Long-term treatment of diabetic rats with vanadyl sulfate or insulin attenuate acute focal cerebral ischemia/reperfusion injury via their antiglycemic effect.

It is well-known that patients with diabetes mellitus have worse clinical outcomes following acute ischemic stroke. The intensifying effects of diabetes on ischemic brain injury have been shown to be mostly due to hyperglycemia, rather than the lack of insulin direct effects on brain. It is also well-approved that vanadium compounds have insulin-like and anti-diabetic effects, and the present study was designed to compare the protective effects of diabetes treatment with vanadium or insulin on ischemic/reperfused brain injury. Male Sprague-Dawley rats were divided into 4 groups (n = 21). Two groups of streptozotocin-induced diabetic rats were treated with either vanadyl sulfate or insulin at proper doses to similarly attenuate hyperglycemia during 45 days, while there was no treatment in the control diabetic and non-diabetic sham groups. Thereafter, all treated and non-treated diabetic rats were subjected to 60-min of the right middle cerebral artery occlusion followed by 12-h reperfusion, and then their brains were removed for evaluating blood-brain barrier leakage, tissue swelling, infarct size and oxidant status in both hemispheres. Vanadium and insulin that equally reduced blood glucose and water intake had some differences in their antidiabetic effects of ameliorating weight loss and hypertension during 45-days treatment period. However, they caused similar decrements in levels of Evans blue dye extravastion, edema, infarct volume and malondialdehyde in ischemic/reperfused cerebral hemisphere. Therefore, it can be suggested that insulin and vanadium via their antiglycemic effect cause reduction in cerebral production of oxidants following acute focal ischemia/reperfusion, which attenuate BBB disruption and brain tissue injury.

Cerebral Ischemia-Reperfusion Injuries in Vanadyl-Treated Diabetic Rats.

BACKGROUND: Ischemic stroke recovery is poor in diabetic mellitus (DM). Vanadium compounds (vanadium) relieve DM signs, but their influences on cerebral ischemia/reperfusion injury (I/RI) are inconclusive. Herein, the intensity of I/RI was inspected in vanadium-treated DM rats. METHODS: Rats made diabetic with a single intravenous dose of streptozocin (39 mg/kg). Normal and DM rats used water or vanadyl solution for 45 days. Under isoflurane anesthesia, right middle cerebral artery occlusion was performed for 60 minutes and 12 hours reperfusion. Ischemic rats were divided into untreated-control normal (ICN) and diabetic (ICD), vanadium-treated normal (IVTN) and diabetic (IVTD) groups (n=14 each). After neurological deficit score (NDS) test, the rats were sacrificed and their brain removed and stained with triphenyltetrazolium chloride (TTC) to measure cerebral infarct volume (CIV, mm3) or Evans blue extravasation (EBE, µg/g wet-tissue). Data analysis was performed using one-way ANOVA and Tukey's test (SPSS software, version 21.0) and P values <0.05 were considered statistically significant. RESULTS: Blood glucose (BG, mg/dL) was similar in ICN and IVTN, elevated in IVTD and ICD (245±6 vs. 344±2, P<0.001). The increased CIV in ICN and IVTN was similar (48±2 and 34±5), very high in ICD but lower in IVTD (249±37 vs. 110±16, P<0.001). EBE was absent in non-lesioned hemispheres, similarly increased in lesioned hemispheres of ICN and IVTN (14±1 and 13±1). EBE in IVTD was significantly lower than ICD (21±2 vs. 33±5, P=0.01). CONCLUSION: I/RI was moderate in normoglycemia and did not change with vanadium. Hyperglycemia robustly intensified I/RI. Vanadium ameliorated hyperglycemia and reduced I/RI. Nonetheless, more investigations are required to link the mechanisms of vanadium on DM and stroke injuries.

Cerebral Ischemia/Reperfusion Injury in the Hyperthyroid Rat.

