Anil Gulati

Role of endothelin in the cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin.

OBJECTIVES Diaspirin crosslinked hemoglobin is a resuscitative solution with excellent oxygen-carrying capacity. Diaspirin crosslinked hemoglobin produces an immediate increase in blood pressure and marked regional circulatory changes in rats and pigs. Our objective was to determine the role of endothelin in the cardiovascular actions of diaspirin crosslinked hemoglobin (modified) and (unmodified) stroma reduced hemoglobin solutions. DESIGN Prospective, randomized comparison of cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin in control rats and in rats pretreated with cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), an endothelin-A receptor antagonist. SETTING Research laboratory. SUBJECTS Male Sprague-Dawley rats. INTERVENTIONS Modified, highly purified, and heat pasteurized (diaspirin crosslinked) and unmodified (stroma reduced) hemoglobin in control (untreated) and BQ-123 (5 mg/kg/hr iv)-treated rats. MEASUREMENTS AND MAIN RESULTS Infusion of stroma reduced hemoglobin (400 mg/kg iv) in control rats produced an increase in blood pressure (43%) and total peripheral resistance (65%) without any change in heart rate, cardiac output, and stroke volume. Stroma reduced hemoglobin decreased blood flow to the kidneys and liver, increased blood flow to the heart, and had no effect on blood flow to the brain, gastrointestinal tract, spleen, musculoskeletal system, skin, and mesentery and pancreas. Infusion of stroma reduced hemoglobin in rats treated with BQ-123 (5 mg/kg/hr iv) increased the blood pressure to a similar degree when compared with control rats, but the increase in total peripheral resistance was significantly attenuated. The stroma reduced hemoglobin-induced decrease in blood flow to the kidneys and liver was significantly attenuated in BQ-123-treated rats as compared with control rats. However, the stroma reduced hemoglobin-induced increase in blood flow to the heart of BQ-123-treated rats was similar to the increase in control rats. Infusion of diaspirin crosslinked hemoglobin (400 mg/kg iv) produced increases in blood pressure (81%), cardiac output (36%), stroke volume (30%), and total peripheral vascular resistance (45%), along with increases in blood flow to the heart, spleen, gastrointestinal tract, and skin of control rats. The blood flows to the brain, kidneys, liver, musculoskeletal system, and mesentery and pancreas were not altered by diaspirin crosslinked hemoglobin in control rats. The increases in blood pressure, cardiac output, stroke volume, and total peripheral vascular resistance by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. The increases in blood flow to the heart, spleen, and skin by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. Diaspirin crosslinked hemoglobin produced an increase in the blood flow to the brain and a decrease in blood flow to the kidney and musculoskeletal system of BQ-123-treated rats as compared with control rats. Blood plasma endothelin-1-like immunoreactivity was found to be significantly increased after treatment with diaspirin crosslinked hemoglobin or stroma reduced hemoglobin. CONCLUSIONS The endothelin-A receptor antagonist, BQ-123, could attenuate the systemic hemodynamic and regional circulatory effects of diaspirin crosslinked hemoglobin and stroma reduced hemoglobin. However, the increase in blood flow to the heart induced by stroma reduced hemoglobin could not be attenuated by BQ-123.

Effect of stroma-free hemoglobin and diaspirin cross-linked hemoglogbin on the regional circulation and systematic hemodynamics

The effects of unmodified stroma-free hemoglobin (SFHb) and diaspirin cross-linked hemoglobin (DCLHb) on the regional blood circulation and systemic hemodynamics were studied in rats using a radioactive microsphere technique. SFHb and DCLHb increased mean arterial blood pressure without affecting heart rate. SFHb produced a 24.9% decrease in the cardiac output while DCLHb produced an 44.8% increase in the cardiac output. Stroke volume was decreased (-27.3%) by SFHb and increased (+36.4%) by DCLHb. Total peripheral resistance increased with both SFHb and DCLHb. DCLHb increased blood flow to the heart, spleen, stomach, small intestine and skin, and had no effect on blood flow to the brain, kidneys, liver, mesentery, pancreas, caecum, large intestine and musculo-skeletal system. In contrast, in animals infused with SFHb, blood flow decreased to the kidneys, liver and spleen, increased to the heart, small intestine and skin, and had no effect to the brain, caecum, large intestine and musculo-skeletal system. DCLHb had no effect on vascular resistance in any organ except for an increase in the musculo-skeletal system. In contrast, SFHb increased vascular resistance in the kidneys, liver, spleen, skin, mesentery and pancreas, and had no effect on vascular resistance in the musculo-skeletal system, brain, heart, stomach, small intestine, caecum and large intestine. SFHb had no effect on distribution of cardiac output to the brain, gastrointestinal tract (GIT), kidneys, skin, musculo-skeletal and portal system, while DCLHb significantly decreased the percent cardiac output to the musculo-skeletal system. DCLHb did not affect the distribution of cardiac output to the brain, GIT, kidneys, skin and portal system. SFHb and DCLHb increased the percent cardiac output to the heart. It is concluded that similar concentrations and doses of DCLHb and SFHb produce different effects on the regional blood circulation and systemic hemodynamics.

