Male NIH Swiss Mice Research Articles

Novel Small Molecule, UTS-1401, as a Radioprotector for Total-Body Irradiation.

We report on a new radioprotector, UTS-1401, a small molecule that was synthesized (by one of us, JS) and evaluated here for its radioprotective effect against total-body irradiation (TBI). Female and male NIH Swiss mice were subjected to TBI at doses of 6.5, 7.5 and 8.5 Gy either with or without a 24 h pretreatment of UTS-1401 given ip and observed for 30 days. Survival rates were significantly increased when mice were treated with UTS-1401 compared to those not treated. The radioprotective effect of UTS-1401 was drug-dose dependent for male mice exposed to 8.5 Gy TBI with 150 mg/kg of UTS-1401 as the optimal dose. The radioprotective effect of UTS-1401 on female mice exposed to 8.5 Gy TBI was observed at 50, 100, and 150 mg/kg, with no dose response relationship noted. Female mice were more radioresistant than male mice with LD50/30 values of 7.8 Gy vs. 6.8 Gy, respectively. Weight changes after UTS-1401 alone showed a significant body weight increase at 150 mg/kg. Both the ip and iv route for UTS-1401 were similarly effective for male mice exposed to 8 Gy TBI. Further analysis using an endogenous spleen colony assay demonstrated that pretreatment of UTS-1401 for up to 72h prior to TBI protected both spleen weight and hematopoietic stem cells with a treated/untreated ratio between 2.0 and 3.2 for the latter for times between 0.5 h and 72 h. A separate invivo study showed that pretreatment of UTS-1401 protected bone marrow CFU-GM for mice exposed to TBI. In summary, UTS-1401 is a promising small-molecule radioprotective agent as demonstrated by whole animal, hematopoietic stem cell and bone marrow myeloid progenitor cell survival.

Convulsant doses of abused synthetic cannabinoid receptor agonists AB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA and JWH-018 do not elicit electroencephalographic (EEG) seizures in male mice.

Synthetic cannabinoid receptor agonists (SCRAs) are found in illicit smoking products, such as "K2" or "Spice." Convulsions are commonly reported adverse effects of SCRAs but are poorly understood. We determined convulsant effects of SCRAs AB-PINACA, and 5F-ADB-PINACA in adult male NIH Swiss mice, and then determined if convulsant effects of AB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, and JWH-018 elicited seizure-like effects using EEG. Mice were administered SCRAs or pentylenetetrazole (PTZ) and placed in observation chambers where convulsant effects were scored. The capacity of the CB1R antagonist rimonabant, the benzodiazepine diazepam, or the non-specific CYP450 inhibitor 1-aminobenzotriazole (1-ABT) to attenuate convulsant effects was determined. Other mice were prepared with EEG headmounts to ascertain whether observed convulsions occurred concurrently with seizure-like effects by assessing root-mean-square (RMS) power, high amplitude EEG spike analysis, and videography. Mice receiving AB-PINACA or 5F-ADB-PINACA exhibited dose-dependent convulsant effects that were blocked by 10mg/kg rimonabant pretreatment but not by pretreatment with 10mg/kg diazepam; these convulsant effects were not altered in the presence of 100mg/kg 1-ABT. Repeated administration of 10mg/kg AB-PINACA and 3mg/kg 5F-ADB-PINACA produced partial tolerance to convulsant effects but did not lead to cross-tolerance to PTZ-induced convulsions. In EEG studies, convulsant doses of AB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, and JWH-018 did not produce seizures concomitantly with convulsions. These data extend previous findings of convulsant effects of SCRAs and suggest that convulsant effects of AB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, and JWH-018 are CB1R-mediated but are not associated with electroencephalographic seizures. These results further suggest that benzodiazepines may not effectively treat convulsions elicited by SCRA use in humans.

Cannabimimetic effects of abused indazole-carboxamide synthetic cannabinoid receptor agonists AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA in mice: Tolerance, dependence and withdrawal

