Quantification of Skeletal Muscle Perfusion in Peripheral Artery Disease Using 18F-Sodium Fluoride Positron Emission Tomography Imaging.

BACKGROUND: Perfusion deficits contribute to symptom severity, morbidity, and death in peripheral artery disease (PAD); however, no standard method for quantifying absolute measures of skeletal muscle perfusion exists. This study sought to preclinically test and clinically translate a positron emission tomography (PET) imaging approach using an atherosclerosis-targeted radionuclide, fluorine-18-sodium fluoride (18F-NaF), to quantify absolute perfusion in PAD. METHODS AND RESULTS: Eight Yorkshire pigs underwent unilateral femoral artery ligation and dynamic 18F-NaF PET/computed tomography imaging on the day of and 2 weeks after occlusion. Following 2-week imaging, calf muscles were harvested to quantify microvascular density. PET methodology was validated with microspheres in 4 additional pig studies and translated to patients with PAD (n=39) to quantify differences in calf perfusion across clinical symptoms/stages and perfusion responses in a case of revascularization. Associations between PET perfusion, ankle-brachial index, toe-brachial index, and toe pressure were assessed in relation to symptoms. 18F-NaF PET/computed tomography quantified significant deficits in calf perfusion in pigs following arterial occlusion and perfusion recovery 2 weeks after occlusion that coincided with increased muscle microvascular density. Additional studies confirmed that PET-derived perfusion measures agreed with microsphere-derived perfusion measures. Translation of imaging methods demonstrated significant decreases in calf perfusion with increasing severity of PAD and quantified perfusion responses to revascularization. Perfusion measures were also significantly associated with symptom severity, whereas traditional hemodynamic measures were not. CONCLUSIONS: 18F-NaF PET imaging quantifies perfusion deficits that correspond to clinical stages of PAD and represents a novel perfusion imaging strategy that could be partnered with atherosclerosis-targeted 18F-NaF PET imaging using a single radioisotope injection. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03622359.

Evaluation of Disinfection Methods for Artificial Plants in Zebrafish (Danio rerio) Recirculating Water Systems.

The use of artificial plants as environmental enrichment for zebrafish (Danio rerio) in biomedical research facilities has been shown to provide benefits in animal welfare and care. Despite the benefits of artificial plants to zebrafish welfare, some research facilities are hesitant to incorporate them into their routine husbandry practices due to concerns about disease transmission and a lack of guidance on effective disinfection practices between tanks. Limited published information is available on how to adequately disinfect artificial plants, which creates concerns regarding their reuse between tanks in recirculating water systems. Proper sanitation and disinfection of these items is crucial to preventing the spread of disease in the system. We evaluated 2 disinfection methods- a commercial-grade laboratory glassware dishwasher and an ethylene oxide (ETO) sterilizer-by using ATP detection and bacterial culture of the artificial plants before and after the disinfection process. Plants were placed in the dirty sump of 2 separate recirculating systems (2,500 to 3,000 fish per system) for 2 wk before the start of the study. High ATP levels and various bacterial organisms were detected prior to disinfection. The commercial-grade labo- ratory glassware dishwasher and ETO sterilizer both significantly reduced ATP levels and resulted in complete eradication of live bacteria that were present before treatment. This study demonstrates 2 effective methods for disinfecting artificial plants in zebrafish facilities.

Computational analysis of serum-derived extracellular vesicle miRNAs in juvenile sheep model of single stage Fontan procedure.

Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Supporting diversity, equity, inclusion, and belonging to strengthen and position the veterinary profession for service, sustainability, excellence, and impact.

