Implantation Of Osmotic Pump Research Articles

Increased incidence of weight-loss-associated humane endpoints in rats administered buprenorphine slow-release LAB formulation following traumatic brain injury: a retrospective study.

Traumatic brain injury (TBI) remains a significant global public health epidemic with adverse health and cost implications. Due to its complex, heterogeneous nature and wide-ranging impacts, definitive TBI treatments remain elusive. As such, continued laboratory research using animal models is warranted. In accordance with guidelines set forth for the humane treatment of research animals, TBI animal models are often administered analgesics for pain management. The choice of drug, timing, dose, and formulation of analgesic can vary depending on the study's unique needs and can potentially and unintentionally influence experimental results. In TBI studies utilizing rats as animal models, buprenorphine is a common analgesic administered. In addition to pain management in such studies, investigators must also monitor the research animals post-operatively and make the decision for humane euthanasia before intended experimental survival timepoint if the animals are assessed to be excessively suffering. This study investigated the differences in adult, male Sprague Dawley rats used for various TBI studies that reached weight-loss-induced humane endpoints following a single administration of buprenorphine slow-release LAB (bup-SR-LAB) or buprenorphine slow-release HCl (bup-SR-HCl). Our findings indicate that TBI-induced rats receiving bup-SR-LAB in conjunction with a secondary surgical insult such as artificial intracranial pressure elevation and/or osmotic pump implantation reach a weight-loss-induced humane euthanasia endpoint more often compared to sham-injured rats. When stratifying into the same groups, we did not find this pattern to hold true for rats administered bup-SR-HCl. Overall, this study contributes to the limited body of literature addressing different analgesic formulations' effects on laboratory animals.

Acyl-CoA binding protein for the experimental treatment of anorexia.

Extracellular acyl-coenzyme A binding protein [ACBP encoded by diazepam binding inhibitor (DBI)] is a phylogenetically ancient appetite stimulator that is secreted in a nonconventional, autophagy-dependent fashion. Here, we show that low ACBP/DBI plasma concentrations are associated with poor prognosis in patients with anorexia nervosa, a frequent and often intractable eating disorder. In mice, anorexia induced by chronic restraint stress (CRS) is accompanied by a reduction in circulating ACBP/DBI concentrations. We engineered a chemical-genetic system for the secretion of ACBP/DBI through a biotin-activatable, autophagy-independent pathway. In transgenic mice expressing this system in hepatocytes, biotin-induced elevations in plasma ACBP/DBI concentrations prevented anorexia induced by CRS or chemotherapeutic agents including cisplatin, doxorubicin, and paclitaxel. ACBP/DBI reversed the CRS or cisplatin-induced increase in plasma lipocalin-2 concentrations and the hypothalamic activation of anorexigenic melanocortin 4 receptors, for which lipocalin-2 is an agonist. Daily intravenous injections of recombinant ACBP/DBI protein or subcutaneous implantation of osmotic pumps releasing recombinant ACBP/DBI mimicked the orexigenic effects of the chemical-genetic system. In conclusion, the supplementation of extracellular and peripheral ACBP/DBI might constitute a viable strategy for treating anorexia.

Glucocorticoid modulates oxidative and thermogenic function of rat brown adipose tissue and human brown adipocytes.

Chronic and excessive glucocorticoid (GC) exposure can cause Cushing's syndrome, resulting in fat accumulation in selected body areas. Particularly in the brown adipose tissue (BAT), GC acts negatively, resulting in whitening of the tissue. We hypothesized that dysregulation of microRNAs by GC could be an additional mechanism to explain its negative actions in BAT. Male Wistar rats were divided into two groups: (1) Control sham and (2) GC group that was administered dexamethasone 6.25 mg/200 μL via osmotic pump implantation over 28 days. After this period, the animals were euthanized and BAT tissue was properly stored. Human fat cells treated with dexamethasone were used to translate the experimental results found in animals to human biology. GC-treated rat BAT presented with large lipid droplets, severely impaired thermogenic activation, and reduced glucose uptake measured by 18F-FDG PET/CT. GC exposure induced a reduction in the mitochondrial OXPHOS system and oxygen consumption. MicroRNA profiling of BAT revealed five top-regulated microRNAs and among them miR-21-5p was the most significantly upregulated in GC-treated rats compared to the control group. Although upregulation of miR-21-5p in the tissue, differentiated primary brown adipocytes from GC-treated rats had decreased miR-21-5p levels compared to the control group. To translate these results to the clinic, human brown adipocytes were treated with dexamethasone and miR-21-5p inhibitor. In human brown cells, inhibition of miR-21-5p increased brown adipocyte differentiation and prevented GC-induced glucose uptake, resulting in a lower glycolysis rate. In conclusion, high-dose GC therapy significantly impacts brown adipose tissue function, with a notable association between glucose uptake and miR-21-5p.

