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While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344). Male and female, 10- to 14-wk-old F344 and SD rats were used to assess withdrawal thresholds in 3 different modalities: the Randall-Selitto test (RST), Hargreaves test (HT), and tail flick test (TFT). The RST was performed by using an operator-controlled handheld instrument to generate a noxious pressure stimulus to the left hind paw. The HT and the TFT used an electronically controlled light source to deliver a noxious thermal stimulus to the left hind paw or tail tip, respectively. Rats of each sex and genetic background underwent one type of test on day 0 and day 7. Withdrawal thresholds and thermal latencies were compared among tests. No significant differences were observed. Our findings can serve as a guide for researchers considering these nociceptive tests for their experiments.

Management of critically ill patients requires simultaneous administration of many medications. Treatment for patient comorbidities may lead to drug-drug interactions which decrease drug efficacy or increase adverse reactions. Current practices rely on a one-size-fits-all dosing approach. Pharmacogenetic testing is generally reserved for addressing problems rather than used proactively to optimize care. We hypothesized that burn and surgery patients will have one or more genetic variants in drug metabolizing pathways used by one or more medications administered during the patient's hospitalization. The aim of this study was to determine the frequency of variants with abnormal function in the primary drug pathways and identify which medications may be impacted. Genetic (19 whole exome and 11 whole genome) and medication data from 30 pediatric burn and surgery patients were analyzed to identify pharmacogene-drug associations. Nineteen patients were identified with predicted altered function in one or more of the following genes: CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The majority had decreased function, except for several patients with CYP2C19 rapid or ultrarapid variants. Some drugs administered during hospitalization that rely on these pathways include hydrocodone, oxycodone, methadone, ibuprofen, ketorolac, celecoxib, diazepam, famotidine, diphenhydramine, and glycopyrrolate. Approximately one-third of the patients tested had functionally impactful genotypes in each of the primary drug metabolizing pathways. This study suggests that genetic variants may in part explain the vast variability in drug efficacy and suggests that future pharmacogenetics research may optimize dosing regimens.

PurposeDiphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).MethodsMolecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants.ResultsDPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.ConclusionWe provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

The Mutant Mouse Resource and Research Center (MMRRC) Program is the pre-eminent public national mutant mouse repository and distribution archive in the USA, serving as a national resource of mutant mice available to the global scientific community for biomedical research. Established more than two decades ago with grants from the National Institutes of Health (NIH), the MMRRC Program supports a Consortium of regionally distributed and dedicated vivaria, laboratories, and offices (Centers) and an Informatics Coordination and Service Center (ICSC) at three academic teaching and research universities and one non-profit genetic research institution. The MMRRC Program accepts the submission of unique, scientifically rigorous, and experimentally valuable genetically altered and other mouse models donated by academic and commercial scientists and organizations for deposition, maintenance, preservation, and dissemination to scientists upon request. The four Centers maintain an archive of nearly 60,000 mutant alleles as live mice, frozen germplasm, and/or embryonic stem (ES) cells. Since its inception, the Centers have fulfilled 13,184 orders for mutant mouse models from 9591 scientists at 6626 institutions around the globe. Centers also provide numerous services that facilitate using mutant mouse models obtained from the MMRRC, including genetic assays, microbiome analysis, analytical phenotyping and pathology, cryorecovery, mouse husbandry, infectious disease surveillance and diagnosis, and disease modeling. The ICSC coordinates activities between the Centers, manages the website (mmrrc.org) and online catalog, and conducts communication, outreach, and education to the research community. Centers preserve, secure, and protect mutant mouse lines in perpetuity, promote rigor and reproducibility in scientific experiments using mice, provide experiential training and consultation in the responsible use of mice in research, and pursue cutting edge technologies to advance biomedical studies using mice to improve human health. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest standards of rigor and reproducibility, while donating investigators benefit by having their mouse lines preserved, protected, and distributed in compliance with NIH policies.

