Microglia polarization in heat-induced early neural injury.

Introduction: In the occurrence and development of heat stroke (HS), factors such as hyperthermia, ischemia and hypoxia are essential to the central nervous system (CNS) inflammatory response, but the main mechanism underlying CNS inflammation remains unclear. The aim of the study was to observe the polarization of microglia in response to heat-induced early nerve injury and to explore its possible mechanism of action. Material and methods: To establish a heatstroke animal model in Beagle dogs, 18 Beagle dogs were divided into control (group A) and experimental groups (group B, group C and group D) according to a random numbers table. The animals in the experimental groups were placed on an electric blanket of an animal body temperature maintaining apparatus. The temperature was set at 40 ±0.5°C, and the rectal temperature was monitored every 5 min until the target body temperature was reached. Once the target temperature was reached, the dogs were transferred to an environment of 26 ±0.5°C and 60 ±0.5% humidity. Western blot analysis was used to detect the expression of microglia-specific markers CD45, iNOS, arginase, and CD206 in normal and heat-damaged brain tissues at different time points (1 h, 6 h, 24 h). The expression of CD45 and arginase was further determined by co-localization with immunofluorescence. Results: CD45 and iNOS protein expression was detected in group A. The two protein markers in group B were significantly higher than those in group A (p < 0.05), and the protein markers in group C were still higher than those in group A (p < 0.05). There was no statistically significant difference among the animals in group A (p > 0.05). Arginase and CD206 protein expression was also detected in group A. Levels of the two protein markers in group B were higher than those in group A (p < 0.05), and the protein marker levels in group C were even higher than those in group A (p < 0.05). Further analysis of the two groups of protein markers in group D showed significantly higher levels than those in group A (p < 0.001). Immunofluorescence co-localization of CD45 and arginase showed significantly increased fluorescence density at 6 h and 24 h after thermal injury (p < 0.001). Conclusions: After heat-induced disease, microglia were found to be active in the brain tissues of dogs. The microglia activated in the early 1-6 h of CNS injury were mainly the M1 type, which were then converted to the M2 type after 6 h. The 24 h M2 type was dominant. The relationship between M1/M2 polarization trends and early brain injury in heat-induced disease may be a key to understanding CNS injury in heat-induced disease.

Genomic analysis and functional properties of Lactobacillus johnsonii GJ231 isolated from healthy beagles.

Probiotics are one of the management tools to improve the host's healthy microbiota. The positive effects of probiotics on host health are species-specific, so probiotics isolated from host's own gut may be most beneficial. Many of the metabolites (e.g., short-chain fatty acids, bacteriocins, and hydrogen peroxide) produced by Lactobacillus johnsonii have specific inhibitory profiles against invading pathogens. In this study, we isolated L. johnsonii GJ231 from the intestinal tract of healthy female beagles. The genome size of 1.763 M encoded a total of 1,691 predicted genes. Many carbohydrate-active enzymes responsible for carbohydrate degradation and the production of short-chain fatty acids were also predicted. The metabolic profile of short-chain fatty acids in L. johnsonii GJ231 was determined using LC-MS/MS. The bacteriocin-producing gene bacteriocin (lactacin F) in L. johnsonii GJ231 was also predicted. In vitro, experiments demonstrated that GJ231 can thrive in weak acids, 0.3% bile salts, and artificial gastrointestinal fluid models. It was tolerant of to high temperatures up to 70°C, was non- hemolytic, inhibited pathogenic bacteria, and had a high antioxidant capacity. In vivo safety experiments conducted in mice revealed that oral administration of GJ231 not only had no toxic side effect but also increased their antioxidant capacity. In conclusion, combining the above test results, which collectively demonstrate that canine-derived L. johnsonii GJ231 was a non-pathogenic, acid-tolerant and bile-salt-tolerant probiotic strain that inhibits pathogenic bacteria and improves host antioxidant function. This may make it a promising candidate for the development of innovative functional foods for pets.

High-frequency irreversible electroporation ablation for the prostate in Beagle dogs.

