia and reoxygenation in vivo, and also studied the relationship between HIF1, PPP, and PPP-mediated functions in vivo and in vitro.
In vivo studies were carried out on adult male Wistar rats in models of severe hypobaric hypoxia (SH, 3 hours stay at 180 mm Hg (5% O2)) and mild hypobaric hypoxia (MHH, 3 consecutive 2-hour sessions at 360 mm Hg st (10% O2) with an interval of 24 hours) (Vetrovoy et al., 2020c). All data were taken in the hippocampus. The role of HIF1 in the studied processes was assessed using the topotecan, which is inhibitor of the translation of this transcription factor.
In vitro studies were performed on the human embryonic kidney cell line HEK293T. To test the relationship between dose-dependent CoCl2 (inducer of
HIF1a accumulation) and topotecan (inhibitor of HIF1a translation), changes in HIF1a content and HIF1-dependent stimulation of transcription, HEK293T cells were transfected with a vector containing the luciferase gene under the control of the HIF1-dependent chromium luminescence method, and its aminesimines were determined by the method. The relationship between HIF1a accumulation and G6PD content was assessed by Western blotting (Vetrovoy et al., 2021a). The results of this block of studies are summarized in Figure 2.
We have shown that SH and subsequent reoxygenation, causing a short-term increase in the amount of the regulatory alpha subunit HIF1 (HIF1a) in the CA1 region of the rat hippocampus, induce a decrease in the amount and activity of G6PD and the amount of NADPH, which is accompanied by oxidative stress and the initiation of apoptosis. The HIF1 inhibitor topotecan injection before SH prevents an increase in the amount of HIF1a, which is normalizes the amount and activity of G6PD and increases the level of NADPH. It is accompanied by a normalization of the redox status and a decrease in free radical oxidation in the hippocampus, as well as prevention of apoptotic processes and neuronal death (Vetrovoy et al., 2020c).
Figure 2. Scheme of regulation of the pentose phosphate pathway and processes associated with the pentose phosphate pathway in the hippocampus of rats survived severe hypoxia or severe hypoxia in combination with HIF1 inhibition.
In addition, using the MHH model in vivo, an inverse relationship was revealed between the HIF1a protein content and the amount of g6pd mRNA (Vetrovoy et al., 2020c).
We also shown the inducing of G6PD expression in response to mild hypoxia for the neuroprotective method of hypoxic postconditioning (Fig. 3) (Vetrovoy et al., 2014, 2016, 2017a, 2019b). This effect is probably associated with the involvement of another extremely interesting transcriptional
regulator, factor NRF2, located on the other side of the HIF1 medal and being the main regulator ensuring the work of antioxidant systems.
Figure 3. Effects of hypoxic postconditioning on the regulation of the pentose phosphate pathway and processes associated with the pentose phosphate pathway in the hippocampus of rats survived severe hypoxia.
The universality of the investigated mechanism of HIF1-dependent negative regulation of G6PD expression was tested in in vitro experiments on human HEK293T cell culture (Vetrovoy et al., 2021a). To assess HIF1-dependent expression, HEK293T cells were transfected with a luciferase vector under an HIF1-dependent promoter (the transfection efficiency was tested using a plasmid containing the GFP gene (Fig. 4, B)). The intensity of HIF1-dependent expression was measured by the chemiluminescence method in the presence of CoCl2 (HIF1 activator) and topotecan (HIF1 inhibitor) at
various concentrations (Fig. 4, A). It was found that CoCl2 at a concentration of 50 |M caused an increase in luciferase activity up to 180% of the control, and CoCl2 at a concentration of 100 |M - up to 190%. Topotecan at a concentration of 0.2 |M did not affect HIF1-dependent expression in the absence of CoCl2 (96% of control) but did not show the stimulating effect of CoCl2 at concentrations of 50 |M and 100 |M (105 and 150% of control, respectively). Topotecan at a concentration of 1 |M reduced the expression from the HIF1-dependent promoter both in the absence (60% of control) and in the presence of CoCl2 (60 and 50% of the control for CoCl2 at a concentration of 50 and 100 |M, respectively). Thus, we obtained a number of probes differing from each other in the efficiency of HIF1-dependent expression, both upward and downward (Vetrovoy et al., 2021a).
Figure 4. HEK293T cells transfected with the plasmid containing the luciferase gene under the control of the HIF1-dependent promoter (pHRE-LUCPP) and normalizing luciferase of the marine polyp Renilla reniformis under the control of the constitutive CMV promoter (pCMV-LUCRR).