BackgroundCeroid lipofuscinosis type 8 belongs to a heterogenous group of vision and life-threatening neurodegenerative diseases, neuronal ceroid lipofuscinosis (NCL). Effective therapy is limited to a single drug for treatment of ceroid lipofuscinosis type 2, necessitating animal disease models to facilitate further therapeutic development. Murine models are advantageous for therapeutic development due to easy genetic manipulation and rapid breeding, however appropriate genetic models need to be identified and characterized before being used for therapy testing. To date, murine models of ocular disease associated with ceroid lipofuscinosis type 8 have only been characterized in motor neuron degeneration mice.MethodsCln8-/- mice were produced by CRISPR/Cas9 genome editing through the International Mouse Phenotyping Consortium. Ophthalmic examination, optical coherence tomography, electroretinography, and ocular histology was performed on Cln8-/- mice and controls at 16 weeks of age. Quantification of all retinal layers, retinal pigmented epithelium, and the choriocapillaris was performed using images acquired with ocular coherence tomography and planimetry of histologic sections. Necropsy was performed to investigate concurrent systemic abnormalities. Clinical correlation with human patients with CLN8-associated retinopathy is provided.ResultsRetinal degeneration characterized by retinal pigment epithelium mottling, scattered drusen, and retinal vascular attenuation was noted in all Cln8-/- mice. Loss of inner and outer photoreceptor segment demarcation was noted on optical coherence tomography, with significant thinning of the whole retina (P=1e-9), outer nuclear layer (P=1e-9), and combined photoreceptor segments (P=1e-9). A global reduction in scotopic and photopic electroretinographic waveforms was noted in all Cln8-/- mice. Slight thickening of the inner plexiform layer (P=0.02) and inner nuclear layer (P=0.004), with significant thinning of the whole retina (P=0.03), outer nuclear layer (P=0.01), and outer photoreceptor segments (P=0.001) was appreciated on histologic sections. Scattered lipid vacuoles were noted in splenic red pulp of all Cln8-/- mice, though no gross systemic abnormalities were detected on necropsy. Retinal findings are consistent with those seen in patients with ceroid lipofuscinosis type 8.ConclusionsThis study provides detailed clinical characterization of retinopathy in adult Cln8-/- mice. Findings suggest that Cln8-/- mice may provide a useful murine model for development of novel therapeutics needed for treating ocular disease in patients with ceroid lipofuscinosis type 8.

The Mutant Mouse Resource and Research Center (MMRRC) Program is the pre-eminent public national mutant mouse repository and distribution archive in the USA, serving as a national resource of mutant mice available to the global scientific community for biomedical research. Established more than two decades ago with grants from the National Institutes of Health (NIH), the MMRRC Program supports a Consortium of regionally distributed and dedicated vivaria, laboratories, and offices (Centers) and an Informatics Coordination and Service Center (ICSC) at three academic teaching and research universities and one non-profit genetic research institution. The MMRRC Program accepts the submission of unique, scientifically rigorous, and experimentally valuable genetically altered and other mouse models donated by academic and commercial scientists and organizations for deposition, maintenance, preservation, and dissemination to scientists upon request. The four Centers maintain an archive of nearly 60,000 mutant alleles as live mice, frozen germplasm, and/or embryonic stem (ES) cells. Since its inception, the Centers have fulfilled 13,184 orders for mutant mouse models from 9591 scientists at 6626 institutions around the globe. Centers also provide numerous services that facilitate using mutant mouse models obtained from the MMRRC, including genetic assays, microbiome analysis, analytical phenotyping and pathology, cryorecovery, mouse husbandry, infectious disease surveillance and diagnosis, and disease modeling. The ICSC coordinates activities between the Centers, manages the website (mmrrc.org) and online catalog, and conducts communication, outreach, and education to the research community. Centers preserve, secure, and protect mutant mouse lines in perpetuity, promote rigor and reproducibility in scientific experiments using mice, provide experiential training and consultation in the responsible use of mice in research, and pursue cutting edge technologies to advance biomedical studies using mice to improve human health. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest standards of rigor and reproducibility, while donating investigators benefit by having their mouse lines preserved, protected, and distributed in compliance with NIH policies.