Use of magnesium in traumatic brain injury

SummaryDepletion of magnesium is observed in animal brain and in human blood after brain injury. Treatment with magnesium attenuates the pathological and behavioral changes in rats with brain injury; however, the therapeutic effect of magnesium has not been consistently observed in humans with traumatic brain injury (TBI). Secondary brain insults are observed in patients with brain injury, which adversely affect clinical outcome. Systemic administration studies in rats have shown that magnesium enters the brain; however, inducing hypermagnesemia in humans did not concomitantly increase magnesium levels in the CSF. We hypothesize that the neuroprotective effects of magnesium in TBI patients could be observed by increasing its brain bioavailability with mannitol. Here, we review the role of magnesium in brain injury, preclinical studies in brain injury, clinical safety and efficacy studies in TBI patients, brain bioavailability studies in rat, and pharmacokinetic studies in humans with brain injury. Neurodegeneration after brain injury involves multiple biochemical pathways. Treatment with a single agent has often resulted in poor efficacy at a safe dose or toxicity at a therapeutic dose. A successful neuroprotective therapy needs to be aimed at homeostatic control of these pathways with multiple agents. Other pharmacological agents, such as dexanabinol and progesterone, and physiological interventions, with hypothermia and hyperoxia, have been studied for the treatment of brain injury. Treatment with magnesium and hypothermia has shown favorable outcome in rats with cerebral ischemia. We conclude that coadministration of magnesium and mannitol with pharmacological and physiological agents could be an effective neuroprotective regimen for the treatment of TBI.

Functional and biochemical evidence indicating beneficial effect of Melatonin and Nicotinamide alone and in combination in experimental diabetic neuropathy

Oxidative stress resulting in excessive generation of ROS is a compelling initiator of DNA damage along with damage to various cellular proteins and other macromolecules. Poly(ADP-ribose) polymerase (PARP) activation in response to DNA damage, stirs an energy-consuming cellular metabolic cycle; culminating into cell death. The present study was designed to determine the effect of combining an antioxidant, Melatonin and a PARP inhibitor, Nicotinamide on the hallmark deficits developing in diabetic neuropathy (DN). Streptozotocin (STZ, 55 mg/kg, i.p.) was administered to induce diabetes. Six weeks post diabetes induction, two week treatment with Melatonin (3 and 10 mg/kg) and Nicotinamide (100 and 300 mg/kg) either alone or in combination was given. Effect of these interventions on the functional, behavioral and biochemical changes caused by hyperglycemia were studied in treated animals. Melatonin and Nicotinamide alone as well as in combination ameliorated the functional deficits along with improvement in pain parameters. The combination also demonstrated an essential reversal of biochemical alterations. Nitrotyrosine and Poly ADP Ribose (PAR) immunopositivity was significantly decreased in sciatic nerve micro-sections of treatment group. The results of this study advocate that simultaneous inhibition of oxidative stress-PARP activation cascade may prove useful for the pharmacotherapy of DN.

