Daily Injections Of Saline Research Articles

Sex-Specific Gene Expression in the Mouse Nucleus Accumbens Before and After Cocaine Exposure.

The nucleus accumbens plays a major role in the response of mammals to cocaine. In animal models and human studies, the addictive effects of cocaine and relapse probability have been shown to be greater in females. Sex-specific differential expression of key transcripts at baseline and after prolonged withdrawal could underlie these differences. To distinguish between these possibilities, gene expression was analyzed in four groups of mice (cycling females, ovariectomized females treated with estradiol or placebo, and males) 28 days after they had received seven daily injections of saline or cocaine. As expected, sensitization to the locomotor effects of cocaine was most pronounced in the ovariectomized mice receiving estradiol, was greater in cycling females than in males, and failed to occur in ovariectomized/placebo mice. After the 28-day withdrawal period, RNA prepared from the nucleus accumbens of the individual cocaine- or saline-injected mice was subjected to RNA sequencing analysis. Baseline expression of 3% of the nucleus accumbens transcripts differed in the cycling female mice compared with the male mice. Expression of a similar number of transcripts was altered by ovariectomy or was responsive to estradiol treatment. Nucleus accumbens transcripts differentially expressed in cycling female mice withdrawn from cocaine exhibited substantial overlap with those differentially expressed in cocaine-withdrawn male mice. A small set of transcripts were similarly affected by cocaine in the placebo- or estradiol-treated ovariectomized mice. Sex and hormonal status have profound effects on RNA expression in the nucleus accumbens of naive mice. Prolonged withdrawal from cocaine alters the expression of a much smaller number of common and sex hormone-specific transcripts.

Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: Subsequent behavioral outcomes

From 30 days after birth until the completion of the study, male and female rats were caged in same-sexed twos or threes either with (enriched cages, EC) or without several objects for them to explore (standard cages, SC). From 41 to 50 days of age (late adolescence), they received a daily intraperitoneal injection of saline, or 10 or 20 mg/kg of the monoaminergic agonist drug of abuse, 1-benzylpiperazine (BZP). Ten days later (PND60+), their behavior was observed over several days in an open field, an elevated plus maze, a light-dark box and (to assess short-term memory) a Y maze in which one of the arms had been changed in brightness between two trials. These tests were repeated from 40 days after PND60+, namely PND100+. While open-arm occupancy at PND100+ in the plus maze was lower following both doses of the drug for SC rats only, other examples of BZP-related heightened anxiety were confined to EC rats. This suggested that enrichment had enhanced rather than reduced any anxiogenic effects of the drug treatment. There was no plausible evidence of BZP-associated impaired spatial memory required to recognize the changed novel Y-maze arm. Instead, changes in novelty preferences or neophobia-related anxiety were most likely. While there were also some examples of sex and age differences in the later effects of BZP, in most cases these were evident at both ages following treatment with both BZP doses. A number of overall BZP, cage, sex and age differences, independent of enrichment effects, were also observed.

Exposure to synthetic glucocorticoids during pregnancy alters the expression of p73 gene variants in fetal brains in a sex-specific manner

Fetal exposure to dexamethasone (DEX) alters brain plasticity and cognitive functions during adulthood in a sex-dependent manner. The mechanisms underlying such long-lasting sex-dependent change of prenatal DEX is not well understood. The p73 gene plays an important role in brain development. It encodes for two protein variants; the neural cell death protein (TAp73) and the anti-neural cell death protein (ΔNp73). Therefore, we sought to determine how prenatal exposure to DEX alters the expression of these p73 gene variants in the brain of male and female fetuses.Pregnant dams received daily injections of either DEX (0.4 mg/kg, i.p.) or saline from gestation day (GD) 14 until GD21. On GD21, body and brain weights were monitored and mRNA and protein levels of TAp73 and ΔNp73 were measured in male and female fetal brains using RT-PCR, Western blot, and immunohistochemistry.Prenatal exposure to DEX significantly reduced the body and brain weights of both male and female fetuses, although reduction in brain weight was less severe than that of the body weight. Administration of DEX to pregnant dams led to enhanced expression of both TAp73 and ΔNp73 gene/protein variants in the brain of male but not in that of female fetuses.Dexamethasone induced a sex-dependent effect on the expression of p73 gene variants. DEX-induced growth restriction in the brain of female fetuses is independent of p73 gene. This study strongly suggests that survival/death programs operate differently during the development of male and female brains.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Valproate is protective against 6-OHDA-induced dopaminergic neurodegeneration in rodent midbrain: A potential role of BDNF up-regulation