BackgroundChronic abuse of synthetic cannabinoid receptor agonists (SCRAs), known as “K2″ or “Spice”, threatens public health and safety. Recently, SCRAs of the indazole-carboxamide structural class have become more prevalent. Preclinical studies investigating the tolerance and dependence potentially involved in chronic SCRA abuse is limited. The present study determined the in vivo effects of chronic exposure to indazole-carboxamide SCRAs, AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA compared to the first-generation SCRA, JWH-018. MethodsAdult male C57Bl/6 mice were used for dose-effect determinations of hypothermic effects. Adult male NIH Swiss mice were used in biotelemetry studies to assess tolerance to hypothermic effects following repeated SCRA administration over 5 consecutive days, and to determine the role of Phase I drug metabolism via acute CYP450 inhibition in the presence of 1-ABT, a nonspecific CYP450 inhibitor. SCRA dependence was determined in adult male NIH Swiss mice via assessment of rimonabant-precipitated observable sign of withdrawal (i.e., front paw tremors). ResultsAll SCRAs elicited dose-dependent hypothermia mediated through cannabinoid CB1 receptors (CB1Rs). 1-ABT increased duration of hypothermia for all SCRAs tested, and increased the magnitude of hypothermia for all SCRAs except 5F-ADB-PINACA. Upon repeated administration, tolerance to hypothermic effects of AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA was much less than that of JWH-018. Similarly, rimonabant-precipitated front paw tremors were much less frequent in mice treated with 5F-AB-PINACA and 5F-ADB-PINACA than in mice treated with JWH-018. ConclusionsThese findings suggest a decreased potential for tolerance and withdrawal among indazole-carboxamide SCRAs, and may imply structural class-dependent profiles of in vivo effects among SCRAs.

Effects of Laboratory Housing Conditions on Core Temperature and Locomotor Activity in Mice.

Drug developers worldwide assess compound safety and efficacy using measures that include mouse core temperature and locomotor activity. Subtle differences in animal housing conditions between institutions can alter these values, impacting scientific rigor and reproducibility. In these studies, adult male NIH Swiss mice were surgically implanted with radiotelemetry probes that simultaneously monitored core temperature and locomotor activity across various housing conditions. In the first study, ambient temperature was varied between 20 °C and 28°C in groups of singly housed mice. Additional studies held the mice at a constant ambient temperature and examined the effects of cage density (housing animals singly or in groups of 3 or 6), bedding change and provision of nesting material, and the availability of a running wheel on core temperature and locomotor activity. Mice overwhelmingly maintained species-typical core temperatures across all ambient temperatures, across all housing conditions, when bedding was fresh or old, and with or without the provision of cotton squares as nesting material. However, engaging in wheel running and the combination of fresh bedding and cotton squares transiently increased core temperatures beyond the species-typical range. Similarly, the circadian distribution of locomotor activity was significantly disrupted by placing animals in cages with fresh bedding or nesting material, or by performing both of these manipulations concurrently during the light period. These findings suggest that standard husbandry practices and common housing conditions may transiently affect core temperature in adult mice. Furthermore, these practices may have profound and relatively long-lasting effects on motor activity and the regulation of circadian rhythms.

In Vitro and In Vivo Effects of Palmaria palmata Derived Peptides on Glucose Metabolism

Three synthetic peptides, ILAP, LLAP and MAGVDHI, derived from a Palmaria palmata protein hydrolysate were assessed for their antidiabetic potential in vitro and in vivo. In addition to inhibiting dipeptidyl peptidase-IV in a cell-based in situ assay all three peptides significantly increased the half-life of the incretin hormone glucagon-like peptide-1 (GLP-1). ILAP and LLAP mediated a significant increase (p < 0.001) in insulin secretion from BRIN-BD11 cells compared to the glucose control, while MAGVDHI had no insulinotropic activity at an eqimolar concentration (10–6 M). A significant increase in the concentration of cyclic adenosine monophosphate production in BRIN-BD11 cells mediated by ILAP (p < 0.001) and LLAP (p < 0.01) compared to the basal control, would indicate that insulin secretion may be mediated by membrane based activation. Furthermore, ILAP and LLAP acted as glucose-dependent insulinotropic polypeptide (GIP) secretagogues, stimulating a significant increase (p < 0.01) in the concentration of GIP released from enteroendocrine STC-1 cells compared to the glucose control. When tested in vivo in healthy male NIH Swiss mice, ILAP and LLAP, mediated a significant increase (p < 0.01) in plasma insulin and decrease (p < 0.05) in blood glucose, respectively, compared to the control. MAGVDHI mediated a significant (p < 0.001) sustained reduction in food intake in food deprived trained mice. These results demonstrate that the Palmaria palmata peptides studied herein have prospective antidiabetic activity and have the potential to act as agents that can be used alone or in combination with drugs, to aid in the prevention and management of Type 2 diabetes mellitus.

