Tail Snip Research Articles

(051) METHAMPHETAMINE-INDUCED VAGINAL LUBRICATION IN RATS AND THE ESTROUS CYCLE

Abstract Introduction In an interview-based study performed by our lab of women who use methamphetamine (meth), we previously found that the drug, when delivered intravenously (IV), produces a feeling indistinguishable from an orgasm, which is accompanied by a flushing sensation and the production of excessive vaginal lubrication. We have also reported meth-induced vaginal lubrication in an anesthetized animal model. This production of vaginal transudate was dependent on the dose of meth used and the potent vasodilator nitric oxide. Objective The objective of the current study was to assess meth-induced vaginal lubrication across different stages of the estrous cycle and following ovariectomy (ovx) in anesthetized rats, and to assess plasma levels of two signaling molecules associated with sexual arousal. Methods Anesthetized rats, each previously implanted with a chronic indwelling jugular catheter, were anesthetized and given one IV infusion of meth (0.24 mg/kg) and vaginal lubrication was measured. A pre-weighed cotton-tipped swab, inserted into the vaginal canal, collected fluid secreted following the administration of meth. Blood was withdrawn before and at ten and fifteen minutes after meth administration either via the jugular catheter or via a small tail snip to measure plasma levels of estradiol and nitric oxide. Testing was completed at each stage of the estrous cycle as well as before and after ovariectomy. Results Estradiol and nitric oxide increased following meth infusion similarly across all stages of the estrous cycle. Vaginal lubrication increased more with meth treatment than with saline treatment. This was true across all stages of the estrous cycle and despite ovariectomy. Conclusions These findings have far-reaching implications for those who suffer from vaginal dryness. The mechanism underlying this phenomenon may present a novel pharmacotherapeutic target to treat vaginal dryness. Since meth still produced vaginal secretions in anesthetized rats, these results suggest that targeting this mechanism may even prove beneficial in post-menopausal women, a patient group likely at highest risk for experiencing vaginal dryness. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Embera Neuurotherapeutics & JanOne.

Charcoal-frying process and quality markers of Sanguisorbae Radix based on hemostatic effect

Studies have reported that the hemostatic effect of Sanguisorbae Radix(SR) is significantly enhanced after processing with charcoal. However, the standard components(tannins and gallic acid) specified in the Chinese Pharmacopeia decrease in charcoal-fried Sanguisorbae Radix(CSR), which is contrast to the enhancement of the hemostatic effect. Therefore, this study aimed to optimize the charcoal-frying process of SR based on its hemostatic efficacy and comprehensively analyze the components of SR and its processed products, thus exploring the material basis for the hemostatic effect. The results indicated that SR processed at 250 ℃ for 14 min(14-min CSR) not only complied with the description in the Chinese Pharmacopeia but also demonstrated improved blood-coagulating and blood-adsorbing effects compared with raw SR(P<0.05). Moroever, 14-min CSR reduced the bleeding time in the rat models of tail snipping, liver bleeding, and muscle injury, surpassing both raw and excessively fried SR(16 min processed) as well as tranexamic acid(P<0.05). Ellagitannin, ellagic acid, methyl gallate, pyrogallic acid, protocatechuic acid, Mg, Ca, Mn, Cu, and Zn contributed to the hemostatic effect of CSR over SR. Among these substances, ellagitannin, ellagic acid, Mg, and Ca had high content in the 14 min CSR, reaching(106.73±14.87),(34.86±4.43),(2.81±0.23), and(1.21±0.23) mg·g~(-1), respectively. Additionally, the color difference value(ΔE~*ab) of SR processed to different extents was correlated with the content of the aforementioned hemostatic substances. In summary, this study optimized the charcoal-frying process as 250 ℃ for 14 min for SR based on its hemostatic effect. Furthermore, ellagic acid and/or the powder chromaticity are proposed as indicators for the processing and quality control of CSR.

