Video laryngoscopic oral intubation in rats: a simple and effective method.

Endotracheal intubation is a vital component of many rat in vivo experiments to secure the airway and allow controlled ventilation. Even in the hands of experienced researchers, however, the procedure remains technically challenging. The safest and most reliable way for human intubation is by video laryngoscopy. Previous attempts to apply this technique in rodents have been complicated and expensive. We, hereby, describe a novel, noninvasive method to safely intubate rats orally by video laryngoscopy, thus avoiding the need for a surgical tracheostomy. By repurposing a commercially available ear wax removal device, visualization of the rat larynx can be significantly enhanced. Because of its small diameter, integrated illumination, and a powerful camera with adequate focal length, the device has all of the necessary properties for exploring the upper airway of a rat. After identifying the vocal cords by video laryngoscopy, the insertion of an endotracheal tube (a 14G intravenous catheter) into the trachea under constant visual control is facilitated by using PE50 polyethylene tubing as a stylet (Seldinger technique). The procedure has been performed more than 60 times in our laboratory; all intubations were successful on the first attempt, and no adverse events were observed. We conclude that the described procedure is a simple and effective way to intubate a rat noninvasively, using inexpensive and commercially available equipment.

Dose optimization of early high-dose valproic acid for neuroprotection in a swine cardiac arrest model.

AIM: High-dose valproic acid (VPA) improves the survival and neurologic outcomes after asphyxial cardiac arrest (CA) in rats. We characterized the pharmacokinetics, pharmacodynamics, and safety of high-dose VPA in a swine CA model to advance clinical translation. METHODS: After 8 â€‹min of untreated ventricular fibrillation CA, 20 male Yorkshire swine were resuscitated until return of spontaneous circulation (ROSC). They were block randomized to receive placebo, 75 â€‹mg/kg, 150 â€‹mg/kg, or 300 â€‹mg/kg VPA as 90-min intravenous infusion (n â€‹= â€‹5/group) beginning at ROSC. Animals were monitored for 2 additional hours then euthanized. Experimental operators were blinded to treatments. RESULTS: The mean(SD) total CA duration was 14.8(1.2) minutes. 300 â€‹mg/kg VPA animals required more adrenaline to maintain mean arterial pressure ≥80 â€‹mmHg and had worse lactic acidosis. There was a strong linear correlation between plasma free VPA Cmax and brain total VPA (r2 â€‹= â€‹0.9494; p â€‹< â€‹0.0001). VPA induced dose-dependent increases in pan- and site-specific histone H3 and H4 acetylation in the brain. Plasma free VPA Cmax is a better predictor than peripheral blood mononuclear cell histone acetylation for brain H3 and H4 acetylation (r2 â€‹= â€‹0.7189 for H3K27ac, r2 â€‹= â€‹0.7189 for pan-H3ac, and r2 â€‹= â€‹0.7554 for pan-H4ac; p â€‹< â€‹0.0001). CONCLUSIONS: Up to 150 â€‹mg/kg VPA can be safely tolerated as 90-min intravenous infusion in a swine CA model. High-dose VPA induced dose-dependent increases in brain histone H3 and H4 acetylation, which can be predicted by plasma free VPA Cmax as the pharmacodynamics biomarker for VPA target engagement after CA.

Combined intra- and post-cardiac arrest hypothermic-targeted temperature management in a rat model of asphyxial cardiac arrest improves survival and neurologic outcome compared to either strategy alone.