Effects of Ambient Temperature and Social Housing on Locomotor Activity, Thermoregulation, and Weight Loss Following “Binge” Administration of Structurally‐Related Abused Psychostimulants

3,4‐methylenedioxypyrovalerone (MDPV) and α‐pyrrolidinopentiophenone (α‐PVP) are common active constituents of abused “bath salts” products. The structures of these synthetic cathinone analogues differ only in that MDPV contains a methylenedioxy ring. Additionally, MDPV is structurally similar to the amphetamine analogue 3,4‐methylenedioxymethamphetamine (MDMA), which differs fromthe structure of methamphetamine (METH) only by the addition of a methylenedioxy ring. MDPV, α‐PVP, MDMA, and METH are four psychostimulants often used interchangeably by humans in crowded, hot environments. The effects of MDMA are known to be dramatically impacted by crowding and ambient temperature and because of the structural similarities and use patterns in humans, it was of interest to investigate the modulatory effects of such environmental variables on the in vivo pharmacology of the four compounds in mice. In the present studies, adult male NIH Swiss mice were housed singly or in groups of 3 or 6, at 20°C, then administered a “binge” regimen of 1.0, 3.0, or 10.0 mg/kg MDPV, α‐PVP, MDMA or METH, or repeated saline, via intraperitoneal injection, every 2 hours for a total of 4 injections. Locomotor activity and core temperature were continuously collected throughout drug administration, continuing 2 hours after the 4th injection. The lowest dose per social housing condition different from saline was repeated at an elevated temperature of 28°C. Doses were limited by lethal effects which were potentiated both by the increased ambient temperature and social housing conditions. Increased ambient temperature resulted in increased locomotor activity for all compounds, and an increased hyperthermic effect occurred in animals administered MDPV or MDMA, both methylenedioxy ring containing compounds. No pronounced differences regarding body weight and core temperature as a function of social housing were observed. Brain regions (including hippocampus and striatum) were collected for neurochemical analysis 10 days after the last injection. Regions from one hemisphere will be processed and analyzed for tissue content of monoamines and their primary metabolites by HPLC, while samples from the other hemisphere will be utilized to determine GLUT2 concentration via western blotting. These studies suggest that the addition of specific moieties, in this case a methylenedioxy ring, to structurally similiar cathinones and amphetamines can result in same effect compared to the parent compound.Support or Funding InformationThese studies supported by DA039195, DA022981 and the UAMS Center for Translational Neuroscience.

Establishing a time course for spontaneous opioid withdrawal in male and female outbred mice using an abbreviated dependence paradigm

Opioid withdrawal is often a limiting factor in recovery from opioid dependence due to negative reinforcement caused by continued use of opioids in order to avoid the distressing withdrawal symptoms. In animal models of dependence, opioid withdrawal is frequently precipitated using opioid antagonists and the severity of withdrawal is related to the level of dependence. However, this model lacks face validity when investigating new treatment possibilities for opioid withdrawal because most human addicts are not exposed to an antagonist in order to precipitate withdrawal. Furthermore, results from animal experiments suggest that spontaneous and precipitated withdrawal involve different mechanisms and may favor different symptoms of withdrawal. These experiments were conducted to investigate the timeline of spontaneous withdrawal in an outbred mouse model using an abbreviated dependence paradigm. Male and female NIH Swiss mice were injected with escalating doses of morphine twice daily (50–125 mg/kg morphine) over a period of four days. On the fifth day, mice were given a final dose of morphine and withdrawal symptoms were assessed at 5, 10, 24, 34, 48, 58, 72, 82, 96, and 106 hrs after the final injection. Withdrawal symptoms were assessed by global scores for an overall picture and by counting the number of tremors, wet dog shakes, and jumps. Results showed a robust withdrawal time course that peaked at 24 hrs in both sexes as assessed by global score. When broken down, wet dog shakes and tremors proved to be the most reliable indicator of severity of withdrawal and females showed more tremors than males for the 1st 24 hours of the time course. These results establish a spontaneous withdrawal time course in an outbred strain of mouse that shows that dependence can be reliably established over a four‐day period using fairly high escalating doses of morphine. Furthermore, these results suggest that individual symptoms of withdrawal should be assessed over time as well as global score.Support or Funding InformationSupported in part by the State of Washington Initiative Measure No.171.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(251) A Time Course of the Development of Hyperalgesia during Spontaneous Withdrawal in NIH Swiss Mice

