Veterinary anesthesia: an opportunity to reduce the environmental footprint of clinical care.

Volatile anesthetic agents are potent greenhouse gases with warming potential hundreds to thousands of times greater than CO2. As health systems, both human and veterinary, seek to reduce their environmental impacts, responsible anesthetic stewardship is a topic of great interest. Through an online survey, we explored the levels of awareness, beliefs, interest, needs, and current actions of veterinary anesthesia professionals around the climate impacts of anesthetic care. We found that even within a respondent group with specialized training and experience, there were significant knowledge gaps about anesthesia's environmental impacts. We also found there is much interest in learning more about climate-friendly anesthesia and broader sustainability initiatives for the veterinary profession. Fortunately, there already exist many ways for the profession to reduce our environmental impact while still providing excellent patient care. In this article, we explore 5 broad categories of action: (1) reducing the overall quantity of anesthetic agent used; (2) choosing lower-impact anesthetics; (3) considering the fate of the anesthetic end product; (4) expanding learning through formal education, experience, and research; and (5) reaching beyond anesthesia to implement a range of sustainability initiatives at veterinary workplaces. Together, we have an opportunity to create a healthier future for our world, our patients, and each other.

The Edwards Acumen IQ system using peripheral arterial catheter-based waveforms to estimate cardiac output is not accurate as compared to thermodilution in dogs.

OBJECTIVE: To assess the agreement between cardiac output (CO) estimated via evaluation of the arterial pressure waveform by a novel monitoring system (Edwards Acumen IQ sensor and HemoSphere Advanced Monitor Platform [HS-IQ]; Edwards LifeSciences) and measured by thermodilution (TD) in anesthetized, normovolemic, and hypovolemic dogs. To assess the agreement between the HS-IQ CO measurements in the radial artery and dorsal metatarsal artery. ANIMALS: 8 purpose-bred Beagles. METHODS: Dogs were placed under general anesthesia. CO was measured via TD and via the HS-IQ at radial and dorsal metatarsal arterial catheters. CO measurements were obtained at 4 time points including normovolemic and multiple hypovolemic states. Paired measurements of CO were evaluated via the method of Bland and Altman with acceptable limits of agreement (LOA) defined as < 30%. RESULTS: A total of 24 (dorsal metatarsal) and 21 (radial) paired measurements were collected in 8 dogs. The overall bias (CI) for comparison of TD to radial arterial HS-IQ CO measurements was -0.09 L/min. LOA and proportional LOA were -2.66 to 2.49 L/min and -140.72% to 104.94%. The overall bias (CI) for comparison of TD to dorsal metatarsal arterial HS-IQ CO measurements was -0.26 L/min. LOA and proportional LOA were -2.76 to 2.24 L/min and -135.96% to 93.25%. The overall proportional error for radial arterial was -17.9% and for dorsal metatarsal was -21.4%. CLINICAL RELEVANCE: CO measurements with the HS-IQ were easy to obtain but did not produce results within a clinically acceptable range for either measurement site, with a very wide LOA. The CO estimations from the HS-IQ are not appropriate for clinical use at this time.

Review of intra-articular local anaesthetic administration in horses: Clinical indications, cytotoxicity, and outcomes.

Equine practitioners frequently inject local anaesthetics (LA) intra-articularly in both diagnosis of lameness and for pain management intra- or post-operatively with synovial endoscopy. Recent reviews of the human and veterinary literature support the concept that chondrotoxicity of LA on joint tissues depends on the type of drug, dose administered, and duration of exposure. The purpose of this review is to summarise the current literature describing intra-articular local anaesthetic use, including both in vitro and in vivo studies, and to draw some comparisons to literature from other species where potential toxicity and duration of effect have been evaluated with the goal of advancing the field's understanding of intra-articular local anaesthetic use in horses, and indicating future directions for the field. The aggregate data available from all species, while generally sparse for horses, indicate that LA are rapidly cleared from the synovial fluid after injection, often within 30 min. In vitro data strongly suggest that lidocaine and bupivacaine are likely more chondrotoxic than other LA, although to what extent is still unknown, and cytotoxicity of LA may be mitigated through concurrent injection with HA, PRP, and drug combinations including nonsteroidal anti-inflammatories and opioids. The current body of in vitro research is not reflective of the in vivo environment, and further in vitro work, if performed, should focus on mimicking the native joint environment, utilising PK data and joint/injection volumes to replicate the native environment more accurately within the joint and the expected exposures to LA.