Priming antibody responses to the fusion peptide in rhesus macaques

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

Direct thrombin inhibitors fail to reverse the negative effects of heparin on lung growth and function after murine left pneumonectomy.

Neonates with congenital diaphragmatic hernia (CDH) frequently require cardiopulmonary bypass and systemic anticoagulation. We previously demonstrated that even subtherapeutic heparin impairs lung growth and function in a murine model of compensatory lung growth (CLG). The direct thrombin inhibitors (DTIs) bivalirudin and argatroban preserved growth in this model. Although DTIs are increasingly used for systemic anticoagulation clinically, patients with CDH may still receive heparin. In this experiment, lung endothelial cell proliferation was assessed following treatment with heparin-alone or mixed with increasing concentrations of bivalirudin or argatroban. The effects of subtherapeutic heparin with or without DTIs in the CLG model were also investigated. C57BL/6J mice underwent left pneumonectomy and subcutaneous implantation of osmotic pumps. Pumps were preloaded with normal saline, bivalirudin, or argatroban; treated animals received daily intraperitoneal low-dose heparin. In vitro, heparin-alone decreased endothelial cell proliferation and increased apoptosis. The effect of heparin on proliferation, but not apoptosis, was reversed by the addition of bivalirudin and argatroban. In vivo, low-dose heparin decreased lung volume compared with saline-treated controls. All three groups that received heparin demonstrated decreased lung function on pulmonary function testing and impaired exercise performance on treadmill tolerance testing. These findings correlated with decreases in alveolarization, vascularization, angiogenic signaling, and gene expression in the heparin-exposed groups. Together, these data suggest that bivalirudin and argatroban fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of low-dose heparin with DTIs on CDH outcomes are warranted.NEW & NOTEWORTHY Infants with pulmonary hypoplasia frequently require cardiopulmonary bypass and systemic anticoagulation. We investigate the effects of simultaneous exposure to heparin and direct thrombin inhibitors (DTIs) on lung growth and pulmonary function in a murine model of compensatory lung growth (CGL). Our data suggest that DTIs fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of heparin with DTIs on clinical outcomes are thus warranted.

Impact of isorhamnetin for suppression of action potential aberrations associated with atrial fibrillation

Abstract Background Isorhamnetin, a natural flavonoid, has potent antioxidant, antifibrotic and L-type Cav1.2 channel modulation effects, but its antiarrhythmic effect has not been reported. Purpose The purpose of this study is to investigate the effect of isorhamnetin on angiotensin II (Ang II)-induced atrial fibrillation (AF) in mice. Methods Wild-type male mice (C57BL/6J, 8 weeks old) were randomly divided into the following three groups: 1) control group, 2) Ang II treated group, 3) Ang II+isorhamnetin treated group. Ang II (1000 ng/kg/min) was administered by an implantable osmotic pump for two consecutive weeks, and isorhamnetin (5 mg/kg) was administered intraperitoneally for three weeks, one week before the initiation of Ang II administration. HL-1 cells were also assigned three groups. Ang II (1 μM) was treated for one day, and isorhamnetin (10 μM) was pretreated one hour before the beginning of Ang II treatment. To evaluate the effect of isorhamnetin on AF, electrophysiologic study including AF induction and action potential duration (APD) measurement with atrial tissue and HL-1 cells was performed. The expression of ion channel genes and proteins, including Cav1.2, were also evaluated. Results AF induction rate was markedly increased by Ang II and significantly decreased by isorhamnetin. AF duration was remarkably prolonged by Ang II and significantly shortened by isorhamnetin. Atrial effective refractory period (AERP) shortened by Ang II and reversed by isorhamnetin. Ang II administration significantly prolonged APD; that was alleviated by isorhamnetin. In addition, Ang II increased the frequency of delayed afterdepolarization (DAD), whereas isorhamnetin reduced its frequency. Cav1.2 channel expression was enhanced by Ang II and normalized by isorhamnetin. On the other hand, Ang II and isorhamnetin had no effect on potassium channels. Conclusion The findings in this study suggest that isorhamnetin prevents APD prolongation via the modulation of Cav1.2 channel and reduced the frequency of DAD, which is one of the mechanisms to prevent AF vulnerability.Effect of isorhamnetin on APD

In Vivo Validation of Modulated Acoustic Radiation Force-Based Imaging in Murine Model of Abdominal Aortic Aneurysm Using VEGFR-2-Targeted Microbubbles.