Pain patients may experience heightened sensitivity to pain while trying to discontinue opioid use which may contribute to resumption of opioid use in order to avoid more pain. However, most studies in animals that investigate hyperalgesia during opioid withdrawal focus on hyperalgesia during precipitated opioid withdrawal and include only male animals. The present study was conducted to assess hyperalgesia during a protracted withdrawal state in female and male NIH Swiss mice. Mice were rendered opioid-dependent by twice daily s.c. injections of morphine in escalating doses (50-125 mg/kg) or saline vehicle for four consecutive days; there was a final dose of morphine on day 5. Body weight was taken prior to injections as well as mornings and evenings prior to testing. Paw withdrawal thresholds to increasing paw pressures were recorded prior to the first injection and at 5, 10, 24, 34, 48, 58, 72, 82, 96 and 106 h after the final injection. Mice consistently lost weight during morphine administration; however, both sexes gained weight immediately following cessation of drug treatment. Morphine-dependent animals exhibited pronounced hyperalgesia starting at 10 h after the final morphine injection and subsiding at roughly 58 h for females and 82 h for males. A main effect of morphine administration showed that animals that received morphine had significantly reduced paw withdrawal thresholds in percentage of baseline. A main effect of sex indicates that males had generally lower scores over time than females in hyperalgesia data. These results indicate that spontaneous withdrawal causes hyperalgesia in NIH Swiss mice. This is significant because while it is known that withdrawal can cause hyperalgesia, most animal models that investigate this phenomenon use a precipitated withdrawal model, while our study demonstrated hyperalgesia also occurs in a spontaneous withdrawal model. This research was supported in part by State of Washington Initiative Measure 171. Pain patients may experience heightened sensitivity to pain while trying to discontinue opioid use which may contribute to resumption of opioid use in order to avoid more pain. However, most studies in animals that investigate hyperalgesia during opioid withdrawal focus on hyperalgesia during precipitated opioid withdrawal and include only male animals. The present study was conducted to assess hyperalgesia during a protracted withdrawal state in female and male NIH Swiss mice. Mice were rendered opioid-dependent by twice daily s.c. injections of morphine in escalating doses (50-125 mg/kg) or saline vehicle for four consecutive days; there was a final dose of morphine on day 5. Body weight was taken prior to injections as well as mornings and evenings prior to testing. Paw withdrawal thresholds to increasing paw pressures were recorded prior to the first injection and at 5, 10, 24, 34, 48, 58, 72, 82, 96 and 106 h after the final injection. Mice consistently lost weight during morphine administration; however, both sexes gained weight immediately following cessation of drug treatment. Morphine-dependent animals exhibited pronounced hyperalgesia starting at 10 h after the final morphine injection and subsiding at roughly 58 h for females and 82 h for males. A main effect of morphine administration showed that animals that received morphine had significantly reduced paw withdrawal thresholds in percentage of baseline. A main effect of sex indicates that males had generally lower scores over time than females in hyperalgesia data. These results indicate that spontaneous withdrawal causes hyperalgesia in NIH Swiss mice. This is significant because while it is known that withdrawal can cause hyperalgesia, most animal models that investigate this phenomenon use a precipitated withdrawal model, while our study demonstrated hyperalgesia also occurs in a spontaneous withdrawal model. This research was supported in part by State of Washington Initiative Measure 171.

Sitagliptin Alters Bone Composition in High-Fat-Fed Mice.

Type 2 diabetes mellitus is recognized as a significant risk factor for fragility of bone. Among the newer anti-diabetic agents, dipeptidyl peptidase-4 inhibitors (DPP4i) have been reported to decrease the occurrence of bone fractures although the reason is unclear. The main aim of this study was to evaluate the impact of sitagliptin treatment on tissue bone strength and compositional parameters in the high-fat-fed mouse model. Male NIH swiss mice were allowed free access to high-fat diet for 150days to induce chronic hyperglycemia and insulin resistance. Sitagliptin was administered once daily for 3weeks. High-fat-fed mice administered with saline were used as controls. Bone strength was assessed at the organ and tissue level by three-point bending and nanoindentation, respectively. Bone microarchitecture was investigated by microcomputed tomography and bone composition was evaluated by Fourier transform infrared imaging and quantitative backscattered electron imaging. Administration of sitagliptin increased non-fasting insulin, improved glucose tolerance and increased insulin sensitivity. This was associated with clear ameliorations in bone strength at the organ and tissue level. No changes in trabecular or cortical microarchitectures were observed. On the other hand, higher values of Camean, Caturn, collagen maturity, mineral/matrix ratio, mineral maturity and crystal size index were evidenced after sitagliptin treatment. Correlation analysis significantly linked the modifications of bone strength to changes in bone compositional parameters. These results bring new light on the mode of action of sitagliptin on bone physiology and demonstrate a benefit of DPP4i.

Long-term treatment with acylated analogues of apelin-13 amide ameliorates diabetes and improves lipid profile of high-fat fed mice.

