Vehicle Rats Research Articles

#4394TACROLIMUS BUT NOT VOCLOSPORIN INHIBITS KIDNEY TUBULAR CALCIUM AND MAGNESIUM REABSORPTION IN RATS AT CLINICALLY THERAPEUTIC DOSES

Abstract Background and Aims Clinically, the calcineurin inhibitor tacrolimus frequently causes hypercalciuria and hypomagnesemia by inhibiting kidney tubular calcium and magnesium reabsorption. Voclosporin, a novel calcineurin inhibitor approved in the USA and Europe for the treatment of adults with active lupus nephritis, has fewer side-effects including less hypomagnesemia. However, the differences between the kidney tubular effects of tacrolimus and voclosporin are unknown. To address this, we compared the effects of tacrolimus, voclosporin, and vehicle in rats. Method Tacrolimus (0.5 mg/kg) and voclosporin (0.5 mg/kg) were administrated by daily intraperitoneal injections in male Wistar rats and were compared against vehicle (Cremophor EL: 95% ethanol:saline (5:5:90, v:v:v)) for 28 days (n = 8–9/group). Dosages were determined based on pharmacokinetic studies in rats and aimed to achieve the area under the concentration-time curve observed in clinical studies. At day 18, blood and 24 h urine were collected to measure trough levels of the drugs and to analyze the fractional excretions of calcium and magnesium. At sacrifice, kidneys were harvested for immunoblotting of tubular proteins. Results Both tacrolimus and voclosporin reached clinically therapeutic doses with trough levels of 2.4 μg/L ± 0.6 μg/L and 25.8 μg/L ± 9.6 μg/L, respectively. Compared to vehicle, tacrolimus caused significantly higher fractional excretions of calcium (+348% ± 127%, P < 0.001) and magnesium (+60% ± 38%, P < 0.01) and also caused hypomagnesemia (plasma magnesium 0.65 mmol/L ± 0.04 mmol/L vs. 0.81 mmol/L ± 0.02 mmol/L, P < 0.001). Compared to vehicle, voclosporin only caused a slight, but non-statistically significant, increase in fractional calcium excretion (+ 44% ± 38%, P = 0.08) and did not cause higher fractional magnesium excretion (+5% ± 30%, P = 0.96) or hypomagnesemia (plasma magnesium 0.8 mmol/L ± 0.04 mmol/L, P = 0.9). Compared to vehicle, tacrolimus caused an 11-fold decrease in the protein abundance of the cytosolic calcium-binding protein calbindin-D28K and a 2-fold decrease in the abundance of the sodium-chloride cotransporter (NCC). In contrast, voclosporin did not decrease the protein abundances of calbindin-D28K and NCC. No differences were observed between vehicle, tacrolimus, and voclosporin in the calcium channel TRPV5 and magnesium channel TRPM6 (Figure 1). Conclusion In contrast to the calcineurin inhibitor tacrolimus, voclosporin does not inhibit the kidney tubular reabsorption of calcium and magnesium and therefore does not cause hypercalciuria or hypomagnesemia. A possible explanation for this difference is that tacrolimus but not voclosporin affects tubular transport in the distal convoluted tubule, which was further supported by the selective inhibition of calbindin-D28K and NCC by tacrolimus. Our data show that tubulotoxicity of tacrolimus is not apparent with voclosporin treatment, at clinically relevant doses.

Chronic Semaglutide Treatment in Rats Leads to Daily Excessive Concentration-Dependent Sucrose Intake.

The glucagon-like peptide-1 receptor (GLP-1R) agonist semaglutide (SEMA) produces 15% weight loss when chronically administered to humans with obesity. In 2 separate experiments, rats received daily injections of either vehicle (VEH) or SEMA starting at 7 µg/kg body weight (BW) and increasing over 10 days to the maintenance dose (70 µg/kg-BW), emulating clinical dose escalation strategies. During dose escalation and maintenance, SEMA rats reduced chow intake and bodyweight. Experiment 2 meal pattern analysis revealed that meal size, not number, mediated these SEMA-induced changes in chow intake. This suggests SEMA affects neural processes controlling meal termination and not meal initiation. Two-bottle preference tests (vs water) began after 10 to 16 days of maintenance dosing. Rats received either an ascending sucrose concentration series (0.03-1.0 M) and 1 fat solution (Experiment 1) or a 4% and 24% sucrose solution in a crossover design (Experiment 2). At lower sucrose concentrations, SEMA-treated rats in both experiments drank sometimes >2× the volume consumed by VEH controls; at higher sucrose concentrations (and 10% fat), intake was similar between treatment groups. Energy intake of SEMA rats became similar to VEH rats. This was unexpected because GLP-1R agonism is thought to decrease the reward and/or increase the satiating potency of palatable foods. Despite sucrose-driven increases in both groups, a significant bodyweight difference between SEMA- and VEH-treated rats remained. The basis of the SEMA-induced overconsumption of sucrose at lower concentrations relative to VEH controls remains unclear, but the effects of chronic SEMA treatment on energy intake and BW appear to depend on the caloric sources available.

