Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Increased drug concentration and repeated eye drop administration as strategies to optimize topical drug delivery: A fluorophotometric study in healthy dogs.

OBJECTIVES: Determine tear film kinetics with different fluorescein concentrations and repeated eye drop administration at various time intervals. ANIMALS STUDIED: Six healthy Beagles. PROCEDURES: Six experiments were conducted on separate days: single eye drop administration (control) or two separate eye drops administered at 30 s, 1, 2, 5, and 10 min intervals. For each experiment, one eye received 0.3% fluorescein solution while the other eye received 1% fluorescein solution, and tear fluid was collected with capillary tubes at 0, 1, 5, 10, 20, 30, 40, 50, 60, 90, 120, and 180 min. Fluorescein concentrations were measured using automated fluorophotometry. RESULTS: Compared with 0.3% solution, eyes receiving 1% fluorescein solution had significantly higher tear film concentrations (p ≤ .046) and the area-under-the-fluorescein-time curve was twofold greater (p = .005). Compared with control: (i) Tear film concentrations were significantly higher for up to 20 min when repeating administration 30 s to 5 min after the first drop (p ≤ .006); (ii) The highest increase in area-under-the-curve was obtained with 2 and 5 min intervals for 0.3% (+109%-130%) and 1% solutions (+153%-157%); (iii) The highest increase in median precorneal retention time (defined as tear film concentration < 5% from baseline values) was obtained with 5 min intervals for 0.3% (55 min vs. 15 min in control) and 2-5 min intervals for 1% solutions (50 min vs. 25 min in control). CONCLUSIONS: Drug delivery to the ocular surface can be enhanced by using more concentrated formulations and/or by repeating eye drop administration 2-5 min after the first dose.

Pharmacokinetics of intramuscular maropitant in pigs (Sus scrofa domesticus).

Pigs are at risk of vomiting from medical conditions as well as the emetic side effects of drugs administered for peri-operative manipulations, but there is a lack of pharmacokinetic data for potential anti-emetic therapies, such as maropitant, in this species. The main objective of this study was to estimate plasma pharmacokinetic parameters for maropitant in pigs after a single intramuscular (IM) administration dosed at 1.0 mg/kg. A secondary objective was to estimate pilot pharmacokinetic parameters in pigs after oral (PO) administration at 2.0 mg/kg. Maropitant was administered to six commercial pigs at a dose of 1.0 mg/kg IM. Plasma samples were collected over 72 h. After a 7-day washout period, two pigs were administered maropitant at a dose of 2.0 mg/kg PO. Maropitant concentrations were measured via liquid chromatography/mass spectrometry (LC-MS/MS). A non-compartmental analysis was used to derive pharmacokinetics parameters. No adverse events were noted in any of the study pigs after administration. Following single IM administration, maximum plasma concentration was estimated at 412.7 ± 132.0 ng/mL and time to maximum concentration ranged from 0.083 to 1.0 h. Elimination half-life was estimated at 6.7 ± 1.28 h, and mean residence time was 6.1 ± 1.2 h. Volume of distribution after IM administration was 15.9 L/kg. Area under the curve was 1336 ± 132.0 h*ng/mL. The relative bioavailability of PO administration was noted to be 15.5% and 27.2% in the two pilot pigs. The maximum systemic concentration observed in the study pigs after IM administration was higher than what was observed after subcutaneous administration in dogs, cats, or rabbits. The achieved maximum concentration exceeded the concentrations for anti-emetic purposes in dogs and cats; however, a specific anti-emetic concentration is currently not known for pigs. Further research is needed into the pharmacodynamics of maropitant in pigs to determine specific therapeutic strategies for this drug.

Linear external skeletal fixation applied in minimally invasive fashion for stabilization of nonarticular tibial fractures in dogs and cats.

