The enteroinsular axis during hospitalization in newborn foals.

The enteroinsular axis (EIA) is an energy regulatory system that modulates insulin secretion through the release of enteroendocrine factors (incretins). Despite the importance of energy homeostasis in the equine neonate, information on the EIA in hospitalized foals is lacking. The goals of this study were to measure serum insulin and plasma incretin (glucose-dependent insulinotropic polypeptide [GIP], glucagon-like peptide-1 [GLP-1] and glucagon-like peptide-2 [GLP-2]) concentrations, to determine the insulin and incretin association, as well as their link to disease severity and outcome in hospitalized foals. A total of 102 newborn foals ≤72 h old were classified into hospitalized (n = 88) and healthy groups (n = 14). Hospitalized foals included septic (n = 55) and sick non-septic (SNS; n = 33) foals based on sepsis scores. Blood samples were collected over 72 h to measure serum insulin and plasma GIP, GLP-1 and GLP-2 concentrations using immunoassays. Data were analyzed by nonparametric methods and univariate logistic regression. At admission, serum glucose and insulin and plasma GIP were significantly lower in hospitalized and septic compared to healthy foals (P < 0.01), while plasma GLP-1 and GLP-2 concentrations were higher in hospitalized and septic foals than healthy and SNS foals, and decreased over time in septic foals (P < 0.05). As a percent of admission values, GLP-1 and GLP-2 concentrations dropped faster in healthy compared to hospitalized foals. Serum insulin concentrations were lower in hospitalized and septic non-survivors than survivors at admission (P < 0.01). Hospitalized foals with serum insulin < 5.8 µIU/mL, plasma GLP-1 >68.5 pM, and plasma GLP-2 >9 ng/mL within 24 h of admission were more likely to die (OR = 4.2; 95% CI = 1.1-16.1; OR = 13.5, 95% CI = 1.4-123.7; OR = 12.5, 95% CI = 1.6-97.6, respectively; P < 0.05). Low GIP together with increased GLP-1 and GLP-2 concentrations indicates that different mechanisms may be contributing to reduced insulin secretion in critically ill foals, including impaired intestinal production (GIP, proximal intestine) and pancreatic endocrine resistance to enhanced incretin secretion (GLP-1, GLP-2; distal intestine). These imbalances could contribute to energy dysregulation in the critically ill equine neonate.

Influence of digital hypothermia on lamellar events related to IL-6/gp130 signalling in equine sepsis-related laminitis.

BACKGROUND: Interleukin-6 (IL-6) is consistently increased in the digital lamellae in different studies of sepsis-related laminitis (SRL). IL-6 signalling through the gp130 receptor activates similar signalling (i.e. mTORC1-related signalling) previously reported to be activated in models of endocrinopathic laminitis. OBJECTIVES: To assess the activation state of signalling proteins downstream of IL-6/gp130 receptor complex activation in an experimental model of SRL. STUDY DESIGN: Randomised experimental study. METHODS: Lamellar phospho-(P) protein concentrations downstream of the IL-6/gp130 receptors were assessed in the oligofructose (OF) model of SRL. Fifteen Standardbred horses were administered water (CON, n = 8) or oligofructose (OF, n = 7) via a nasogastric tube. At 12 h post-OF/water administration, one randomly assigned forelimb was exposed to continuous digital hypothermia (CDH) by placement in ice water (ICE, maintained at <7°C); the other forelimb was maintained at ambient temperature (AMB). Lamellar tissue samples were collected after 24 h of CDH from both ICE and AMB forelimbs and immediately snap-frozen. Lamellar proteins of interest were assessed by immunoblotting and immunofluorescence. RESULTS: Immunoblotting revealed increase (P<0.05) in the phosphorylation states of Akt (Ser 473), RPS6 (Ser235/236), RPS6 (Ser240/244), STAT3 (Ser727) and STAT3 (Tyr705) in lamellar tissue from OF-treated animals (AMB OF vs. AMB CON limbs); CDH resulted in decreased (P<0.05) lamellar concentrations of phosphorylated Akt, p70S6K, RPS6 (235/236), RPS6 (240/244) and STAT3 (S727) in OF-treated animals (AMB OF vs. ICE OF). Immunofluorescence showed that activated/phosphorylated forms of RPS6 and STAT3 were primarily localised to lamellar epithelial cells. MAIN LIMITATIONS: The nature, sequence and timing of sub-cellular events in this experimental model may differ from those that accompany naturally occurring sepsis. CONCLUSIONS: There were increased lamellar concentrations of activated signalling proteins downstream of the IL-6/Gp130 receptor complex in OF-treated horses; CDH inhibited this activation for the majority of the proteins assessed. These results demonstrate similar lamellar signalling (e.g. mTORC1-related signalling) and, therefore, possible therapeutic targets occurring in sepsis-related laminitis as previously reported in models of endocrinopathic laminitis.