The objective of this study is to validate the modulated acoustic radiation force (mARF)-based imaging method in the detection of abdominal aortic aneurysm (AAA) in murine models using vascular endothelial growth factor receptor 2 (VEGFR-2)-targeted microbubbles (MBs). The mouse AAA model was prepared using the subcutaneous angiotensin II (Ang II) infusion combined with the β-aminopropionitrile monofumarate solution dissolved in drinking water. The ultrasound imaging session was performed at 7 days, 14 days, 21 days, and 28 days after the osmotic pump implantation. For each imaging session, 10 C57BL/6 mice were implanted with Ang II-filled osmotic pumps, and 5 C57BL/6 mice received saline infusion only as the control group. Biotinylated lipid MBs conjugated to either anti-mouse VEGFR-2 antibody (targeted MBs) or isotype control antibody (control MBs) were prepared before each imaging session and were injected into mice via tail vein catheter. Two separate transducers were colocalized to image the AAA and apply ARF to translate MBs simultaneously. After each imaging session, tissue was harvested and the aortas were used for VEGFR-2 immunostaining analysis. From the collected ultrasound image data, the signal magnitude response of the adherent targeted MBs was analyzed, and a parameter, residual-to-saturation ratio ( Rres - sat ), was defined to measure the enhancement in the adherent targeted MBs signal after the cessation of ARF compared with the initial signal intensity. Statistical analysis was performed with the Welch t test and analysis of variance test. The Rres - sat of abdominal aortic segments from Ang II-challenged mice was significantly higher compared with that in the saline-infused control group ( P < 0.001) at all 4 time points after osmotic pump implantation (1 week to 4 weeks). In control mice, the Rres - sat values were 2.13%, 1.85%, 3.26%, and 4.85% at 1, 2, 3, and 4 weeks postimplantation, respectively. In stark contrast, the Rres - sat values for the mice with Ang II-induced AAA lesions were 9.20%, 20.6%, 22.7%, and 31.8%, respectively. It is worth noting that there was a significant difference between the Rres - sat for Ang II-infused mice at all 4 time points ( P < 0.005), a finding not present in the saline-infused mice. Immunostaining results revealed the VEGFR-2 expression was increased in the abdominal aortic segments of Ang II-infused mice compared with the control group. The mARF-based imaging technique was validated in vivo using a murine model of AAA and VEGFR-2-targeted MBs. Results in this study indicated that the mARF-based imaging technique has the ability to detect and assess AAA growth at early stages based on the signal intensity of adherent targeted MBs, which is correlated with the expression level of the desired molecular biomarker. The results may suggest, in very long term, a pathway toward eventual clinical implementation for an ultrasound molecular imaging-based approach to AAA risk assessment in asymptomatic patients.

Osmotic pump maintains plasma concentrations of meloxicam for 6 days after orthopedic surgery in pigeons (Columba livia).

To determine the plasma concentration of meloxicam delivered via an osmotic pump in pigeons undergoing orthopedic surgery and if an osmotic pump is a suitable alternative to repeated oral administration of this drug. 16 free-ranging pigeons presented for rehabilitation with a wing fracture. An osmotic pump filled with 0.2 mL of 40 mg/mL meloxicam injectable solution was implanted subcutaneously in the inguinal fold of 9 pigeons under anesthesia for orthopedic surgery. The pumps were removed 7 days postsurgery. Blood samples were collected before pump implantation (time 0) and 3, 24, 72, and 168 hours after pump implantation in 2 pigeons in a pilot study then at 12, 24, 72, and 144 hours in the 7 pigeons of the main study. The blood of 7 other pigeons receiving meloxicam at 2 mg/kg, PO, every 12 hours was also sampled between 2 to 6 hours after the last meloxicam administration. Plasma meloxicam concentrations were measured via high-performance liquid chromatography. The plasma concentration of meloxicam was maintained at significant levels from 12 hours to 6 days after osmotic pump implantation. Median and minimum plasma concentrations in implanted pigeons were maintained at the same or higher level than those measured in pigeons that received meloxicam at a dose known to be analgesic in this species. No adverse effects attributable to either osmotic pump implantation and removal or meloxicam delivery were observed in this study. Plasma concentrations levels of meloxicam in pigeons implanted with osmotic pumps were maintained at a similar concentration or higher than the suggested analgesic meloxicam plasma concentration in this species. Thus, osmotic pumps could represent a suitable alternative to the frequent capture and handling of birds for analgesic drug administration.

