Surgical Closure of Equine Abdomen, Prevention, and Management of Incisional Complications.

Although rarely fatal, complications of ventral midline laparotomy incision in equine patients increase hospitalization cost and duration and may jeopardize return to athletic function. Therefore, many techniques have been developed to reduce their occurrence and expedite their resolution when they occur. Our technique of celiotomy incision closure includes the use of tension sutures (vertical U mattress) of polyglactin 910 on the linea alba, which is then apposed by polyglactin 910 interrupted sutures or a simple continuous pattern suture with a stop midway before routine closure of the superficial layers. The celiotomy incision is protected by an elastic bandage during the immediate postoperative period. This technique has been associated with favorable results: 5.3% confirmed incisional infections after a single celiotomy and 26.7% after repeat celiotomy. The overall incisional complication (serous/sanguineous discharge, hematoma, infection, hernia formation, and complete wound breakdown) occurrence was 9.5% and 33.3% after single and repeat laparotomy, respectively. In cases considered more susceptible to infection (early relaparotomy or laparotomy incisions longer than 30 cm), negative pressure therapy was found easy to apply on closed incisions. No detrimental effects were observed. However, the potential prophylactic benefit of this therapy needs to be confirmed in a larger group. In infected laparotomy wounds requiring drainage, the use of negative pressure therapy seemed to have a positive effect on the formation of granulation tissue. However, there was no control group to allow statistical confirmation. Finally, one case of complete breakdown of the laparotomy incision was managed by stainless steel retention sutures, the application of negative pressure therapy, and a hernia belt. At re-evaluation 15 months post-surgery, several small hernias were detected, but the horse had returned to his previous level of sports performance and had not shown any episode of colic.

Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis.

Experimental laminitis, characterized by a failure of the dermal-epidermal interface of the foot, can be induced in horses by the oral administration of a black walnut extract (BWE). In the early phase of this severe and painful disease, an activation of neutrophil occurs, with the release of myeloperoxidase (MPO), a pro-oxidant enzyme of neutrophils, in plasma, skin, and laminar tissue. Juglone, a naphthoquinone derivative endowed with redox properties, is found in walnuts and has been incriminated in this neutrophil activation. We report for the first time the inhibitory activity of juglone on the degranulation of neutrophils induced by cytochalasin B and formyl-methionyl-leucyl-phenylalanine as monitored by the MPO release (>90% inhibition for 25 and 50 µM). Moreover, it also acts on the peroxidase activity of MPO by interacting with the intermediate "π cation radical," as evidenced by the classical and specific immunological extraction followed by enzymatic detection (SIEFED) assays. These results are confirmed by a docking study showing the perfect positioning of juglone in the MPO enzyme active site and its interaction with one of the amino acids (Arg-239) of MPO apoprotein. By chemiluminescence and electron paramagnetic resonance techniques, we demonstrated that juglone inhibited reactive oxygen species (ROS) and superoxide anion free radical produced from phorbol myristate acetate (PMA)-activated polymorphonuclear neutrophils (PMNs). These results indicate that juglone is not the trigger for equine laminitis, at least if we focus on the modulation of neutrophil activation.

Complications associated with closure of the linea alba using a combination of interrupted vertical mattress and simple interrupted sutures in equine laparotomies.

OBJECTIVES: (1) Evaluate the occurrence and variables associated with incisional morbidities (IMs) after ventral median laparotomy when using interrupted vertical mattress sutures (IVMS) and (2) determine the occurrence of abdominal bandage-associated complications in horses. METHODS: Occurrence of IM and bandage-associated complications were determined after single laparotomies (SL group; n=546 horses) and repeat laparotomies (RL group: multiple laparotomies within four weeks; n=30 horses) in horses that survived ≥7 days postoperatively. Univariate analysis and multivariate logistic regression were performed to evaluate variables associated with IM. RESULTS: The IM rate was 9.52 per cent in the SL group and 33.33 per cent in the RL group. The actual infection rate was 5.31 per cent in the SL group and 26.67 per cent in the RL group. Overall, long-term clinically relevant wound complications was 1.68 per cent. After multivariate analysis, increased anaesthesia duration was associated with IM and performing an enterotomy and postoperative intravenous lidocaine administration were associated with incisional infection in the SL group; no parameter remained significant in the RL group. Bandage-related complications were recorded in 2.95 per cent of the cases. CONCLUSIONS: These results suggest that the use of IVMS for closure of the linea alba is another viable option for closure and that an abdominal bandage does not appear to cause significant complications.