The main purpose of this study was to extend previously reported showing potent neuroprotective effect of valproic acid (VPA) in primary midbrain neuro-glial cultures to investigate whether VPA could protect dopamine (DA) neurons invivo against 6-hydroxydopamine (6-OHDA)-induced neurodegeneration and to determine the underlying mechanism. Male adult rats received a daily intraperitoneal injection of VPA or saline for two weeks before and after injection of 5, 10, or 15μg of 6-OHDA into the brain. All rats were evaluated for motor function by rotarod performance. Brain samples were prepared for immunohistochemical staining and for determination of levels of dopamine, dopamine metabolites, and neurotrophic factors. 6-OHDA injection showed significant and dose-dependent damage of dopaminergic neurons and decrease of striatal dopamine content. Rats in the VPA-treated group were markedly protected from the loss of dopaminergic neurons and showed improvements in motor performance, compared to the control group at the moderate 6-OHDA dose (10μg). VPA-treated rats also showed significantly increased brain-derived neurotrophic factor (BDNF) levels in the striatum and substantia nigra compared to the levels in control animals. Our studies demonstrate that VPA exerts neuroprotective effects in a rat model of 6-OHDA-induced Parkinson's disease (PD), likely in part by up-regulation BDNF.

A recreational dose of methylphenidate, but not methamphetamine, decreases anxiety-like behavior in female rats

Methylphenidate (MPH) and methamphetamine (METH) are two commonly abused psychomotor stimulants that impact anxiety, but in a manner that is currently unclear. This study adds to the literature by testing the effects of MPH and METH on anxiety in adult female rats, which has not previously been studied. In Experiment1, changes in anxiety-like behavior were determined using the Elevated Plus Maze (EPM) following either an acute injection of saline, METH (1 mg/kg), or MPH (10 mg/kg). Changes in general locomotion were measured using the open field test. MPH, but not METH, significantly decreased anxiety; MPH and METH were associated with increased activity in the open field. In Experiment2, we compared the effects of three once daily injections of saline to MPH (10 mg/kg) or METH (1 mg/kg). As with the acute dosing, repeated exposure to MPH, but not METH, decreased anxiety, and both drugs increased locomotion. Neither acute nor chronic dosing produced a change in locomotion during the EPM, indicating that the anxiolytic effects of MPH are independent of changes in locomotor behavior. These findings add further clarification to the literature investigating the psychoactive properties of MPH, with a special and needed emphasis on female behavior.

Vascular endothelial growth factor accelerates compensatory lung growth by increasing the alveolar units.

BackgroundDeficiency of vascular endothelial growth factor (VEGF) is associated with hypoplastic lung diseases such as congenital diaphragmatic hernia (CDH). Provision of VEGF has been demonstrated to be beneficial in hyperoxia-induced bronchopulmonary dysplasia and thus could induce lung growth and improve outcome in hypoplastic lung diseases. We aimed to determine the effects of exogenous VEGF in a rodent model of compensatory lung growth after left pneumonectomy.MethodsEight-ten-week-old C57Bl6 male mice underwent left pneumonectomy followed by daily intra-peritoneal injections of saline or VEGF (0.5 mg/kg). Lung volume measurement, pulmonary function tests, and morphometric analyses were performed on post-operative day (POD) 4 and 10. Pulmonary expression of angiogenic factors was analyzed by quantitative polymerase chain reaction and western blot.ResultsLung volume on POD 4 was higher in the VEGF-treated mice (P = 0.03). On morphometric analyses, VEGF increased parenchymal volume (P = 0.001), alveolar volume (P = 0.0003), and alveolar number (P < 0.0001) on POD 4. The VEGF group displayed higher levels of phosphorylated-VEGFR2/VEGFR2 (P = 0.03) and epidermal growth factor (EGF) mRNA (P = 0.01).ConclusionVEGF accelerated compensatory lung growth in mice by increasing alveolar units. These changes may be mediated by VEGFR2 and EGF-dependent mechanisms.

