Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ± 0.19 and Dex = 0.35 ± 0.13 vs saline (S) = 2.85 ± 0.59; fMLP: Ptx = 0.43 ± 0.09 and Dex 0.01 ± 0.01 vs S = 1.08 ± 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ± 1.4 and Dex = 3.06 ± 0.76 vs S = 15.94 ± 3.97; fMLP: Ptx = 3.85 ± 0.56 and Dex = 0.40 ± 0.16 vs S = 7.15 ± 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ± 0.38 vs S = 4.20 ± 1.01; fMLP: Dex = 0.25 ± 0.11 vs S = 2.20 ± 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ± 2.25 vs S = 4.20 ± 1.01; fMLP: Ptx = 4.66 ± 1.24 vs S = 2.20 ± 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.

The carotid body of the spontaneous insulin-dependent diabetic rat

The carotid bodies from adult spontaneous insulin-dependent diabetic rats (strain BB/S) were perfusion-fixed at normal arterial blood pressure with 3% phosphate-buffered glutaraldehyde and compared with the organs from control rats (strain BB/Sc) prepared in the same way. Serial 5-µm sections were cut, stained, and using an interactive image analysis system, were analysed to determine the volumes of the carotid body and its vascular and extravascular compartments. There was no evidence of systemic arterial disease in the carotid stem arteries in either group of animals, and the microvasculature of the organs appeared normal by light microscopy. The volume of the carotid body was unchanged 3 months after the onset of diabetes but was increased at 6 months. The total vascular volume of the organ was unchanged, but the volume of the small vessels (5-12 µm) was increased. In the control group the small vessels comprised 5% of the total volume of the carotid body, or about 44% of the vascular compartment. The percentage of small vessels increased at 3 months in the diabetic group, but had returned to normal at 6 months. The extravascular volume followed the same pattern as the total carotid body volume and so did not change appreciably when expressed as a percentage of the total volume of the organ. The increase in size of the carotid body in diabetic rats is due, therefore, to an augmented extravascular volume. In one diabetic specimen the carotid sinus nerve showed signs of diabetic neuropathy, axonal swelling and intramyelinic oedema. The clinical implications of these results are discussed.

Effects of microcystin-LR in isolated perfused rat kidney

Microcystin is a hepatotoxic peptide which inhibits protein phosphatase types 1 and 2A. The objective of the present study was to evaluate the physiopathologic effects of microcystin-LR in isolated perfused rat kidney. Adult Wistar rats (N = 5) of both sexes (240-280 g) were utilized. Microcystin-LR (1 µg/ml) was perfused over a period of 120 min, during which samples of urine and perfusate were collected at 10-min intervals to determine the levels of inulin, sodium, potassium and osmolality. We observed a significant increase in urinary flow with a peak effect at 90 min (control (C) = 0.20 ± 0.01 and treated (T) = 0.32 ± 0.01 ml g-1 min-1, P<0.05). At 90 min there was a significant increase in perfusate pressure (C = 129.7 ± 4.81 and T = 175.0 ± 1.15 mmHg) and glomerular filtration rate (C = 0.66 ± 0.07 and T = 1.10 ± 0.04 ml g-1 min-1) and there was a significant reduction in fractional sodium tubular transport at 120 min (C = 78.6 ± 0.98 and T = 73.9 ± 0.95%). Histopathologic analysis of the perfused kidneys showed protein material in the urinary space, suggestive of renal toxicity. These data demonstrate renal vascular, glomerular and urinary effects of microcystin-LR, indicating that microcystin acts directly on the kidney by probable inhibition of protein phosphatases.

Quantitative studies of the vasculature of the carotid body in the chronically hypoxic rat

The carotid bodies of rats made chronically hypoxic by breathing 12% O2 in a normobaric chamber (inspired PO2 91 mmHg) were compared with those of controls. Serial 5-µm sections of the organs were examined using an interactive image analysis system. The total volume of the carotid bodies was increased by 64%. The total vascular volume rose by 103% and was likely due to an increase in size of the large vessels (>12 µm lumen diameter) because the small vessel (5-12 µm lumen diameter) volume did not increase significantly while the small vessel density tended to decrease. The extravascular volume was increased by 57%. Expressed as a percentage of the total volume of the organ, the total vascular volume did not change, but the small vessel volume was significantly decreased from 7.83 to 6.06%. The large vessel volume must therefore have been increased. The proportion occupied by the extravascular volume was virtually unchanged (84 vs 82%). In accordance with these findings, the small vessel endothelial surface area per unit carotid body volume was diminished from 95.2 to 76.5 mm-1, while the extravascular area per small vessel was increased from 493 to 641 µm2 or by 30%. In conclusion, the enlargement of the carotid body in chronic hypoxia is most likely due to an increase in total vascular volume, mainly involving the "large" vessels, and to an increase in extravascular volume. This is in contrast to our previously published findings indicating that in the spontaneous insulin-dependent diabetic rat the enlargement of the carotid body is due solely to an increase in extravascular volume.