Abstract 6654: Oncolytic virotherapy targeting high-grade glioma tumor antigens

Abstract Glioblastoma, the most common primary brain malignancy in adults, is an aggressive cancer with five-year median survival of ~6.8%. New treatment options are urgently needed for glioblastoma and other high-grade gliomas (HGGs). Oncolytic viruses (OVs) are an emerging class of cancer therapy currently being evaluated as treatments for a variety of malignancies, including glioblastoma. Our labs developed OV C134, now under evaluation in a phase I clinical trial of recurrent glioblastoma (NCT03657576). In addition to directly infecting and lysing tumor cells, OVs are capable of stimulating anti-tumor immune responses. We previously showed in the CT2A syngeneic mouse model of HGG that treatment with a version of C134 engineered to express the shared tumor antigen Ephrin-A2 (EPHA2; commonly upregulated in glioblastoma) significantly increased survival and produced EPHA2-specific T cell responses. This finding led us to hypothesize that C134 could be further engineered to stimulate antigen-specific immune responses against tumor-specific neoantigens. We first assessed the impact of C134 treatment on immune cell dynamics in HGGs, by performing single cell RNA sequencing of CD45+ immune cell infiltrate from CT2A HGGs treated with C134 or saline (control). Clustering of these data identified 17 distinct immune cell types, and differential expression analyses revealed several C134-induced changes. These included increased expression of genes involved in antigen presentation and STAT signaling in microglia and macrophage subsets, the proliferation marker Mki67 in germinal center B cells, the chemokine Ccl12 from microglia, and tissue-resident memory marker genes Fos and Jun in T cells. These data suggest C134 treatment poises the tumor immune microenvironment for antigen presentation and antigen-specific immune responses. Next, we evaluated CT2A to identify candidate tumor neoantigens by isolating DNA and RNA from orthotopic tumors and DNA from tail snips of mice treated with saline or C134. We carried out RNA sequencing (tumors) and whole exome sequencing (tumor and normal), and employed the Personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) computational pipeline. This pipeline takes somatic variant calls from the exome comparison of tumor to normal DNA, validates RNA expression of called variants, and feeds corresponding peptide sequences into algorithms that predict the binding of variant-containing peptides to major histocompatibility complex (MHC), thereby assessing for potential neoantigens. Our pVAC-Seq analysis revealed a conserved set of 1,264 tumor-exclusive variants expressed at the RNA level with high (ic50<500nm) predicted binding to mouse MHC. I will present details of C134-induced immune remodeling in syngeneic HGG models, and describe a plan to engineer C134 to express a prioritized set of neoantigens to evaluate how efficacy from OV therapy may be further enhanced in a patient-specific manner. Citation Format: Jack Hedberg, Alexia Martin, Doyeon Kim, Jason Navarro, Adithe Rivaldi, Yeaseul Kim, Ilse Hernandez-Aguirre, Elizabeth Garfinkle, Katherine Miller, Kevin Cassady, Elaine Mardis. Oncolytic virotherapy targeting high-grade glioma tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6654.

A Mouse Tail Snip Procedure for Peripheral Blood Collection and Plasma Isolation

A Mouse Tail Snip Procedure for Peripheral Blood Collection and Plasma Isolation

A Mouse Tail Snip Procedure for Peripheral Blood Collection and Plasma Isolation

A Mouse Tail Snip Procedure for Peripheral Blood Collection and Plasma Isolation

Impact of acute stress on murine metabolomics and metabolic flux

Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.