A - Effect of HIF1 inducer CoCl2 (0, 50, and 100 |M) and HIF1 blocker topotecan (0, 0.2, and 1 |M) on HIF1-dependent expression. The results are expressed in arbitrary units of chemiluminescence intensity (CHL; LUCPP/LUCRR). B - HEK293T cells transfected with the plasmid containing pHRE-LUCPP and pCMV-LUCRR or a plasmid containing the GFP gene (pCMVGFP) to control the transfection efficiency. Fluorescence microscopy, ob. 10x.
Western blot analysis of the content of the oxygen-regulated subunit of this factor (HIF1a) (Fig. 5 A, B) confirmed the cause of the previously identified changes in expression from the HIF1-dependent promoter. Figure 5, B presents the results of quantitative analysis, indicating that the accumulation of HIF1a protein is directly dependent on the dose of CoCl2 and inversely on the dose of topotecan (Vetrovoy et al., 2021a).
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Figure 5. The content of HIF1a (A, B) and G6PD (A, C) proteins according to the results of Western blotting (A-C) in HEK293T cells in the presence of HIF1 inducer CoCl2 (0, 50, and 100 |M) and HIF1 inhibitor topotecan (0, 0.2, and 1
|M). *, statistically significant differences from the group Topotecan (0 |M) and CoCl2 (0 |M) (p < 0.05, a = 0.05); #, statistically significant differences from the group Topotecan (0 |M) and CoCl2 (50 |M) (p < 0.05, a = 0.05); $, statistically significant differences from the group Topotecan (0.2 |M) and CoCl2 (0 |M) (p < 0.05, a = 0.05).
The quantitative Western blot study of G6PD (Fig. 5 A, C) showed that the content of this protein is inversely related to the accumulation of HIF1 and the efficiency of HIF1-dependent expression. Thus, under conditions of the maximum amount of HIF1a, in the presence of CoCl2 at a concentration of 100 | M and in the absence of topotecan, the amount of G6PD was minimal and amounted to 75% (Fig. 5, C) of the control. In the absence of CoCl2, topotecan at concentrations of 0.2 and 1 |M did not affect the amount of G6PD. A decrease in the amount of G6PD upon the addition of CoCl2 at a concentration of 50 |M was effectively prevented by topotecan at a concentration of 0.2 and 1 |M. Topotecan at a concentration of 1 |M (but not 0.2 |M) prevented a decrease in the amount of G6PD in the presence of 100 |M CoCl2 (Vetrovoy et al., 2021a).
The pentose phosphate pathway of glucose metabolism, being the main source of reduced NADP, plays the role of a key regulator of antioxidant systems and ensuring the normal redox status of cells (Fernandez-Fernandez et al., 2012), as well as pathways for detoxification of xenobiotics (Spencer, Stanton, 2017). A decrease in the efficiency of the pentose phosphate pathway mediates a wide range of unfavorable conditions (Tang, 2019), such as impaired functioning of mixed-function oxidases (Stincone et al., 2014), a
decrease in the efficiency of steroid hormone synthesis (Dhur et al., 1989), and a decrease in the rate of thioredoxin recovery. and glutathione (Fernandez-Fernandez et al., 2012), which is reflected both in a defect in individual cell functions and in a violation of the integrative regulation of the whole organism. We have shown that the pathogenesis of posthypoxic reoxygenation in the rat brain is largely determined by a decrease in the efficiency of the pentose phosphate pathway. At the same time, inhibition of the transcription factor HIF1 prevented the disruption of this metabolic pathway, which was accompanied by the prevention of oxidative stress and apoptotic processes (Vetrovoy et al., 2020c).
Downregulation of the expression of the key enzyme of the pentose phosphate pathway of glucose metabolism G6PD by the transcription factor HIF1 is a mechanism characteristic not only of rat neuronal cells, but also, at least, of human kidney cells. Such reprogramming of metabolism due to the role of the pentose phosphate pathway as a source of NADPH in the processes of antioxidant protection and anabolism is an important homeostatic response in adaptation to prolonged tissue hypoxia, when there is a need to increase the efficiency of catabolism. However, in the absence of oxygen deficiency, such as various iron deficiency states (Dhur et al., 1989) or with rapid reoxygenation after ischemia (Vetrovoy et al., 2020c), for example, in short-term severe hypoxia, post-stroke conditions, suppression of the pentose phosphate pathway may be a serious pathogenetic factor. Accordingly, the possibility of its pharmacological (Hwang et al., 2018) or non-drug (Vetrovoy et al., 2017b; Vetrovoy et al., 2019b) activation, as well as the use of
substitution therapy (Li et al., 2015), are potential therapeutic a strategy for correcting such metabolic disorders.