Mesh closure of epiploic foramen by ventral laparotomy in 17 horses with entrapment.

BACKGROUND: Epiploic foramen entrapment (EFE) of small intestine is a severe cause of strangulating small intestinal obstruction (SSI) with long-term survival seeming lower than for other causes of SSI in horses. Different techniques via laparoscopy or laparotomy for epiploic foramen (EF) closure have been developed. METHODS: This study describes a technique of peroperative mesh closure of the EF in clinical cases and their long-term follow up. RESULTS: In the study period of 5.5 years, 36 horses were admitted to the clinic with EFE. Of these, 17 horses had peroperative mesh closure, with resection anastomosis in 4 cases and enterotomy in 4 other cases. Fifteen of these survived to discharge. Long-term follow-up (one to three years, median three years) was favourable in all 15 horses not showing recurrence of EFE nor other related signs of colic. Laparoscopic evaluation of the EF was performed in two cases and showed integration of the mesh. One horse was euthanased 3.5 years after mesh placement for an unrelated cause and the mesh was well adherent obliterating the EF. CONCLUSION: Mesh closure of EF during emergency coeliotomy did not cause short-term complications even in horses with resection or enterotomy and may reduce the risk of recurrence of EFE in horses.

Nephrosplenic space closure significantly decreases recurrent colic in horses: a retrospective analysis.

BACKGROUND: Nephrosplenic space closure is commonly used to reduce relapse of nephrosplenic space entrapment in cases of left dorsal displacement of the left colon (LDDLC). Nevertheless, studies documenting the effectiveness of this surgery are sparse in the literature. The aim of this study was to analyse clinical data of horses presented for LDDLC and evaluate the effect of nephrosplenic space closure on the incidence of colic recurrence. METHODS: Medical records of 156 horses diagnosed with LDDLC at the Equine Clinic of the University of Liège between 2004 and 2016 were retrieved for analysis. Extracted data included horse breed, sex, age and weight, initial treatment, and if a subsequent preventive surgical closure of the nephrosplenic space was carried out or not. Follow-up information was obtained by telephone interview. Statistical analyses were performed using a chi-squared test with significance set at P<0.05. RESULTS: Follow-up data were available for 65 per cent of horses. The mean follow-up was 35 months. There was a significant decrease in the total incidence of colic after closure surgery compared with non-operated horses. No horse was diagnosed with LDDLC after closure of the nephrosplenic space. CONCLUSION: Closure of the nephrosplenic space significantly decreases recurrent colic in horses compared with non-operated horses.

Gastrointestinal effects of general anaesthesia in horses undergoing non abdominal surgery: focus on the clinical parameters and ultrasonographic images.

The ultrasonographic images of the gastrointestinal tract in horses can be influenced by fasting and sedation but the proper effect of general anaesthesia (GA) on them has not been determined yet. This study aimed to evaluate the effects of GA on ultrasonographic images of the gastrointestinal tract in horses and to compare these effects with a clinical evaluation. Twenty horses undergoing non-abdominal surgeries were evaluated by ultrasonography before and 4 times within 24 h after GA. Each ultrasonographic exam focused on the stomach, the duodenum and on 5 locations on the jejunum. The four-quadrant auscultation and the postoperative faecal output were also recorded. Pre and post anaesthetic values were compared using linear mixed effects models. None of the horses presented colic signs or reduced faecal output. During the first 2 post anaesthetic evaluations, the gut sounds were significantly decreased and, when taking all jejunal locations together, the jejunal diameter and visualisation frequency significantly increased. No intestinal loop appeared thickened and most of their diameters remained within the normal range. Our results suggest that the effects of GA on the ultrasonographic images of the small intestine are mild and of short duration and can therefore be differentiated from a pathological process.

Assessment of systemic inflammation by time-trends of blood granulocyte count and plasma myeloperoxidase and elastase concentrations following colic surgery in horses.

