Standing laparoscopy combined with a conventional inguinal approach to treat extended septic funiculitis in 12 horses.

OBJECTIVE: To describe a 2-step surgical procedure combining standing laparoscopy with a conventional inguinal approach to treat deep intra-abdominal funiculitis (extended septic funiculitis) after castration. STUDY DESIGN: Retrospective case series. SAMPLE POPULATION: Twelve client-owned horses. METHODS: Medical records of horses treated for extended septic funiculitis with the 2-step surgical procedure were reviewed. Data collected included the initial castration technique, number of surgical interventions for septic funiculitis prior to presentation, clinical signs, diagnostic and surgical procedure, and short-term and long-term survival. RESULTS: Complete resection of the infected spermatic cord was achieved without intraoperative complications. Intra-abdominal adhesions were documented in 6 horses, involving the ascending colon (4 cases) and descending colon (2 cases). Postoperatively, minor incisional swelling (2/12) and emphysema (3/12) at the laparoscopic portals were observed. All horses survived to discharge. At 3 months, wounds had completely healed without complications. No recurrence of signs was recorded at long-term follow up (4-36 months after discharge). CONCLUSION: In cases of extended septic funiculitis, complete resection of the infected spermatic cord can be safely performed using a laparoscopic-assisted surgical approach, reducing postoperative complications and risk of recurrence of infection.

Sex Hormone Binding Globulin (SHBG) Mitigates ER Stress in Hepatocytes In Vitro and Ex Vivo.

Despite multiple research studies regarding metabolic syndrome and diabetes, the full picture of their molecular background and pathogenies remains elusive. The latest studies revealed that sex hormone-binding globulin (SHBG)-a serum protein released mainly by the liver-may participate in metabolic dysregulation, as its low serum level correlates with a risk for obesity, metabolic syndrome, and diabetes. Yet, the molecular phenomenon linking SHBG with these disorders remains unclear. In the presented study, we investigate how exogenous SHBG affects metabolically impaired hepatocytes with special attention to endoplasmic reticulum stress (ER stress) and lipid metabolism both in vitro and ex vivo. For that reason, palmitate-treated HepG2 cells and liver tissue samples collected post mortem were cultured in the presence of 50 nM and 100 nM SHBG. We found that SHBG protects against ER stress development and its progression. We have found that SHBG decreased the expression levels of inositol-requiring enzyme 1 (IRE1α), activating transcription factor 6 (ATF6), DNA damage-inducible transcript 3 (CHOP), and immunoglobulin heavy chain-binding protein (BIP). Furthermore, we have shown that it regulates lipolytic gene expression ex vivo. Additionally, herein, we deliver a novel large-animal model to study SHBG in translational research. Our data provide new insights into the cellular and molecular mechanisms by which SHBG modulates hepatocyte metabolism and offer a new experimental approach to study SHBG in human diseases.

Therapeutic mesenchymal stromal stem cells: Isolation, characterization and role in equine regenerative medicine and metabolic disorders.

Mesenchymal stromal cells (MSC) have become a popular treatment modality in equine orthopaedics. Regenerative therapies are especially interesting for pathologies like complicated tendinopathies of the distal limb, osteoarthritis, osteochondritis dissecans (OCD) and more recently metabolic disorders. Main sources for MSC harvesting in the horse are bone marrow, adipose tissue and umbilical cord blood. While the acquisition of umbilical cord blood is fairly easy and non-invasive, extraction of bone marrow and adipose tissue requires more invasive techniques. Characterization of the stem cells as a result of any isolation method, is also a crucial step for the confirmation of the cells' stemness properties; thus, three main characteristics must be fulfilled by these cells, namely: adherence, expression of a series of well-defined differentiation clusters as well as pluripotency. EVs, resulting from the paracrine action of MSCs, also play a key role in the therapeutic mechanisms mediated by stem cells; MSC-EVs are thus largely implicated in the regulation of proliferation, maturation, polarization and migration of various target cells. Evidence that EVs alone represent a complex network 0involving different soluble factors and could then reflect biophysical characteristics of parent cells has fuelled the importance of developing highly specific techniques for their isolation and analysis. All these aspects related to the functional and technical understanding of MSCs will be discussed and summarized in this review.

Laparoscopic resection of an exostosis of the os pubis in a horse.