OBJECTIVE: The objective of this study was to evaluate the use of linear external skeletal fixation (ESF) applied using minimally invasive techniques in dogs and cats. STUDY DESIGN: Retrospective study. ANIMALS: Forty-nine dogs and 6 cats. METHODS: Medical records of cases with nonarticular tibial fractures, repaired using linear ESF at a single academic institution between July 2010 and 2020, were reviewed. All records of cases that had nonarticular tibial fractures repaired using linear ESF were included. Information was collected regarding signalment, surgical procedures performed, perioperative care, radiographic evaluation, and postoperative complications. RESULTS: Intraoperative imaging was used in 40/55 (72%) of cases. Tibal plateau angle (TPA), tibial mechanical medial proximal and distal tibial angles (mMPTA and mMDTA, respectively) were not affected by intraoperative imaging (P = .344, P = .687, P = .418). A total of 22 (40%) complications occurred. Of these, 18 were considered minor and 4 were considered major. Open fractures had more major complications than closed fractures (P = .019). All fractures reached radiographic union of the fracture. The mean ± SD time to external fixator removal was 71 ± 48 days. CONCLUSION: Linear ESF applied using minimally invasive techniques with or without intraoperative imaging was an effective treatment for nonarticular tibial fractures. CLINICAL SIGNIFICANCE: Closed application of linear ESF should be considered as a minimally invasive option for stabilizing nonarticular tibial fractures.

Retrospective evaluation of toceranib phosphate (Palladia) use in the treatment of feline pancreatic carcinoma.

Objective: To retrospectively assess the biological response in cats with pancreatic carcinoma treated with toceranib phosphate. Animals: Twenty-six client-owned cats. Procedure: Patient information from multiple institutions was solicited via an emailed REDCap survey. For inclusion, cats were required to have a confirmed diagnosis of exocrine pancreatic carcinoma either by histopathology, cytology, or both; to have received treatment with toceranib phosphate; and to have adequate follow-up data for analysis. Results: Twenty cats were treated for gross disease and 6 for microscopic disease/incomplete margins. Clinical benefit (complete response, partial response, or stable disease ≥ 10 wk) was observed in 9/20 cats treated in the gross disease setting (45%; complete response: n = 1, stable disease: n = 8). The remaining 11 cats with gross disease did not respond to toceranib phosphate. In the cats with microscopic disease, response was mixed. The median survival time for all cats was 97 d (range: 1 to 1666 d). Conclusion: Toceranib phosphate was well-tolerated and provided modest clinical benefit to a subset of cats treated. Clinical relevance: Although feline exocrine pancreatic carcinoma continues to be a challenging disease to treat, toceranib phosphate appeared to provide potential clinical benefit.

Évaluation rétrospective de l'utilisation du phosphate de tocéranib (Palladia) dans le traitement du carcinome pancréatique félin. Objectif: Évaluer rétrospectivement la réponse biologique chez les chats atteints d'un carcinome pancréatique traités par le phosphate de tocéranib. Animaux: Vingt-six chats appartenant à des clients. Procédure: Les informations sur les patients de plusieurs institutions ont été sollicitées via une enquête REDCap envoyée par courriel. Pour être inclus, les chats devaient avoir un diagnostic confirmé de carcinome pancréatique exocrine, soit par histopathologie, soit par cytologie, soit par les deux; avoir reçu un traitement par phosphate de tocéranib; et disposer de données de suivi adéquates pour l'analyse. Résultats: Vingt chats ont été traités pour une maladie macroscopique et six pour une maladie microscopique/marges incomplètes. Un bénéfice clinique (réponse complète, réponse partielle ou maladie stable ≥ 10 semaines) a été observé chez 9 chats sur 20 traités dans le cadre d'une maladie macroscopique (45 %; réponse complète: n = 1, maladie stable: n = 8). Les 11 chats restants atteints d'une maladie macroscopique n'ont pas répondu au phosphate de tocéranib. Chez les chats atteints d'une maladie microscopique, la réponse était mitigée. La durée médiane de survie pour tous les chats était de 97 jours (écart: 1 à 1666 jours). Conclusion: Le phosphate de tocéranib a été bien toléré et a apporté un bénéfice clinique modeste à un sous-ensemble de chats traités. Pertinence clinique: Bien que le carcinome pancréatique exocrine félin continue d'être une maladie difficile à traiter, le phosphate de tocéranib semble offrir un bénéfice clinique potentiel.(Traduit par Dr Serge Messier).

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Inflammation, negative affect, and amyloid burden in Alzheimer's disease: Insights from the kynurenine pathway.