Advancing equality and equity in society is creating positive change, and the time has come to critically evaluate veterinary medicine, which, by all metrics, lacks diversity. To keep pace with increasingly diverse demographics and recent surges in pet ownership among all racial/ethnic groups, significant efforts to enhance diversity, equity, inclusion, and belonging (DEIB) must occur in veterinary colleges and the profession. Recruiting more underrepresented students, building pipelines for diverse faculty/staff, and creating inclusive, welcoming environments where all can thrive are critical steps toward enhancing DEIB within our organizations and profession. Our goal is to share experiences and lessons learned from our intentional commitment to strengthen DEIB, with the hope that our journey will be helpful to others. Increasing diversity in the veterinary profession will be facilitated through removing barriers, creating inclusive work environments where all people feel they belong, and ensuring fair and equitable hiring and personnel management practices. These steps should in turn improve access and quality of veterinary care, ensure we are more representative of the communities we serve, increase revenue, and preserve the human-animal bond. "You cannot change any society unless you take responsibility for it, unless you see yourself belonging to it, and responsible for changing it." - Grace Lee Boggs.

Internal Temperatures of Packaging for Overnight Cross-country Shipping of Zebrafish (Danio Rerio).

As the use of zebrafish (Danio rerio) as a research model continues to rise, so too will the shipping and sharing of zebrafish strains across collaborating institutions. If done incorrectly, shipping can result in significant mortality, welfare concerns, and loss of valuable resources for researchers and research institutions. Here we introduce a novel method to track temperatures of zebrafish containers during shipping and show that internal packaging temperatures are directly affected by the external temperatures. We used temperature logging Thermochron iButtons to track the temperatures of 2 packages containing adult zebrafish that were shipped overnight from Dallas, TX to Columbus, OH during winter following recommended fish shipping guidelines. We found that the external packaging of both boxes of fish were exposed to temperatures that had previously been shown to be lethal to zebrafish. However, internal temperatures and, more specifically, water temperature, stayed within 24.0 to 26.5°C during shipment, resulting in 100% survival of adult zebrafish. This novel method of tracking packaging temperatures of live fish during shipping can help to inform fish health status on arrival.

Detection of Zoonotic Bacteria and Paragonimus kellicotti in Red Swamp Crayfish (Procambarus clarkii) and the Assessment of Traditional Crayfish Boils.

ABSTRACT: Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P < 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Anesthesia with Tricaine Methanesulfonate (MS222) and Propofol and Its Use for Computed Tomography of Red Swamp Crayfish (Procambarus clarkii).

Crayfish (Decapoda: Astacoidea and Parastacoidea) are among the few animals that have stem cells in hemolymph, with the capacity to continuously produce differentiated neuronal structures throughout life. As the use of crayfish and other invertebrates increases in biomedical research, we must develop laboratory standards and guidelines for performing clinical procedures. This manuscript presents introductory protocols for anesthesia in crayfish during diagnostic imaging. Five anesthetic protocols were evaluated: immersion in buffered tricaine methanesulfonate (MS222; 50 mg/L); immersion in buffered MS222 (150 mg/L); immersion in propofol (65 mg/L); injection of propofol (50 mg/kg); and injection of propofol (100 mg/kg) into the ventral surface of an abdominal somite. MS222 immersion (50 and 150 mg/L) had no observable effect on crayfish. After an extended period of time, immersion in propofol (65 mg/L) created a sedative effect suitable for short-term handling. Propofol injection (50 mg/kg) into the ventral surface of an abdominal somite created an effective plane of anesthesia without adverse effects during or after recovery. Propofol injection at 100 mg/kg had adverse effects and is not recommended for use in crayfish. CT imaging was performed successfully as proof of concept for handling anesthetized crayfish. These findings provide initial data for the anesthetization of crayfish used in research settings.

Continual cerebrospinal fluid sampling in the neonatal domestic piglet for biomarker and discovery studies.