Inhibition of Microbiota-dependent Trimethylamine N-Oxide Production Ameliorates High Salt Diet-Induced Sympathetic Excitation and Hypertension in Rats by Attenuating Central Neuroinflammation and Oxidative Stress.

Excessive dietary salt intake induces neuroinflammation and oxidative stress in the brain, which lead to sympathetic excitation, contributing to hypertension. However, the underlying mechanisms remain elusive. Accumulating evidence reveals that trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite, is implicated in the pathogenesis of multiple cardiovascular diseases. The present study sought to determine whether central TMAO is elevated and associated with neuroinflammation and oxidative stress in the brain after long-term high salt (HS) diet intake and, if so, whether inhibition of TMAO generation ameliorates HS-induced sympathetic excitation and hypertension. Sprague–Dawley rats were fed either a HS diet or a normal salt (NS) diet and simultaneously treated with vehicle (VEH) or 1.0% 3,3-Dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) for 8 weeks. HS + VEH rats, compared with NS + VEH rats, had elevated TMAO in plasma and cerebrospinal fluid (CSF), increased blood pressure (BP), and increased sympathetic drive as indicated by the BP response to ganglionic blockade and plasma norepinephrine levels. HS-induced these changes were attenuated by DMB, which significantly reduced TMAO in plasma and CSF. Neuroinflammation as assessed by proinflammatory cytokine expression and NF-κB activity and microglial activity, and oxidative stress as measured by NAD(P)H oxidase subunit expression and NAD(P)H activity and reactive oxygen species (ROS) production in the hypothalamic paraventricular nucleus (PVN) were increased in HS + VEH rats but were decreased by DMB. DMB had no effects on above measured parameters in NS rats. The results suggest that long-term HS diet intake causes elevation in TMAO in the circulation and brain, which is associated with increased neuroinflammation and oxidative stress in the PVN, an important cardiovascular regulatory center. Inhibition of TMAO generation ameliorates HS-induced sympathetic excitation and hypertension by reducing neuroinflammation and oxidative stress in the PVN.

Intranasally Administered L-Myc-Immortalized Human Neural Stem Cells Migrate to Primary and Distal Sites of Damage after Cortical Impact and Enhance Spatial Learning.

As the success of stem cell-based therapies is contingent on efficient cell delivery to damaged areas, neural stem cells (NSCs) have promising therapeutic potential because they inherently migrate to sites of central nervous system (CNS) damage. To explore the possibility of NSC-based therapy after traumatic brain injury (TBI), isoflurane-anesthetized adult male rats received a controlled cortical impact (CCI) of moderate severity (2.8 mm deformation at 4 m/s) or sham injury (i.e., no cortical impact). Beginning 1-week post-injury, the rats were immunosuppressed and 1 × 106 human NSCs (LM-NS008.GFP.fLuc) or vehicle (VEH) (2% human serum albumen) were administered intranasally (IN) on post-operative days 7, 9, 11, 13, 15, and 17. To evaluate the spatial distributions of the LM-NSC008 cells, half of the rats were euthanized on day 25, one day after completion of the cognitive task, and the other half were euthanized on day 46. 1 mm thick brain sections were optically cleared (CLARITY), and volumes were imaged by confocal microscopy. In addition, LM-NSC008 cell migration to the TBI site by immunohistochemistry for human-specific Nestin was observed at day 39. Acquisition of spatial learning was assessed in a well-established Morris water maze task on six successive days beginning on post-injury day 18. IN administration of LM-NSC008 cells after TBI (TBI + NSC) significantly facilitated spatial learning relative to TBI + VEH rats (p < 0.05) and had no effect on sham + NSC rats. Overall, these data indicate that IN-administered LM-NSC008 cells migrate to sites of TBI damage and that their presence correlates with cognitive improvement. Future studies will expand on these preliminary findings by evaluating other LM-NSC008 cell dosing paradigms and evaluating mechanisms by which LM-NSC008 cells contribute to cognitive recovery.

Preventing or controlling periodontitis reduces the occurrence of osteonecrosis of the jaw (ONJ) in rice rats (Oryzomys palustris).