The effects of gonadotropin-releasing hormone agonist (buserelin) and orchidectomy on bone turnover markers and histomorphometry in rats

This study aimed to compare the skeletal effect between GnRH agonist therapy and orchidectomy in male rats assessed using serum turnover markers and bone histomorphometry. Three-month-old male Sprague-Dawley rats (n = 46) were divided into three experimental arms, baseline, buserelin, and orchidectomy. In the buserelin arm, the rats received a daily subcutaneous injection of either normal saline or buserelin acetate at 25 µg/kg or 75 µg/kg. In the orchidectomy arm, the rats were either sham-operated or orchidectomized. The rats were euthanized after the three-month treatment. Blood was collected for the evaluation of bone turnover markers. Femurs were harvested for bone histomorphometry examination. A significant increase in serum C-telopeptide of type 1 collagen was observed in the orchidectomized group compared with the sham group (p < .05). Structural histomorphometry analysis showed that both buserelin (25 µg/kg and 75 µg/kg) and orchidectomy significantly decreased the trabecular bone volume, number and significantly increased trabecular separation in rats compared with their respective controls (p < .05). Osteoclast number and eroded surface were significantly increased in both buserelin (25 µg/kg and 75 µg/kg) and orchidectomized group compared with their respective controls (p < .05). As a conclusion, buserelin causes deterioration of bone microarchitecture and increased bone resorption similar to orchidectomy after three months.

Arbutin attenuates behavioral impairment and oxidative stress in an animal model of Parkinson's disease.

Arbutin has been shown to have antioxidant and free-radical scavenging properties. The aim of this study was to investigate the effects of arbutin administration on behavioral impairment, and oxidative and nitrosative stress in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal model of Parkinson's disease (PD). PD model was developed by 4 intraperitoneal (i.p.) injections of MPTP (20 mg/kg) with 2 h intervals in mice. Experimental groups received once daily injection of saline as vehicle (control group) or arbutin (50 mg/kg, i.p.) one week before MPTP injections and this protocol was continued seven days post lesion. Behavioral deficits were evaluated using locomotion test, hanging wire test and forepaw stride length. Parameters indicating the oxidation levels including lipid peroxidation marker (TBARS), nitrite, protein carbonyl levels and antioxidant activity including ferric reducing antioxidant power (FRAP) were assessed in serum and midbrain samples. Treatment with arbutin improved motor functions in an MPTP-induced PD model compared to control group (p<0.001). Mice treated with MPTP showed reduced levels of FRAP (p<0.001) and increased levels of TBARS (p<0.001), nitrite (p<0.001) and protein carbonyl (p<0.01), compared to the control group. In contrast to the MPTP group, arbutin treatment decreased the levels of TBARS (p<0.05), nitrite (p<0.05), protein carbonyl (p<0.05), and increased FRAP levels (p<0.05) in mice with PD. These findings suggest that arbutin attenuates the behavioral impairment and oxidative stress in a PD animal model.

Phencyclidine dose optimisation for induction of spatial learning and memory deficits related to schizophrenia in C57BL/6 mice.

Phencyclidine (PCP) has been used to model cognitive deficits related to schizophrenia in rats and mice. However, the model in mice is not consistent in terms of the PCP effective dose reported. Furthermore, most of the previous studies in mice excluded the presence of drug washout period in the regime. Thus, we aimed to optimize the dose of PCP in producing robust cognitive deficits by implementing it in a PCP regime which incorporates a drug washout period. The regimen used was 7 days’ daily injection of PCP or saline for treatment and vehicle groups, respectively; followed by 24 h drug washout period. After the washout period, the test mice were tested in water maze (5 days of acquisition + 1 day of probe trial) for assessment of spatial learning and memory. Initially, we investigated the effect of PCP at 2mg/kg, however, no apparent impairment in spatial learning and memory was observed. Subsequently, we examined the effect of higher doses of PCP at 5, 10 and 20 mg/kg. We found that the PCP at 10 mg/kg produced a significant increase in “latency to reach the platform” during the acquisition days and a significant increase in “latency of first entry to previous platform” during the probe day. There was no significant change observed in “swim speed” during the test days. Thus, we concluded that PCP at 10 mg/kg produced robust deficits in spatial learning and memory without being confounded by motor disturbances.

