Institutional Animal Care Research Articles

Iron Chelators Augment Large Osteochondral Allograft Osseointegration in a Preclinical Canine Model.

Osteochondral allograft transplantation (OCAT) can be a successful joint restoration treatment option for large post-traumatic articular defects but is still associated with significant revision and failure rates. Despite recent advances that have improved OCAT success, insufficient osteochondral allograft (OCA) osseointegration remains a major cause of failure. Deferoxamine (DFO) is an effective angiogenic and osteo-anabolic iron chelator that consistently promotes bone neovascularization and regeneration. This study was designed to investigate local delivery of DFO for augmenting OCA osseointegration using a preclinical canine model for OCAT in the knee and hip as commonly affected joints. On Institutional Animal Care and Use Committee (IACUC) approval, 12 purpose-bred dogs underwent OCAT of the femoral head or femoral condyles with DFO or DFO-free (controls) microspheres in recipient sites. OCA revascularization, cellular repopulation, and integration were evaluated based on functional, diagnostic imaging, microcomputed tomography, histology, and immunohistochemistry outcome measures. Local delivery of DFO into OCAT recipient sites was associated with maintained or improved joint function, superior radiographic appearance, significantly greater trabecular thickness, higher bone volume, and new bone ingrowth compared with DFO-free controls. OCA osseointegration is dependent on cellular repopulation and neovascularization, resulting in new bone ingrowth through creeping substitution, and insufficient osseointegration with resorption and subsidence of the OCA remains a major cause of failure after transplantation. The results of this study suggest that local delivery of DFO using a controlled microsphere release system may reduce resorption and improve revascularization and cellular repopulation to increase new bone ingrowth, potentially expediting OCA osseointegration after transplantation.

Cyanide Medical Countermeasure Development: Assessing the Efficacy of Intramuscular Sodium Tetrathionate for the Treatment of Acute, Severe Cyanide Toxicity in Swine (Sus scrofa).

Given its availability and lethality, cyanide has potential for weaponization and thus has the attention of several governmental agencies. In large scale exposure scenarios, an effective countermeasure that can be administered quickly and in low volume intramuscularly may prove valuable because IV medications may have limited practical applications in these situations. Sodium tetrathionate, a potential cyanide antidote, is a compound that provides sulfur to rhodanese, the enzyme that detoxifies cyanide endogenously. Additionally, sodium tetrathionate has been reported to directly react with cyanide and is effective when administered intramuscularly. In this study, we assess the efficacy of sodium tetrathionate, when administered intramuscularly for the treatment of acute, oral cyanide poisoning in swine. We conducted a prospective trial approved by the 59th Medical Wing Institutional Animal Care and Use Committee comparing intramuscular sodium tetrathionate (n=6) to no treatment control (n=4) in animals (Sus scrofa) exposed to a lethal dose of oral potassium cyanide. Survival at 120 minutes was the primary outcome. Lactate, a cyanide toxicity biomarker, was measured. At the study end, all animals were euthanized in compliance with the Animal Welfare Act and the American Association for Accreditation of Laboratory Animal Care. Survival between groups was summarized using a Kaplan-Meier survival curve after comparing survival by log-rank, Mantel-Cox analysis. The Mann-Whitney U test was used for comparison of other variables between groups. At baseline animals were similar. There was 100% survival in the treatment group and 0% survival in the control group (P=0.0011). Serum lactate significantly increased in the control group (control: 5±0.9 vs. treatment: 2.1 ± 0.5 mmol/L at 20 minutes). Sodium tetrathionate (intramuscular) significantly improved survival in a large, swine model of acute, oral cyanide poisoning. Future studies will be directed at further assessing sodium tetrathionate as a potential medical countermeasure for cyanide poisoning.

Comparative Electroencephalographic Profile of a New Anesthetic and Anticonvulsant That Is Selective for the GABAAR Slow Receptor Subtype.