OBJECTIVE: To report the diagnostic findings and laparoscopic removal of an exostosis of the os pubis in a horse. STUDY DESIGN: Case report. ANIMAL: One 12-year-old Black Forest draught gelding. METHODS: History included recurrent colic before and during urination and poor performance. Findings at rectal examination included a pointed osseous prominence adjacent to the symphysis of the pecten ossis pubis. Cystoscopy revealed that this prominence caused a protrusion of the bladder wall into the lumen. Standing laparoscopy and laparoscopy under general anesthesia were performed. RESULTS: After a failed attempt at standing laparoscopy, the horse was anesthetized, and the exostosis of the os pubis was removed laparoscopically without complications. No recurrence of clinical signs associated with the exostosis was detected 12 months postoperatively. CONCLUSION: Minimally invasive surgical resection of an exostosis of the os pubis was achieved under general anesthesia with appropriately designed instruments. This treatment alleviated symptoms associated with the exostosis, including potential injury of the urinary bladder wall.

Nortropane alkaloids as pharmacological chaperones in the rescue of equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome through mitochondrial potentiation, endoplasmic reticulum stress mitigation and insulin resistance alleviation.

OBJECTIVES: Equine metabolic syndrome (EMS) refers to a cluster of associated abnormalities and metabolic disorders, including insulin resistance and adiposity. The numerous biological properties of mesenchymal stem cells (MSCs), including self-renewal and multipotency, have been the subject of many in-depth studies, for the management of EMS; however, it has been shown that this cell type may be affected by the condition, impairing thus seriously their therapeutic potential. Therefore, an attempt to rescue EMS adipose-derived stem cells (ASCs) with calystegines (polyhydroxylated alkaloids) that are endowed with strong antioxidant and antidiabetic abilities was performed. METHODS: ASCs isolated from EMS horses were subsequently treated with various concentrations of total calystegines. Different parameters were then assessed using flow cytometry, confocal as well as SE microscopy, and RT-qPCR. RESULTS: Our results clearly demonstrated that calystegines could improve EqASC viability and proliferation and significantly reduce apoptosis, via improvement of mitochondrial potentiation and functionality, regulation of pro- and anti-apoptotic pathways, and suppression of ER stress. Furthermore, nortropanes positively upregulated GLUT4 and IRS transcripts, indicating a possible sensitizing or mimetic effect to insulin. Most interesting finding in this investigation lies in the modulatory effect of autophagy, a process that allows the maintenance of cellular homeostasis; calystegines acted as pharmacological chaperones to promote cell survival. CONCLUSION: Obtained data open new perspectives in the development of new drugs, which may improve the metabolic dynamics of cells challenged by MS.

Osteochondritis dissecans (OCD) in Horses - Molecular Background of its Pathogenesis and Perspectives for Progenitor Stem Cell Therapy.

Osteochondrosis (osteochondrosis dissecans; OCD) is a disease syndrome of growing cartilage related to different clinical entities such as epiphysitis, subchondral cysts and angular carpal deformities, which occurs in growing animals of all species, including horses. Nowadays, these disorders are affecting increasing numbers of young horses worldwide. As a complex multifactorial disease, OCD is initiated when failure in cartilage canals because of existing ischemia, chondrocyte biogenesis impairment as well as biochemical and genetic disruptions occur. Recently, particular attention have been accorded to the definition of possible relations between OCD and some metabolic disorders; in this way, implication of mitochondrial dysfunctions, endoplasmic reticulum disruptions, oxidative stress or endocrinological affections are among the most considered axes for future researches. As one of the most frequent cause of impaired orthopaedic potential, which may result in a sharp decrease in athletic performances of the affected animals, and lead to the occurrence of complications such as joint fragility and laminitis, OCD remains as one of the primary causes of considerable economic losses in all sections of the equine industry. It would therefore be important to provide more information on the exact pathophysiological mechanism(s) underlying early OC(D) lesions, in order to implement innovative strategies involving the use of progenitor stem cells, which are considered nowadays as a promising approach to regenerative medicine, with the potential to treat numerous orthopaedic disorders, including osteo-degenerative diseases, for prevention and reduction of incidence of the disease, not only in horses, but also in human medicine, as the equine model is already widely accepted by the scientific community and approved by the FDA, for the research and application of cellular therapies in the treatment of human conditions.

Cladophora glomerata methanolic extract decreases oxidative stress and improves viability and mitochondrial potential in equine adipose derived mesenchymal stem cells (ASCs).