Establishing an Animal Model of Secondary Osteoporosis by Using a Gonadotropin-releasing Hormone Agonist

Introduction: Orchidectomy is currently the preferred method to induce bone loss in preclinical male osteoporosis model. Gonadotropin-releasing hormone (GnRH) agonists used in prostate cancer treatment can induce testosterone deficiency but its effects on bone in preclinical male osteoporosis model are less studied.Objective: This study aimed to evaluate the skeletal effect of buserelin (a GnRH agonist) in male rats and compare it with orchidectomy.Methods: Forty-six three-month-old male Sprague-Dawley rats were divided into three experimental arms. The baseline arm (n=6) was sacrificed at the onset of the study. In the buserelin arm, the rats received a daily subcutaneous injection of either normal saline (n=8), buserelin acetate at 25 µg/kg (n=8) or 75 µg/kg (n=8). In the orchidectomy arm, the rats were either sham-operated (n=8) or orchidectomized (n=8). All groups underwent in-vivo X-ray micro-computed tomography scanning at the left proximal tibia every month. Blood was collected at the beginning and the end of the study for testosterone level evaluation. The rats were euthanized after the three-month treatment. The femurs were harvested for biomechanical strength and bone calcium determination.Results: The results showed that buserelin at both doses caused a significant decline in testosterone level and deterioration in bone microstructure (p<0.05), but did not affect bone calcium content (p>0.05). Buserelin at 25 µg/kg decreased displacement and strain of the femur significantly (p<0.05). Similar changes were observed in the orchidectomized group compared to the sham-operated group but without any significant changes in biomechanical strength (p>0.05).Conclusion: Buserelin can induce testosterone deficiency and the associated deterioration of bone microarchitecture similar to orchidectomy in three months. However, it may require a longer time to show significant effects on bone strength and mineral content.

The effects of hydroalcoholic extract of Allium elburzense Wendelbo bulb on dexamethasone-induced dyslipidemia, hyperglycemia, and oxidative stress in rats.

Recent evidences have suggested the beneficial cardiovascular effects of some plants belonging to the genus Allium. The present study is an attempt to investigate the effects of hydroalcoholic extract of Allium elburzense bulb on dexamethasone-induced dyslipidemia in rats. Total phenolic content of A. elburzense bulb hydroalcoholic extract was determined using Folin-Ciocalteu method. Thirty-six male Wistar albino rats in 6 groups were studied. Group 1 (dyslipidemic control) received dexamethasone (10 mg/kg/day, s.c.) for 7 days, groups 2-4 (treated) received dexamethasone and simultaneously treated orally with 100, 200, or 400 mg/kg of A. elburzense extract, group 5 (normal control) received a single daily injection of normal saline (1 mL/kg, s.c.) and the vehicle orally, and group 6 (reference) received dexamethasone and atorvastatin (40 mg/kg) orally. At the end of experiment, blood glucose, lipid profile, and malondialdehyde (MDA) levels were assessed in serum samples. Livers were processed for histopathological examination. Total phenolic content of A. elburzense extract was estimated to be 33.52 ± 1.3% mg gallic acid equivalent/g of the dried plant extract. The plant extract significantly reduced serum blood glucose, triglyceride, total cholesterol, low-density lipoprotein-cholesterol, and MDA levels and increased the high density lipoprotein-cholesterol level and also improved liver steatosis compared to the dyslipidemic control group. These results suggest the hydroalcoholic extract of A. elburzense bulb has anti-dyslipidemic, anti-hyperglycemic, and antioxidant effects on rats receiving high doses of dexamethasone.

Propolis supplementation attenuates the negative effects of oxidative stress induced by paraquat injection on productive performance and immune function in turkey poults

Paraquat (PQ) is used as a herbicide in agriculture and causes oxidative and inflammatory damage to animal tissues. The current study was conducted to investigate the positive effects of dietary propolis (PR), as a potent naturally produced antioxidant, on growth performance and immune function of turkey poults exposed to oxidative stress induced by PQ injection. Native male turkey poults (n = 120, 49-d-old) were randomly assigned into 4 groups: poults received a basal diet with a daily subcutaneous PQ injection of 5 mg/kg BW for 7 consecutive days (PQ group), an experimental diet containing 1 g/kg PR with a daily subcutaneous PQ injection for 7 days (PR+PQ group), or received the experimental PR diet with a daily subcutaneous injection of 0.5 mL sterile saline for 7 days (PR group); while the control poults received a basal diet with a daily subcutaneous saline injection for 7 consecutive days (C group). The productive performance in the PQ group showed a significant (P < 0.05) reduction in the weight gain (WG) and feed intake (FI), and impaired feed conversion ratio (FCR). Propolis supplementation in the PR+PQ group significantly ameliorated the PQ effects on WG and FCR. Turkey poults of the PQ and PR+PQ groups had a significant augmentation in the blood malondialdehyde (MDA), tumor necrosis factor-α (TNFα), and corticosterone levels, and in contrast, a significant reduction in the triiodothyronine (T3), when compared to the C group. While propolis significantly reduced the MDA and corticosterone, and increased the T3 levels in the PR+PQ group compared to the PQ group. Furthermore, the dietary PR supplementation significantly limited the PQ-suppressive effects on cell- and humoral-mediated immunity and lymphocyte proliferation of turkey poults. In addition, propolis supplementation in the PR and PR+PQ groups markedly reversed the PQ-induced DNA fragmentation and heat shock protein 70 (Hsp70) over-expression in blood cells. It can be concluded that PR could improve turkey immunity and performance, particularly under inflammation and oxidative stress induced by PQ exposure.