Background: Ablation procedures have garnered significant attention as a minimally invasive treatment of prostate diseases. However, the feasibility and safety of applying high-frequency irreversible electroporation (H-FIRE) to ablate the Beagle prostate for benign prostatic hyperplasia (BPH) treatment have not been thoroughly explored, the appropriate range of parameters has not been determined. In order to ensure the feasibility and safety of prostate ablation surgery, we conducted a study using Beagle dogs as subjects to investigate prostate tissue. Methods: We utilized a composite steep pulse therapy device to perform ablations on 26 lateral lobes of the prostate in 13 Beagles, employing various parameters for different needle distances. The effectiveness of this device was assessed through the observation of the ablation area, intraoperative muscle tremors, postoperative hematological examination, and gross inspection. Results: The findings of our study revealed that 1,000 to 2,000 v/cm in electric field strength, combined with 5 µs pulse width and pulse number 100, is a safe parameter range for ablation of prostate tissue. At the same time, the large electric field strength (2,000 v/cm) has the best ablation effect with the biggest continuous and thorough ablation area. All parameters of H-FIRE were safe for Beagles. Conclusions: H-FIRE ablation for prostate is safe and effective in dogs, which has the potential to be a useful addition to the range of minimally invasive treatments available for the treatment of BPH against this backdrop of increasing surgical practice.

Hyperbranched Poly-l-Lysine Modified Titanium Surface With Enhanced Osseointegration, Bacteriostasis, and Anti-Inflammatory Properties for Implant Application: An Experimental In Vivo Study.

OBJECTIVES: This study aimed to explore multiple effects of hyperbranched poly-l-lysine (HBPL) titanium (Ti) surfaces on osseointegration, bacteriostasis, and anti-inflammation across three different animal models. METHODS: Ti surfaces were covalently modified with HBPL, with uncoated surfaces as controls. Characterization included scanning electron microscopy (SEM) and surface chemistry and elemental analysis (EDX). Ti and Ti-HBPL implants were placed in conventional canine edentulous sites, post-operative infection canine edentulous sites, and diabetic rat tibias. Implants from canine edentulous models were analyzed using micro-CT and histomorphometry to assess osseointegration at 8 weeks. Post-operative infection beagles were used to evaluate antibacterial efficacy through clinical parameters and bacterial cultures at 1 week. In diabetic rats, micro-CT and histomorphometry were performed at 8 weeks. RESULTS: HBPL was uniformly grafted on Ti-HBPL surfaces. Ti-HBPL surfaces showed higher bone volume/total volume (BV/TV, p < 0.001), bone-implant contact (BIC%, p < 0.001), and trabecular number (Tb.N, p < 0.01) in beagles. Besides, it displayed higher BIC% (p < 0.001) and bone area fraction occupancy (BAFO%, p < 0.01) in hard tissue sections. In an infected model, Ti-HBPL surfaces exhibited lower bleeding on probing (BOP, p < 0.001), and plaque index (DI, p < 0.01), with reduced bacterial colony formation (p < 0.001) compared to the control group. In diabetic rats, Ti-HBPL surfaces showed an increase in BV/TV (p < 0.01) and Tb.N (p < 0.001), downregulated TNF-α and IL-1ß (p < 0.01), and upregulated IL-10 (p < 0.01) and osteocalcin (OCN) expression (p < 0.01). CONCLUSIONS: HBPL-Ti surfaces demonstrated enhanced osseointegration, bacteriostasis, and anti-inflammatory effects in vivo.

Development of a Simple and Validated LC-MS/MS Method for Quantitative Determination of Ketotifen in Beagle Dog Plasma and Its Application to Bioequivalence Study of Ketotifen Syrup Dosage Form.

A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC-MS/MS system. Analytes were separated on a Luna® Hilic column (50 × 2.0 mm i.d., 3 µm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC-MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup.

Shelf-life of cooked meat of southern king crab (Lithodes santolla) and false king crab (Paralomis granulosa) during refrigerated storage.