Anesthetics like propofol increase electroencephalography (EEG) power in delta frequencies (0.1-4 Hz), with a decrease of power in bandwidths >30 Hz. Propofol is nonselective for gamma amino butyric acid type A receptor subtypes (GABAAR) as it enhances all 3 GABAAR subtypes (slow, fast, and tonic). Our newly developed anesthetic class selectively targets GABAAR-slow synapses to depress brain responsiveness. We hypothesized that a selective GABAAR-slow agonist, KSEB 01-S2, would produce a different EEG signature compared to the broad-spectrum GABAAR agonist (propofol), and tested this using rat EEG recordings. Male rats were studied after Institutional Animal Care and Use Committees (IACUC) approval from the US Army Medical Research Institute of Chemical Defense and the University of Michigan. Rats were anesthetized using isoflurane (3%-5% induction, 1%-3% maintenance) with oxygen at 0.5 to 1.0 L/min. Stainless steel screws were placed in the skull and used to record subcranial cortical EEG signals. After recovery, either propofol or KSEB 01-S2 was administered and effects on EEG signals were analyzed. As previously reported, propofol produced increased power in delta frequencies (0.1-4 Hz) compared to predrug recordings and produced a decrease in EEG power >30 Hz but no significant changes were seen within ±20 seconds of losing the righting reflex. By contrast, KSEB 01-S2 produced a significant increase in theta frequency percent power (median 14.7%, 16.2/13.8, 75/25 confidence interval; to 34.7%, 35/31.8; P < .015) and a significant decrease in low gamma frequency percent power (16.9%, 18.6/15.8; to 5.45%, 5.5/5.39; P < .015) for all rats at ± 20 seconds of loss of consciousness (LOC). Both anesthetics produced a flattening of chaotic attractor plots from nonlinear dynamic analyses, like that produced by volatile and dissociative anesthetics at LOC. KSEB 01-S2 produced a markedly different EEG pattern, with a selective increase observed in the theta frequency range. KSEB 01-S2 also differs markedly in its activity at the GABAAR-slow receptor subtype, suggesting a possible mechanistic link between receptor subtype specificity and EEG frequency band signatures. Increased theta together with depressed gamma frequencies is interesting because GABAAR slow synapses have previously been suggested to underlie theta frequency oscillations, while fast synapses control gamma activity. These reciprocal effects support a previous model for theta and nested gamma oscillations based on inhibitory connections between GABAAR fast and slow interneurons. Although each anesthetic produced a unique EEG response, propofol and KSEB 01-S2 both increased slow wave activity and flattened chaotic attractor plots at the point of LOC.

PSIX-5 Performance of black soldier fly larvae meal as secondary protein sources in extruded dog diets

Abstract Black soldier fly larvae meal (BSFL) has gained market exposure with ties to sustainability and novelty. Graded inclusions of BSFL were assessed for nutrient digestibility, stool quality, and palatability in adult dogs. The BSFL was included at 0%, 5% and 10% in chicken-based extruded dog diets (formulated to 22% crude protein, 13% crude fat, 3% crude fiber). The study was approved by the facility’s Institutional Animal Care and Use Committee. A standard 2-bowl palatability test over a 2-d period was executed (n = 30 dogs each) to determine intake ratios between diets (0% vs. 5%, 0% vs. 10%, and 5% vs. 10%). Total tract nutrient digestibility was evaluated (n = 6 dogs per diet) with 5 d acclimation followed by 5 d total fecal collection. Stool quality was evaluated on a 1 to 5 scale. Palatability data were analyzed using paired t-test. To test for mean differences in digestibility and stool quality outcomes between 0%, 5%, and 10% BSFL dogs, we applied the Kruskal-Wallis test. Statistical significance was set at 5% type 1 error rate. Dry matter, protein, fat, and energy digestibility, and stool quality were similar between all diets (P &amp;gt; 0.05). Palatability was improved (P &amp;lt; 0.05) with inclusion of BSFL (i.e., preference for 5% and 10% vs. 0% inclusion) and improved (P &amp;lt; 0.05) at 10% vs. 5% inclusion. The BSFL tested showed merit as a palatable ingredient source that maintains stool quality and digestibility in adult dogs.

PSIX-2 Skin microbial compositions differ among dog body sites and are affected by dietary probiotics

Abstract The study aimed to characterize the skin microbiota across four distinct body sites and investigate the impact of dietary probiotics on the skin microbiota of healthy adult dogs. Healthy adult dogs [n = 60; mean age = 6.2 ± 2.8 yr; mean body weight (BW) = 13.1 ± 4.0 kg] were randomly assigned into three treatment groups and fed for 60 d: a control kibble diet (CON; n = 20; 29.0% crude protein, 10.1% crude fat, 9.9% crude fiber, 7.1% ash, as-fed), CON supplemented with Bifidobacterium longum 548 (CON-B; n = 20), or CON supplemented with Bifidobacterium longum 548 and Lactobacillus rhamnosus 451 (CON-B-L; n = 20). All dogs were individually housed in a temperature- and humidity-controlled research facility. Protocols were approved by the facility’s Institutional Animal Care and Use Committee before the study. On d 60, skin swab samples were collected from the dorsal lumbar area, right axilla, right groin, and right ear concave pinnae of each dog using sterile swabs. These samples were placed into bead tubes and stored at -20°C until analysis. DNA extraction from skin swab samples was followed by sequencing the V4 region of 16S rRNA using Illumina sequencing on a MiSiq. Sequence data were processed through QIIME 2.0, and statistical analysis was performed using R 4.3.1. to test differences between body sites and diets, with a significance threshold set at P &amp;lt; 0.05. The predominant bacterial phyla observed across all body sites were Actinobacteria (40 ± 21.7%), Firmicutes (31.2 ± 17.9 %), Proteobacteria (17.9 ± 13.3%), and Bacteroidetes (6.6 ± 6.1%). Additionally, Acidobacteria, Gemmatimonadetes, and Verrucomicrobia were exclusive to the groin region. Considering data from all body sites, diets affected relative abundances of phyla and genera in dogs. At phylum level, dogs fed the CON-B diet displayed greater (P &amp;lt; 0.05) relative abundance of Actinobacteria compared with those on the CON diet. The CON-B diet also resulted in a greater (P &amp;lt; 0.05) relative abundance of Firmicutes than dogs on either the CON or CON-B-L diets. At genus level, dogs on the CON-B diet showed significantly greater (P &amp;lt; 0.05) relative abundance of Clostridium, Clostridiaceae unclassified, and Lachnospiraceae unclassified compared with the other diet groups. Dogs on the CON diet displayed a greater (P &amp;lt; 0.05) relative abundance of Dietzia compared with those on probiotic-supplemented diets. These findings underscore the variation in skin microbial composition across distinct body sites, contributing valuable data to limited information available on the skin microbiota of dogs. Moreover, consumption of probiotic influenced the skin microbial compositions in dogs, which warrant further studies to elucidate the relationship between probiotics, skin microbiota, and skin health in dogs.