AIM: Post-cardiac arrest hypothermic-targeted temperature management (HTTM) improves outcomes in preclinical cardiac arrest studies. However, inadequate understanding of the mechanisms and therapeutic windows remains a barrier to optimization. We tested the hypothesis that combined intra- and post-cardiac arrest HTTM provides a synergistic outcome benefit compared to either strategy alone. METHODS: Rats subjected to 8-min asphyxial cardiac arrest were block randomized to 4 treatment groups (n=12/group): NTTM) normothermic-targeted temperature management; 1-24 HTTM) HTTM initiated 1h post-ROSC and maintained for 24h; Intra-1 HTTM) HTTM initiated at CPR onset and maintained for 1h; and Intra-24 HTTM) HTTM initiated at CPR onset and maintained for 24h. HTTM was induced by nasopharyngeal cooling and maintained using an automated temperature regulation system. Target temperature range was 36.5-37.5°C for NTTM and 32.0-34.0°C for HTTM. Post-arrest neurologic function score (NFS) was measured daily, and rats surviving 72h were euthanized for histological analysis of neurodegeneration. RESULTS: Target brain temperature was achieved 7.8±3.3min after initiating intra-arrest cooling. The survival rate was 42%, 50%, 50%, and 92% in the NTTM, 1-24 HTTM, Intra-1 HTTM, and Intra-24 HTTM groups, respectively (p<0.05, Intra-24 group vs. all other groups). The rate of survival with good neurologic function (NFS≥450) was 33% in the Intra-24 HTTM group vs. 0% in all other groups (mid p<0.05). Hippocampal CA1 sector neurodegeneration was significantly reduced in the Intra-24 HTTM group compared to all other groups (p<0.05). CONCLUSION: Combined intra- and post-cardiac arrest HTTM has greater outcome benefits than either strategy alone.

Translating drug-induced hibernation to therapeutic hypothermia.

Therapeutic hypothermia (TH) improves prognosis after cardiac arrest; however, thermoregulatory responses such as shivering complicate cooling. Hibernators exhibit a profound and safe reversible hypothermia without any cardiovascular side effects by lowering the shivering threshold at low ambient temperatures (Ta). Activation of adenosine A1 receptors (A1ARs) in the central nervous system (CNS) induces hibernation in hibernating species and a hibernation-like state in rats, principally by attenuating thermogenesis. Thus, we tested the hypothesis that targeted activation of the central A1AR combined with a lower Ta would provide a means of managing core body temperature (Tb) below 37 °C for therapeutic purposes. We targeted the A1AR within the CNS by combining systemic delivery of the A1AR agonist (6)N-cyclohexyladenosine (CHA) with 8-(p-sulfophenyl)theophylline (8-SPT), a nonspecific adenosine receptor antagonist that does not readily cross the blood-brain barrier. Results show that CHA (1 mg/kg) and 8-SPT (25 mg/kg), administered intraperitoneally every 4 h for 20 h at a Ta of 16 °C, induce and maintain the Tb between 29 and 31 °C for 24 h in both naïve rats and rats subjected to asphyxial cardiac arrest for 8 min. Faster and more stable hypothermia was achieved by continuous infusion of CHA delivered subcutaneously via minipumps. Animals subjected to cardiac arrest and cooled by CHA survived better and showed less neuronal cell death than normothermic control animals. Central A1AR activation in combination with a thermal gradient shows promise as a novel and effective pharmacological adjunct for inducing safe and reversible targeted temperature management.

Ethical considerations in hibernation research.

Ethical research practices are a key component of scientific integrity and of public support for research. Hibernation research presents specific ethical issues in regard to animal welfare. In this article, the authors apply the '3Rs' principles of humane experimental technique (replacement, reduction and refinement) to hibernation research. They provide recommendations for hibernation researchers and suggest future directions for addressing issues specific to hibernation research. They discuss the use of appropriate behavioral and physiological monitoring procedures, the development of species-specific brain atlases for placement of brain probes, the provision of environmental enrichment and the management of studies involving pharmacological induction of torpor. Addressing these issues in hibernation research will lead to improvements in research outcomes and in welfare of hibernating species.

Circannual rhythm in body temperature, torpor, and sensitivity to A1 adenosine receptor agonist in arctic ground squirrels.