Lithodes santolla (SKC) and Paralomis granulosa (FSKC) are economically important resources exploited in southern South America. The effect of refrigerated storage (4 °C on flake ice) on physico-chemical (pH, thiobarbituric reactive substances (TBARs), total volatile basic nitrogen (TVB-N), water holding capacity (WHC), and water content (WC)), microbiological (total viable mesophilic bacteria (TVMC), psychrotrophic bacteria (TVPC), Staphylococcus spp, coliforms, enterobacteria, molds and yeasts) and sensory (odor, appearance, texture, juiciness, and taste) parameters was analyzed in the cooked SKC and FSKC merus. For each species, cooked merus from 36 animals were randomly distributed into 6 groups, corresponding to 0, 2, 5, 8, 11, and 14 days of storage. On each day, samples were taken for physico-chemical (n = 6), microbiological (n = 3), and sensory (n = 15) analyses. The pH values increased over time (P < 0.01 in both species), the TBARs only increased in FSKC (P = 0.008), whereas the TVB-N significantly rose only in SKC (P = 0.001). The WHC and the WC did not change over time for any of the king crab species (P > 0.05) in all cases. The presence of TVCM, TVCP, and Staphylococcus spp. in both species was observed from day 0. Furthermore, pathogenic microorganisms (S. aureus, coliforms, and enterobacteria) were not detected, and only the TVCP in SFKC reached the suggested microbial limit after 11 days. All sensory scores significantly decreased (P < 0.001) over time, but the quality of both king crab species remained acceptable until the 11th day. These findings suggest that the shelf-life of cooked merus was 11 and 8 days for SKC and SFKC, respectively, when stored at 4 °C with the presence of flake ice. These contributions consist of elucidating the shelf-life of these economically important seafood products and providing insights into their quality maintenance during storage.

A preliminary study of the mechanical properties of 3D-printed personalized mesh titanium alloy prostheses and repair of hemi-mandibular defect in dogs.

This study is a preliminary investigation exploring the mechanical properties of three-dimensional (3D)-printed personalized mesh titanium alloy prostheses and the feasibility of repairing hemi-mandibular defects. The ANSYS 14.0 software and selective laser melting (SLM) were used to produce personalized mesh titanium alloy scaffolds. Scaffolds printed using different parameters underwent fatigue property tests and scanning electron microscopy (SEM) of the fracture points. Models of hemi-mandibular defects (encompassing the temporomandibular joint) were created using beagle dogs. Freeze-dried allogeneic mandibles or 3D-printed personalized mesh titanium alloy prostheses were used for repair. Gross observation, computed tomography (CT), SEM, and histological examinations were used to compare the two repair methods. The prostheses with filament diameters of 0.5 and 0.7 mm could withstand 14,000 times and >600,000 cycles of alternating stresses, respectively. The truss-structure scaffold with a large aperture and large aperture ratio could withstand roughly 250,000 cycles of alternating forces. The allogeneic mandible graft required intraoperative shaping, while the 3D-printed mesh titanium alloy prostheses were personalized and did not require intraoperative shaping. The articular disc on the non-operated sides experienced degenerative changes. No liver and kidney toxicity was observed in the two groups of animals. The 3D-printed mesh titanium alloy prostheses could effectively restore the shape of the mandibular defect region and reconstruct the temporomandibular joint.

Development of a UHPLC-MS/MS method for the quantification of Pristinamycin â A and â ¡A in beagle dog plasma and its pharmacokinetic application.

