Chronic Systemic Administration Research Articles

6-Methoxyflavone antagonizes chronic constriction injury and diabetes associated neuropathic nociception expression

Neuropathy is a disturbance of function or a pathological change in nerves causing poor health and quality of life. A proportion of chronic pain patients in the community suffer persistent neuropathic pain symptoms because current drug therapies may be suboptimal so there is a need for new therapeutic modalities. This study investigated the neuroprotective flavonoid, 6-methoxyflavone (6MF), as a potential therapeutic agent and gabapentin as the standard comparator, against neuropathic models. Thus, neuropathic-like states were induced in Sprague-Dawley rats using sciatic nerve chronic constriction injury (CCI) mononeuropathy and systemic administration of streptozotocin (STZ) to induce polyneuropathy. Subsequent behaviors reflecting allodynia, hyperalgesia, and vulvodynia were assessed and any possible motoric side-effects were evaluated including locomotor activity, as well as rotarod discoordination and gait disruption. 6MF (25–75 mg/kg) antagonized neuropathic-like nociceptive behaviors including static- (pressure) and dynamic- (light brushing) hindpaw allodynia plus heat/cold and pressure hyperalgesia in the CCI and STZ models. 6MF also reduced static and dynamic components of vulvodynia in the STZ induced polyneuropathy model. Additionally, 6MF reversed CCI and STZ suppression of locomotor activity and rotarod discoordination, suggesting a beneficial activity on motor side effects, in contrast to gabapentin. Hence, 6MF possesses anti-neuropathic-like activity not only against different nociceptive modalities but also impairment of motoric side effects.

181-OR: Pancreatic Gene Therapy Durably Improves Glycemia and Delays Disease Progression in a Murine Model of Type 2 Diabetes

Beta-cell (β-cell) failure is a root cause of the progression of type 2 diabetes (T2D). GLP-1 receptor agonists (GLP1RA) have proven clinical efficacy and improve β-cell function. However, systemic side effects limit patient adherence and long-term efficacy. Localized pancreatic GLP1RA production may engender durable islet cell benefit while diminishing side effects. We developed a novel adeno-associated virus (AAV) gene therapy delivery platform to enable local and durable production of therapeutic proteins by the pancreas. We assessed to what degree a β-cell restricted GLP1RA gene therapy impacts disease progression in the db/db mouse compared to semaglutide (Sema). To establish a baseline for chronically administered systemic GLP1RA, Sema (10 nmol/kg) or vehicle were given daily to 5-week-old mice for 10 weeks (n=8/group). Sema treated mice had a 64% decrease in fasting blood glucose (FBG; p<0.0001) and a 3.5-fold increase in fasting insulin (FI; p=0.01) across the time course. Glucose tolerance was significantly improved during IPGTT at weeks 3, 6, and 9 (p<0.001). To determine the impact of a single dose GLP1RA gene therapy, 1) β-cell restricted AAV-GLP1RA, 2) AAV control, or 3) vehicle were given to 5-week-old mice (n=8/group). Remarkably, AAV-GLP1RA treatment decreased FBG levels by 70% (p<0.0001) and elevated FI by 1.9-fold (p<0.01) compared to controls for 10 weeks. Glucose tolerance was improved during IPGTT at weeks 3, 5, 7, and 9 (p<0.01). AAV-GLP1RA protein was undetectable in serum and restricted to the islet, suggesting improved glycemic control was due to intra-islet GLP1RA production and signaling. These results demonstrate that a single dose β-cell restricted GLP1RA gene therapy can comparably improve glycemia and delay disease progression in a murine model of T2D when compared to chronic systemic GLP1RA administration. These data provide proof of concept that a gene therapy has the potential to durably treat metabolic diseases, including T2D. Disclosure A.L.Fitzpatrick: Employee; Fractyl Health, Inc. E.Cozzi: Employee; Fractyl Health, Inc. J.Wainer: Employee; Fractyl Health, Inc. R.Reese: Employee; Fractyl Health, Inc. N.Khanna: Employee; Fractyl Health, Inc. J.Caplan: Employee; Fractyl Health, Inc. H.Rajagopalan: Employee; Fractyl Health, Inc. J.A.West: Employee; Fractyl Health, Inc. Funding Fractyl Health, Inc.