Cardiovascular images: vascular hamartoma of the mitral valve in a horse.

An 8-month-old Hanoverian gelding was presented with a history of cardiac murmurs that were not apparent as a foal nor reported at the time of castration. Major echocardiographic findings included mitral valvular thickening, functional stenosis, and mitral regurgitation of sufficient severity to cause diastolic and systolic cardiac murmurs, left-sided volume overload, and pulmonary hypertension. Due to the hemodynamic severity of the lesion and poor prognosis for future performance and longevity, euthanasia was elected. On gross postmortem examination, there was focal fibrous epicarditis affecting the heart base, and the left atrium was moderately dilated. The mitral valve surface was irregular and contained several nodules along the atrial face of the cusp. Histologically, this lesion was diagnosed as a vascular hamartoma, which is rarely reported in veterinary species and has not been described in heart valves. This benign proliferative lesion, and concurrent valvular dysfunction, was associated with an unusual manifestation of clinically evident disease and should be differentiated from common incidental valvular lesions such as hematocysts.

Steroids, steroid precursors, and neuroactive steroids in critically ill equine neonates.

Hypothalamic-pituitary-adrenal axis (HPAA) dysfunction has been associated with sepsis and mortality in foals. Most studies have focused on cortisol, while other steroids have not been investigated. The objectives of this study were to characterise the adrenal steroid and steroid precursor response to disease and to determine their association with the HPAA response to illness, disease severity, and mortality in hospitalised foals. All foals (n=326) were classified by two scoring systems into three categories: based on the sepsis score (septic, sick non-septic [SNS] and healthy) and the foal survival score (Group 1: 3-18%; Group 2: 38-62%; Group 3: 82-97% likelihood of survival). Blood concentrations of adrenocorticotropic hormone (ACTH) and steroids were determined by immunoassays. ACTH-cortisol imbalance (ACI) was defined as a high ACTH/cortisol ratio. Septic foals had higher ACTH, cortisol, progesterone, 17α-OH-progesterone, pregnenolone, and androstenedione concentrations as well as higher ACTH/cortisol, ACTH/progesterone, ACTH/aldosterone, and ACTH/DHEAS ratios than SNS and healthy foals (P<0.01). Foals with DHEAS of 0.4-5.4ng/mL were more likely to have ACI (OR=2.5). Foals in Group 1 had higher ACTH, aldosterone, progesterone, and cortisol concentrations as well as ACTH/cortisol, ACTH/progesterone, and ACTH/DHEAS ratios than foals in Groups 2 and 3 (P<0.01). High progesterone concentrations were associated with non-survival and the cutoff value below which survival could be predicted was 23.5ng/mL, with 75% sensitivity and 72% specificity. In addition to cortisol, the response to the stress of illness in foals is characterised by the release of multiple adrenal steroids. DHEAS and progesterone were good predictors of HPAA dysfunction and outcome in hospitalised foals.