BACKGROUND: Hyperthyroidism as a risk factor for stroke is not conclusive. There are no definite data on the relationship between ischemic cerebrovascular injury and hyperthyroidism. This study was designed to define whether the outcomes of post-ischemic stroke injury are influenced by chronic hyperthyroidism. METHODS: Two groups of hyperthyroid (HT) and control euthyroid rats of equal numbers (n=22) were included in the study. Hyperthyroidism was induced for 4 weeks by adding L-thyroxine (300 µg/kg) to drinking water. The middle cerebral artery occlusion technique was used to induce focal cerebral ischemia. Neurological disability (neurological deficit score [NDS]) was evaluated after 24 hours, and the rats were sacrificed to obtain their brain. Triphenyl Tetrazolium Chloride (TTC) staining and Evans Blue (EB) extravasation were used to quantify cerebral infarct volume and cerebrovascular integrity disruption. Data analysis was done using SPSS, version 21. RESULTS: Thyroid hormones levels, T3 (314±7 vs. 198±3 ng/dL;P=0.001) and T4 (9.8±0.3 vs. 3.08±0.07 µg/dL;P=0.001), were significantly higher in the HT group than in the controls. Furthermore, most clinical signs seen in hyperthyroid patients were also present in the HT group. Comparison of the data on cerebral ischemia between the HT and control groups showed significant increases in the NDS (2.76±0.16 vs. 2.23±0.09;P=0.03), cerebral infarct volume (479±12 vs. 266±17 mm3;P=0.001), and EB extravasation (50.08±2.4 vs. 32.6±1.2 µg/g;P=0.001) in the former group. CONCLUSION: The intensified cerebral infarct size and cerebrovascular integrity disruption suggested that chronic hyperthyroidism aggravated post-stroke injury in the rats. More investigation is required to analyze the pathological mechanisms underlying the association between cerebrovascular disease and hyperthyroidism.

Quantitative Assessment of Proliferative Effects of Oral Vanadium on Pancreatic Islet Volumes and Beta Cell Numbers of Diabetic Rats.

BACKGROUND: Oral vanadyl sulfate (vanadium) induces normoglycemia, proliferates beta cells and prevents pancreatic islet atrophy in streptozotocin-induced diabetic rats. Soteriological method is used to quantitate the proliferative effects of vanadium on beta-cell numbers and islet volumes of normal and diabetic rats. METHODS: Adult male Sprague-Dawley rats were made diabetic with intravenous streptozotocin injection (40 mg/kg). Normal and diabetic rats were divided into four groups. While control normal and diabetic (CD) groups used water, vanadium-treated normal (VTN) and diabetic (VTD) groups used solutions containing vanadyl sulfate (0.5-1 mg/mL, VOSO4+5H2O). Tail blood samples were used to measure blood glucose (BG) and plasma insulin. Two months after treatment, rats were sacrificed, pancreata prepared, and stereology method was used to quantitatively evaluate total beta cell numbers (TBCN) and total islet volumes (TISVOL). RESULTS: Normoglycemia persisted in VTN with significantly decreased plasma insulin (0.19±0.08 vs. 0.97±0.27 ng/dL, P<0.002). The respective high BG (532±49 vs. 144±46 mg/dL, P<0.0001) and reduced plasma insulin (0.26±0.15 vs. 0.54±0.19 ng/dL, P<0.002) seen in CD were reversed in VTD during vanadium treatment or withdrawal. While the induction of diabetes, compared to their control, significantly decreased TISVOL (1.9±0.2 vs. 3.03±0.6 mm3, P<0.003) and TBCN (0.99±0.1 vs. 3.2±0.2 x 106, P<0.003), vanadium treatment significantly increased TISVOL (2.9±0.8 and 4.07±1.0 mm3, P<0.003) and TBCN (1.5±0.3 and 3.8±0.6 x 106, P<0.03). CONCLUSION: Two-month oral vanadium therapy in STZ-diabetic rats ameliorated hyperglycemia by partially restoring plasma insulin. This action was through proliferative actions of vanadium in preventing islet atrophy by increasing beta-cell numbers.

Trophic actions of oral vanadium and improved glycemia on the pancreatic beta-cell ultrastructure of streptozotocin-induced diabetic rats.