Effects of glycogen synthase kinase-3β activity inhibition on cognitive, behavioral, and hippocampal ultrastructural deficits in adulthood associated with adolescent methamphetamine exposure.

Glycogen synthase kinase-3β (GSK3β) has been implicated in the maintenance of synaptic plasticity, memory process, and psychostimulant-induced behavioral effects. Hyperactive GSK3β in the Cornu Ammonis 1 (CA1) subregion of the dorsal hippocampus (DHP) was associated with adolescent methamphetamine (METH) exposure-induced behavioral and cognitive deficits in adulthood. This study aimed to evaluate the possible therapeutic effects of GSK3β inhibition in adulthood on adolescent METH exposure-induced long-term neurobiological deficits. Adolescent male mice were treated with METH from postnatal day (PND) 45-51. In adulthood, three intervention protocols (acute lithium chloride systemic administration, chronic lithium chloride systemic administration, and chronic SB216763 administration within CA1) were used for GSK3β activity inhibition. The effect of GSK3β intervention on cognition, behavior, and GSK3β activity and synaptic ultrastructure in the DHP CA1 subregion were detected in adulthood. In adulthood, all three interventions reduced adolescent METH exposure-induced hyperactivity (PND97), while only chronic systemic and chronic within CA1 administration ameliorated the induced impairments in spatial (PND99), social (PND101) and object (PND103) recognition memory. In addition, although three interventions reversed the aberrant GSK3β activity in the DHP CA1 subregion (PND104), only chronic systemic and chronic within CA1 administration rescued adolescent METH exposure-induced synaptic ultrastructure changes in the DHP CA1 subregion (PND104) in adulthood. Rescuing synaptic ultrastructural abnormalities in the dHIP CA1 subregion by chronic administration of a GSK3β inhibitor may be a suitable therapeutic strategy for the treatment of behavioral and cognitive deficits in adulthood associated with adolescent METH abuse.

Simultaneous Antagonism at H3R/D2R/D3R Reduces Autism-like Self-Grooming and Aggressive Behaviors by Mitigating MAPK Activation in Mice.

Dysregulation in brain neurotransmitters underlies several neuropsychiatric disorders, e.g., autism spectrum disorder (ASD). Also, abnormalities in the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway pave the way for neuroinflammation, neurodegeneration, and altered learning phenotype in ASD. Therefore, the effects of chronic systemic administration of the multiple-targeting antagonist ST-713 at the histamine H3 receptor (H3R) and dopamine D2/D3 receptors (D2/D3R) on repetitive self-grooming, aggressive behaviors, and abnormalities in the MAPK pathway in BTBR T + Itpr3tf/J (BTBR) mice were assessed. The results showed that ST-713 (2.5, 5, and 10 mg/kg, i.p.) mitigated repetitive self-grooming and aggression in BTBR mice (all p < 0.05), and the ameliorative effects of the most promising dose of ST-713 (5 mg/kg, i.p.) on behaviors were completely abrogated by co-administration of the H3R agonist (R)-α-methylhistamine or the anticholinergic drug scopolamine. Moreover, the elevated levels of several MAPK pathway proteins and induced proinflammatory markers such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and IL-6 were significantly suppressed following chronic administration of ST-713 (5 mg/kg, i.p.) (all p < 0.01). Furthermore, ST-713 significantly increased the levels of histamine and dopamine in hippocampal tissue of treated BTBR mice (all p < 0.01). The current observations signify the potential role of such multiple-targeting compounds, e.g., ST-713, in multifactorial neurodevelopmental disorders such as ASD.

Multiple Pyogenic Granulomas in the External Auditory Canal.

Pyogenic granuloma is a benign vascular tumor of the skin or mucous membrane. One-third of pyogenic granulomas have been reported in the head and neck, but it is rarely present in the external auditory canal. Pyogenic granuloma mostly presents as a solitary granuloma, and only a few cases of multiple forms have been reported. This report describes a rare case of multiple pyogenic granulomas in the external auditory canal of a 36-year-old man along with a review of the literatures. Although it is very rare for PGs to occur in the EAC, it can be suspected in conditions such as after acute and/or chronic trauma, hormonal changes or systemic drug administration, rapid growth, and easy bleeding tendency with a friable surface. Some PGs may spontaneously resolve, but when they cause symptoms, excision is recommended for treatment and diagnosis. In the case of excision, the tumor should be excised down to the perichondrium level to prevent recurrence. Although PG mainly occurs in a solitary form, the present case shows a new clinical variation where multiple PGs were present in the EAC.