Novel preventive effect of isorhamnetin on electrical and structural remodeling in atrial fibrillation.

Isorhamnetin, a natural flavonoid, has strong antioxidant and antifibrotic effects, and a regulatory effect against Ca2+-handling. Atrial remodeling due to fibrosis and abnormal intracellular Ca2+ activities contributes to initiation and persistence of atrial fibrillation (AF). The present study investigated the effect of isorhamnetin on angiotensin II (AngII)-induced AF in mice. Wild-type male mice (C57BL/6J, 8 weeks old) were assigned to three groups: (1) control group, (2) AngII-treated group, and (3) AngII- and isorhamnetin-treated group. AngII (1000 ng/kg/min) and isorhamnetin (5 mg/kg) were administered continuously via an implantable osmotic pump for two weeks and intraperitoneally one week before initiating AngII administration, respectively. AF induction and electrophysiological studies, Ca2+ imaging with isolated atrial myocytes and HL-1 cells, and action potential duration (APD) measurements using atrial tissue and HL-1 cells were performed. AF-related molecule expression was assessed and histopathological examination was performed. Isorhamnetin decreased AF inducibility compared with the AngII group and restored AngII-induced atrial effective refractory period prolongation. Isorhamnetin eliminated abnormal diastolic intracellular Ca2+ activities induced by AngII. Isorhamnetin also abrogated AngII-induced APD prolongation and abnormal Ca2+ loading in HL-1 cells. Furthermore, isorhamnetin strongly attenuated AngII-induced left atrial enlargement and atrial fibrosis. AngII-induced elevated expression of AF-associated molecules, such as ox-CaMKII, p-RyR2, p-JNK, p-ERK, and TRPC3/6, was improved by isorhamnetin treatment. The findings of the present study suggest that isorhamnetin prevents AngII-induced AF vulnerability and arrhythmogenic atrial remodeling, highlighting its therapeutic potential as an anti-arrhythmogenic pharmaceutical or dietary supplement.

Direct thrombin inhibitors as alternatives to heparin to preserve lung growth and function in a murine model of compensatory lung growth

Infants with congenital diaphragmatic hernia (CDH) may require cardiopulmonary bypass and systemic anticoagulation. Expeditious lung growth while on bypass is essential for survival. Previously, we demonstrated that heparin impairs lung growth and function in a murine model of compensatory lung growth (CLG). We investigated the effects of the direct thrombin inhibitors (DTIs) bivalirudin and argatroban. In vitro assays of lung endothelial cell proliferation and apoptosis were performed. C57BL/6 J mice underwent left pneumonectomy and subcutaneous implantation of osmotic pumps. Pumps were pre-loaded with normal saline (control), bivalirudin, argatroban, or heparin and outcomes were assessed on postoperative day 8. Heparin administration inhibited endothelial cell proliferation in vitro and significantly decreased lung volume in vivo, while bivalirudin and argatroban preserved lung growth. These findings correlated with changes in alveolarization on morphometric analysis. Treadmill exercise tolerance testing demonstrated impaired exercise performance in heparinized mice; bivalirudin/argatroban did not affect exercise tolerance. On lung protein analysis, heparin decreased angiogenic signaling which was not impacted by bivalirudin or argatroban. Together, this data supports the use of DTIs as alternatives to heparin for systemic anticoagulation in CDH patients on bypass. Based on this work, clinical studies on the impact of heparin and DTIs on CDH outcomes are warranted.

Skeletal muscle phenotypic switching in heart failure with preserved ejection fraction.

