Effect of estrogen replacement treatment on ischemic preconditioning in isolated rat hearts

In the present study, we tested the effects of long-term estrogen replacement treatment on myocardial ischemia-reperfusion injury and on the cardioprotection of ischemic preconditioning in isolated hearts from ovariectomized rats. Ovariectomized rats were treated with 17beta-estradiol (30 micro g/kg/d, s.c.) for 12 weeks. Isolated rat hearts were perfused in the Langendorff mode. Heart rate, coronary flow, left ventricular pressure and its first derivative (+/-LVdp/dtmax) were recorded. Fifteen-min global ischemia and 30-min reperfusion caused a significant decrease of cardiac mechanical function, which were not affected by ovariectomy or estrogen replacement treatment. The isolated hearts in all groups could be preconditioned, and the cardioprotection afforded by preconditioning in the sham-operated rats was greater compared with ovariectomized rats with or without estrogen treatment. These results suggest that long-term estrogen replacement treatment exerts no effect on the inhibition of mechanical function after ischemia-reperfusion, and this study also suggests that estrogen does not affect ischemic preconditioning in isolated hearts of ovariectomized rats.     

High-soy diet decreases infarct size after permanent middle cerebral artery occlusion in female rats

Estrogen is a powerful neuroprotective agent in rodent models of ischemic stroke. However, in humans, estrogen treatment can increase risk of stroke. Health risks associated with hormone replacement have led many women to consider alternative therapies including high-soy diets or supplements containing soy isoflavones, which act as estrogen receptor ligands to selectively mimic some of estrogen's actions. We hypothesized that a high-soy diet would share the neuroprotective actions of estrogen in focal cerebral ischemia. Female Sprague-Dawley rats were ovariectomized and divided into three groups: isoflavone-free diet + placebo (IF-P), isoflavone-free diet + estradiol (IF-E), or high-soy diet + placebo (S-P). Two weeks after being placed on diets, rats underwent left permanent middle cerebral artery occlusion (MCAO). Reductions in ipsilateral cerebral blood flow were equivalent across groups ( approximately 50%). Twenty-four hours later neurological deficit was determined, and brains were collected for assay of cerebral infarct by TTC staining. In the IF-P rats MCAO produced a 50 +/- 4% cerebral infarct. Estrogen and high-soy diet both significantly reduced the size of the infarcts to 26 +/- 5% in IF-E rats and to 37 +/- 5% in S-P rats. Analysis at five rostro-caudal levels revealed that estrogen treatment was slightly more effective at reducing infarct size than high soy diet. Overall neurological deficit scores at 24 h correlated with infarct size; however, there were no statistically significant differences among the treatment groups. These data show that 2 wk of a high-soy diet is an effective prophylactic strategy for reducing stroke size in a rat model of focal cerebral ischemia.     

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning (IP) was investigated in pentobarbital-anesthetized rats. In 5 unpreconditioned control rats, 30 min of occlusion of the left coronary artery elicited ventricular tachycardia (VT) and fibrillation (VF), with an average duration of VT and VF of 51 +/- 6 and 43 +/- 4 s, respectively. Frequent ventricular premature beats (VPBs; average 1,249 +/- 145) were also documented in these animals. Thirty minutes of reperfusion after the prolonged coronary occlusion in these animals caused more severe arrhythmias, including irreversible VF. In animals pretreated with IP (n = 5), which was achieved by 3 cycles of 3 min of occlusion followed by 5 min of reperfusion, 30 min of coronary artery occlusion caused neither VT nor VF, but occasional VPBs (average 2 +/- 1, p < 0.001 vs. control). Only occasional VPBs were observed during 30 min of reperfusion in this group. In animals pretreated with indomethacin (1 mg/kg i.v., n = 5) followed by IP, prolonged ischemia and reperfusion led to frequent VPBs but no VT or VF. The average number of VPBs during ischemia and reperfusion in this indomethacin-treated group was less than that of the controls but greater than the IP-only group (p < 0.01). In conclusion, prostaglandins appear to play a role in the protective effect of IP against VPBs during acute ischemia and reperfusion.     

