High positive end-expiratory pressure ventilation mitigates the progression from unilateral pulmonary contusion to ARDS: An animal study.

BACKGROUND: Pulmonary contusion (PC) is common in severely traumatized patients and can lead to respiratory failure requiring mechanical ventilation (MV). Ventilator-induced lung injury (VILI) might aggravate lung damage. Despite underrepresentation of trauma patients in trials on lung-protective MV, results are extrapolated to these patients, potentially disregarding important pathophysiological differences. METHODS: Three MV protocols with different positive end-expiratory pressure (PEEP) levels: ARDSnetwork lower PEEP (ARDSnet-low), ARDSnetwork higher PEEP (ARDSnet-high), and open lung concept (OLC) were applied in swine for 24 hours following PC. Gas exchange, lung mechanics, quantitative computed tomography, and diffuse alveolar damage (DAD) score were analyzed. Results are given as median (interquartile range) at 24 hours. Statistical testing was performed using general linear models (group effect) over all measurement points and pairwise Mann-Whitney U tests for DAD. RESULTS: There were significant differences between groups: PEEP ( p < 0.0001) ARDSnet-low (8 [8-10] cmH 2 O), ARDSnet-high (12 [12-12] cmH 2 O), OLC (21 [20-22] cmH 2 O). The fraction of arterial partial pressure of oxygen and inspired oxygen fraction ( p = 0.0016) was lowest in ARDSnet-low (78 (73-111) mm Hg) compared with ARDSnet-high (375 (365-423) mm Hg) and OLC (499 (430-523) mm Hg). The end-expiratory lung volume (EELV) differed significantly ( p < 0.0001), with highest values in OLC (64% [60-70%]) and lowest in ARDSnet-low (34% [24-37%]). Costa's surrogate for mechanical power differed significantly ( p < 0.0001), with lowest values for ARDSnet-high (73 [58-76]) compared with OLC (105 [108-116]). Diffuse alveolar damage was lower in ARDSnet-high compared with ARDSnet-low (0.0007). CONCLUSION: Progression to ARDS, 24 hours after PC, was mitigated by OLC and ARDSnet-high. Both concepts restored EELV. ARDSnet-high had the lowest mechanical power surrogate and DAD. Our data suggest, that ARDSnet-high restored oxygenation and functional lung volume and reduced physiological and histological surrogates for VILI. ARDSnet-low generated unfavorable outcomes, such as loss of EELV, increased mechanical power and DAD after PC in swine. The high respiratory rate in the OLC may blunt favorable effects of lung recruitment.

Treatment of Naturally Occurring Tendon Disease with Allogeneic Multipotent Mesenchymal Stromal Cells: A Randomized, Controlled, Triple-Blinded Pilot Study in Horses.

The treatment of tendinopathies with multipotent mesenchymal stromal cells (MSCs) is a promising option in equine and human medicine. However, conclusive clinical evidence is lacking. The purpose of this study was to gain insight into clinical treatment efficacy and to identify suitable outcome measures for larger clinical studies. Fifteen horses with early naturally occurring tendon disease were assigned to intralesional treatment with allogeneic adipose-derived MSCs suspended in serum or with serum alone through block randomization (dosage adapted to lesion size). Clinicians and horse owners remained blinded to the treatment during 12 months (seven horses per group) and 18 months (seven MSC-group and five control-group horses) of follow-up including clinical examinations and diagnostic imaging. Clinical inflammation, lameness, and ultrasonography scores improved more over time in the MSC group. The lameness score difference significantly improved in the MSC group compared with the control group after 6 months. In the MSC group, five out of the seven horses were free of re-injuries and back to training until 12 and 18 months. In the control group, three out of the seven horses were free of re-injuries until 12 months. These results suggest that MSCs are effective for the treatment of early-phase tendon disease and provide a basis for a larger controlled study.

Successful intra-abdominal resection of a 24 kg ovarian granulosa cell tumor in a Warmblood mare.