The Effects of Antimicrobial Protocols and Other Perioperative Factors on Postoperative Complications in Horses Undergoing Celiotomy: A Retrospective Analysis, 2008-2021.

Recognition of antimicrobial resistance in equine practice has increased over the past decade. The objective of this study was to provide an updated retrospective review of antimicrobial regimens in one tertiary referral hospital and to evaluate the association with postoperative complications. A secondary objective was to evaluate other perioperative factors including surgical procedure, anesthetic and recovery parameters, and the effect of perioperative medications on complications and outcomes. A computerized search of medical records was performed to identify horses undergoing exploratory celiotomy from 1 January 2008 to 31 December 2021. A total of 742 celiotomies were performed (608 completed, 134 terminated intraoperatively). Factors recorded were evaluated using logistic regression for the presence of either incisional infection, postoperative ileus, or other complications postoperatively. Antimicrobial type or timing (pre-, intra-, or postoperative) were not associated with decreased risk of incisional infection or postoperative ileus; however, the duration of NSAID use was positively associated with incisional infection (OR 1.14 per day). Lidocaine and alpha-2-agonist administration postoperatively were also associated with increased incidence of postoperative ileus (OR 21.5 and 1.56, respectively). Poor recovery quality (OR 4.69), the addition of other antimicrobials besides penicillin/gentamicin postoperatively (OR 3.63), and an increased number of different NSAID classes used (OR 1.46 per additional) were associated with other complications. Implementation of enterotomy was associated with decreased risk of other complications (OR 0.64). These findings provide an updated summary of factors associated with postoperative complications in horses undergoing celiotomy.

Current Antimicrobial Use in Horses Undergoing Exploratory Celiotomy: A Survey of Board-Certified Equine Specialists.

In the past decade, there has been a considerable increase in the recognition of antimicrobial resistance in equine practice. The objective of this study was to survey the current clinical use of antimicrobials for a commonly performed surgical procedure (exploratory celiotomy) with the goal of understanding how recent literature and changes in microbial resistance patterns may have impacted antimicrobial selection practices. An electronic survey was distributed to veterinary professionals within the American College of Veterinary Internal Medicine (ACVIM) and the American College of Veterinary Surgery (ACVS). A total of 113 completed surveys were returned. Practitioners reported antimicrobials were most frequently given 30-60 min preoperatively (63.1%). Two antimicrobial classes were typically administered (95.5%), with gentamicin (98.2%) and potassium penicillin (74.3%) being the most common. Antimicrobials were typically not re-dosed intraoperatively (78.6%). Factors that affected overall treatment length postoperatively included resection (81.4%), bloodwork (75.2%), enterotomy (74.3%), fever (85.0%), incisional complications (76.1%), and thrombophlebitis (67.3%). The most common duration of antimicrobial use was 1-3 d for non-strangulating lesions (54.4% of cases) and inflammatory conditions such as enteritis or peritonitis (50.4%), and 3-5 d for strangulating lesions (63.7%). Peri-incisional and intra-abdominal antimicrobials were used by 24.8% and 11.5% of respondents, respectively. In summary, antimicrobial usage patterns were highly variable among practitioners and, at times, not concordant with current literature.

Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system.

Introduction: Multiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP). Methods: Equine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages. Results: All treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling. Discussion: These findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build.

Waste not want not: piloting a clinical waste audit at a United States university veterinary teaching hospital.

