2 курс / Нормальная физиология / VII_Сибирский_съезд_физиологов_Афтанас_Л_И_,_Труфакин_В_А_,_Манчук

глюкозы в крови у сытых животных), которые зависят от захвата глюкозы в мышцах и жире под влиянием инсулина.

Согласно нашим данным, мутация yellow не влияет на чувствительность к эстрадиолу репродуктивных органов, гипоталамических центров, регулирующих потребление пищи, и чувствительность к эстрадиолу печени, но препятствует участию эстрадиола в регуляции метаболизма глюкозы и жиров в мышечной и жировой тканях. Можно предположить, что развитие ожирения и диабета 2 типа у самок мышей с мутацией yellow в локусе агути отчасти обусловлено нарушением регуляторного действия эстрадиола на углеводно-жировой обмен.

Работа выполнена при поддержке РФФИ (грант 10-04-00331, НШ,

6477.2010.4).

АПОПТОТИЧЕСКИЕ ПРОЦЕССЫ НЕЙТРОФИЛЬНЫХ ГРАНУЛОЦИТОВ КРОВИ ПРИ ОБОСТРЕНИИ ЗАБОЛЕВАНИЙ ВЕРХНИХ ДЫХАТЕЛЬНЫХ ПУТЕЙ

С.Н. Якушкина, О.А. Ставинская, Л.К. Добродеева Институт физиологии природных адаптаций УрО РАН, Архангельск,

ifpa-svetlana@mail.ru

Известно, что апоптоз обеспечивает сохранение постоянства клеточной популяции. Данных по апоптозу нейтрофилов очень мало. Нет сведений о взаимодействии апоптоза гранулоцитов с уровнем их пролиферации, а также с пролиферацией лимфоцитов и моноцитов. В связи с вышесказанным, целью данного исследования явилось выяснение особенностей апоптоза и пролиферации нейтрофильных гранулоцитов крови при обострении заболеваний верхних дыхательных путей.

Методы. Проведено обследование 30 человек с обострением хронических назофарингитов (гаймориты, отиты) и 30 практически здоровых людей. Исследование проводили с соблюдением основных норм биомедицинской этики. В мазках крови, окрашенных по Романовскому−Гимзе, кроме лейкограммы определяли нейтрограмму по Й. Т. Тодорову (1968). Фагоцитарную активность нейтрофилов исследовали с помощью частиц латекса. Содержание лимфоцитов CD10+, CD25+ определяли с помощью непрямой иммуннопероксидазной реакции с использованием моноклональных антител на препаратах лимфоцитов типа «высушенной капли» (НПЦ «МедБиоСпектр», г. Москва). Концентрации цитокинов IL-1, IL-2, TNF-α, РЭА в сыворотке крови определяли методом иммуноферментного анализа (Bender MedSystems, Австрия; ConAg Diagnostics). Реакцию оце-

нивали с помощью фотометра Multiskan MS (Labsystems, Финляндия) при длине волны 450 нм. Полученные результаты обработаны с использованием пакета прикладных программ Statistica 6,1 (StatSoft, США).

Результаты. Общее содержание лейкоцитов, в том числе уровень нейтрофилов и лимфоцитов, в периферической венозной крови у людей с

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заболеваниями верхних дыхательных путей практически не отличаются от таковых у практически здоровых людей: 7,0(5,3-8,4) и 6,7(5,4-8,4)×109 кл/л,

и соответственно 3,19(2,46-5,01) и 3,68(2,96-5,18)×109 кл/л; 2,5(2,02-2,89) и

2,6(1,8-3,4)×109 кл/л. Однако значительно увеличивается количество моно-

цитов с 0,17(0,09-0,34) до 0,41(0,30-0,46)×109 кл/л и CD25+ с 0,5(0,39-0,64)

до 0,74(0,62-0,84)×109 кл/л. У больных снижается концентрация лимфоци-

тов CD10+ с 0,57(0,41-0,68) до 0,27(0,23-0,35)×109 кл/л и уровень РЭА с

1,71(0,92-2,60) до 1,06(0,44-1,54) нг/мл. При сравнении нейтрограмм не установлено изменения количества клеток с пятью и более сегментами яд-

