Structural and functional characteristics of rat hearts with and without myocardial infarct. Initial experience with doppler echocardiography

OBJECTIVE: To assess by Doppler echocardiography the structural and functional alterations of rat heart with surgical induced extensive myocardial infarction. METHODS: Five weeks after surgical ligature of the left coronary artery, 38 Wistar-EPM rats of both sexes, 10 of them with extensive infarction, undergone anatomical and functional evaluation by Doppler echocardiography and then euthanized for anatomopathological analysis. RESULTS: Echocardiography was 100% sensible and specific to anatomopathological confirmed extensive miocardial infarction. Extensive infarction lead to dilatation of left ventricle (diastolic diameter: 0.89cm vs.0.64cm; systolic: 0.72cm vs. 0.33cm) and left atrium (0.55cm vs. 0.33cm); thinning of left ventricular anterior wall (systolic: 0.14cm vs. 0.23cm, diastolic: 0.11cm vs. 0.14cm); increased mitral E/ A wave relation (6.45 vs. 1.95). Signals of increased end diastolic ventricle pressure, B point in mitral valve tracing in 62.5% and signs of pulmonary hypertension straightening of pulmonary valve (90%) and notching of pulmonary systolic flow (60%) were observed in animals with extensive infarction. CONCLUSION: Doppler echocardiography has a high sensitivity and specificity for detection of chronic extensive infarction. Extensive infarction caused dilatation of left cardiac chambers and showed in Doppler signals of increased end diastolic left ventricular pressure and pulmonary artery pressure.

Opiatergic participation in the thirst-inhibiting effect of acute third ventricle injections of cadmium (Cd2+) and lead (Pb2+)

We have previously demonstrated that acute third ventricle injections of both lead and cadmium prevent the dipsogenic response elicited by dehydration or by central injections of dipsogenic agents such as angiotensin II, carbachol and isoproterenol in rats. We have also shown that the antidipsogenic action of cadmium may be due, at least in part, to activation of thirst-inhibitory central serotonergic pathways. In the present paper we show that in Wistar male rats the antidipsogenic effect of both lead acetate (3.0 nmol/rat) and cadmium chloride (3.0 nmol/rat) may be partially dependent on the activation of brain opiatergic pathways since central injections of naloxone (82.5 nmol/rat), a non-selective opioid antagonist, blunt the thirst-inhibiting effect of these metals. One hundred and twenty minutes after the second third ventricle injections, dehydrated animals (14 h overnight) receiving saline + sodium acetate displayed a high water intake (7.90 ± 0.47 ml/100 g body weight) whereas animals receiving saline + lead acetate drank 3.24 ± 0.47 ml/100 g body weight. Animals receiving naloxone + lead acetate drank 6.94 ± 0.60 ml/100 g body weight. Animals receiving saline + saline drank 8.16 ± 0.66 ml/100 g body weight whilst animals receiving saline + cadmium chloride drank 1.63 ± 0.37 ml/100 g body weight. Animals receiving naloxone + cadmium chloride drank 8.01 ± 0.94 ml/100 g body weight. It is suggested that acute third ventricle injections of both lead and cadmium exert their antidipsogenic effect by activating thirst-inhibiting opioid pathways in the brain.

Differential effects of isoproterenol on the activity of angiotensin-converting enzyme in the rat heart and aorta