Previous studies have shown that modified apelin analogues exhibited enzyme resistance in plasma and improved circulating half-life compared to apelin-13. This study investigated the antidiabetic effects of chronic administration of stable long acting fatty acid modified apelin analogues, namely, (Lys8GluPAL)apelin-13 amide and pGlu(Lys8GluPAL)apelin-13 amide, in high-fat fed obese-diabetic mice. Male NIH Swiss mice (groups n = 8) were maintained either on a high-fat diet (45% fat) from 8 to 28 weeks old, or control mice were fed a normal diet (10% fat). When diet induced obesity-diabetes was established after high-fat feeding, mice were injected i.p. once daily with apelin analogues, liraglutide (25 nmol/kg) or saline (controls). Administration of (Lys8GluPAL)apelin-13 amide and pGlu(Lys8GluPAL)apelin-13 amide for 28 days significantly reduced food intake and decreased body weight. Non-fasting glucose was reduced (p<0.01 to p<0.001) and plasma insulin concentrations increased (p<0.01 to p<0.001). This was accompanied by enhanced insulin responses (p<0.01 to p<0.001) and significant reductions in glucose excursion after oral (p<0.01) or i.p. (p<0.01) glucose challenges and feeding. Apelin analogues also significantly improved HbA1c (p<0.01), enhanced insulin sensitivity (p<0.01), reduced triglycerides (p<0.001), increased HDL-cholesterol (p<0.01) and decreased LDL-cholesterol (p<0.01), compared to high-fat fed saline treated control mice. Cholesterol levels were decreased (p<0.01) by pGlu(Lys8GluPAL)apelin-13 amide and both apelin treated groups showed improved bone mineral content, reduced fat deposits and increased plasma GLP-1. Daily treatment with liraglutide mirrored many of these changes (not on bone or adipose tissue), but unlike apelin analogues increased plasma amylase. Consumption of O2, production of CO2, respiratory exchange ratio and energy expenditure were improved by apelin analogues. These results indicate that long-term treatment with acylated analogues (Lys8GluPAL)apelin-13 amide and particularly pGlu(Lys8GluPAL)apelin-13 amide resulted in similar or enhanced therapeutic responses to liraglutide in high-fat fed mice. Fatty acid derived apelin analogues represent a new and exciting development in the treatment of obesity-diabetes.

Evaluation of hyperbaric oxygen (HBO2) as a treatment for hyperalgesia associated with naloxone‐precipitated withdrawal in morphine‐dependent mice

Opioid dependence has become a major problem in the United States. Withdrawal from opioid drugs is associated with hyperalgesia (increased sensitivity to a pain stimulus) which may contribute to relapse [Carcoba et al., J Addict Dis 30(3): 258–70, 2012]. Hyperbaric oxygen (HBO2) is 100% oxygen at higher than atmospheric pressure. HBO2 has recently been shown to be effective at decreasing physical signs of withdrawal in morphine‐dependent mice, and there is a growing body of research showing that HBO2 is effective at decreasing pain in clinical conditions and in preclinical modeling [Nicoara et al., Brain Res. 1648:434–7, 2016; Efrati et al., PLOS One 10(5)1–25, 2015; Gibbons at al., Brain Res. 1537:111–6, 2013]. The effectiveness of HBO2 to decrease hyperalgesia caused by opioid withdrawal in an animal model of drug dependence was evaluated in this study. Male and female NIH Swiss mice were made dependent upon morphine by injecting animals s.c. with twice‐daily escalating doses of morphine for 4 consecutive days. On the 5th day, animals were given a final injection of the highest dose of morphine and injected i.p. with 1.0 mg/kg naloxone 8 hours after the final morphine injection. Animals were treated with 30 min of HBO2 at 3.5 atmospheres absolute or room air 60 min prior to naloxone injection. Nociception was evaluated using the tail withdrawal, hot plate and paw pressure tests every 10 min for 30 min after naloxone injection. Baseline levels of nociception for each endpoint were taken prior to induction of morphine dependence. Male and female animals exhibited hyperalgesia in the hot plate and paw pressure tests but not the tail withdrawal test. HBO2 significantly decreased the amount of hyperalgesia in the hot plate and paw pressure tests in male animals. In female animals, HBO2 had no effect on hyperalgesia in the hot plate test but significantly decreased the level of hyperalgesia associated with the paw pressure test. While no hyperalgesia was seen in the tail withdrawal test, HBO2 did produce significant antinociception in male and female animals. These intriguing results show that HBO2 is capable of reducing hyperalgesia associated with opioid withdrawal and warrant further investigation. Furthermore, these results help support previous findings showing that HBO2 may be effective at decreasing symptoms of opioid withdrawal.Support or Funding InformationThis research was supported by the Marchionne 12‐month Fellowship from the Department of Psychology at Washington State University, NIH Grant AT‐007222, and the Honors College Distinguished Professorship from Washington State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuroinflammatory Responses in a Mouse Model of Tetramethylenedisulfotetramine‐Induced Status Epilepticus