Background: Granulosa cell tumors (GCTs) are the most common ovarian tumors in mares. The classical presentation of a GCT is a unilaterally enlarged ovary appearing as a multicystic honeycomb mass. In rare cases, GCTs cause hemoperitoneum as a result of the rapid growth of the tumor. The clinical diagnosis of GCT is usually based on history, rectal examination, ultrasonographic examination, and serum hormone analysis, and surgical removal of the affected ovary is the treatment of choice. The different surgical approaches are based on the dimension of the GCT. Case Description: A 7-year-old mare was referred to the department for horses due to suspicion of a large colon impaction. The mare presented with clinical signs of colic, fever, and signs of hypovolemic shock. Rectal and ultrasonographic examination showed hemoperitoneum and a honey-comb mass within the abdomen, and a GCT as the cause of an acute hemoperitoneum was diagnosed based on the serum level of anti-Müllerian hormone. After stabilization of the mare, the GCT was removed through a ventral midline incision. Because of the enormous dimensions of the GCT, intra-abdominal partial resection of the tumor using a tenotomy knife was necessary to exteriorize the ovarian pedicle. At 3 months follow-up, the mare was ridden for her intended use. Conclusion: This report provides an approach to an uncommon case of a very large and heavy GCT.

Pyelonephritic end-stage kidney and ureterocutaneous fistula in a Shetland pony.

A 12-y-old Shetland Pony was presented with a mucus-secreting fistula in the right paralumbar fossa. Surgery was performed to unravel the origin of the fistula. The horse died under anesthesia and was forwarded to autopsy. The right kidney was markedly atrophic and fibrotic, consistent with unilateral end-stage kidney. The right ureter was markedly thickened, but with luminal continuity leading into the urinary bladder where a partial obstruction caused by nodular para-ureteral fat necrosis was evident. The lumen of the cutaneous fistula was continuous with the right ureter; therefore, we diagnosed the lesion as a ureterocutaneous fistula. Anomalies of the ureter are uncommon, and ureterocutaneous fistula formation in equids has not been reported previously to our knowledge.

Synovial Fluid Analysis and Microscopic Assessment of Macrophage Quantities and Morphology in Equine Septic Arthritis.

OBJECTIVE: Research and provision of data on macrophages by cytological synovial fluid analysis and light microscopy in horses with septic arthritis MATERIAL AND METHODS: Records of 167 synovial fluid samples were evaluated and subdivided into different groups: (1) non-septic, (2) haematogenous septic arthritis in foals and (3) traumatic/iatrogenic septic arthritis. The effect of joint lavage on synovial fluid cytology and on the occurrence of macrophage phenotypes was investigated. RESULTS: Regardless of aetiology and age of the horse, macrophage concentrations in synovial sepsis are decreased to a median of 5-6 % (unaffected joints: 23.5 %) and further diminished by joint lavage. Microscopic assessment led to the identification of 4 phenotypes. Morphological characteristics of type 1 showed similarities to monocytes and predominated in unaffected and in septic joints after lavage. CONCLUSION AND CLINICAL RELEVANCE: Macrophages are highly versatile by altering their phenotype. A morphological assessment by light microscopy is easily applicable. Type 1 presumably contributes to joint homeostasis.

Xylazine or detomidine in dairy calves: a comparison of clinically relevant pharmacodynamic parameters under sedation.