304 Amino acid digestibility and protein quality of fermented soybean-based ingredients using the precision-fed cecectomized rooster assay

Abstract Fermented soybean-based ingredients may serve as an alternative protein source for pet foods because of their high-quality protein content. The amino acid (AA) content and protein quality of fermented soybean-based ingredients may vary depending on their composition and processing, however, so testing is required. Our objective was to measure the AA composition, AA digestibility, and protein quality of fermented soybean-based ingredients using the precision fed cecectomized rooster assay. The University of Illinois Institutional Animal Care and Use Committee approved all animal procedures prior to experimentation. Cecectomized roosters (n = 36; six roosters per substrate) were randomly allotted to one of five test substrates: 1) autoclaved soybean (ASB); 2) fermented soybeans (FSB); 3) fermented soybeans with probiotics (FSBP); 4) fermented soybean meal (FSBM); 5) fermented soybean meal with probiotics (FSBMP). After 26 h of feed withdrawal, roosters were tube-fed test substrates. Following crop intubation excreta samples were collected 48 h. Endogenous loss corrections for AA were made by using five additional cecectomized roosters. All data were analyzed using SAS version 9.4. All substrates had increased AA digestibilities, with all indispensable AA digestibilities being &amp;gt;80% except for lysine (78.1% to 86.9%). Tryptophan digestibility was less (P &amp;lt; 0.05) in FSB than other substrates, but other AA digestibilities were not different. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to nutritional guidelines for adult dogs, adult cats, growing puppies, and growing kittens. The most limiting AA was the same for all ingredients. In adult dogs, methionine was the most limiting AA based on National Research Council (NRC; DIAAS: 33.4-35.1), European Pet Food Industry Nutritional Guidelines (FEDIAF; DIAAS: 49.6-52.1), and Association of American Feed Control Officials (AAFCO; DIAAS: 60.2-63.2) guidelines. In adult cats, phenylalanine was the most limiting AA (DIAAS: 59.5-63.2) when using NRC guidelines, but DIAAS were above 100 for FEDIAF and AAFCO guidelines. In growing puppies, methionine was most limiting according to NRC (DIAAS: 56.7-59.6) and FEDIAF (DIAAS: 78.8-82.7) guidelines, but threonine was most limiting according to AAFCO (DIAAS: 68.8-72.3) guidelines. In growing kittens, methionine was most limiting according to NRC (DIAAS: 70.9-74.4) and AAFCO (53.4-56.0) guidelines, but phenylalanine was most limiting according to FEDIAF (DIAAS: 66.7-70.8) guidelines. Our results demonstrate that fermented soybean-based ingredients are high-quality protein sources. Even though they have one or more limiting AA, they would serve as strong complementary proteins for pet foods. Further research in dogs and cats is necessary to confirm sufficient palatability and digestibility, but these data suggest that they are valuable sources of protein for pet food.

According to Which Criteria Should We Determine Whether and When IACUCs Are Sufficient for Protecting the Welfare of Nonhuman Animals Used in Research?

Nonhuman animals used in biomedical research frequently suffer and are harmed as part of their use as experimental models. The Institutional Animal Care and Use Committee (IACUC) of a given institution is meant to ensure that research protocols follow federal guidelines, but research protocols such as those described in this case can generate unnecessary suffering; this problem suggests limitations of IACUCs' capacity to protect nonhuman animals' welfare. This commentary on the case considers how to more fully protect nonhuman animals used in scientific research and identifies barriers to more comprehensive protection of nonhuman animals' welfare.