The anorexic effect of DL-fenfluramine is dependent on animals' habituation to different food types.

As rates of obesity and diabetes have increased dramatically over the past few decades, the use of anti-obesity drugs has now become a routine therapeutic measure. However, the pharmacological effects of chronic use of these drugs in humans frequently lead to reduced efficacy in reducing appetite and body weight through as-yet-unidentified mechanisms. An example of this can be found in animal studies where the appetite suppressant DL-fenfluramine (FEN) is chronically administered and its tolerance develops in animals and humans. The appetite effects of FEN are typically measured in several animal studies by the feeding changes in a balanced standard diet. To determine whether FEN differentially altered appetite suppression in animals with long-term expression with different macronutrient diet compositions, its anorexic effects were measured specifically in male rats that had previously been chronically maintained on normal chow (NC) or a high-fat and high-carbohydrate western diet (WD). Three experiments were conducted by feeding the animals either NC or WD for 1 month to habituate them with their diet. Animals maintained on either NC or WD were subsequently offered both diet options ad libitum for a 2- or 7-day adaptation period while receiving daily systemic FEN treatment. The results suggested that long-term habituated food affected the food preference of animals and their appetite even after chronic systemic FEN administration. Therefore, the effectiveness and success or failure of repeated use of chronic anti-obesity drugs may depend on habituated food type. The appetite suppressant effect was found to be determined by the palatability of a specific macronutrient and the habituated food rather than by a change in the concentration of the administered FEN. This results in a critical analysis of the rationale for taking medication considering the patient's past dietary habits to achieve successful weight loss.

Morphine Accumulates in the Retina Following Chronic Systemic Administration.

Opioid transport into the central nervous system is crucial for the analgesic efficacy of opioid drugs. Thus, the pharmacokinetics of opioid analgesics such as morphine have been extensively studied in systemic circulation and the brain. While opioid metabolites are routinely detected in the vitreous fluid of the eye during postmortem toxicological analyses, the pharmacokinetics of morphine within the retina of the eye remains largely unexplored. In this study, we measured morphine in mouse retina following systemic exposure. We showed that morphine deposits and persists in the retina long after levels have dropped in the serum. Moreover, we found that morphine concentrations (ng/mg tissue) in the retina exceeded brain morphine concentrations at all time points tested. Perhaps most intriguingly, these data indicate that following chronic systemic exposure, morphine accumulates in the retina, but not in the brain or serum. These results suggest that morphine can accumulate in the retina following chronic use, which could contribute to the deleterious effects of chronic opioid use on both image-forming and non-image-forming visual functions.

The interactive effects of verapamil and CB1 cannabinoid receptor antagonist/inverse agonist, AM251 on passive avoidance learning and memory in rat.

There are reports regarding the effects of intracellular Ca2+ and synthesis and release of endocannabinoids. The secretion of endocannabinoids depends on the L-type calcium channel. The present study evaluated the involvement of the cannabinoid CB1 receptors in the effect of L-type calcium channel blocker verapamil on passive avoidance learning (PAL) in adult male rats. In this study, we examined the effects of an acute administration of the cannabinoid CB1 receptors antagonist/inverse agonist AM251 following a chronic administration of the Ca2+ channel blocker verapamil on PAL. Male Wistar rats were administered verapamil (10, 25 and 50 mg/kg) or saline intraperitoneally (i.p) daily for 13 days (n = 10/group). After this treatment period, a learning test (acquisition) was performed, and a retrieval test was performed the following day. The results indicated that chronic systemic administration of verapamil (in a dose-dependent manner) impaired memory acquisition and retrieval. Pre-training acute administration of a selective CB1 antagonist/inverse agonist, AM251 (5 mg/kg, i.p.) did not change memory acquisition and retrieval. Co-administration of the verapamil and AM251 significantly reversed verapamil-induced amnesia, suggesting a functional interaction between AM251 and verapamil. The results indicated the interactive effects of cannabinoid CB1 receptors and L-type calcium channel in passive avoidance learning and AM251 can counter the effects of verapamil on memory.

