EARLY AND REMOTE RESPONSE OF HIF-1Α PROTEIN IN THE HIPPOCAMPUS FIELDS TO ISCHEMIA-REPERFUSION IN RATS WITH DIABETES MELLITUS .

Conclusion. DM restricts Hif-1α protein response to ischemia-reperfusion in the neurons of СА1-СА3 field at the early ischemic-reperfusion period and in the neurons of СА2-СА4 fields — on the 12 th day of the observation.


Introduction
Inadequacy between oxygen supply and its requirement is universally known to initiate a series of biochemical and molecular events resulting in death of neurons in the brain during its ischemicreperfusion lesion. In this situation protective cellular mechanisms are activated in the cerebral tissue and induction of different transcription factors in particular. Hif-1α factor induced by hypoxia plays an important role among them. Hif-1α factor is a transcription regulator of oxygen homeostasis and a key factor promoting formation of an adaptive response [1,2]. Hif-1α is a powerful regulator of various target genes which increase erythropoiesis, stimulate angiogenesis through the activation of the vascular endothelial growth factor, provide an adequate metabolism of glucose and its transport into the neurons, promote maintenance of the mitochondrial structure and survival of cells [3,4].
Meanwhile, not all the researchers identically estimate the role of Hif-1α. At the same time, not only neuroprotective but neurotoxic effects of Hif-1α have been experimentally obtained. The latter are realized through the increased activity of p53 gene product -p53 protein and other factors of apoptosis activation [5]. Moreover, Hif-1α participates in killing cells by means of necrosis interacting with calcium and calpain; it is able to intensify brain edema increasing permeability of the hematoencephalic barrier [6][7][8][9]. Protective effects of Hif-1α are considered to be realized mainly in case of mild hypoxia and neurotoxic effectsin severe one.
In addition to hypoxia, hyperglycemia is a powerful regulator of Hif-1α activity [8]. In their turn, hypoxia and hyperglycemia are the main factors determining chronic complications of diabetes. Scientific works in recent years have been indicative of the fact that Hif-1α destabilization transduced by hyperglycemia is manifested by the loss of cellular response to hypoxia with diabetes complications, which in turn produces a negative effect on adaptation of cells and tissues to low oxygen content [10,11]. The mechanisms of Hif-1α stabilization by hypoxia are substantially studied, but destabilization and reduced activity of this factor under conditions of hyperglycemia remain disputable. Methylglyoxal effect is one of the recently found mechanisms of destabilization and functional repression of Hif-1α with DM. It is accumulated under conditions of high glucose level and leads to quick protease-dependent degradation of Hif-1α under hypoxic conditions [10]. Inconsiderable hyperglycemia activates Hif-1α signalization in certain specific types of cells, but high glucose content inhibits it [12].
The role of the transcription factor Hif-1α in the pathogenesis of hypoxic lesions and DM seems to be confirmed, though the mechanisms of its activation and destabilization are still actively investigated. At the same time, molecular mechanisms underlying dysfunctions of this factor in combination of DM with ischemic-reperfusion lesion of the brain remain uncertain.
Objective of the study: to investigate the parameters of the transcription factor Hif-1α activity in the neurons of the hippocampus fields of rats with experimental DM in the dynamics of ischemic-reperfusion lesion of the brain.
Materials and methods. The study was conducted under conditions of simulated bilateral carotid ischemia by means of 20-minute clipping of both common carotid arteries with reperfusion of a different duration in rats without DM and with it. DM was simulated by means of streptozotocin (Sigma, USA) injection (60 mg/kg) into the peritoneum of albino nonlinear male rats at the age of two months [13]. Diabetes duration was 4 months which is enough to form diabetic encephalopathy in rats [13]. Early consequences of ischemic-reperfusion lesion of the hippocampus were studied after one-hour reperfusion, and remote oneson the 12 th day of the post-ischemic period.
The concentration of Hif-1α protein, its specific content and the area of Hif-1α-immunoreactive material (IRM) were determined. The processed histological sections were examined under the fluorescent microscope Axioskop. The images were introduced into the computer system of the digital analysis Vidas-386 (Kontron Elektronik, Germany) [15].
