Avadhesh C. Sharma

Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice

1. The effect of drugs affecting learning and memory was investigated using transfer latency (TL) as parameter for acquisition and retention of memory process on elevated plus-maze both in rats and mice. Further the validity of the procedure was envisaged. 2. The results provide an evidence for utility of shortened TL on 2nd day trial in old rats and mice as a parameter for retention or consolidation of memory, while treatment of drugs 30 min prior to 1st day may also be utilised for acquisition related action of drugs. 3. The drugs producing acquisition deficits namely scopolamine (0.1-0.5 mg/kg) and MK 801 (0.05-0.1 mg/kg) did not affect the shortened TL on elevated plus-maze in rats while nootropics, like piracetam (150 mg/kg) and captopril (30 mg/kg) reduced the shortened TL. The memory enhancing effect of these agent was reversed by scopolamine (0.3 mg/kg) and MK 801 (0.1 mg/kg) both in rats and mice. The results suggested the acquisition affecting drugs, however, did not show any effect on retention parameter (shortened TL) but can reverse the retention facilitatory action of nootropics. The results also provide indirect evidence for participation of cholinergic and NMDA-receptor blockade in the mechanism of these drugs. 4. Scopolamine and MK 801 produced acquisition deficits in mice, as they increased the TL on 1st and 2nd day trial while physostigmine (0.05 mg/kg) decreased the 2nd day TL. Physostigmine (0.1 mg/kg) reversed scopolamine and MK 801 induced acquisition deficits suggested participation of cholinergic and NMDA- receptor in learning process. 5. The results validate the utility of the elevated plus-maze for evaluation of possible nootropic action of drugs.

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.

Acute lung injury:apoptosis and signaling mechanisms.

Acute lung injury (ALI) has been documented clinically following several pathological states such as trauma, septic shock and pneumonia. The histopathological characteristics, paired with the production of a number of cellular pro-inflammatory mediators, play a crucial role in the progression of ALI. During ALI, polymorphonuclear neutrophil (PMN)-mediated apoptosis is delayed by macrophages, possibly via effects on the Fas/FasL mediated pathway, leading to the accumulation of these cells at the site of injury and inflammation. The transcriptional regulation of NFκB, CREB, and AP-1 also regulates the pathogenesis of ALI. During sepsis and septic shock, we found evidence of infiltrating leukocytes in the alveolar spaces along with an increased number of TUNEL-positive cells in the lung sections. We also observed an increased expression of TRADD and Bax/Bcl2 ratio at 7 days post-sepsis. In contrast, the NFκB/IκB ratio increased at 1 day post-sepsis. Together, these data provide evidence illustrating the induction of apoptosis in lung tissues subsequent to the onset of polymicrobial sepsis. The results support the concept that the upregulation of apoptosis following lung inflammation plays a crucial role in the development of acute lung injury and related disorders such as ARDS.

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.

Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK) phosphorylation in renal and vascular dysfunction

BackgroundIn the present study we tested the hypothesis that progression of streptozotocin (STZ)-induced diabetes (14-days to 28-days) would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK) phosphorylation in kidneys and thoracic aorta.MethodsMale Sprague Dawley rats (350–400 g) were randomized into three groups: sham (N = 6), 14-days diabetic (N = 6) and 28-days diabetic rats (N = 6). Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V.) on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat.ResultsThe diabetic rats lost body weight at both 14-days (-10%) and 28-days (-13%) more significantly as compared to sham (+10%) group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS), prepro endothelin-1 (preproET-1) and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group.ConclusionThe study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

Sepsis-induced depressed contractile function of isolated ventricular myocytes is due to altered calcium transient properties.

Chronic peritoneal sepsis in a rodent model produces myocardial dysfunction characterized by decreased rates of ventricular contraction and relaxation in the isolated heart preparation. However, it remains controversial whether the ventricular contractility is altered during sepsis. In the present study, we determined the effect of chronic peritoneal sepsis on the mechanical properties and intracellular Ca2+ handling of cardiac myocytes isolated from septic rats at 24 or 48 h. Mechanical properties were evaluated by use of an IonOptix MyoCam system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), and maximal velocities of shortening and relengthening (±dL/d t). Intracellular Ca2+ handling was evaluated with fura-2 fluorescent dye. Myocytes obtained from 24-h postseptic animals exhibited a depressed PS (85% of control), normal TPS, prolonged TR90 (147% of control), and reduced ±dL/d t (both 79% of control). Myocytes from 48-h postseptic animals also exhibited a reduced peak of intracellular Ca2+ sequestration (55% of control), but resting intracellular Ca2+ and Ca2+-transient decay were comparable with the values seen in myocytes from untreated rats. Myocytes from septic and control animals were equally responsive over a range of stimulation frequencies (0.1–5 Hz). Myocytes from septic animals were unresponsive (5% of control) to increase of extracellular Ca2+ (0.5–3 mM). These results demonstrate that sepsis produces substantial deficits in cardiac myocytes function that can be attributed to altered calcium transient properties.

