Anti-apoptotic effects of 2-pyrrolidone derivatives in cerebral ischemia

INTRODUCTION: Uncontrolled course of apoptosis reactions underlies a wide range of pathological processes, including ischemic events. AIM: To evaluate the anti-apoptotic properties of some racetams in experimental brain ischemia in rats. MATERIALS AND METHODS: Cerebral ischemia was modeled in Wistar rats by irreversible occlusion of the middle cerebral artery. The test-compounds and the reference drug piracetam were administered per os at a dose of 250 mg/kg. After 72 hours of the ischemic period, the activity of apoptotic systems in the brain tissue was evaluated by determining the concentration of the apoptotic-inducing factor (AIF), caspase-3, ionized calcium, the latent opening time of the mitochondrial transition permeability pore and the zone of brain necrosis. RESULTS: The study showed that the use of the studied compounds contributed to a decrease in the intensity of reactions, both caspase-dependent and caspase-independent apoptosis, which was reflected in a decrease in the concentration of AIF and caspase-3 by 32.4% (p < 0.05); 34.6% (p < 0.05); 31.1% (p < 0.05), and 41.9% (p < 0.05); 39.1% (p < 0.05); 34.5% (p < 0.05) when PirPr, PirAc and PirBut were administered, respectively. Also, the use of the studied substances led to an increase in the latent period of opening the mitochondrial transition permeability pore, a decrease in the concentration of intracellular calcium and the zone of brain necrosis. At the same time, the pharmacological effect of the administration of the compound PirAc exceeded the effect of piracetam and other test substances. CONCLUSIONS: Based on the results obtained, it can be assumed that the studied racetams have neuroprotective action, realized through suppression of the reactions of apoptosis.

Promotion of angiogenesis toward transplanted ovaries using nitric oxide releasing nanoparticles in fibrin hydrogel

Transplantation of ovary is one method of facilitating fertility preservation to increase the quality of life of cancer survivors. Immediately after transplantation, ovaries are under ischemic conditions owing to a lack of vascular anastomosis between the graft and host tissues. The transplanted ovaries can suffer damage because of lack of oxygen and nutrients, resulting in necrosis and dysfunction. In the technique proposed in this paper, the ovary is encapsulated with nitric oxide-releasing nanoparticles (NO-NPs) in fibrin hydrogels, which form a carrying matrix to prevent ischemic damage and accelerate angiogenesis. The low concentration of NO released from mPEG-PLGA nanoparticles elicits blood vessel formation, which allows transplanted ovaries in the subcutis to recover from the ischemic period. In experiments with mice, the NO-NPs/fibrin hydrogel improved the total number and quality of ovarian follicles after transplantation. The intra-ovarian vascular density was 4.78 folds higher for the NO-NPs/fibrin hydrogel groups compared to that for the nontreated groups. Finally, in vitro fertilization revealed a successful blastocyst formation rate for NO-NPs/fibrin hydrogel coated ovaries. Thus, NO-NPs/fibrin hydrogels can provide an appropriate milieu to promote angiogenesis and be considered as adjuvant surgery materials for fertility preservation.

State of proteolytic and fibrinolytic processes in the brain of rats with experimental diabetes mellitus complicated by cerebrovascular accident in the basin of the carotid arteries

Introduction. The imbalance in the protease-antiprotease system is an integral part of the pathogenesis of acute disorder of cerebrovascular circulation and diabetes mellitus (DM), but its manifestations in the complication of diabetes by ischemia-reperfusion of the brain have not been investigated yet. The objective of the work – to study the dynamics of carotid ischemia-reperfusion effect on the proteo- and fibrinolytic activity in brain structures of rats with experimental DM. Rats with the four-month streptozotocin DM were modeled bilateral carotene ischemia during 20 minutes. In homogenates of brain structures, indicators of proteo- fibrinolytic activity were determined after 1 hour from the beginning of reperfusion and during the 12th day of the post-ischemic period. In the absence of DM, the proteolytic activity of all or individual indicators il increased in both periods of the post-ischemic period in the cortex of the frontal and occipital lobes, the fields of hippocampus CA2 and CA3, and during the 12th day in the field CA1. Results. In rats with diabetes, in all brain structures, there are no changes in the lysis of azo-albumin and azo-casein in both terms of observation and lysis of collagen progressively decreases. In rats without DM in the cortex of the studied particles, fields of the hippocampus CA1 and CA2, all or separate indices of fibrinolytic activity are increased in both periods of the post-ischemic period, in the field CA3 – all indices at the 12th day. In the presence of diabetes in the cortex of both studied lobes and the field CA1, the fibrinolytic activity decreases in the late post-ischemic period, in the fields of CA2 and CA3 – during both observation periods. Conclusion. In the brain structures under investigation at both time intervals, the DM eliminates the reaction of low and high molecular weight proteins to the ischemia-reperfusion and suppresses fibrinolytic activity.