BACKGROUND: Depressive symptoms in Alzheimer's disease (AD) predict worse cognitive and functional outcomes. Both AD and major depression inflammatory processes are characterized by shunted tryptophan metabolism away from serotonin (5-HT) and toward the neuroinflammatory kynurenine (Kyn) pathway. The present study assessed associations between Kyn and behavioral, neuroanatomical, neuropathological, and physiological outcomes common to both AD and negative affect across the AD continuum. METHODS: In 58 cognitively normal, 396 mild cognitive impairment, and 112 AD participants from the Alzheimer's Disease Neuroimaging Initiative-1 (ADNI1) cohort, serum markers of 5-HT, tryptophan, and Kyn were measured and their relationships investigated with immunologic markers, affect and functional outcomes, CSF markers of beta-amyloid (Aß) and tau, and regional gray matter. RESULTS: A higher Kyn/Tryptophan ratio was linked to many inflammatory markers, as well as lower functional independence and memory scores. A higher Kyn/5-HT ratio showed similar associations, but also strong relationships with negative affect and neuropsychiatric disturbance, executive dysfunction, and global cognitive decline. Further, gray matter atrophy was seen in hippocampus, anterior cingulate, and prefrontal cortices, as well as greater amyloid and total tau deposition. Finally, using moderated-mediation, several pro-inflammatory factors partially mediated Kyn/5-HT and negative affect scores in participants with subclinical Aß (i.e., Aß-), whereas such associations were fully mediated by Complement 3 in Aß+ participants. CONCLUSION: These findings suggest that inflammatory signaling cascades may occur during AD, which is associated with increased Kyn metabolism that influences the pathogenesis of negative affect. Aß and the complement system may be critical contributing factors in this process.

Population modeling of tumor growth curves and the reduced Gompertz model improve prediction of the age of experimental tumors.

Tumor growth curves are classically modeled by means of ordinary differential equations. In analyzing the Gompertz model several studies have reported a striking correlation between the two parameters of the model, which could be used to reduce the dimensionality and improve predictive power. We analyzed tumor growth kinetics within the statistical framework of nonlinear mixed-effects (population approach). This allowed the simultaneous modeling of tumor dynamics and inter-animal variability. Experimental data comprised three animal models of breast and lung cancers, with 833 measurements in 94 animals. Candidate models of tumor growth included the exponential, logistic and Gompertz models. The exponential and-more notably-logistic models failed to describe the experimental data whereas the Gompertz model generated very good fits. The previously reported population-level correlation between the Gompertz parameters was further confirmed in our analysis (R2 > 0.92 in all groups). Combining this structural correlation with rigorous population parameter estimation, we propose a reduced Gompertz function consisting of a single individual parameter (and one population parameter). Leveraging the population approach using Bayesian inference, we estimated times of tumor initiation using three late measurement timepoints. The reduced Gompertz model was found to exhibit the best results, with drastic improvements when using Bayesian inference as compared to likelihood maximization alone, for both accuracy and precision. Specifically, mean accuracy (prediction error) was 12.2% versus 78% and mean precision (width of the 95% prediction interval) was 15.6 days versus 210 days, for the breast cancer cell line. These results demonstrate the superior predictive power of the reduced Gompertz model, especially when combined with Bayesian estimation. They offer possible clinical perspectives for personalized prediction of the age of a tumor from limited data at diagnosis. The code and data used in our analysis are publicly available at https://github.com/cristinavaghi/plumky.

Serum albumin and total protein concentration in the tear film of horses with healthy or diseased eyes.

OBJECTIVE: To determine total protein content (TPC) and serum albumin levels in the tears of horses with healthy or diseased eyes. ANIMALS STUDIED: Forty-two horses with healthy eyes and 11 horses with unilateral (n = 10) or bilateral (n = 1) ocular disease. PROCEDURE: Each eye underwent an ophthalmic examination including detailed conjunctivitis scoring and tear collection with Schirmer strips. TPC and serum albumin levels were quantified in tear samples and compared among healthy eyes, affected eyes, and contralateral unaffected eyes. The impact of the following variables on lacrimal protein levels were assessed: age, breed, and sex (healthy eyes), as well as conjunctivitis score (diseased eyes). RESULTS: Lacrimal TPC ranged from 7.0 to 19.5 mg/mL in healthy eyes, while serum albumin ranged from 71.1 to 711.3 µg/mL (~1.6% of TPC) and was higher in tears of aged and female horses (P ≤ .033). Eyes with ocular disease had significantly greater (P ≤ .001) serum albumin in tears (median 679.6 µg/mL) compared to contralateral unaffected eyes (130.0 µg/mL) and eyes of the reference population (200.7 µg/mL). However, lacrimal TPC did not differ significantly among the 3 groups. Scoring of palpebral conjunctival hyperemia trended toward a positive association with serum albumin in tears (r = 0.49, P = .062). CONCLUSIONS: The protein profile in equine tears differs in health and disease. Serum albumin in tears increases with ocular disease and, similar to other species, might serve as a biomarker for ocular insult in horses. Future studies could investigate the protein levels in horses with specific ocular conditions and help determine the biological importance of albumin on the equine ocular surface.