Numerical data were statistically processed with the applied programs Statistica 6.0 and SPSS 13 using parametric Student t-criterion. The critical level of significance in checking statistical hypotheses was considered as 0.05.
Results. Results of the study are presented in Table 1. 20-minute carotid ischemia with one-hour reperfusion was found to cause an increase of the concentration and specific content of Hif-1α protein in СА1 field of rats without DM 2.1 and 1.9 times, respectively comparing to parameters in control group (p<0.05). Under this interference in СА2 field the area of Hif-1α-(IRM) was 1.6 times larger, the concentration and specific content of Hif-1α protein was 1.5 and 1.8 times higher, respectively. The cells in CA3 field demonstrated the similar response: the area of Hif-1α-(IRM), the concentration and specific content of Hif-1α protein in it were 1.7; 1.3 and 2.3 times greater, respectively than the similar parameters in control group (p<0.05). In СА4 field of rats from this experimental group the concentration and specific content of Hif-1α protein was 1.8 and 1.7 times higher than in control group. Notes: difference probability: *compared with the control; ^ischemia-reperfusion (20 min/1 hour) in control animals; #diabetes; &ischemia-reperfusion (20 min/1 hour) in animals with diabetes Analysis of the obtained results is indicative of the fact that at the early ischemic-reperfusion period the activity of the transcription factor Hif-1α intensifies in the hippocampus fields (considering changes of Hif-1α protein, a product of its activity).
On the 12 th day after modeling 20-minute carotid ischemia the concentration of Hif-1α protein in СА1 field remained 20% higher in comparison with the parameter of rats from the control group (p<0.05), though it decreased reliably concerning the previous term 1.8 times. Specific content of Hif-1α protein was also reduced concerning the previous term 1.8 times and reached the values peculiar for the control group. At the late ischemicreperfusion period all the three examined parameters remained increased in СА2 field concerning the values of the control animals: the area of Hif-1α-ІRМ -2.2 times, the concentration and specific content of Hif-1α -1.9 and 2.1 times respectively. Though their dynamics differed: concerning the early post-ischemic period the concentration of Hif-1α protein 1,3 times decreased, its specific content did not change, and the area of Hif-1α-ІRМ 1,4 times increased. On the 12 th day of observation all the examined parameters in СА3 filed increased those of the control: the area of Hif-1α-ІRМtwice as much, the concentration and specific content of Hif-1α protein -1.8 and 3.9 times respectively. At the same time, the two latter parameters concerning the early postischemic period were 1.4 and 1.7 times higher respectively, and the area of Hif-1α-ІRМ remained on the level similar to that of the preliminary term. The area of Hif-1α-ІRМ, the concentration and specific content of Hif-1α protein in СА4 filed concerning the parameters of animals from the control group were 1.6; 2.0, 2.4 times higher. The dynamic was observed in increase compared to the parameters at the early term of observation concerning the area of Hif-1α-ІRМ and specific content of Hif-1α protein 1.7 and 1.4 times.

Conclusions
1. 20-minute ischemia with one-hour reperfusion increases the content of Hif-1α protein in all the examined hippocampus fields in rats without DM. On the 12 th day of ischemicreperfusion period the values of certain examined parameters of the transcription factor Hif-1α activity in СА2-СА4 hippocampus fields continue to increase, and in СА1 field they normalize or reach the values of the animals from the control group.
2. Rats with 4-month DM demonstrate higher values of Hif-1α activity in all the examined hippocampus fields than those of the control group.
3. In rats with diabetes level of Hif-1α protein hasn't been changed in CA1 field at the early ischemic-reperfusion period, and in СА2 field its activity has been decreased, in СА3 field changes are limited by the response of this parameter, in СА4 field they are of the same character as the controls have under the given experimental conditions. On the 12 th day of ischemic-reperfusion period all the parameters of the transcription factor Hif-1α activity increase in CA1 field, becoming higher by their absolute values than corresponding parameters in the control group under similar experimental conditions; changes of the examined parameters are limited in СА2 and СА3 fields in comparison with the corresponding ones in the animals from the control group; the parameters increasing in the control group decreased in СА4 field.