Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

OBJECTIVE: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). ANIMALS: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. METHODS: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. RESULTS: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. CLINICAL RELEVANCE: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Dorsal-to-palmar branch neuroanastomosis in horses undergoing palmar digital neurectomy does not reduce neuroma formation or improve outcome.

OBJECTIVE: Chronic foot pain, a common cause of forelimb lameness, can be treated by palmar digital neurectomy (PDN). Complications include neuroma formation and lameness recurrence. In humans, neuroanastomoses are performed to prevent neuroma formation. The aim of the study was to evaluate the outcome of horses undergoing dorsal-to-palmar branch neuroanastomosis following PDN. STUDY DESIGN: Retrospective case series. ANIMALS: Eighty-five horses with PDN and dorsal-to-palmar branch neuroanastomosis. METHODS: Medical records for horses undergoing this procedure at two hospitals between 2015 and 2020 were reviewed. Palmar and dorsal nerve branches of the PDN were transected and end-to-end neuroanastomosis was performed by apposition of the perineurium. Follow-up was obtained from medical records and telephone interviews. Success was defined as resolution of lameness for at least one year. RESULTS: Lameness resolved following surgery in 81/85 (95%) horses with 57/84 (68%) sound at one year. Postoperative complications occurred in 19/85 (22%) cases. The main limitations of the study were an incomplete data set, inaccurate owner recall, and variations in procedure. CONCLUSION: Compared to previous studies, this technique resulted in similar numbers of horses sound immediately after surgery, a comparable rate of postoperative neuroma formation but a higher recurrence of lameness rate at 1 year postoperatively. CLINICAL SIGNIFICANCE: End-to-end neuroanastomosis of the dorsal and palmar branches of the PDN does not reduce the rate of neuroma formation in horses. Long-term outcome was less favorable compared to previously reported PDN techniques.

Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways.

OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. ANIMALS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). CLINICAL RELEVANCE: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Little Brown Bats (Myotis lucifugus) Support the Binding of SARS-CoV-2 Spike and Are Likely Susceptible to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), believed to have originated from a bat species, can infect a wide range of non-human hosts. Bats are known to harbor hundreds of coronaviruses capable of spillover into human populations. Recent studies have shown a significant variation in the susceptibility among bat species to SARS-CoV-2 infection. We show that little brown bats (LBB) express angiotensin-converting enzyme 2 receptor and the transmembrane serine protease 2, which are accessible to and support SARS-CoV-2 binding. All-atom molecular dynamics (MD) simulations revealed that LBB ACE2 formed strong electrostatic interactions with the RBD similar to human and cat ACE2 proteins. In summary, LBBs, a widely distributed North American bat species, could be at risk of SARS-CoV-2 infection and potentially serve as a natural reservoir. Finally, our framework, combining in vitro and in silico methods, is a useful tool to assess the SARS-CoV-2 susceptibility of bats and other animal species.

A potentially fatal cranial pathology in a specimen of Tarchia.

Skulls of the Mongolian ankylosaurids Shamosaurus, Tarchia, and Saichania were scanned for information about their internal anatomy. Computed tomography (CT) imaging of the Tarchia skull revealed substantial internal anatomical differences from known Campanian North American taxa, particularly in the morphology of the airway. In addition, unexpected anomalies were detected within the airway and sinuses. The anomalies include multiple bilaterally distributed, variably sized hyperdense (mineralized) concretions within the airway and sinuses, the largest of which, positioned in the right nasal cavity medial to the supraorbitals, has an asymmetric ovoid shape that tapers caudally and which is partially encased within a hemispherical trabeculated osseous proliferation (sinus exostosis). Immediately adjacent to the exostosis is a subcircular transosseous defect in the prefrontal region of the skull roof that is partially filled with trabeculated ossified material with similar architectural features as the larger exostosis. Irregularities along the internal and external surfaces of the cranial vault may be associated. The radiologic features of the hemicircumferential exostosis suggest a chronic reactive osteoproliferation, possibly in response to an ongoing inflammatory reaction to primary sinus infection or, in combination with the unilateral transosseous defect, traumatically introduced infection with potentially fatal consequences. This report underscores the value of CT scanning of fossil vertebrate specimens, which in this case revealed large internal lesions of the skull that, at the time the scan was performed, were otherwise indiscernible.