Effect of diaspirin crosslinked and stroma-reduced hemoglobin on mean arterial pressure and endothelin-1 concentration in rats

The effect of unmodified stroma reduced (SRHb) and modified diaspirin crosslinked (DCLHb) hemoglobin solutions on the mean arterial pressure and endothelin-1 (ET-1) concentration in blood plasma and various tissues was studied. Infusion of DCLHb or SRHb increased mean arterial blood pressure by 96% and 39%, respectively. Heart rate was not significantly affected by DCLHb or SRHb. A significant increase (P < 0.003) in the ET-1 levels in blood plasma after DCLHb and SRHb infusion was observed. The increase in plasma ET-1 concentration was significantly more marked with SRHb (141%) as compared to DCLHb (78%) treated rats. The concentration of ET-1 in the heart and brain regions was not altered in DCLHb or SRHb treated rats as compared to control. However, ET-1 concentration was significantly increased in the thoracic aorta (151%) and renal medulla (272%) of DCLHb treated rats. SRHb treated rats also showed a significant increase in ET-1 concentration in the thoracic aorta (141%) and renal medulla (429%). The effect of SRHb on the renal medulla was found to be significantly greater than that of DCLHb. ET may be one of the factors responsible for the cardiovascular effects of hemoglobin solutions.

Sepsis-induced myocardial dysfunction

ABSTRACT Despite the fact that septic patients exhibit altered cardiac function, it is not considered a major pathology during sepsis. Thus, the molecular mechanisms underlying sepsis-induced myocardial dysfunction have not been studied extensively. In a polymicrobial septic rat model, +dP/dt and −dP/dt on day 1 were not altered but found depressed later, i.e., at 3 and 7 days postsepsis. Diastolic dysfunction characterized by an elevation of the time constant of left ventricular relaxation, tau, was evident at 1, 3, and 7 days postsepsis. Recent data from our laboratory demonstrated that sepsis-induced cardiodynamic alterations correlated with upregulation of TNF receptor-associated death domain, Bax, Smac (both mitochondrial and cytosolic fractions), total nuclear factor &kgr;B expression, p38-mitogen-activated protein kinase and c-Jun N-terminal kinase phosphorylation, and cytochrome c levels in the rat heart at 3 and 7 days postsepsis. Data from various laboratories emphasized that molecular myocardial alteration, which occurs during early and late stages of sepsis, needs to be elucidated thoroughly. A poor understanding of myocardial signaling during early sepsis could be one of the main reasons for limited success of pharmacotherapeutic options for sepsis. We anticipate that an increased understanding of pathophysiological mechanisms leading to sepsis-induced myocardial dysfunction would generate new enthusiasm among various research groups in this area of research.

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.

Steroid hormone alterations following induction of chronic intraperitoneal sepsis in male rats.

The influence of sepsis on male reproductive function in chronic animal models has not been extensively investigated. On the basis of earlier clinical studies, it was hypothesized that chronic intraperitoneal (i.p.) sepsis in rats would modulate the circulating levels of steroid reproductive hormones. Male Sprague-Dawley rats (300-375 g) were randomized to septic and nonseptic groups. Sepsis was induced with cecal slurry (200 mg/kg/5 mL 5% dextrose in water (D5W); i.p.) in septic rats, while nonseptic rats received only sterile D5W. The rats (n = 8-12) were catheterized to measure systemic hemodynamics and to collect blood at 0, 12, 24, and 48 h after induction of sepsis/sham sepsis. A separate group of normal rats was included to serve as an unoperated control group. The plasma concentration of corticosterone, progesterone, and testosterone in serum was determined using radioimmunoassay. The heart rate was significantly increased at t = 12, 24, and 48 h following induction of sepsis. However, septic rats did not display any significant alterations in the mean arterial pressure and pulse pressure. Basal circulating concentrations of serum corticosterone, progesterone, and testosterone were 356 +/- 124 ng/mL, 2.37 +/- 1.03 ng/mL, and 1.88 +/- .29 ng/mL, respectively, in the unoperated rats. At t = 0 h there was a significant increase in the levels of corticosterone in septic rats and in the levels of progesterone in both septic and nonseptic rats. The elevations in the concentrations of corticosterone and progesterone returned to basal values after 24 and 48 h. The septic animals had significantly decreased levels of testosterone at t = 24 and 48 h as compared with basal values and nonseptic groups. Our model of sepsis produced a time-dependent decrease in levels of testosterone, an end product of male steroidogenesis. This, along with unchanged levels of corticosterone and progesterone at 24 and 48 h following sepsis, indicates that separate mechanisms for steroidogenesis regulating synthesis of these steroid hormones (progesterone and testosterone) occur with sepsis. It is concluded that in our chronic septic rat model, induction of i.p. sepsis produced dysfunction in steroidogenesis, which selectively affected the synthesis of testosterone.