ра: 0,13(0,11-0,23) и 0,15(0,07-0,28)×109 кл/л. Содержание зрелых диффе-

ренцированных клеток с тремя сегментами ядра также остается без изме-

нения и составляет 1,43(0,99-1,79) и 1,41(1,12-2,07)×109 кл/л. При этом у больных снижается абсолютное количество палочкоядерных нейтрофилов с 0,18(0,11-0,31) до 0,09(0,05-0,27)×109 кл/л и увеличивается интенсивность фагоцитоза с (4,16 ± 0,27) до (10,33 ± 0,64) шт., p<0,05. Количество актив-

ных фагоцитов наоборот уменьшается: (51,13 ± 2,78) и (42,03 ± 1,10) %. При обострении хронических заболеваний верхних дыхательных путей обнаружено увеличение концентрации регуляторных цитокинов IL-1 у (55,00

± 3,67) % и IL-2 у (26,66 ± 3,42) % обследуемых лиц. Выявлено повышение содержания в крови TNF-α с 22,30(20,75-25,0) до 34,16(21,16-48,49) пг/мл по сравнению с практически здоровыми людьми.

Заключение. Таким образом, снижение пролиферативной активности нейтрофилов при хроническом воспалении верхних дыхательных путей ассоциировано с низкой пролиферативной способностью лимфоцитов. Не установлены взаимосвязи между уровнями пролиферации и апоптоза гранулоцитов и моноцитов. В данной ситуации апоптоз регулирует общее содержание нейтрофилов и может являться мерой предотвращения избыточного некробиоза в инфицированных тканях.

РОЛЬ ЦИКЛИЧЕСКИХ НУКЛЕОТИДОВ В МЕХАНИЗМАХ ВЛИЯНИЯ ЭНДОТЕЛИНА-1 НА ЭЛЕКТРИЧЕСКУЮ

И СОКРАТИТЕЛЬНУЮ АКТИВНОСТЬ ГЛАДКОМЫШЕЧНЫХ КЛЕТОК ВОРОТНОЙ ВЕНЫ

П.И. Янчук, П.Ф. Пелюх НИИ физиологии им. ак. Петра Богача Учебно-научного центра «Институт биологии»

Киевского национального университета им. Тараса Шевченко, Киев, Украина, yanchuk49@ukr.net

Эндотелин-1 (ЭТ-1) относится к наиболее мощным вазоконстрикторным факторам. Он синтезируется не только эндотелиоцитами, но и гладкомышечными клетками (ГМК) сосудов, нейронами, астроцитами, гепатоцитами. В нашей лаборатории изучено влияние ЭТ-1 на кровообращение, термо- и кислородный баланс печени. Однако его влияние на элек-

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трофизиологические показатели ГМК воротной вены (ВВ) изучены недостаточно.

Исследования проведены на отрезках гладких мышц ВВ крыс. Электрическую активность ВВ регистрировали при помощи методики двойных сахарозных промежутков, а ее сократительную активность, используя механоэлектрический преобразователь 6МX1C и усилитель. Использовали стандартный нормальный раствор Кребса (НРК) с постоянной температу-

рой (36,5°С).

ЭТ-1 в концентрациях 2·10-9-2·10-6 моль/л в НРК вызывал кратковременную (1-2 мин) гиперполяризацию плазматических мембран ГМК ВВ и угнетение ее сократительной активности, после чего наступала резкая деполяризация мембран, возникал потенциал действия и сокращение ГМК ВВ. При этом с увеличением концентрации ЭТ-1 наблюдалось увеличение амплитуды и продолжительности мембранных потенциалов действия и сокращения препаратов ВВ. Явление десенситизации не наблюдалось.

Каждое из используемых веществ отдельно: 8-Вr цГМФ в концентрации 1·10-5-1·10-4 моль/л, метиленовый синий (блокатор синтеза гуанилатциклазы) в концентрации 1·10-7-1·10-6 моль/л, а также нитропруссид натрия (1·10-7-1·10-6 моль/л) в нормальном растворе Кребса угнетали электрическую и сократительную активность ГМК ВВ, тогда как дб-цАМФ (1·10-5-1·10-4 моль/л) в НРК слабо (на 4-5%) увеличивала электрическую и сократительную активность ГМК ВВ. Причем метиленовый синий вызывал гиперполяризацию мембран ГМК ВВ и увеличение анэлектротонических потенциалов, а нитропруссид натрия также вызывал гиперполяризацию мембран ГМК, но уменьшение их анэлектротонических потенциалов. Введение в эти растворы ЭТ-1 вызывало увеличение параметров электрической и сократительной активности ГМК аналогичное тому, что наблюдалось в предыдущей серии опытов (с одним лишь ЭТ-1 в НРК).