The Effect of Gold Nanoparticles and Apricot Kernel Extract on the Osseointegration of Dental Implants - A Rabbit Model

Introduction The osseointegration of dental implants is critical to their longevity and success. Various factors such as surface modifications, both additive and subtractive, systemic drug administration, growth factors, and plasma derivatives can influence osseointegration. Aims and Objectives This study aimed to study the effects of gold nanoparticles and aqueous extract of apricot kernels on the osseointegration of titanium implants in the tibia of rabbits. Materials and Methods Apricot kernels were dried, grounded, and dissolved in water to obtain an aqueous extract. It was filtered and stored in the refrigerator at 4oC. Gold nanoparticles (AuNPs) were prepared by mixing 3 ml of 0.02 mM hydrogen tetrachloroaurate with 1 ml of olive leaf extract. The mixture was stirred vigorously for 15 minutes on a heater stirrer at 50oC. Titanium screw implants were sterilized in an autoclave at 120oC with 15 pounds per square inch for 30 minutes. Sterilized implants were stored in sealed containers until use. The study was carried out at the College of Veterinary Medicine, University of Mosul, Iraq. The ethical approval for the study was obtained from the institutional animal care and use committee, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, on 15/04/2023 on reference no UM.VET.2023.013. Nine healthy male adult white New Zealand rabbits aged between 10-12 months were utilized for the study. Three pure titanium implants, each of a type, were placed in each tibia. The implants were categorized as follows: control group (18 uncoated implants) and experimental group (36 coated implants). The implant placement was divided into three groups: controls with uncoated implants (n=18), AuNPs-coated implants (n=18), and apricot kernel extract-coated implants (n=18). General anesthesia was induced by intramuscular injection of ketamine hydrochloride 50mg (1ml /kg of body weight) plus xylazine 2% (0.2 ml /kg of body weight). The AuNPs-coated implants were implanted in the superior osteotomy site in the tibia. The apricot kernel extract-coated implants were implanted in the inferior osteotomy site and the uncoated implants were implanted in the middle osteotomy sites. Histological and histomorphometric tests were performed on all bone implant blocks at one-week, two-week, and three-week intervals after implant placement. The results were evaluated based on the amount of bone formation, cellular components, and inflammation. Tabulated data were statistically analyzed using SPSS v23.0 (IBM, Armonk, NY) using ANOVA with a statistical significance of p &lt;0.05 Results We observed a significant amount of osteogenesis with reduced inflammation in AuNPs-coated implants and apricot kernel extract-coated implants compared to untreated implants. The osteoclastic activity was significantly higher in untreated implants than the treated implant sites. A statistically significant association was observed between coated implants and untreated implants. Conclusion The application of gold nanoparticles and aqueous extract of apricot kernels appears to be promising in reducing inflammation and improving bone apposition on the titanium implant surface. However, this preliminary investigation warrants further research.

Incidence of Intra-abdominal Adhesions Following Intraperitoneal Injection of Hemostatic Products in Rabbits.

Definitive management of non-compressible intra-abdominal hemorrhage (NCIAH) currently requires a surgeon and operating room capable of performing damage control surgery. In a wartime scenario or a geographically remote environment, these may not be readily available. In this study, we sought to test the safety of 2 emerging injectable hemostatic agents (CounterFlow and Fast Onset Abdominal Management, or FOAM, poloxamer component) versus normal saline control over a prolonged monitoring duration following administration by a non-surgical provider. The Institutional Animal Care and Use Committee approved all research conducted in this study. We randomized male New Zealand white rabbits into 2 monitoring cohorts of 24 hours and 2 weeks. Each cohort contained 3 treatment groups (n = 4 rabbits/group): CounterFlow, the testable poloxamer component of FOAM, and normal saline control. We injected each treatment intraperitoneally in the left lower abdominal quadrant. Doses were 15 mL/kg for CounterFlow, 6.3 mL/kg for the poloxamer component of FOAM, and 15 mL/kg for normal saline. We conducted all injections under isoflurane anesthesia monitored by trained veterinary staff. Animals were euthanized at each cohort end point, and a veterinary pathologist blinded to treatment type performed necropsy. The primary outcome was incidence of intra-abdominal adhesions at necropsy. Quantitatively, adhesions when present were graded by the veterinary pathologist on a 1 to 4 scale, where "1" represented adhesions involving from 1 to 25% of the examined abdomen, "2" represented from 26 to 50%, "3" represented from 51 to 75%, and "4" represented from 76 to 100%. Qualitatively, adhesions present were graded by degree ("1" = minimal, "2" = mild, "3" = moderate, and "4" = severe) and chronicity ("1" = acute, "2" = subacute, and "3" = chronic). We also drew d-dimer blood values and measured body weights for each animal. Statistical analysis included either repeated measures 2-way ANOVA or a mixed-effects model (in the case of missing data) with Geisser-Greenhouse correction. We adjusted multiple comparisons using Tukey statistical hypothesis tests. In the 2-week cohort, 3 CounterFlow animals showed adhesions judged to be "1" quantitatively. Qualitatively, 2 of these were assessed as "1" for degree of adhesions and the other demonstrated a "2." On the chronicity of adhesions scale, 1 animal demonstrated a "2" and 2 demonstrated a "3." No animals in other groups (FOAM and control) demonstrated adhesions. CounterFlow-treated animals showed a statistically significant rise in d-dimer values in the 24-hour cohort only. In the 2-week cohort, CounterFlow-treated animals showed a decrease in body weight at 24 hours after injection but returned to their baseline (normal) body weights at 7 days. Findings from this study demonstrate that the tested ingredients of FOAM poloxamer component are safe for intraperitoneal injection and hold potential for further study directed toward prehospital non-compressible intra-abdominal hemorrhage management by non-surgical providers. Although CounterFlow produced abdominal adhesions in 3 of 4 rabbits in the 2-week cohort, these were determined to be "minimal" or "mild" in degree.