The content of hypoxia-inducible factor HIF1a in the hippocampus of rats survived prenatal hypoxia. To assess the effect of PSH on the activity of the HIF1 system in the hippocampus of 1-day-old rat pups, we analyzed the distribution of the HIF1a protein between the nuclear and cytosolic fractions by Western blotting and quantitatively analyzed the mRNA of the HIF1-dependent genes for lactate dehydrogenase (Idha) and G6PD (g6pd) (Vetrovoy et al., 2020b).
PSH induces persistent accumulation of HIFla in the cytosolic fraction of the hippocampus of 1-day old rat pups (Fig. 6, A, C), which is accompanied by an increase in its amount in the nuclear fraction (Fig. 6, B, D). More efficient translocation of HIFla to the nucleus is accompanied by a decrease in the mRNA level of the HIF1-suppressed gene g6pd (Fig. 6, E) and, on the contrary, an increase in the mRNA level of the HIF1-inducible gene Idha (Fig. 6, F) (Vetrovoy et al., 2020b).
Figure 6. Content of protein HIF1a in the cytosolic (A, C) and nuclear (B, D) fractions of the hippocampus, the content of mRNA of the HIF1-suppressed gene g6pd (E) and the HIF1-activated gene Idha (F) in the hippocampus of 1-day-old control rats and rats survived PSH on days 14-16 of embryogenesis. The data are presented as % of the control. White bars, control, 100% (n = 6); black bars, PSH (n = 6). *, differences with the control are statistically significant, p < 0.05
Hypoxia is one of the most important and clinically significant stress effects on the fetus. Until now, many researchers believe that the basis of the pathology of brain development under the action of hypoxia during prenatal ontogenesis is the release of maternal stress hormones in response to damaging effects, which affect both the state of the placenta and directly on the development of the fetus. In a model of prenatal hypoxia, a significant
increase in the HIF1a protein in the brain of a 21-day-old fetus (on the eve of delivery) was previously shown, which is an indicator of fetal brain tissue hypoxia (Gonzalez-Rodriguez et al., 2014).
Our data show that hypoxia presented during the period of hippocampus initiation on the 14-16th day of embryogenesis (Rice, Barone, 2004; Golan, Huleihel, 2006) causes a prolonged increase in the content and activity of the transcription factor HIF1a, which persists in newborn animals. An increase in the amount and activity of HIF1 in immature precursors of hippocampal neurons through the involvement of epigenetic mechanisms can contribute to a stable change in the expression of genes for energy metabolism (Sharp and Bernaudin, 2004; Vetrovoy et al., 2020c), thereby disrupting the functional activity of nerve cells in further ontogenesis (Togher et al., 2014; Tyulkova et al., 2020).
Figure 7. Photographs (A) and results of quantitative analysis (B) of HIFla protein expression in the hippocampus of 2-week-old, 3-month-old and 18-month-old control rats and rats survived PSH on days 14-16 of embryogenesis, obtained by Western blotting. Data are presented as % of control. White bars - control, 100% (n = 6); black bars - PSH (n = 6). *, differences with the control of the corresponding age are statistically significant, p < 0.05
When analyzing the hippocampus of rats during their further postnatal ontogenesis by Western blotting, we found that PSH causes a delayed decrease in the expression of the HIF1a protein at the age of 2 weeks (Fig. 7, A, B). By the age of 3 months, the content of HIF1a in the hippocampus of rats that survived PSH does not differ from the control, however, during aging (age 18 months), the content of HIF1a in the hippocampus of animals of this group increases (Fig. 7, A, B), which may involve age-associated disorders of the energy metabolism of the brain.
The results of this block of studies are published in the articles:
1) O.V. Vetrovoy, E.A. Rybnikova, T.S. Glushchenko, K.A. Baranova, M.O. Samoilov. Mild hypobaric hypoxic postconditioning increases the expression of HIF-1a and erythropoietin in the CA1 field of the hippocampus of rats that survive after severe hypoxia. Neurochemical Journal, vol. 8, 2014, pp. 103108.