Microglia and their LAG3 checkpoint underlie the antidepressant and neurogenesis-enhancing effects of electroconvulsive stimulation.

Despite evidence implicating microglia in the etiology and pathophysiology of major depression, there is paucity of information regarding the contribution of microglia-dependent molecular pathways to antidepressant procedures. In this study, we investigated the role of microglia in a mouse model of depression (chronic unpredictable stress-CUS) and its reversal by electroconvulsive stimulation (ECS), by examining the effects of microglia depletion with the colony stimulating factor-1 antagonist PLX5622. Microglia depletion did not change basal behavioral measures or the responsiveness to CUS, but it completely abrogated the therapeutic effects of ECS on depressive-like behavior and neurogenesis impairment. Treatment with the microglia inhibitor minocycline concurrently with ECS also diminished the antidepressant and pro-neurogenesis effects of ECS. Hippocampal RNA-Seq analysis revealed that ECS significantly increased the expression of genes related to neurogenesis and dopamine signaling, while reducing the expression of several immune checkpoint genes, particularly lymphocyte-activating gene-3 (Lag3), which was the only microglial transcript significantly altered by ECS. None of these molecular changes occurred in microglia-depleted mice. Immunohistochemical analyses showed that ECS reversed the CUS-induced changes in microglial morphology and elevation in microglial LAG3 receptor expression. Consistently, either acute or chronic systemic administration of a LAG3 monoclonal antibody, which readily penetrated into the brain parenchyma and was found to serve as a direct checkpoint blocker in BV2 microglia cultures, rapidly rescued the CUS-induced microglial alterations, depressive-like symptoms, and neurogenesis impairment. These findings suggest that brain microglial LAG3 represents a promising target for novel antidepressant therapeutics.

Iduronate-2-sulfatase transport vehicle rescues behavioral and skeletal phenotypes in a mouse model of Hunter syndrome.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in cellular accumulation of glycosaminoglycans (GAGs) throughout the body. Treatment of MPS II remains a considerable challenge as current enzyme replacement therapies do not adequately control many aspects of the disease, including skeletal and neurological manifestations. We developed an IDS transport vehicle (ETV:IDS) that is engineered to bind to the transferrin receptor; this design facilitates receptor-mediated transcytosis of IDS across the blood-brain barrier and improves its distribution into the brain while maintaining distribution to peripheral tissues. Here we show that chronic systemic administration of ETV:IDS in a mouse model of MPS II reduced levels of peripheral and central nervous system GAGs, microgliosis, and neurofilament light chain, a biomarker of neuronal injury. Additionally, ETV:IDS rescued auricular and skeletal abnormalities when introduced in adult MPS II mice. These effects were accompanied by improvements in several neurobehavioral domains, including motor skills, sensorimotor gating, and learning and memory. Together, these results highlight the therapeutic potential of ETV:IDS for treating peripheral and central abnormalities in MPS II. DNL310, an investigational ETV:IDS molecule, is currently in clinical trials as a potential treatment for patients with MPS II.