The excessive stimulation of beta-adrenergic receptors in the heart induces myocardial hypertrophy. There are several experimental data suggesting that this hypertrophy may also depend, at least partially, on the increase of local production of angiotensin II secondary to the activation of the cardiac renin-angiotensin system. In this study we investigated the effects of isoproterenol on the activity of angiotensin-converting enzyme (ACE) in the heart and also in the aorta and plasma. Male Wistar rats weighing 250 to 305 g were treated with a dose of (±)-isoproterenol (0.3 mg kg-1 day-1, N = 8) sufficient to produce cardiac hypertrophy without deleterious effects on the pumping capacity of the heart. Control rats (N = 7) were treated with vehicle (corn oil). The animals were killed one week later. ACE activity was determined in vitro in the four cardiac chambers, aorta and plasma by a fluorimetric assay. A significant hypertrophy was observed in both ventricular chambers. ACE activity in the atria remained constant after isoproterenol treatment. There was a significant increase (P<0.05) of ACE activity in the right ventricle (6.9 ± 0.9 to 8.2 ± 0.6 nmol His-Leu g-1 min-1) and in the left ventricle (6.4 ± 1.1 to 8.9 ± 0.8 nmol His-Leu g-1 min-1). In the aorta, however, ACE activity decreased (P<0.01) after isoproterenol (41 ± 3 to 27 ± 2 nmol His-Leu g-1 min-1) while it remained unchanged in the plasma. These data suggest that ACE expression in the heart can be increased by stimulation of beta-adrenoceptors. However, this effect is not observed on other local renin-angiotensin systems, such as the aorta. Our data also suggest that the increased sympathetic discharge and the elevated plasma concentration of catecholamines may contribute to the upregulation of ACE expression in the heart after myocardial infarction and heart failure.

A routine electrocardiogram cannot be used to determine the size of myocardial infarction in the rat

Nine lead electrocardiograms of non-infarcted (N = 61) and infarcted (N = 71) female Wistar rats (200-250 g) were analyzed in order to distinguish left ventricle myocardial infarction (MI) larger than 40% (LMI) from MI smaller than 40% (SMI). MI larger than 40% clearly caused a deviation of ÂQRS and ÂT from normal values of 270-360 degrees to 90-270 degrees. Infarcted rats showed Q wave in D1 larger than 1 mm with 94% sensitivity and 100% specificity. The sum of QRS positivity in V1, V2 and V6 lower than 10 mm identified MI with 82% sensitivity and 100% specificity. The data showed that MI can be easily and reliably diagnosed by electrocardiogram in the rat. However, contradicting what is frequently believed, when specificity and sensitivity were analyzed focusing on MI size, none of these current electrocardiographic indices of MI size adequately discriminates LMI from SMI.

Anabolic steroid- and exercise-induced cardiac stress protein (HSP72) in the rat

The present study investigated the effects of exercise and anabolic-androgenic steroids on cardiac HSP72 expression. Male Wistar rats were divided into experimental groups: nandrolone exercise (NE, N = 6), control exercise (CE, N = 6), nandrolone sedentary (NS, N = 6), and control sedentary (CS, N = 6). Animals in the NE and NS groups received a weekly intramuscular injection (6.5 mg/kg of body weight) of nandrolone decanoate, while those in the CS and CE groups received mineral oil as vehicle. Animals in the NE and CE groups were submitted to a progressive running program on a treadmill, for 8 weeks. Fragments of the left ventricle were collected at sacrifice and the relative immunoblot contents of HSP72 were determined. Heart weight to body weight ratio was higher in exercised than in sedentary animals (P < 0.05, 4.65 ± 0.38 vs 4.20 ± 0.47 mg/g, respectively), independently of nandrolone, and in nandrolone-treated than untreated animals (P < 0.05, 4.68 ± 0.47 vs 4.18 ± 0.32 mg/g, respectively), independently of exercise. Cardiac HSP72 accumulation was higher in exercised than in sedentary animals (P < 0.05, 677.16 ± 129.14 vs 246.24 ± 46.30 relative unit, respectively), independently of nandrolone, but not different between nandrolone-treated and untreated animals (P > 0.05, 560.88 ± 127.53 vs 362.52 ± 95.97 relative unit, respectively) independently of exercise. Exercise-induced HSP72 expression was not affected by nandrolone. These levels of HSP72 expression in response to nandrolone administration suggest either a low intracellular stress or a possible less protection to the myocardium.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 µL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 µg/kg: control (C), 5.3 ± 0.6 vs AEA, 2.7 ± 0.6 mg; MC 3 µg/kg: C, 17.6 ± 1.0 vs AEA, 8.7 ± 0.9 mg; MC 10 µg/kg: C, 37.4 ± 1.2 vs AEA, 22.9 ± 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 µg/kg: C, 3.8 ± 0.8 vs AEA, 1.4 ± 0.6 mg; MC 3 µg/kg: C, 14.7 ± 2.4 vs AEA, 6.9 ± 1.2 mg; P < 0.05; MC 10 µg/kg: C, 39.5 ± 1.0 vs AEA, 22.3 ± 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 µL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 µL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Bone mineral density of rat femurs after hindlimb unloading and different physical rehabilitation programs