Study Protocol: Molecular Imaging of Mouse Brain to Investigate Effect of Pre-made Creatine for Neurodevelopmental Disorders in Neonates

Abstract Objectives The global incidence of preterm births is 11% of all births worldwide each year. With advancement of perinatal care, an increasing number of preterm babies survive. However, this increasing survival does not couple with a reduction in the impaired neurodevelopment. Preterm babies receive parenteral nutrition (PN) in the first few weeks to months of their life. Current PN does not contain creatine. In my previous work, 2-weeks of PN reduced the plasma creatine and its intermediate, guanidinoacetate (GAA) compared to neonatal piglets fed PN with premade creatine. L-arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) synthesize GAA and creatine. In-vitro activities of AGAT and GAMT in the whole brain of neonates were negligible suggesting neonatal brains may rely on exogenous creatine. Prefrontal cortex (PFC) and hippocampus (Hip) are implicated in cognitive development and memory such as those associated with neurodevelopmental disorders of preterm neonates. We optimized mRNA molecular imaging technique to measure multiple mRNAs in a single brain section. Objective 1: measure distribution pattern and co-expression of AGAT and GAMT enzymes in PFC and Hip. Creatine is a neurotransmitter and an agonist for postsynaptic Gamma-aminobutyric acid (GABA) a receptor. Altered GABAa receptor signalling is implicated in neurodevelopmental disorders. Objective 2: how lack of creatine affects GABAa receptor mRNA expression in PFC and Hip regions of neonatal brain. Methods Methods: Sixteen C57BL6 postpartum dams will be randomly assigned to 2 groups and administer an intraperitoneal injection of 1) creatine (160 mg/kg of body weight) or 2) saline once a week. The blood will be sampled from pups once a week from tail snip to measure circulating GAA and creatine concentrations of pups. Pups will be sacrificed after 4 weeks of nursing and extracted brains will be used for molecular imaging of mRNAs (AGAT, GAMT and GABAa receptor). Results This is proposed study protocol. Conclusions Investigating the co-expression of AGAT and GAMT in brain regions will be novel finding from this proposed protocol. Pre-made creatine will reduce brain AGAT and GAMT mRNA expression but increase GABAa receptor mRNA expression. This will suggest that exogenous creatine is important for Hip and PFC GABAa receptor expression. Funding Sources Not decided.

Brain-specific PAPP-A knock-out mice?

PAPP-A knock-out (KO) mice are a valuable model for investigating the effects of down-regulating localized insulin-like growth factor (IGF) action, which has been shown to extend lifespan and healthspan when the PAPP-A gene is globally deleted. Based on previous mouse models of brain-specific reduction in IGF signaling associated with longevity, we sought to generate brain-specific PAPP-A KO mice and determine effects on metabolism and lifespan. Mice with the PAPP-A gene floxed (fPAPP-A) were crossed with Nestin promoter-driven Cre recombinase transgenic mice. This cross-breeding of mice for Nestin-Cre and mice with other floxed target alleles has been used extensively to investigate brain-specific effects. Our cross-breeding generated four genotypes for study: fPAPP-A/Nestin positive (brain-specific PAPP-A KO); fPAPP-A/Nestin negative (Control for floxed PAPP-A); WT/Nestin positive (Control for Nestin-Cre); WT/Nestin negative (Wild-type Control).The basic genotype screen of neonatal tail snip DNA clearly indicated PAPP-A gene status and the presence (pos) or absence (neg) of Nestin-Cre. We then determined tissue specificity of PAPP-A gene excision. We had expected fPAPP-A/pos mice to be relatively brain-specific for PAPP-A gene deletion and the controls (fPAPP-A/neg, WT/neg and WT/pos mice) to show no effect on PAPP-A expression in brain or other tissues. However, in fPAPP-A/neg mice we found evidence of PAPP-A excision in all tissues examined, i.e., in the presumed absence of Nestin-Cre, indicating germline recombination. We further found that fPAPP-A/pos mice showed near complete excision of the PAPP-A gene in brain, but some also showed germline recombination affecting all tissues tested.To determine if the level of excision indicated by tissue genotyping approximated PAPP-A mRNA expression, we performed RT-qPCR. fPAPP-A/pos mice that showed markedly decreased whole brain PAPP-A mRNA expression (~80%), with little or no effect on expression in the other tissues tested, were designated as “brain-specific” PAPP-A KO. fPAPP-A/pos mice that showed germline recombination had similar decreases in PAPP-A expression in brain but also showed 40–65% decreased PAPP-A mRNA expression in other tissues as well, which was especially striking in kidney, tibia, thymus and spleen. These were designated as “non-specific” PAPP-A KO mice.With unknown and unpredictable specificity until harvest, we chose to assess a surrogate marker of lifespan i.e., thymic involution, in 15- to 18-month-old fPAPP-A/pos and WT/pos mice, the latter an important control for a possible effect of Nestin-Cre per se. Diminished thymic involution as indicated by increased thymic weight (135%, P = 0.035) and decreased histological disruption was seen in “non-specific” PAPP-A KO mice, similar to what was previously reported in 18-month-old global PAPP-A KO mice. There was no significant difference between “brain-specific” PAPP-A KO and control mice.This study highlights the importance of thorough characterization of assumed tissue-specific mouse models and awareness of potential germline recombination for proper data interpretation.