Osteonecrosis of the jaw (ONJ) is an adverse event that requires association of both systemic risk factors, such as powerful anti-resorptives (pARs; e.g. zoledronic acid [ZOL]), and local oral risk factors (e.g. tooth extraction, periodontitis). Whereas optimal oral health prior to initiate pARs is recognized as critically important for minimizing ONJ risk, the efficacy of preventive/maintenance measures in patients who are taking pARs is understudied. Rice rats fed a standard diet (STD), rich in insoluble fiber, develop localized periodontitis. STD-rats with localized periodontitis treated with ZOL for 18-24 wk develop ONJ. Hence, we hypothesized that controlling/preventing localized periodontitis in the ZOL-treated rats, reduces ONJ occurrence. We used two approaches to attempt reducing periodontitis prevalence: 1) periodontal cleaning (PC); and 2) replacing the STD-diet with a nutritionally-equivalent diet high in soluble fiber (SF). 75 four-week-old male rats were weight-randomized into five groups (n=15) in a 24-week experiment. Three groups ate the STD-diet and two the high SF-diet. STD-diet groups received intravenous (IV) vehicle (VEH) q4wks (STD+VEH), 80μg/kg ZOL q4wks IV (STD+ZOL), or ZOL plus PC q2wks (STD+ZOL+PC). The SF-diet groups received VEH (SF+VEH) or ZOL (SF+ZOL). Jaws were processed for histopathology and evaluated for ONJ prevalence and tissue-level periodontitis. 1) 40% of STD+VEH rats developed maxillary localized periodontitis with no ONJ; 2) 50% of STD+ZOL rats developed ONJ; 3) 7% of STD+ZOL+PC rats developed ONJ (p<0.01 vs. STD+ZOL); and 4) one SF+ZOL rat developed localized periodontitis, and no SF+VEH or SF+ZOL rats developed ONJ (p<0.001 vs. STD+ZOL). 1) Periodontal cleaning in ZOL-treated rats decreases localized periodontitis severity and reduces ONJ prevalence; and 2) feeding a SF-diet to ZOL-treated rats reduces both incidence of localized periodontitis and ONJ. Our data indicates strong oral microbial community shifts according to oral health condition and trends in the shifts associated with diet.

Activated Microglia in the Rat Spinal Cord Following Peripheral Axon Injury Promote Glial and Neuronal Plasticity Which is Necessary for Long-Term Neuronal Survival.

Following the transection of peripheral sympathetic preganglionic axons comprising the cervical sympathetic trunk (CST), we observe robust glial and neuronal plasticity at 1week post-injury in the rat spinal cord intermediolateral cell column (IML), which houses the injured parent neuronal cell bodies. This plasticity contributes to neuroprotection, as no neuronal loss in the IML is present at 16weeks post-injury. Here, we administered the antibiotic minocycline or vehicle (VEH) daily for 1week after CST transection to investigate the role of activated microglia in IML glial and neuronal plasticity and subsequent neuronal survival. At 1week post-injury, minocycline treatment did not alter microglia number in the IML, but led to a dampened microglia activation state. In addition, the increases in oligodendrocyte (OL) lineage cells and activated astrocytes following injury in VEH rats were attenuated in the minocycline-treated rats. Further, the normal downregulation of choline acetyltransferase (ChAT) in the injured neurons was blunted. At 16weeks post-injury, fewer ChAT+ neurons were present in the minocycline-treated rats, suggesting that activated microglia together with the glial and neuronal plasticity at 1week post-injury contribute to the long-term survival of the injured neurons. These results provide evidence for beneficial crosstalk between activated microglia and neurons as well as other glial cells in the cord following peripheral axon injury, which ultimately leads to neuroprotection. The influences of microglia activation in promoting neuronal survival should be considered when developing therapies to administer minocycline for the treatment of neurological pathologies.

Different pharmaceutical preparations of Δ9 -tetrahydrocannabinol differentially affect its behavioral effects in rats.

Based on the contribution of the endocannabinoid system to the pathophysiology of schizophrenia, the primary pro-psychotic ingredient of Cannabis sativa, Δ-9-tetrahydrocannabinol (Δ-9-THC), is used in preclinical as well as clinical research to mimic schizophrenia-like symptoms. While it is common to administer lipid-based formulations of Δ-9-THC in human studies orally, intraperitoneal injections of water-based solutions are used in animal models. Because of the poor water solubility of Δ-9-THC, solubilizers such as ethanol and/or emulsifiers are needed for these preparations. In order to test whether a lipid-based solvent would be superior over a water-based vehicle in rats, we compared the effects on locomotor activity and prepulse inhibition (PPI) of the acoustic startle reaction, as well as pharmacokinetic data obtained from rats' serum and brain tissue samples. Up to 50mg/kg Δ-9-THC in the lipid-based formulation was not able to induce any behavioral alterations, while already 5mg/kg of the water-based Δ-9-THC preparation significantly reduced locomotor activity. This also induced a small but significant PPI reduction, which was prepulse intensity dependent. Interestingly, the reflexive motor response to the startle stimulus was not affected by the water-based Δ-9-THC solution. Analysis of serum and brain Δ-9-THC levels by high-performance liquid chromatography/mass spectrometry revealed that although the final concentration reached in the brain was comparable for both pharmaceutical preparations, the water-based formulation achieved a faster kinetic. We, therefore, conclude that the slope of the Δ-9-THC concentration-time curve and the resulting cannabinoid receptor type 1 activation per time unit are responsible for the induction of behavioral alterations.