Laminar inflammatory events in lean and obese ponies subjected to high carbohydrate feeding: Implications for pasture-associated laminitis.

REASONS FOR PERFORMING STUDY: Acute, massive enteral carbohydrate overload is associated with laminar inflammation in equids; it is unclear if the same is true for a more prolonged period of moderate dietary carbohydrate intake. OBJECTIVES: To characterise laminar inflammation in ponies exposed to a dietary carbohydrate challenge meant to mimic acute pasture exposure. STUDY DESIGN: In vivo experiment. METHODS: Mixed-breed ponies (n = 22) received a diet of hay chop (nonstructural carbohydrate [NSC] ∼7% on a dry matter [DM] basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following dietary acclimation, ponies were stratified into either Lean (n = 11, body condition score [BCS] ≤4) or Obese (n = 11, BCS ≥7) groups and each group further stratified to either remain on the control, low NSC diet (n = 5 each for Obese and Lean) or receive a high NSC diet (hay chop supplemented with sweet feed and oligofructose, total diet ∼42% NSC; n = 6 each for Obese and Lean) for a period of 7 days. Laminar samples were collected following euthanasia and sections stained immunohistochemically for CD163, MAC387/calprotectin and cyclo-oxygenase-2 (COX-2) using commercially available antibodies. The number of CD163 (+) and MAC387(+) cells was quantified for each section; the distribution of COX-2 expression was qualitatively assessed. Laminar mRNA concentrations of several proinflammatory molecules (interleukin-1ß [IL-1ß], IL-6, tumour necrosis factor-α [TNFα], IL-8, IL-10, monocyte chemoattractant protein-1 [MCP-1], MCP-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, plasminogen activator inhibitor-1 (PAI-1) and COX-2 were evaluated using real-time quantitative polymerase chain reaction (qPCR). RESULTS: High carbohydrate feeding resulted in no increase in laminar proinflammatory cytokine expression; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. CONCLUSIONS: These results suggest that the marked laminar inflammation observed in models of sepsis-associated laminitis may not play a central role in the pathophysiology of pasture-associated laminitis.

Effect of dietary nonstructural carbohydrate content on activation of 5'-adenosine monophosphate-activated protein kinase in liver, skeletal muscle, and digital laminae of lean and obese ponies.

BACKGROUND: In EMS-associated laminitis, laminar failure may occur in response to energy failure related to insulin resistance (IR) or to the effect of hyperinsulinemia on laminar tissue. 5'-Adenosine-monophosphate-activated protein kinase (AMPK) is a marker of tissue energy deprivation, which may occur in IR. HYPOTHESIS/OBJECTIVES: To characterize tissue AMPK regulation in ponies subjected to a dietary carbohydrate (CHO) challenge. ANIMALS: Twenty-two mixed-breed ponies. METHODS: Immunohistochemistry and immunoblotting for total AMPK and phospho(P)-AMPK and RT-qPCR for AMPK-responsive genes were performed on laminar, liver, and skeletal muscle samples collected after a 7-day feeding protocol in which ponies stratified on body condition score (BCS; obese or lean) were fed either a low-CHO diet (ESC + starch, approximately 7% DM; n = 5 obese, 5 lean) or a high-CHO diet (ESC + starch, approximately 42% DM; n = 6 obese, 6 lean). RESULTS: 5'-Adenosine-monophosphate-activated protein kinase was immunolocalized to laminar keratinocytes, dermal constituents, and hepatocytes. A high-CHO diet resulted in significantly decreased laminar [P-AMPK] in lean ponies (P = .03), but no changes in skeletal muscle (lean, P = .33; obese, P = .43) or liver (lean, P = .84; obese, P = .13) [P-AMPK]. An inverse correlation existed between [blood glucose] and laminar [P-AMPK] in obese ponies on a high-CHO diet. CONCLUSIONS AND CLINICAL IMPORTANCE: Laminar tissue exhibited a normal response to a high-CHO diet (decreased [P-AMPK]), whereas this response was not observed in liver and skeletal muscle in both lean (skeletal muscle, P = .33; liver, P = .84) and obese (skeletal muscle, P = .43; liver, P = .13) ponies.