OBJECTIVE: Chemical restraint in dairy calves is necessary to enable diagnostic and surgical procedures. It is unclear whether xylazine or detomidine differ with regard to desirable and unwanted effects. MATERIAL AND METHODS: In a prospective randomized interventional study, 10 healthy Holstein-Friesian calves (age range 3-6 month) were sedated with either xylazine (0.1 mg/kg, Group X, n = 5) or detomidine (0.03 mg/kg, Group D, n = 5) intravenously, followed by butorphanol (0.1 mg/kg i. v.) in all animals. Characteristics of sedation and selected pharmacodynamic parameters were compared between groups using a non-parametric Mann-Whitney U test. RESULTS: All calves (5/5) in Group X and (3/5) calves in Group D became laterally recumbent within 5 minutes. Two calves (40 %) in Group D remained standing and could not been positioned in lateral recumbency 15 minutes after initial administration of the sedation agents. Sedation scores, onset and duration of sedation did not differ between groups. Heart and respiratory rate decreased in both groups. Mean arterial pressure was with around 30 mmHg significantly higher in Group D (t25, t30, t35, t40 with p = 0.018, 0.036, 0.029 and 0.016, respectively). In Group X, glucose level (t60) and packed cell volume (t30) were significantly lower (p = 0.032 and 0.048, respectively). CONCLUSION: and clinical relevance The xylazine-butorphanol combination provided reliable recumbent chemical restraint. With detomidine-butorphanol recumbency failed in some individuals, but a sufficient clinical sedation was achieved. Based on the limited monitoring used in this study, the side effects are of minor clinical relevance in healthy individuals.

Surgical hand preparation in an equine hospital: Comparison of general practice with a standardised protocol and characterisation of the methicillin-resistant Staphylococcus aureus recovered.

Presurgical hand asepsis is part of the daily routine in veterinary medicine. Nevertheless, basic knowledge seems to be low, even among specialised veterinary surgeons. The major objectives of our study were to assess current habits for presurgical hand preparation (phase 1) among personnel in a veterinary hospital and their effectiveness in reducing bacteria from hands in comparison to a standardised protocol (phase 2). Assessment of individual habits focused on time for hand washing and disinfection, the amount of disinfectant used, and the usage of brushes. The standardised protocol defined hand washing for 1 min with liquid neutral soap without brushing and disinfection for 3 min. All participants (2 surgeons, 8 clinic members, 32 students) used Sterillium®. Total bacterial counts were determined before and after hand washing, after disinfection, and after surgery. Hands were immersed in 100 ml sterile sampling fluid for 1 min and samples were inoculated onto Columbia sheep blood agar using the spread-plate method. Bacterial colonies were manually counted. Glove perforation test was carried out at the end of the surgical procedure. Differences in the reduction of relative bacterial numbers between current habits and the standardised protocol were investigated using Mann-Whitney-Test. The relative increase in bacterial numbers as a function of operation time (≤60 min, >60 min) and glove perforation as well as the interaction of both was investigated by using ANOVA. Forty-six and 41 preparations were carried out during phase 1 and phase 2, respectively. Individual habits differed distinctly with regard to time (up to 8 min) and amount of disinfectant (up to 48 ml) used both between participants and between various applications of a respective participant. Comparison of current habits and the standardised protocol revealed that the duration of hand washing had no significant effect on reducing bacteria. Contrary, the reduction in bacterial numbers after disinfection by the standardised protocol was significantly higher (p<0.001) compared to routine every-day practice. With regard to disinfection efficacy, the standardised protocol completely eliminated individual effects. The mean reduction in phase 1 was 90.72% (LR = 3.23; right hand) and 89.97% (LR = 3.28; left hand) compared to 98.85% (LR = 3.29; right hand) and 98.92% (LR = 3.47; left hand) in phase 2. Eight participants (19%) carried MRSA (spa type t011, CC398) which is well established as a nosocomial pathogen in veterinary clinics. The isolates could further be assigned to a subpopulation which is particularly associated with equine clinics (mainly t011, ST398, gentamicin-resistant). Glove perforation occurred in 54% (surgeons) and 17% (assistants) of gloves, respectively, with a higher number in long-term invasive procedures. Overall, bacterial numbers on hands mainly increased over time, especially when glove perforation occurred. This was most distinct for glove perforations on the left hand and with longer operating times. Our results demonstrate that standardised protocols highly improve the efficacy of hand asepsis measures. Hence, guiding standardised protocols should be prerequisite to ensure state-of-the-art techniques which is essential for a successful infection control intervention.