Prior exposure to repeated peripheral LPS injections prevents further accumulation of hippocampal beta-amyloid

Alzheimer’s disease (AD) is characterized by the accumulation of beta-amyloid plaques and neurofibrillary tangles. Our laboratory has previously demonstrated that repeated bouts of LPS-induced inflammation increase beta-amyloid within the hippocampus of non-transgenic mice. Given the relationship between inflammation and AD pathology onset and progression, we sought to explore how a second series of peripheral lipopolysaccharide (LPS) would impact beta-amyloid levels. Male C57BL6/J mice were given daily injections of LPS (250 μg/kg) or sterile saline for seven days. Fourteen days following the last injection, animals were administered another series of daily injections of LPS or sterile saline. Hippocampal tissue was collected and assayed to quantify total beta-amyloid levels. Results demonstrated that animals given one round of LPS injections had significantly more beta-amyloid than saline injected controls, whereas animals administered two rounds of LPS had intermediary levels of beta-amyloid. This effect appears to be driven by the reduced inflammatory response to the secondary exposure to LPS following a fourteen-day recovery. More specifically, four hours following the first LPS injection of the second series, animals previously exposed to LPS produced significantly less pro-inflammatory cytokine mRNA than animals exposed to LPS for the first time. The mechanisms behind this diminished inflammatory response may be related to antibody production against LPS or cellular tolerance, both of which are currently being explored.

Anti-Inflammatory Effect of Recombinant Human Alpha-fetoprotein (rhAFP) in the Model of TNBS-induced Colitis

Introduction: Management of IBD relies primarily on steroids, immunosuppressors and anti-TNF agents. Although effective, these drugs are associated with side effects and reduced quality of life. Safer and more effective therapies are needed. In a small placebo-controlled trial (78 pts) human AFP (hAFP) was reported to effectively reduce symptoms of IBD. After 30 days of treatment 100% of hAFP pts showed clinical improvement. 47% gained weight. Over 30% of Crohn's and colitis pts had regained normal bowel movement. Endoscopy confirmed decreased number of ulcerative lesions. Almost half of hAFP pts were able to reduce or stop steroid use while no changes occurred in placebo patients*. Phase I and 2 clinical trials of rhAFP demonstrated an excellent safety profile with no serious adverse events or immune reactions. Present study aimed to investigate the efficacy and mode of action of rhAFP in a model of TNBS-induced colitis. Methods: Colitis was induced by intrarectal infusion of TNBS in C57bl6 mice. Mice received either daily injections of saline IV, anti-TNFα 0.1 mg/mouse, IP starting 3 days prior to TNBS infusion or rhAFP 200 μg/day starting 1 day before colitis induction and until euthanasia on day 2 after induction (20 mice/group). Colonic inflammation was evaluated using Wallace and Ameho scores.Colonic MPO level as well as gene expression of pro- and anti-inflammatory cytokines (IL-1 β IL-6, IFNγ, TNFα, and IL-10) were also measured. Results: rhAFP administration significantly decreased colonic inflammation with a 40% reduction in Wallace score compared to saline group (Fig. 1). Ameho's score was significantly lower in rhAFP group than in anti-TNFα or saline groups (Fig. 2), while MPO level in the colon was reduced from 40.72±0.64 to 29.59±3.11 and 25.42±4.22 ng/mg protein in control, anti-TNFα and rhAFP groups, respectively. The effect of rhAFP on cytokine production was similar to that of anti-TNFα with significant reduction in gene expression for IL-1β, IL-6, IFNγ, and TNFα and a significant increase for IL-10 (Table 1).Figure: Evaluation of inflammation at the macroscopic level: Wallace's score.Figure: Evaluation of inflammation at the histological level: Ameho's score.Table: Table. Cytokine Gene Expression Levels (mRNA/GAPDH ratio)Conclusion: This study confirms strong anti-inflammatory activity of rhAFP in the TNBS model of colitis with efficacy similar or greater than anti-TNFα. Considering the exceptional safety profile of rhAFP this molecule offers the possibility of novel safe and effective treatment for patients with IBD. *Chereshnev V, et al. Alpha-Fetoprotein. Russian Academy of Sciences, 2004, 239-245.

