Krishna Kishore

Non invasive real-time monitoring of bacterial infection & therapeutic effect of anti-microbials in five mouse models

Background & objectives: In vivo imaging system has contributed significantly to the understanding of bacterial infection and efficacy of drugs in animal model. We report five rapid, reproducible, and non invasive murine pulmonary infection, skin and soft tissue infection, sepsis, and meningitis models using Xenogen bioluminescent strains and specialized in vivo imaging system (IVIS). Methods: The progression of bacterial infection in different target organs was evaluated by the photon intensity and target organ bacterial counts. Genetically engineered bioluminescent bacterial strains viz. Staphylococcus aureus Xen 8.1, 29 and 31; Streptococcus pneumoniae Xen 9 and 10 and Pseudomonas aeruginosa Xen-5 were used to induce different target organs infection and were validated with commercially available antibiotics. Results: The lower limit of detection of colony forming unit (cfu) was 1.7-log10 whereas the lower limit of detection of relative light unit (RLU) was 4.2-log10. Recovery of live bacteria from different target organs showed that the bioluminescent signal correlated to the live bacterial count. Interpretation & conclusions: This study demonstrated the real time monitoring and non-invasive analysis of progression of infection and pharmacological efficacy of drugs. These models may be useful for pre-clinical discovery of new antibiotics.

Adenosine A1 receptor antagonist mitigates deleterious effects of sleep deprivation on adult neurogenesis and spatial reference memory in rats.

Sleep deprivation (SD) upsurges intracellular levels of adenosine, impairs adult neuronal cell proliferation (NCP) and cognition while caffeine, a non-selective adenosine A1 receptor (A1R) antagonist improves cognition and adult NCP during SD. We examined the selective antagonistic effects of adenosine A1R using 8-cyclopentyl-1,3-dimethylxanthine (8-CPT) on impairment of spatial reference memory and adult NCP during 48h SD. Adult male Sprague Dawley rats were sleep deprived for 48h, using an automatic cage vibrating stimulus based on animal activity. Spatial reference memory was tested as a measure of cognitive performance employing Morris Water Maze. Rats were given 8-CPT dissolved in 50% dimethyl sulfoxide (DMSO), twice daily (10mg/kg, i.p.) along with 5-bromo-2-deoxyuridine (BrdU) (50mg/kg/day, i.p.). The rats treated with 8-CPT showed significantly short mean latency and path-length to reach the platform compared to the SD rats. Consistent with these findings, 8-CPT-treated group was found to have significantly increased the number of BrdU, Ki-67 and doublecortin (DCX) positive cells. However, no significant difference was seen in NeuN expression in the Dentate Gyrus (DG). Brain-derived neurotropic factor (BDNF) expression in the DG and CA1 region was observed to decrease significantly after SD and be rescued by 8-CPT treatment. Furthermore, latency to reach platform showed a negative correlation with number of BrdU, DCX type-1 cells and BDNF expression in DG. Thus, it may be concluded that treatment with 8-CPT, an adenosine A1R antagonist during SD mitigates SD induced decline in spatial reference memory and adult NCP possibly via up regulation of BDNF levels in DG and CA1 regions.

Tyrosine ameliorates heat induced delay in event related potential P300 and contingent negative variation

The efficacy of tyrosine, a catecholamine precursor, as a countermeasure in the reduction of cognitive decline during heat exposure (HE) using event-related potential P300, and contingent negative variation (CNV) was evaluated. Ten healthy males, age 20-30years participated in the study. Volunteers received placebo or tyrosine (6.5g) 90min prior to HE (1.5h in 45°C+30% RH). P300 latency was significantly increased (p<0.01) during exposure with placebo, which was reduced significantly (p<0.01) after tyrosine supplementation. There was an increase in CNV M100 latency (p<0.05) and reaction time (p<0.01) and decrease in M100 amplitude (p<0.01) during HE with placebo, which returns to near normal level with the tyrosine administration. A significantly higher plasma norepinephrine (p<0.05), dopamine and epinephrine levels were detected in tyrosine supplemented group post heat exposure. HE increases the brain catecholamine activity thereby reduces the plasma norepinephrine and dopamine level leading to a reduction in cognitive performances. Tyrosine supplementation increases the catecholamine level and reduces the impairment of cognitive performance during HE.