Intermittent hypoxic training protects canine myocardium from infarction

This investigation examined cardiac protective effects of normobaric intermittent hypoxia training. Six dogs underwent intermittent hypoxic training for 20 consecutive days in a normobaric chamber ventilated intermittently with N2 to reduce fraction of inspired oxygen (FiO2) to 9.5%-10%. Hypoxic periods, initially 5 mins and increasing to 10 mins, were followed by 4-min normoxic periods. This hypoxia-normoxia protocol was repeated, initially 5 times and increasing to 8 times. The dogs showed no discomfort during intermittent hypoxic training. After 20 days of hypoxic training, the resistance of ventricular myocardium to infarction was assessed in an acute experiment. The left anterior descending (LAD) coronary artery was occluded for 60 mins and then reperfused for 5 hrs. At 30 mins of LAD occlusion, radioactive microspheres were injected through a left atrial catheter to assess coronary collateral blood flow into the ischemic region. After 5 hrs reperfusion, the heart was dyed to delineate the area at risk (AAR) of infarction and stained with triphenyl tetrazolium chloride to identify infarcted myocardium. During LAD occlusion and reperfusion, systemic hemodynamics and global left ventricular function were stable. Infarction was not detected in 4 hearts and was 1.6% of AAR in the other 2 hearts. In contrast, 6 dogs sham-trained in a chamber ventilated with compressed air and 5 untrained dogs subjected to the same LAD occlusion/reperfusion protocol had infarcts of 36.8% +/- 5.8% and 35.2% +/- 9.5% of the AAR, respectively. The reduction in infarct size of four of the six hypoxia-trained dogs could not be explained by enhanced collateral blood flow to the AAR. Hypoxia-trained dogs had no ventricular tachycardia or ventricular fibrillation. Three sham-trained dogs had ventricular tachycardia and two had ventricular fibrillation. Three untrained dogs had ventricular fibrillation. In conclusion, intermittent hypoxic training protects canine myocardium from infarction and life-threatening arrhythmias during coronary artery occlusion and reperfusion. The mechanism responsible for this potent cardioprotection merits further study.     

Cardiac spinal deafferentation reduces the susceptibility to sustained ventricular tachycardia in conscious rats

The response to myocardial ischemia is complex and involves the cardio-cardiac sympathetic reflex. Specifically, cardiac spinal (sympathetic) afferents are excited by ischemic metabolites and elicit an excitatory sympathetic reflex, which plays a major role in the genesis of ventricular arrhythmias. For example, brief myocardial ischemia leads to ATP release, which activates cardiac spinal afferents through stimulation of P2 receptors. Clinical work with patients and preclinical work with animals document that disruption of this reflex protects against ischemia-induced ventricular arrhythmias. However, the role of afferent signals in the initiation of sustained ventricular tachycardia has not been investigated. Therefore, we tested the hypothesis that cardiac spinal deafferentation reduces the susceptibility to sustained ventricular tachycardia in adult (12-15 wk of age), conscious, male Sprague-Dawley rats. To test this hypothesis, the susceptibility to ventricular tachyarrhythmias produced by occlusion of the left main coronary artery was determined in two groups of conscious rats: 1) deafferentation (bilateral excision of the T1-T5 dorsal root ganglia) and 2) control (sham deafferentation). The ventricular arrhythmia threshold (VAT) was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. Results document a significantly higher VAT in the deafferentation group (7.0 ± 0.7 min) relative to control (4.3 ± 0.3 min) rats. The decreased susceptibility to tachyarrhythmias with deafferentation was associated with a reduced cardiac metabolic demand (lower rate-pressure product and ST segment elevation) during ischemia.     