Evaluation of locking compression plate fixation of the distal phalanx to the hoof wall as a potential therapy for laminitis.

BACKGROUND: Surgical stabilisation of the distal phalanx (DP) is a potential therapeutic strategy for severe acute laminitis. OBJECTIVE: To evaluate the effects of locking compression plate (LCP) fixation of the DP to the dorsal hoof wall. STUDY DESIGN: Ex vivo and in vivo experiments. METHODS: A T-shaped LCP was applied to one limb per pair in six pairs of cadaver forelimbs subjected to a combination of thermally induced lamellar failure and vertical load to simulate severe acute laminitis. Standard radiographic measurements were used to compare DP displacement. The LCP was then applied to one forefoot in 12 healthy Standardbred horses either standing (n = 6) or under general anaesthesia (n = 6). Lameness was evaluated daily, then horses were euthanised (day 8) and lamellar tissue analysed using light microscopy, histomorphometery and molecular markers of apoptosis. RESULTS: In the cadaver limb model, LCP fixation prevented the significant changes in hoof-distal phalanx distance, coronary extensor process distance and sole depth that characterised DP displacement in untreated limbs (p < 0.05). Application of the construct in vivo was well tolerated with minimal lameness (10/12 horses were sound at the trot on day 8); however, histology revealed dorsal lamellar pathology consistent with laminitis, but with extensive keratinocyte apoptosis. Adjacent to the LCP, caspase-3 positive cell counts were approximately 20-fold higher than control (p < 0.001). MAIN LIMITATIONS: Pathology was evaluated at a single time point. Microvascular perfusion was not evaluated. CONCLUSIONS: Rigid fixation of the DP to the hoof capsule was achieved with the LCP construct in a cadaver limb laminitis model. In live horses, LCP fixation caused regional lamellar pathology with extensive apoptosis, likely due to disturbed lamellar microvascular perfusion and/or mechanostasis. Understanding these mechanisms is critical for refinement of the technique in order to avoid iatrogenic lamellar damage.

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.

Gross, histopathologic, microbiologic, and radiologic characterization of lesions associated with clinical lameness in a cohort of group-housed sows euthanized for lameness.

Lameness in sows is reported as the most frequent cause of early culling from commercial farms and results in reduced productivity, economic losses, and a negative impact on animal welfare. Osteochondrosis was reported as the leading cause of lameness in North American sows and, although more recent European studies report infectious arthritis as the leading cause, lameness in US production facilities using group housing for gestating sows has not yet been evaluated. This study's aim was to characterize lesions associated with lameness in the appendicular musculoskeletal system of 26 sows euthanized for lameness using pathologic, radiologic, and microbiologic analyses. Of 178 total lesions, infectious lesions were most common (54%), predominated in distal limb segments (ie, at or distal to carpi and tarsi) and more often correlated with the clinically lame limb, whereas osteochondrosis and degenerative osteoarthritis predominated in proximal limb segments (ie, at or proximal to cubital and stifle joints) and rarely correlated with the clinically lame limb. The location and characteristics of infectious lesions, including mixed bacterial growth isolated from 22/22 orthopedic sites representing 19 sows with Trueperella pyogenes isolated in 16/22 (73%) of samples, suggest an etiologic component involving trauma. Radiography had a 70.6% sensitivity and 93.9% specificity for detecting infectious lesions affecting tarsocrural, antebrachiocarpal, and digital (ie, claw) regions combined. The frequency, type, and location of infectious lesions identified in this cohort of sows euthanized for lameness differ from previous reports, indicating the need for further investigation of the etiopathogenesis, earlier detection methods, and prevention.