The aim of this study was to develop and fully validate a sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of pristinamycin ⅠA (PⅠA) and pristinamycin ⅡA (PⅡA) in plasma of beagle dogs after oral administration of pristinamycin tablets. PⅠA, PⅡA and quinupristin (internal standard, IS) were separated on an Agilent Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 µm particle size) by using gradient elution consisting of methanol and water (0.1 % formic acid) at a flow rate of 0.4 mL/min in 4.0 min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 867.6→134.1, 548.4→287.1 and 1022.7→133.9 for PⅠA, PⅡA and IS at positive ion mode, respectively. The method was developed at linearity ranging from 1.0 to 1000 ng/mL for all analytes.The accuracy of PⅠA and PⅡA was observed to range between -10.6 % and 7.1 %, while the precision was found to be within 8.9 %. No significant matrix effect was observed. PⅠA and PⅡA demonstrated stability during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile of PⅠA and PⅡA in beagle dogs after oral administration of pristinamycin tablets (75 mg for PⅠA and 175 mg for PⅡA). The biological half-life (t1/2) was determined to be 1.75 ± 0.07 h and 1.44 ± 0.31 h for PⅠA and PⅡA, respectively. The areas under curves (AUC0-t) of PⅠA and PⅡA were 80.7 ± 24.6 and 230 ± 94.8 µg/L·h, respectively.

Characterization of an Uncommon Finding in the Basal Nuclei in Beagle Dogs: A Novel Potential Background Lesion in the Canine Brain.

Degenerative lesions specific to the basal nuclei have not been described as a background finding in Beagle dogs. This report comprises a documentation of seven cases. In the context of a nonclinical safety studies, the authors suggest documenting the lesion descriptively as degeneration neuropil, basal nuclei, bilateral as it is characterized by (1) vacuolation, neuropil; (2) gliosis (astro- and/or microgliosis); and (3) demyelination. This novel lesion is considered a potential new background change for several reasons: (1) It occurred in animals from test item-treated and also vehicle-treated groups; (2) no dose dependency was observed; (3) in one of six affected test item-treated dogs, the given compound was shown not to penetrate the blood-brain barrier; and (4) statistical comparison between the proportions of affected dogs in the treatment and control groups did not yield a statistically significant difference. The etiology remains unknown and is subject to further investigations.

Preliminary study of a new magnetic compression technique for circumcision in dogs: An experimental animal model.

Introduction: Traditional/ritual/medical circumcision can be associated with considerable intraoperative blood loss and a prolonged postoperative healing course. This study investigated the feasibility of the magnetic compression technique (MCT) for circumcision in beagle dogs. Methods: A set of magnetic rings including a daughter magnetic ring (DMR) and a parent magnetic ring (PMR) were designed for circumcision. In eight beagle dogs as the animal model, the DMR was placed between the penis and the foreskin through the glans, and then the PMR was placed outside the penis. The DMR and PMR automatically attracted together to compress the foreskin. The necrosis of the prepuce of the anterior penis was observed daily. The operation time and time to magnetic ring shedding were recorded. Healing of the foreskin stump was visually observed. Results: The magnetic rings were successfully installed in all eight dogs, and the operation process was without complication. The average operation time was 3.13 ± 0.92 min (range, 2-4.5 min). Postoperative X-rays showed good attraction of the magnetic rings. Daily post-operative observation showed progressive ischemic necrosis of the anterior foreskin and mild edema of the proximal foreskin. The dogs were generally in good condition and urinated freely. The magnetic rings fell off spontaneously 8-12 days after the operation, and the stump of the foreskin healed well. Conclusion: The MCT may be a new approach for circumcision in a canine model, which suggests its potential for use in humans.

Effect of particle size on gastric emptying of enteric-coated granules in fasted beagle dogs: Relationship with interdigestive migrating motor complex.

This study investigates the particle size threshold at which the interdigestive migrating motor complex (IMMC) becomes active in gastric emptying for fasted beagle dogs. Enteric-coated granules containing cetirizine dihydrochloride (CET) were prepared in three particle sizes, 200, 660, and 1,200 µm (D50). To mark IMMC timing and water movement from the stomach, enteric-coated aspirin tablets and acetaminophen solution were used. To six fasted beagle dogs with 50 mL of acetaminophen solution was administered each granule size as a multiple-unit and a single enteric-coated aspirin tablet (3-period crossover study). No significant difference in pharmacokinetic parameters of CET after oral administration of different particle sizes was observed. However, the appearance time of CET in plasma with smaller granules (200 and 660 µm) was significantly faster than that of salicylic acid (a major metabolite of aspirin) in all dogs. In the case of the largest granules (1,200 µm), no significant time difference was observed in the appearance of both compounds in plasma. Furthermore, in two dogs, both compounds appeared at the same time, implying IMMC-regulated gastric emptying for the largest CET granules. These results support a particle size threshold between 660 and 1,200 µm for gastric emptying without IMMC action in fasted beagle dogs.