Pioglitazone prevents sevoflurane‑induced neuroinflammation and cognitive decline in a rat model of chronic intermittent hypoxia by upregulating hippocampal PPAR‑γ

Post‑operative cognitive dysfunction is a common complication after anesthesia and surgery. Sevoflurane(SEV), a widely used inhalational anesthetic, can exaggerate neuroinflammation and cause cognitive dysfunction under chronic intermittent hypoxia(CIH) conditions by downregulating hippocampal peroxisome proliferator‑activated receptor‑γ(PPAR‑γ). In the present study, it was examined whether treatment with PPAR‑γ agonist pioglitazone(PIO) is beneficial in counteracting SEV‑induced neuroinflammation and cognitive decline in a rat model of CIH. Rats were exposed to CIH for 4weeks. After 2weeks of CIH, these animals underwent either 2.6%SEV or control(CON) exposure for 4h. PIO (60mg/kg) or vehicle (VEH) was administered orally twice daily for 2weeks, starting one day prior to SEV or CON exposure. Compared with CIH‑CON+VEH rats, CIH‑SEV+VEH rats exhibited significant cognitive decline as indicated by increased latency to locate the hidden platform and shorter dwell‑time in the goal quadrant in the Morris Water Maze task. Molecular studies revealed that CIH‑SEV+VEH rats had increased proinflammatory cytokine expression and microglial activation in the hippocampus, which were associated with decreased PPAR‑γ activity. Notably, SEV‑induced cognitive decline and increases in proinflammatory cytokine expression and microglial activation were prevented by PIO, which increased hippocampal PPAR‑γ activity. PIO also increased hippocampal PPAR‑γ activity in CIH‑CON rats but did not alter proinflammatory cytokine expression and microglial activation as well as cognitive function. Additionally, expression of hippocampal PPAR‑α and PPAR‑β, two other PPAR isotypes, were comparable among the groups. These data suggest that PIO prevents SEV‑induced exaggeration of neuroinflammation and cognitive decline under CIH conditions by upregulating hippocampal PPAR‑γ. PIO may have the potential to prevent anesthetic SEV‑induced cognitive decline in surgical patients with obstructive sleep apnea.

TPX-100 leads to marked, sustained improvements in subjects with knee osteoarthritis: pre-clinical rationale and results of a controlled clinical trial

Purpose: TPX-100 is a 23-amino acid peptide derived from Matrix Extracellular Phosphoglycoprotein (MEPE), a Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING) family protein. TPX-100 has been shown to induce articular cartilage proliferation in goats (N = 8/ dose group) after a standardized full-thickness chondral defect and treatment with 4 weekly intra-articular (IA) injections of TPX-100 vs. vehicle (25, 125 or 250 mg/injection). After 6 months, histopathological staining in TPX-100-treated joints indicated robust articular (hyaline) cartilage formation. Additionally, IA TPX-100 reduced joint damage and improved osteoarthritis scores vs. vehicle in rats after standardized ACL transection and partial medial meniscectomy. This Phase 2 study evaluated safety, tolerability and preliminary efficacy of TPX-100 by IA administration in subjects with bilateral patellofemoral osteoarthritis (PFOA). Each subject served as his/her own control, intended to minimize effects of age, sex, genetic factors, and activity levels on outcome measures. Methods: Subjects with ICRS grade 2-3 PFOA, confirmed by centrally-read screening MRI, were enrolled at 15 sites. One knee was randomly assigned to receive 4 weekly injections of TPX-100, while the contralateral knee received placebo injections. Investigator, subject, site and sponsor were blinded to treatment assignment. In Part A of the study, 4 dose cohorts (n = 6–9 subjects/cohort) received 20, 50, 100 or 200 mg/injection. Safety/tolerability of each cohort was evaluated by a Safety Review Committee (SRC). In Part B, all subjects received 200 mg/injection. Quantitative MRIs, obtained at baseline, 6 and 12 months, were read centrally. Validated patient-reported outcomes (PROs) included the Knee Osteoarthritis Outcome Score (KOOS), appropriate for younger, more athletic subjects, and the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC). Results: All subjects who received 4 weekly 200 mg injections, with at least one follow-up MRI, were analyzed (n = 93 subjects, 186 knees). The study population was typical of the knee OA population in the U.S. (median age, 58.4 years; BMI 30.3), and 68 of 93 subjects (73%) also had ICRS grade 2 – 4 mild to severe bilateral tibio-femoral osteoarthritis (TFOA). Safety: There were no drug-related SAEs and no dose-limiting toxicities across doses. Common adverse events such as knee pain had virtually identical incidences in control and TPX-100-treated knees. Efficacy: Only 14% of knees changed in cartilage thickness/volume over the 12 months of the study. Quantitative MRI revealed no measurable between-knee structural differences in this small sample. However, statistically significant (P<0.05) and clinically meaningful differences in KOOS and WOMAC scores were demonstrated compared with placebo-exposed knees at 6 or 12 months or both, including activities of daily living, sports activities, and knee-related quality of life. Comparing TPX-100 treated knees vs. control knees, at 12 months; there was a significant difference in pain ascending and descending stairs, a chief complaint in PFOA. Overall, use of analgesics, including non-steroidal anti-inflammatory medications, declined markedly (62.5%) during the study. Conclusions: TPX-100, administered in 4 weekly intra-articular injections (200 mg/injection), is safe, well tolerated, and associated with statistically significant and clinically meaningful functional benefits sustained to at least 12 months. Improvement in knee functional status with reduction in disease burden represents a key therapeutic goal in OA therapeutic development. The small sample size of knees with MRI changes in cartilage thickness/volume limited the power of this study to detect differences between knees in these measures. However, pre-clinical large and small animal data indicate the structure-modifying potential of TPX-100, which may be demonstrated with a longer study involving a larger sample size.