Transforming Growth Factor Beta 3-Loaded Decellularized Equine Tendon Matrix for Orthopedic Tissue Engineering.

Transforming growth factor beta 3 (TGFß3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGFß3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGFß3 in an in vitro model. TGFß3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGFß3 were not detected in the rinsing fluid of the TGFß3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGFß3 to assess its bioactivity. Both scaffold-associated TGFß3 and TGFß3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGFß3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaffold-associated TGFß3 is preserved, thus TGFß3 application via absorption in decellularized tendon scaffolds is a feasible approach.

A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays.

Impact of body weight gain on hepatic metabolism and hepatic inflammatory cytokines in comparison of Shetland pony geldings and Warmblood horse geldings.

BACKGROUND: Non-alcoholic fatty liver disease is known as determining part of human obesity. The impact of body weight (BW) gain on liver metabolism has not been extensively investigated yet. OBJECTIVES: To investigate hepatic alterations caused by increasing BW in ponies and horses. ANIMALS: A total of 19 non-obese equines (10 Shetland ponies, geldings; nine Warmblood horses, geldings). METHODS: Animals received 200% of their metabolizable maintenance energy requirements for 2 years. Serum alkaline phosphatase, glutamate dehydrogenase (GLDH), aspartate aminotransferase (AST), and gamma-glutamyl transferase activities and bile acids were analyzed several times during 2 years of hypercaloric diet. Hepatic lipid content and hepatic levels of the interleukin (IL)-6, tumor necrosis factor α (TNFα), cluster of differentiation (CD) 68, IL-1ß, lipoprotein lipase (LPL), fatty acid-binding protein 1, chemerin and nuclear factor-κB mRNAs were assessed at the start of the study and after 1 and 2 years of excess energy intake. RESULTS: The mean (±SD) BW gain recorded during 2 years of excess energy intake was 29.9 ± 19.4% for ponies and 17 ± 6.74% for horses. The hepatic lipid content was not profoundly affected by increasing BW. Levels of the IL-6, TNFα, CD68 and IL-1ß mRNAs did not change during BW gain. Levels of the chemerin mRNA increased significantly in both breeds (ponies: P = 0.02; horses: P = 0.02) in response to BW gain. Significant differences in serum GLDH and AST activities, serum bile acid concentrations and hepatic levels of the LPL mRNA were observed between ponies and horses at the end of the study. CONCLUSIONS: Chemerin might represent an interesting marker for future equine obesity research. Interestingly, steatosis caused by increasing BW may occur later in the development of obesity in equines than in humans. Additionally, the hepatic metabolism exhibits differences between ponies and horses, which may explain in part the greater susceptibility of ponies to obesity-associated metabolic dysregulations.

[Intravitreal injection of low-dose gentamicin for the treatment of ERU-affected horses]. / Intravitreale Low-Dose-Gentamicininjektion zur Behandlung ERU-erkrankter Pferde.

OBJECTIVE: Aim of the study was the retrospective evaluation regarding vision and recurrence in equine recurrent uveitis (ERU)-affected horses treated by intravitreal low-dose gentamicin injection in Tierklinik Alt Sammit during the years 2006-2013. MATERIAL AND METHODS: The study included 50 equine patients (61 ERU-affected eyes) of different age and breed with different stages of ERU. The solution for the injection contained 4.0 mg sterile gentamicin per eye. The injection was performed according to the procedure described by Pinard et al. in 2005 under general anaesthesia in lateral recumbency of the patient and within a non-inflammatory period. The patients were monitored over a postoperative period of 2-96 months (mean 25.8 months, median 15 months). RESULTS: A total of 56 out of 61 treated eyes displayed no recurrence of ERU during the follow-up period. Recurrence of a single uveitic attack occurred in five eyes (8.2 %) within a period of 2 weeks to 28 months after surgery. Long-term stable vision was achieved in 43 out of 61 eyes (70.5 %). Postoperative blindness developed in 18 eyes, the majority hereof had already preoperatively displayed obvious pathologic ophthalmic findings with partly highly impaired vision. The main reason for a vision loss was the progression of a preoperatively existing cataract formation. Perioperative complications were limited to vitreous reflux into the subconjunctival space (two cases), subconjunctival haemorrhage in the area of the injection site (two cases), vitreous haemorrhage (one case) and two cases of immediate postoperative pain reaction associated with the intravitreal injection. CONCLUSION AND CLINICAL RELEVANCE: The results of the therapy regarding the reduction of the recurrence rate and preservation of vision are comparable to those of vitrectomy. The complication rate is extremely low when using both the correct injection technique and amount of gentamicin solution. Therefore, the intravitreal injection of low-dose gentamicin is an inexpensive and uncomplicated alternative to vitrectomy.