Reactive oxygen species (ROS) are key mediators of several cellular damage and thus associated with equine diseases such as inflammation and metabolic syndrome. This study aimed to evaluate the protective and antioxidant activities of methanolic extract prepared from Cladophora glomerata (C. glomerata) biomass, on equine adipose derived mesenchymal stem cells (EqASCs), under experimental oxidative stress induced by H2O2. Pre-treatment of EqASCs cells with different concentrations of C. glomerata methanolic extract (1% and 5%) provided a clear protection against cellular damage triggered by H2O2. The cell's apoptotic status was significantly regulated, with promotion of cell viability, down-regulation of pro-apoptotic (p21, p53, Bax and Casp-9) genes expression, concomitant to up-regulation of the survival gene Bcl-2, this being supported by a mitigation of the endoplasmic reticulum (ER) stress and significant minimization of mitochondrial dysfunction. The results also showed that C. glomerata extract significantly increased the antioxidant enzymes Superoxide dismutase (SOD) and Catalase (CAT) activities, positively regulated the enzymes genes expression, and markedly reduced the protein carbonyls derivatives production. Finally, RT-qPCR analysis of the inflammatory related genes allowed to highlight a promising anti-inflammatory and immunomodulatory effect of this extract. Due to the valuable antioxidant and anti-inflammatory activities, C. glomerata may have potential benefits for the prevention of equine diseases associated with oxidative stress, including metabolic syndrome.

Immunomodulatory Properties of Adipose-Derived Stem Cells Treated with 5-Azacytydine and Resveratrol on Peripheral Blood Mononuclear Cells and Macrophages in Metabolic Syndrome Animals.

Endocrine disorders, including equine metabolic syndrome (EMS), are a serious issue in veterinary medicine and horse breeding. Furthermore, EMS was shown to affect the cytophysiological properties of adipose-derived stem cells, reducing their therapeutic potential. However, it was shown that those cells can be rejuvenated while using a combination of two chemicals: 5-azacytydine (AZA) and resveratrol (RES). In the present study, we decided to evaluate the immunomodulatory properties of AZA/RES-treated adipose-derived stem cells (ASC) isolated from EMS horses (ASCEMS). Thus, we co-cultured ASC with peripheral blood mononuclear cells (PBMC) and RAW264.7 macrophages. Most attention was placed on regulatory T lymphocytes (TREG), as well as the messenger RNA (mRNA) and protein levels of several cytokines (tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-10, and IL-1ß). Moreover, we also investigated the expression of genes related to auto- and mitophagy in both PBMCs and ASCs. PBMCs were obtained from healthy and EMS-suffering individuals and were co-cultured with ASCs that were isolated from healthy and EMS horses cultured in control conditions and with AZA/RES. We discovered that cells treated with AZA/RES increase the TREG number while co-cultured with PBMCs. Moreover, the co-culture of PBMCs with AZA/RES-treated ASCEMS induced mitophagy in PBMCs. Furthermore, ASCEMS pre-treated with AZA/RES displayed anti-inflammatory properties, as decreased levels of TNF-α, nitric oxide (NO), and IL-6 were observed in those cells in comparison with their untreated counterparts in the co-culture with RAW264.7 macrophages. In summary, we demonstrated that ASCEMS treated with AZA/RES displayed increased anti-inflammatory properties, and was able to regulate and activate the TREG-related anti-inflammatory response.

Thoracoscopic diaphragmatic hernia repair in a warmblood mare.

OBJECTIVE: To describe successful repair of a diaphragmatic hernia in a standing sedated horse using a minimally invasive thoracoscopic technique. STUDY DESIGN: Clinical report. ANIMALS: Warmblood mare with a diaphragmatic hernia. RESULTS: An 18-year-old Warmblood mare with severe colic was referred for surgical treatment of small intestinal strangulation in a diaphragmatic defect. Twelve days after initial conventional colic surgery, left-sided laparoscopy in the standing sedated mare for diaphragmatic herniorrhaphy failed because the spleen obscured the hernia. One week later, a left-sided thoracoscopy was performed in the standing sedated horse and the hernia repaired by an intrathoracic suture technique. No long-term complications occurred (up to 4 years) and the mare returned to her previous athletic activity, followed by use as a broodmare. CONCLUSIONS: To avoid the high risks associated with general anesthesia, and to reduce surgical trauma and postoperative recovery, central diaphragmatic hernias are amenable to repair using a minimally invasive thoracoscopic technique in the standing sedated horse.

Endoscopic-assisted electrohydraulic shockwave lithotripsy in standing sedated horses.