Influence of Schirmer strip wetness on volume absorbed, volume recovered, and total protein content in canine tears.

OBJECTIVE: Determine the protein content and volume of tears sampled by Schirmer strips wetness ranging from 20 to 35 mm. ANIMALS STUDIED: Ten healthy beagle dogs. PROCEDURES: Each dog underwent 20 tear collections per day (10 sessions in each eye, spaced by ≥1 h) for 4 separate days, providing 200 tear samples for each length of wetness evaluated: 20, 25, 30, and 35 mm. A Schirmer strip was placed in each eye until the selected mm-mark was reached, calculating the volume absorbed (VA) as the difference between the post- and pre-collection weight (assuming 1 mg~1 µL for tear fluid), and the volume recovered (VR) as the amount pipetted from the tube following centrifugation. Total protein content (TPC) was measured with infrared spectroscopy. Outcome measures were compared with the Kruskal-Wallis test. RESULTS: Median values for VA (µL), VR (µL) and TPC (mg/mL) were as follows: 20 mm (18, 10, 5.94), 25 mm (22, 12.5, 5.97), 30 mm (25.5, 16, 5.89), and 35 mm (31, 22.5, 7.13). Both VA and VR were significantly greater (p < .001) for Schirmer strips wetness of 35¼30¼25¼20 mm. TPC was significantly greater (p < .001) for 35 > 20-30 mm, but not among other groups (p = 1.000). CONCLUSIONS: The study established normative data to consider when canine studies use Schirmer strips to collect tears for bioanalytical purposes (eg, proteomics, pharmacokinetics). Although 35 mm yielded higher VA and VR, the higher TPC could be explained by greater disruption of ocular surface homeostasis. Absorption to 20-30 mm is the suggested length of strip wetness for bioanalytical tear collection in dogs.

Computed tomography evaluation of proposed implant corridors in canine thoracic vertebrae.

OBJECTIVE: Identify acceptable implant corridors in the normal canine thoracic vertebrae (T) from T1 to T9. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Computed tomographic (CT) studies of normal canine thoracic spines (n = 39). METHODS: CT imaging studies of normal T1-T9 canine spines were evaluated by five independent observers. Each identified a proposed corridor, measured the width, length, and angle off mid-sagittal that the corridor occupied. RESULTS: CT studies were from 39 dogs weighing 3.19-60 kg (mean 10.72, SD 9.9 kg). Vertebral corridors ranged in average width from 3.8 to 5.2 mm, the widest being located at T1. They ranged in average length from 13.3 to 17.5 mm, shortest being T1 and longest being T6. The angle of corridors varied the most between individual vertebrae at T1-T3. The average corridor angles were: T1 = 38°, T2 = 32°, T3 = 27°, T4 = 26°. T5-T9 angle ranged from 23° to 24°. CONCLUSION: The average dimensions of corridors measured in dogs weighing 3.1-60 kg were consistent with those of commercially available cortical screws and pins. CLINICAL SIGNIFICANCE: Corridor trajectories identified in this population can be achieved from a dorsal approach between T5 and T9. A dorsal approach for implant placement would be challenging for T1-T4 due to the variability found in these vertebrae as well as regional anatomical constraints.

Short-term outcomes of 59 dogs treated for ilial body fractures with locking or non-locking plates.