Skeletal muscle (SkM) phenotypic switching is associated with exercise intolerance in heart failure with preserved ejection fraction (HFpEF). Patients with HFpEF have decreased type-1 oxidative fibers and mitochondrial dysfunction, indicative of impaired oxidative capacity. The SAUNA (SAlty drinking water/Unilateral Nephrectomy/Aldosterone) mice are commonly used in HFpEF pre-clinical studies and demonstrate cardiac, lung, kidney, and white adipose tissue impairments. However, the SkM (specifically the oxidative-predominant, soleus muscle) has not been described in this preclinical HFpEF model. We sought to characterize the soleus skeletal muscle in the HFpEF SAUNA mice and investigate its translational potential. HFpEF was induced in mice by uninephrectomy, d-aldosterone or saline (Sham) infusion by osmotic pump implantation, and 1% NaCl drinking water was given for 4 weeks. Mice were euthanized, and the oxidative-predominant soleus muscle was collected. We examined fiber composition, fiber cross-sectional area, capillary density, and fibrosis. Molecular analyses were also performed. To investigate the clinical relevance of this model, the oxidative-predominant, vastus lateralis muscle from patients with HFpEF was biopsied and examined for molecular changes in mitochondrial oxidative phosphorylation, vasculature, fibrosis, and inflammation. Histological analyses demonstrated a reduction in the abundance of oxidative fibers, type-2A fiber atrophy, decreased capillary density, and increased fibrotic area in the soleus muscle of HFpEF mice compared to Sham. Expression of targets of interest such as a reduction in mitochondrial oxidative-phosphorylation genes, increased VEGF-α and an elevated inflammatory response was also seen. The histological and molecular changes in HFpEF mice are consistent and comparable with changes seen in the oxidative-predominant SkM of patients with HFpEF. The HFpEF SAUNA model recapitulates the SkM phenotypic switching seen in HFpEF patients. This model is suitable and relevant to study SkM phenotypic switching in HFpEF.

Preclinical Development and Screening of a Quinone-Nitroalkene Molecule for Sickle Cell Anemia

Background: Sickle cell anemia (SCA) is a hereditary disorder caused by the formation of hemoglobin S (HbS). HbS undergoes polymerization and generates sickled red blood cells (RBCs). Along with having reduced oxygen carrying capacity, sickled RBCs are also prone to frequent hemolysis releasing the pro-oxidant heme in the circulation. Oxidative stress created by the free heme leads to sterile inflammation, vaso-occlusion and cardiovascular complications. The prevalence of SCA is greatest in the poorer regions of Sub-Saharan Africa and there are around 100,000 new incidents of SCA every year in the USA alone. Although a number of therapeutic options are currently FDA-approved for the treatment of SCA patients, there is considerable interpatient variability in terms of their efficacy and safety, along with high morbidity and mortality rates. We have developed a novel quinone-nitroalkene hybrid molecule called CP50 (figure 1), which has been shown to activate two important proteins: 1) nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of cellular resistance to oxidants and 2) cytochrome b5 reductase 3 (CYB5R3), an anti-stress enzyme in the cardiovascular system. Methodology and results: Primary human aortic endothelial cells (HAECS) were incubated with 5 µM CP50 for 6 hours to perform a genomic screening. The mRNA expression profile revealed that CP50 differentially regulated pathways associated with oxidative balance, inflammation, and erythropoiesis. CP50 was associated with upregulation of genes responsible for counteracting stress, including heat shock protein family, HSPA6, HSPA1A, HSPA1B and heme-oxygenase 1 (HMOX-1) (n=5, p<0.05). CP50 downregulated genes that inhibit effective erythropoiesis, such as ZFP36 Ring Finger Protein Like 2 (ZFP36L2) and Growth/differentiation factor 15 (GDF15), thus imparting a positive effect on the process (n=5, p<0.05). Immunoblotting for heme-oxygenase 1 (HO-1) using wild type and Nrf2-knockdown HAECs demonstrated that HO-1 was induced by 5 µM CP50 by 5 fold (±1.36) after 24 hours of exposure, in a Nrf2 dependent manner (n=3, p<0.0001). CYB5R3 activity measured by its impact on myoglobin reduction rate, was also found to increase dose-dependently by CP50 (n=3, p<0.001). Afterwards, CD34+ hematopoietic stem cells (HSCs, n=3) were cultured with 5 µM CP50 and 50 µM hydroxyurea (HU) to observe the effect on erythropoiesis, using CD71, CD235a and fetal hemoglobin (HbF) as markers for flow cytometry. CP50 augmented the percentage of HbF-expressing F cells by 1.7 fold (±0.54) in comparison to 2.21 fold (±0.89) by HU on day 9. F cells are known to impart resilience against sickling and hemolysis in sickle cell anemia. We then observed the effect of CP50 administered by osmotic pump implantation on hematopoiesis in Townes HbSS mice (vehicle, 5mg/kg/day, 15mg/kg/day, n=3/4 in each group). After a period of 4 weeks, we collected whole bone marrow from the mice and using flow cytometry checked for HSCs. CP50 (15 mg/kg/day) significantly increased (p<0.001) the HSC pool by 59% in comparison to vehicle. Additionally, it also enhanced the differentiation of megakaryocyte-erythroid progenitors (MEP) in bone marrow cells by 11% (p<0.05). In a separate experiment, peripheral blood samples from Townes HbSS mice were collected and incubated with 5 µM CP50 before chemically inducing oxidative hemolysis. CP50 reduced hemolysis by 73.68% in comparison to vehicle. Conclusion: Together, our studies indicate that CP-50 has the potential to correct oxidative imbalance by activating antioxidant genes, reverse anemia by inducing hematopoiesis, and inhibit hemolysis by increasing F cell percentage. Thus, we can conclude that CP50 is a prospective drug candidate with the promise to satisfy the unmet need for additional safe and effective therapeutic options for patients with sickle cell anemia. Legend (figure 1): The quinone head group in CP50 serves as an antioxidant, whereas the nitro group in the fatty acid side chain acts as an electrophile. The electrophile modifies the cysteine maintaining the Keap1-Nrf2 interaction and releases the Nrf2. Nrf2 enters the nucleus and initiates the expression of genes that counteract oxidative stress. Keap1: Kelch-like ECH-associated protein 1 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A TNF receptor 2 agonist ameliorates neuropathology and improves cognition in an Alzheimer’s disease mouse model