OBJECTIVE: To report use of transendoscopic electrohydraulic shockwave lithotripsy for fragmentation of urinary calculi in horses. STUDY DESIGN: Case series. ANIMALS: Male horses (n = 21). METHODS: Fragmentation of cystic calculi (median, 6 cm diameter; range, 4-11 cm diameter) was achieved by transurethral endoscopy in standing sedated horses using an electrohydraulic shockwave fiber introduced through the biopsy channel of an endoscope. The fiber was advanced until it contacted the calculus. Repeated activation of the fiber was used to disrupt the calculus into fragments <1 cm diameter. Visibility within the bladder was maintained by repeated lavage with saline solution. RESULTS: Complete calculus removal was achieved in 20 horses (95%) with mean total surgical time of 168.6 minutes (range, 45-450). In the 20 horses with single calculi, 1-6 sessions were required to completely fragment the calculus. Except for 1 horse, in which perineal urethrotomy was eventually performed for complete fragment removal, fragments calculi were excreted via the urethra. Postoperative complications included hematuria because of severe mucosal erosion (n = 2), dysuria because of a trapped urethral fragment (2), small amount of urinary debris (1). One horse was euthanatized because of bladder rupture. Complete clearance of calculi and urinary debris was confirmed endoscopically 20 (3-45) days after the last session. Telephone follow-up (mean, 18.8 months; range, 7-24 months) revealed that horses had returned to previous activity levels without recurrence of clinical signs. CONCLUSIONS: Transendoscopic electrohydraulic lithotripsy appears to be an effective method for fragmentation of low-density calcium carbonate cystic calculi in male horses.

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells.

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.

Unilateral and bilateral laparoscopic ovariectomy in 157 mares: a retrospective multicenter study.

OBJECTIVE: To evaluate surgical techniques, perioperative complications and outcome of laparoscopic surgery for unilateral and bilateral ovariectomy in mares. STUDY DESIGN: Retrospective multicenter case series. ANIMALS: Mares (n = 157). METHODS: Indications for surgery were neutering, fertility problems, elimination of hormone-related abnormal behavior, ovarian tumors as well as nonregressive ovarian hematomas or cysts. Ovariectomy (n = 206) were performed by a standard laparoscopic procedure in 157 standing sedated mares. Dissection and hemostasis were achieved by using either (1) a linear stapling device, (2) bipolar electrosurgical instruments, with or without a modified Roeder knot, (3) a vessel-sealing system, or (4) a diode-laser with ligation. For some larger ovarian masses (>20 cm diameter), a 2-step surgical procedure was used with standing flank laparoscopic ovariectomy followed by ovarian retrieval through a median celiotomy. Surgical techniques, outcome, and perioperative complications of each method were recorded and analyzed. RESULTS: Laparoscopic dissection of the ovary was accomplished in all mares. Because of the size of the ovarian tumor (n = 7) or in 1 mare with behavioral problems (n = 1), the dissected ovary was removed through a median celiotomy under general anesthesia. Seventeen mares (10.8%) developed postoperative complications (eg, incisional drainage, incisional infection, seroma formation, dehiscence, transient fever and mild abdominal discomfort). All mares with flank incisional problems had ovarian size >12 cm and in 15 (88%) of these mares electrosurgical instruments were used for mesovarial dissection. CONCLUSIONS: Laparoscopic ovariectomy in standing sedated mares provides good surgical access and is associated with low morbidity.

Characterization of adipose-derived equine and canine mesenchymal stem cells after incubation in agarose-hydrogel.

Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive application. Isolated ASCs were expanded and 2.5 × 10(6) cells were combined with agarose-hydrogel to build a 0.4% hydrogel-cell solution which was stored at two temperatures (room temperature (RT) vs. 37 °C). Cell viability was investigated (live-dead assay) at different time points (0, 1, 6 and 24 h) in order to determine i) the effect of different temperatures on the cell survival as well as ii) the maximum possible time span before implantation. CFU-assay and WST-1 assay were performed after 24 h incubation in agarose-hydrogel and the cells were induced into adipogenic and osteogenic differentiation to analyze the effects of the incubation on the cell behaviour. No negative effect of the agarose-hydrogel incubation was determined on the different species' cell behaviour at either RT or 37 °C with any of the assays used. We can recommend agarose-hydrogel as a cell carrier for cell implantation with a storage period of up to 24 h at room temperature or at 37 °C prior to implantation.

Left- and right-sided laparoscopic-assisted nephrectomy in standing horses with unilateral renal disease.