2) O.V. Vetrovoi, E.A. Rybnikova, T.S. Glushchenko, M.O. Samoilov. Effects of hypobaric hypoxia in various modes on expression of
neurogenesis marker NeuroD2 in the dentate gyrus of rats hippocampus. Bulletin of Experimental Biology and Medicine, vol. 160, 2016, pp. 510-513.
3) O. Vetrovoy, E. Tulkova, K. Sarieva, E. Kotryahova, M. Zenko, E. Rybnikova. Neuroprotective effect of hypobaric hypoxic postconditioning is accompanied by DNA protection and lipid peroxidation changes in rat hippocampus. Neuroscience Letters, vol. 369, 2017, pp. 49-52.
4) O.V. Vetrovoy, E.A. Rybnikova, M.O. Samoilov. Cerebral mechanisms of hypoxic/ischemic postconditioning. Biochemistry (Moscow), vol. 82, 2017, pp. 392-400.
5) O. Vetrovoy, K. Sarieva, O. Galkina, N. Eschenko, A. Lyanguzov, T. Gluschenko, E. Tyulkova, E. Rybnikova. Neuroprotective mechanism of hypoxic post-conditioning involves HIF1-associated regulation of the pentose phosphate pathway in rat brain. Neurochemical Research, vol. 44, 2019, pp. 1425-1436
6) O. Vetrovoy, E. Rybnikova. Neuroprotective action of PHD inhibitors is predominantly HIF-1-independent: An Editorial for 'Sex differences in neonatal mouse brain injury after hypoxia-ischemia and adaptaquin treatment' on page 759. Journal of Neurochemistry, vol. 150, 2019, pp. 645647
7) O.V. Vetrovoy, P.P. Nimiritsky, E.I. Tyulkova, and E.A. Rybnikova. The Content and activity of hypoxia-inducible factor HIF1a increased in the hippocampus of newborn rats that were subjected to prenatal hypoxia on days 14-16 of embryogenesis. Neurochemical Journal, vol. 14, 2020, 286-289
8) O. Vetrovoy, K. Sarieva, E. Lomert, P. Nimiritsky, N. Eschenko, O. Galkina, A. Lyanguzov, E. Tyulkova, E. Rybnikova. Pharmacological HIF1 inhibition eliminates downregulation of the pentose phosphate pathway and prevents neuronal apoptosis in rat hippocampus caused by severe hypoxia. Journal of Molecular Neuroscience, vol. 70, 2020, pp. 635-646
9) O.V. Vetrovoy, P.P. Nimiritsky, E.I. Tyulkova, E.A. Rybnikova. Transcription factor HIF1 negatively regulates the content of glucoses-phosphate dehydrogenase in HEK293T. Cell and Tissue Biology, vol. 15, 2021, pp. 181-188
10) O.V. Galkina, O.V. Vetrovoy, N.D. Eschenko. The role of lipids in implementing specific functions in the central nervous system. Russian Journal of Bioorganic Chemistry, vol. 47, 2021, pp. 1004-1013
Chapter 3. Central and peripheral disturbances of the glucocorticoid system in rats survived prenatal hypoxia
Neurodevelopmental pathologies caused by prenatal hypoxia are primarily determined by the maternal stress response to external influences (Li et al., 2012; Tomalski, Johnson, 2010). In response to stress, glucocorticoid hormones are released into the blood. The effects of glucocorticoids are mainly mediated by binding to glucocorticoid (GR) and mineralocorticoid (MR) receptors, which act as transcription factors (de Kloet ER et al., 2005, 2008; Viho et al., 2019). MR is involved in the initial stress response, while the management of the later adaptive phase primarily depends on GR (Champagne et al., 2009).
During pregnancy, excess glucocorticoids can cause abnormalities in the maternal and fetal parts of the placenta and affect fetal development (Togher et al., 2014; Glover et al., 2018). It has been found in rats that the expression level and corticosterone-dependent transcriptional activity of GR is of great importance for the developing brain (Lee et al., 2007; Prevot, Millar, 2019; Provençal et al., 2019). Depending on the modality and duration of exposure, the effect of glucocorticoids can be both neuroprotective and neurodegenerative (Abraham et al., 2001; Viho et al., 2019; Franco et al., 2020).