Tumor necrosis factor-α (TNF-α) is a pleiotropic, proinflammatory cytokine related to different neurodegenerative diseases, including Alzheimer's disease (AD). Although the linkage between increased TNF-α levels and AD is widely recognized, TNF-α-neutralizing therapies have failed to treat AD. Previous research has associated this with the antithetic functions of the two TNF receptors, TNF receptor 1, associated with inflammation and apoptosis, and TNF receptor 2 (TNFR2), associated with neuroprotection. In our study, we investigated the effects of specifically stimulating TNFR2 with a TNFR2 agonist (NewStar2) in a transgenic Aβ-overexpressing mouse model of AD by administering NewStar2 in two different ways: centrally, via implantation of osmotic pumps, or systemically by intraperitoneal injections. We found that both centrally and systemically administered NewStar2 resulted in a drastic reduction in amyloid β deposition and β-secretase 1 expression levels. Moreover, activation of TNFR2 increased microglial and astrocytic activation and promoted the uptake and degradation of Aβ. Finally, cognitive functions were also improved after NewStar2 treatment. Our results demonstrate that activation of TNFR2 mitigates Aβ-induced cognitive deficits and neuropathology in an AD mouse model and indicates that TNFR2 stimulation might be a potential treatment for AD.

Effects of CRN04894, a Nonpeptide Orally Bioavailable ACTH Antagonist, on Corticosterone in Rodent Models of ACTH Excess

CRN04894 is an orally administered nonpeptide that is a potent and selective antagonist for adrenocorticotropic hormone (ACTH) acting at the melanocortin 2 receptor (MC2R) and is currently under development for the treatment of diseases of ACTH excess such as Cushing’s disease, congenital adrenal hyperplasia, and ectopic ACTH-secreting tumors. Cushing’s disease results from an adenoma derived from pituitary corticotropic cells that secrete excess ACTH, whereas ectopic ACTH syndrome arises from nonpituitary ACTH secreting tumors. Congenital adrenal hyperplasia is a genetic disease that results in cortisol deficiency leading to high levels of ACTH and adrenal androgens. Each of these indications is characterized by high ACTH levels that act on MC2R expressed in the adrenal cortex to drive pathological elevations of adrenally derived steroid hormones. CRN04894 blocks the action of ACTH at MC2R, providing a potential novel treatment for these diseases. Preclinical models of chronic hypercortisolemia include implantation of ACTH-secreting pituitary tumor cells in mice and continuous administration of ACTH via subcutaneously implanted osmotic pumps in rats. These models induce features consistent with human diseases of ACTH excess including hypercortisolemia and hypertrophy of the adrenal glands. We employed both rodent models to examine the pharmacodynamic effects of CRN04894 on corticosterone levels and adrenal gland morphology. In the mouse pituitary tumor model, subcutaneous inoculation of the ACTH-secreting mouse pituitary tumor cell line, AtT-20, into immunodeficient mice resulted in formation of tumors and increased plasma ACTH and corticosterone levels. Repeated daily oral administration of CRN04894 for 14 days dose-dependently and robustly suppressed plasma corticosterone levels in mice with AtT-20 tumors. In the rat model, subcutaneous implantation of osmotic pumps delivering ACTH resulted in increased corticosterone levels, reduction in body weight, and hypertrophy of the adrenal glands after 7 days. Daily oral administration of CRN04894 over 7 days dose-dependently suppressed corticosterone levels, mitigated the effect of ACTH excess on body weight, and rescued the adrenal gland hypertrophy. These findings provide evidence that CRN04894 functions as an effective ACTH antagonist at MC2R to suppress adrenal corticosterone secretion in both mouse and rat models of ACTH excess and hypercortisolemia, thus providing a strong rationale for its potential therapeutic utility in diseases of ACTH excess. This work was supported in part by an SBIR grant from the NIH awarded to Dr. Struthers (R43- DK115245)