Palmar arthroscopic approach and intra-articular anatomy of the bovine carpal joints.

OBJECTIVE: To describe the palmar arthroscopic approaches to the bovine carpus and document the visible intra-articular structures in each approach. STUDY DESIGN: Ex vivo study. SAMPLE POPULATION: Thirty fresh cadaveric bovine forelimbs. METHODS: Computed tomography (CT) and gross dissection were performed on 4 carpi. Latex models were produced from 6 carpi to define the gross anatomy and arthroscopic portals to the palmar carpal pouches. Ultrasonography and arthroscopy were performed on 20 carpi to document visualization of the local anatomy. RESULTS: A single palmar pouch was present on both sides of each joint on gross dissection and evaluation of latex models. The relationship between pouches and palmar structures was observed by CT. Small anechoic areas identified on ultrasonography increased after joint distension. Palmar arthroscopy of the antebrachiocarpal joint enabled visualization of the palmar surfaces of radial, intermediate, and ulnar carpal bones; distal radius; ulnar styloid process; palmar radiocarpal ligament; and articulation of accessory and ulnar carpal bones. Palmar arthroscopy of the middle carpal joint enabled inspection of the palmar aspects of ulnar, intermediate, radial, fused second and third and fourth carpal bones, palmar carpometacarpal ligament, and the short lateral collateral ligament. Palmar aspects of the fourth and fifth metacarpal bones were explored through the palmarolateral approach to the middle carpal joint. Instrument portals for each pouch were created under arthroscopic guidance. CONCLUSION: Palmar arthroscopy of the adult bovine carpus enabled visualization of the clinically relevant palmar intra-articular structures. CLINICAL SIGNIFICANCE: Palmar carpal arthroscopy should be considered as an adjunct to dorsal approach for the diagnosis and treatment of bovine carpal diseases.

Growth Factor-Mediated Tenogenic Induction of Multipotent Mesenchymal Stromal Cells Is Altered by the Microenvironment of Tendon Matrix.

Age-related degenerative changes in tendon tissue represent a common cause for acute tendon pathologies. Although the regenerative potential of multipotent mesenchymal stromal cells (MSC) was reported to restore functionality in injured tendon tissue, cellular mechanisms of action remain partly unclear. Potential tenogenic differentiation of applied MSC is affected by various intrinsic and extrinsic factors. The current study presents an in vitro model to evaluate the combined extrinsic effects of decellularized equine tendon matrix, transforming growth factor beta 3 (TGFß3) and bone morphogenetic protein 12 (BMP12) on the tenogenic fate of equine adipose tissue-derived MSC. Monolayer MSC cultures supplemented with TGFß3 and BMP12 as well as MSC cultured on tendon matrix scaffolds preloaded with the growth factors were incubated for 3 and 5 days. Histological evaluation and real time reverse transcription polymerase chain reaction (RT-PCR) revealed that growth factor-mediated tenogenic induction of MSC was modified by the conditions of the surrounding microenvironment. While the gene expression pattern in monolayer cultures supplemented with TGFß3 or TGFß3 and BMP12 revealed an upregulation for collagen 1A2, collagen 3A1, tenascin c, scleraxis and mohawk ( p < 0.05 ), the presence of tendon matrix led to an upregulation of decorin and osteopontin as well as to a downregulation of smad8 ( p < 0.05). Preloading of scaffolds with either TGFß3, or with TGFß3 and BMP12 promoted a tenocyte-like phenotype and improved cell alignment. Furthermore, gene expression in scaffold culture was modulated by TGFß3 and/or BMP12, with downregulation of collagen 1A2, collagen 3A1, decorin, scleraxis, smad8 and osteopontin, whereas gene expression of tenascin c was increased. This study shows that growth factor-induced tenogenic differentiation of equine MSC is markedly altered by topographical constraints of decellularized tendon tissue in vitro. While TGFß3 represents an effective mediator for tenogenic induction, the role of BMP12 in tenogenesis may be of modulatory character and needs further evaluation.