Yohimbine modulates diaspirin crosslinked hemoglobin-induced systemic hemodynamics and regional circulatory effects

OBJECTIVE Diaspirin crosslinked hemoglobin, a hemoglobin-based blood substitute, is proposed to be an effective resuscitative solution. It produces an immediate, but limited increase in blood pressure when administered to conscious or anesthetized rats. This vasoactivity is associated with an increase in blood flow to several major organs. It has been shown that alpha-adrenergic receptors in the peripheral vascular system are sensitized by diaspirin crosslinked hemoglobin in rats. The present study was conducted to determine the effect of yohimbine, an alpha 2-adrenergic receptor antagonist on systemic hemodynamics and regional circulatory effects of diaspirin crosslinked hemoglobin. DESIGN Prospective, randomized comparison of cardiovascular effects of diaspirin crosslinked hemoglobin in control and yohimbine-pretreated rats. SETTING Laboratory of experimental medicine. SUBJECTS Male Sprague-Dawley rats weighing 300 to 350 g. INTERVENTIONS Modified, highly purified, and heat-pasteurized hemoglobin (diaspirin crosslinked hemoglobin) in control and yohimbine-treated (2 mg/kg i.v.) rats. MEASUREMENTS AND MAIN RESULTS The systemic hemodynamics and regional circulation were measured using a radioactive microsphere technique. Diaspirin crosslinked hemoglobin (400 mg/kg i.v.) produced an increase in blood pressure and total peripheral resistance, while heart rate, cardiac output, and stroke volume were not significantly altered in control rats. In yohimbine-pretreated (2 mg/kg i.v.) animals, diaspirin crosslinked hemoglobin did not produce any change in heart rate, stroke volume, cardiac output, and total peripheral resistance, but a slight increase in blood pressure was observed compared with baseline values obtained after the administration of yohimbine. The increase in blood pressure induced by diaspirin crosslinked hemoglobin was significantly blocked by pretreatment with yohimbine. Yohimbine (2 mg/kg i.v.) per se decreased blood pressure, while other systemic hemodynamic parameters were not affected. Diaspirin crosslinked hemoglobin increased blood flow to the heart, gastrointestinal tract (stomach, small intestine, cecum, and large intestine), portal (spleen, mesentery, and pancreas) and skin, while blood flow to the brain (cerebral hemispheres, diencephalon, cerebellum, and brain stem), liver, kidneys, and musculoskeletal system was not affected in control rats. In yohimbine-pretreated animals, diaspirin crosslinked hemoglobin produced an increase in blood flow to the heart, brain (cerebellum and brain stem), liver, small intestine, cecum, spleen, mesentery and pancreas, kidneys, skin and musculoskeletal system, while blood flow to the stomach and large intestine was not affected. Yohimbine pretreatment significantly attenuated the diaspirin crosslinked hemoglobin-induced increase in blood flow to the large intestine, mesentery, and pancreas. CONCLUSIONS The cardiovascular actions of diaspirin crosslinked hemoglobin are partially mediated through alpha 2-adrenergic receptors. Adrenergic receptor antagonists may be useful in attenuating the pressor effect of diaspirin crosslinked hemoglobin while maintaining the regional perfusion.

Repeated administration of exendin-4 reduces focal cerebral ischemia-induced infarction in rats

Exendin-4 is a GLP-1 agonist that is clinically used for the treatment of diabetes mellitus and may also have neuroprotective effect. We explored the effect of repeated administration of exendin-4 (0.5 μg/kg, intraperitoneal twice a day for 7 days) on infarct volume, neurological deficit (neurological score, grip test, foot fault and rota rod tests), oxidative stress parameters (malondialdehyde, reduced glutathione, and superoxide dismutase) and expression of endothelin (ET) ET(A) and ET(B) receptors following cerebral ischemia produced in rats by permanent middle cerebral artery occlusion (MCAO). Since ET(A) receptors in the central nervous system (CNS) are involved in cerebral ischemia, we determined the effect of a specific ET(A) receptor antagonist, BQ123 (1mg/kg, intravenously administered thrice: 30 min, 2h and 4h after MCAO for a total dose of 3 mg/kg) on cerebral ischemia in control and exendin-4 treated rats. Results indicate that exendin-4 treated rats had significant protection following MCAO induced cerebral ischemia. The infarct volume was 27% less compared to vehicle treated rats. The neurological deficit following MCAO was lower and oxidative stress parameters were improved in exendin-4 treated rats compared to control. BQ123 significantly improved infarct volume, oxidative stress parameters and neurological deficit in ischemic rats treated with vehicle or exendin-4. BQ123 induced protection from cerebral ischemia was similar in vehicle or exendin-4 treated rats. Expression of ET(A) receptors was significantly increased following cerebral ischemia which was not affected by exendin-4 treatment or by BQ123 administration. No change in expression of ET(B) receptors was observed following cerebral ischemia or any treatment. It is concluded that exendin-4 protects the CNS from damage due to cerebral ischemia by reducing oxidative stress and is independent of ET receptor involvement.