The sustained-release formulation of meloxicam (MSR) is a compounded NSAID that may provide pain relief for as long as 72 h after administration. MSR injection-site skin reactions have occurred in several species but have not previously been observed in mice. We investigated the development and progression of localized skin reactions after a single injection of MSR in Crl:CD1(ICR), C57BL/6J, and BALB/cJ mice. Each mouse received a subcutaneous injection of MSR (n = 60), standard-formulation meloxicam (MEL; n = 24) or saline (control; SC; n = 24) and was scored daily according to a 5-point system for erythema and mass characteristics. Mice were euthanized at either 7 or 14 d after injection and underwent postmortem analysis. MSR-treated mice had more erythematous and mass reactions than did MEL and SC mice. Mass lesions developed in 49 MSR mice (82%; 95% CI, 70% to 90%), 5 MEL animals (21%; 95% CI, 7% to 42%), and 1 SC mouse. MSR-treated BALB/cJ developed erythematous lesions less frequently than similarly treated Crl:CD1(ICR) or C57BL/6J. Lesions often were ventrolateral to the injection site. The median times to the appearance of mass and erythematous lesions were 2 d and 3 d, respectively. Histologically, the erythematous and mass reactions correlated with necrotizing to pyogranulomatous injection-site panniculitis. Inflammation severity scores at 7 and 14 d after injection were greater in the MSR-treated group than the other 2 groups. No strain- or sex-associated differences emerged except that inflammation severity scores at day 14 were higher in Crl:CD1(ICR) females than males. The character of the inflammatory response in MSR-treated mice did not differ between 7 and 14 d after injection, indicating that MSR-induced inflammation is slow to resolve. The ventral migration and delayed onset of MSR injection-site reactions could result in their being attributed to another cause or not being identified. Researchers and clinicians should be aware of the potential for slowly resolving injection-site reactions with MSR.

An extended-release formulation of the NSAID meloxicam (MSR) is used to provide 72 h of continuous analgesia in many species, including rodents. Although standard formulations of meloxicam are frequently used in rats with no observable injection-site reactions, the potential adverse effects from MSR have not been characterized sufficiently nor has a prospective study of these effects been performed in rats. To address this deficiency, we evaluated injection-site reactions after a single subcutaneous administration of MSR (n = 16) or sterile saline (SC, n = 6) in the flank of age- and sex-matched Sprague-Dawley rats. Mass and erythema scores were measured daily for 2 wk, and injection sites were collected for histopathology after euthanasia. Rats were randomly selected for euthanasia at 7 d (n = 12) or 14 d (n = 10) after injection to capture the subacute and chronic phases of mass and erythematic lesion formation. No rats in the SC group developed lesions, whereas all 16 MSR-treated rats developed masses. The median time to first mass in the MSR treatment group was 3 d (95% CI, 2-3 d), and nearly 8 d for erythema (95% CI, 6.7-9.1 d). The trajectory of mass lesion severity showed rapid progression from score 1 at onset (day 2 or 3) to score 2 for almost all animals by day 5 or 6. Histopathology was characterized by localized inflammation with central necrosis and peripheral fibrosis, with some sections showing developing draining tracts. Given the high prevalence and severity of localized skin reactions, MSR analgesia should be considered carefully for Sprague-Dawley rats.

BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P

The Roux-en-Y Gastric Bypass (RYGB) mouse model is a vital tool for studying the pathophysiology of bariatric surgery and contributes greatly to research on obesity and diabetes. However, complications including postsurgical hypoglycemia can have profoundly negative effects. Unlike in humans, blood glucose (BG) is not typically managed in postoperative rodents, despite their critical role as translational models; without this management, rodents can experience hypoglycemia, potentially impairing wound healing, decreasing survivability, complicating interpretation of research data, and limiting translational utility. In this project, we sought to identify an optimal method for minimally invasive administration of dextrose in C57BL/6N (n = 16; 8 male, 8 female) mice. To do so, we characterized BG pharmacokinetic profiles after subcutaneous and oral-transmucosal (OTM) administration of dextrose. Compared with OTM dosage, the subcutaneous route provided more consistent and reliable delivery of glucose and did not cause significant adverse reactions. We then evaluated the frequency of hypoglycemic events after RYGB in C57BL/6N mice (n = 16; 8 male, 8 female) and the effects of subcutaneous dextrose supplementation on morbidity and mortality. BG measurement and behavioral pain assessment (grimace test) were performed for 3 d after surgery. Hypoglycemic (BG ≤ 60 mg/dL) animals were assigned to dose (5% dextrose SC) or no-dose treatment groups. Nearly all (87%) mice became hypoglycemic; 2 of these mice died. No significant intergroup difference in grimace score or mortality was detected. Overall, our results demonstrate that hypoglycemia is a frequent adverse event after RYGB in mice and that subcutaneous injection of dextrose is a safe and effective way to manage hypoglycemia. Further studies are necessary to optimize the intervention threshold and optimal dosage; regardless, we recommend glycemic management after RYGB surgery in mice.

Opioids are essential first line analgesics for pain management after burn injury. Opioid dosing remains challenging in burn patients, particularly in children, due to the immense variability in efficacy between patients. Opioid pharmacokinetics are altered in burned children, increasing variability and obviating dosing regimens extrapolated from adult-data. The present study aimed to characterize variability in fentanyl pharmacokinetics and identify significant contributors to variability in children with ≥10% total body surface area burn requiring fentanyl during routine wound care. We recorded patient demographics and clinical data. Blood samples were collected following fentanyl administration for pharmacokinetics at time 0, 30, 60, 120, and 240 minutes on day of admission and repeated on days 3 and 7. Serum fentanyl concentrations were quantified using tandem liquid chromatography mass spectrometry. Population analysis was used to estimate pharmacokinetics parameters. Fourteen patients, 1.2-17 years, with burns from 10-50.5% were included in analysis. A two-compartment model with body weight as a covariate best described fentanyl pharmacokinetics for the overall population. The population clearance and intercompartmental clearance were 7.19 and 2.16 L/hour, respectively, and the volume of distribution for the central and peripheral compartments was 4.01 and 25.1 L, respectively. Individual patient parameter estimates had extensive variability. This study confirmed the high variability in pediatric burn patient fentanyl pharmacokinetics and demonstrates similarities and differences to other populations reported in literature. Further research is needed with a larger number of patients to extensively investigate the impact of burns, genetic polymorphisms, and other factors on fentanyl efficacy and patient outcomes.

Pain management is critical for burn care. Unfortunately, interindividual variation in pharmacokinetics (PK) due to burn hypermetabolism and genetic polymorphisms can lead to treatment failures in this at-risk population. Analgesics may be affected by genetic polymorphisms affecting cytochrome P450 (CYP) drug metabolizing enzymes. Fentanyl is a common opiate primarily metabolized by CYP3A4 subtypes. Recent studies demonstrate CYP2D6 variants, affecting fentanyl PK. Functional CYP polymorphisms can significantly alter opiate levels resulting in inadequate analgesia or life-threatening toxicity. The goal of our study was to evaluate fentanyl PK and assess associations with CYP polymorphisms. We obtained samples from the previously banked blood of 13 patients (eight males and five females) with >20% TBSA burns. Mean (SD) patient age was 41.7 (14.5) years, and mean burn size was 25.8 (15.3) %TBSA. Plasma fentanyl was quantified, and CYP genotyping was performed. Pharmacokinetic analysis was performed using Monolix software (Lixsoft, France) with a two-compartment population model best-representing fentanyl profiles. Three CYP slow-metabolizing genotypes were identified, which included CYP2D6*9, CYP2D6*29, and CYP3A4*1B. All three patients with variant polymorphisms had increased serum fentanyl concentrations due to impaired clearance. This pilot study supports the need for further research in this topic, and CYP genotyping of individual patients prior to receiving opiate analgesics to inform precision-guided decisions, improve therapeutic efficacy, and, most importantly, increase patient well-being and safety.