OBJECTIVE: To determine the influence of plating systems on the clinical outcomes in dogs treated for ilial fractures. DESIGN: Retrospective study. ANIMALS: Fifty-nine dogs (63 hemipelves). METHODS: Radiographs and medical records of dogs with ilial fractures presented to Iowa State University between 2003 and 2019 were reviewed. After fracture reduction, fractures were fixed with a locking plate system (LPS) or non-locking plate system (NLS). Perioperative, long-term complications, and follow-up data were recorded. The frequency of implant failure and pelvic collapse were compared using a logistic and linear regression analysis, respectively. Where the univariate test was statistically significant, a multivariate analysis across categories was performed to identify statistically different categories. RESULTS: LPS and NLS implants were used in 25/63 and 38/63 hemipelves, respectively. Median follow-up time was 8 weeks (3-624 weeks). Implant failure occurred in 18/63 (29%) of fracture repairs, consisting of 17 with NLS and 1 with LPS. Revision surgery was recommended in five cases of implant failure, all with NLS. The probability of implant failure was higher when fractures were fixed with NLS (p = .0056). All other variables evaluated did not seem to influence outcome measures. CONCLUSION: The variable with the most influence on the outcomes of dogs treated for ilial fractures consisted of the fixation method (NLS vs. LPS). Fractures repaired with NLS were nearly 20 times more likely to fail than those repaired with LPS. CLINICAL RELEVANCE: Surgeons should consider repairing ilial body fractures in dogs with LPS to reduce the risk of short-term implant failure.

Radiation exposure associated with percutaneous fluoroscopically guided lag screw fixation for sacroiliac luxation in dogs.

OBJECTIVE: To determine radiation exposure to surgical personnel and to evaluate the accuracy of a modified percutaneous lag screw fixation technique for sacroiliac luxation (SIL) under fluoroscopic guidance in dogs. STUDY DESIGN: Cadaveric experimental study. SAMPLE POPULATION: Seventeen beagle cadavers with iatrogenic SIL. METHODS: Seventeen beagles with iatrogenic SIL underwent reduction and stabilization with 3.5-mm screws. Hypodermic needles (14 gauge) and fluoroscopy were used to orient two Kirschner wires for temporary stabilization and to guide drilling of glide and pilot holes using cannulated drill bits. Duration of surgery and radiation exposure were recorded. Postoperative computed tomographic evaluation of screw position and angulation was performed. RESULTS: Average time for fixation was 15.85 minutes (range, 6.37-33.5). Cumulative radiation doses of 0.4 mrem for the dominant arm of the assistant and 0 mrem for the primary surgeon were recorded. The mean dorsoventral and craniocaudal screw angles were 0.68° ± 3.4° (range - 5.4° to 9.5°) and 1.9° ± 3.2° (range - 4.3° to 9.1°), respectively. Sixteen of the 17 dogs had 100% sacral screw purchase, with the remaining case achieving 93.4% purchase. CONCLUSION: Fluoroscopy-assisted percutaneous placement of 3.5-mm cortical screws in lag fashion performed with 14-gauge needles in conjunction with Kirschner wires and cannulated drill bits yielded repeatable accurate screw placement with low levels of ionizing radiation exposure to the surgical team. CLINICAL SIGNIFICANCE: The described technique may be a viable method for minimally invasive osteosynthesis fixation of SIL with low levels of radiation exposure to the surgical team. These results provide evidence to support further evaluation of radiation exposure in clinical cases and can aid in study design and sample size determination.

An eye on the dog as the scientist's best friend for translational research in ophthalmology: Focus on the ocular surface.

Preclinical animal studies provide valuable opportunities to better understand human diseases and contribute to major advances in medicine. This review provides a comprehensive overview of ocular parameters in humans and selected animals, with a focus on the ocular surface, detailing species differences in ocular surface anatomy, physiology, tear film dynamics and tear film composition. We describe major pitfalls that tremendously limit the translational potential of traditional laboratory animals (i.e., rabbits, mice, and rats) in ophthalmic research, and highlight the benefits of integrating companion dogs with clinical analogues to human diseases into preclinical pharmacology studies. This One Health approach can help accelerate and improve the framework in which ophthalmic research is translated to the human clinic. Studies can be conducted in canine subjects with naturally occurring or noninvasively induced ocular surface disorders (e.g., dry eye disease, conjunctivitis), reviewed herein, and tear fluid can be easily retrieved from canine eyes for various bioanalytical purposes. In this review, we discuss common tear collection methods, including capillary tubes and Schirmer tear strips, and provide guidelines for tear sampling and extraction to improve the reliability of analyte quantification (drugs, proteins, others).

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Impact of acute conjunctivitis on ocular surface homeostasis in dogs.