Biomedical waste is a well-recognized environmental problem, yet less is known about the waste generated in the delivery of veterinary care compared to human medicine. The objective of this project was to develop and pilot a waste audit protocol for veterinary medicine that could inform waste management at a US university veterinary teaching hospital and the broader veterinary community. We conducted a multi-day review of the Colorado State University Veterinary Teaching Hospital's small animal surgery and anesthesia units to measure the types and amounts of waste generated during routine surgeries. Metrics included total weight, number of bags, and individual counts for specific items of concern and items with sustainable alternatives. We calculated frequencies and percentages of waste by waste audit material category and noted any erroneous materials sorted. Despite waste being a prioritized sustainability issue in veterinary medicine, this work highlighted opportunities for better education on managing and optimizing existing resources through behavior modification. This article explores ways the 5Rs (Rethink, Reduce, Reuse, Recycle, Research) could be better operationalized in veterinary hospitals.

Liposomal bupivacaine is both safe and effective when administered via local infiltration at surgical site and mesovarium for laparoscopic ovariectomy in mares.

BACKGROUND: Liposomal local anaesthetic solutions may provide extended-duration analgesia postoperatively but have not been assessed following intra-peritoneal local infiltration in any species. OBJECTIVES: To evaluate two doses of 1.33% liposomal bupivacaine (LB) versus 0.75% bupivacaine HCL (BHCl) for analgesia following laparoscopic ovariectomy in mares. STUDY DESIGN: Prospective cohort study. METHODS: Fifteen healthy Quarter Horse mares (age 2-20 years) with normal bilateral ovarian palpation and appearance were enrolled. Horses were restrained in standing stocks and administered an α-2 agonist, butorphanol, and flunixin meglumine, followed by a variable rate infusion of sedation with α-2 agonists. Bilateral paralumbar fossa ovariectomies were performed. Treatment with either 30 ml 0.75% BHCl followed by 20 or 40 ml LB 13.3% (LB20 and LB40) volume expanded with saline to 80 ml total (n = 6/group) or 80 ml BHCl alone (n = 3, BCHL) was infused around incision sites and each mesovarium (LB or BHCl) prior to ovariectomy. Horses were monitored for 72 h by physical examination, algometry, and pain scoring (composite pain scale by Bussieres et al., Horse Grimace Scale). Abdominocentesis with peritoneal fluid analysis was performed at 72 h. RESULTS: Analgesia achieved with all treatment protocols allowed completion of ovariectomy procedures. Pressure algometry scores were lower in BHCl-treated horses versus both LB groups overall. Pain scores were improved with LB-treated horses in a dose-dependent fashion (Horse Grimace Scale scores LB40 < LB20 < BHCL; composite pain scale scores LB40 < BHCL, LB20 < BHCL, BHCL, and LB20 did not differ). Peritoneal fluid total protein was lower in LB40 versus LB20 and BHCL horses. No complications from LB administration were appreciated. MAIN LIMITATIONS: Small patient sample size, lack of follow-up past 72 h or histopathology. CONCLUSIONS: Analgesia duration was extended and pain scores improved postoperatively with LB versus BHCl in a dose-dependent fashion. Further clinical evaluation of extended-duration local anaesthetics in horses for improved postoperative pain management is warranted.

Equine blood flow restriction training: Safety validation.