MP39-18 AN AUTOCHTHONOUS MOUSE MODEL OF KIDNEY CANCER WITH T CELL INFILTRATION

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-18 AN AUTOCHTHONOUS MOUSE MODEL OF KIDNEY CANCER WITH T CELL INFILTRATION Nitika Sharma, Bo Xu, Jacky Chow, Jason Muhitch, and Eric Kauffman Nitika SharmaNitika Sharma More articles by this author , Bo XuBo Xu More articles by this author , Jacky ChowJacky Chow More articles by this author , Jason MuhitchJason Muhitch More articles by this author , and Eric KauffmanEric Kauffman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immunocompetent animal models that recapitulate human renal cell carcinoma (RCC) and its tumor-immune microenvironment are critical for the preclinical study of novel immunotherapeutic strategies. Abundant CD8+ T cell lymphocyte infiltration is a defining feature of human clear cell RCC but is lacking from many mouse models. Iron is a known carcinogen with increased cellular levels in human RCC. In the FeNTA mouse model, chronic systemic administration of iron (Fe) with a chelator (nitrilotriacetic acid, NTA) induces renal carcinogenesis, however the tumor biology is largely uncharacterized. We studied whether FeNTA mouse model cancers have similar biology as human RCC, including an abundance of tumor-infiltrating lymphocytes. METHODS: 60 male mice (AJ strain) were treated for 12 weeks (5X/week) with intraperitoneal FeNTA injection to induce renal tumorigenesis. Renal tumors were harvested at 12-15 months post-treatment and underwent H&E histologic evaluation by a clinical genitourinary pathologist. Tumor lysates were analyzed for PAX8 and HIF2-alpha protein levels by Western Blot, while tumor single-cell suspensions were analyzed for immune cell antigens (CD45, CD3, CD4, CD8) by imaging flow cytometry to quantify tumor-infiltrating T cell lymphocyte populations. RESULTS: Of 60 FeNTA-treated mice, 14 (23.3%) developed renal tumors as confirmed by magnetic resonance imaging and histology. Mean tumor size was 1.5 cm (interquartile range 1.1-1.8 cm). Tumor histology was most similar to clinical clear cell RCC with eosinophilic cytoplasm. The clinical RCC biomarker, PAX8, was expressed in murine tumors, as was the HIF2-alpha protein that typifies human clear cell RCC. Infiltrating immune cells (CD45+) comprised on average 33.2% (+/- standard error, 4.6%) of all tumor cells, including 11.9% (+/- 3.9%) of tumor cells that were specifically T cell lymphocytes (CD45+/CD3+). Most tumor-infiltrating T cells were CD8+/CD4- (74.3+/- 3.7%), while the remainder were CD4+/CD8- (25.7+/- 3.7%). CONCLUSIONS: The FeNTA autochthonous immunocompetent mouse model recapitulates key clinical RCC biology, including a tumor-infiltrating T cell profile similar to that of the human disease. This model may provide a valuable resource for preclinical evaluation of novel immunotherapeutic strategies for RCC. Source of Funding: American Cancer Society © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e711-e712 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nitika Sharma More articles by this author Bo Xu More articles by this author Jacky Chow More articles by this author Jason Muhitch More articles by this author Eric Kauffman More articles by this author Expand All Advertisement Loading ...

The long-acting amylin/calcitonin receptor agonist ZP5461 suppresses food intake and body weight in male rats.

The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.

The histone methyltransferase G9a mediates stress-regulated alcohol drinking.

The epigenetic enzyme G9a is a histone methyltransferase that dimethylates lysine 9 on histone H3 (H3K9me2), and in the adult nucleus accumbens (NAc), G9a regulates multiple behaviors associated with substance use disorder. We show here that chronic intermittent ethanol (CIE) exposure in male mice reduced both G9a and H3K9me2 levels in the adult NAc, but not dorsal striatum. Viral-mediated reduction of G9a in the NAc had no effects on baseline volitional ethanol drinking or escalated alcohol drinking produced by CIE exposure; however, NAc G9a was required for stress-regulated changes in ethanol drinking, including potentiated alcohol drinking produced by activation of the kappa-opioid receptor. In addition, we observed that chronic systemic administration of a G9a inhibitor, UNC0642, also blocked stress-potentiated alcohol drinking. Together, our findings suggest that chronic alcohol use, similar to other abused substances, produces a NAc-selective reduction in G9a levels that serves to limit stress-regulated alcohol drinking. Moreover, our findings suggest that pharmacological inhibition of G9a might provide a novel therapeutic approach to treat stress-induced alcohol drinking, which is a major trigger of relapse in individuals suffering from AUD.

Role of 5-HT1A and 5-HT2C receptors of the dorsal periaqueductal gray in the anxiety- and panic-modulating effects of antidepressants in rats