Intracoronary infusion of Gd3+ into ischemic region does not suppress phase Ib ventricular arrhythmias after coronary occlusion in swine

Increased mechanical tension in the ischemic region during acute coronary occlusion might favor the occurrence of phase Ib ventricular arrhythmias. We aimed to investigate whether intracoronary administration of Gd(3+), a stretch-activated channel blocker, into the ischemic zone reduces the incidence of these arrhythmias. In thiopental-anesthetized, open-chest pigs, the left anterior descending coronary artery (LAD) was ligated for 45 or 48 min. Phosphate-free, HEPES-buffered saline bubbled with 100% N(2) was infused into the ischemic region for 4 min, starting 5 min (series A; n = 16) or 20 min (series B; n = 16) after coronary occlusion, at a rate doubling the baseline blood flow. Animals were blindly allocated to receive 40 muM Gd(3+) or only the buffer during the final 2 min of the infusion. There were no differences between groups with respect to hemodynamic variables, plasma K(+) levels, or size of the ischemic region. In neither series was the number of phase Ib premature ventricular beats reduced by Gd(3+) (46 +/- 20 in untreated vs. 91 +/- 37 in Gd(3+)-treated animals in series A and 19 +/- 7 vs. 22 +/- 13, respectively, in series B; both P = not significant). The occurrence of ventricular tachycardia or fibrillation was significantly associated with the magnitude of early ischemic expansion of the LAD region, as measured by ultrasonic crystals, but was also not prevented by Gd(3+). These results argue against a major role of stretch-activated channels inside the area at risk in the genesis of phase Ib ischemic ventricular arrhythmias.     

Sex differences to myocardial ischemia and beta-adrenergic receptor blockade in conscious rats

We recently documented sex differences in the susceptibility to reperfusion-induced sustained ventricular tachycardia and beta-adrenergic receptor blockade in conscious rats. However, the effect of sex on ischemia-induced ventricular arrhythmias and beta-adrenergic receptor blockade is under-investigated. Therefore, we tested the hypothesis that gonadal hormones influence the ventricular arrhythmia threshold (VAT) induced by coronary artery occlusion as well as the response to beta-adrenergic receptor blockade. The VAT was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. Male and female intact and gonadectomized (GnX) rats were instrumented with a radiotelemetry device for recording arterial pressure, temperature, and ECG, as well as a Doppler ultrasonic flow probe to measure cardiac output and a snare around the left main coronary artery. The VAT was determined in conscious rats by pulling on the snare. The VAT was significantly longer in intact females (5.56 +/- 0.19) vs. intact males (4.31 +/- 0.14 min). This sex difference was abolished by GnX. Specifically, GnX decreased the VAT in females (4.55 +/- 0.22) and increased the VAT in males (5.14 +/- 0.30 min). Thus male sex hormones increase and female sex hormones decrease the susceptibility to ischemia-induced sustained ventricular tachycardia. beta-Adrenergic receptor blockade increased the VAT in intact males and GnX females only. Thus gonadal hormones influence the response to beta-adrenergic receptor blockade. Uncovering major differences between males and females in the pathophysiology of the cardiovascular system may result in sex-specific optimization of patient treatments.     

Bradykinin mediates cardiac preconditioning at a distance

Preconditioning the heart by brief coronary (CAO) or mesenteric artery occlusion (MAO) can protect against damage during subsequent prolonged CAO and reperfusion. The role of bradykinin (BK) in remote cardiac preconditioning by MAO is investigated by antagonizing the BK B(2) receptor [Hoechst 140 (HOE-140)] or simulating local BK release by mesenteric intra-arterial infusion. Anesthetized male Wistar rats (n = 6-8) were treated with HOE-140 or saline before starting the preconditioning protocol, CAO, MAO, or non-preconditioned control. Infarct size related to risk area [ratio of infarct area to area at risk (IA/AR)] was determined after 3 h of reperfusion following a 60-min CAO. IA/AR was 62 +/- 5% in controls and not affected by HOE-140 (58 +/- 6%). CAO as well as MAO significantly protected the heart (IA/AR, 37 +/- 3 and 35 +/- 5%), which was prevented by HOE-140 (IA/AR, 71 +/- 6 and 65 +/- 7%, respectively). Brief intramesenteric BK infusion mimicked MAO (IA/AR, 26 +/- 3%). Pretreatment with hexamethonium could abolish this protection (IA/AR, 67 +/- 4%). These data indicate an important role for BK in remote preconditioning by MAO. Results support the hypothesis that remote preconditioning acts through sensory nerve stimulation in the ischemic organ.     