CONTEXT: Oral vanadyl sulfate (vanadium) has potent hypoglycemic effects in diabetes animals, but data about its actions on pancreatic beta-cells (BC) ultrastructure is limited. OBJECTIVE: Partial diabetic rats were treated with vanadium and insulin injection and their effects on BC ultrastructure are studied. METHODS: Male rats were made diabetic with intravenous streptozotocin injection (STZ, 40 mg/kg). Animals were randomly divided to control (CD), vanadium-treated (1 mg/mL VOSO4 + 5H2O in base solution, VTD) and insulin-treated (80 U/kg/day NPH insulin injection, ITD) diabetic groups. Treatments started 10 days after STZ injection and terminated after 2 months. Intermittent tail blood samples were taken for measurements of blood glucose (BG) and plasma insulin (PI). Finally animals were sacrificed and pancreata prepared for assessments of BC ultrastructure, islets histology and insulin immunoreactivity (IIR). RESULTS: Vanadium decreased BG (P<0.0001), elevated the reduced PI (P<0.001), prevented islet atrophy and restored BC ultrastructure. Low BG seen during treatment in VTD and ITD only persisted in VTD after vanadium withdrawal. Hyperglycemia worsened in CD and repaired in ITD shortly after insulin withdrawal. CD islets were atrophied, had scattered IIR spots. BC had pyknotic nuclei, vacuolated cytoplasm and few tiny insulin secretory granules. VTD islets looked normal with compact centered IIR spots. BC had well-developed endoplasmic reticulum, many insulin secretory granules and mitochondria. ITD islet structure was slightly better than CD and BC had some immature insulin secretory granules. CONCLUSION: The trophic actions of vanadium in diabetic rats effectively renovated beta cell ultrastructure and prevented pancreatic islets atrophy, whereas the relief of glucotoxicity seen with insulin treatment could repair some beta cells and partially prevented islet atrophy.

Blockade of Central Angiotensin II AT1 Receptor Protects the Brain from Ischemia/Reperfusion Injury in Normotensive Rats.

BACKGROUND: Stroke is the third leading cause of invalidism and death in industrialized countries. There are conflicting reports about the effects of Angiotensin II on ischemia-reperfusion brain injuries and most data have come from chronic hypertensive rats. In this study, hypotensive and non-hypotensive doses of candesartan were used to investigate the effects of angiotensin II AT1 receptor blockade by transient focal cerebral ischemia in normotensive rats. METHODS: In this experimental study, 48 male Sprague-Dawley rats were randomly divided into four groups (n=12). Sham group, the control ischemic group, and two ischemic groups received candesartan at doses of 0.1 or 0.5 mg/kg at one hour before ischemia. Transient focal cerebral ischemia was induced by 60 minutes occlusion of the middle cerebral artery, followed by 24 h reperfusion. The neurological deficit score was evaluated at the end of the reperfusion period. The total cortical and striatal infarct volumes were determined using triphenyltetrazolium chloride staining technique. Tissue swelling was calculated for the investigation of ischemic brain edema formation. RESULTS: In comparison with the control ischemic group, AT1 receptor blockade with both doses of candesartan (0.1 or 0.5 mg/kg) significantly improved neurological deficit and lowered cortical and striatal infarct sizes. In addition, pretreatment with candesartan significantly reduced ischemia induced tissue swelling. CONCLUSION: Angiotensin II by stimulating AT1 receptors, participates in ischemia-reperfusion injuries and edema formation. AT1 receptor blockade with candesartan decreased ischemic brain injury and edema and improved neurological outcome.

Candesartan attenuates ischemic brain edema and protects the blood-brain barrier integrity from ischemia/reperfusion injury in rats.

