Influence of cerebral ischemia-reperfusion on the condition of bcl-2- dependent antiapoptotic mechanisms in the hippocampus of rats with diabetes mellitus

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Article:

Influence of cerebral ischemia-reperfusion on the condition of bcl-2- dependent antiapoptotic mechanisms in the hippocampus of rats with diabetes mellitus

Nika O.M., Higher State Educational Establishment of Ukraine «Bukovinian State Medical University», Chernivtsi

Abstract

Influence of cerebral ischemia-reperfusion on the condition of bcl-2- dependent antiapoptotic mechanisms in the hippocampus of rats with diabetes mellitus

Nika O.M., Higher State Educational Establishment of Ukraine «Bukovinian State Medical University», Chernivtsi

In animals without diabetes mellitus (DM), after 20 min ischemia/1 hour reperfusion, the activity of Bcl-2 anti-apoptotic mechanisms increases in the hippocampal fields CA1, CA2, CA4, and reduces - in the field CA3. On the 12th day of the post-ischemic period, anti-apoptotic activity in the fields CA1 and CA4 returns to the level of activity in animals of the control group; in the field CA2 it decreases compared to the controls; and, in the field CA3 - depression of the anti-apoptotic activity becomes deeper. Four-month DM activates Bcl-2 anti- apoptotic mechanisms in the fields CA3 and CA; suppresses them in the CA1 field and does not affect them - in the field CA4. In rats with four-month diabetes, in early post-ischemic period, the activity of Bcl-2 anti-apoptotic processes increases in the fields CA1 and CA4, and reduces - in the fields CA2 and CA3. On the 12th day of ischemia-reperfusion period in rats with diabetes, Bcl-2 anti-apoptotic activity returns to level of activity in diabetic rats without disturbing the cerebral blood flow in the field CA1; decreases - in the CA4, and undergoes even greater depression than in the early period, in the fields CA2 and CA3.

Key words: hippocampus, diabetes mellitus, brain ischemia-reperfusion, Bcl-2 protein.

Резюме

Влияние ишемии-реперфузии головного мозга на состояние bcl-2- зависимых антиапоптотических механизмов гиппокампа крыс с сахарным диабетом

Ника О.М.

У животных без сахарного диабета (СД) после 20-минутной ишемии/одночасовой реперфузии активность Bcl-2-антиапоптотических механизмов повышается в полях гиппокампа СА1, СА2, СА4 и снижается - в поле СА3. На 12-е сутки постишемического периода антиапоптотическая активность в полях СА1 и СА4 возвращается к уровню у животных контрольной группы, в поле СА2 - снижается относительно контроля, а в поле СА3 ее депрессия нарастает. Четырехмесячный СД активирует Bcl-2- антиапоптотические механизмы в полях СА2 и СА3, подавляет их в поле СА1 и не влияет на них - в поле СА4. У крыс с четырехмесячным СД в раннем постишемическом периоде активность Bcl-2- антиапоптотических процессов в полях СА1 и СА4 повышается, в полях СА2 и СА3 - снижается. На 12-е сутки ишемически-реперфузионного периода у крыс с СД Bcl-2-антиапоптотическая активность возвращается к уровню у крыс с диабетом без нарушения церебрального кровообращения в поле СА1, снижается - в поле СА4 и подвергается еще большей, чем в раннем периоде, депрессии в полях СА2 и СА3.

Ключевые слова: сахарный диабет, ишемия-реперфузия мозга, белок Bcl-2.

Introduction

Numerous experimental studies are indicative of the fact that proteins of Bcl-2 family are key determinants of the cellular survival or death under effect of unfavorable factors [5, 8]. Among their number, protein Bcl-2 and related with it protein Bcl-x-l are widely found in the brain of mammals and possess the ability to protect neurons against apoptosis in case of ischemic effect both in vivo and in vitro [3, 9, 10, 12]. The hippocampus is known to be one of the brain structures most susceptible to ischemic-reperfusion damage [1, 2, 12]. Both focal and global cerebral ischemia caused by heart arrest is found to intensify apoptosis in the hippocampus [2, 13]. Structural changes in the hippocampus are also found in animals with experimental diabetes [14]. Cognitive disorders are indicated to occur in DM patients and animals with simulated diabetes due to structural-functional changes in the hippocampus, which is a key portion of the brain for these forms of higher thinking processes, and especially susceptible to changes of glucose homeostasis [4, 11]. Experimental and clinical studies determined that DM causes apoptosis of the hippocampus neurons due to oxidative stress, caspase inhibition, mitochondrial dysfunction, disorders of expression of apoptotic genes-regulators [14]. The patterns of type 1 and 2 diabetes demonstrates imbalance between pro- and anti-apoptotic signals in the hippocampus resulting in apoptosis induction and loss of nerve cells in its different fields [14]. Therefore, a considerable amount of scientific facts have been accumulated concerning apoptosis role in pathogenesis of ischemic-reperfusion brain injuries and diabetic encephalopathy. Though, we have found only single works dealing with investigation of anti-apoptotic hippocampal potential under conditions of DM complicated by acute disorders of cerebral circulation at the early post-ischemic period [1], and these processes in dynamics remain understudied.