Repeated exposure to methamphetamine induces sex-dependent hypersensitivity to ischemic injury in the adult rat heart.

BackgroundWe previously reported that adult female, but not male rats that were prenatally exposed to methamphetamine exhibit myocardial hypersensitivity to ischemic injury. However, it is unknown whether hypersensitivity to ischemic injury develops when rats are exposed to methamphetamine during adulthood. The goal of this study was to determine whether methamphetamine exposure during adulthood sensitizes the heart to ischemic injury.MethodsAdult male and female rats received daily injections of methamphetamine (5 mg/kg) or saline for 10 days. Their hearts were isolated on day 11 and subjected to a 20 min ischemic insult on a Langendorff isolated heart apparatus. Cardiac contractile function was measured by an intraventricular balloon, and infarct size was measured by triphenyltetrazolium chloride staining.ResultsHearts from methamphetamine-treated females exhibited significantly larger infarcts and suppressed postischemic recovery of contractile function compared to hearts from saline-treated females. In contrast, methamphetamine had no effect on infarct size or contractile recovery in male hearts. Subsequent experiments demonstrated that hypersensitivity to ischemic injury persisted in female hearts following a 1 month period of abstinence from methamphetamine. Myocardial protein kinase C-ε expression, Akt phosphorylation, and ERK phosphorylation were unaffected by adult exposure to methamphetamine.ConclusionsExposure of adult rats to methamphetamine sex-dependently increases the extent of myocardial injury following an ischemic insult. These data suggest that women who have a heart attack might be at risk of more extensive myocardial injury if they have a recent history of methamphetamine abuse.

Microbial Metabolite, Butyrate, Increases Blood Pressure in Rodent Models of Hypertension

IntroductionThe emerging link between gut dysbiosis and hypertension is characterized by a significant reduction in butyrate‐producing bacteria in the spontaneously hypertensive rat (SHR) compared with the Wistar Kyoto (WKY) control. Butyrate, a major end product of dietary fiber fermentation by gut microbiota, reportedly produces beneficial effects in multiple dysbiosis‐related diseases. However, butyrate has also been linked with activation of the sympathetic drive, elevated heart rate and release of norepinephrine. Considering the established link between sympathetic drive and hypertension, we investigated the role of butyrate in blood pressure (BP) control in hypertension.MethodsMale SHR (5 wo) were placed on either the fiber‐rich (butyrolytic) diet (SHR‐BD) or control diet (SHR‐CD) for 13 weeks ad libitum. Fecal/cecal butyrate levels were determined using high performance liquid chromatography (HPLC) throughout the study. Following this, fecal matter transplant (FMT) from both groups was performed separately to male WKY recipients (7 wo) with antibiotic‐depleted endogenous gut bacteria, to create two groups: WKY FMT‐BD and WKY FMT‐CD. Five weeks following FMT, both groups were infused with a subpressor dose of angiotensin II (Ang II; 50ng/kg/min) for three weeks. Separate male SHR (9 wo) were administered with daily injections of either saline or butyrate (500mg/kg/day I.P.) for four weeks. BPs were measured in all groups using tailcuff or telemetry throughout the study.ResultsElevated cecal butyrate in SHR‐BD (by 2 fold vs. SHR‐CD; P&lt;0.001) was associated with significantly higher SBP (SHR‐CD: 183.3mmHg vs SHR‐BD: 206.6 mmHg; P&lt;0.01). FMT from SHR‐CD and SHR‐BD caused a similar BP increase in the WKY recipients (ΔSBP=11.5 mmHg, P&lt;0.001), However, a subsequent administration of subpressor Ang II further elevated SBP in the WKY FMT‐BD group only (by 7.7 mmHg vs. WKY FMT‐CD; P=0.06). Finally, chronic IP injection of butyrate in the SHR resulted in significant SBP increase compared to saline‐injected SHR (ΔSBP=14.5 mmHg, P&lt;0.05).ConclusionSHR gut microbiota is pro‐hypertensive in the WKY. Moreover, subpressor Ang II exacerbates this hypertension in the presence of excess butyrate. Thus, elevated butyrate may interact with Ang II in the pathophysiology of hypertension, indicating the disadvantageous role of butyrate in the context of hypertension.Support or Funding Information1. American Heart Association 2. College of Veterinary Medicine, University of Florida