Cardiomyocyte TRPV4 deletion preserves cardiac function following pressure overload‐induced pathological hypertrophy independent of cardiac fibrosis

Left ventricular hypertrophy is compensatory adaptive response towards physical stimuli. However, prolonged changes in hemodynamic pressure due to pathological insults disrupts the electrical and bio-mechanical coupling between cardiomyocytes which leads to heart failure. Previous studies have demonstrated the role of soluble factors in the regulation of cardiomyocyte function, however, mechanosensor and mechanotransduction pathways in cardiomyocytes are remain unknown. We have recently shown that global deletion of mechanosensitive, transient receptor potential vanilloid 4 ion channel (TRPV4) preserves cardiac function and structure via reducing cardiac fibrosis after MI. Here we investigated cardiomyocyte specific role of TRPV4 following pressure overload induced hypertrophy. First, we found that TRPV4 expression is increased following pressure overload induced hypertrophy in wildtype (WT) mice. Global deletion of TRPV4 preserved myocardial structure and function compared to WT after 28 days of TAC. To understand the cardiomyocyte TRPV4 role, we have generated cardiomyocyte specific TRPV4 knockout mice (TRPV4CMKO) by crossing TRPV4lox/lox with Myh6cre mice. The littermates were confirmed for TRPV4 deletion in cardiomyocytes through genotyping and Real time PCR in tail snips and isolated cardiomyocytes. Next, we subjected the TRPV4lox/lox and TRPV4CMKO to TAC and then measured cardiac function. After 28 days of TAC, absence of TRPV4 in cardiomyocyte preserved the cardiac function compared to TRPV4lox/lox animals. Surprisingly, histological analysis revealed increased cardiac hypertrophy (cardiomyocyte cross sectional area) and cardiac fibrosis in TRPV4CMKO mice compared to TRPV4lox/lox mice or sham controls. Taken together our results suggest that cardiomyocyte specific deletion of TRPV4 preserves cardiac function despite of increased pathological remodeling of the heart and identify TRPV4 as new therapeutic target for the heart failure.

Effect of telmisartan on blood glucose levels in euglycemic albino rats

Introduction: Diabetes is considered primary risk factor for heart disease and comorbidities in older people and in children. Due to escalating epidemic, diabetes is concerned high profile public health problem. Materials and Methods: Swiss albino rats weighing around 150-200gms of either sex were randomly selected from the central animal facility. Animals were randomly divided into 2 groups, six animals in each group. A group of 3 rats were kept in individual cages. The control was given 0.5 ml 1% Gum acacia, test group was given Telmisartan at the dose of 7.2mg/kg/day. All the drugs were given orally and for the period of 5 days. All rats were fasted overnight before the 5th day. On the 5th day glucose (0.6gm/kg body weight) was given orally using oral feeding tube and oral glucose tolerance test (OGTT) was performed. Blood was collected by tail snipping method and capillary blood glucose was assessed at 0, 60, and 150 minutes using a glucometer. Results: Fall in capillary blood glucose level was seen in Telmisartan group at all-time intervals when compared to control group. Conclusion:Telmisartan when given orally for 5 days showed fall in blood glucose level in swiss albino rats. Keywords: Adipokines, Capillary blood glucose, Diabetes, Euglycemic