Antidepressant drugs are first-line treatment for panic disorder. Facilitation of 5-HT1A receptor-mediated neurotransmission in the dorsal periaqueductal gray (dPAG), a key panic-associated area, has been implicated in the panicolytic effect of the selective serotonin reuptake inhibitor fluoxetine. However, it is still unknown whether this mechanism accounts for the antipanic effect of other classes of antidepressants drugs (ADs) and whether the 5-HT interaction with 5-HT2C receptors in this midbrain area (which increases anxiety) is implicated in the anxiogenic effect caused by short-term treatment with ADs. The results showed that previous injection of the 5-HT1A receptor antagonist WAY-100635 in the dPAG blocked the panicolytic-like effect caused by chronic systemic administration of the tricyclic AD imipramine in male Wistar rats tested in the elevated T-maze. Neither chronic treatment with imipramine nor fluoxetine changed the expression of 5-HT1A receptors in the dPAG. Treatment with these ADs also failed to significantly change ERK1/2 (extracellular-signal regulated kinase) phosphorylation level in this midbrain area. Blockade of 5-HT2C receptors in the dPAG with the 5-HT2C receptor antagonist SB-242084 did not change the anxiogenic effect caused by a single acute injection of fluoxetine or imipramine in the Vogel conflict test. These results reinforce the view that the facilitation of 5-HT1A receptor-mediated neurotransmission in the dPAG is a common mechanism involved in the panicolytic effect caused by chronic administration of ADs. On the other hand, the anxiogenic effect observed after short-term treatment with these drugs does not depend on 5-HT2C receptors located in the dPAG.

Toward a Medical Gastric Bypass: Chronic Feeding Studies With Liraglutide + PYY3-36 Combination Therapy in Diet-Induced Obese Rats.

BackgroundCombination therapies of anorectic gut hormones partially mimic the beneficial effects of bariatric surgery. Thus far, the effects of a combined chronic systemic administration of Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine 3-36 (PYY3-36) have not been directly compared to Roux-en-Y gastric bypass (RYGB) in a standardized experimental setting.MethodsHigh-fat diet (HFD)-induced obese male Wistar rats were randomized into six treatment groups: (1) RYGB, (2) sham-operation (shams), (3) liraglutide, (4) PYY3-36, (5) PYY3-36+liraglutide (6), saline. Animals were kept on a free choice high- and low-fat diet. Food intake, preference, and body weight were measured daily for 4 weeks. Open field (OP) and elevated plus maze (EPM) tests were performed.ResultsRYGB reduced food intake and achieved sustained weight loss. Combined PYY3-36+liraglutide treatment led to similar and plateaued weight loss compared to RYGB. Combined PYY3-36+liraglutide treatment was superior to PYY3-36 (p ≤ 0.0001) and liraglutide (p ≤ 0.05 or p ≤ 0.01) mono-therapy. PYY3-36+liraglutide treatment and RYGB also reduced overall food intake and (less pronounced) high-fat preference compared to controls. The animals showed no signs of abnormal behavior in OF or EPM.ConclusionsLiraglutide and PYY3-36 combination therapy vastly mimics reduced food intake, food choice and weight reducing benefits of RYGB.

Metoprolol Impairs β1-Adrenergic Receptor-Mediated Vasodilation in Rat Cerebral Arteries: Implications for β-Blocker Therapy.

The practice of prescribing β-blockers to lower blood pressure and mitigate perioperative cardiovascular events has been questioned because of reports of an increased risk of stroke. The benefit of β-blocker therapy primarily relies on preventing activation of cardiac β1-adrenergic receptors (ARs). However, we reported that β1ARs also mediate vasodilator responses of rat cerebral arteries (CAs), implying that β-blockers may impair cerebral blood flow under some conditions. Here, we defined the impact of metoprolol (MET), a widely prescribed β1AR-selective antagonist, on adrenergic-elicited diameter responses of rat CAs ex vivo and in vivo. MET (1-10 µmol/l) prevented β1AR-mediated increases in diameter elicited by dobutamine in cannulated rat CAs. The β1AR-mediated dilation elicited by the endogenous adrenergic agonist norepinephrine (NE) was reversed to a sustained constriction by MET. Acute oral administration of MET (30 mg/kg) to rats in doses that attenuated resting heart rate and dobutamine-induced tachycardia also blunted β1AR-mediated dilation of CAs. In the same animals, NE-induced dilation of CAs was reversed to sustained constriction. Administration of MET for 2 weeks in drinking water (2 mg/ml) or subcutaneously (15 mg/kg per day) also resulted in NE-induced constriction of CAs in vivo. Thus, doses of MET that protect the heart from adrenergic stimulation also prevent β1AR-mediated dilation of CAs and favor anomalous adrenergic constriction. Our findings raise the possibility that the increased risk of ischemic stroke in patients on β-blockers relates in part to adrenergic dysregulation of cerebrovascular tone. SIGNIFICANCE STATEMENT: β-Blocker therapy using second-generation, cardioselective β-blockers is associated with an increased risk of stroke, but the responsible mechanisms are unclear. Here, we report that either acute or chronic systemic administration of a cardioselective β-blocker, metoprolol, mitigates adrenergic stimulation of the heart as an intended beneficial action. However, metoprolol concomitantly eliminates vasodilator responses to adrenergic stimuli of rat cerebral arteries in vivo as a potential cause of dysregulated cerebral blood flow predisposing to ischemic stroke.