Bone weakening can occur due to the absence of load on the skeleton or even short periods of decreased physical activity. Therefore, musculoskeletal diseases that involve temporary immobilization by casts, inactivity or tension increases the risk of fractures. Physical activity is the most studied procedure both to prevent damage and to restore bone structure. The present study aimed at evaluating, by bone densitometry on rat femurs, the influence of hindlimb unloading and later running activity on treadmill or free movement. Sixty-four Wistar rats were used, aged 65 days with a mean corporal mass of 316.11g, randomly divided into eight experimental groups: group 1, the suspended control with seven animals under hindlimb unloading regimen for 28 days, then euthanized; groups 2 and 3, the trained suspended comprising of 7 and five animals, respectively, subjected to hindlimb unloading for 28 days, followed by treadmill exercise for 28 days (group 2) or 56 days (group 3), then euthanized; groups 4 and 5, designated free suspended, comprised of 7 animals each under hindlimb unloading regimen for 28 days followed by free activity in cages for 28 days (group 4) or 56 days (group 5), then euthanized; groups 6, 7 and 8, negative controls, each with 8 animals allowed to free activity in cages and euthanized at the ages of 93, 121 and 149 days, respectively. Bone mineral density (BMD) of the left femur was analyzed by bone densitometry. Unloading by tail-suspension decreased BMD while treadmill training and free activity in cages promoted its recovery in a similar way and over time.

Cyclophosphamide effect on coccidioidomycosis in the rat

Coccidioidomycosis is a systemic mycosis, endemic in arid areas of the American continent. The rat was employed as an experimental host, since it had been shown to reproduce human lesions and present a chronic course of disease with granulomas mainly restricted to lungs. Given the influence of immunosuppressive therapy on the clinical course of human coccidioidomycosis, we studied the effect of cyclophosphamide (CY) in the experimental rat model. Accordingly, animals were inoculated with 400 Coccidioides immitis arthroconidia of the Acosta strain, by intracardiacal route. As single CY doses failed to alter the course of disease, three schedules were used: A) 4 daily doses of 20 mg/kg each, prior to C. immitis inoculation; B) 4 similar daily doses after infection; and C); 6 doses of 20 mg/kg each, given from day +1 to +4, then on days +8 and +9, post infection (pi), taking day 0 as the time of fungal inoculation. The first two schedules inhibited antibody formation up to day 28 pi, without modifying cellular response to coccidioidin as measured by foodpad swelling. Initially, there was greater fungal spread than in controls receiving C. immitis alone, which proved self-limiting in the latter. In contrast, schedule C led to 559r mortality, with both humoral and cellular response abrogation, accompanied by extensive C. immitis dissemination. Histology disclosed significant alterations, such as the persistence of primary infection sporangia, corresponding to the acute stage of coccidioidomycosis in the absence of granuloma development. Therefore, the observed depression in cellular immunity seems responsible for the lack of inflammatory reaction capable of restricting sporangia proliferation in tissues which, in turn, enhances pathogen spread and mortality rate.

Pentavalent antimonial nephrotoxicity in the rat

Aspects of the renal function were assessed in rats treated with the pentavalent antimonials Glucantime (Meglumine Antimoniate, Rhodia) or Pentostam (Sodium Stibogluconate, Wellcome). In dose of 30 mg of Sb v (Glucantime or Pentostam) by 100 mg of weight by day for 30 days, renal functional changes were observed consisting of disturbances in urine concentrating capacity. Such disturbances were expressed by significantly low values of urine osmolality as compared to the basal values previous to the drugs. The decrease in urine osmolality was associated to a significant increase in urinary flow and in negative free-water clearance. There was no alteration in osmolar clearance and in fractional excretion of sodium. These observations suggest an interference of the drugs in the action of the antidiuretic hormone. The disturbance in urine concentration was reversible after a seven days period without the drugs administration. No significant histopathological alterations were observed in the kidneys of the rats treated with the drugs. On the other hand, the rats treated with a high dose of Pentostam (200 mg/100 grams of weight/day) showed the functional and the histopathological alterations of the acute tubular necrosis.