Tetramethylenedisulfotetramine (TETS) is a GABAA receptor antagonist that is considered a chemical threat agent due to its ease of synthesis and its potent bioactivity in humans. Acute exposure to TETS can trigger seizures that evolve into status epilepticus (SE), and death. The current medical countermeasure for treating acute intoxication with seizurogenic chemicals like TETS includes the administration of benzodiazepines such as diazepam or midazolam. When administered 40 min post‐exposure in a mouse model of TETS‐induced SE, these positive allosteric modulators of the GABAA receptor are effective in stopping seizures and reducing mortality from ~90% to ~25%. The goal of this project was to determine the efficacy of this intervention in mitigating the neuroinflammatory response following TETS‐induced SE, with the objective of determining a feasible window of time for therapeutic intervention. In the current study, adult male NIH Swiss mice were pretreated with riluzole (10 mg/kg, i.p.) 10 min prior to receiving a lethal dose of TETS (0.2 mg/kg, i.p.). Control animals received vehicle in place of TETS and benzodiazepine. Animals were continuously monitored by EEG and/or scored for seizure behavior to confirm that TETS exposure elicited electrographic and/or behavioral seizures. At 10 or 40 min after the first myoclonic body twitch, animals were administered either diazepam (1.8 mg/kg, i.p.) or midazolam (1.8 mg/kg, i.m.) to terminate the persistent seizures. At 72h post‐intoxication, animals were perfused with fixative and brains were removed for FluoroJade C (FJC) labeling to assess neurodegeneration, and for GFAP and IBA1 immunohistochemistry to quantify neuroinflammation. Relative to vehicle control, GFAP immunoreactivity was significantly increased in the piriform cortex of all TETS‐exposed groups regardless of time of benzodiazepine administration. Midazolam was not superior to diazepam in protecting against neurodegeneration or neuroinflammation. These findings do not support the hypothesis that earlier treatment with either benzodiazepine is more effective in mitigating TETS‐induced neuropathology.Support or Funding InformationSupported by the NINDS CounterACT Program (grant # U54 NS079202).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

1,2-Dichloroethane Induces Reproductive Toxicity Mediated by the CREM/CREB Signaling Pathway in Male NIH Swiss Mice.

1,2-Dichloroethane (1,2-DCE) is a widely used chlorinated organic toxicant but little is known about the reproductive disorders induced by its excessive exposure. To reveal 1,2-DCE-induced male reproductive toxicity and to elucidate the underlying mechanisms, we exposed male National Institutes of Health Swiss mice to 1,2-DCE by inhalation at 0, 100, 350, and 700 mg/m3 for 6 h/day, for 1 and 4 weeks. Our findings showed a significant decrease in body weight with increased testis/body weight ratio, reduced sperm concentration and induced malformation of spermatozoa, and vacuolar degeneration of germ cells in the seminiferous tubules of testes in mice exposed to 1,2-DCE. Cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) and cAMP-response element modulator (CREM) were significantly inhibited by 1,2-DCE. This is consistent with the declines in the transducer of regulated CREB activity 1 and activator of CREM in testis, which results in the decrease in lactate dehydrogenase C and testis-specific kinase 1 in the testes. Moreover, the activation of p53 and Bax with the inhibition of Bcl-2 might be the reason for the upregulation of caspase-3 in the apoptosis, as detected by TdT-mediated dUTP nick-end labeling assay in the testes induced by 1,2-DCE. Finally, elevated testosterone levels were found along with increased levels of gonadotropin-releasing hormone, cAMP, luteinizing hormone (LH), and LH receptors in the testes. These findings suggest that 1,2-DCE inhibits CREM/CREB signaling cascade and subsequently induces apoptosis associated with p53 activation and mitochondrial dysfunction. This also results in induced malformation of spermatozoa, reduced sperm concentration, and pathological impairment of the testes.

Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice.