Preliminary Promising Findings for Manganese Chloride as a Novel Radiation Countermeasure Against Acute Radiation Syndrome.

Military members and first responders may, at moment's notice, be asked to assist in incidents that may result in radiation exposure such as Operation Tomadachi in which the U.S. Navy provided significant relief for the Fukushima Daiichi Nuclear Reactor accident in Japan after an earthquake and tsunami in 2011. We are also currently facing potential threats from nuclear power plants in the Ukraine should a power disruption to a nuclear plant interfere with cooling or other safety measures. Exposure to high doses of radiation results in acute radiation syndrome (ARS) characterized by symptoms arising from hematopoietic, gastrointestinal, and neurovascular injuries. Although there are mitigators FDA approved to treat ARS, there are currently no FDA-approved prophylactic medical interventions to help protect persons who may need to respond to radiation emergencies. There is strong evidence that manganese (Mn) has radiation protective efficacy as a promising prophylactic countermeasure. All animal procedures were approved by the Institutional Animal Care and Use Committee. Male and female B6D2F1J mice, 10 to 11 weeks old, were used for neurotoxicity studies and temporal effects of Mn. Four groups were evaluated: (1) vehicle injection, (2) dose of 4.5 mg/kg for 3 days, (3) dose of 13.5 mg/kg, and (4) sham. Irradiated mice were exposed to 9.5 Gy whole body Co60 γ-radiation. MRI was performed with a high dose of manganese chloride (MnCl2) (150 mg/kg) to assess the distribution of the MnCl2. The mice have promising survival curves (highest survival-13.5 mg/kg dose over 3 days of MnCl2 at 80% [87% female, 73% male] P = 0.0004). The complete blood count (CBC) results demonstrated a typical hematopoietic response in all of the irradiated groups, followed by mildly accelerated recovery by day 28 in the treated groups. No difference between groups was measured by Rota Rod, DigiGait, and Y-maze. Histologic evaluation of the bone marrow sections in the group given 13.5 mg/kg dose over 3 days had the best return to cellularity at 80%. MRI showed a systemic distribution of MnCl2. The preliminary data suggest that a dose of 13.5 mg/kg of MnCl2 given over 3 days prior to exposure of radiation may have a protective benefit while not exhibiting the neurobehavioral problems. A countermeasure that can prophylactically protect emergency personnel entering an area contaminated with high levels of radiation is needed, especially in light that nuclear accidents are a continued global threat. There is a need for a protective agent with easy long-term storage, easy to transport, easy to administer, and low cost. Histologic evaluation supports the promising effect of MnCl2 in protecting tissue, especially the bone marrow using the dose given over 3 days (4.5 mg/kg per day) of MnCl2. Initial experiments show that MnCl2 is a promising safe and effective prophylactic countermeasure against ARS. MRI data support the systemic distribution of MnCl2 which is needed in order to protect multiple tissues in the body. The pathology data in bone marrow and the brain support faster recovery from radiation exposure in the treated animals and decreased organ damage.

Novel High-tech, Low-cost Ventilation for Military Use, Mass Casualty Incidents, Stockpiling, and Underserved Communities.