BACKGROUND: Blood flow restriction (BFR) has become a key rehabilitative tool for human orthopaedic conditions. With modernised technology and evolution of clinical application, patient-specific delivery of occlusion percentages is now considered the standard of care in human patients due to improved therapeutic outcomes and minimised safety risks. Safety validation and limb occlusion pressure (LOP) data for horses, however, are lacking. OBJECTIVE: (1) To determine if BFR exposure resulted in forelimb biomechanical gait dysfunction as safety validation and (2) to investigate inter-horse and inter-limb LOP differences. STUDY DESIGN: Controlled in vivo experiment. METHODS: Daily unilateral forelimb BFR was performed in four horses over 56 days. Clinical examinations and objective gait analyses were performed on Days 0, 28 and 56. Daily LOP values were determined by Doppler evaluation to deliver 80% vascular occlusion at a walk. A linear mixed model evaluated for differences in lameness, kinetic and kinematic gait parameters. RESULTS: There were no significant differences in forelimb lameness (range of Grades 0-2 across all forelimbs), kinematic or kinetic gait parameters over time or between BFR-exposed and control (contralateral) limbs (p > 0.05). Clinically apparent complications related to BFR such as thrombosis or dermatitis were not appreciated. Significant differences in mean LOP values between various horses (p < 0.001) and measured left (204.48 mmHg) and right (173.78 mmHg) forelimbs (p < 0.001) were observed. Mean LOP and standard deviation across all readings was 189.1 ± 22.2 mmHg. MAIN LIMITATIONS: Optimal BFR occlusion percentages and protocols with documented clinical efficacy are unknown. Small study population. CONCLUSIONS: Exposure to BFR did not result in forelimb biomechanical dysfunction in four horses. Applied pressures of 75-151 mmHg would likely simulate a range of 50%-80% vascular occlusion in horses, but inherent physiological variation between horses and forelimbs warrants incorporation of individual pressures.

TLR-activated mesenchymal stromal cell therapy and antibiotics to treat multi-drug resistant Staphylococcal septic arthritis in an equine model.

Background: Rapid development of antibiotic resistance necessitates advancement of novel therapeutic strategies to treat infection. Mesenchymal stromal cells (MSC) possess antimicrobial and immunomodulatory properties, mediated through antimicrobial peptide secretion and recruitment of innate immune cells including neutrophils and monocytes. TLR-3 activation of human, canine and equine MSC has been shown to enhance bacterial killing and clearance in vitro, in rodent Staphylococcal biofilm infection models and dogs with spontaneous multi-drug-resistant infections. The objective of this study was to determine if intra-articular (IA) TLR-3-activated MSC with antibiotics improved clinical parameters and reduced bacterial counts and inflammatory cytokine concentrations in synovial fluid (SF) of horses with induced septic arthritis. Methods: Eight horses were inoculated in one tarsocrural joint with multidrug-resistant Staphylococcus aureus (S. aureus). Bone marrow-derived MSC from three unrelated donors were activated with TLR-3 agonist polyinosinic, polycytidylic acid (pIC). Recipient horses received MSC plus vancomycin (TLR-MSC-VAN), or vancomycin (VAN) alone, on days 1, 4, 7 post-inoculation and systemic gentamicin. Pain scores, quantitative bacterial counts (SF, synovium), SF analyses, complete blood counts, cytokine concentrations (SF, plasma), imaging changes (MRI, ultrasound, radiographs), macroscopic joint scores and histologic changes were assessed. Results were reported as mean ± SEM. Results: Pain scores (d7, P=0.01, 15.2±0.2 vs. 17.9±0.5), ultrasound (d7, P=0.03, 9.0±0.6 vs. 11.8±0.5), quantitative bacterial counts (SF d7, P=0.02, 0±0 vs. 3.4±0.4; synovium P=0.003, 0.4±0.4 vs. 162.7±18.4), systemic neutrophil (d4, P=0.03, 4.6±0.6 vs. 7.8±0.6) and serum amyloid A (SAA) (d4, P=0.01, 1,106.0±659.0 vs. 2,858.8±141.3; d7, P=0.02, 761.8±746.2 vs. 2,357.3±304.3), and SF lactate (d7, P<0.0001, 5.4±0.2 vs. 15.0±0.3), SAA (endterm, P=0.01, 0.0 vs. 2,094.0±601.6), IL-6 (P=0.03, 313.0±119.2 vs. 1,328.2±208.9), and IL-18 (P=0.02, 11.1±0.5 vs. 13.3±3.8) were improved in TLR-MSC-VAN vs. VAN horses. Study limitations include the small horse sample size, short study duration, and lack of additional control groups. Conclusions: Combined TLR-activated MSC with antibiotic therapy may be a promising approach to manage joint infections with drug resistant bacteria.