A low-cost mouse cage warming system provides improved intra-ischemic and post-ischemic body temperature control – application for reducing variability in experimental stroke studies

Experimental guidelines have been proposed to improve the rigor and reproducibility of experimental stroke studies in rodents. As brain temperature is a strong determinant of ischemic injury, tight management of brain or body temperature (Tcore) during the experimental protocol is highly recommended. However, little guidance is provided regarding how or for how long temperature support should be provided. We compared a commonly used heat support method (cage on heating pad) with a low-cost custom built warm ambient air cage (WAAC) system. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra-ischemic period (60 min) and post-ischemic period (3 hrs). Neurologic deficit score showed significantly less variance at post-stroke day 1 (PSD1) in WAAC system mice. Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54% lower in the WAAC system group. In summary, we provide a simple low-cost heat support system that provides superior Tcore management in mice during the intra-ischemic and post-ischemic periods, which results in reduced variability of experimental outcomes.HighlightsWe describe the fabrication of a low-cost mouse cage warming system (warmed ambient air cage; WAAC system) that can be assembled and applied in any stroke laboratory.The WAAC system provides more precise control of post-stroke mouse body temperature compared with traditional heating pad warming system.The more precise control of post-stroke core temperature reduces variability in some experimental measures in more severely injured mice.

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

The role of the transcription factor Hif-1α in pathogenesis of hypoxic lesions and diabetes mellitus (DM) has been confirmed, though molecular mechanisms underlying dysfunctions of the factor in the association of DM with ischemic-reperfusion lesion of the brain remain unknown. Objective: the investigation of Hif-1α protein content in the neurons of the hippocampus fields of rats with experimental DM in the dynamics of ischemic-reperfusion lesion of the brain. The study was conducted on 60 6-month rats with DM simulated at the age of 2 months by means of a single administration of streptozotocin (60 mg/kg of the body weight) (Sigma, USA). Disorders of the cerebral circulation were simulated by means of occlusion of both carotid arteries for 20 minutes. The content of Hif1-α protein was determined by means of fluoroimmunoassay after 20-minute ischemia with one hour reperfusion, and on the 12th day of the post-ischemic period in the hippocampus fields: СА1, СА2, СА3, СА4. In rats without DM 20-minute ischemia with one hour reperfusion increases the content of Hif-1α protein in all the hippocampus fields. On the 12th day of ischemic-reperfusion period in СА2-СА4 hippocampus fields the values of certain examined activity indices of the transcription factor Hif-1α continue to increase, and in СА1 field they are normalized or come closer to the values of animals from the control group. In rats with DM at the early post-ischemic period changes of Hif-1α protein content are lacking in СА1 field, the signs of its reduced activity are found in СА2 field, in СА3 field they are limited by the response of one index, and in СА4 field they are similar to those of the control rats under the experimental conditions. On the 12th day of ischemic-reperfusion period all the activity indices of the transcription factor Hif-1α increase in СА1 filed. They are higher than the corresponding indices in animals from the control group by absolute values under similar experimental conditions; changes of the examined parameters are limited in СА2 and СА3 fields in comparison with those from the control group; the parameters, which increased in the control group of animals, decreased in СА4 field. 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 12th day of the observation.