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

Hyperbaric oxygen treatment suppresses withdrawal signs in morphine-dependent mice

Hyperbaric oxygen (HBO2) therapy reportedly reduces opiate withdrawal in human subjects. The purpose of this research was to determine whether HBO2 treatment could suppress physical signs of withdrawal in opiate-dependent mice. Male NIH Swiss mice were injected s.c. with morphine sulfate twice a day for 4 days, the daily dose gradually increasing from 50mg/kg on day 1 to 125mg/kg on day 4. On day 5, withdrawal was precipitated by i.p. injection of 5.0mg/kg naloxone. Mice were observed for physical withdrawal signs, including jumping, forepaw tremor, wet-dog shakes, rearing and defecation for 30min. Sixty min prior to the naloxone injection, different groups of mice received either a 30-min or 60-min HBO2 treatment at 3.5atm absolute. HBO2 treatment significantly reduced naloxone-precipitated jumping, forepaw tremor, wet-dog shakes, rearing and defecation. Based on these experimental findings, we concluded that treatment with HBO2 can suppress physical signs of withdrawal syndrome in morphine-dependent mice.

I10W]tigerinin-1R enhances both insulin sensitivity and pancreatic beta cell function and decreases adiposity and plasma triglycerides in high-fat mice.

We have previously described the insulinotropic activities of [I10W]tigerinin-1R (RVCSAIPLPWCH.NH2) in vitro. In this study, we investigated the effects of the peptide on nutrient homoeostasis in mice with diet-induced obesity and insulin resistance. Male NIH Swiss mice were maintained on a high-fat diet for 12 weeks prior to the study. Twice-daily intraperitoneal injections of [I10W]tigerinin-1R (75nmol/kg body weight) were administered for 28days. Body weight, energy intake, body fat content, and plasma concentrations of triglyceride, cholesterol, non-fasting glucose and insulin were monitored. Effects of the peptide on glycaemic control were measured by glucose tolerance and insulin sensitivity tests. Pancreatic hormone content and insulin secretory responses of islets isolated from treated and untreated mice were examined. Immunohistochemical analysis was performed to study possible changes in islet morphology. Administration of [I10W]tigerinin-1R to high-fat-fed mice produced significant (P<0.05) decreases in plasma glucose, glucagon and triglyceride concentrations and an increase in plasma insulin compared to high-fat-fed controls. No changes in body weight or energy intake were observed with peptide treatment, but glycaemic control was significantly improved in response to oral or intraperitoneal glucose. Insulin sensitivity and secretory responses of islets to established insulin secretagogues were also significantly improved in peptide-treated mice. Total body fat, pancreatic insulin and glucagon contents, islet, beta and alpha cell areas were all significantly decreased in treated mice. This study shows that [I10W]tigerinin-1R improves insulin sensitivity, islet function and glycaemic control in high-fat-fed mice and has potential as a template for development of novel anti-diabetic agents.

The frog skin host-defense peptide CPF-SE1 improves glucose tolerance, insulin sensitivity and islet function and decreases plasma lipids in high-fat fed mice

The frog skin host-defense peptide CPF-SE1 has previously been shown to stimulate the in vitro release of insulin from clonal BRIN-BD11 β-cells. In this study, the in vivo effects of the peptide were investigated in male NIH Swiss mice maintained on a high-fat diet to induce obesity and insulin resistance. Insulin-secretory responses of islets isolated from treated and untreated mice and changes in islet morphology were also examined. Total body fat, plasma glucagon, triglyceride and cholesterol concentrations were measured at the end of the treatment period. Acute intraperitoneal administration of CPF-SE1 (75nmol body weight) to high-fat fed mice together with glucose (18mmol/kg body weight) improved glucose tolerance and insulin responses compared to high-fat fed controls. Long term administration of CPF-SE1 (twice-daily administration of 75nmol/kg body weight for 28 days) did not affect body weight or energy intake but decreased circulating glucose and increased insulin concentrations. Insulin sensitivity and insulin-secretory responses of islets to secretagogues were also significantly improved at 28 days in peptide-treated mice. In addition, significant decreases in plasma glucagon concentrations, pancreatic insulin and glucagon content, islet and beta cell area, body fat and plasma triglyceride levels were observed in CPF-SE1 treated with mice. In conclusion, CPF-SE1 improves beta cell function, insulin sensitivity and glycaemic control whilst reducing total body fat and circulating triglyceride levels. The peptide shows potential for development into an agent for treatment of patients with metabolic syndrome and type 2 diabetes.