OBJECTIVE: To investigate the effects of acute conjunctivitis on tear film characteristics and corneal sensitivity in dogs. ANIMALS STUDIED: Eight female spayed Beagle dogs (1.5-2 years old, 7.5-10 kg). PROCEDURES: On two consecutive days, one randomly selected eye in each dog received 1 or 375 mg/mL histamine solution to induce mild or severe conjunctivitis, while the contralateral eye served as control. Diagnostic tests were performed in the following order: fluorescein instillation and repeated tear collection over 20 minutes (to determine tear volume [TV] and turnover rate [TTR] by fluorophotometry), Schirmer tear test-1 (STT-1), tear ferning, corneal esthesiometry, and tear film breakup time (TFBUT). RESULTS: Results are presented as median values for severe conjunctivitis, mild conjunctivitis, and control eyes. Eyes with severe conjunctivitis had significantly higher STT-1 (24, 19.5, 17.5 mm/min; P = .002) and significantly lower TFBUT (10.5, 13.5, 15.5 s; P = .002), but no changes were noted in corneal tactile sensation (2, 2.5, 2.5 cm) or tear ferning (grades 2, 2, 2.5). Severe conjunctivitis significantly increased TV by nearly 10-fold (631, 97, 65 µL) initially (reflex tearing), although basal TV returned rapidly (<5 minutes) in all eyes (46, 58, 48 µL). Finally, there was a nonsignificant trend for higher reflex TTR in the conjunctivitis vs control eyes (68, 58, 43%/min). CONCLUSIONS: Experimentally induced conjunctivitis increases tear quantity and decreases tear quality in dogs, but has no impact on corneal sensitivity. Changes in tear film dynamics could affect ocular pharmacology (eg, precorneal retention time), although homeostasis of lacrimal volume and drainage is rapidly restored.

Nerve growth factor in dogs: Assessment of two immunoassays and selected ocular parameters following a nicergoline challenge per os.

Impairment of corneal nerves can result in the development of ocular surface diseases such as aqueous tear deficiency and neurotrophic keratopathy. This study investigates oral nicergoline, an α-adrenoceptor antagonist shown to enhance endogenous secretion of nerve growth factor (NGF) by the lacrimal gland, as a potential therapy for these conditions. Five female spayed Beagle dogs received a 2-week course of oral nicergoline (10 mg twice daily). Drug safety was evaluated with ophthalmic and physical examinations, blood pressure monitoring, bloodwork, and urinalysis. The effect of nicergoline on the ocular surface was assessed with corneal esthesiometry, Schirmer tear test-1, and tear film breakup time. Drug effect on NGF levels was assessed by collecting tears and blood at baseline and completion of therapy using a bead-based immunoassay and an enzyme-linked immunosorbent assay. Although nicergoline was well tolerated in all dogs, it did not have a significant impact on corneal sensitivity, tear production, or tear stability. Of note, NGF was below the limit of quantification in all tear samples and was only detected in 8/20 serum samples with no significant difference between levels at baseline (189.4 ± 145.1 pg/mL) and completion of therapy (149.4 ± 79.4 pg/mL). Further validation of NGF analytical assays is warranted before nicergoline is investigated in clinical patients.

Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs.

OBJECTIVE: To investigate the effects of various biological factors on total protein concentration (TPC) and serum albumin levels in canine tears. ANIMALS STUDIED: 10 healthy beagles (5 female, 5 male) were used. PROCEDURES: Experiments were conducted on separate days, collecting tears with either capillary tubes or Schirmer strips, as follows: (i) Tear collection at 3 hours intervals (from 6 am to 12 am); and (ii) Tear collection before and 20 minutes following topical histamine application (1, 10, 375 mg/mL) to induce mild, moderate, and severe conjunctivitis, respectively. TPC and serum albumin were measured with infrared spectroscopy and ELISA, respectively. RESULTS: Tear film TPC and serum albumin ranged from 9.7-26.1 mg/mL and 6.4-1662.6 µg/mL, respectively. Protein levels did not differ significantly among time points (P ≥ .080). Median coefficient of variation (CV%) was lower with Schirmer strips compared to capillary tubes for both TPC (12% vs 15%, P = .020) and serum albumin (57% vs 78%, P = .232). TPC (P < .001), but not serum albumin was greater in male vs. female dogs. Serum albumin, but not TPC (P ≥ .099), increased significantly with each grade of conjunctivitis severity (P < .001), with no differences between collection devices (P ≥ .322); median increase was 106%, 1389%, and 2871% in eyes with mild, moderate, and severe conjunctivitis, respectively. CONCLUSIONS: There is no apparent diurnal variation in canine tear protein levels. Blood-tear barrier breakdown with conjunctivitis allows serum albumin to leak into the tear film at high concentrations. Schirmer strips compare well with capillary tubes for bioanalytical purposes in healthy and diseased eyes, and this collection method may offer improved reproducibility for protein quantification.