Fibrosis regression is induced by AdhMMP8 in a murine model of chronic kidney injury

Adenoviral vector AdhMMP8 (human Metalloproteinase-8 cDNA) administration has been proven beneficial in various experimental models of liver injury improving liver function and decreasing fibrosis. In this study, we evaluated the potential therapeutic AdhMMP8 effect in a chronic kidney damage experimental model. Chronic injury was induced by orogastric adenine administration (100mg/kg/day) to Wistar rats for 4 weeks. AdhMMP8 (3x1011vp/kg) was administrated in renal vein during an induced-ligation-ischemic period to facilitate kidney transduction causing no-additional kidney injury as determined by histology and serum creatinine. Animals were sacrificed at 7- and 14-days post-Ad injection. Fibrosis, histopathological features, serum creatinine (sCr), BUN, and renal mRNA expression of αSMA, Col-1α, TGF-β1, CTGF, BMP7, IL-1, TNFα, VEGF and PAX2 were analyzed. Interestingly, AdhMMP8 administration resulted in cognate human MMP8 protein detection in both kidneys, whereas hMMP8 mRNA was detected only in the left kidney. AdhMMP8 significantly reduced kidney tubule-interstitial fibrosis and glomerulosclerosis. Also, tubular atrophy and interstitial inflammation were clearly decreased rendering improved histopathology, and down regulation of profibrogenic genes expression. Functionally, sCr and BUN were positively modified. The results showed that AdhMMP8 decreased renal fibrosis, suggesting that MMP8 could be a possible therapeutic candidate for kidney fibrosis treatment.

Rapid development of strong, persistent, spatiotemporally extensive cortical synchrony and underlying oscillations following acute MCA focal ischemia

Stroke is a leading cause of death and the leading cause of long-term disability, but its electrophysiological basis is poorly understood. Characterizing acute ischemic neuronal activity dynamics is important for understanding the temporal and spatial development of ischemic pathophysiology and determining neuronal activity signatures of ischemia. Using a 32-microelectrode array spanning the depth of cortex, electrophysiological recordings generated for the first time a continuous spatiotemporal profile of local field potentials (LFP) and multi-unit activity (MUA) before (baseline) and directly after (0–5 h) distal, permanent MCA occlusion (pMCAo) in a rat model. Although evoked activity persisted for hours after pMCAo with minor differences from baseline, spatiotemporal analyses of spontaneous activity revealed that LFP became spatially and temporally synchronized regardless of cortical depth within minutes after pMCAo and extended over large parts of cortex. Such enhanced post-ischemic synchrony was found to be driven by increased bursts of low multi-frequency oscillations and continued throughout the acute ischemic period whereas synchrony measures minimally changed over the same recording period in surgical sham controls. EEG recordings of a similar frequency range have been applied to successfully predict stroke damage and recovery, suggesting clear clinical relevance for our rat model.

Histological changes of the arterial bed of the hind limbs of the rats under condition of the acute ischemia-reperfusion and correction with the carbacetam