Objective: to study dynamics of Bcl-2- dependent anti-apoptotic activity in the hippocampal fields of rats with DM complicated by incomplete global ischemia- reperfusion of the brain.

Materials and methods. DM was simulated by intraperitoneal administration of streptozotocin («Sigma», USA, 60 mg/kg) to albino nonlinear male rats, two months of age [1]. Incomplete global cerebral ischemia was simulated in a part of animals with DM four months later and six-month rats from the control group by means of applying clips on both common carotid arteries during 20 minutes. The early consequences of ischemic-reperfusion injury of the hippocampus were investigated 1 hour after the initiation of reperfusion, and remote ones - on the 12th day of the post-ischemic period. Surgery and euthanasia of animals were performed under calipsol narcosis (75 mg/kg intraperitoneally). The brain was quickly removed at a cold temperature, and using the stereotaxic atlas coordinates [7] the hippocampal fields CA1, CA2, CA3 and CA4 were isolated. The samples were fixed in 10% Bouin solution (a compound fixative used in histology) during 24 hours. After standard histological processing they were placed into paraffin blocks, histological sections were made 5 mcm thick. The content of Bcl-2 protein was determined by means of the immune indirect fluorescent method. Rehydrated histological sections of the thymus were placed into the incubator for 18 hours in a moist chamber at the temperature of 40 C with primary murine monoclonal antibodies to Bcl-2 of a rat (mouse IgG1 isotype) produced by «Sigma Chemical» (USA). After washing off the excess of the primary antibodies in 0,1 M phosphate buffer the sections were placed into the incubator for 60 minutes at the temperature of 370 C with secondary antibodies (goat antibodies to a complete murine molecule IgG conjugated with FITC («Sigma Chemical», USA) diluted in ratio 1:64). Then the sections were washed in 0,1 M phosphate buffer and placed into the mixture of glycerin and phosphate buffer in the ratio 9:1 for further luminescence microscopy. Identified Bcl-2 -cells of the hippocampus by means of the fluorescent microscope AXIOSKOP (Zeiss, Germany) and high-sensitivity video camera COHU-4722 (COHU Inc., USA) were entered into the computer system of image analysis VIDAS-386 («Kontron Elektronik», Germany) [6]. The experiments were conducted keeping to the main principles of GLP (1981) the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (1986); EU Directives № 609 dated 24.11.1986, and the Order of the Ministry of Health of Ukraine № 690 dated 23.09.2009. Numeric data were statistically processed in the applied programs “Statistica 6.0” and “SPSS 13” using parametric Student t-criterion. Critical significance level while checking statistical hypotheses was equal 0,05.

Results and discussion

diabetes mellitus rat brain

The studies conducted demonstrated that in rats without DM 20-minute ischemia with 1-hour reperfusion in the hippocampal fields CA1 and CA2 resulted in decreased concentration of Bcl-2 protein (22 and 25 % respectively) with simultaneous increase of the area of immune-reactive material (Bcl-2-IPM) (40 and 63 %), and specific content of Bcl- 2 protein (24 and 32 %). In the field CA4 the latter two parameters increased (1,6 times for both parameters), and in the field CA3 - decreased (28 and 25 %). Thus, at the early stage of ischemic-reperfusion injury of the hippocampus a selective sensitivity of its different portions to this effect is found, which is manifested by intensification of anti-apoptotic potential in the fields CA1, CA2, CA4 and its decrease - in the field CA3. On the 12th day of observation in the filed CA1 all the changed parameters returned to the level peculiar for the control rats. As to the early post-ischemic period the concentration of Bcl-2 protein in creased reliably (5 %) and the area of Bcl-2-IPM decreased (7%).