Lyophilized Human Platelets Restore Hemostasis in the Presence of the P2Y12 Inhibitors Cangrelor, Ticagrelor and Clopidogrel

• Background: P2Y12 inhibitors block platelet ADP receptors thereby reducing clotting capacity. In an emergency, these agents must be reversed or therapeutically overcome to stop bleeding. Additionally, prior to and during elective procedures these inhibitors must be withdrawn, thereby increasing thrombotic risk. Lyophilized human platelets (Thrombosomes®) are stabilized platelet derived hemostatic agents currently under Phase 2 clinical development for thrombocytopenia and have potential as antiplatelet reversal agents. • Aims: The aim of this study was to determine if lyophilized human platelets are resistant to the antiplatelet effect of P2Y12 inhibitors and therefore restore hemostasis in the presence of P2Y12 inhibitors. • Methods: Fresh drawn platelet rich plasma (PRP) was treated with pharmacological concentrations of P2Y12 inhibitors cangrelor or ticagrelor and confirmed to inhibit ADP-stimulated aggregation on the PAP-8E Platelet Aggregometer. Changes in hemostatic properties of the P2Y12 inhibitor treated PRP were also tested under shear force on the Total Thrombus formation Analysis System (T-TAS®). P2Y12 inhibitor treated PRP samples were dosed with lyophilized platelets and tested for return of hemostatic properties by T-TAS. NOD SCID mice were used in a tail snip model to determine hemostatic efficacy of Thrombosomes® after super pharmacological doses of clopidogrel treatment. • Results: Fresh PRP derived platelet aggregation response to ADP was inhibited by cangrelor or ticagrelor whereas lyophilized platelets did not respond to either inhibitor. Normal thrombus formation of PRP as measured by T-TAS occurred at 19.5± 1.5 minutes (n=4) but increases to 28.0±3.0 minutes with cangrelor (n=4) or 28.0±3.0 minutes with ticagrelor (n=5) treatment. The addition of 150k/µL lyophilized platelets to P2Y12-inhibited PRP reduced time to thrombus formation to lower than PRP alone; 15.5 ±0.5 minutes in the presence of cangrelor (n=3) versus 17.5 ±1.5 minutes in the presence of ticagrelor (n=5). In the in vivo tail snip mouse model animals treated with super pharmacological dose of clopidogrel bled for 18.0±10.0 minutes (n=5) unlike those not treated that only bleed for 9.5±2.5 minutes (n=6). Lyophilized platelet treated clopidogrel animals stopped bleeding at 12.5 ±4.5 minutes (n=5) after tail snip. • Conclusion: Lyophilized human platelets, unlike normal platelets were resistant to the antiplatelet effects of P2Y12 inhibitors. In vitro and in vivo studies reveal that lyophilized human platelets can be used to recover the anti-thrombotic effect of P2Y12 antagonists, potentially allowing continued drug compliance prior to elective procedures and as a possible treatment for emergent acute bleeding while on P2Y12 antagonist therapy. Disclosures Dickerson: Cellphire Inc.: Current Employment. Lee:Cellphire, Inc.: Current Employment. Hale:Cellphire: Current Employment. Moskowitz:Cellphire Inc.: Current Employment.

Efficacy of Levofloxacin Loaded Nonionic Surfactant Vesicles (Niosomes) in a Model of Pseudomonas aeruginosa Infected Sprague Dawley Rats.