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.

A comparison of patient comfort during cataract surgery with topical anesthesia versus topical anesthesia and intracameral lidocaine.

OBJECTIVE: To determine whether intraocular lidocaine increases patient comfort during cataract surgery while under topical anesthesia. DESIGN: Prospective, randomized, double-masked, placebo-controlled clinical trial. PARTICIPANTS: Both men and women between 45 and 85 years of age who were scheduled for elective cataract surgery while under topical anesthesia participated. Sixty-eight patients were randomized to each group. INTERVENTION: Patients were randomized to receive either topical anesthesia plus intracameral 1% preservative-free lidocaine or intracameral balanced salt solution. MAIN OUTCOME MEASURES: Patient assessment of pain during delivery of the anesthesia, surgery, and after surgery using a visual analog pain scale was measured. Patients also recorded the degree to which they were bothered by tissue manipulation and the microscope light. Surgeon assessments of operative conditions, patient cooperation, and intraoperative complications were recorded. The attending anesthesiologist recorded any required supplemental intravenous sedation and any increase in pulse or increase in blood pressure. RESULTS: There was no significant difference in patient-reported pain scores for delivery of anesthesia (P = 0.902), surgery (P = 0.170), or after surgery (P = 0.680). Patients in the lidocaine group reported being less bothered by tissue manipulation (P = 0.021). The surgeon assessment showed more patient cooperation in the lidocaine group (P = 0.043). CONCLUSIONS: Both topical anesthesia alone and topical anesthesia plus intracameral lidocaine provide good operative conditions for the surgeon and comfortable surgical circumstances for the patient. Injection of intraocular lidocaine increases patient cooperation and decreases the degree to which patients are bothered by tissue manipulation, two outcomes that justify its use.

Prospective evaluation of topical versus retrobulbar anesthesia: a converting surgeon's experience.

PURPOSE: To evaluate and compare the efficacy of topical versus retrobulbar anesthesia for cataract surgery performed by a surgeon newly converting to the topical technique. SETTING: Department of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. METHODS: Ninety patients were prospectively assigned by permuted block restricted randomization to receive topical (Group 1; n = 45) or retrobulbar (Group 2; n = 45) anesthesia. Group 1 received topical bupivacaine 0.75% and intravenous midazolam and fentanyl for anesthesia. Group 2 received intravenous methohexital followed by retrobulbar block with an equal mixture of lidocaine 2% and bupivacaine 0.75% plus hyaluronidase 150 units. A visual pain analog scale was used to assess the degree of pain during anesthesia administration and surgery and postoperatively. The degree to which eye movement, touch, and light caused patient discomfort was assessed. Intraoperative conditions and complications were recorded. RESULTS: Intraoperative operating conditions were significantly better in Group 2 (P < .05). There was a small but statistically significant difference in the degree of discomfort during anesthesia administration and surgery (P < .05). There was no difference in postoperative discomfort. Chemosis, subconjunctival hemorrhage, and eyelid hemorrhage occurred only in Group 2, in which there was one retrobulbar hemorrhage. Although eyelid squeezing and ocular motility were present more frequently in Group 1, neither was a problem to the surgeon. CONCLUSION: Cataract surgery was safely performed by a surgeon converting to topical anesthesia. After a distinct learning curve, the procedure was performed with minimal patient discomfort. Surgical training and patient preparation are the key to safe use of topical anesthesia.

Propofol and alfentanil for sedation during placement of retrobulbar block for cataract surgery.