Metabotropic glutamate 2,3 receptor stimulation desensitizes agonist activation of G-protein signaling and alters transcription regulators in mesocorticolimbic brain regions.

Metabotropic glutamate (mGlu) receptors are regulators of glutamate release and targets for development of therapies for hyperactive glutamatergic signaling. However, the effects of long-term stimulation of mGlu receptors on cellular signaling in the brain have not been described. This study investigated the effects of 2-day and 14-day osmotic mini-pump administration of the mGlu2,3 agonist LY379268 (3.0mgkg-1 day-1 ) to rats on receptor-mediated G-protein activation and signaling in mesocorticolimbic regions in rat brain sections. A significant reduction in LY379268-stimulated [35 S]GTPγS binding was observed in the 14-day group in some cortical regions, prefrontal cortex, nucleus accumbens, and ventral pallidum. The 14-day LY379268 treatment group exhibited mGlu2 mRNA levels significantly lower in hippocampus, nucleus accumbens, caudate, and ventral pallidum. In both 2-day and 14-day treatment groups immunodetectable phosphorylated cAMP Response Element-Binding protein (CREB) was significantly reduced across all brain regions. In the 2-day group, we observed significantly lower immunodetectable CREB protein across all brain regions, which was subsequently increased in the 14-day group but failed to achieve control values. Neither immunodetectable extracellular signal-regulated kinase (ERK) protein nor phosphorylated ERK from 2-day or 14-day treatment groups differed significantly from control across all brain regions. However, the ratio of phosphorylated ERK to total ERK protein was significantly greater in the 14-day treatment group compared with the control. These results identify compensatory changes to mGlu2,3 signal transduction in rat brains after chronic systemic administration of agonist, which could be predictive of the mechanism of action in human pharmacotherapies.

Genetic and pharmacological inactivation of astroglial connexin 43 differentially influences the acute response of antidepressant and anxiolytic drugs

Astroglial connexins (Cxs) 30 and 43 are engaged in gap junction and hemichannel activities. Evidence suggests that these functional entities contribute to regulating neurotransmission, thereby influencing brain functions. In particular, preclinical and clinical findings highlight a role of Cx43 in animal models of depression. However, the role of these proteins in response to currently available psychotropic drugs is still unknown. To investigate this, we evaluated the behavioural effects of the genetic and pharmacological inactivation of Cx43 on the antidepressant- and anxiolytic-like activities of the selective serotonin reuptake inhibitor fluoxetine and the benzodiazepine diazepam, respectively. A single administration of fluoxetine (18mg/kg; i.p.) produced a higher increase in hippocampal extracellular serotonin levels, and a greater antidepressant-like effect in the tail suspension test in Cx43 knock-down (KD) mice bred on a C57BL/6 background compared to their wild-type littermates. Similarly, in outbred Swiss wild-type mice, the intra-hippocampal injection of a shRNA-Cx43 or the acute systemic injection of the Cxs inhibitor carbenoxolone (CBX: 10mg/kg; i.p.) potentiated the antidepressant-like effects of fluoxetine. Evaluating the effects of such strategies on diazepam (0.5mg/kg; i.p.), the results indicate that Cx43 KD mice or wild-types injected with a shRNA-Cx43 in the amygdala, but not in the hippocampus, attenuated the anxiolytic-like effects of this benzodiazepine in the elevated plus maze. The chronic systemic administration of CBX mimicked the latter observations. Collectively, these data pave the way to the development of potentiating strategies in the field of psychiatry based on the modulation of astroglial Cx43.