This study examined the effectiveness of niosomes loaded with levofloxacin in treating Pseudomonas aeruginosa (American Type Culture Collection—ATCC 27853) infections in Sprague Dawley rats since these infections are becoming more common and resistant to treatment. Levofloxacin entrapped in niosomes was prepared using the thin-film hydration method and was assessed for in vitro release and stability. Three groups of six (6) animals were infected with a lethal dose of Pseudomonas aeruginosa via the intraperitoneal (Ip) route. At six (6) hours postinfection, the animals were treated with either drug-free niosomes (control), free levofloxacin (conventional), or levofloxacin trapped in niosomes (Ip) at a dose of 7.5 mg/kg/once daily. Blood was collected via tail snips on days 0, 1, 3, 5, 7, and 10 for complete blood counts and viable bacterial counts (CFU/μl). At day 10, the animals were sacrificed, and the kidney, liver, and spleen were harvested for bacterial counts. The niosomes showed a sustained drug release profile and were most stable at 4°C. All animals in the control group succumbed to the infection; one animal from the conventional group died, and all niosome treated animals survived at day 10. The mean lymphocyte count (×109) was lower for the niosome (7.258 ± 1.773) versus conventional group (17.684 ± 10.008) (p < 0.03) at day ten (10). Neutrophil counts (×109) were lower for the niosome (2.563 ± 1.609) versus conventional (6.2 ± 6.548) (p < 0.02) groups. Though CFUs in the bloodstream were comparable for both treatment groups, the niosome treated group showed a significant reduction of CFUs in the liver, kidney, and spleen versus the conventional group (1.33 ± 2.074) vs (5.8 ± 3.74) (p < 0.043), (1.5 ± 2.35) vs (9.6 ± 8.65) (p < 0.038) and (3.8 4.71) vs (25.6 14.66) (p < 0.007), respectively. These findings indicate that niosome is promising as a drug delivery system in treating systemic infections, but further work using niosomes with surface modification is recommended.

Elucidation of the active ingredients of Lamiophlomis herba against hemorrhage based on network pharmacology and tail snipping model in mice.

This study aimed to elucidate the active ingredients of Lamiophlomis herba (LH), the overground part of Lamiophlomis rotata (Benth.) Kudo, against hemorrhage based on network pharmacology and tail snipping model in mice. A total of 118 hemorrhage-related target genes were identified by retrieving public databases, and 39 genes were identified as the hub genes of hemorrhage based on protein-protein interaction and module analyses. The interactions between 67 potentially active ingredients in LH and 7 genes in the 39 hub genes were established and analyzed through molecular docking and Cytoscape. A total of 21 ingredients were involved in the interactions, and were divided into three categories: iridoid (15 ingredients), flavonoid (2 ingredients) and other category (4 ingredients). Based on the "multi-ingredient, multi-target" characteristic of traditional Chinese medicines (TCMs), the results of network pharmacology indicated that iridoid might be the key active ingredient group of LH against hemorrhage. The contribution of iridoid to the hemostatic effect of LH was investigated by the tail snipping model in mice. The results showed that iridoid was the key active ingredient group of LH against hemorrhage, which confirmed the prediction in network pharmacology. Additionally, the previous reports also supported this prediction. In conclusion, the finding of the present study indicates that iridoid is the key hemostatic ingredient group of LH. This work provides valuable references for investigation of the hemostatic ingredients of LH based on the holistic theory of TCMs. Meanwhile, this work also provides further insight into the development of hemostatic drugs based on LH.

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of Protein N-Glycosylation.

SummaryDehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Hypoglycemic effects of leaf extracts of Moringa oleifera and Vitex simplicifolia on blood glucose level of Wistar rat

Diabetes mellitus (DM) is as a disorder that is characterized by increase in blood sugar levels, as well as disturbances in the metabolism of the three main classes of food. Whole plants or their parts serve as alternatives in the treatment of many diseases and, have, for a time immemorial, been very important in the health care delivery in many countries. The objective of this study was to compare the relative antihyperglycemic properties of Moringa oleifera and Vitex simplicifolia, as well as the reference drug glibenclamide, on blood sugar levels. DM was induced via intraperitoneal injection of alloxan monohydrate; levels of glucose in circulating blood were measured using a glucometer, by tail snip method. Potent oral hypoglycemic properties and thus, a fast onset of anti-diabetic activity was more pronounced in Vitex simplicifolia than in Moringa oleifera. The two were more potent than the reference drug, glibenclamide. The studied leaf extracts are relatively safe and could be used in the treatment of diabetes mellitus.