OBJECTIVE: To describe a technique for, and outcome after, left- or right-sided laparoscopic-assisted nephrectomy in standing horses with unilateral renal disease. STUDY DESIGN: Clinical report. ANIMALS: Horses (n=3) with unilateral renal disease. METHODS: Horses were sedated with detomidine (0.01 mg/kg intravenously [IV]) and levomethadone (0.05 mg/kg IV). Paravertebral anesthesia and infiltration-anesthesia with 2% lidocaine were used to create a surgical field incorporating the 17th intercostal space and paralumbar fossa. Two separate, ipsilateral portals and a mini-laparotomy were used. The perirenal peritoneum was horizontally incised (10-15 cm) using endoscissors and the incision digitally enlarged for manual dissection of the perirenal fat and kidney mobilization. The renal vessels and ureter were individually dissected, ligated, and transected under laparoscopic observation and the kidney removed. The perirenal and laparotomy peritoneal defects were not closed; and the laparotomy was closed in a multilayered fashion. The transverse abdominal muscle was apposed in a continuous pattern using 1 polyglactin 910, the subcutaneous tissue (simple continuous pattern) and skin (simple interrupted pattern) with 2-0 polyglactin 910. RESULTS: Left (2) and right (1) sided laparoscopic-assisted nephrectomy (1 nephrolithiasis, 2 hydronephrosis) was performed successfully. Sedation and local anesthesia was adequate for intraoperative immobilization and analgesia. No intraoperative complications occurred. Incisional seroma formation and fever occurred on days 3 and 4 in 1 horse and resolved with medical management. CONCLUSION: Laparoscopic-assisted nephrectomy can be used for removal of the left or right kidney in standing horses with unilateral kidney disease. CLINICAL RELEVANCE: To avoid risks associated with general anesthesia and to reduce surgical trauma, laparoscopic-assisted nephrectomy can be performed in the standing sedated horse using a 2 portal technique and a mini-laparotomy.

Laparoscopic-assisted cystotomy for urolith removal in geldings.

OBJECTIVE: To describe a technique for laparoscopic-assisted removal of cystic calculi in geldings and report outcome. STUDY DESIGN: Clinical report. ANIMALS: Four geldings with cystic calculi. METHODS: Laparoscopic-assisted cystotomy and urolith retrieval was performed in 4 anesthetized geldings positioned in dorsal recumbency. With a laparoscope portal located at the umbilicus, the abdomen was insufflated and then the surgical table was tilted (30 degrees head-down position) before an instrumental portal was created parallel and 2-3 cm medial to the left external inguinal ring. Laparoscopic grasping forceps were inserted to grasp the cranial aspect of the bladder and elevate it to the ventral abdominal wall. With the instrumental portal as mid-point, the parainguinal skin incision was longitudinally extended cranial and caudal (approximately 8-10 cm) to accommodate the size of the urolith. The apex of the bladder was exteriorized and sharply incised, the urolith extracted, and after cystotomy closure, the bladder was repositioned. The mini-laparotomy and trocar incisions were closed in layers. RESULTS: There were no intra- or post-operative complications. All horses had minor incisional swelling for 3-4 days. No signs of abdominal or incisional pain were observed. Hematuria and slight stranguria occurred until the 3rd or 4th day. Surgical time (skin incision to skin closure) was 35-40 minutes. On long-term follow-up (up to 12 months) no recurrence of clinical signs associated with cystic calculi occurred. CONCLUSION: Uroliths (6-8 cm diameter) can be removed by laparoscopic-assisted cystotomy in geldings. CLINICAL RELEVANCE: Laparoscopic-assisted cystotomy combines the advantages of the parainguinal laparocystotomy with laparoscopic technique for removal of cystic calculi while avoiding their disadvantages.

Indications, surgical technique, and long-term experience with laparoscopic closure of the nephrosplenic space in standing horses.

OBJECTIVE: To describe surgical technique and outcome after laparoscopic closure of the nephrosplenic space for treatment of recurrent left dorsal displacement of the left colon (LDDLC) in standing horses. STUDY DESIGN: Retrospective study. ANIMALS: Forty-four horses with LDDLC. METHODS: Laparoscopic portals were located in the left flank region. After administration of etilefrin intravenously to contract the spleen, the dorsal splenic margin was sutured to the nephrosplenic ligament to obliterate the nephrosplenic space. Horses were re-examined (< or = 3 years) for history of recurrence. In addition, medical records of 4,852 horses treated for colic over 16 years were evaluated to establish incidence of LDDLC and recurrence after treatment. RESULTS: Splenic contraction facilitated suture placement and closure of the nephrosplenic space was achieved without complications. LDDLC recurrence did not occur although 5 horses had subsequent episodes of colic; 4 horses had displacement of the ascending colon between the spleen and body wall. Review of medical records revealed an incidence of LDDLC of 6% and recurrence of 21% in a population of horses with signs of colic. CONCLUSION: Laparoscopic closure of the nephrosplenic space, facilitated by etilefrin-induced splenic contraction can be efficiently performed in standing horses. CLINICAL RELEVANCE: Laparoscopic ablation of the nephrosplenic space should be considered in horses that are predisposed to recurrent LDDLC.