A phytic acid analogue INS-3001 prevents ectopic calcification in an Abcc6-/- mouse model of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic calcification disorders, affects the skin, eyes and the cardiovascular system due to inactivating mutations in the ABCC6 gene. There is no effective treatment for the systemic manifestations of PXE. In this study, the efficacy of INS-3001, an analogue of phytic acid, was tested for inhibition of ectopic calcification in an Abcc6-/- mouse model of PXE. In prevention study, Abcc6-/- mice, at 6weeks of age, the time of onset of ectopic calcification, were treated with INS-3001 with 0.16, 0.8, 4, 20 or 100mg/kg/day administered by subcutaneous implantation of osmotic pumps, as well as 4mg/kg/day by subcutaneous injection thrice weekly or 14, 4 and 0.8mg/kg/day once weekly subcutaneous injection. Mice were necropsied at 12weeks of age. Histologic examination and quantitative calcium assay revealed that mice receiving 6weeks of continuous INS-3001 administration via osmotic pumps showed dose-dependent inhibition of muzzle skin calcification with complete response at 4mg/kg/day and a minimum effective dose at 0.8mg/kg/day. INS-3001 plasma concentrations were dose-dependent and largely consistent during treatment for each dose. thrice weekly and once weekly subcutaneous injections of INS-3001 also prevented calcification. In established disease study, 12-week-old Abcc6-/- mice with extensive calcification were continuously administered INS-3001 at 4mg/kg/day for a follow-up of 12weeks. INS-3001 treatment was found to stabilize existing calcification that had developed at start of treatment. These results suggest that INS-3001 may provide a promising preventive treatment strategy for PXE, a currently intractable ectopic calcification disorder.

Local continuous glial cell derived neurotrophic factor release using osmotic pump promotes parasympathetic nerve rehabilitation in an animal model of cavernous nerve injury induced erectile dysfunction.

BackgroundNerve injury-related erectile dysfunction (ED) is one of the types that respond poorly to conventional ED treatments. Our previous experiments have demonstrated the paracrine of various neurotrophic factors (NTFs) by stem cells or other treatment modalities as a potential mechanism in the recovery of nerve injury-related ED. Glial cell derived neurotrophic factor (GDNF) is one of the essential NTFs for the regeneration of nerve fibers, especially for parasympathetic nerves. The aim of this study is to explore if local continuous GDNF administration is beneficial for the functional and histological recovery of nerve injury induced ED.MethodsEight-week-old male Sprague-Dawley rats were used for this study. Rats were randomly grouped into 5: Sham surgery (Sham), bilateral cavernous nerve injury (BCNI) and placebo treatment, BCNI and 0.1 µg/100 µL GDNF treatment (BCNI+GDNF 0.1), BCNI and 1 µg/100 µL GDNF treatment (BCNI+GDNF 1), BCNI and 10 µg/100 µL GDNF treatment (BCNI+GDNF 10). GDNF was administered using an osmotic pump technique which would deliver GDNF locally and continuously for 28 days without the need for external connections or frequent handling of animals. Recovery of sexual function, nerve fibers regeneration, and expression of neurotrophic receptors were examined and compared among groups after the treatment.ResultsLocal continuous GDNF release treatment increased the average number of intromissions in the sexual behavior test and intracavernous pressure (ICP) in the erectile function test in a dose dependent manner. Osmotic pump implantation induced increased local GDNF concentration and mild inflammatory response. Gene expression of GDNF receptors in major pelvic ganglion (MPG) and nerve regeneration along the urethra were partially promoted by GDNF. These changes were associated with increased nerve fibers especially the parasympathetic nerve fibers in dorsal nerve of penis (DNP) in GDNF treated groups.ConclusionsIn conclusion, our project illustrated the promising effects of local continuous GDNF administration for the functional and histological recovery of nerve injury-induced ED.