Optimization of tissue sampling for Borrelia burgdorferi in white-footed mice (Peromyscus leucopus)

Peromyscus leucopus (the white-footed mouse) is a known reservoir of the Lyme disease spirochete Borrelia burgdorferi. Sampling of white-footed mice allows for year-round B. burgdorferi surveillance as well as opportunities to establish the diversity of the different variants in a geographic region. This study explores the prevalence of B. burgdorferi infections in the tissues of white-footed mice, investigates the correlations between B. burgdorferi infected tissues, and determines the optimum field methods for surveillance of B. burgdorferi in P. leucopus. A total of 90 mice and 573 tissues (spleen, liver, ear, tongue, tail, heart, and kidney) were screened via nested PCR for B. burgdorferi infections. A large number of infections were found in the 90 mice as well as multiple infections within individual mice. Infections in a single mouse tissue (spleen, liver, ear, tongue and tail) were predictive of concurrent infection in other tissues of the same mouse at a statistically significant level. Ear tissue accounted for 68.4% of detected infections, which increased to 78.9% of the infected mice with the inclusion of tail samples. The use of ear punch or tail snip samples (used individually or in tandem) have multiple advantages over current Lyme disease ecological studies and surveillance methodologies, including lower associated costs, minimization of delays, year-round B. burgdorferi testing opportunities, as well as longitudinal monitoring of B. burgdorferi in defined geographic regions. In the absence of an effective vaccine, personal prevention measures are currently the most effective way to reduce Lyme disease transmission to humans. Thus, the identification and monitoring of environmental reservoirs to inform at-risk populations remains a priority. The sampling methods proposed in this study provide a reasonable estimate of B. burgdorferi in white-footed mice in a timely and cost-effective manner.

Downregulation of Sprr1a Contributes to the Pathobiology of Sickle Cell Disease

Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p&amp;lt;0.001); significant increase in Evan blue leakage in the skin in response to substance P and capsaicin (p&amp;lt;0.001 and 0.003, respectively); 30-40% increase in calcium release from DRG neurons in response to capsaicin and menthol (p&amp;lt;0.05 and 0.01, respectively); and increased phospho-p38 MAPK in the spinal cords (p&amp;lt;0.003). Complementary to these observations, Sprr1a-KO mice evinced heightened mechanical, heat and cold sensitivity, which increased with age from 1.2 to 7.0 months of age in both male and female mice compared to C57BL/6 WT. These data suggest that loss of Sprr1a leads to skin thinning, increased permeability and activation of peripheral and central nervous system leading to mechanical and thermal hyperalgesia (pain). To identify the molecular pathways involved in different functions of Sprr1a, we performed mass-spectrometry on the DRG of WT and Sprr1a-KO mice on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to a Prominence Nano LC (Shimadzu) using the Xcalibur version 2.7.0 (Thermo Scientific) and Proteome Discoverer 2.2 software. In parallel, we genetically overexpressed Sprr1a in HbSS-BERK mice (Sprr1a-OE-SS) using the Gateway cloning system combined with PiggyBac transposon: We obtained 1 cell embryo from HbSS mice, and carried out pronuclear injection with Sprr1a cDNA. The resulting HbSS mice harboring the Sprr1a transgene were screened by appropriate genotyping of tail snips. Proteomic analysis of DRG and subsequent network analysis using the STRING database tool of the significantly modified proteins (p&amp;lt;0.05) revealed that their interactions with Sprr1a have never been investigated. Many of the significantly altered proteins in Sprr1a-KO are associated with SCD pathobiology, including, but not limited to, SPIC (7-fold increase, p&amp;lt;0.0001), associated with red blood cell (RBC) recycling and VCAM1 regulation; CD9 (22-fold increase, p&amp;lt;0.0001), involved in platelet activation and cell adhesion; SPTBN4 (2-fold decrease, p&amp;lt;0.05), neuropathy, and kininogen (3-fold decrease, p&amp;lt;0.001). Decreased kininogen is observed in sickle cell patients, which is further reduced during crisis (Verma et al., Res Commun Chem Pathol Pharmacol 1983). Thus, downregulation of Sprr1a may contribute to SCD pathobiology, which may in turn lead to pain. This appears to be the case, because overexpression of Sprr1a in HbSS-BERK sickle mice (Sprr1a-OE-SS) led to amelioration of mechanical and cold hyperalgesia (p&amp;lt;0.01 &amp; 0.05, respectively) at 2.8 months of age compared to age/gender matched HbSS-BERK mice. This decrease was maintained up to 9.8 months of age (last period of observation) in Sprr1a-OE-SS, which was similar to the sensitivity in control HbAA-BERK mice and significantly lower than HbSS-BERK mice. Together, these data suggest a causal link between Sprr1a downregulation and pain in SCD. It is likely that Sprr1a loss contributes to complex sickle pathobiology leading to dehydration due to loss of water from thin and permeant skin, non-healing skin ulceration, neuropathy and pain. In conclusion, through genetic and proteomic approaches, we have identified Sprr1a as a novel treatable target for several consequences of SCD. Disclosures Gupta: 1910 Genetics: Research Funding.