Evaluation of factors associated with surgical site infection in equine proximal interphalangeal joint arthrodesis: 54 cases (2010-2019).

BACKGROUND: The frequency of surgical site infection (SSI) following orthopaedic implant placement in horses has been reported but not compared with respect to specific antibiotic protocols administered. OBJECTIVES: To determine factors associated with SSI in horses undergoing proximal interphalangeal joint (PIPJ) arthrodesis including perioperative antibiotic protocols. METHODS: Records were evaluated (2010-2019), and horses undergoing PIPJ arthrodesis were identified. Patient signalment, supervising surgeon, reason for surgery, limb, implants placed, anaesthetic time, duration casting/coaptation postoperatively, antibiotic regimen and incidence/onset SSI were recorded. Bayesian and frequentist logistic regressions were used to estimate the contribution of covariates to infection occurrence. RESULTS: Fifty-four PIPJ arthrodeses were performed. SSI occurred in 2/54 (3.7%) on day 15,30. Arthrodesis was performed most commonly for osteoarthritis (33/54, 61.1%), fracture (11/54, 20.4%), and subluxation (5/54, 9.3%). Perioperative systemic antibiotics were administered 1-3 days (15/54, 27.8%) or > 3 days (39/54, 72.2%). Antibiotic protocols included cefazolin/gentamicin (20/54, 37%), cefazolin/gentamicin/doxycycline (14/54, 25.9%) and potassium penicillin/gentamicin (10/54, 18.5%). Regional limb perfusion was performed preoperatively 31/54 (57.4%) and postoperatively 7/54 (13%). Survival to dismissal was 98.1% (53/54 horses) with one horse euthanized due to support limb laminitis. No association was identified between antibiotic selection or duration (1-3 vs. > 3 days), pre-operative regional antibiotic perfusion, intraoperative antibiotic lavage or anaesthetic time (< or > 3 h) and SSI; however, modelling was complicated by quasi-complete or complete separation of the data. Bayesian analysis (but not frequentist analysis) indicated an association between post-operative regional antibiotic perfusion and SSI. Limitations include the retrospective nature of data collection and the low rate of infection overall. CONCLUSIONS: The prevalence of SSI in this population was lower than that in previous reports of equine orthopaedic internal fixation. There was no difference in SSI rate in cases administered systemic antibiotics for 1-3 days or >3 days, or for those horses that did or did not receive preoperative regional antibiotic perfusion.

Liposomal bupivacaine provides longer duration analgesia than bupivacaine hydrochloride in an adjustable sole-pressure model of equine lameness.

OBJECTIVE: To compare the efficacy and duration of action for perineural analgesia with liposomal bupivacaine (LB) versus bupivacaine hydrochloride (BHCl) in a sole-pressure induced model of forelimb lameness in horses. ANIMALS: 6 healthy adult research horses. PROCEDURES: In 1 randomly assigned forelimb, grade 3/5 lameness was induced by use of a sole-pressure lameness model. Objective lameness (vector sum [VS]) was determined with an inertial sensor system at 0, 1, 6, and 24 hours after lameness induction to evaluate the model. Mechanical nociceptive thresholds (MNTs) and objective lameness (VS and force platform kinetics) were recorded prior to and at 1, 6, 24, 48, and 72 hours after perineural anesthesia of the palmar nerves at the level of the proximal sesamoid bones with LB or BHCl in random order, with a 1-week washout period between crossover treatments. Data analysis was performed with mixed-model ANOVA. RESULTS: When evaluating the lameness model, there was a decrease in lameness at 24 hours in at least 1 limb of each horse (7/12 limbs); thus, screw length was increased by 1 to 2 mm at each 24-hour interval to maintain lameness. Compared with results at baseline, horses treated with BHCl had significant improvements in median MNT and VS identified at only 1 hour after injection, whereas treatment with LB yielded significantly improved median MNT, VS score, and peak vertical force for up to 24 hours. DISCUSSION: In this experimental model of forelimb lameness, LB provided longer analgesia when compared with BHCl and should be further investigated for treatment of pain in horses.