A Platelet-Rich Plasma-Derived Biologic Clears Staphylococcus aureus Biofilms While Mitigating Cartilage Degeneration and Joint Inflammation in a Clinically Relevant Large Animal Infectious Arthritis Model.

The leading cause of treatment failure in Staphylococcus aureus infections is the development of biofilms. Biofilms are highly tolerant to conventional antibiotics which were developed against planktonic cells. Consequently, there is a lack of antibiofilm agents in the antibiotic development pipeline. To address this problem, we developed a platelet-rich plasma (PRP)-derived biologic, termed BIO-PLY (for the BIOactive fraction of Platelet-rich plasma LYsate) which has potent in vitro bactericidal activity against S. aureus synovial fluid free-floating biofilm aggregates. Additional in vitro studies using equine synoviocytes and chondrocytes showed that BIO-PLY protected these cells of the joint from inflammation. The goal of this study was to test BIO-PLY for in vivo efficacy using an equine model of infectious arthritis. We found that horses experimentally infected with S. aureus and subsequently treated with BIO-PLY combined with the antibiotic amikacin (AMK) had decreased bacterial concentrations within both synovial fluid and synovial tissue and exhibited lower systemic and local inflammatory scores compared to horses treated with AMK alone. Most importantly, AMK+BIO-PLY treatment reduced the loss of infection-associated cartilage proteoglycan content in articular cartilage and decreased synovial tissue fibrosis and inflammation. Our results demonstrate the in vivo efficacy of AMK+BIO-PLY and represents a new approach to restore and potentiate antimicrobial activity against synovial fluid biofilms.

A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition.

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes that influence Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection and impact on the viability of host cells in the context of parasite infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection.

Comparison of the zip skin closure system with conventional suture for skin closure of ventral midline incisions in horses.

OBJECTIVE: To compare the application and healing of the zip skin closure system (ZSCS) with sutured closure by use of a split-scar model of ventral midline incisions in horses in a prospective, randomized experimental study. ANIMALS: 8 adult horses. PROCEDURES: All horses underwent an exploratory ventral midline celiotomy with a standardized 30-cm skin incision. Each horse was randomized to have either the cranial 15 cm closed with suture and caudal 15 cm with the ZSCS or vice versa (split-scar model). Skin closure time was recorded and compared. Photography and skin biopsies were taken preoperatively and 14 days postoperatively. Cosmetic appearance was assessed by use of a proposed equine celiotomy incision score. Healing at 14 days was assessed by histopathology. RESULTS: Skin closure times were faster with the ZSCS compared to sutured incisions. At 14 days postoperatively, the cosmetic appearance (equine celiotomy incision scores) for ZSCS incisions were better than sutured closure and histologic healing scores were not different between methods of closure. Subcuticular sutures were associated with deep dermal inflammation and necrosis independent of epidermal closure methods. CLINICAL RELEVANCE: While limitations to the utility of the ZSCS are recognized, the potential benefits of expedient closure, good cosmetic outcome, and satisfactory healing make this method viable for closure of linear wounds or incisions in horses.

Phlegmonous gastritis in 2 yearling horses.

Phlegmonous gastritis was diagnosed in 2 yearling fillies that were presented with a 1-wk history of fever, lethargy, and hypoproteinemia, associated with a previous diagnosis of equine proliferative enteropathy based on clinical signs and PCR assay detection of Lawsonia intracellularis in fecal samples. Abdominal ultrasound revealed enlargement of the stomach and expansion of its submucosal layer with hypoechoic fluid, as well as thickened hypomotile small intestinal segments. Given the poor prognosis and poor response to treatment, both horses were euthanized, one on the day of presentation and the other after 3 wk of intensive medical management including a combination of antimicrobials, analgesics, and intravenous colloids. At autopsy, acute mural gastritis characterized by severe submucosal edema with suppurative inflammation (i.e., phlegmonous gastritis) and necroulcerative enteritis compatible with the necrotizing form of equine proliferative enteropathy were identified in both horses. The gastric inflammation was associated with thrombosis and mixed bacterial populations, including Clostridium perfringens, that were confined to the submucosa without evidence of mucosal involvement; toxin genes compatible with C. perfringens type C were identified in one case. Human phlegmonous gastritis is an uncommon, often-fatal pyogenic infection that is often associated with mucosal injury, bacteremia, or immunocompromise. Our finding of this unusual gastric lesion in 2 horses with similar signalment, clinical disease, and spectrum of postmortem lesions suggests a similar etiopathogenesis that possibly involves local, regional, or distant hematogenous origin, and should be considered a potential complication of gastrointestinal mucosal compromise in horses.