Experimental coccidioidomycosis in the immunosuppressed rat

C. immitis inoculated rats are known to develop infection restricted to lung whereas cyclophosphamide (CY) treatment leads to widespread dissemination with considerable mortality. In this study, an attempt was made to elucidate the mechanisms involved in such behaviour. With this aim, spleen cells were transferred from infected CY-treated to infected untreated rats, achieving significant specific inhibition in footpad swelling to coccidioidin in recipients, attributable to a suppressor T cell subpopulation induced by greater fungal antigen concentration arising from widespread C. immitis dissemination in immunosuppressed animals. NK activity proved similar regardless of CY treatment. Lastly, chronically infected rats presented increased colony forming units count after several weekly doses of CY, as happens in immunosuppressed patients harbouring a previous infection.

Cyclophosphamide effect on paracoccidioidomycosis in the rat

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

Efficacy of voriconazole in experimental rat paracoccidioidomycosis

INTRODUCTION: Amphotericin B, azole or sulfamide drugs are used for treatment of patients with paracoccidioidomycosis. Among the azole drugs, voriconazole was active in vitro against Paracoccidioides brasiliensis and showed efficacy in the treatment of patients infected with this fungus.In the present study the antifungal activity of voriconazole and of other drugs was compared in a rat model of paracoccidioidomycosis. METHODS: Wistar rats were inoculated intravenously with the BOAS strain of P. brasiliensis and antifungal drugs were administered to the animals by gavage at the following doses (mg/kg weight/day): voriconazole (5 to 20), ketoconazole (12 to 15), fluconazole (6), itraconazole (4), and sulfamethoxazole-trimethoprim (120 to 150). The antifungal activity of the drugs was assessed by determining the P. brasiliensis colony forming units in the lungs and spleen of the animals at the end of treatment and by a survival study. RESULTS: Voriconazole reduced the total tissue fungal burden of P. brasiliensis, particularly at doses of ≥10mg/kg weight/day but its antifungal activity was less intense than that of fluconazole, itraconazole and sulfamethoxazole-trimethoprim. The mean survival of animals treated with the last three drugs, 29.1±10.7, 26.1± 10.1 and 28.4±9.6 days, respectively, was higher than that achieved with voriconazole 10mg/kg weight/day (18.5±8.3 days) and that observed in untreated animals (15.7±3.6 days). CONCLUSIONS: At doses similar to those used for clinical treatment, voriconazole showed lower antifungal activity in experimental rat paracoccidioidomycosis than that obtained with itraconazole and sulfamethoxazole-trimethoprim.

Rat allotransplantation of epigastric microsurgical flaps: a study of rejection and the immunosuppressive effect of cyclosporin A

The rejection of allotransplantation of epigastric microsurgical flaps and the effect of immunosuppression have been studied in 58 rats. Three sets of experiments were planned: (1) Wistar Furth isogenic donors and receptors (control set); (2) Brown Norway donors and Wistar Furth receptors (rejection set); and (3) Brown Norway donors and Wistar Furth immunosuppressed receptors (cyclosporin A set). Cyclosporin A (10 mg/kg/d) treated rats had a transplantation survival rate of up to 30 days: 83.3% among isogenic animals and 60% among allogeneic. There was 100% rejection by the 9th day after the transplantation in allogeneic non-immunosuppressed rats. Biopsies embedded with historesin were taken from the flap and normal contralateral skin (used as control) on the 3rd, 7th, 15th, and 30th days after the surgery. A quantitative study of infiltrating lymphocytes in the flaps, with and without cyclosporin A, was done by evaluating the local inflammatory infiltrate. A significant increase in the number of lymphocytes among the rejection and immunosuppressed groups was seen, as compared to the isogenic set. Local lymphocytosis in allogeneic non-immunosuppressed transplantations reached its highest level on the 3rd day after surgery, before gross findings of rejection, which could only be seen by naked eye on the 5th or 6th day. Therefore, we conclude that cyclosporin A is effective in preserving allogenic transplantation in rats. Biopsies of transplanted areas may contribute to earlier diagnosis of the need for immunosuppressive therapy.