In the filed CA2 at this term of ischemic-reperfusion injury of the brain decreased concentration of Bcl-2 protein remained on the level of the early period. The parameter of Bcl-2-IPM area returned to the control level. And increased specific content of Bcl-2 protein changed into its reliable decrease (1,4 times). Analysis of the dynamics of changes demonstrated decrease of Bcl2-IPM area (1,9 times) concerning the previous term of observation and specific content of the examined protein (1,8 times). Remote changes of Bcl-2-anti-apoptotic activity in the field CA3 consisted of a reliable increase of Bcl-2 protein (31 %), decreased Bcl-2-IPM area (2,1 times) and specific content of cl-2 protein (1,7 times) in comparison with the control. A significant dynamics of changes was observed: concerning the previous term of observation the concentration of Bcl-2 protein 23 % increased, specific content of this protein and Bcl-2-IPM area decreased 1,3 and 1,5 times respectively, which in general is indicative of anti-apoptotic potential of the cells in this field at tis term. In the field CA4 on the 12th day of observation reliable changes of the examined parameters were not found concerning the control, though concerning the early period of the experiment Bcl-2- IPM area and specific content of Bcl-2 protein decreased twice as much and it returned these parameters to the control level. Thus, changes of the examined parameters on the whole were indicative of decreased anti-apoptotic capacity in the hippocampal fields CA1- CA3 at the expense of decreased Bcl-2-IPM area in the field CA1, concentration and specific content of Bcl-2 protein in the field CA2 and Bcl-2-IPM area, and specific content of Bcl-2 protein in the field CA3. Sampled sensitivity of the hippocampal fields by the examined parameters was determined before DM as well.

In the field CA1 of rats with this pathology in comparison with the parameters of animals from the control group the following was found: decrease of the concentration and specific content of Bcl-2 protein (29 and 25 % respectively), in the field CA2 -19 % decrease of Bcl-2 protein concentration, increase of its specific content and Bcl-2-IPM area (69 and 55 % respectively), in the field CA3 - increase of Bcl-2-IPM area and specific content of Bcl-2 protein (60 and 61 % respectively), in the filed CA4 - any changes were lacking. At the early ischemic-reperfusion period the neurons of the field CA1 of rats with DM responded by the increase of Bcl- 2-IPM area and specific content of Bcl-2 protein (twice and 1,95 times respectively), the field CA2 - decreased concentration (19 %) and specific content (18 %) of Bcl-2 protein, filed CA3 - decreased Bcl-2-IPM area and specific content of Bcl-2 protein (1,5 and 1,6 times respectively), field CA4 - decreased concentration of Bcl-2 protein (1,4 times), 30% increase of its specific content and 68% -Bcl-2-IPM area. Therefore, at this term of observation in rats with DM response of Bcl-2- dependent anti-apoptotic mechanisms to ischemia- reperfusion in the hippocampal fields CA1, CA3 and СА4 did not differ much from that of the control rats. Considerable changes were observed in the field СА2. In rats with DM on the 12th day of the post-ischemic period in the filed СА1 intensification of Bcl-2- anti- apoptotic activity changed into its return to the level of rats with diabetes and without cerebral circulation disorders at the expense of decrease concerning the parameters in the previous term of observation of Bcl-2-ІРМ area (2,2 times) and specific content of Bcl-2 protein (twice). In the fields СА2 and СА3 depression of the anti-apoptotic material became deeper, which was evidenced by a negative dynamics of the examined parameters in comparison with the previous term of observation (decrease of the specific content of Bcl-2 protein and Bcl-2-ІРМ area in the field СА2 - 1,9 and 2 times, in the field СА3 -1,6 and 1,4 times respectively). In the field СА4 diverse changes of the examined parameters were found in comparison with those during the early ischemic-reperfusion period - 20% increase of Bcl-2 protein concentration against the ground of its decreased specific content (twice) and Bcl-2+ -ІРМ area (2,3 times). The two last parameters became 1,5 and 1,4 times lower than those in rats with DM and without cerebral circulatory disorders, which is indicative of depression of Bcl-2-anti-apoptotic potential.

Cocnlusions

1. In animals without diabetes mellitus (DM), after 20 min ischemia/1 hour reperfusion, the activity of Bcl-2 anti-apoptotic mechanisms increases in the hippocampal fields CA1, CA2, CA4, and reduces - in the field CA3.

2. On the 12th day of the post-ischemic period, anti-apoptotic activity in the fields CA1 and CA4 returns to the level of activity in animals of the control group; in the field CA2 it decreases compared to the controls; and, in the field CA3 - depression of the anti-apoptotic activity becomes deeper.

3. Four-month DM activates Bcl-2 anti-apoptotic mechanisms in the fields CA3 and CA; suppresses them in the CA1 field and does not affect them - in the field CA4.

4. In rats with four-month diabetes, in early post-ischemic period, the activity of Bcl-2 anti-apoptotic processes increases in the fields CA1 and CA4, and reduces - in the fields CA2 and CA3.

5. On the 12th day of ischemia- reperfusion period in rats with diabetes, Bcl-2 anti-apoptotic activity returns to level of activity in diabetic rats without disturbing the cerebral blood flow in the field CA1; decreases - in the CA4, and undergoes even greater depression than in the early period, in the fields CA2 and CA3.

Prospects of further studies. Apoptotic activity in the hippocampal fields under condition of associated action of experimental diabetes mellitus and ischemia-reperfusion of the brain is planned to be investigated.

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