Dose-dependent effect of diaspirin cross-linked hemoglobin on regional blood circulation of severely hemorrhaged rats.

Diaspirin cross-linked hemoglobin (DCLHbTM), a hemoglobin-based blood substitute, has been found to improve systemic hemodynamics, cutaneous oxygen tension, and normalization of blood lactate levels and acid-base equilibrium after hemorrhage in animals. The present study was conducted to determine the dose-dependent effect of a 10% solution of DCLHb (20, 50, and 100% of shed blood volume; SBV) on regional blood circulation in hemorrhaged rats. Hemorrhage was induced in urethane-anesthetized rats by bleeding them at a rate of approximately .5 to 1 mL/min until a mean arterial pressure of 35–40 mmHg was achieved. This was maintained for up to 90 min to reach a base deficit of more than −12 mmol/L. Hemorrhage significantly decreased oxygen consumption, mean arterial pressure, cardiac output, stroke volume, and regional blood circulation, but increased total peripheral resistance. The vehicle Ringers lactate (RL at 20% of SBV, intravenously) did not produce any improvements in oxygen consumption, base deficit, systemic hemodynamics, and regional blood circulation. DCLHb increased oxygen consumption, decreased base deficit, and produced significant improvements in systemic hemodynamics and regional blood flow in a dose-dependent manner. The increase in blood flow was highly significant until 60 min, but was less marked at 120 min, after resuscitation with DCLHb. Resuscitation with RL (300% of SBV) significantly improved systemic and regional blood circulation. However, the improvement was greater after resuscitation with DCLHb (50 or 100% of SBV) as compared with RL at 300% SBV. DCLHb in the dose of 50% of SBV produced maximal resuscitative effects, which were comparable to a DCLHb dose of 100% of SBV. The effect of DCLHb at 50% of SBV on renal cortical blood perfusion, concentration of moving red blood cells (CMBC), and blood velocity was also studied using laser Doppler flowmetry. Hemorrhage produced a decrease in renal cortical blood perfusion (85.3%), which was due to a decrease in the CMBC (61.0%) and their velocity (64.2%). Resuscitation with the RL did not produce any improvement in renal cortical perfusion. However, resuscitation with DCLHb significantly increased renal cortical perfusion (364.7%) due to an increase in both CMBC (123.4%) and their velocity (109.9%). It is concluded that DCLHb in a dose of 50% of SBV produces maximal improvement in regional blood circulation of hemorrhaged rats.

Neuroprotective and anti-apoptotic effects of liraglutide in the rat brain following focal cerebral ischemia.

Stroke is a leading cause of death and serious, long-term disability worldwide. We report that rats receiving liraglutide show markedly attenuated infarct volumes and neurological deficit following ischemic insult. We have also investigated the effect of liraglutide on apoptosis and oxidative stress pathways after ischemic injury in diabetic and non-diabetic rats. Male Sprague-Dawley rats weighing 300-350g were used. Diabetes was induced by streptozotocin. Rats were pretreated with either vehicle or liraglutide (50μg/kg, s.c.) for 14days and thereafter subjected to middle cerebral artery occlusion (MCAO). Twenty-four hours after occlusion, rats were assessed for neurological deficit, motor function and subsequently sacrificed for estimation of infarct volume, oxidative stress and apoptotic markers. Vehicle-treated non-diabetic and diabetic rats showed significant (p<0.001) neurological deficit following cerebral ischemia. Liraglutide pretreatment resulted in significantly (p<0.001) less neurological deficit compared to vehicle-treated MCAO rats. Cerebral ischemia produced significant (p<0.0001) infarction in vehicle-treated rats; however, the infarct volume was significantly (p<0.001) less in liraglutide-pretreated rats. Oxidative stress markers were increased following ischemia but were attenuated in liraglutide-treated rats. Anti-apoptotic protein Bcl-2 expression was decreased and pro-apoptotic protein Bax expression was increased in vehicle-treated MCAO rats compared to sham (p<0.0001). On the other hand liraglutide pretreatment showed significantly (p<0.01) increased expression of Bcl-2 and decreased expression of Bax in MCAO rats. In vehicle-treated group, the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells significantly (p<0.0001) increased in the ischemic hemisphere compared to sham-operated group. The number of TUNEL-positive cells in vehicle group was 73.5±3.3 and 85.5±5.2/750μm(2) in non-diabetic and diabetic vehicle-treated MCAO rats, respectively. Following liraglutide treatment the number of TUNEL-positive cells was remarkably attenuated to 25.5±2.8 and 41.5±4.1/750μm(2) (p<0.001) in non-diabetic and diabetic rats, respectively. The results demonstrate that glucagon-like peptide 1 (GLP-1) agonist, liraglutide, is a neuroprotective agent and attenuates the neuronal damage following cerebral ischemia in rats by preventing apoptosis and decreasing oxidative stress.