Despite the significant need for mechanical ventilation in- and out-of-hospital, mechanical ventilators remain inaccessible in many instances because of cost or size constraints. Mechanical ventilation is especially critical in trauma scenarios, but the impractical size and weight of standard mechanical ventilators restrict first responders from carrying them in medical aid bags, leading to reliance on imprecise manual bag-mask ventilation. This is particularly important in combat-related injury, where airway compromise and respiratory failure are leading causes of preventable death, but medics are left without necessary mechanical ventilation. To address the serious gaps in mechanical ventilation accessibility, we are developing an Autonomous, Modular, and Portable Ventilation platform (AMP-Vent) suitable for austere environments, prolonged critical care, surgical applications, mass casualty incidents, and stockpiling. The core system is remarkably compact, weighing <2.3 kg, and can fit inside a shoebox (23.4 cm × 17.8 cm × 10.7 cm). Notably, this device is 65% lighter than standard transport ventilators and astoundingly 96% lighter than typical intensive care unit ventilators. Beyond its exceptional portability, AMP-Vent can be manufactured at less than one-tenth the cost of conventional ventilators. Despite its reduced size and cost, the system's functionality is uncompromised. The core system is equipped with closed-loop sensors and advanced modes of ventilation (pressure-control, volume-control, and synchronized intermittent mandatory ventilation), enabling quality care in a portable form factor. The current prototype has undergone preliminary preclinical testing and optimization through trials using a breathing simulator (ASL 5000) and in a large animal model (swine). This report aims to introduce a novel ventilation system and substantiate its promising performance through evidence gathered from preclinical studies. Lung simulator testing was performed using the ASL 5000, in accordance with table201.105 "pressure-control inflation-type testing" from ISO 80601-2-12:2020. Following simulations, AMP-Vent was tested in healthy 10-kg female domestic piglets. The Children's Hospital of Philadelphia Institutional Animal Care and Use Committee approved all animal procedures. Swine received 4-min blocks of alternating ventilation, where AMP-Vent and a conventional anesthesia ventilator (GE AISYS CS2) were used to titrate to varied end-tidal carbon dioxide (EtCO2) goals with the initial ventilator switching for each ascending target (35, 40, 45, 50, 55 mmHg). During ASL 5000 simulations, AMP-Vent exhibited consistent performance under varied conditions, maintaining a coefficient of variation of 2% or less within each test. In a large animal study, AMP-Vent maintained EtCO2 and SpO2 targets with comparable performance to a conventional anesthesia ventilator (GE AISYS CS2). Furthermore, the comparison of minute ventilation (Ve) distributions between the conventional anesthesia ventilator and AMP-Vent at several EtCO2 goals (35, 40, 45, 50, and 55 mmHg) revealed no statistically significant differences (p = 0.46 using the Kruskal-Wallis rank sum test). Preclinical results from this study highlight AMP-Vent's core functionality and consistent performance across varied scenarios. AMP-Vent sets a benchmark for portability with its remarkably compact design, positioning it to revolutionize trauma care in previously inaccessible medical scenarios.

The partnership between statisticians and the Institutional Animal Care and Use Committee (IACUC)

AbstractIn this tutorial we explore the valuable partnership between statisticians and Institutional Animal Care and Use Committees (IACUCs) in the context of animal research, shedding light on the critical role statisticians play in ensuring the ethical and scientifically rigorous use of animals in research. Pharmaceutical statisticians have increasingly become vital members of these committees, contributing expertise in study design, data analysis, and interpretation, and working more generally to facilitate the integration of good statistical practices into experimental procedures. We review the “3Rs” principles (Replacement, Reduction, and Refinement) which are the foundation for the humane use of animals in scientific research, and how statisticians can partner with IACUC to help ensure robust and reproducible research while adhering to the 3Rs principles. We also highlight emerging areas of interest, such as the use of virtual control groups.

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

204 Detecting subclinical mastitis in meat-breed ewes using infrared thermography

Abstract Mastitis, the inflammation of the mammary gland most commonly due to bacterial infection, is an important welfare concern in meat-breed sheep. The objective of our study was to evaluate udder surface temperature obtained from infrared thermography as a diagnostic tool for subclinical mastitis. We predicted that udder halves with subclinical mastitis would have greater surface temperatures than healthy udder halves. We enrolled 39 meat-breed ewes (10 Hampshire, 24 Polypay, 5 Targhee; 2 to 9 yr of age) at the University of Wisconsin-Madison sheep unit. All procedures were approved by the UW-Madison Institutional Animal Care and Use Committee. Milk samples from each udder half were collected in duplicate weekly beginning 2 to 4 d postpartum until 8 wk postpartum. Immediately before milk sampling, the udder was photographed from the rear using a thermal camera. A sample was considered a true infection if at least 100 CFU/mL of a bacterial species were isolated. From each photo, we obtained values for minimum, maximum, and mean skin surface temperature for the right and left udders. Three linear mixed models were fit using minimum, maximum, and mean temperatures as the response variables. Fixed effects were infection status (infected or not), weeks postpartum (wk 1-8), and ambient temperature, with a random effect of udder half nested within animal ID. Contrary to our predictions, infection status did not predict udder skin surface temperature (P &amp;gt; 0.12). Healthy udder halves had a mean temperature of 35.5 ± 0.1°C (mean ± SE), minimum temperature of 29.9 ± 0.3°C, and maximum temperature of 37.8 ± 0.1°C. Infected udder halves had a mean temperature of 35.4 ± 0.1°C, minimum temperature of 30.5 ± 0.3°C, and maximum temperature of 37.7 ± 0.1°C. Our results contradict findings in dairy cows, and suggest more research is needed to evaluate the relationship between udder surface temperature and subclinical mastitis in sheep.