Assessment of remdesivir and its nucleoside metabolite in beagle dogs and healthy humans by liquid chromatography coupled with triple quadrupole mass spectrometry.

The aim of this study was to assess the pharmacokinetics of the existing remdesivir intravenous formulation (100 mg dose) against the newly developed oral formulation (20 mg dose) for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dogs followed by healthy human volunteers. A quantification method for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dog and human plasma has been developed and validated using liquid chromatography coupled to triple quadrupole mass spectrometry detection. The analytical methods for beagle dogs and human differ in the calibration curve range, plasma matrix, processing volume, reconstitution volume and injection volume; however all other parameters were same in both methods. A simple protein precipitation extraction was carried out using acetonitrile containing the internal standard remdesivir D5. Remdesivir and GS-441524 were separated on an Endurus C-18P, 100 × 4.6 mm, 3 µm column and detected using a mass spectrometer with electrospray ionization in positive ion mode. The ion transitions used were m/z 603.1 → m/z 200.0 for remdesivir, m/z 292.0 → m/z 202.2 for GS-441524 and m/z 608.2 → m/z 205.1 for remdesivir D5. The calibration curve results were linear in beagle dog plasma (2.0-2,000.8 ng/ml range for remdesivir and 2.0-1,500.4 ng/ml for GS-441524) and human plasma (30.0-4,503.9 ng/ml range for remdesivir and 2.0-200.4 ng/ml for GS-441524). The recovery was >90% in beagle dog and human plasma. These methods were successfully used to determine the pharmacokinetic parameters of the intravenous injection and subcutaneous tablets dosage forms in beagle dogs and healthy humans.

Inflammatory Reprogramming Mediates Changes in Three-Dimensional Strain Capacity and Cardiac Function in Beagle Dogs with Doxorubicin-Related Cardiomyopathy.

Background: The cardiotoxicity of doxorubicin (DOX) limits its use in cancer treatment. To address this limitation, we developed a novel animal model that uses beagle dogs to investigate DOX-induced cardiac disorders. Unfortunately, the lack of effective cardioprotection strategies against DOX-induced cardiotoxicity poses a significant challenge. To establish a canine model for low-mortality DOX-induced cardiac dysfunction and explore the relationship between inflammatory reprogramming and DOX-related cardiotoxicity. Methods: Twenty male beagle dogs aged two years were randomly assigned into the DOX (N = 10) and control (CON) (N = 10) groups. DOX was infused (1.5 mg/kg) every two weeks until doses cumulatively reached 12 mg/kg. Serum biomarkers and myocardial pathology were evaluated, while real-time fluorescence-based quantitative polymerase chain reaction (RTFQ-PCR), two- and three-dimensional echocardiography (2DE and RT3DE), functional enrichment, and matrix correlation were also performed. Results: In the DOX group, high-sensitive cardiac troponin T (hs cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were significantly increased. Myocardial pathology indicated early to medium myocardial degeneration via a decreased cardiomyocyte cross-sectional area (CSA). Increased levels of inflammatory gene transcripts (interleukin 6 (IL6), tumor necrosis factor (TNF), transforming growth factor ß (TGF ß ), intercellular adhesion molecule 1 (ICAM1), interleukin 1 (IL1), interleukin 1 ß (IL1 ß ), and interleukin 8 (IL8)), of collagen metabolism and deposition regulatory genes (matrix metalloproteinase (MMP) family and tissue inhibitor of matrix metalloproteinase (TIMP) family), and the natriuretic peptide family (NPS) (natriuretic peptide A, B and C (NPPA, NPPB, and NPPC)) were observed. Strain abnormalities in the right ventricular longitudinal septal strain (RVLSS), right ventricular longitudinal free-wall strain (RVLFS), left ventricular global longitudinal strain (LVGLS), and left ventricular global circumferential strain (LVGCS) were detected at week 28 (vs. week 0 or CON group, p < 0.05, respectively). A significant decline in RVLSS and RVLFS occurred at week 16, which was earlier than in the corresponding left ventricular areas. A significant right ventricular ejection fraction (RVEF) decline was noted at week 16 (vs. week 0, 33.92 ± 3.59% vs. 38.58 ± 3.58%, p < 0.05), which was 12 weeks earlier than for the left ventricular ejection fraction (LVEF), which occurred at week 28 (vs. week 0, 49.02 ± 2.07% vs. 54.26 ± 4.38%, p < 0.01). The right ventricular strain and functional damages correlated stronger with inflammatory reprogramming (most R from 0.60 to 0.90) than the left ones (most R from 0.30 to 0.65), thereby indicating a more pronounced correlation. Conclusions: Inflammatory reprogramming mediated disorders of strain capacity and cardiac function predominantly in the right side of the heart in the newly established DOX-related cardiomyopathy beagle dog model.