Role of capsaicin-sensitive peripheral sensory neurons in anorexic responses to intravenous infusions of cholecystokinin, peptide YY-(3-36), and glucagon-like peptide-1 in rats.

Cholecystokinin (CCK)-induced suppression of feeding is mediated by vagal sensory neurons that are destroyed by the neurotoxin capsaicin (CAP). Here we determined whether CAP-sensitive neurons mediate anorexic responses to intravenous infusions of gut hormones peptide YY-(3-36) [PYY-(3-36)] and glucagon-like peptide-1 (GLP-1). Rats received three intraperitoneal injections of CAP or vehicle (VEH) in 24 h. After recovery, non-food-deprived rats received at dark onset a 3-h intravenous infusion of CCK-8 (5, 17 pmol·kg⁻¹·min⁻¹), PYY-(3-36) (5, 17, 50 pmol·kg⁻¹·min⁻¹), or GLP-1 (17, 50 pmol·kg⁻¹·min⁻¹). CCK-8 was much less effective in reducing food intake in CAP vs. VEH rats. CCK-8 at 5 and 17 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 39 and 71% in VEH rats and 7 and 18% in CAP rats. In contrast, PYY-(3-36) and GLP-1 were similarly effective in reducing food intake in VEH and CAP rats. PYY-(3-36) at 5, 17, and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 15, 33, and 70% in VEH rats and 13, 30, and 33% in CAP rats. GLP-1 at 17 and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 48 and 60% in VEH rats and 30 and 52% in CAP rats. These results suggest that anorexic responses to PYY-(3-36) and GLP-1 are not primarily mediated by the CAP-sensitive peripheral sensory neurons (presumably vagal) that mediate CCK-8-induced anorexia.

Nociceptor-selective Peripheral Nerve Block Induces Delayed Mechanical Hypersensitivity and Neurotoxicity in Rats

Long-lasting, sensory-specific peripheral nerve blockade would advance perioperative analgesia. Perineural injection of a combination of transient receptor potential vanilloid 1 channel agonists and lidocaine or its hydrophilic derivative, QX-314, produces prolonged sensory or nociceptor-selective nerve block in rodents. In this study, the authors tested the efficacy of these combinations in peripheral nerve block after incisional surgery in rats. The authors administered perisciatic lidocaine (2%), QX-314 (0.2%) followed by dilute capsaicin (0.05%, 10 min later), or vehicle in rats and the duration of motor and sensory block to thermal and mechanical stimuli assessed in normal animals and those after incisional surgery to the hind paw. Other animals receiving these injections were evaluated 7 weeks later by behavior and histology for potential neurotoxicity. Perineural injection of the combination not only attenuated mechanical hypersensitivity for 72 h after incision but also resulted in delayed onset mechanical hypersensitivity several weeks later, accompanied by degeneration of central terminals of isolectin B4 (nonpeptidergic) and calcitonin gene-related peptide-containing (peptidergic) afferents in the ipsilateral spinal cord. Dorsal root ganglia ipsilateral to injection of the combination showed increased expression of activating transcription factor-3 and satellite cell activation. Combined administration of local anesthetics with the transient receptor potential vanilloid 1 agonist capsaicin induced a near complete blockade of incision-induced hypersensitivity for several days. However, the same combination induced delayed mechanical hypersensitivity and neurotoxicity in naïve rats. Combination of these drugs in these concentrations is likely to result in neurotoxicity, and the safety of other concentrations warrants further study.