Effect of Maternal High Fat Diet on the Hypothalamus-Pituitary-Adrenal Gland (HPA) Axis of the Offspring in Sheep

Maternal consumption of a high fat diet (HFD) during pregnancy is associated with metabolic dysfunction and altered HPA axis function in the offspring. These effects are well established in species with short gestation length (e.g., rodents), but less established in species with longer gestation periods more comparable with humans. The objective of the study was to determine the effect of maternal HFD on basal and stimulated cortisol secretion in the offspring at 16 weeks of age in sheep. All experiments were approved by the Institutional Animal Care and Use Committee at Cal Poly Pomona. Pregnant ewes were assigned to control (n=3) and HFD (n=3) groups. The control group was fed farmers best complete ewe and ram pellets. The HFD group was fed the same diet with an additional 30% rumen protected fat (Megalac, Volac Wilmar Feed Ingredients Ltd). After birth lambs were weaned at 12 weeks of age and given free access to control pellet diet with grains. To assess adrenal cortisol secretion at 16 weeks, lambs were infused intravenously with adrenocorticotropic hormone (ACTH; 2 ug/kg cortrosyn in 1 mL saline). Blood samples (1 mL) were taken through the jugular catheter at −30, −15, and 0 min (immediately before) and 5, 15, 30, 60, 90, and 120 min after ACTH administration for measurement of plasma cortisol by ELISA. Data were analyzed by two-way repeated measures ANOVA with a Tukey posthoc test. Basal plasma cortisol tended to be higher in HFD (32±8 ng/mL) than control (13±3 ng/mL) lambs, but this did not reach statistical significance. Plasma cortisol was higher than baseline from 15 to 90 minutes after ACTH administration in all lambs (p&lt;0.05). The increment in plasma cortisol was higher in HFD than control lambs from 15-90 minutes (p&lt;0.05). Accordingly, the maximum increase in plasma cortisol at 60 minutes and was higher in HFD (245±8 ng/mL) than control lambs (121±50 ng/mL p&lt;0.05). These preliminary data suggest that maternal consumption of a high fat diet during pregnancy may increase basal and stimulated activity of the HPA axis in sheep. The study was funded by a Strategic Interdisciplinary Research Grant (SIRG) at California State Polytechnic University, Pomona. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shenqi Pill Mitigates Renal Interstitial Fibrosis ThroughWeakening Notch1/Jag1 Pathway.

Shenqi pill (SQP) can be used to treat various kidney related diseases, but its exact mechanism of action remains unclear. We intended to analyze the role and mechanism of SQP on renal interstitial fibrosis (RIF). After performing unilateral ureteral obstruction (UUO) surgery following the Institutional Animal Care and Use Committee guidelines, all rats were assigned into the sham group, UUO group, UUO + SQP 1.5 g/kg, UUO + SQP 3 g/kg, and UUO + SQP 6 g/kg groups. After treatment with SQP for 4 weeks, the appearance of kidney, serum creatinine (SCr), and blood urea nitrogen (BUN) levels were monitored in each group. The pathological injury, extracellular matrix (ECM), and Notch1 pathway-related protein levels were measured using H&E staining, Masson staining, immunohistochemistry, and Western blot, respectively. SQP could obviously ameliorate the appearance of the kidney as well as the levels of SCr and BUN in UUO rats (SCr: 67.6 ± 4.64 μM, 59.66 ± 4.96 μM, 48.76 ± 4.44 μM, 40.43 ± 3.02 μM for UUO, low, medium, and high SQP treatment groups; BUN: 9.09 ± 0.97 mM, 7.72 ± 0.61 mM, 5.42 ± 0.42 mM, 4.24 ± 0.34 mM for UUO, low, medium, and high SQP treatment groups; P < .05). SQP also effectively mitigated renal tissue injury in UUO rats (P < .05). Moreover, we uncovered that SQP significantly inhibited Collagen I, α-SMA, Collagen IV, TGF-B1, Notch1, and Jag1 protein expressions in UUO rats kidney (P < .05). Our data elucidated that SQP can alleviate RIF, and the mechanism may be related to the Notch1/Jag1 pathway. DOI: 10.52547/ijkd.7703.

Indian Gooseberry and Barley Sprout Complex Prevent Oxidative Stress and Photoaging of the Skin in Ultraviolet B-Irradiated SHK-I Mice.

This study investigated the protective effects of a complex of Indian gooseberry and barley sprout (IB complex) on oxidative stress and skin damage caused by ultraviolet B irradiation in SHK-I hairless mice. The study examined the impact of IB complex on skin hydration, wrinkle formation, and melanogenesis using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot analysis. The IB complex reduced skin hydration loss and wrinkle formation, while also demonstrating enhanced antioxidant activities. The IB complex maintained skin hydration via upregulation of hyaluronic acid and ceramide synthesis, including the regulation of hyaluronic acid synthase, long-chain ceramide formation, dihydroceramide desaturase 1 activity, and type I collagen production. The IB complex prevented wrinkle formation via downregulating JNK and upregulating TGF-β pathways. Moreover, IB complex blocked melanin production via inhibition of protein kinase A, cAMP response element-binding protein, and microphthalmia-associated transcription factor pathways. These results suggest that IB complex is a potential agent to protect the skin against photodamage caused by exposure to UVB radiation. The research protocols underwent approval from the Institutional Animal Care and Use Committee of Kyung Hee University (KHGASP-21-577), ensuring compliance with ethical standards.