Preventing High Fat Diet-Induced Obesity and Related Hepatic Steatosis by Chlorin e6-Mediated Photodynamic Therapy.

Obesity and its associated hepatic steatosis have become a global concern, posing numerous health hazards. Photodynamic therapy (PDT) is a unique approach that promotes anti-obesity by releasing intracellular fat. Chlorin e6 (Ce6)-PDT was tested for its anti-obesity properties in male ovariectomized (OVX) beagle dogs, as well as male C57BL/6 and Balb/c mice. The 12 OVX beagles were randomly assigned to one of four groups: high-fat diet (HFD) only, Ce6 only, Ce6 + 10 min of light-emitting diode light (LED) treatment, and Ce6 + 15 min of light treatment. We assessed several parameters, such as body weight, adipose tissue morphology, serum biochemistry, and body fat content analysis by computed tomography (CT) scan in HFD-fed beagle dogs. At the end of the study period, dogs that were treated for 35 days with Ce6 and exposed to LED irradiation (660 nm) either for 10 min (Ce6 + 10 min of light) or for 15 min (Ce6 + 15 min of light) had decreased body weight, including visceral and subcutaneous fats, lower aspartate transaminase (AST)/alanine transaminase (ALT) ratios, and a reduction in the area of individual adipocytes with a concomitant increase in the number of adipocytes. Furthermore, C57BL/6 male mice following an HFD diet were effectively treated by Ce6-PDT treatment through a reduction in weight gain and fat accumulation. Meanwhile, Ce6-PDT attenuated hepatocyte steatosis by decreasing the epididymal adipose tissue and balloon degeneration in hepatocytes in HFD-fed Balb/c mice. Taken together, our results support the idea that Ce6-PDT is a promising therapeutic strategy for the recovery of obesity and obesity-related hepatic steatosis.

Dog leukocyte antigen genotyping across class I and class II genes in beagle dogs as laboratory animals.

Dog leukocyte antigen (DLA) polymorphisms have been found to be associated with inter-individual variations in the risk, susceptibility, and severity of immune-related phenomena. While DLA class II genes have been extensively studied, less research has been performed on the polymorphisms of DLA class I genes, especially in beagle dogs commonly used as laboratory animals for safety evaluations in drug development. We genotyped four DLA class I genes and four DLA class II genes by locus-specific Sanger sequencing using 93 laboratory beagle dogs derived from two different strains: TOYO and Marshall. The results showed that, for DLA class I genes, 11, 4, 1, and 2 alleles, including a novel allele, were detected in DLA-88, DLA-12/88L, DLA-64, and DLA-79, while, for DLA class II genes, 1, 10, 6, and 7 alleles were detected in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. It was estimated that there were 14 DLA haplotypes, six of which had a frequency of ≥ 5%. Furthermore, when comparing the DLA diversity between TOYO and Marshall strains, the most common alleles and haplotypes differed between them. This is the first study to genotype all DLA loci and determine DLA haplotypes including all DLA class I and class II genes in dogs. Integrating information on the DLA diversity of laboratory beagle dogs should reinforce their benefit as an animal model for understanding various diseases associated with a specific DLA type.