A1 adenosine receptor (A1AR) activation within the central nervous system induces torpor, but in obligate hibernators such as the arctic ground squirrel (AGS; Urocitellus parryii), A1AR stimulation induces torpor only during the hibernation season, suggesting a seasonal increase in sensitivity to A1AR signaling. The purpose of this research was to investigate the relationship between body temperature (Tb) and sensitivity to an adenosine A1 receptor agonist in AGS. We tested the hypothesis that increased sensitivity in A1AR signaling would lead to lower Tb in euthermic animals during the hibernation season when compared with the summer season. We further predicted that if a decrease in euthermic Tb reflects increased sensitivity to A1AR activation, then it should likewise predict spontaneous torpor. We used subcutaneous IPTT-300 transponders to monitor Tb in AGS housed under constant ambient conditions (12:12 L:D, 18 °C) for up to 16 months. These animals displayed an obvious rhythm in euthermic Tb that cycled with a period of approximately 8 months. Synchrony in the Tb rhythm within the group was lost after several months of constant L:D conditions; however, individual rhythms in Tb continued to show clear sine wave-like waxing and waning. AGS displayed spontaneous torpor only during troughs in euthermic Tb. To assess sensitivity to A1AR activation, AGS were administered the A1AR agonist N(6)-cyclohexyladenosine (CHA, 0.1 mg/kg, ip), and subcutaneous Tb was monitored. AGS administered CHA during a seasonal minimum in euthermic Tb showed a greater drug-induced decrease in Tb (1.6 ± 0.3 °C) than did AGS administered CHA during a peak in euthermic Tb (0.4 ± 0.3 °C). These results provide evidence for a circannual rhythm in Tb that is associated with increased sensitivity to A1AR signaling and correlates with the onset of torpor.

Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii).

Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5 mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5 mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200 µg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.

Season primes the brain in an arctic hibernator to facilitate entrance into torpor mediated by adenosine A(1) receptors.

Torpor in hibernating mammals defines the nadir in mammalian metabolic demand and body temperature that accommodates seasonal periods of reduced energy availability. The mechanism of metabolic suppression during torpor onset is unknown, although the CNS is a key regulator of torpor. Seasonal hibernators, such as the arctic ground squirrel (AGS), display torpor only during the winter, hibernation season. The seasonal character of hibernation thus provides a clue to its regulation. In the present study, we delivered adenosine receptor agonists and antagonists into the lateral ventricle of AGSs at different times of the year while monitoring the rate of O(2) consumption and core body temperature as indicators of torpor. The A(1) antagonist cyclopentyltheophylline reversed spontaneous entrance into torpor. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) induced torpor in six of six AGSs tested during the mid-hibernation season, two of six AGSs tested early in the hibernation season, and none of the six AGSs tested during the summer, off-season. CHA-induced torpor within the hibernation season was specific to A(1)AR activation; the A(3)AR agonist 2-Cl-IB MECA failed to induce torpor, and the A(2a)R antagonist MSX-3 failed to reverse spontaneous onset of torpor. CHA-induced torpor was similar to spontaneous entrance into torpor. These results show that metabolic suppression during torpor onset is regulated within the CNS via A(1)AR activation and requires a seasonal switch in the sensitivity of purinergic signaling.

Altered thermoregulation via sensitization of A1 adenosine receptors in dietary-restricted rats.

RATIONALE: Evidence links longevity to dietary restriction (DR). A decrease in body temperature (T(b)) is thought to contribute to enhanced longevity because lower T(b) reduces oxidative metabolism and oxidative stress. It is as yet unclear how DR decreases T(b). OBJECTIVE: Here, we test the hypothesis that prolonged DR decreases T(b) by sensitizing adenosine A(1) receptors (A(1)AR) and adenosine-induced cooling. METHODS AND RESULTS: Sprague-Dawley rats were dietary restricted using an every-other-day feeding protocol. Rats were fed every other day for 27 days and then administered the A(1)AR agonist, N(6)-cyclohexyladenosine (CHA; 0.5 mg/kg, i.p.). Respiratory rate (RR) and subcutaneous T(b) measured using IPTT-300 transponders were monitored every day and after drug administration. DR animals displayed lower RR on day 20 and lower T(b) on day 22 compared to animals fed ad libitum and displayed a larger response to CHA. In all cases, RR declined before T(b). Contrary to previous reports, a higher dose of CHA (5 mg/kg, i.p.) was lethal in both dietary groups. We next tested the hypothesis that sensitization to the effects of CHA was due to increased surface expression of A(1)AR within the hypothalamus. We report that the abundance of A(1)AR in the membrane fraction increases in hypothalamus, but not cortex of DR rats. CONCLUSION: These results suggest that every-other-day feeding lowers T(b) via sensitization of thermoregulatory effects of endogenous adenosine by increasing surface expression of A(1)AR. DISCUSSION: Evidence that diet can modulate purinergic signaling has implications for the treatment of stroke, brain injury, epilepsy, and aging.