BACKGROUND: Longitudinal access to cerebrospinal fluid (CSF) is useful for biomarker discovery in neurological disorders or diseases affecting CSF composition. Here, we aim to test a new method for insertion of a permanent intrathecal catheter, facilitating longitudinal collection of CSF. NEW METHOD: We surgically placed a permanent intrathecal catheter into the cisterna magna of anesthetized neonatal piglets. The thecal sac was accessed at the L5-S1 spinal level and a radiopaque catheter was inserted under fluoroscopic x-ray guidance to position the tip at the cisterna magna. A titanium access port was connected to the catheter and anchored subcutaneously. Immediately after surgery, we confirmed CSF flow through the catheter and port via needle aspiration. Catheter patency over a two-month study period was determined through periodic CSF collection from the port. RESULTS: Frequent (up to 3 times weekly), longitudinal sampling of CSF was achievable in neonatal piglets up to 60 days after implantation. CSF was readily accessible through the port without major adverse events. Catheterized piglets demonstrated slower, but normal, weight gain compared to control piglets. Post-operative complications were managed with standard access precautions and medications. There were no complications involving the implanted hardware. COMPARISON WITH EXISTING METHOD(S): This method fills a critical gap in the existing methods for longitudinal CSF sampling through an implanted intrathecal catheter system in neonatal piglets. CONCLUSIONS: This novel method is both safe and effective for longitudinal CSF access in the domestic piglet. Catheter patency and access to CSF is maintained over multiple months without major adverse events.

Premature delivery in the domestic sow in response to in utero delivery of AAV9 to fetal piglets.

Numerous pediatric neurogenetic diseases may be optimally treated by in utero gene therapy (IUGT); but advancing such treatments requires animal models that recapitulate developmental physiology relevant to humans. One disease that could benefit from IUGT is the autosomal recessive motor neuron disease spinal muscular atrophy (SMA). Current SMA gene-targeting therapeutics are more efficacious when delivered shortly after birth, however postnatal treatment is rarely curative in severely affected patients. IUGT may provide benefit for SMA patients. In previous studies, we developed a large animal porcine model of SMA using AAV9 to deliver a short hairpin RNA (shRNA) directed at porcine survival motor neuron gene (Smn) mRNA on postnatal day 5. Here, we aimed to model developmental features of SMA in fetal piglets and to demonstrate the feasibility of prenatal gene therapy by delivering AAV9-shSmn in utero. Saline (sham), AAV9-GFP, or AAV9-shSmn was injected under direct ultrasound guidance between gestational ages 77-110 days. We developed an ultrasound-guided technique to deliver virus under direct visualization to mimic the clinic setting. Saline injection was tolerated and resulted in viable, healthy piglets. Litter rejection occurred within seven days of AAV9 injection for all other rounds. Our real-world experience of in utero viral delivery followed by AAV9-related fetal rejection suggests that the domestic sow may not be a viable model system for preclinical in utero AAV9 gene therapy studies.

PATET ratio by Doppler echocardiography: noninvasive detection of pediatric pulmonary arterial hypertension.

Pulmonary artery acceleration time (PAT) and PAT: ejection time (PATET) ratio are echocardiographic measurements of pulmonary arterial hypertension (PAH). These noninvasive quantitative measurements are ideal to follow longitudinally through the clinical course of PAH, especially as it relates to the need for and/or response to treatment. This review article focuses on the current literature of PATET measurement for infants and children as it relates to the shortening of the PATET ratio in PAH. At the same time, further development of PATET as an outcome measure for PAH in preclinical models, particularly mice, such that the field can move forward to human clinical studies that are both safe and effective. Here, we present what is known about PATET in infants and children and discuss what is known in preclinical models with particular emphasis on neonatal mouse models. In both animal models and human disease, PATET allows for longitudinal measurements in the same individual, leading to more precise determinations of disease/model progression and/or response to therapy. IMPACT: PATET ratio is a quantitative measurement by a noninvasive technique, Doppler echocardiography, providing clinicians a more precise/accurate, safe, and longitudinal assessment of pediatric PAH. We present a brief history/state of the art of PATET ratio to predict PAH in adults, children, infants, and fetuses, as well as in small animal models of PAH. In a preliminary study, PATET shortened by 18% during acute hypoxic exposure compared to pre-hypoxia. Studies are needed to establish PATET, especially in mouse models of disease, such as bronchopulmonary, as a routine measure of PAH.

Endovascular repair and open repair surgery of thoraco-abdominal aortic aneurysms cause drastically different types of spinal cord injury.

