Dark-field microscopy in the assessment of large colon microperfusion and mucosal injury in naturally occurring surgical disease of the equine large colon.

REASON FOR PERFORMING STUDY: Intraoperative assessment of colonic viability can be challenging and largely subjective. Objective methods are often impractical. Viability is related to re-establishment of colonic perfusion; particularly microvascular perfusion. This study evaluated the utility of dark-field microscopy (DFM) of the colonic serosa as an objective method of assessing microperfusion. OBJECTIVES: To measure microvascular perfusion indices (MPI) of the pelvic flexure serosa in horses with surgical colonic lesions and correlate these with macroperfusion indices (MaPI) and histomorphometry. STUDY DESIGN: Prospective, clinical, case-control study. METHODS: Control horses and horses with colonic volvulus (LCV), displacement, and/or simple obstruction undergoing surgery had DFM video loops performed on the pelvic flexure. Total vessel density, perfused vessel density, proportion of perfusion vessels and microvascular flow index were calculated from video analysis. Macroperfusion indices (arterial blood pressure and heart rate) were recorded. Histomorphometry was used to determine a mucosal injury score. Differences between lesions for MPI, MaPI and histomorphometry were compared using ANOVA or Kruskal-Wallis statistic. Spearman correlations between MPI with MaPI were performed. Linear regression was used to assess the relationship between MPI and histomorphometry. P<0.05 was significant. RESULTS: Horses with LCV had lower perfused vessel density, proportion of microvascular perfusion vessels and flow index than horses with nonstrangulating obstructions and control horses. Macroperfusion indices were not correlated with MPI but MPI were correlated with histomorphometry. CONCLUSIONS: Dark-field microscopy is achievable in the operating room and can quantify MPI from the colonic serosa in different colonic lesions. Macroperfusion indices were not related with colonic MPI. Microvascular perfusion indices can predict the severity of histopathological change at the pelvic flexure. Derangements of MPI may be more useful indicators of colonic pathology and viability and offer a more objective assessment of intestinal injury than subjective methods. Further study is needed to determine the utility of DFM in predicting survival in horses with LCV.

Naturally occurring compensated insulin resistance selectively alters glucose transporters in visceral and subcutaneous adipose tissues without change in AS160 activation.

Although the importance of adipose tissue (AT) glucose transport in regulating whole-body insulin sensitivity is becoming increasingly evident and insulin resistance (IR) has been widely recognized, the underlying mechanisms of IR are still not well understood. The purpose of the present study was to determine the early pathological changes in glucose transport by characterizing the alterations in glucose transporters (GLUT) in multiple visceral and subcutaneous adipose depots in a large animal model of naturally occurring compensated IR. AT biopsies were collected from horses, which were classified as insulin-sensitive (IS) or compensated IR based on the results of an insulin-modified frequently sampled intravenous glucose tolerance test. Protein expression of GLUT4 (major isoform) and GLUT12 (one of the most recently discovered isoforms) were measured by Western blotting in multiple AT depots, as well as AS160 (a potential key player in GLUT trafficking pathway). Using a biotinylated bis-mannose photolabeled technique, active cell surface GLUT content was quantified. Omental AT had the highest total GLUT content compared to other sites during the IS state. IR was associated with a significantly reduced total GLUT4 content in omental AT, without a change in content in other visceral or subcutaneous adipose sites. In addition, active cell surface GLUT-4, but not -12, was significantly lower in AT of IR compared to IS horses, without change in AS160 phosphorylation between groups. Our data suggest that GLUT4, but not GLUT12, is a pathogenic factor in AT during naturally occurring compensated IR, despite normal AS160 activation.

Insulin resistance selectively alters cell-surface glucose transporters but not their total protein expression in equine skeletal muscle.

BACKGROUND: Insulin resistance (IR) has been widely recognized in humans, and more recently in horses, but its underlying mechanisms are still not well understood. The translocation of glucose transporter 4 (GLUT4) to the cell surface is the limiting step for glucose uptake in insulin-sensitive tissues. Although the downstream signaling pathways regulating GLUT translocation are not well defined, AS160 recently has emerged as a potential key component. In addition, the role of GLUT12, one of the most recently identified insulin-sensitive GLUTs, during IR is unknown. HYPOTHESIS/OBJECTIVES: We hypothesized that cell-surface GLUT will be decreased in muscle by an AS160-dependent pathway in horses with IR. ANIMALS: Insulin-sensitive (IS) or IR mares (n = 5/group). METHODS: Muscle biopsies were performed in mares classified as IS or IR based on results of an insulin-modified frequently sampled IV glucose tolerance test. By an exofacial bis-mannose photolabeled method, we specifically quantified active cell-surface GLUT4 and GLUT12 transporters. Total GLUT4 and GLUT12 and AS160 protein expression were measured by Western blots. RESULTS: IR decreased basal cell-surface GLUT4 expression (P= .027), but not GLUT12, by an AS160-independent pathway, without affecting total GLUT4 and GLUT12 content. Cell-surface GLUT4 was not further enhanced by insulin stimulation in either group. CONCLUSIONS AND CLINICAL IMPORTANCE: IR induced defects in the skeletal muscle glucose transport pathway by decreasing active cell-surface GLUT4.