Tenogenic Properties of Mesenchymal Progenitor Cells Are Compromised in an Inflammatory Environment.

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1ß and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.

Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P < 0.05), with lower percentages of CD90+ cells found in equine MSC samples cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29+ (98.60-99.90%), CD73+ (94.60-98.40%), CD90+ (99.60-99.90%), and CD105+ (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species-specific, indicating a need for serum-free media to be optimized for MSCs from relevant animal species. © 2017 International Society for Advancement of Cytometry.

Incisional complications following ventral median coeliotomy in horses. Does suturing of the peritoneum reduce the risk? / Wundheilungsstörungen nach ventraler medianer Laparotomie beim Pferd. Reduziert die Bauchfellnaht Wundheilungsstörungen?

OBJECTIVE: Despite advances in surgical technique in abdominal surgery, incisional complications are frequently reported following ventral midline laparotomy in horses. The aim of this study was to determine the incidence of incisional complications at our clinic and to identify possible risk factors. Furthermore, we investigated whether suturing the peritoneum leads to a reduction of incisional complications. MATERIAL AND METHODS: In this retrospective study, records of patients of the Large Animal Clinic for Surgery of the University of Leipzig from January 2010 to December 2015 were analysed. Horses with ventral midline laparotomy following colic were included in the study. Evaluated parameters comprised breed, sex, age of the horse, localisation of the colic disease, invasiveness of the surgical intervention, duration of general anaesthesia, suture pattern of the laparotomy wound and postoperative fever or leukopenia. Incisional complications included suture dehiscence, exudation from the wound and incisional hernia formation. Wound oedema formation was evaluated separately. RESULTS: Incisional complications in the form of exudation and suture dehiscence occurred in 8.9% (18/202) of the horses. Postoperative hernia formation was observed in 5.2% (9/173) of patients. Breed, sex, localisation of the colic disease, invasiveness of the surgical intervention, duration of general anaesthesia and postoperative leukopenia did not influence the frequency of incisional complications. An increased risk of incisional complications was found in horses aged 20 years and older (odds ratio [OR] 17.90), in animals with postoperative fever (OR 7.48) and in horses with unsutured peritoneum (OR 7.68). Furthermore, patients with moderate and severe wound oedema displayed a significantly increased risk for the development of incisional complications. CONCLUSION AND CLINICAL RELEVANCE: Suture pattern is the only risk factor that can be directly influenced by the surgeon. Because a peritoneal suture is associated with a decreased risk of incisional complications, it should be standard practice when closing a laparotomy wound in the horse.

Retrospective Evaluation of Hemithyroidectomy in 14 Horses.