Ovariectomy is protective against renal injury in the high-salt-fed older mRen2. Lewis rat

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2. Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2. Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2. Lewis rat conveys protection against salt-dependent increase in renal injury.     

T5 spinal cord transection increases susceptibility to reperfusion-induced ventricular tachycardia by enhancing sympathetic activity in conscious rats

We recently documented that paraplegia (T(5) spinal cord transection) alters cardiac electrophysiology and increases the susceptibility to ventricular tachyarrhythmias induced by programmed electrical stimulation. However, coronary artery occlusion is the leading cause of death in industrially developed countries and will be the major cause of death in the world by the year 2020. The majority of these deaths result from tachyarrhythmias that culminate in ventricular fibrillation. beta-Adrenergic receptor antagonists have been shown to reduce the incidence of sudden cardiac death. Therefore, we tested the hypothesis that chronic T(5) spinal cord transection increases the susceptibility to clinically relevant ischemia-reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity. Intact and chronic (4 wk after transection) T(5) spinal cord-transected (T(5)X) male rats were instrumented to record arterial pressure, body temperature, and ECG. In addition, a snare was placed around the left main coronary artery. The susceptibility to sustained ventricular tachycardia produced by 2.5 min of occlusion and reperfusion of the left main coronary artery was determined in conscious rats by pulling on the snare. Reperfusion culminated in sustained ventricular tachycardia in 100% of T(5)X rats (susceptible T(5)X, 10 of 10) and 0% of intact rats [susceptible intact, 0 of 10 (P < 0.05, T(5)X vs. intact)]. Beta-adrenergic receptor blockade prevented reperfusion-induced sustained ventricular tachycardia in T(5)X rats [susceptible T(5)X 0 of 8, 0% (P < 0.05)]. Thus paraplegia increases the susceptibility to reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity.     

Acute, nongenomic vasodilatory action of estradiol is attenuated by chronic estradiol treatment

Deficiency of estradiol or chronic estrogen treatment may alter the responses to this hormone in many tissues. A possible interaction between the acute nongenomic and the chronic effects of estradiol on microvessels have not been investigated yet. In the present study we have investigated whether acute in vitro vasodilatory action of estradiol on a small artery is altered by chronic estradiol pretreatment. Female rats were surgically ovariectomized and subjected to either estradiol replacement therapy (estradiol propionate, 450 micrograms/kg/week) or vehicle administration for 5 weeks. Cylindrical segments of the saphenous artery were studied using videocomputerized microarteriography in vitro. Estradiol, in concentrations of 10(-6) to 10(-4) M relaxed norepinephrine precontracted vessel segments in a dose-dependent manner. Magnitude of relaxation observed in arteries of estradiol replaced animals was significantly smaller at all concentrations than that of nonreplaced ovariectomized rats; maximal relaxation in the control ovariectomized group was 64.5% +/- 3.6%, while it was 34.3% +/- 4.2% only in the ovariectomized and estradiol replaced group (P < 0.001). Comparison of acute relaxations in response to papaverine and nifedipine failed to prove a reduced activity of the general relaxation machinery in estradiol replaced animals. We conclude that chronic estradiol replacement can downregulate the acute nongenomic vasorelaxation effect of this hormone in small arteries of ovariectomized rats.     