Aging-related changes in fluid intelligence, muscle and adipose mass, and sex-specific immunologic mediation: A longitudinal UK Biobank study.

BACKGROUND: Obesity in midlife and early late-life is associated with worse normal cognitive aging. Dual-energy X-ray absorptiometry (DEXA) suggests that visceral adipose mass (VAM) plays a predominant role, whereas non-visceral adipose mass (NVAM) and lean muscle mass (LMM) have shown conflicting relationships. It is unknown how longitudinal, cognitive changes in age-sensitive domains like fluid intelligence (FI) correspond to VAM, NVAM, and LMM in women and men. Furthermore, changes over time in blood leukocyte sub-populations may partially or fully account for sex-specific associations. METHODS: Data on 4431 late middle-aged, cognitively unimpaired adults (mean = 64.5 y) was obtained from the UK Biobank prospective cohort across 22 centers. FI scores, blood leukocyte counts, and covariates (age, social class, education) were measured at three 2-year intervals over 6 years. DEXA collection overlapped with these intervals. Sex-stratified growth curves, structural equations, and Preacher-Hayes mediation were used to estimate direct and indirect effects. ß-weights were standardized. RESULTS: More LMM predicted gains in FI scores among women (ß = 0.130, p < .001) and men (ß = 0.089, p < .001). Conversely, more VAM and NVAM independently predicted FI decline equally among sexes (e.g., NVAM: women: ß = -0.082, p < .001; men: ß = -0.076, p < .001). Among women, FI associations were fully mediated by higher eosinophil counts via VAM (λ = 30.8%, p = .028) and lower lymphocyte counts via LMM (λ = 69.2%, p = .021). Among men, FI associations were partially mediated by lower basophils counts via LMM (λ = 4.5%, p = .042) and higher counts via VAM (λ = 50%, p = .037). CONCLUSION: The proportion of LMM and VAM equally influenced male FI changes over 6 years, whereas higher LMM among women appeared to more strongly influence. FI changes. Leukocyte counts strongly mediated VAM- and LMM-related FI changes in a sex-specific manner, but not for NVAM. For clinical translation, exercise studies in older adults may benefit from assessing sex-specific values of DEXA-based tissue mass, FI, and leukocyte sub-populations to gauge potential cognitive benefits of less VAM and more LMM.

Paper spray high-resolution accurate mass spectrometry for quantitation of voriconazole in equine tears.

Paper spray high-resolution accurate mass spectrometry is a fast and versatile analysis method. This ambient ionization technique enables the quantitation of xenobiotics in complex biological matrices without chromatography or conventional sample extraction. The simplicity, rapidity, and affordability of the paper spray mass spectrometry (PS-MS) method make the technique especially attractive for clinical investigations where fast and affordable sample analysis is crucial. A new PS-MS method for the quantitation of voriconazole in equine tears was developed and validated. For a concentration range of 10 to 1000 ng/mL, good linearity (R2 > 0.99), inter- and intra-run precision (coefficient of variation (CV) max. 11.9%), accuracy (bias of the nominal concentration ± 13.9%), and selectivity (signal areas of the double blanks represent 0.13 ± 0.05% of the lower limit of quantitation (LLOQ) signal in equine tears) were observed. The quantitation of voriconazole was based on three product ions and calculated relative to the isotope-labeled internal standard, voriconazole-d3, which had a final concentration of 250 ng/mL in the standards and samples. The matrix effect of the method showed an ionization suppression by reduction of the voriconazole response to 63.6%, 70.2%, and 81.9% for 30 ng/mL, 450 ng/mL, and 900 ng/mL in equine tears compared with voriconazole in solvent (methanol:water, 50:50, v:v). The method was used to analyze 126 study samples collected for a pharmacokinetic study investigating a novel approach for treatment of fungal keratitis in horses. Therefore, the integrity of the sample dilution (n = 6, CV 6.90%, and bias of nominal concentration + 8.40%) and the carryover effect (increase from 0.33 ± 0.21% to 1.33 ± 0.89% of the signal of the LLOQ) was further investigated. To our knowledge, this method is the first application of PS-MS for quantitation of drug concentrations in tears from any species.