Effects of 3,4‐Methylenedioxypyrovalerone (MDPV) and 3,4‐Methylenedioxymethamphetamine (MDMA) on Place Conditioning in Mice

Recently, there has been an increase in abuse of new synthetic psychostimulant drugs derived from the cathinone structure. These products are typically referred to as “bath salts”, and one of the most common psychoactive constituents of these preparations is 3,4‐methylenedioxypyrovalerone (MDPV). In these studies, we used the conditioned place preference (CPP) paradigm to explore the apparent appetitive effects of MDPV in mice, as compared to those of the structurally similar psychostimulant 3,4‐methylenedioxymethamphetamine (MDMA). The primary aims of this study were to investigate whether MDPV and MDMA induce CPP in mice, to determine the dose‐responsiveness of the CPPs elicited by these compounds, to compare the magnitude of CPP between drugs, and explore whether binge exposure to either MDPV or MDMA one week before the first CPP pairing increased preference scores for either drug, perhaps consistent with the concept of “sensitization” to abuse‐related effects of these compounds. Adult male NIH Swiss mice were conditioned with MDPV and MDMA at doses of 1, 3, or 10 mg/kg (i.p.), with mice receiving two pairing sessions (one saline‐one drug) per day for 3 days. Binge exposure animals were treated with 3 mg/kg MDPV or MDMA every two hours over an eight hour period, and then conditioned for CPP one week later. Both MDPV and MDMA elicited significant place preference across the doses studied, with MDPV producing CPP equal to or higher than that of MDMA. The effects of binge exposure on CPP will be discussed. Given the public health concerns related to “bath salts” use, future studies of both the acute and chronic effects of these compounds are necessary to fully understand the effects of these drugs and their potential for abuse. Studies supported by T32 DA022981.

(336) Role of serotonin in the acute antinociceptive effect of hyperbaric oxygen (HBO2) in mice

Hyperbaric oxygen (HBO2) causes an antinociceptive effect in both humans and animals. Our laboratory has been elucidating the mechanisms that underlie HBO2-induced antinociception and has reported that this effect is opioid- and nitric oxide-dependent. Since 5-hydroxytryptamine (5HT, serotonin) has been widely implicated in descending pain modulation, this study was conducted to investigate the role of serotonin receptors in the acute antinociceptive effect of HBO2. Male NIH Swiss mice were pretreated intracerebroventricularly (i.c.v.) and intrathecally (i.t.) with increasing doses of the 5HT1A receptor antagonist WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexane carboxamide maleate) 30 min prior to receiving HBO2 at 3.5 atmospheres absolute for 11 min.

Behavioral assessment of NIH Swiss mice acutely intoxicated with tetramethylenedisulfotetramine

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison that is thought to trigger seizures by inhibiting the function of the type A gamma-aminobutyric acid receptor (GABAAR). Acute intoxication with TETS can cause vomiting, convulsions, status epilepticus (SE) and even death. Clinical case reports indicate that individuals who survive poisoning may exhibit long-term neuropsychological issues and cognitive deficits. Therefore, the objective of this research was to determine whether a recently described mouse model of acute TETS intoxication exhibits persistent behavioral deficits. Young adult male NIH Swiss mice received a seizure-inducing dose of TETS (0.15mg/kg, ip) and then were rescued from lethality by administration of diazepam (5mg/kg, ip) approximately 20min post-TETS-exposure. TETS-intoxicated mice typically exhibited 2 clonic seizures prior to administration of diazepam with no subsequent seizures post-diazepam injection as assessed using behavioral criteria. Seizures lasted an average of 72s. Locomotor activity, anxiety-like and depression-relevant behaviors and cognition were assessed at 1week, 1month and 2months post-TETS exposure using open field, elevated-plus maze, light↔dark transitions, tail suspension, forced swim and novel object recognition tasks. Interestingly, preliminary validation tests indicated that NIH Swiss mice do not respond to the shock in fear conditioning tasks. Subsequent evaluation of hot plate and tail flick nociception tasks revealed that this strain exhibits significantly decreased pain sensitivity relative to age- and sex-matched C57BL/6J mice, which displayed normal contextual fear conditioning. NIH Swiss mice acutely intoxicated with TETS exhibited no significant anxiety-related, depression-relevant, learning or memory deficits relative to vehicle controls at any of the time points assessed with the exception of significantly increased locomotor activity at 2months post-TETS intoxication. The general absence of long-term behavioral deficits in TETS-intoxicated mice on these six assays suggests that the neurobehavioral consequences of TETS exposure described in human survivors of acute TETS intoxication are likely due to sustained seizure activity, rather than a direct effect of the chemical itself. Future research efforts are directed toward developing an animal model that better recapitulates the SE and seizure duration reported in humans acutely intoxicated with TETS.