Synthesis and pharmacological evaluation of [(4-arylpiperazin-1-yl)-alkyl]-carbamic acid ethyl ester derivatives as potential anxiolytic agents

On the basis of our earlier studies, a series of N-{4-[4-(aryl) piperazin-1-yl]-phenyl}-amine derivatives containing terminal carbamoyl fragment with alkyl spacer of different lengths (15–20) were synthesized as ligands, for 5-hydroxytryptamine-1A (5-HT1A) receptor. Molecular modeling studies were undertaken to explain the influence of spacer length on ligands affinity towards 5-HT1A receptor. Compound 19 showed all the specific interactions responsible for recognition. The protonated amine of the ligand forms an ionic hydrogen bond with the negatively charged Asp116 of transmembrane3 helix (TM3), while the carbamoyl moiety interacts with Asn386 and Tyr390 of TM7. The aryl group is involved in forming a CH-π interaction with Phe362. The strong interaction of compound 19 with 5-HT1A receptor in docking studies was confirmed by radio ligand binding studies. Compound 19 showed high affinity for the receptor (Ki = 0.018 nM). In vivo pharmacological testing of compound 19 (3 mg/kg body weight) showed increased open arm entries, as well as time spent in Elevated plus Maze test. Toxicological analysis also revealed no significant biochemical or morphological alterations in the vital organs of experimental animals. Furthermore our results suggest that these compounds share some pharmacological effects with established anxiolytics and might prove to be effective compounds for the treatment of anxiety.

Caffeine and Modafinil Ameliorate the Neuroinflammation and Anxious Behavior in Rats during Sleep Deprivation by Inhibiting the Microglia Activation

Background: Sleep deprivation (SD) plagues modern society due to the professional demands. It prevails in patients with mood and neuroinflammatory disorders. Although growing evidence suggests the improvement in the cognitive performance by psychostimulants during sleep-deprived conditions, the impending involved mechanism is rarely studied. Thus, we hypothesized that mood and inflammatory changes might be due to the glial cells activation induced modulation of the inflammatory cytokines during SD, which could be improved by administering psychostimulants. The present study evaluated the role of caffeine/modafinil on SD-induced behavioral and inflammatory consequences. Methods: Adult male Sprague-Dawley rats were sleep deprived for 48 h using automated SD apparatus. Caffeine (60 mg/kg/day) or modafinil (100 mg/kg/day) were administered orally to rats once every day during SD. Rats were subjected to anxious and depressive behavioral evaluation after SD. Subsequently, blood and brain were collected for biochemical, immunohistochemical and molecular studies. Results: Sleep deprived rats presented an increased number of entries and time spent in closed arms in elevated plus maze test and decreased total distance traveled in the open field (OF) test. Caffeine/modafinil treatment significantly improved these anxious consequences. However, we did not observe substantial changes in immobility and anhedonia in sleep-deprived rats. Caffeine/modafinil significantly down-regulated the pro- and up-regulated the anti-inflammatory cytokine mRNA and protein expression in the hippocampus during SD. Similar outcomes were observed in blood plasma cytokine levels. Caffeine/modafinil treatment significantly decreased the microglial immunoreactivity in DG, CA1 and CA3 regions of the hippocampus during SD, however, no significant increase in immunoreactivity of astrocytes was observed. Sholl analysis signified the improvement in the morphological alterations of astrocytes and microglia after caffeine/modafinil administration during SD. Stereological analysis demonstrated a significant improvement in the number of ionized calcium binding adapter molecule I (Iba-1) positive cells (different states) in different regions of the hippocampus after caffeine or modafinil treatment during SD without showing any significant change in total microglial cell number. Eventually, the correlation analysis displayed a positive relationship between anxiety, pro-inflammatory cytokines and activated microglial cell count during SD. Conclusion: The present study suggests the role of caffeine or modafinil in the amelioration of SD-induced inflammatory response and anxious behavior in rats. Highlights - SD induced mood alterations in rats. - Glial cells activated in association with the changes in the inflammatory cytokines. - Caffeine or modafinil improved the mood and restored inflammatory changes during SD. - SD-induced anxious behavior correlated with the inflammatory consequences.

Inhibitory effect of seabuckthorn (Hippophea rhamnoides) on platelet aggregation and oxidative stress.