Both endovascular repair (EVR) and open repair (OR) surgery of thoraco-abdominal aortic aneurysms cause spinal cord (SC) injury that can lead to paraparesis or paraplegia. It has been assumed that mechanisms responsible for SC damage after EVR are similar to those after OR. This pilot study compared the pathophysiology of SC injury after EVR versus OR using a newly developed EVR dog model. An increasing number of stents similar to those used in patients were inserted in the aorta of three dogs to ensure thoracic or thoracic plus lumbar coverage. The aorta of OR dogs was cross-clamped for 45 min. Behavior assessment demonstrated unique patterns of proprioceptive ataxia and evolving paraparesis in EVR versus irreversible paraplegia in OR. MRI showed posterior signal in lumbar SC after EVR versus central cord edema after OR. Histopathology showed white matter edema in L3-L5 localized to the dorsal column medial lemniscus area associated with loss of myelin basic protein but not neurons after EVR, versus massive neuronal loss in the gray matter in L3-L5 after OR. Metabolome analysis demonstrates a distinctive chemical fingerprint of cellular processes in both interventions. Our results call for the development of new therapeutics tailored to these distinct pathophysiologic findings.

Evaluation of the Sterility of Reynolds Wrap Aluminum Foil for Use During Rodent Surgery.

In biomedical research, surgeons are often responsible for simultaneously conducting rodent surgical procedures, monitoring anesthesia, and adjusting nonsterile equipment. Maintaining appropriate aseptic technique can be challenging when working under these conditions. Applying a sterile barrier material such as aluminum foil to nonsterile surfaces in these circumstances offers an innovative, inexpensive option to improve asepsis. The purpose of this study was to validate the sterility of foodgrade aluminum foil for use as a sterile barrier on nonsterile equipment during rodent surgery. In this investigation, 10 boxes of aluminum foil were assessed for sterility by using ATP swabs and replicate organism detection and counting (RODAC) plates at 0, 14, and 28 d and 6 mo. At 6 mo, foil was applied to surgical equipment, and sterility was assessed by using ATP swabs and RODAC plates. Results revealed no ATP-positive results at any time point. During assessment of samples obtained directly from boxes, RODAC plates yielded minimal bacterial growth (1 cfu per plate) in 2 of the 10 boxes at initial testing and in 1 box at the day 0, day 14, and 6 mo time points. No growth was observed at day 28 (tested directly from the box) or at 6-mo apparatus testing. Our data revealed minimal bacterial growth on tested samples and support the use of Reynolds Wrap aluminum foil as a sterile barrier on nonsterile surfaces during aseptic rodent surgery.

Effects of Cling Film Draping Material on Body Temperature of Mice During Surgery.

General anesthesia induces many systemic effects, including thermoregulatory impairment and subsequent perioperative hypothermia. Due to the animals' small size, monitoring and maintaining body temperatures in laboratory rodents during anesthesia is important for successful surgical outcomes and prompt anesthetic recovery. Draping materials have the potential to aid in thermal support during surgical anesthesia. In this study, rectal and surface (infrared) temperatures were measured in C57BL/6 mice under isoflurane anesthesia every 5 min for the duration of a 35-min sham surgery. In addition to placement on a circulating water bath, mice (n = 6/group) were draped with commercial cling film (CF; Press'n Seal, Glad, Oakland, CA), a conventional paper drape (PD), or no drape (ND) during surgery. Results demonstrated that CF-draped animals had significantly higher rectal temperatures than nondraped animals. Furthermore, surface temperatures of CF-draped mice were considerably higher than those of both paper-draped and undraped animals. The data indicate that cling film is an effective material to help minimize hypothermia in mice and potentially in other laboratory rodents requiring general anesthesia.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Improvement of osseointegration by recruiting stem cells to titanium implants fabricated with 3D printing.