Следовательно, полученные нами данные свидетельствуют о том, что эндотелин-1 вызывает зависимое от его концентрации увеличение амплитуды и продолжительности мембранных потенциалов действия и сокращения гладкомышечных клеток воротной вены крыс. Эти эффекты ЭТ- 1 не опосредованы изменением внутриклеточной концентрации циклических нуклеотидов, а обусловлены, вероятнее всего, изменением проницаемости плазмолем гладкомышечных клеток воротной вены к ионам кальция.

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EFFECTS OF GLUTAMATERGIC BLOCKADE ON BEHAVIORAL

AND NEURONAL CHANGES IN PARKINSON’S DISEASE RAT

MODEL

Ying-Jui Ho1, Ying-Cih Hung1, Chin-Yen Chou1, Shih-Chun Ho1, Guo-Joe Huang1,

Tan-Yu Liao1, Guan-Da Huang1, Shih-Hui Hsu2, Sheng-Huei Wu2

1 School of Psychology, 2School of Medical Laboratory and Biotechnology, Chung Shan

Medical University (Taichung City, Taiwan, ROC) yjho@csmu.edu.tw

In addition to motor dysfunction, up to 20-30% of patients with Parkinson’s disease (PD) suffer from dementia also, which is known as Parkinson’s disease dementia (PDD). The features of PDD include impairments of working memory and recognition. Hyper-glutamatergic activity has been implicated in the pathophysiology of PD. Although NMDA receptorand metabotropic glutamate receptor (mGluR)-mediated excitotoxicity may take part in neuronal degeneration in PD, role of these two kinds of receptors in PDD remains unclear. To address this issue, systemic administration with 2-methyl-6-(phenylethynyl)- pyridine (MPEP), a selective antagonist of mGluR5, and MK-801, a noncompetitive antagonist of NMDA receptors, were used to evaluate the role of glutamatergic activity in neurodegeneration and behavioral deficits in 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced rat PD model. One day after MPTP lesioning, the rats received daily treatment with MPEP (2 mg/kg, i.p.) or MK-801 (0.2 mg/kg/day, i.p.) for 14 days. Motor function and working memory were measured by using the bar test and T-maze test, respectively. MPTP caused significant motor deficit, compared to the sham-operated group, on day 1. But this impairment recovered spontaneously on day 7. A decrease in working memory was observed in MPTP-lesioned rats, which was restored by both MPEP and MK-801 treatment. Further, the administration of MPEP and MK801 prevented the disturbance of object recognition in MPTP-lesioned rats. Moreover, the administration of MPEP and MK-801 significantly ameliorated MPTP-induced neuroinflammation, dopaminergic degeneration in the nigrostriatal system, and neuronal loss in the hippocampal CA1 area. These results suggest that suppression of glutamatergic activity may have clinical potential in the treatment of dementia associated with PD.

SIMULTANEOUS FILTRATION AND FOCUSING OF BIOLOGICAL CELLS ON AN INSULATOR-BASED DIELECTROPHORETIC MICRODEVICE

Chun-Ping Jen1, Wei-Fu Chen1 and Tamara G. Amstislavskaya2

1 National Chung Cheng University (Chia-Yi 621, Taiwan, R.O.C.)

2 Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia) imecpj@ccu.edu.tw

Manipulating and separating biological cells of interest using microfluidic and micro total analysis system (μTAS) devices has potential applications in clinical diagnostics and medicine. For example, in investigations of the nervous