Endothelin-A Receptor Antagonists Prevent Amyloid-β-Induced Increase in ETA Receptor Expression, Oxidative Stress, and Cognitive Impairment

Alzheimers disease is a neurodegenerative disorder associated with abnormal accumulation of amyloid-β (Aβ) which can release endothelin (ET). The present study was conducted to investigate the effect of ET antagonists on Aβ-induced changes in ETA and ETB receptor expression, oxidative stress, and cognitive impairment. Male Sprague-Dawley rats were treated with Aβ1-40 in the lateral cerebral ventricles and were administered vehicle or ET antagonists for 14 days. Aβ treatment produced an increase in ETA receptor expression in the cerebral cortex, hippocampus, and brain stem by 72%, 85%, and 90%, respectively. No change in ETB receptor expression was observed. There was an increase in malondialdehyde (MDA) and decrease in reduced glutathione (GSH) and superoxide dismutase (SOD) levels in Aβ-treated rats. In the Morris swim task, Aβ treated rats showed a significant impairment in spatial memory. ET receptor antagonists, BQ123, BMS182874, and TAK-044, significantly decreased Aβ-induced increase in ETA expression in the cortex, hippocampus, and brain stem. Rats treated with ET antagonists showed significant attenuation of Aβ-induced changes in the brain MDA, GSH, and SOD levels. Rats treated with specific ETA receptor antagonists, BQ123 and BMS182874, significantly reduced the cognitive impairment induced by Aβ. However, nonspecific ETA/ETB receptor antagonist TAK-044 did not show any improvement in the learning and memory parameter. This study demonstrates that ETA receptor antagonists are effective in preventing cognitive impairment, changes in ETA expression and oxidative stress induced by Aβ. It is concluded that ETA receptor antagonists may be useful in improving cognitive impairment due to Alzheimers disease.

Changes in the Concentration of Endothelin-1 during Development of Hypertensive Rats

The concentration of endothelin-1 (ET-1) in the brain regions, heart, and throacic aorta of 1-, 4-, 6- and 8-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined using radioimmunoassay. ET-1-like immunoreactivity in the brain regions of 1-week-old WKY and SHR rats was lower compared to older (6 and 8 weeks) rats. ET-1 levels in the central nervous system gradually increased with age in both SHR and WKY rats. However, the concentration of ET-1 in 8-week-old rats was lower in the brain regions of SHR compared to WKY rats. The concentration of ET-1 in the thoracic aorta of SHR (224 +/- 43 pg/g tissue) rats was lower than that of WKY (452 +/- 11 pg/g tissue) rats at 1 week of age. However, ET-1 levels gradually increased with age in SHR rats. By 8 weeks of age, levels of ET-1 in SHR (623 +/- 33 pg/g tissue) rats were higher compared to WKY (439 +/- 62 pg/g tissue) rats. In the heart, ET-1 levels were similar in WKY and SHR rats at 4 weeks of age, but at 8 weeks of age ET-1 levels were higher in SHR rats (364 +/- 33 pg/g tissue) compared to WKY rats (260 +/- 31 pg/g tissue). It appears that at 8 weeks of age when hypertension is fully expressed in rats, ET-1 levels are lower in the central nervous system and are higher in the thoracic aorta and heart of SHR compared to WKY rats.