Raman needle arthroscopy for in vivo molecular assessment of cartilage.

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key extracellular matrix (ECM) biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include (1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2 O] scores), and (2) multiplexed polarized Raman spectroscopy to quantify superficial zone (SZ) COL anisotropy via a partial least squares-discriminant analysis-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: (1) chemically GAG-depleted bovine cartilage explants (n = 40), (2) mechanically abraded bovine cartilage explants (n = 30), (3) aging human cartilage explants (n = 14), and (4) anatomical-site-varied ovine osteochondral explants (n = 6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p < 0.001). RCAF values were significantly different for explants with abrasion-induced SZ COL loss (p < 0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2 O scores) predicted 94% of the variation in elastic modulus of ovine explants (p < 0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This study advances Raman arthroscopy toward a transformative low-cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

Enterocyte-innate lymphoid cell crosstalk drives early IFN-γ-mediated control of Cryptosporidium.

The intestinal parasite, Cryptosporidium, is a major contributor to global child mortality and causes opportunistic infection in immune deficient individuals. Innate resistance to Cryptosporidium, which specifically invades enterocytes, is dependent on the production of IFN-γ, yet whether enterocytes contribute to parasite control is poorly understood. In this study, utilizing a mouse-adapted strain of C. parvum, we show that epithelial-derived IL-18 synergized with IL-12 to stimulate innate lymphoid cell (ILC) production of IFN-γ required for early parasite control. The loss of IFN-γ-mediated STAT1 signaling in enterocytes, but not dendritic cells or macrophages, antagonized early parasite control. Transcriptional profiling of enterocytes from infected mice identified an IFN-γ signature and enrichment of the anti-microbial effectors IDO, GBP, and IRG. Deletion experiments identified a role for Irgm1/m3 in parasite control. Thus, enterocytes promote ILC production of IFN-γ that acts on enterocytes to restrict the growth of Cryptosporidium.

Use of Polyamide (Nylon) Cable Ties for Vascular Ligation of Healthy Equine Jejunal Mesentery.

Jejunal vascular ligation is an essential step in performing jejunojejunostomy. Hand sewn ligation is typically used and can increase operative time with long sections of bowel to be removed. Nylon cable ties (NCT) have been used for vascular ligation in horses but are yet to be investigated for application on the mesenteric vasculature of the gastrointestinal tract. Our objective was to evaluate the efficacy and short-term safety of NCT jejunal mesenteric vessel ligation in healthy horses. Eight healthy adult horses underwent midline celiotomy. A segment of jejunal mesentery was identified (≥4 arcades). Briefly, three fenestrations (proximal, middle, distal) were made 5-10 mm apart adjacent to the first and last vascular arcade to be ligated. Two sterilized NCT were passed to encircle the mesentery through the proximal and middle fenestrations, separated by intact mesentery. NCT were closed tightly and the vascular pedicle transected with Mayo scissors through the distal fenestration. Jejunojejunostomy was then performed and the mesentery sutured closed. The number of vascular arcades and time to ligate using NCT were recorded. At 2 weeks, horses underwent repeat celiotomy to assess the healing of the NCT ligation site and an equal number of vascular arcades were hand sewn double ligated using 2-0 Polyglactin 910 as a timed comparison. NCT mesenteric ligation was significantly faster than hand sewn methods (P < 0.01). Effective hemostasis was achieved in all cases. There was no evidence of local infection or adhesions at 14 days post-operatively. Further investigation in the long-term effects in horses as well as horses with strangulating jejunal lesions are needed for clinical application.