Computer-aided design and 3D printing for a stable construction of segmental bone defect model in Beagles: a short term observation.

OBJECTIVE: Segmental bone defect animal studies require stable fixation which is a continuous experimental challenge. Large animal models are comparable to the human bone, but with obvious drawbacks of housing and costs. Our study aims to utilize CAD and 3D printing in the construction of a stable and reproducible segmental bone defect animal mode. METHODS: CAD-aided 3D printed surgical instruments were incorporated into the construction of the animal model through preoperative surgical emulation. 20 3D printed femurs were divided into either experimental group using 3D surgical instruments or control group. In Vitro surgical time and accuracy of fixation were analysed and compared between the two groups. A mature surgical plan using the surgical instruments was then utilized in the construction of 3 segmental bone defect Beagle models in vivo. The Beagles were postoperatively assessed through limb function and imaging at 1, 2 and 3 months postoperatively. RESULTS: In vitro experiments showed a significant reduction in surgical time from 40.6 ± 14.1 (23-68 min) to 26 ± 4.6 (19-36 min) (n = 10, p < 0.05) and the accuracy of intramedullary fixation placement increased from 71.6 ± 23.6 (33.3-100) % to 98.3 ± 5.37 (83-100) %, (n = 30, p < 0.05) with the use of CAD and 3D printed instruments. All Beagles were load-bearing within 1 week, and postoperative radiographs showed no evidence of implant failure. CONCLUSION: Incorporation of CAD and 3D printing significantly increases stability, while reducing the surgical time in the construction of the animal model, significantly affecting the success of the segmental bone defect model in Beagles.

H3N2 canine influenza virus-like particle vaccine with great protection in beagle dogs.

In 2016, a distinct branch of H3N2 canine influenza virus (CIV) emerged, which has mutations related to mammalian adaptation and has replaced previously prevalent strains. This branch poses a risk of zoonotic infection. To prevent and control H3N2 CIV, an H3N2 virus-like particle (VLP) vaccine based on the insect cell baculovirus expression system has been developed in the study. The H3N2 VLP vaccine induced high titers of hemagglutination inhibition (HI) antibodies in nasal and muscular immunized beagle dogs. Meanwhile, the VLP vaccine provided effective protection against homologous virus challenge comparable to inactivated H3N2 canine influenza virus. In addition, the intranasal H3N2 VLP vaccine induced significantly higher Th1, Th2, and Th17 immune responses, respectively (p,0.05). Importantly, intramuscular injection of VLP and inactivated H3N2 virus has complete protective effects against homologous H3N2 virus attacks. Nasal immunization with H3N2 VLP can partially protect beagles from H3N2 influenza. IMPORTANCE: A new antigenically and genetically distinct canine influenza virus (CIV) H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and substituted previously circulating strains. This clade poses a risk for zoonotic infection. In our study, intramuscular injection of the H3N2 virus-like particle (VLP) vaccine and inactivated H3N2 CIV confer completely sterilizing protection against homologous H3N2 canine influenza virus challenge. Our results provide further support for the possibility of developing VLP vaccines that can reliably induce immunity in animal species.

13-Week Repeated Oral Toxicity and Toxicokinetic Studies of Rabeprazole Sodium and Sodium Bicarbonate Combination in Dogs.