Abstract P2011: Chronic Systemic Administration of Pro-Inflammatory Cytokines Causes Cardiovascular Dysfunction and Downregulates Brain Expression of Regulator of G-Protein Signaling-10 in Rats

Systemic pro-inflammatory cytokines (PICs) promote central oxidative stress and neuroinflammation that contribute to sympathetic excitation and cardiovascular dysfunction in hypertension and heart failure. However, the key molecular regulators of neuroinflammatory responses in the brain remain unclear. Regulator of G-protein Signaling-10 (RGS10), a GTPase-accelerating protein that is expressed predominantly in the brain, has recently emerged as an important counter regulator of PICs-induced downstream signaling in the central nervous system. RGS10 negatively regulates NF-kB-mediated inflammatory responses in microglia and antagonizes neurotoxic effects of peripheral inflammation. The present study examined whether peripherally administered PICs alter RGS10 expression in cardiovascular/autonomic brain regions like subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN). Male SD rats received a 2-week subcutaneous infusion of vehicle, interleukin (IL)-1β (400 ng/day) and tumor necrosis factor (TNF)-α (600 ng/day). Rats treated with IL-1β (n=6) or TNF-α (n=6) had significantly (*P&lt;0.05) reduced mRNA level (fold change) of RGS10 in SFO (0.60±0.15* or 0.69±0.16* vs 1.03±0.28; IL-1β or TNF-α, vs vehicle treatment) and PVN (0.72±0.15* or 0.71±0.19* vs 1.02±0.24), and lessoned mRNA level of NF-kB inhibitor IkB-α (SFO: 0.60±0.21* or 0.55±0.16* vs 1.07±0.46; PVN: 0.70±0.31* or 0.82±0.16* vs 1.00±0.07). Treatments with IL-1β or TNF-α increased mRNA level of NADPH oxidase 2 (SFO: 1.96±0.91* or 1.62±0.47* vs 1.03±0.28; PVN: 2.00±0.82* or 1.74±0.70* vs 1.00±0.15) and cyclooxygenase-2 (SFO: 1.78±0.76* or 1.98±1.04* vs 1.00±0.13; PVN: 1.68±0.34* or 1.62±0.57* vs 1.02±0.24). Rats treated with IL-1β or TNF-α had elevated mean blood pressure (98±11* or 100±9* vs 84±5 mmHg) and increased sympathetic activity as indicated by pressure response to ganglionic blockade (38±7 * or 36±6* vs 28±4 mmHg). Additionally, IL-1β or TNF-α-treated rats had increased cardiac fibrotic markers and impaired cardiac diastolic function. These results suggest that circulating PICs reduce RGS10 expression in the brain, which may contribute to sympathetic excitation and cardiovascular dysfunction in inflammatory conditions.

Role of angiotensin II in chronic blood pressure control of heterozygous Ren-2 transgenic rats: Peripheral vasoconstriction versus central sympathoexcitation

Our previous studies demonstrated that chronic systemic blockade of renin-angiotensin system (RAS) lowered blood pressure (BP) of Ren-2 transgenic rats (TGR) by the attenuation of both angiotensin II-dependent and sympathetic vasoconstriction. Since systemic RAS blockade also inhibits brain RAS, we were interested which effects on these two types of vasoconstriction will have the central RAS blockade in hypertensive TGR rats. Adult male heterozygous TGR rats and their Hannover Sprague Dawley (HanSD) controls were subjected to chronic systemic or intracerebroventricular administration of either angiotensin type 1 receptor blocker losartan or direct renin inhibitor aliskiren for 4 weeks. Additional groups of TGR and HanSD rats were used for the evaluation of acute peripheral and brain effects of angiotensin II. Both chronic systemic and intracerebroventricular administrations of losartan or aliskiren normalized BP of TGR animals. BP effect of brain RAS blockade was based solely on the reduced sympathetic vasoconstriction, while systemic RAS blockade attenuated both angiotensin II-dependent and sympathetic vasoconstriction. Surprisingly, neither peripheral nor central pressor effects of acute angiotensin II administration were enhanced in TGR compared to HanSD rats. In conclusion, sympathoinhibition represents the main mechanism of BP reduction in heterozygous TGR rats subjected to chronic brain or systemic RAS blockade, while peripheral attenuation of angiotensin II-dependent vasoconstriction during systemic RAS blockade is less important. Our data suggest that the participation of angiotensin II in BP control of adult heterozygous TGR rats is shifted from peripheral vasoconstriction to central sympathoexcitation. Similar mechanisms cannot be excluded in human essential hypertension.