Role of delta-opioid receptor agonists on infarct size reduction in swine

Opioids are involved in cardiac ischemic preconditioning. Important species differences in cellular signaling mechanisms, antiarrhythmic, and antistunning effects have been described. The role of the delta-opioid receptor activation in swine remains unknown. Forty minutes before a 45-min occlusion and 180-min reperfusion of the left anterior descending coronary artery, open-chest, pentobarbital-anesthetized swine received either 1) saline (controls); 2) [D-Ala(2),D-Leu(5)]enkephalin (DADLE); 3) [D-Pen(2,5)]enkephalin (DPDPE); 4) deltorphin-D, a novel delta(2)-opioid agonist; or 5) ischemic preconditioning (IP). Assessed were 1) infarct size to area at risk (IS, triphenyltetrazolium staining), 2) regional and global myocardial function (sonomicrometry, ventricular pressure catheters), and 3) arrhythmias (electrocardiogram analyses). It was found that DPDPE and deltorphin-D pretreatment reduced IS from 64.7 +/- 5 to 36.5 +/- 6% and 27.4 +/- 11% (P < 0.01), respectively, whereas DADLE had no effect (66.8 +/- 3%). Both IP and DADLE had a proarrhythmic effect (P < 0.01). However, no differences in global or regional myocardial function or arrhythmia scores were observed between groups. This suggests that delta-receptor-specific opioids provide cardioprotection in swine.     

Sex influences the susceptibility to reperfusion-induced sustained ventricular tachycardia and beta-adrenergic receptor blockade in conscious rats

Reperfusion after a brief period of cardiac ischemia can lead to potentially lethal arrhythmias. Importantly, there are sex-related differences in cardiac physiology and in the types and severity of cardiac arrhythmias. Therefore, we tested the hypothesis that gonadal hormones influence the susceptibility to reperfusion-induced sustained ventricular tachycardia (VT), as well as the response to beta-adrenergic receptor blockade. Male and female intact and gonadectomized rats were instrumented, and arterial pressure, temperature, ECG, and cardiac output were recorded. In addition, a snare was placed around the left main coronary artery. Tension was applied to the snare for determination of susceptibility to sustained VT produced by 3 min of occlusion and reperfusion of the left main coronary artery in conscious rats. Reperfusion culminated in sustained VT in 77% (10 of 13 susceptible) of female rats and 56% (9 of 16 susceptible) of male rats (P > 0.05, male vs. female). beta-Adrenergic receptor blockade prevented sustained VT in females only [1 of 9 susceptible females (11%) vs. 6 of 9 susceptible males (67%), P < 0.05]. Ovariectomy did not significantly reduce the susceptibility to reperfusion arrhythmias [5 of 9 susceptible (56%)]. In sharp contrast, orchidectomy significantly increased the susceptibility to reperfusion arrhythmias [9 of 9 susceptible (100%)]. Finally, beta-adrenergic receptor blockade prevented sustained VT in ovariectomized females [0 of 4 susceptible (0%)] and orchidectomized males [0 of 7 susceptible (0%)], but the protective effect of beta-blockade was due to a reduction in heart rate in males only. Thus gonadal hormones influence the susceptibility to reperfusion-induced arrhythmias, as well as the effects and mechanisms of beta-adrenergic receptor blockade.     

Is Performance Time a Prerequisite for the Onset and Intensity of the Occlusion-Reperfusion Arrhythmias in Anaesthetised Rats?

The aim of the present study is to investigate the onset and the intensity of arrhythmias in anaesthetized rats as a function of time under a standardized experimental condition, which is composed of 30 min occlusion and 60 min reperfusion. Local bred rats (250-350 g) housed in a 12-h light-dark cycle (lights on at 09.00 h, lights off at 21.00 h) were anaesthetized by sodium thiopentone (60 mg kg^-1 i.p.) and left anterior descending coronary artery ligation method using 6/0 braided silk ligature was used to induce 30 min occlusion and 60-min reperfusion. Animals were randomly allocated into three groups to exposure to 30-min occlusion at 9.00 h and 60 min reperfusion at 9:30 h (Group I, n = 6); to 30 min occlusion at 15.00 h and 60 min reperfusion at 15:30 h (Group II, n = 6); and to 30 min occlusion at 21.00 h and 60 min reperfusion at 21.30 h (Group III, n = 6). ECG and haemodynamic parameters were recorded throughout the experiments. The onset of ventricular ectopic beats (VEBs), number of VEBs, incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF) during the periods of occlusion-reperfusion were analysed. Total VF incidence during occlusion were lower than the VT incidence in all groups. Either VT or VF incidences during reperfusion showed same profiles in all groups but VT incidence was 2-fold higher than VF. Time-dependent application of occlusion-reperfusion induced by coronary artery ligation method in the anaesthetized rats did not result in a variation in the onset and the intensity of arrhythmias. The duration of the experimental ischaemia was the principal factor, which determines the time of onset and intensity of the occlusion-reperfusion arrhythmias.     