Abstract 15127: Isorhamnetin Reverses Angiotensin Ii-induced Atrial Fibrillation Vulnerability Through Electrophysiological and Structural Reverse Remodeling in Mice

Background: Isorhamnetin (ISO), a natural flavonoid compound, has been shown to have strong antioxidant and antifibrotic effects in vivo and in vitro; however, no report has been shown on the effect of ISO on arrhythmic disorders. This study aimed to investigate the effect of ISO on angiotensin-II (Ag-II) induced atrial fibrillation (AF) in mice. Methods: Wild-type male mice (C57BL/6J, 8 weeks old) were assigned in 3 groups as follows: control group (Cont), Ag-II group treated with Ag-II and Ag-II &amp; ISO group treated with Ag-II and ISO. Ag-II (1000 ng/kg/min) was continuously administered using an implantable osmotic pump for 2 weeks, and ISO (5 mg/kg) was administered intraperitoneally one week before starting Ag-II administration. We performed AF induction and electrophysiologic study via transvenous electrode catheter and Ca 2+ imaging with isolated cardiomyocyte. We also assessed the gene expression levels of AF-related molecules, and histological examination of atrial fibrosis. Results: Compared with Cont, AF inducibility was dramatically increased by Ag-II and significantly decreased by ISO. AF duration was also remarkably prolonged by Ag-II and significantly reduced by ISO. Atrial effective refractory period (A-ERP) (BCL=150msec) was reduced by Ag-II and recovered by ISO. Incidences of diastolic intracellular Ca 2+ abnormal activities (sarcoplasmic reticulum [SR] Ca 2+ leakage) was observed in Ag-II group; however, ISO treatment eliminated these abnormalities. Ag-II induced elevated expression of fibrosis-related molecules (Col1a1, Tgfb1 and Tgfb2) and Ca 2+ -handling related molecules (Camk2 and Ryr2), those were partially normalized by ISO. Histological examination revealed that cell size and the fibrosis ratio were increased by Ag-II, and significantly attenuated by ISO. Conclusion: These results suggest that ISO prevented against Ag-II induced AF vulnerability through both electrophysiological and structural reverse remodeling.

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the α3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the α3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Chronic Nicotine Exposure Alters Uninjured Tendon Vascularity and Viscoelasticity.

Background:Although nicotine and tobacco use are well established to have adverse health effects, the effects on tendons are less well understood. The purpose of this study was to investigate the effect of chronic nicotine exposure on Achilles tendon (AT) and supraspinatus tendon (SS) physiologic and mechanical properties in a rodent model.Methods:Sprague-Dawley rats (n = 20) were exposed to either 0.9% saline or 36 mg/mL nicotine through an implantable osmotic pump for 12 weeks. At 12 weeks, the AT was imaged in vivo with contrast-enhanced ultrasound (CE-US) to assess vascularity. Bilateral AT and SS were then harvested for ex vivo biomechanical analysis. Statistical analysis was performed using Student t test and Mann Whitney U test.Results:AT CE-US demonstrated a significantly increased wash-in rate in the nicotine group compared to the saline group, indicating an increase in tissue perfusion rate in the nicotine group. AT percent stress relaxation, a measure of tendon viscoelasticity, was significantly increased in the nicotine group compared to the saline group. Similarly, there was a trend toward increased SS percent stress relaxation in the nicotine group compared to the saline group. No differences in other mechanical properties were observed.Conclusion:AT perfusion rate increased and both AT and SS viscoelasticity were altered with chronic nicotine exposure at a clinically relevant dose that models the average smoker in the United States (14 cigarettes per day). Further studies are necessary to link these properties with tendon degeneration and injury.Clinical Relevance:Chronic nicotine exposure affects tendon health. Patients should be counseled as such.