Studies indicate that the gut microbiota (GM) can significantly influence both local and systemic host physiologic processes. With rising concern for optimization of experimental reproducibility and translatability, it is essential to consider the GM in study design. However, GM profiles can vary between rodent producers making consistency between models challenging. To circumvent this, we developed outbred CD1 mouse colonies with stable, complex GM profiles that can be used as donors for a variety of GM transfer techniques including rederivation, co-housing, cross-foster, and fecal microbiota transfer (FMT). CD1 embryos were surgically transferred into CD1 or C57BL/6 surrogate dams that varied by GM composition and complexity to establish four separate mouse colonies harboring GM profiles representative of contemporary mouse producers. Using targeted 16S rRNA amplicon sequencing, subsequent female offspring were found to have similar GM profiles to surrogate dams. Furthermore, breeding colonies of CD1 mice with distinct GM profiles were maintained for nine generations, demonstrating GM stability within these colonies. To confirm GM stability, we shipped cohorts of these four colonies to collaborating institutions and found no significant variation in GM composition. These mice are an invaluable experimental resource that can be used to investigate GM effects on mouse model phenotype.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Special populations, including women (non-pregnant and pregnant), pediatrics, and the elderly, require additional consideration with regard to clinical research. There are very specific regulatory laws, which protect these special populations, that need to be understood and adhered to in order to perform clinical research. This review provides a broad overview of some of the physiological differences in special populations and discusses how these differences may affect study design and regulatory considerations. These various special populations, with respect to regulatory affairs, are clearly defined within the Code of Federal Regulations. The definition of "special population" exists to provide enhanced awareness of their vulnerabilities, thereby allowing the creation of regulatory guidance aimed to decrease injury or outright harm. Currently, progress is being made to be more inclusive of special populations in clinical trials. This reflects changing attitudes towards drug information, with it being more representative of those patients that will ultimately be prescribed or exposed to the therapy. However, all research undertaken in these populations should be performed in a manner that ensures all protections of each participant are upheld.

ObjectiveTo determine if binge ethanol consumption before ovulation affects oocyte quality, gene expression, and subsequent embryo development.DesignBinge levels of ethanol were given twice weekly for 6 months, followed by a standard in vitro fertilization cycle and subsequent natural mating.SettingNational primate research center.Animal(s)Adult female rhesus monkeys.Intervention(s)Binge levels of ethanol, given twice weekly for 6 months before a standard in vitro fertilization cycle with or without embryo culture. With in vivo development, ethanol treatment continued until pregnancy was identified.Main outcome measure(s)Oocyte and cumulus/granulosa cell gene expression, embryo development to blastocyst, and pregnancy rate.Result(s)Embryo development in vitro was reduced; changes were found in oocyte and cumulus cell gene expression; and spontaneous abortion during very early gestation increased.Conclusion(s)This study provides evidence that binge drinking can affect the developmental potential of oocytes even after alcohol consumption has ceased.

While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344). Male and female, 10- to 14-wk-old F344 and SD rats were used to assess withdrawal thresholds in 3 different modalities: the Randall-Selitto test (RST), Hargreaves test (HT), and tail flick test (TFT). The RST was performed by using an operator-controlled handheld instrument to generate a noxious pressure stimulus to the left hind paw. The HT and the TFT used an electronically controlled light source to deliver a noxious thermal stimulus to the left hind paw or tail tip, respectively. Rats of each sex and genetic background underwent one type of test on day 0 and day 7. Withdrawal thresholds and thermal latencies were compared among tests. No significant differences were observed. Our findings can serve as a guide for researchers considering these nociceptive tests for their experiments.