Protein kinase C is involved in cardioprotective effects of ischemic preconditioning on infarct size and ventricular arrhythmia in rats in vivo

Protein kinase C (PKC) has been known to play an important role in ischemic preconditioning (IP). This study was designed to examine whether the translocation of PKC is associated with the cardioprotective effects of IP in vivo on infarct size and ventricular arrhythmias in a rat model.Using anesthetized rats, heart rate, systolic blood pressure, infarct size and ventricular arrhythmias during 45 min of coronary occlusion were measured. PKC activity was assayed in both the cytosolic and cell membrane fraction . Brief 3-min periods of ischemia followed by 10 min of reperfusion were used to precondition the myocardium. Calphostin C was used to inhibit PKC.Infarct size was significantly reduced by IP (68.1 (2.5)%, mean (S.E.) vs. 45.2 (3.4)%, p < 0.01). The reduction in infarct size by IP was abolished by pretreatment with calphostin C. The total number of ventricular premature complex (VPC) during 45 min of coronary occlusion was reduced by IP (1474 (169) beats/45 min vs. 256 (82) beats/45 min, p < 0.05). The reduction the total number of VPC induced by IP was abolished by the administration of calphostin C before the episode of brief ischemia. The same tendency was observed in the duration of ventricular tachycardia and the incidence of ventricular fibrillation. PKC activity in the cell membrane fraction transiently increased immediately after IP (100 vs. 142%, p < 0.01) and returned to baseline 15 min after IP. Pretreatment with calphostin C prevented the translocation of PKC.The translocation of PKC plays an important role in the cardioprotective effect of IP on infarct size and ventricular arrhythmias in anesthetized rats.     

17beta-estradiol prevents oxidative stress and decreases blood pressure in ovariectomized rats

In this study, we tested whether estrogen deficiency is associated with oxidative stress and decreased nitric oxide (NO) production, which could be responsible for an increased blood pressure in ovariectomized rats. Hemodynamic studies were performed on conscious, chronically instrumented rats. Chronic estrogen replacement on ovariectomized rats lowered blood pressure approximately 13 mmHg, from 119 +/- 3 mmHg in ovariectomized rats to 106 +/- 3 mmHg in ovariectomized-treated rats; it was also accompanied by an increase in cardiac index and vascular conductance, achieving hemodynamic values similar to those shown by sham-operated rats. N(G)-nitro-L-arginine methyl ester administration lowered significantly less the vascular conductance (0.14 +/- 0.01 vs. 0.22 +/- 0.03 and 0.26 +/- 0.01 ml. min(-1). mmHg(-1)/100 g; P < 0.05) in ovariectomized rats than in the sham-operated and estrogen-treated ovariectomized rats, respectively. Estrogen replacement prevented the lower plasma levels of nitrites/nitrates observed in ovariectomized rats. The lower plasma total antioxidant status and reduced thiol groups and the increase in plasma lipoperoxides presented in ovariectomized animals were reestablished with the estrogen treatment. These results show that estrogen administration decreases blood pressure and increases vascular conductance in ovariectomized rats. This effect may be related to an increase in NO synthesis and/or preventing oxidative stress, then improving endothelial function.     