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system, glial cells can enwrap neuronal cells and thus shield neurons from a direct extracellular access. Cellular focusing in microfluidic devices is a prerequisite for medical applications, such as cell sorting, cell counting or flow cytometry. In the present study, an insulator-based dielectrophoretic microdevice is designed for the simultaneous filtration and focusing of biological cells. The cells are introduced into the microchannel and pre-confined hydrodynamically by the funnel-shaped insulating structures close to the inlet. Dead and viable human carcinoma (HeLa) cells were cultured for the experiment demonstrating hydrodynamic separation herein. There are ten sets of X-patterned insulating structures in the microfluidic channel. The main function of the first five sets of insulating structures is to guide the cells by negative dielectrophoretic responses (viable HeLa cells) into the center region of the microchannel. The positive dielectrophoretic cells (dead HeLa cells) are attracted to regions with a high electricfield gradient generated at the edges of the insulating structures. The remaining five sets of insulating structures are mainly used to focus negative dielectrophoretic cells that have escaped from the upstream region. Experiments employing a mixture of dead and viable HeLa cells are conducted to demonstrate the effectiveness of the proposed design. The results indicate that the performance of both filtration and focusing improves with the increasing strength of the applied electric field and a decreasing inlet sample flow rate, which agrees with the trend predicted by the numerical simulations. The filtration efficiency, which is quantitatively investigated, is up to 88% at an applied voltage of 50 volts peak- to-peak (1 kHz) and a sample flow rate of 0.5 μL/min. The proposed device can focus viable cells into a single file using a voltage of 35 volts peak-to-peak (1 kHz) at a sample flow rate of 1.0 μL/min.

This work was supported by grant No. 148 SB RAS, Russia - NSC, Taiwan Research Cooperation.

LONG-TERM ADVERSE EFFECT OF NEONATAL

GLUCOCORTICOID TREATMENT ON AMYGDALA

Kwok-Tung Lu 1, Meng-Chang Ko1, Yu-Hui Hung1, Yi-Ling Yang2

1 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan

2 Department of Biochemical Science and Technology, National Chia-Yi University, Chia-Yi,

Taiwan ktlu@ntnu.edu.tw

In addition to neonatal adversity, some neonatal medications are suspected to produce subtle changes in brain maturation, the impact of which may become apparent only in the late period of life. Dexamethasone (DEX) is a synthetic glucocorticoid and is frequently used to lessen the progression of chronic lung disease in extremely low birth weight (ELBW) infants. Even though clinical trials failed to consistently demonstrate improvement in mortality or length of hospitalization, exposures of up to 42 days were frequently used as a therapeutic agent to lessen the morbidity of chronic lung disease in premature infants.

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Recent studies pointed out that the neonatal DEX treatment alters hippocampal synaptic plasticity in juvenile. Little is known about the long-term effect of neonatal DEX treatment on amygdale function. The current study was performed to evaluate the long-term effect of neonatal DEX treatment on amygdale synaptic plasticity. Male adult Wister rats were subjected to receive subcutaneous injection (sc) of tapering doses of DEX (0.5 mg/kg, 0.3 mg/kg and 0.1mg/kg) from postnatal day 1 to 3, PN1~PN3. Animals were then subjected for fearpotentiated startle (FST) and electrophysiological recording at the age of 6 or 8 weeks. Results showed that neonatal DEX treatment temporary decrease body weight in the young rats, but not affected in the later life when compared with the control group. In addition, neonatal DEX treatment affected the formation of amygdaloid long-term potentiation. Furthermore, neonatal DEX treated animals showed deficit on the acquisition of fear-potentiated startle (FST) which revealed neonatal DEX treatment has long-term adverse effect on amygdale function and resulted in adverse consequence in the later life.

MATERNAL SEPARATION OF DIFFERENT INTERVALS INDUCES SEX DIFFERENCES IN ANXIETY RESPONSES IN POST-WEANING RATS

Yuan-Feen Tsai1, Yi-Sian Wu1, Hsiao-Fung Pu2, Mei-Ying Yeh1,

Su-Ping Wang1, Mei-Yun Tai1

1 Institute of Physiology, College of Medicine, National Taiwan University, Taiwan, ROC 2Department of Physiology, School of Medicine, National Yang-Ming University, Taiwan, ROC

yftsai@ntu.edu.tw

Early life experience has prolonged effects on emotionality and endocrine functions at adulthood. Maternal separation has been described to induce different anxiety responses at adult age, but rare studies have investigated the effects of maternal separation in post-weaning rats. In the present study, we used both male and female Long-Evans rats at 22 days of age to reveal the effects of maternal separation on anxiety-like behavior, the basal levels of corticosterone and adrenal gland weights. Neonatal pups were randomly assigned to three groups: control, maternal separation 15 minutes (MS-15), and maternal separation 180 minutes (MS-180). Infant rats were deprived of maternal contact between the 4th and the 21st postnatal days. The elevated plus-maze was used to establish the anxiety-like behavior profile of animals. In female rats, the MS-15 group showed significantly lower anxiety-like behavior, serum corticosterone levels and lighter adrenal glands than control rats. However, the female MS-180 animals had significantly lower serum corticosterone levels compared to the female controls, but their anxiety-like performance was not significantly different from that in the controls. In contrast, although there was no significant difference in the anxiety-like behavior between these three male groups, the male MS-15 rats had lighter adrenal glands than those of the male MS-180 rats. Both the male

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MS-15 and MS-180 groups had lower serum corticosterone levels than the male controls. Together, these results suggest that neonatally maternal separation of different intervals may cause sex differences in anxiety-like behavior in postweaning rats.