OBJECTIVE: To describe the presentation, presurgical diagnostic findings, treatment, and outcome of horses with histologically confirmed, unilateral thyroid neoplasia. The complications, particularly laryngeal hemiplegia, were investigated. STUDY DESIGN: Retrospective case series. ANIMALS: Client-owned horses (n=14). METHODS: Medical records of horses presenting with a unilateral thyroid mass due to neoplasia from 2003-2015 were reviewed. Horses must have undergone preoperative clinical evaluations that included ultrasound examination of the mass and upper airway endoscopy. Short-term follow-up at 2 weeks after surgery and owner questionnaire for long-term follow-up at >6 months were completed. RESULTS: Fourteen horses aged 6-21 years were included. The majority of tumors were adenocarcinomas (11/14), mainly diagnosed in mares (9/14). Intraoperative complications included hemorrhage (1/14) and postoperative complications included seroma formation (4/14). No horse developed postoperative laryngeal hemiplegia. No horses developed clinical signs of metastases or a thyroid disorder long term (mean follow-up 4.9 years). All owners reported a successful long-term outcome. CONCLUSION: The clinical findings of thyroid neoplasia in horses are not associated with the diagnosis of malignancy. Complete surgical resection of the abnormal lobe prevents local recurrence of neoplastic thyroid tissue. The modified hemithyroidectomy technique preserves the function of the recurrent laryngeal nerve.

Comparison of humoral insulin-like growth factor-1, platelet-derived growth factor-BB, transforming growth factor-ß1, and interleukin-1 receptor antagonist concentrations among equine autologous blood-derived preparations.

OBJECTIVE To compare humoral insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-ß1, and interleukin-1 receptor antagonist (IL-1Ra) concentrations in plasma and 3 types of equine autologous blood-derived preparations (ABPs). SAMPLE Blood and ABP samples from 12 horses. PROCEDURES Blood samples from each horse were processed by use of commercial systems to obtain plasma, platelet concentrate, conditioned serum, and aqueous platelet lysate. Half of the platelet concentrate samples were additionally treated with a detergent to release intracellular mediators. Humoral IGF-1, PDGF-BB, TGF-ß1, and IL-1Ra concentrations were measured with ELISAs and compared statistically. RESULTS Median IGF-1 concentration was highest in conditioned serum and detergent-treated platelet concentrate, followed by platelet concentrate and plasma; IGF-1 was not detected in platelet lysate. Mean PDGF-BB concentration was highest in platelet lysate, followed by detergent-treated platelet concentrate and conditioned serum; PDGF-BB was not detected in plasma and platelet concentrate. Median TGF-ß1 concentration was highest in detergent-treated platelet concentrate, followed by conditioned serum, platelet lysate, and platelet concentrate; TGF-ß1 was not detected in most plasma samples. Median IL-1Ra concentration was highest in platelet lysate, followed by conditioned serum; IL-1Ra was not detected in almost all plasma, detergent-treated platelet concentrate, and platelet concentrate samples. CONCLUSIONS AND CLINICAL RELEVANCE Each ABP had its own cytokine profile, which was determined by the specific processing method. Coagulation and cellular lysis strongly increased humoral concentrations of cell-derived cytokines. No ABP had the highest concentrations for all cytokines. Further studies are needed to assess clinical relevance of these findings.

Long-Term Cell Tracking Following Local Injection of Mesenchymal Stromal Cells in the Equine Model of Induced Tendon Disease.

Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10106 Molday ION Rhodamine B-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and histology. Cell tracking based on the hypointense artifacts induced by the superparamagnetic iron oxide (SPIO) labeling agent in MRI as well as based on Rhodamine B fluorescence was feasible. However, Prussian blue staining for assessment of histology was not entirely specific for SPIO. Labeled cells could be traced at their injection site by MRI as well as histology for the whole follow-up period of 24 weeks. Although the numbers of labeled cells within the injected tendon lesions decreased over time, part of the applied cells appeared to remain viable and integrated within the injured tissue. Furthermore, small numbers of labeled cells were identified in peripheral blood within the first 24 h after cell injection and could also be found until week 24 within the contralateral control tendon lesions that had been injected with serum. The present findings unveil details on MSC biodistribution and persistence after their local application, which are of clinical relevance with regard to MSC safety and mechanisms of action.