Slow and incomplete osseointegration and loss of osseointegration are major problems in dental and bone implants. We designed implants with interconnected 3D-tubulous structures and hypothesized that such interconnecting 3D (I3D) structures would serve as a repository for chemoattractants to recruit stem cells to promote osseointegration. A concept Laser Mlab-cusing-R laser-powder-bed-fusion (LPBF) 3D printing system was used to produce titanium implants with designed features. The implants were loaded (coated) with stromal cell-derived factor-1 alpha (SDF-1α), and subjected to stem cell recruitment. Implants were then surgically transplanted into the rabbit skull bone. After 12 weeks, osseointegration was analyzed by reverse-torque test and the implants were examined for calcium deposition by Alizarin Red staining. The I3D implants attracted significantly more stem cells than solid implants when coated (loaded) with SDF-1α. Greater torque force was needed to extract the I3D implants with 200 and 300 µm I3D structures than to extract solid implants from the skull. Generally, more calcium deposition was observed on the I3D implants than on the solid counterparts. LPBF 3D printing can be used to fabricate implants with complex structures. I3D-tubulous structures of implants can retain chemoattractant for recruitment of stem cells to enhance osseointegration.

Evaluation of the Sterility of Press'n Seal Cling Film for Use in Rodent Surgery.

level and improve surgical outcomes. Recently, some institutions have approved the use of Press'n Seal cling film (CF; Glad Products, Oakland, CA) as a practical, cost-effective alternative to sterile drapes for rodent surgeries. The purpose of this study was to evaluate the sterility of CF by using ATP and replicate organism detection and counting (RODAC) plates. We tested 10 boxes of CF at days 0, 14, and 28 after opening the box and compared the results with traditional packaged sterile drapes. Our data indicated that CF ATP bioluminescence remained at or below 10 relative light units for 28 d after opening the box. In addition, RODAC plates had no growth for 70% of CF boxes at day 0, 100% at day 14, and 90% at day 28. The mean growth for the positive plates was 0.024 cfu/cm² sampled after contacting locations on the front and back of the CF. The results of this study support the use of CF as an acceptable alternative to traditional sterile drapes during rodent aseptic surgery.

Scrotal ablation and orchiectomy in the domestic laboratory goat (Capra hircus).

Domestic goats (Capra hircus) have been used as animal models in biomedical research, and for numerous years for agricultural research and models for human diseases. This column describes an improved surgical technique, for castration of male goats, similar to companion animal techniques for use in the laboratory setting. The technique discussed supports a more in-depth perioperative protocol for performing scrotal ablation and orchiectomy in the male goat.

The effects of high fat diet and estradiol on hypothalamic prepro-QRFP mRNA expression in female rats.

Estradiol (E2) is a potent regulator of feeding behavior, body weight and adiposity in females. The hypothalamic neuropeptide, QRFP, is an orexigenic peptide that increases the consumption of high fat diet (HFD) in intact female rats. Therefore, the goal of the current series of studies was to elucidate the effects of E2 on the expression of hypothalamic QRFP and its receptors, QRFP-r1 and QRFP-r2, in female rats fed a HFD. Alterations in prepro-QRFP, QRFP-r1, and QRFP-r2 expression across the estrous cycle, following ovariectomy (OVX) and following estradiol benzoate (EB) treatment were assessed in the ventral medial nucleus of the hypothalamus/arcuate nucleus (VMH/ARC) and the lateral hypothalamus. In intact females, consumption of HFD increased prepro-QRFP and QRFP-r1 mRNA levels in the VMH/ARC during diestrus, a phase associated with increased food intake and low levels of E2. To assess the effects of diminished endogenous E2, rats were ovariectomized. HFD consumption and OVX increased prepro-QRFP mRNA in the VMH/ARC. Ovariectomized rats consuming HFD expressed the highest levels of QRFP. In the third experiment, all rats received EB replacement every 4days following OVX to examine the effects of E2 on QRFP expression. Prepro-QRFP, QRFP-r1 and QRFP-r2 mRNA were assessed prior to and following EB administration. EB replacement significantly reduced prepro-QRFP mRNA expression in the VMH/ARC. Overall these studies support a role for E2 in the regulation of prepro-QRFP mRNA in the VMH/ARC and suggest that E2's effects on food intake may be via a direct effect on the orexigenic peptide, QRFP.