Pharmacokinetics and Pharmacodynamics of Afamelanotide and its Clinical Use in Treating Dermatologic Disorders.

Afamelanotide, the first α-melanocyte-stimulating hormone (MSH) analogue, synthesized in 1980, was broadly investigated in all aspects of pigmentation because its activity and stability were higher than the natural hormone. Afamelanotide binds to the melanocortin-1 receptor (MC1R), and MC1R signaling increases melanin synthesis, induces antioxidant activities, enhances DNA repair processes and modulates inflammation. The loss-of-function variants of the MC1R present in fair-skinned Caucasians are less effectively activated by the natural hormone. Afamelanotide was the first α-MSH analogue to be applied to human volunteers. Ten daily doses of between 0.08 and 0.21mg/kg in saline injected subcutaneously resulted in long-lasting skin pigmentation and enabled basic pharmacokinetics. Subcutaneous application had full bioavailability, but neither oral nor transdermal application resulted in measurable plasma concentrations or pigmentation response. Two trials in human volunteers showed that neither MC1R variants nor fair skin reduced the afamelanotide-induced increase in skin pigmentation. A controlled-release formulation optimizes administration in man and is effective at a lower dose than the daily saline injections. Promising therapeutic results were published in polymorphic light eruption, erythropoietic protoporphyria (EPP), solar urticaria, Hailey-Hailey disease and vitiligo. In 2014, afamelanotide was approved by the European Medicines Agency for the prevention of phototoxicity in adult patients with EPP. No late effects were reported in volunteers 25years after the first exposure or after continuous long-term application of up to 8years in EPP patients, and an immunogenic potential has been excluded. Generally, adverse effects were benign in all trials.

The Impact of Exercise on Statin-Associated Skeletal Muscle Myopathy.

HMG-CoA reductase inhibitors (statins) are the most effective pharmacological means of reducing cardiovascular disease risk. The most common side effect of statin use is skeletal muscle myopathy, which may be exacerbated by exercise. Hypercholesterolemia and training status are factors that are rarely considered in the progression of myopathy. The purpose of this study was to determine the extent to which acute and chronic exercise can influence statin-induced myopathy in hypercholesterolemic (ApoE-/-) mice. Mice either received daily injections of saline or simvastatin (20 mg/kg) while: 1) remaining sedentary (Sed), 2) engaging in daily exercise for two weeks (novel, Nov), or 3) engaging in daily exercise for two weeks after a brief period of training (accustomed, Acct) (2x3 design, n = 60). Cholesterol, activity, strength, and indices of myofiber damage and atrophy were assessed. Running wheel activity declined in both exercise groups receiving statins (statin x time interaction, p<0.05). Cholesterol, grip strength, and maximal isometric force were significantly lower in all groups following statin treatment (statin main effect, p<0.05). Mitochondrial content and myofiber size were increased and 4-HNE was decreased by exercise (statin x exercise interaction, p<0.05), and these beneficial effects were abrogated by statin treatment. Exercise (Acct and Nov) increased atrogin-1 mRNA in combination with statin treatment, yet enhanced fiber damage or atrophy was not observed. The results from this study suggest that exercise (Nov, Acct) does not exacerbate statin-induced myopathy in ApoE-/- mice, yet statin treatment reduces activity in a manner that prevents muscle from mounting a beneficial adaptive response to training.