STRESS EFFECT OF THYROIDECTOMY ON YHE SECRETION OF

GLUCOCORTICOIDS IN MALE RATS

Paulus S. Wang1,2,3,4,5, Yung-Hsing Yeh1, Cai-Yun Jian1, Shyi-Wu Wang6 1 Dept. Physiology, Sch. Med., National Yang-Ming University, Taipei, 2 Dept. Med. Res. Edu., Taipei Veteran General Hospital, Taipei,

3 Dept. Med. Res. Edu., Taipei City Hospital, Taipei,

4 Grad. Inst. Exerc. Sports Sci., Taipei Physical Edu. Coll., Taipei, 5 Ph.D. Program for Aging, China Med. University, Taichung,

6 Dept. Physiol. Pharmacol., Coll. Med., Chang-Gung University, Taoyuan, Taiwan,

Republic of China pswang@ym.edu.tw

Both adrenocorticotropin (ACTH) and glucocorticoids are stress hormones for regulating psychoneuroendocrine functions. It has been shown that the pituitary-adrenal functions are modulated by hypothyroidism. Thyroid hormones increases the secretion of ACTH and consequently corticosterone production. Hypothyoid men have higher levels of plasma cortisol. However, the detail mechanisms of the effects of thyroid hormones on glucocorticoid production are unclear. In the present studies, the effects of thyroid hormones and thyroidectomy on glucocorticoid secretion were studied. Blood samples were collected from male rats after receiving intravenous 3,5,3’-triiodothyronine (T3) or thyroxine (T4). Zona fasciculata-reticularis (ZFR) cells were treated with

ACTH, T3, T4, ACTH plus T3, or ACTH plus T4 at 37°C for 2 h. Male rats were thyroidectomized (Tx) or sham Tx for 2 weeks before challenging with ACTH. Blood samples were collected at different time intervals. In another in vitro study, rat ZFR cells were incubated with or without forskolin, 8-Br-cAMP, or ACTH at 37 for 1 h. The concentrations of corticosterone in plasma and medium samples were measured by radioimmunoassay. Administration of T3 and T4 suppressed the basal and the ACTH-stimulated levels of plasma corticosterone. In ZFR cells, both thyroid hormones inhibited ACTH-stimulated corticosterone secretion, but the basal corticosterone was inhibited only with T3>10-10M or T4 >10-8M. Likewise, T3 or T4 inhibited the basaland ACTHstimulated levels of intracellular cAMP. Thyroidectomy resulted in an increase of the basal level of plasma corticosterone and spontaneous release of corticosterone from rat ZFR cells. The maximal response of plasma corticosterone to ACTH challenge was attenuated by thyroidectomy. Thyroidectomy caused an increased response of corticosterone production in vitro to the administration of forskolin but not 8-Br-cAMP. These results suggest that removal of thyroid hormones is a stress to increase the production of glucocorticoids via a mechanism at least in part associated with an activation of adenylyl cyclase. Mean-

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while, thyroid hormones counteract ACTH in adrenal steroidogenesis through their inhibition of cAMP production in ZFR cells.

RESVERATRON PROTECTS RATS FROM Aβ-INDUCED NEUROTOXICITY BY THE REDUCTION OF iNOS EXPRESSION AND LIPID PEROXIDATION

Yi-Ling Yang1, T.C. Huang2, K. T. Lu2, Y.Y. P. Wo1, Y.J. Wu2

1 Department of Biochemical Science and Biotechnology, National Chia-Yi University, ChiaYi, Taiwan, 2Department of Life Science, National Taiwan Normal University, Taipei, Taiwan ylyang@mail.ncyu.edu.tw

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β–amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Ab peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβinduced hippocampal neuron loss and memory impairment. On adult SpragueDawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygen- ase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβinduced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.

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