The ischemic-reperfusion lesion is a complex multifactorial damage of the primary ischemic tissues as a result of restoration of the arterial blood circulation in them, which is accompanied by local morpho-functional reorganization of the vascular bed of the hind limbs of the rats. One of the promising means in the treatment and prevention of the reperfusion disorders is a carbacetam, which smooths the phenomena of hypo- and hyperperfusion in the post-ischemic period. The aim of the study was to established the manifestations of the morpho-functional remodeling of the vascular bed of the hind limbs of the rats in ischemia-reperfusion and under conditions of correction with carbacetam. Histological examination of the vascular bed of the hind limbs of 30 rats under conditions of ischemia-reperfusion (group I) and 30 rats in the simulation of ischemia-reperfusion, which in the post-ischemic period administered carbacetam once a day (5 mg/kg) for 14 days (group II) were done. There were 6 intact animals in the control group. Simulation of ischemia was performed by applying SWAT rubber tourniquets on the hind limbs for 2 hours, and reperfusion – by removing of the tourniquet. The animals of the experimental groups were divided into 5 subgroups with reperfusion terms after 1, 2 hours and 1 day, as well as after 7 and 14 days. Histological examination was performed according to generally accepted methods. The vascular bed in the middle third of the thigh and the shin below the tourniquet was examined using a Bresser Trino Researcher 40x–1000x microscope. Analyzing of the obtained results, was established that after 1 hour of the reperfusion the histological changes became a systemic, and after 1 day it were more significant. It should be noted that the thickness of the vessel walls increased, and the elastic membranes were partially eligned, thinned and torned. The stepwise clarity of the arterials walls structure was lost. The edema acquired a total nature. The histological examination of the vessels after 7 days revealed that the swelling of the walls decreased and the condition of the elastic frame was improved. There was a proliferation of collagen fibers in the adventitia, which was a response to ischemic effects. It is noted that after 14 days in all wall membranes the proliferative activity of fiboblasts was remained. Under the conditions of the correction with the carbacetam after 2 hours, the structural positive dynamics became more pronounced and increased to a maximum level after 7 days of the experiment. The number of the modified and exfoliated endothelial cells decreased, and the condition of smooth myocytes increased. Histologically, the gradual restoration of endothelial coverage of the intima was established. As follows, ischemia and reperfusion cause vascular remodeling after 1 hour with a peak of the manifestations after 1 day of the reperfusion, which includes edematous syndrome, dystrophic-degenerative changes with an inflammatory response to the damage, and in the late reperfusion period increased a fibroblasts activity. Gradual return of morphological changes occurs after 14 days of the experiment. Under the conditions of correction, the acceleration of the remodeling with stabilization of the process and the most possible structural restoration after 7 days of the study was noted.

P1603Nicorandil suppresses ischemia-induced cardiac interstitial norepinephrine enhancement and ventricular arrhythmia in hypertrophic hearts

Background Myocardial infarction (MI) is a major cause of death in western countries and Japan, and hypertension is a major risk factor of MI. In hypertensive heart, acute myocardial infarction often leads to lethal ventricular arrhythmia. Nicorandil, an ATP sensitive potassium channel (KATP) opener, is usually used in the treatment of acute myocardial infarction. The effects of nicorandil on ischemic myocyte are fully defined. On the other hand, KATP in neuroterminals is known to regulate norepinephrine release, but the effect of nicorandil on ischemic norepinephrine release in cardiac tissue has remained unexplored. Purpose We examined whether nicorandil suppressed norepinephrine release via neuronal KATP and ventricular arrhythmia during acute ischemia in pressure overload-induced hypertrophic hearts. Methods SD Rats were divided into two groups; abdominal aortic constriction (AAC) group and sham-operated (Sham) group. Four weeks after constriction, cardiac geometry and function were examined using echocardiography. Then, myocardial ischemia was induced by the left anterior descending artery occlusion for 100 minutes in the presence or absence of intravenous infusion of nicorandil. Cardiac interstitial norepinephrine concentration in ischemic region was measured using the microdialysis method and concentration of cyclic AMP, a second messenger of norepinephrine, in cardiac tissue was measured by ELISA. Ventricular arrhythmias were monitered by ECG during whole ischemic period. Results Four weeks after constriction, remarkable left ventricular wall thickening was observed in AAC group. Before ischemia, ventricular arrhythmia was not found in both groups. Number of ventricular arrhythmia, including ventricular tachycardia and ventricular fibrillation, was increased in early ischemic period (- 40 min) in both groups, and was grater in AAC group. Before ischemia, interstitial norepinephrine concentration in cardiac tissue was higher level in AAC group than in Sham group. Ischemia obviously increased norepinephrine concentration in both groups time dependently and AAC further increased norepinephrine than Sham group. Concentration of cyclic AMP in cardiac tissue was raised in early ischemic period (- 40 min) and then gradually decreased. Nicorandil significantly suppressed the number of ventricular arrhythmias, and abolished the ventricular tachycardia and fibrillation without hemodynamic alterations. Nicorandil also attenuated norepinephrine and cAMP enhancement in acute ischemic period in both groups. Conclusion Ischemia-induced ventricular arrhythmia was more frequent and severe in hypertrophic hearts and interstitial norepinephrine enhancement may play a role in this ischemic arrhythmia. Nicorandil suppressed ischemia-induced interstitial norepinephrine release by neuronal KATP opening, which attenuated ventricular arrhythmias in normal and hypertrophic hearts.