Proinflammatory cytokine and chemokine gene expression profiles in subcutaneous and visceral adipose tissue depots of insulin-resistant and insulin-sensitive light breed horses.

BACKGROUND: Insulin resistance has been associated with risk of laminitis in horses. Genes coding for proinflammatory cytokines and chemokines are expressed more in visceral adipose tissue than in subcutaneous adipose tissue of insulin-resistant (IR) humans and rodents. HYPOTHESIS/OBJECTIVES: To investigate adipose depot-specific cytokine and chemokine gene expression in horses and its relationship to insulin sensitivity (SI). ANIMALS: Eleven light breed mares. METHODS: Animals were classified as IR (SI=0.58+/-0.31x10(-4) L/min/mU; n=5) or insulin sensitive (IS; SI=2.59+/-1.21x10(-4) L/min/mU; n=6) based on results of a frequently sampled intravenous glucose tolerance test. Omental, retroperitoneal, and mesocolonic fat was collected by ventral midline celiotomy; incisional nuchal ligament and tail head adipose tissue biopsy specimens were collected concurrently. The expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) in each depot was measured by real-time quantitative polymerase chain reaction. Data were analyzed by 2-way analysis of variance for repeated measures (P<.05). RESULTS: No differences in TNF-alpha, IL-1beta, IL-6, PAI-1, or MCP-1 mRNA concentrations were noted between IR and IS groups for each depot. Concentrations of mRNA coding for IL-1beta (P=.0005) and IL-6 (P=.004) were significantly higher in nuchal ligament adipose tissue than in other depots. CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest that the nuchal ligament depot has unique biological behavior in the horse and is more likely to adopt an inflammatory phenotype than other depots examined. Visceral fat may not contribute to the pathogenesis of obesity-related disorders in the horse as in other species.

Clinical assessment and outcome of a single-layer technique for anastomosis of the small intestine in horses.

In order to assess postoperative outcome in horses undergoing end-to-end anastomosis of the small intestine, performed using a one-layer technique, 15 horses that underwent exploratory coeliotomy, resection of the small intestine and end-to-end anastomosis using a continuous Lembert pattern were studied. Information on the age, breed, sex, diagnosis, treatment, complications and outcome of each case were obtained from medical records. Follow-up information was obtained via telephone conversations with clients and trainers. Five of the horses had short-term postoperative complications: one had postoperative ileus (POI), colic and peritonitis, one had POI and colic, two had POI only and one had diarrhoea only. A second exploratory coeliotomy was recommended in two of the 15 horses (13 per cent). The short-term survival rate, defined as survival up to the time of discharge from the hospital, was 93.3 per cent (14 of 15 horses). The long-term survival rate, defined as survival for at least 12 months after the surgery, was 84.6 per cent (11 of 13 horses followed up).

Calcium regulating hormones and serum calcium and magnesium concentrations in septic and critically ill foals and their association with survival.

BACKGROUND: Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival. HYPOTHESIS: Blood concentrations of ionized calcium (Ca(2+)) and magnesium (Mg(2+)) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival. ANIMALS: Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of or=14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays. RESULTS: Septic foals had decreased Ca(2+) (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg(2+), PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82). CONCLUSIONS AND CLINICAL IMPORTANCE: Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.

Blood arginine vasopressin, adrenocorticotropin hormone, and cortisol concentrations at admission in septic and critically ill foals and their association with survival.

BACKGROUND: Sepsis is an important cause for neonatal foal mortality. The hypothalamic-pituitary-adrenal axis (HPAA) responses to sepsis are well documented in critically ill humans, but limited data exist in foals. The purpose of this study was to evaluate the HPAA response to sepsis in foals, and to associate these endocrine changes with survival. HYPOTHESIS: Blood concentrations of arginine vasopressin (AVP), adrenocorticotropin hormone (ACTH), and cortisol will be higher in septic foals as compared with sick nonseptic and healthy foals. The magnitude of increase in hormone concentration will be negatively associated with survival. ANIMALS: Fifty-one septic, 29 sick nonseptic, and 31 healthy foals of < or =7 days of age were included. METHODS: Blood was collected at admission for analysis. Foals with positive blood culture or sepsis score > or =14 were considered septic. Foals admitted with disease other than sepsis and healthy foals were used as controls. AVP, ACTH, and cortisol concentrations were measured using validated immunoassays. RESULTS: AVP, ACTH, and cortisol concentrations were increased in septic foals. Septic nonsurvivor foals (n = 26/51) had higher plasma ACTH and AVP concentrations than did survivors (n = 25/51). Some septic foals had normal or low cortisol concentrations despite increased ACTH, suggesting relative adrenal insufficiency. AVP, ACTH, and cortisol concentrations were higher in sick nonseptic foals compared with healthy foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased plasma AVP and ACTH concentrations in septic foals were associated with mortality. Several septic foals had increased AVP : ACTH and ACTH : cortisol ratios, which indicates relative adenohypophyseal and adrenal insufficiency.