Vcam1 Is a "Don't-Eat-Me" Signal on Healthy Hematopoietic and Leukemic Stem Cells

▪Hematopoietic stem cells (HSCs) possess the ability to maintain the entire population of blood cells throughout life and to replenish the hematopoietic system after transplantation into marrow-ablated recipients. In mammalian adults, HSCs predominantly reside in the bone marrow to generate and maintain all blood cell types. HSCs, however, retain their ability to migrate to ectopic niches via the bloodstream, and traffic back to the bone marrow microenvironment via in part the interactions of alpha4 integrins with Vascular Cell Adhesion Molecule-1 (Vcam1, also known as CD106), constitutively expressed by endothelial and stromal cells. In the course of studies to investigate the role of Vcam1 in the macrophage erythroblastic niche, we have found using Vcam1-floxed mice crossed with a Csf1r-iCre (hereafter Vcam1Δ/Δ) that Vcam1 and Cre, using this transgenic line, were expressed on Lineage− (Lin−) c-Kit+ Sca1+ CD48− CD150+ HSCs. Although Vcam1Δ/Δ mice show no significant steady-state hematopoietic defect, transplantation of Vcam1Δ/Δ bone marrow, both in competitive and non-competitive settings, failed to engraft irradiated wild-type recipients. Indeed, ~80% of animals transplanted with Vcam1Δ/Δ cells died, and the few that survived failed to successfully engraft secondary recipient mice. Vcam1Δ/Δ HSC/progenitors homed to bone marrow as well as wild-type counterparts and showed no detectable change in apoptosis in the bone marrow. However, syngeneic Vcam1Δ/Δ HSC/progenitors were selectively cleared in vivo by bone marrow macrophages over 4 days. The in vivo Vcam1Δ/Δ HSC/progenitor clearance did not require irradiation-induced damage since in steady-state parabiosis (Vcam1Δ/Δ and wild-type co-joined pairs), Vcam1Δ/Δ HSCs did not engraft the partner after G-CSF-induced mobilization whereas wild-type HSCs engrafted the parabiont partner. In addition, in vitro incubation of bone marrow-derived macrophages with Lin− cells revealed rapid phagocytosis by macrophages of Vcam1Δ/Δ, but not wild-type, cells and phagocytosis was also induced when the integrin alpha4 beta1 (also known as VLA-4) was blocked in wild-type Lin− cells. Thus, these results suggest that Vcam1 expressed on HSCs serves as a novel don’t-eat-me signal, controlling the migration in the bone marrow by a macrophage-enabled checkpoint. Since we found that Vcam1 is expressed at higher levels on acute myelogenous leukemia (AML) cells than on healthy HSCs, and high VCAM1 expression correlates with poor prognosis in human AML patients (Ley et. al. NEJM 2013), we examined whether Vcam1 expression by AML cell could affect leukemogenesis in vivo. To test this idea, we generated AML-Vcam1Δ/Δ and AML-Control cells by transduction of bone marrow Lin− c-Kit+ Sca1+ cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, FACS analysis of primary AML recipient bone marrow revealed a marked reduction (>99%) in the number and percentage of phenotypic Lin− IL7Rα− Sca1− MLL-AF9 GFP+ c-Kithigh CD34low FcγRII/IIIhigh leukemic stem cells in AML-Vcam1Δ/Δ mice compared to control. These results were confirmed by imaging analyses, which showed a marked reduction in leukemic infiltration in AML-Vcam1Δ/Δ mice compared to control. Kaplan-Meier survival analysis of secondary recipient mice receiving 20,000 GFP+ leukemic cells revealed a significantly greater survival of mice harboring AML-Vcam1Δ/Δ cells relative to AML-Control (P<0.0001). To assess in a pre-clinical experimental setting whether Vcam1 inhibition can alter the course of AML, we established AML in immunocompetent C57BL/6 recipients and started therapy of moribund leukemic mice (>50% circulating AML-GFP+ cells) with a daily injection of saline, IgG control, anti-Vcam1 blocking antibody (100 μg/day), cytarabine (100 mg/kg), or a combination of anti-Vcam1/cytarabine for 5 days. Remarkably, this short treatment with anti-Vcam1 blocking antibody was able to preferentially and significantly reduce the frequency and absolute number of phenotypic bone marrow leukemic stem cells in vivo. In addition, our results suggest that using a blocking anti-Vcam1 antibody synergizes with cytarabine treatment to decrease leukemic burden in vivo. These studies lay the groundwork for the development of a new therapeutic strategy for eliminating leukemia stem cells by modulating the innate immune response. DisclosuresFrenette:GSK: Research Funding; Pfizer: Consultancy; PHD Biosciences: Research Funding.