Development of a standardized histopathology scoring system for intervertebral disc degeneration and regeneration in rabbit models-An initiative of the ORSspine section.

BACKGROUND: The rabbit lumbar spine is a commonly utilized model for studying intervertebral disc degeneration and for the pre-clinical evaluation of regenerative therapies. Histopathology is the foundation for which alterations to disc morphology and cellularity with degeneration, or following repair or treatment are assessed. Despite this, no standardized histology grading scale has yet been established for the spine field for any of the frequently utilized animal models. AIMS: The purpose of this study was to establish a new standardized scoring system to assess disc degeneration and regeneration in the rabbit model. MATERIALS AND METHODS: The scoring system was formulated following a review of the literature and a survey of spine researchers. Validation of the scoring system was carried out using images provided by 4 independent laboratories, which were graded by 12 independent graders of varying experience levels. Reliability testing was performed via the computation of intra-class correlation coefficients (ICC) for each category and the total score. The scoring system was then further refined based on the results of the ICC analysis and discussions amongst the authors. RESULTS: The final general scoring system involves scoring 7 features (nucleus pulposus shape, area, cellularity and matrix condensation, annulus fibrosus/nucleus pulposus border appearance, annulus fibrosus morphology, and endplate sclerosis/thickening) on a 0 (healthy) to 2 (severe degeneration) scale. ICCs demonstrated overall moderate to good agreement across graders. An addendum to the main scoring system is also included for use in studies evaluating regenerative therapeutics, which involves scoring cell cloning and morphology within the nucleus pulposus and inner annulus fibrosus. DISCUSSION: Overall, this new scoring system provides an avenue to improve standardization, allow a more accurate comparison between labs and more robust evaluation of pathophysiology and regenerative treatments across the field. CONCLUSION: This study developed a histopathology scoring system for degeneration and regeneration in the rabbit model based on reported practice in the literature, a survey of spine researchers, and validation testing.

Development of a standardized histopathology scoring system for intervertebral disc degeneration in rat models: An initiative of the ORS spine section.

BACKGROUND: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions. AIMS: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models. MATERIALS & METHODS: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability. RESULTS: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 µm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories. CONCLUSION: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories.

A comprehensive tool box for large animal studies of intervertebral disc degeneration.

Preclinical studies involving large animal models aim to recapitulate the clinical situation as much as possible and bridge the gap from benchtop to bedside. To date, studies investigating intervertebral disc (IVD) degeneration and regeneration in large animal models have utilized a wide spectrum of methodologies for outcome evaluation. This paper aims to consolidate available knowledge, expertise, and experience in large animal preclinical models of IVD degeneration to create a comprehensive tool box of anatomical and functional outcomes. Herein, we present a Large Animal IVD Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging, and biomechanics), microscopic (histological, biochemical, and biomolecular analyses), and clinical (neurologic state, mobility, and pain). The proposed algorithm encompasses a stepwise evaluation on all three scales, including spinal pain assessment, and relevant structural and functional components of IVD health and disease. This comprehensive tool box was designed for four commonly used preclinical large animal models (dog, pig, goat, and sheep) in order to facilitate standardization and applicability. Furthermore, it is intended to facilitate comparison across studies while discerning relevant differences between species within the context of outcomes with the goal to enhance veterinary clinical relevance as well. Current major challenges in pre-clinical large animal models for IVD regeneration are highlighted and insights into future directions that may improve the understanding of the underlying pathologies are discussed. As such, the IVD research community can deepen its exploration of the molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration, paving the path for clinically relevant therapeutic strategies.

A perspective on the ORS Spine Section initiative to develop a multi-species JOR Spine histopathology series.

This perspective summarizes the genesis, development, and potential future directions of the multispecies JOR Spine histopathology series.