Comparison of equine synovial sepsis rate following intrasynovial injection in ambulatory versus hospital settings.

BACKGROUND: Frequency of synovial sepsis in horses following intrasynovial injection has been reported, but not compared with respect to the environment in which the injection was performed. OBJECTIVES: To describe occurrence of synovial sepsis following intrasynovial injections performed in ambulatory vs hospital settings. STUDY DESIGN: Retrospective cohort study. METHODS: Records from the Colorado State University were evaluated (2014-2018) and horses receiving intrasynovial injections were identified. Patients presenting for septic synovial structures were excluded. Patient signalment, primary supervising service, medications injected, location (field/hospital), whether synovial sepsis resulted, and at what time sepsis was recognised were recorded. Logistic regression was used to estimate the contributions of covariates to the occurrence of synovial sepsis following injection. RESULTS: During the study period, 3866 intrasynovial injections were performed in 1112 horses during 1623 sessions, with 643/1623 sessions performed in the field. The most frequently used medications were hyaluronate (846/1623, 52.1%), triamcinolone acetonide (780 /1623, 48.1%) and amikacin sulfate (684/1623, 42.1%). Four horses developed synovial sepsis (0.2% sessions, 0.1% synovial structures); 3/4 were injected in the field, 2/4 received antibiotics with the injection. The frequency of septic synovitis was 10.4 cases per 10 000 injections, or 1 in 967 injections. All horses recovered following synovial lavage and antibiotic therapy. Performing injections in the field (P = .2) or without antibiotics (P = .7) did not alter the risk of synovial sepsis. MAIN LIMITATIONS: Limitations include the retrospective nature of data collection and low rate of infection overall, which prohibited evaluation of individual medication regimes as factors associated with resultant infection. CONCLUSIONS: The frequency of synovial sepsis in this population of horses was not higher when injections were performed in the field or without concurrent antibiotic administration. These data may help to inform practitioners and clients regarding the relative potential risk of complications following intrasynovial medication in different environmental settings.

Raptor Sedation and Anesthesia.

Sedation and/or anesthesia is routinely and successfully used in raptors for a wide variety of procedures from the routine such as physical examination, radiographs, or venipuncture, to the more complex, such as orthopedic surgeries. Understanding the anatomy and physiology of raptor patients who present for care, and being fully prepared before the start of any procedure, can increase the success of anesthetic procedures. Recent advances in raptor sedation and anesthesia continue to improve the health and welfare of these avian patients.

Evaluation of Intra-Articular Amikacin Administration in an Equine Non-inflammatory Joint Model to Identify Effective Bactericidal Concentrations While Minimizing Cytotoxicity.

Septic arthritis causes significant morbidity and mortality in veterinary and human clinical practice and is increasingly complicated by multidrug-resistant infections. Intra-articular (IA) antibiotic administration achieves high local drug concentrations but is considered off-label usage, and appropriate doses have not been defined. Using an equine joint model, we investigated the effects of amikacin injected at three different doses (500, 125, and 31.25 mg) on the immune and cartilage responses in tibiotarsal joints. Synovial fluid (SF) was sampled at multiple time points over 24 h, the cell counts determined, and amikacin concentrations measured by liquid chromatography-mass spectrometry. Cytokine concentrations and collagen degradation products in SF were measured by ELISA and multiplex immunoassays. The mean amikacin concentrations in SF were greater than or equal to the minimum inhibitory concentration (MIC) (0.004 mg/ml) for most common equine joint pathogens at all time points tested to 24 h for all three amikacin doses evaluated. The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) increased significantly in SF in the highest amikacin dose group, despite the fact that increases in SF cell counts were not observed. Similarly, the biomarkers of cartilage type II collagen cleavage (C2C and C12C) were increased in SF following amikacin injection. Mechanistically, we further demonstrated using in vitro studies that chondrocytes and synoviocytes killed by exposure to amikacin underwent apoptotic cell death and were phagocytosed by macrophages in a non-inflammatory process resembling efferocytosis. Neutrophils and T cells were susceptible to amikacin cytotoxicity at clinically relevant doses, which may result in blunting of cellular inflammatory responses in SF and account for the lack of increase in total nucleated cell counts following amikacin injection. In summary, decisions on whether to inject cytotoxic antibiotics such as aminoglycosides intra-articularly and what doses to use should take into account the potential harm that antibiotics may cause and consider lower doses than those previously reported in equine practice.

Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells.

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte-macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion.

OBJECTIVE: To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. STUDY DESIGN: Controlled laboratory study. SAMPLE POPULATION: Equine bone-marrow-derived MSCs (three horses). METHODS: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 105 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37. RESULTS: MSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 106 cells/ml, 2 h, 10 µg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p < .0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05). CONCLUSION: TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction. CLINICAL SIGNIFICANCE: In vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.

Retrospective evaluation of association between perioperative antimicrobial protocol and complications following elective equine synovial endoscopy.

BACKGROUND: Prophylactic perioperative antimicrobial protocols in equine synovial endoscopy have been described but not compared with respect to post-operative outcomes and complications. Increasing antimicrobial resistance in equine practice and interest in promoting judicious use of antimicrobials has prompted reevaluation of drug selection and dosing strategies. OBJECTIVES: To determine the frequency of and compare post-operative complications following elective synovial endoscopy between horses receiving different perioperative antimicrobial protocols. METHODS: Records from the Colorado State University Veterinary Teaching Hospital were evaluated (2014-2018) and equine patients undergoing elective synovial endoscopy were identified. Patients undergoing endoscopy for sepsis or internal fixation were excluded. Patient signalment, clinician, joint and limb involved, perioperative antimicrobial regimen, number endoscopic portals and closure technique, and post-operative complications including incidence of joint infection were recorded. Generalized linear models were used to estimate the odds of post-operative complications. RESULTS: Elective synovial endoscopies of 516 horses in 537 procedures evaluating 761 synovial structures were performed. No horses developed post-operative septic synovitis. Administration of post-operative antimicrobials, type used and patient sex were all significantly associated with increased risk of complications, which were predominantly gastrointestinal-related. Complication rates in horses receiving a single preoperative dose of cefazolin were lower than in horses receiving potassium penicillin, gentamicin or multiple doses. Complication rates were lower in females compared to castrated or intact males. Other factors evaluated (breed, age, surgeon, anaesthesia duration or hospitalization, joint/limb operated, number endoscopic portals) were not associated with increased risk of complications post-operatively in this case population. CONCLUSIONS: Prophylactic perioperative antimicrobial protocols in equine practice deserve periodic reconsideration due to increased antimicrobial resistance. Prolonged antimicrobial usage beyond the time of surgery was unnecessary to prevent septic synovitis following synovial endoscopy in this case population and was furthermore associated with an increased risk of gastrointestinal complications.

Evaluation of the Effect of Cannabidiol on Naturally Occurring Osteoarthritis-Associated Pain: A Pilot Study in Dogs.

The objective of this study was to provide preliminary data describing the safety and effect of cannabidiol (CBD) for symptom relief of canine osteoarthritis-associated pain in a clinical setting using objective outcome measures. Twenty-three client-owned dogs with naturally occurring osteoarthritis of appendicular joints completed this prospective, double-blinded, crossover, placebo-controlled study. Baseline data were acquired for 4 wk, followed by random allocation to either placebo or CBD treatment for 6 wk, followed by 6 wk with the opposite treatment. Outcome measures included objective gait analysis, activity counts (via accelerometry) and clinical metrology instruments. There were no differences noted between groups at any time point for any of the recorded outcome measures. Adverse events associated with CBD administration included elevation in liver enzymes (n = 14) and vomiting (n = 2).

Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

BACKGROUND: Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. OBJECTIVES: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. STUDY DESIGN: In vitro experimental study. METHODS: Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. RESULTS: Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. MAIN LIMITATIONS: Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. CONCLUSIONS: Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.