STUDY OBJECTIVE: To determine if the addition of alfentanil to propofol is more effective than propofol alone to provide adequate conditions for placement of a retrobulbar block prior to cataract surgery. DESIGN: Randomized, double-blinded study. SETTING: Outpatients at a university hospital. PATIENTS: 40 adult ASA physical status I, II, and III outpatients scheduled for elective cataract surgery. INTERVENTIONS: Patients were randomly assigned to receive one of four drug combinations prior to the placement of a retrobulbar block: Group 1, propofol alone; Group 2, alfentanil 5 micrograms/kg plus propofol; Group 3, alfentanil 10 micrograms/kg plus propofol; Group 4, alfentanil 15 micrograms/kg plus propofol. All patients were preoxygenated by face mask for two minutes prior to drug administration. The quality of conditions for block placement were determined by: (1) assessing the amount of movement by the patients while the block needle was in place, (2) cooperativeness of the patients during the operation, (3) hemodynamic side effects, (4) incidence and severity of respiratory depression, (5) incidence of nausea and vomiting, (6) recall of placement of the block, and (7) time to discharge from the hospital. MEASUREMENTS AND MAIN RESULTS: The addition of alfentanil to propofol for sedation prior to placement of the retrobulbar block resulted in a dose-dependent reduction in movement by the patients. However, the highest dose of alfentanil (15 micrograms/kg) resulted in the greatest frequency (40% of the patients in this group) of respiratory depression (SpO2 < 90%). All patients were cooperative during the operation and responsive to verbal command within 5 minutes of placement of the block. In addition, all of the patients denied being nauseated, having vomited, or recalling block placement in the recovery room or the next day. CONCLUSIONS: The combination of alfentanil and propofol may be used to sedate patients in order to limit movement and provide a cooperative, alert patient with stable hemodynamics and limited respiratory depression during placement of retrobulbar block prior to ophthalmic surgery. However, excessive dosage of these drugs may result in hazardous respiratory depression in this patient population.

A comparison of topical and retrobulbar anesthesia for cataract surgery.

PURPOSE: To evaluate and compare the efficacy of topical and retrobulbar anesthesia for cataract extraction with intraocular lens implantation. METHODS: One hundred thirty-eight patients prospectively were assigned to the topical (group 1; n = 69) or retrobulbar (group 2; n = 69) anesthesia groups by permuted block restricted randomization. Group 1 received topical 0.75% bupivacaine and intravenous midazolam and fentanyl for anesthesia. Group 2 received intravenous methohexital followed by retrobulbar block with an equal mixture of 2% lidocaine and 0.75% bupivacaine plus hyaluronidase (150 U). A visual pain analogue scale was used to assess the degree of pain during the administration of anesthesia, during surgery, and post-operatively. The degree to which eye movement, touch, and light caused patient discomfort was assessed. Complications and surgical conditions were recorded. RESULTS: There was no difference in the surgical conditions (P = 0.5) or pain during surgery (P = 0.35) between the two groups. There was more discomfort during administration of topical anesthesia (P < 0.0001) and postoperatively (P < 0.05) in the topical group. Chemosis, subconjunctival hemorrhage, and eyelid hemorrhage were seen almost exclusively in the retrobulbar group. One patient in group 2 had a retrobulbar hemorrhage. Although eyeball movement and squeezing of the eyelids were present more frequently in the topical group, neither was a problem to the surgeon. CONCLUSION: Topical anesthesia can be used safely for cataract extraction. The degree of patient discomfort is only marginally higher during administration of the anesthesia and postoperatively. However, surgical training and patient preparation are the keys to the safe use of topical anesthesia.

Selection and Characterization of Aspartokinase Feedback-Insensitive Mutants of Azotobacter vinelandii.

Aspartokinase feedback-insensitive mutants of Azotobacter vinelandii were selected as resistant to l-threonine, (beta)-hydroxynorvaline, or S-(2-aminoethyl)-l-cysteine. l-Threonine-resistant strains were classified into three groups based on their ability to transport l-threonine and their growth response to O-methylthreonine and (beta)-hydroxynorvaline. Most of the mutants were transport defective; however, some were desensitized to feedback regulation.