A New Method for Creating Impact-Induced Intra-Articular Fractures in a Rabbit Model Induces Severe Post-Traumatic Osteoarthritis.

The objective of this work was to develop a model of intra-articular fracture (IAF) in a rabbit and document the speed and severity of degenerative joint changes after fracture fixation. With Institutional Animal Care & Use Committee approval, impact-induced IAFs were created in the distal tibia of 16 New Zealand White rabbits. Fractures were fixed with a plate and screws. Pain and function were monitored at regular postoperative intervals with limb loading analysis. Twelve or 26 weeks after fracture, animals were euthanized for histological assessment of cartilage degeneration and micro-computed tomography analysis of bone histomorphometry. Eleven animals successfully completed the study. Maximum foot force in the fractured limb was 41% ± 21% lower than preoperative values ( P = 0.006) 12 weeks after fracture and remained 25% ± 13% lower ( P = 0.081) after 26 weeks. Cortical bone mineral density in micro-computed tomography images was 34% ± 13% lower 12 weeks after fracture ( P < 0.001) and remained (42% ± 8%) lower 26 weeks after fracture ( P < 0.001). Twelve weeks after fracture, Mankin scores of cartilage degeneration were significantly higher in the medial talus ( P = 0.007), lateral talus ( P < 0.001), medial tibia ( P = 0.017), and lateral tibia ( P = 0.002) of the fractured limb compared with the uninjured contralateral limb. Average Mankin scores in the talus increased from 12 to 26 weeks (5.9 ± 0.9 to 9.4 ± 0.4; P < 0.001 lateral; 5.4 ± 1.8 to 7.8 ± 2.0; P = 0.043 medial), indicating substantial and progressive joint degeneration. The ankle joint of the New Zealand White rabbit provides the smallest available model of impact-induced IAF that can be treated with clinically relevant techniques and replicates key features of healing and degeneration found in human patients.

Long-Term Ultrasound Twinkling Detectability and Safety of a Polymethyl Methacrylate Soft Tissue Marker Compared to Conventional Breast Biopsy Markers—A Preclinical Study in a Porcine Model

ObjectiveWe have studied the use of polymethyl methacrylate (PMMA) as an alternative biopsy marker that is readily detectable with ultrasound Doppler twinkling in cases of in vitro, ex vivo, or limited duration in vivo settings. This study investigates the long-term safety and ultrasound Doppler twinkling detectability of a PMMA breast biopsy marker following local perturbations and different dwell times in a 6-mo animal experiment. MethodsThis study, which was approved by our Institutional Animal Care and Use Committee, involved three pigs and utilized various markers, including PMMA (Zimmer Biomet), 3D-printed, and Tumark Q markers. Markers were implanted at different times for each pig. Mesh material or ethanol was used to induce a local inflammatory reaction near certain markers. A semiquantitative twinkling score assessed twinkling for actionable localization during monthly ultrasounds. At the primary endpoint, ultrasound-guided localization of lymph nodes with detectable markers was performed. Following surgical resection of the localized nodes, histomorphometric analysis was conducted to evaluate for tissue ingrowth and the formation of a tissue rind around the markers. ResultsNo adverse events occurred. Twinkling scores of all markers for all three pigs decreased gradually over time. The Q marker exhibited the highest mean twinkling score followed by the PMMA marker, PMMA with mesh, and Q with ethanol. The 3D-printed marker with mesh and PMMA with ethanol had the lowest scores. All wire-localized lymph nodes were successfully resected. Despite varying percentages of tissue rind around the markers and a significant reduction in overall twinkling (p < 0.001) over time, mean PMMA twinkling scores remained clinically actionable at 6 and 5 mo using a General Electric C1-6 probe and 9L-probe, respectively. ConclusionsIn this porcine model, the PMMA marker demonstrates an acceptable safety profile. Clinically actionable twinkling aids PMMA marker detection even after 6 mo of dwell time in porcine lymph nodes. The Q marker maintained the greatest twinkling over time compared to all the other markers studied.

Mouse Anesthesia and Analgesia

AbstractProviding anesthesia and analgesia for mouse subjects is a common and critical practice in the laboratory setting. This practice is necessary for performing invasive procedures, achieving prolonged immobility for sensitive imaging modalities (magnetic resonance imaging, for instance), and providing intra‐ and post‐procedural pain relief. In addition to facilitating the procedures performed by the investigator, the provision of anesthesia and analgesia is crucial for the preservation of animal welfare and for humane treatment of animals used in research. Furthermore, anesthesia and analgesia are important components of animal use protocols reviewed by Institutional Animal Care and Use Committees, requiring careful consideration and planning for the particular animal model. In this article, we provide technical guidance for the investigator, covering the provision of anesthesia by two routes (injectable and inhalant), guidelines for monitoring anesthesia, current techniques for recognition of pain, considerations for administering preventative analgesia, and considerations for post‐operative care. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Injectable anesthesiaBasic Protocol 2: Inhalant anesthesiaBasic Protocol 3: Assessing pain