OBJECTIVE: To determine changes in blood granulocyte counts and in plasma myeloperoxidase (MPO) and elastase (ELT) concentrations in surgical colic cases, and to determine the relationship between these changes and the surgical procedure performed, occurrence of postoperative ileus, and final outcome. DESIGN: Prospective clinical study conducted over a 12-month period. SETTING: University teaching hospital. ANIMALS: Fifty-three horses undergoing emergency laparotomy and surviving at least 12 hours postoperatively. INTERVENTIONS: Blood samples were taken before surgery, during surgery, at the recovery from anesthesia, and then serially until the 150th hour after the first blood sampling. Granulocyte counts were performed by an automated cell hematology analyzer. Specific ELISAs were performed for the MPO and ELT measurements. Mixed models were used to compare the time-trends of the 3 parameters. MEASUREMENTS AND MAIN RESULTS: Taking all horses together, the time-trends of MPO and ELT were not significantly different from each other, but they were significantly different from the granulocyte time-trend. The type of surgical procedure did not influence the time-trends of the 3 parameters. Significant changes in the granulocyte time-trends were associated with postoperative ileus and outcome. Significant changes in the MPO time-trends were associated with outcome. The ELT time-trends were not influenced by ileus or outcome. CONCLUSIONS: Granulocyte counts and MPO change over time and are related to the severity of the inflammatory reaction in surgical colic cases. These time-trends may allow evaluation of treatment efficacy in an effort to modulate excessive granulocyte activation and degranulation.

An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity.

Assessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.

OBJECTIVE: To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. ANIMALS: 5 healthy horses (5 to 15 years old). PROCEDURES: Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3'-diaminobenzidine chromogen solution to detect peroxidase binding. RESULTS: Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation-treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses.

The role of activated neutrophils in the early stage of equine laminitis.

Despite ongoing research and a widening range of treatment options, laminitis remains a severely damaging condition with poorly understood pathophysiology. Results obtained from cytokine regulation studies during the last decade have highlighted the inflammatory nature of laminitis. This review will describe the role of systemic activation and local infiltration of neutrophils in laminar tissues in the induction of laminitis. Particular emphasis is placed on the role of neutrophil activation in subsequent vascular dysfunction and oxidative and proteolysis imbalances that are pathways previously implicated in laminitis. Neutrophils, by the way of their interdependent relationship with endothelial cells and keratinocytes, dramatically increase the inflammatory response culminating in the failure of the laminar dermal-epidermal interface.

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract.

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5-15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5h (n=5), 3h (n=6) or 12h (n=10) after BWHE administration. Control horses (n=7) were euthanized 12h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3h BWHE horses, in 6 of the 12h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p<0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8h compared to the control group and at 8 and 10h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.

Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.

Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4h), was obtained with plasma from EDTA anticoagulated blood. The mean value (+/-SEM) measured in 37 healthy horses was 32.53+/-4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids.

Evaluation of low-molecular-weight heparin for the prevention of equine laminitis after colic surgery.

OBJECTIVES: The aim of this study is to describe the prevalence of postoperative laminitis in colic cases and to determine if low-molecular-weight heparin (LMWH) is effective in preventing this complication. DESIGN: Retrospective clinical study. ANIMALS: Client-owned horses. Interventions- SC administration of enoxaparin during the postoperative period. MEASUREMENTS AND MAIN RESULTS: Medical records of 360 horses undergoing surgery for colic and surviving at least 3 days were evaluated. Fifty-six horses admitted before 1995 did not receive LMWH (control group) and 304 admitted after 1995 received LMWH as a prophylaxis for laminitis (treatment group). Three grades of severity were defined for laminitis. Prevalence and severity of laminitis were compared between the 2 groups. Several parameters recorded on admission (sex, age, breed, site and nature of the disease, heart rate, PCV, gravity score, and shock score) and the administration of LMWH were tested as risk factors in the development of laminitis in a logistic regression procedure. Prevalence and grade of laminitis were significantly lower in the treatment group. Only the absence of LMWH was recognized as a significant risk factor in the logistic regression model. CONCLUSIONS: The administration of LMWH appears to be effective in the prophylaxis of laminitis following colic surgery and may be useful in the postoperative management of these horses.