BACKGROUND: Angiotensin II (Ang II) has an important role on cerebral microcirculation; however, its direct roles in terms of ischemic brain edema need to be clarified. This study evaluated the role of central Ang II by using candesartan, as an AT1 receptor blocker, in the brain edema formation and blood-brain barrier (BBB) disruption caused by ischemia/reperfusion (I/R) injuries in rat. METHODS: Rats were exposed to 60-min middle cerebral artery (MCA) occlusion. Vehicle and non-hypotensive doses of candesartan (0.1 mg/kg) were administered one hour before ischemia. Neurological dysfunction scoring was evaluated following 24 h of reperfusion. Animals were then decapitated under deep anesthesia for the assessments of cerebral infarct size, edema formation, and BBB permeability. RESULTS: The outcomes of 24 h reperfusion after 60-min MCA occlusion were severe neurological disability, massive BBB disruption (Evans blue extravasation = 12.5 ± 1.94 µg/g tissue), 4.02% edema, and cerebral infarction (317 ± 21 mm3). Candesartan at a dose of 0.1 mg/kg, without changing arterial blood pressure, improved neurological dysfunction scoring together with significant reductions in BBB disruption (54.9%), edema (59.2%), and cerebral infarction (54.9%). CONCLUSIONS: Inactivation of central AT1 receptors, if not accompanied with arterial hypotension, protected cerebral micro-vasculatures from damaging effects of acute stroke.

Does the relief of glucose toxicity act as a mediator in proliferative actions of vanadium on pancreatic islet beta cells in streptozocin diabetic rats?

BACKGROUND: Data shows vanadium protects pancreatic beta cells (BC) from diabetic animals. Whether this effect is direct or through the relief of glucose toxicity is not clear. This study evaluated the potential effect of oral vanadyl sulfate (vanadium) on glycemic status and pancreatic BC of normal and diabetic rats. METHODS: Rats were divided into five groups of normal and diabetic. Diabetes was induced with streptozocin (40 mg/kg, i.v.). Normal rats used water (CN) or vanadium (1 mg/ml VOSO4, VTN). Diabetic rats used water (CD), water plus daily neutral protamine Hagedorn insulin injection (80 U/kg, ITD) or vanadium (VTD). Blood samples were taken for blood glucose (BG, mg/dL) and insulin (ng/dL) measurements. After two months, the pancreata of sacrificed rats were prepared for islet staining. RESULTS: Pre-treated normal BG was 88 ± 2, and diabetic BG was 395 ± 9. The final BG in CD, VTD, and ITD was 509 ± 22, 138 ± 14, and 141 ± 14, respectively. Insulin in VTN (0.75 ± 0.01) and VTD (0.78 ± 0.01) was similar, higher than CD (0.51 ± 0.07) but lower than CN (2.51 ± 0.02). VTN islets compared to CN had larger size and denser central core insulin immunoreactivity with plentiful BC. CD and ITD islets were atrophied and had scattered insulin immunoreactivity spots and low BC mass. VTD islets were almost similar to CN. CONCLUSION: Besides insulin-like activity, vanadium protected pancreatic islet BC, and the relief of glucose toxicity happening with vanadium had a little role in this action.

Enalapril attenuates ischaemic brain oedema and protects the blood-brain barrier in rats via an anti-oxidant action.

1. In the present study, we investigated the effects of postischaemic angiotensin-converting enzyme (ACE) inhibition with enalapril on vasogenic oedema formation and blood-brain barrier (BBB) integrity following transient focal cerebral ischaemia in rats. 2. Cerebral ischaemia was induced by 60 min occlusion of the right middle cerebral artery, followed by 24 h reperfusion. Vehicle and a non-hypotensive dose of enalapril (0.03 mg/kg) were administered at the beginning of the reperfusion period. A neurological deficit score (NDS) was determined for all rats at the end of the reperfusion period. Then, brain oedema formation was investigated using the wet-dry weight method and BBB permeability was evaluated on the basis of extravasation of Evans blue (EB) dye. In addition, oxidative stress was assessed by measuring reduced glutathione (GSH) and malondialdehyde (MDA) in brain homogenates. 3. Inhibition of ACE by enalapril significantly reduced NDS and decreased brain oedema formation (P < 0.05 for both). Disruption of the BBB following ischaemia resulted in considerable leakage of EB dye into the brain parenchyma of the ipsilateral hemispheres of vehicle-treated rats. Enalapril significantly (P < 0.05) decreased EB extravasation into the lesioned hemisphere. Enalapril also augmented anti-oxidant activity in ischaemic brain tissue by increasing GSH concentrations and significantly (P < 0.05) attenuating the increased MDA levels in response to ischaemia. 4. In conclusion, inhibition of ACE with a non-hypotensive dose of enalapril may protect BBB function and attenuate oedema formation via anti-oxidant actions.