Extracts from seabuckthorn (Hippophea rhamnoides) leaves, fruit and seed oil were screened for anti-platelet properties in-vitro using human platelets stimulated with 0.2 mM ADP. Half maximal inhibitory concentration was found to be 55 microg/ml platelet rich plasma(PRP) for leaf extract, 47.7 microg/ml PRP for fruit extract and 0.62 microl/ml PRP for seed oil. In-vitro incubation of platelets with increasing concentrations of seed oil was found to inhibit oxidative stress in resting as well as agonist stimulated platelets as evident by decreased formation of peroxide and superoxide radicals. Leaf extract and seed oil were further evaluated for antiplatelet and antioxidant action in-vivo in cholesterol-induced experimental atherosclerosis in rabbits. Rabbits fed on diet supplemented with cholesterol (0.5% or 1%) for 60 days showed statistically significant increase in platelet aggregation stimulated by ADP and collagen as compared to platelet aggregation in control rabbits. Malondialdehyde levels in platelets of cholesterol fed rabbits were significantly higher than those of control rabbits. Both leaf extract and seed oil supplementation prevented cholesterol induced alterations in platelet aggregation and platelet malondialdehyde levels. The study shows that SBT leaf extract and seed oil inhibit oxidative stress and hyperactivity of platelets both in-vitro as well as in-vivo and plausibly attenuate atherogenesis.

Phosphorylated delta sleep inducing peptide restores spatial memory and p-CREB expression by improving sleep architecture at high altitude

Aims: Sleep loss at high altitude (HA) play major role in worsening of neuropsychological functions, such as attention, memory and decision making. This study investigates the role of phosphorylated delta sleep inducing peptide (p‐DSIP) in improving sleep architecture during chronic hypobaric hypoxia (HH) exposure and restoration of spatial navigational memory. Methods: Morris water maze (MWM) trained rats were exposed to HH at 7620 m. p‐DSIP was injected intra‐peritoneally (10 &mgr;g/Kg bw) during HH exposure as an intervention against sleep alteration. Sleep architecture was recorded telemetrically before and during HH exposure. Monoamines were estimated by high performance liquid chromatography from brain stem (BS) and hypothalamus. CREB and p‐CREB level in hippocampus was studied by western blotting and expression of different monoamine regulatory enzymes in BS was measured by flow cytometry. Naloxone (1 mg/kg bw), a &mgr; opioid receptor antagonist of sleep inducing effect of DSIP was also studied. Key findings: p‐DSIP injection daily in circadian active period (18.30 h) during chronic HH enhanced non rapid eye movement sleep, rapid eye movement sleep as well as improved MWM performance of rats. p‐DSIP treatment showed lower monoamine level and tyrosine hydroxylase (TH) expression and increased monoamine oxidase A (MAO A), glutamic acid decarboxylase (GAD) and Choline acetyltransferase (ChAT) expression. Further, naloxone altered navigational memory by decreasing the CREB and p‐CREB level in hippocampus suggesting suppression of sleep inducing effect of p‐DSIP. Significance: Our study demonstrates that improvement of sleep quality by p‐DSIP restores spatial memory by up regulating CREB phosphorylation during simulated high altitude hypoxia. Graphical abstract Figure. No caption available.

Hypobaric hypoxia impairs cued and contextual fear memory in rats

Fear memory is essential for survival, and its dysregulation leads to disorders. High altitude hypobaric hypoxia (HH) is known to induce cognitive decline. However, its effect on fear memory is still an enigma. We aimed to investigate the temporal effect of HH on fear conditioning and the underlying mechanism. Adult male Sprague-Dawley rats were trained for fear conditioning and exposed to simulated HH equivalent to 25,000 ft for different durations (1, 3, 7, 14 and 21 days). Subsequently, rats were tested for cued and contextual fear conditioning. Neuronal morphology, apoptosis and DNA fragmentation were studied in the medial prefrontal cortex (mPFC), hippocampus and basolateral amygdala (BLA). We observed significant deficit in cued and contextual fear acquisition (at 1, 3 and 7 days) and consolidation (cued at 1 and 3 days and contextual fear at 1, 3 and 7 days) under HH. HH exposure with retraining showed the earlier restoration of contextual fear memory. Further, we found a gradual increase in the number of pyknotic and apoptotic neurons together with the increase in DNA fragmentation in mPFC, hippocampus, and BLA up to 7 days of HH exposure. The present study concludes that HH exposure equivalent to 25,000 ft induced cued and contextual fear memory deficit (acquisition and consolidation) which is found to be correlated with the neurodegenerative changes in the limbic brain regions.

Sleep deprivation induces spatial memory impairment by altered hippocampus neuroinflammatory responses and glial cells activation in rats

We aimed to investigate the glial cells activation as a potential mechanism involved in the sleep deprivation (SD) induced cognitive impairment through changes in inflammatory cytokines. We analyzed the spatial memory, inflammatory cytokine levels, and gliosis during SD. SD induced spatial memory impairment, imbalance of inflammatory (increased pro- and decreased anti-) cytokines in both hippocampus and plasma in association with glial cells activation in the hippocampus of sleep-deprived rats were observed. Further analysis of the data presented a correlation between spatial memory impairment and activated microglia induced increased pro-inflammatory cytokines after 48h of SD.