Cardiac and vascular effects of diltiazem, dobutamine and amrinone, drugs used after myocardial revascularization

Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 µM to 10 mM) and amrinone (10 µM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 ± 7%) at its ED50 for aortic relaxation (88 ± 7 µM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 ± 5 µM, ED50 HSV = 72 ± 31 µM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5%) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 µg/ml) inhibited the contractile effects of 1 µM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means ± SEM = 184 ± 6, 185 ± 3 and 188 ± 19 µg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 µM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 ± 7 µg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 µM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca² channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 µg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 µg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

Biomechanics and structural adaptations of the rat femur after hindlimb suspension and treadmill running

We microscopically and mechanically evaluated the femurs of rats subjected to hindlimb unloading (tail suspension) followed by treadmill training. Female Wistar rats were randomly divided into five groups containing 12-14 rats: control I (118 days old), control II (139 days old), suspended (tail suspension for 28 days), suspended-released (released for 21 days after 28 days of suspension), and suspended-trained (trained for 21 days after 28 days of suspension). We measured bone resistance by bending-compression mechanical tests of the entire proximal half of the femur and three-point bending tests of diaphyseal cortical bone. We determined bone microstructure by tetracycline labeling of trabecular and cortical bone. We found that tail suspension weakened bone (ultimate load = 86.3 ± 13.5 N, tenacity modulus = 0.027 ± 0.011 MPa·m vs ultimate load = 101.5 ± 10.5 N, tenacity modulus = 0.019 ± 0.006 MPa·m in control I animals). The tenacity modulus for suspended and released animals was 0.023 ± 0.010 MPa·m vs 0.046 ± 0.018 MPa·m for trained animals and 0.035 ± 0.010 MPa·m for control animals. These data indicate that normal activity and training resulted in recovered bone resistance, but suspended-released rats presented femoral head flattening and earlier closure of the growth plate. Microscopically, we found that suspension inhibited new bone subperiosteal and endosteal formation. The bone disuse atrophy secondary to hypoactivity in rats can be reversed by an early regime of exercising, which is more advantageous than ordinary cage activities alone.

The effect of microgravity on tissue structure and function of rat testis

To explore whether an environment of weightlessness will cause damage to the reproductive system of animals, we used the tail-suspension model to simulate microgravity, and investigated the effect of microgravity on the tissue structure and function of the testis in sexually mature male rats. Forty-eight male Wistar rats weighing 200-250 g were randomly assigned to three groups (N = 16 each): control, tail traction, and tail suspension. After the rats were suspended for 7 or 14 days, morphological changes of testis were evaluated by histological and electron microscopic methods. The expression of HSP70, bax/bcl-2 and AR (androgen receptor) in testis was measured by immunohistochemistry. Obvious pathological lesions were present in the testis after the rats were suspended for 7 or 14 days. We detected overexpression of HSP70 and an increase of apoptotic cells, which may have contributed to the injury to the testis. The expression of AR, as an effector molecule in the testis, was significantly decreased in the suspended groups compared to control (P < 0.01). We also observed that, with a longer time of suspension, the aforementioned pathological damage became more serious and some pathological injury to the testis was irreversible. The results demonstrated that a short- or medium-term microgravity environment could lead to severe irreversible damage to the structure of rat testis.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Deficiency of sex hormones does not affect 17-ß-estradiol-induced coronary vasodilation in the isolated rat heart

The relaxation of coronary arteries by estrogens in the coronary vascular beds of naive and hypertensive rats has been well described. However, little is known about this action in gonadectomized rats. We investigated the effect of 17-ß-estradiol (E2) in coronary arteries from gonadectomized rats, as well as the contributions of endothelium-derived factors and potassium channels. Eight-week-old female and male Wistar rats weighing 220-300 g were divided into sham-operated and gonadectomized groups (n=9−12 animals per group). The baseline coronary perfusion pressure (CPP) was determined, and the vasoactive effects of 10 μM E2 were assessed by bolus administration before and after endothelium denudation or by perfusion with NG-nitro-L-arginine methyl ester (L-NAME), indomethacin, clotrimazole, L-NAME plus indomethacin, L-NAME plus clotrimazole or tetraethylammonium (TEA). The CPP differed significantly between the female and sham-operated male animals. Gonadectomy reduced the CPP only in female rats. Differences in E2-induced relaxation were observed between the female and male animals, but male castration did not alter this response. For both sexes, the relaxation response to E2 was, at least partly, endothelium-dependent. The response to E2 was reduced only in the sham-operated female rats treated with L-NAME. However, in the presence of indomethacin, clotrimazole, L-NAME plus indomethacin or L-NAME plus clotrimazole, or TEA, the E2 response was significantly reduced in all groups. These results highlight the importance of prostacyclin, endothelium-derived hyperpolarizing factor, and potassium channels in the relaxation response of coronary arteries to E2 in all groups, whereas nitric oxide may have had an important role only in the sham-operated female group.