Long- term efficacy and safety of vanadium in the treatment of type 1 diabetes.

BACKGROUND: Vanadium compounds are able to reduce blood glucose in experimentally- induced diabetic rats and type 2 diabetic patients, but data about their long- term safety and efficacy in diabetic patients are scarce. METHODS: Fourteen type 1 diabetic patients received oral vanadyl sulfate (50 - 100 mg TID) for a period of 30 months. Fasting blood sugar (FBS), lipid levels, hematologic, and biochemical parameters were measured before and periodically during the treatment. RESULTS: The daily doses of insulin decreased from 37.2 ± 5.5 to 25.8 ± 17.3 units/day and at the same time the mean FBS decreased from 238 ± 71 to 152 ± 42 mg/dL. Meanwhile, there was a decrease in plasma total cholesterol without any change in triglyceride level. No significant clinical or paraclinical side effects, with the exception for mild diarrhea at the beginning of treatment, were observed during 30 months therapy. CONCLUSION: Vanadium is effective and safe for long- term use in type 1 diabetic patients.

Autonomic regulation of organ vascular resistances during hypoxemia in the cat.

This study aimed to dissect the roles played by the autonomic interoreceptors, the carotid bodies (cbs) and the aortic bodies (abs) on the vascular resistances of several organs in anesthetized, paralyzed, artificially ventilated cats challenged by systemic hypoxemia. Two 15 min challenges stimulated each of 5 animals in two different groups: (1) in the intact group hypoxic hypoxia (10% O2 in N2; HH) stimulated both abs and cbs, increasing neural output to the nucleus tractus solitarius (NTS); (2) in this group carbon monoxide hypoxia (30% O2 in N2 with the addition of CO; COH) stimulated only the abs, increasing neural output to the NTS. (3) In the second group in which their bilateral aortic depressor nerves had been transected only the cbs increased neural output to the NTS during the HH challenge; (4) in this aortic body resected group during COH neither abs nor cbs increased neural traffic to the NTS. CO and 10% O2 reduced Hb saturation to the same level. With the use of radiolabeled microspheres blood flow was measured in a variety of organs. Organ vascular resistance was calculated by dividing the aortic pressure by that organ's blood flow. The spleen and pancreas revealed a vasoconstriction in the face of systemic hypoxemia, thought to be sympathetic nervous system (SNS)-mediated. The adrenals and the eyes vasodilated only when cbs were stimulated. Vasodilation in the heart and diaphragm showed no effect of chemoreceptor stimulated increase in SNS output. Different chemoreceptor involvement had different effects on the organs.

Autonomic control of the cardiovascular system in the cat during hypoxemia.

This study aimed to determine the roles played by the autonomic interoreceptors, the carotid bodies (cbs) and the aortic bodies (abs) in anesthetized, paralyzed, artificially ventilated cats' response to systemic hypoxemia. Four 15min challenges stimulated each of 15 animals: (1) hypoxic hypoxia (10%O2 in N2; HH) in the intact (int) cat where both abs and cbs sent neural traffic to the nucleus tractus solitarius (NTS); (2) carbon monoxide hypoxia (30%O2 in N2 with the addition of CO; COH) in the intact cat where only the abs sent neural traffic to the NTS; (3) HH in the cat after transection of both aortic depressor nerves, resecting the aortic bodies (HHabr), where only the cbs sent neural traffic to the NTS; (4) COH to the abr cat where neither abs nor cbs sent neural traffic to the NTS. Cardiac output (C.O.), contractility (dP/dt(MAX)), systolic/diastolic pressures, aortic blood pressure, total peripheral resistance, pulmonary arterial pressure, and pulmonary vascular resistance (PVR) were measured. When both cbs and abs were active the maximum increases were observed except for PVR which decreased. Some variables showed the cbs to have a greater effect than the abs. The abs proved to be important during some challenges for maintaining blood pressure. The data support the critically important role for the chemoreceptor-sympathetic nervous system connection during hypoxemia for maintaining viable homeostasis, with some differences between the cbs and the abs.