It has been shown in studies on humans and rodents, that disturbances in the intrauterine environment and various kinds of stressful influences during early ontogenesis lead to epigenetic changes that entail a violation of the
expression of GR in the brain, especially in the hippocampus (Weaver et al., 2005, 2007 ; Mueller, Bale, 2008; Oberlander et al., 2008; Turner et al., 2008, 2010; Xiong, Zhang, 2013; Gonzalez-Rodriguez et al., 2014).
This stage aimed to study the effects of the maternal stress response to hypoxia comprehensively during pregnancy on the functioning of the central and peripheral components of the glucocorticoid system of offspring during further ontogenesis, from birth to aging.
The scheme of experiments studying features of the functioning of the glucocorticoid system in rats survived PSH is shown in Figure 8.
Figure 8. Scheme of experiments to study the central and peripheral effects of prenatal hypoxia on the glucocorticoid neuroendocrine system of rats.
The effect of prenatal hypoxia on the content of corticosterone in the blood plasma of pregnant females and offspring. When analyzing glucocorticoids in the blood plasma of pregnant rats, we revealed overproduction of corticosterone in response to the first hypoxic session and a deficiency of its production in late pregnancy (Fig. 9, A) (Vetrovoy et al., 2020d).
The demonstrated increase in the release of corticosterone in response to stressful influences, as well as a delayed decrease in its amount in blood plasma by the mechanism of negative feedback, represent a typical stress curve. However, deviations of glucocorticoid stimulation of the developing fetal brain from the norm can lead to serious impairments of further development. When the content of corticosterone in the blood plasma of the offspring of rats subjected to hypoxia was being examined, we found a decrease in its level in newborns (1 day old) with normalization till 2 weeks of age (Fig. 9, B) and hyperproduction during further ontogenesis (Fig. 9, C) (Vetrovoy et al., 2020d).
Figure 9. The concentration of corticosterone in the blood plasma of pregnant control rats and rats exposed to sessions of severe hypoxia (SH) on days 14, 15, 16 of pregnancy (A). The concentration of corticosterone in the arterial blood plasma of 1-day-old and 2-week-old rats (B) and in the capillary blood plasma of 3-, 11- and 18-month-old (C) control rats and rats survived PSH. *,
differences with the control of the corresponding age are statistically significant, p<0.05.
Effect of prenatal hypoxia on the expression of glucocorticoid receptors and glucocorticoid-dependent processes in the liver. Immunohistochemical analysis of liver glucocorticoid receptors (GR) showed that against the background of corticosterone deficiency in the blood plasma of 1-day-old PSH rats, there is a compensatory increase in the amount of GR at this time point (Fig. 10, A, B), which persists in 2-week-old rats (Fig. 10, C, D) (Vetrovoy et al., 2020d).
Figure 10. Immunohistochemical analysis of the amount of glucocorticoid receptors (GR) in the liver of 1-day (A, B), 2-week, 3- and 18-month (C, D)
control rats and rats survived prenatal hypoxia. *, differences with the control of the corresponding age are statistically significant, p<0.05.
Against the background of an increase in the basal level of corticosterone in blood plasma of PSH rats, the amount of GR in the liver gets equal with the control at the age of 3 months and decreases at the age of 18 months (Fig. 10, C, D) (Vetrovoy et al., 2020d).
We analyzed the content of glycogen in the liver and found that prenatal hypoxia caused an increase in its accumulation in 2-week-old rat pups (Fig. 11) (Vetrovoy et al., 2020d).
Figure 11. The content of glycogen in the liver during lifespan of control rats and rats survived prenatal hypoxia. Results are expressed as % of 3 months control. *, differences with the control of the corresponding age are statistically significant, p<0.05.
At the same time, during aging in PSH rats, in contrast to control animals, glycogen stores do not decrease, which indicates a inefficiency of peripheral glucocorticoid reception.
A decrease in the amount of GR in the liver of 18-month-old PSH rats
also affects the efficiency of glucose excretion into the blood plasma. Thus, the activity of the GR-dependent enzyme glucose-6-phosphatase in the liver of 18-month-old PSH rats is halved compared to the corresponding control (Fig. 12, A). In addition, at this age point, a decrease in the amount of glucose in both venous (Fig. 12, B) and arterial blood (Fig. 12, C) is observed in the blood plasma of rats (Vetrovoy et al., 2020d).
Figure 12. Enzymatic activity of glucose-6-phosphatase in the liver (A), the amount of glucose in the plasma of capillary (B) and arterial (C) blood of 2-week, 3- and 18-month-old control rats and rats survived prenatal hypoxia. Results are expressed as % of 3 months control. *, differences with the control of the corresponding age are statistically significant, p<0.05.