English

Antidesma bunius (L.) Spreng and Mollugo oppositifolia L., also known as “bignay” and “malagoso” respectively, are commonly used medicinal plants in the Philippines to treat diabetes. However, the hypoglycemic effect of A. bunius (L.) Spreng and M. oppositifolia L. has not been reported to date. Therefore, this study was undertaken to investigate the hypoglycemic effects of the fresh and ethanolic extracts of the plants in db/db diabetic male mice. Fresh and ethanolic extracts (12.5 ml/kg and 250 mg/kg, respectively) were administered by gavage in db/db mouse, an animal model of diabetes. Blood from the tail snip was used to measure blood sugar levels on days 1 (D1), 8 (D8), 15 (D15), 22 (D22) and 29 (D29). Fresh extract of M. oppositifolia L. caused a significant reduction of blood glucose levels on D22 compared to untreated db/db mice (P < 0.05). On D29, fresh extract of A. bunius (L.) Spreng (P < 0.01), and fresh (P < 0.01) and ethanolic leaf extracts (P < 0.05) of M. oppossitifolia L. caused a significant reduction of blood glucose compared to untreated db/db mice. Fresh and ethanolic extracts of A. bunius (L.) Spreng and M. oppositifolia L. exhibited a significant hypoglycemic activity in db/db mice. Key words: Antidesma bunius L, Spreng, Mollugo oppositifolia L., diabetes, mouse, hypoglycemic.

Regulation of endothelial cell permeability by platelet-derived extracellular vesicles.

Platelet (Plt)-derived extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular EC permeability, similar to fresh Plts. To investigate this hypothesis, we used in vitro and in vivo models of vascular endothelial compromise and bleeding. In the vitro model, Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function were assessed by transendothelial electrical resistance measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts was assessed by multiple electrode Plt aggregometry. Using an in vivo model, the effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by an established Miles assay of vascular permeability and a tail snip bleeding assay. In the in vitro model, Plt-EVs displayed exosomal size distribution and expressed Plt-specific surface markers. Platelets and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced thrombin receptor-activating peptide-mediated aggregation of whole blood, whereas Plts enhanced thrombin receptor-activating peptide-, arachidonic acid-, collagen-, and adenosine diphosphate-mediated aggregation. In the in vivo model, Plt-EVs are equivalent to Plts in attenuating vascular endothelial growth factor (VEGF)-A-induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability, and mitigate the endotheliopathy of trauma.