Attenuation of focal cerebral ischemic injury following post-ischemic inhibition of angiotensin converting enzyme (ACE) activity in normotensive rat.

BACKGROUND: Central renin angiotensin system has an important role on the cerebral microcirculation and metabolism. Our previous work showed that inhibition of angiotensin converting enzyme (ACE) activity prior to induction of ischemia protected the brain from severe ischemia/reperfusion (I/R) injuries. This study evaluated the impacts of post-ischemic inhibition of ACE, enalapril, on brain infarction in normotensive rats. METHODS: Rats were anesthetized with chloral hydrate (400 mg/kg). Focal cerebral ischemia was induced by 60-min intraluminal occlusion of right middle cerebral artery (MCA). Intraperitoneal injection of enalapril (0.03 or 0.1 mg/kg) was done after MCA reopening (reperfusion). Neurological deficit score (NDS) was evaluated after 24 h and the animals randomly assigned for the assessments of infarction, absolute brain water content (ABWC) and index of brain edema. RESULTS: Severe impaired motor functions (NDS = 2.78 ± 0.28), massive infarction (cortex = 214 ± 19 mm3, striatum = 86 ± 5 mm3) and edema (ABWC = 83.1 ± 0.46%) were observed in non-treated ischemic rats. Non-hypotensive dose of enalapril (0.03 mg/kg) significantly reduced NDS (1.5 ± 0.22), infarction (cortex = 102 ± 16 mm3, striatum = 38 ± 5 mm3) and edema (ABWC = 80.9 ± 0.81%). Enalapril at dose of 0.1 mg/kg significantly lowered arterial pressure could not improve NDS (2.0 ± 0.45) and reduce infarction (cortex = 166 ± 26 mm3, striatum = 71 ± 11 mm3). CONCLUSION: Post-ischemic ACE inhibition in the normotensive rats without affecting arterial pressure protects the brain from reperfusion injuries; however, this beneficial action is masked by hypotension.

Contribution of nitric oxide synthase (NOS) activity in blood-brain barrier disruption and edema after acute ischemia/reperfusion in aortic coarctation-induced hypertensive rats.

BACKGROUND: Nitric oxide synthase (NOS) activity is increased during hypertension and cerebral ischemia. NOS inactivation reduces stroke-induced cerebral injuries, but little is known about its role in blood-brain barrier (BBB) disruption and cerebral edema formation during stroke in acute hypertension. Here, we investigated the role of NOS inhibition in progression of edema formation and BBB disruptions provoked by ischemia/reperfusion injuries in acute hypertensive rats. METHODS: Rats were made acutely hypertensive by aortic coarctation. After 7 days, the rats were randomly selected for the recording of carotid artery pressure, or regional cerebral blood flow (rCBF) using laser Doppler. Ishcemia induced by 60-min middle cerebral artery occlusion (MCAO), followed by 12-h reperfusion. A single i.p. dose of L-NAME (1 mg/kg) was injected before MCAO. After evaluation of neurological disabilities, rats were slaughtered under deep anesthesia to assess cerebral infarction volume, edema, or BBB disruption. RESULTS: A 75-85% reduction in rCBF was occurred during MCAO which returned to pre-occluded levels during reperfusion. Profound neurological disabilities were evidenced after MCAO alongside with severe cerebral infarctions (628 ± 98 mm3), considerable edema (4.05 ± 0.52%) and extensive BBB disruptions (Evans blue extravasation, 8.46 ± 2.03 mug/g). L-NAME drastically improved neurological disabilities, diminished cerebral infarction (264 ± 46 mm3), reduced edema (1.49 ± 0.47%) and BBB disruption (2.93 ± 0.66 mug/g). CONCLUSION: The harmful actions of NOS activity on cerebral microvascular integrity are intensified by ischemia/reperfusion injuries during acute hypertension. NOS inactivation by L-NAME preserved this integrity and diminished cerebral edema.