The hypocretin/orexin antagonist almorexant promotes sleep without impairment of performance in rats

The hypocretin receptor (HcrtR) antagonist almorexant (ALM) has potent hypnotic actions but little is known about neurocognitive performance in the presence of ALM. HcrtR antagonists are hypothesized to induce sleep by disfacilitation of wake-promoting systems whereas GABAA receptor modulators such as zolpidem (ZOL) induce sleep through general inhibition of neural activity. To test the hypothesis that less functional impairment results from HcrtR antagonist-induced sleep, we evaluated the performance of rats in the Morris Water Maze in the presence of ALM vs. ZOL. Performance in spatial reference memory (SRM) and spatial working memory (SWM) tasks were assessed during the dark period after equipotent sleep-promoting doses (100 mg/kg, po) following undisturbed and sleep deprivation (SD) conditions. ALM-treated rats were indistinguishable from vehicle (VEH)-treated rats for all SRM performance measures (distance traveled, latency to enter, time within, and number of entries into, the target quadrant) after both the undisturbed and 6 h SD conditions. In contrast, rats administered ZOL showed impairments in all parameters measured compared to VEH or ALM in the undisturbed conditions. Following SD, ZOL-treated rats also showed impairments in all measures. ALM-treated rats were similar to VEH-treated rats for all SWM measures (velocity, time to locate the platform and success rate at finding the platform within 60 s) after both the undisturbed and SD conditions. In contrast, ZOL-treated rats showed impairments in velocity and in the time to locate the platform. Importantly, ZOL rats only completed the task 23–50% of the time while ALM and VEH rats completed the task 79–100% of the time. Thus, following equipotent sleep-promoting doses, ZOL impaired rats in both memory tasks while ALM rats performed at levels comparable to VEH rats. These results are consistent with the hypothesis that less impairment results from HcrtR antagonism than from GABAA-induced inhibition.

A cornerstone of heart failure treatment is not effective in experimental right ventricular failure

BackgroundRight ventricular (RV) failure due to increased pressure load causes significant morbidity and mortality in patients with congenital heart diseases and pulmonary arterial hypertension. It is unknown whether renin–angiotensin–aldosterone-system (RAAS) inhibition (the cornerstone of left ventricular failure treatment) is effective in RV failure. We investigated the effects of combination treatment of aldosterone-blocker eplerenone+angiotensin II receptor blocker losartan (Ep/Lo) on RV remodeling and function in a model of RV failure due to increased pressure load. Methods and resultsRats (n=48) were randomized for pulmonary artery banding (PAB) or sham surgery and for losartan (20mg/kg/d)+eplerenone (100mg/kg/d) treatment (Ep/Lo) or vehicle (VEH). RV function was assessed by echocardiography and pressure–volume analysis at 5 and 11weeks, or at the occurrence of clinical RV failure symptoms necessitating termination.PAB resulted in RV failure in all rats, as defined by reduced cardiac output, RV stroke volume, increased RV end diastolic pressure and liver congestion as well as RV fibrosis, hypertrophy and reduced capillary density. Clinical RV failure necessitated termination in 5/12 PAB–VEH rats. Angiotensin II type 1-receptor expression in the RV was reduced in PAB rats indicating local RAAS activation. Treatment of PAB rats with Ep/Lo significantly lowered arterial pressures, but had no significant effect on RV function, remodeling or survival compared to PAB–VEH rats. ConclusionsRAAS inhibition does not beneficially affect experimental RV failure due to chronic pressure load. This is of high clinical relevance, because it indicates that the RV response to RAAS inhibition might fundamentally differ from that of the LV.

Nucleus accumbens GLP-1 receptors influence meal size and palatability.

Recent evidence suggests that the glucagon-like peptide-1 (GLP-1) neuronal projection to the nucleus accumbens core (NAcC) contributes to food intake control. To investigate the role of endogenous stimulation of GLP-1 receptors (GLP-1R) in NAcC, we examined the effects of the GLP-1R antagonist exendin-(9-39) (Ex9) on meal pattern and microstructure of ingestive behavior in rats. Intra-NAcC Ex9 treatment selectively increased meal size relative to vehicle in rats consuming 0.25 M sucrose solution or sweetened condensed milk. Microstructural analysis revealed effects of NAcC Ex9 on initial lick rate and the size and duration of licking bursts in rats consuming 0.1 or 0.25 M sucrose, suggesting that blockade of NAcC GLP-1R increases palatability. Because NAcC Ex9 did not affect licking for nonnutritive saccharin (0.1%), we suggest that the presence of nutrients in the gut may be required for endogenous stimulation of NAcC GLP-1R. Consistent with this, we also found that the meal size-suppressive effects of intragastric nutrient infusion were attenuated by NAcC delivery of Ex9 at a dose that had no effect when delivered alone. Analysis of licking patterns revealed that NAcC Ex9 did not reverse intragastric nutrient-induced suppression of burst number but rather blunted the effect of nutrient infusion on meal size primarily by increasing the size and duration of licking bursts. Together, our results suggest that NAcC Ex9 influences taste evaluation. We conclude that GLP-1 released in NAcC in response to gastrointestinal nutrients reduces the hedonic value of food.