Extent of damage in ischemic,nonreperfused, and reperfused myocardium of anesthetized rats

To investigate the localization of the earliest damage in ischemic and ischemic-reperfused myocardium, anesthetized rats were subjected to coronary occlusion for 15, 30, 45, or 90 min. One-half of the animals in each group had no reperfusion, whereas the other half was reperfused for 14 min. With the use of histological methods, preferentially in the periphery of the area at risk, localized zones were detected that lacked the hypoxia-specific increase in NADH fluorescence. The extent of these areas displaying injured tissue was found to be significantly smaller in the ischemic-nonreperfused hearts than in the ischemic-reperfused organs (15-min ischemia: 0.22 +/- 0.12% vs. 43.0 +/- 5.0%; 30-min ischemia: 5.7 +/- 2.7% vs. 64.6 +/- 2.9%; 45-min ischemia: 5.6 +/- 1.2% vs. 66.0 +/- 7.5%; 90-min ischemia: 39.3 +/- 5.5% vs. 86.7 +/- 1.8% of the area at risk). The results point to a localized initiation of the damage close to the surrounding oxygen-supplied tissue during ischemia and an expansion of this injury by intercellular actions into yet-intact areas upon reperfusion.     

Daily exercise-induced cardioprotection is associated with changes in calcium regulatory proteins in hypertensive rats

Epidemiological data document that regular exercise protects against the morbidity and mortality associated with ischemic heart disease. Therefore, we tested the hypothesis that daily exercise (DE) increases the ventricular arrhythmia threshold (VAT) induced by coronary artery occlusion and alters the expression of calcium regulatory proteins. The VAT was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. To test this hypothesis, we recorded the VAT in conscious sedentary normotensive, sedentary hypertensive, and DE hypertensive rats, and we associated these thresholds with the protein expression of the L-type calcium channel, Na+/Ca2+ exchanger, phospholamban, and sarco(endo)plasmic reticulum Ca(2+)-ATPase. Results document a significantly reduced time to ventricular arrhythmias (sedentary hypertensive, 3.7 +/- 0.3 min vs. sedentary normotensive, 4.8 +/- 0.3 min), an increased Na+/Ca2+ exchanger protein expression (47%), and a decreased phospholamban protein expression (-34%) in conscious hypertensive rats. DE increased the VAT (5.9 +/- 0.2 min), decreased the protein expression of the Na+/Ca2+ exchanger, and normalized the protein expression of phospholamban in the hypertensive rats. Thus DE may be a primary prevention approach for reducing the incidence of arrhythmias by altering calcium regulatory proteins in hypertensive rats.     

Conserved aquaporin 4 levels associated with reduction of brain edema are mediated by estrogen in the ischemic brain after experimental stroke

Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.     

Early ischemic preconditioning against focal transient and permanent brain ischemia in rats: role of collateral circulation

We hypothesize that early ischemic preconditioning (IPC) can afford protection against focal brief and prolonged cerebral ischemia with subsequent reperfusion as well as permanent brain ischemia in rats by amelioration of regional cerebral blood flow. Adult male Wistar rats (n=97) were subjected to transient (30 and 60 minutes) and permanent middle cerebral artery (MCA) occlusion. IPC protocol consisted of two episodes of 5-min common carotid artery occlusion + 5-min reperfusion prior to test ischemia either followed by 48 hours of reperfusion or not. Triphenyltetrazolium chloride and Evans blue were used for delineation of infarct size and anatomical area at risk (comprises ischemic penumbra and ischemic core), respectively. Blood flow in the MCA vascular bed was measured with use of Doppler ultrasound. The IPC resulted in significant infarct size limitation in both transient and permanent MCA occlusion. Importantly, IPC caused significant reduction of area at risk after 30 min of focal ischemia as compared to controls [med(min-max) 11.4% (3.59-2 0.35%) vs. 2.47% (0.8-9.31%), p = 0.018] but it failed to influence area at risk after 5 min of ischemia [med(min-max) 7.61% (6.32-10.87%) vs. 8.2% (4.87-9.65%), p > 0.05]. No differences in blood flow were found between IPC and control groups using Doppler ultrasound. This is suggestive of the fact that IPC does not really influence blood flow in the large cerebral arteries such as MCA but it might have some effect on smaller arteries. It seems that, along with well established cytoprotective effects of IPC, IPC-mediated reduction of area at risk by means of improvement in local cerebral blood flow may contribute to infarct size limitation after focal transient and permanent brain ischemia in rats.