Effect of prenatal hypoxia on the expression of glucocorticoid receptors and glucocorticoid-dependent transcription in the hippocampus. It is known that on the 14-16th day of embryonic development of the rat brain, corresponding to the 5-7th week of human pregnancy, a ventral hippocampus begins to develop. The hippocampus is the most important extrahypothalamic structure that controls glucocorticoid negative feedback. Hypoxic exposure and the maternal stress response during this period can lead to permanent disturbances in the functional activity of this structure at the molecular-cellular level.
To assess the features of glucocorticoid reception in the hippocampus of 1-day-old rat pups, we performed Western blot analysis of the distribution of glucocorticoid receptors (GR) between the cytosolic and nuclear fractions and quantitatively analyzed the mRNA of the GR-dependent crhrl and maoa genes.
PSH does not affect the amount of GR in the cytosol of the hippocampus of newborn rat pups (Fig. 13. A, C), however, in the nuclear fraction there is a twofold decrease in the amount of GR compared to the control (Fig. 13, B, D). The decrease in GR content in the nucleus is accompanied by a decrease in the intensity of GR-dependent transcription of crhrl (encodes the corticoliberin receptor) (Fig. 13, E), and maoa (encodes monoamine oxidase A) (Fig. 13, F) mRNA compared to control (Vetrovoy et al ., 2020d).
Figure 13. Photographs (A, B) and the results of quantitative analysis (C, D) of glucocorticoid receptors (GR) in the cytosolic (A, C) and nuclear (B, D) fractions of the hippocampus, obtained by Western blotting. The number of mRNA of glucocorticoid-dependent genes crhrl (D) and maoa (E) in the hippocampus of newborn (1-day-old) control rats and rats survived prenatal hypoxia. Results are expressed as % of 3 months control. *, differences with control are statistically significant, p<0.05.
Immunohistochemical analysis of the CA1 field and the dentate gyrus (DG) of the rat hippocampus during their further ontogenesis showed that PSH causes a steady decrease in the amount of GR (Fig. 14, A-E) (Vetrovoy et al., 2020d).
A lifelong decrease in GR expression in the hippocampus of PSH rats is accompanied by a decrease in transcription of the GR-dependent maoa gene in this brain structure (Fig. 14, E) (Vetrovoy et al., 2020d).
We also showed a decrease in GR expression in the brain structures of adult rats treated with dexamethasone on the 14-16th day of gestation. It demonstrates the key role of the maternal stress response in persistent impairment of the sensitivity of the hippocampus of offspring to glucocorticoids (Vetrovoy et al., 2021b).
Figure 14. Immunohistochemical analysis of the content of glucocorticoid receptors (GR) in the CA1 field (A, C) and dentate gyrus (DG) (B, D) of the hippocampus, the amount of mRNA of the glucocorticoid-dependent gene maoa (E) in the hippocampus during the life of control rats and rats survived prenatal hypoxia. Results are expressed as % of 3 months control. *, differences with the control of the corresponding age are statistically significant, p<0.05.
The Effect of inhibition of glucocorticoid synthesis by metyrapone on gr protein expression in the hippocampus of PSH rats. To test the role of the maternal glucocorticoid stress response to hypoxia in the formation of the revealed peripheral and central dysfunction of the glucocorticoid neuroendocrine system, an additional experiment was performed using the inhibitor of corticosterone synthesis metyrapone (Fig. 15) (Vetrovoy et al., 2020d).
Metyrapone caused a decrease in plasma corticosterone concentration in control rats at 15, 16, 17 and 19 days of gestation, respectively (Fig. 15, A). In the group of rats exposed to SH, metyrapone suppressed excessive corticosterone production in response to the first hypoxia session and prevented a decrease in the amount of corticosterone in response to subsequent hypoxia sessions on the 17th day of gestation; however, on the 19th day, the amount of corticosterone in this group significantly decreased in comparing to the control (Fig. 15, A) (Vetrovoy et al., 2020d).
Western blotting showed that normalization of plasma corticosterone levels in pregnant rats which survived hypoxic episodes with metyrapone was accompanied by the prevention of a decrease in the amount of GR in the hippocampus (Fig. 15, B, C) of one-day-old rat pups. (Vetrovoy et al., 2020d).
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