Neuroprotective properties of the non-peptidyl radical scavenger IAC in rats following transient focal cerebral ischemia

Experimental evidence suggests that reactive free radicals are generated during brain ischemia. We investigated the effect of a novel brain penetrant, low molecular weight, non-peptidyl carbon, oxygen- and nitrogen-centered radical scavenger, IAC, on infarct volume and sensory-motor performance in a rat transient middle cerebral artery occlusion model (tMCAO). Rats received 90 min tMCAO and treated with i.p. or i.v. injections of vehicle or IAC following tMCAO. Sensory-motor performance was evaluated by neuroscore tests (NS). Cerebral infarct volume was evaluated at 72 h after tMCAO. Rats treated with IAC i.p. (1 or 6 h after the onset of tMCAO) or i.v. (1 h after the onset of tMCAO) showed significant improvement in NS during the 3 or 21 day follow-up period when compared to vehicle treated rats. Cerebral infarct volumes were significantly decreased compared to vehicle in rats receiving IAC i.p. 1 h or 6 h after occlusion, ∼30.5% decrease compared to vehicle, or i.v. 1 h after the onset of tMCAO, 48.6% decrease compared to vehicle. These results demonstrate that IAC has neuroprotective properties with a wide therapeutic window following tMCAO in rats. IAC could therefore be a candidate for the treatment of stroke.

Histaminergic modulation of acoustically induced running behavior in rats

Explosive running is a reliable initial component of audiogenic seizures (AS) induced by acoustic stimulation in genetically prone rodents. This profound locomotor activation is usually considered as a convulsive manifestation of AS although some studies attribute running to a panic-like response. Increase in central histamine activity has been shown to suppress clonic and tonic seizures. The present study examined the involvement of histaminergic mechanisms in the expression of running component of AS. Metoprine, an inhibitor of histamine- N-methyltransferase, was used to increase brain histamine level. Running was induced 4 and 24 h after intraperitoneal injection of metoprine or vehicle in rats of different strains. A brief sound stimulation elicited running followed by clonic–tonic convulsions in Krushinsky–Molodkina (KM) rats or running alone in AS-prone Wistar and WAG/Rij rats. In KM rats, metoprine exerted opposite effects on the main phases of AS. It increased the duration of running and decreased the duration and severity of clonic–tonic convulsions. In Wistar rats, metoprine produced a remarkable aggravation of running leading to its 2- to 3-fold prolongation. In WAG/Rij rats with mixed seizures (absence and audiogenic), the drug caused either aggravation or suppression of running behavior. These results suggest specific role for histaminergic system in the expression of behavioral components of AS. Suppressive role of histamine in clonic–tonic seizures is associated with facilitation of running suggesting specific effects of histamine on brainstem neuronal networks underlying these phases of AS. Possible roles of histaminergic mechanisms in seizure, motor and aversive aspects of sound-induced running are discussed.

Neonatal capsaicin causes compensatory adjustments to energy homeostasis in rats

Several mechanisms involved in ingestive behavior and neuroendocrine activity rely on vagal afferent neuronal signaling. Seemingly contradictory to this idea are observations that vagal afferent neuronal ablation by neonatal capsaicin (CAP) treatment has relatively small effects on glucose homeostasis and long-term regulation of energy balance. It may be proposed that humoral endocrine factors and/or their sensitivities compensate for the loss of vagal afferent information, particularly when subjects face disturbances in ambient fuel levels. Therefore, male adult rats neonatally treated with CAP or with the vehicle (VEH) underwent intravenous glucose tolerance tests (IVGTTs) during which blood fuel levels, and circulating adipose, pancreatic, and adrenal hormones were assessed. CAP rats displayed similar hyperglycemia as VEH rats, but with markedly reduced plasma insulin and corticosterone responses. These results indicate that CAP rats have increased insulin sensitivity during hyperglycemic episodes, and lower plasma levels of corticosterone in CAP rats relative to VEH rats could underlie this effect. After the IVGTT, CAP rats had increased plasma adiponectin and reduced plasma resistin levels, and these alterations in adipose hormones might be relevant for post-ingestive metabolic processes. In a second experiment, anorexigenic efficacies of cholecystokinin and leptin were assessed. While VEH rats, but not CAP rats, responded with reduced food intake to i.p. injected cholecystokinin, only CAP rats responded to i.v. infused leptin with a reduction in food intake. It is concluded that reduced HPA axis activity and/or increased leptin signaling could underlie compensations in fuel handling and energy balance following CAP treatment.

Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction

The pleiotropic actions of statins have been largely explored. These drugs have been tested in several models of progressive renal disease, most of them accompanied by hypertension. We sought to investigate more closely the effects of simvastatin on renal interstitial fibrosis due to unilateral ureteral obstruction (UUO). Munich-Wistar rats were submitted to UUO and studied after 14 days. Animals were divided into two groups: vehicle (VH) or simvastatin (SIMV) 2 mg/kg b.i.d. by gavage. At sacrifice kidneys were harvested for morphology, mRNA and protein analysis. RT-PCR was done to assess expression of collagen I and III, fibronectin, MCP-1, TGF-beta1 and bFGF. Protein expression was assessed by western blot (TGF-beta) and immunostaining (macrophage, lymphocyte, PCNA, vimentin and alpha-smooth muscle actin). Contralateral kidneys (CL) were used as controls. SIMV-treated animals had less severe renal inflammation. MCP-1 was markedly expressed in obstructed kidneys and diminished with SIMV (48.9+/- 2.5 vs 64.3+/-3.1 OD in VH, P<0.01). Interstitial fibrosis (IF) was significantly attenuated with SIMV (8.2+/-1.3 vs 13.2+/-0.6%, P<0.01 SIMV vs VH), which was confirmed by a decrease in collagen I and fibronectin renal expression. Vimentin, a marker of dedifferentiation, was expressed in tubular cells of VH and decreased with SIMV treatment. alpha-SMA, a marker of myofibroblast-type cells, was increased in renal interstitium of VH rats and SIMV significantly reduced its expression. PCNA was increased in the UUO kidneys, but SIMV did not decrease tubular or interstitial proliferating cells. TGF-beta1, which was highly induced in the obstructed kidneys, decreased at the post-transcriptional level with SIMV treatment (5.35+/-0.75 vs 13.10+/-2.9 OD in VH, P<0.05). bFGF mRNA was also overexpressed in the obstructed kidneys, although SIMV treatment did not significantly decrease its expression. SIMV had an evident protective effect on renal interstitial inflammation and fibrosis. It is conceivable that by attenuating inflammation, SIMV prevented tubular activation and transdifferentiation, two processes largely involved in the renal fibrosis of the UUO model.

Deafferentation affects short-term but not long-term control of food intake

Deafferentation affects short-term but not long-term control of food intake (PHYSIOL BEHAV XX(X) 000-000, 2005). Rats were treated neonatally with capsaicin (CAP) to investigate the involvement of vagal afferents in food intake control and body weight regulation. In the first set of experiments, rats were offered increasing concentrations of sucrose (10–15–20–40%) in short-term feeding tests of 1 h. At the end, 10% was offered again to see whether CAP rats modified their intake after repeated exposure to different concentrations of sucrose solution. Results demonstrated that CAP animals overconsume persistently compared to vehicle (VEH) controls. This overconsumption is most pronounced and variable at 10% trials. Hypertonic 40% sucrose solution resulted in a small but significant drop in intake in CAP rats. Overall, if the concentration of sucrose solution is more than 10%, sucrose ingestion of CAP and VEH rats does not depend on the concentration of sucrose solution and remains relatively constant during all trials. In another experiment, rats were exposed to a high-fat condensed milk suspension (CMS) for 5 days. CAP rats initially overconsumed from this CMS compared to VEH. This was accompanied by a decreased intake in chow. However, over the 5 day period CAP animals adjusted their CMS and chow intake to control levels. During both experiments there were no differences in body weight gain between CAP and VEH. Together, these results suggest that capsaicin-sensitive vagal C-fibers are involved in the control of volume ingestion and short-term food intake control but are not required for long-term control of energy intake.

Stimulating effect of glucocorticosteroids on intestinal fructose transport in rats is increased by feeding a saturated fatty acid diet.

Glucocorticosteroids enhance absorptive functions of the intestine. This study was undertaken to assess the influence of budesonide (BUD), prednisone (PRED), or control vehicle in rats fed either a saturated fatty acid diet (SFA) or a polyunsaturated fatty acid diet (PUFA), on the uptake of sugars. Steroids increased the uptake of fructose, and these effects were greater with SFA than PUFA. No effect on the abundance or the expression of the mRNAs of SGLT1, GLUT5, or GLUT2 was observed, and yet immunohistochemistry staining in the jejunum for SGLT1 and GLUT5 was greater in SFA than in PUFA. The early response genes and proglucagon expression was enhanced in the ileum of animals fed SFA and given control vehicle. Steroids increased the ileal proglucagon mRNA. In summary, (1) PRED and BUD enhance the uptake of fructose, (2) this enhancement may be increased further by feeding SFA, and (3) signaling may involve early response genes and proglucagon.