The subchronic toxicity and toxicokinetics of a combination of rabeprazole sodium and sodium bicarbonate were investigated in dogs by daily oral administration for 13 consecutive weeks with a 4-week recovery period. The dose groups consisted of control (vehicles), (5 + 200), (10 + 400), and (20 + 800) mg/kg of rabeprazole sodium + sodium bicarbonate, 20 mg/kg of rabeprazole sodium only, and 800 mg/kg of sodium bicarbonate only. Esophageal ulceration accompanied by inflammation was observed in only one animal in the male (20 + 800) mg/kg rabeprazole sodium + sodium bicarbonate group. However, the severity of the ulceration was moderate, and the site of occurrence was focally extensive; thus, it was assumed to be a treatment-related effect of rabeprazole sodium + sodium bicarbonate. In the toxicokinetics component of this study, systemic exposure to rabeprazole sodium (AUClast and Cmax at Day 91) was greater in males than females, suggesting sex differences. AUClast and Cmax at Day 91 were increased compared to those on Day 1 in a dose-dependent manner. A delayed Tmax and no drug accumulation were observed after repeated dosage. In conclusion, we suggest under the conditions of this study that the no-observed-adverse-effect level (NOAEL) of the combination of rabeprazole sodium + sodium bicarbonate in male and female dogs is (10 + 400) and (20 + 800) mg/kg, respectively.

The stereoselective pharmacokinetics of the desmethyl-phencynonate hydrochloride in beagle dogs.

The aim of this study was to investigate the chiral inversion and the stereoselective pharmacokinetic profiles of desmethyl-phencynonate hydrochloride after administration of the single isomer and its racemate to beagle dogs. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was established for determination of the stereoisomers on chiral columns in beagle dog plasma, which met all the requirements. The chiral inversion in dogs of the desmethyl-phencynonate hydrochloride were studied after administration of the single isomer or the racemic modification. The stereoselective pharmacokinetic profiles of the desmethyl-phencynonate hydrochloride were studied by assays for simultaneous isomers after administration of the racemic modification. The results showed that the absorption of the R-configuration dosed as the single isomer was higher than it dosed as the racemic modification. The AUC(0-t), AUC(0-∞), and Cmax of the S-configuration were much higher than those of R-configuration after oral administration of the racemic desmethyl-phencynonate hydrochloride. The chiral inversion of desmethyl-phencynonate isomers could not occur in dogs after administration of the R-configuration.

[Determination of nine components of fried Ziziphi Spinosae Semen extract in beagle dog plasma by UPLC-MS/MS and its application in pharmacokinetic research].

This study established a convenient, rapid, and sensitive ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, swertisin, N-nornuciferine, 6-feruloylspinosin, and jujuboside B in beagle dog plasma after oral administration of fried Ziziphi Spinosae Semen(FZSS) extract. The Waters HSS-T3 C_(18) column(2.1 mm×100 mm, 1.8 µm) was used. The methanol-aqueous solution(containing 0.01% formic acid) was adopted as the mobile phase for gradient elution. The nine components and two internal standards were completely separated within 8 min. The mass spectrometry detection was performed in multiple reaction monitoring(MRM) mode by positive and negative ion switching of electrospray ionization. The analytical method was validated in terms of specificity, selectivity, linear range, accuracy, precision, recovery, matrix effect, and stability. It could meet the requirement of pharmacokinetic research after oral administration of FZSS extract to beagle dogs. The results showed that the time to reach the peak concentration(T_(max)) of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, 6-feruloylspinosin, and jujuboside B was 2.40-3.20 h, and the elimination halflife(t_(1/2)) was 2.08-6.79 h after a single-dose oral administration of FZSS to beagle dogs. The exposure of magnoflorine and spinosin was high, with a peak concentration(C_(max)) of 76.7 and 31.5 ng·mL~(-1) and an area under the curve(AUC_(0-∞)) of 581 and 315 ng·h·mL~(-1), respectively. The exposure of the remaining five compounds was lower, with a C_(max) of 0.81-13.0 ng·mL~(-1) and an AUC_(0-∞) of 6.00-106 ng·h·mL~(-1). This study provides a reference for the follow-up research of FZSS.