Implementation of a prototype dynamic laryngoplasty system in standing sedated horses provided arytenoid abduction control at seven days postoperatively.

OBJECTIVE: Evaluate a prototype dynamic laryngoplasty system (DLPS) in horses; a feasibility study. ANIMALS: 7 healthy Standardbred adult horses. METHODS: This was an in vivo experimental study. Horses had a standing surgical procedure to induce complete laryngeal hemiplegia, which was subsequently treated using the dynamic laryngoplasty system (DLPS). Activation of the DLPS was achieved using an injection port exiting through the skin (n = 2) or a subcutaneous injection port (n = 5). For each horse, endoscopic examinations of the upper respiratory tract were performed preoperatively, intraoperatively, and 7 days postoperatively. Left-to-right quotient ratios calculated during inactivated and activated states were obtained from still images of the rima glottidis acquired during day 7. In 3 horses, the device was intentionally overinflated to evaluate for device failure, and postmortem examinations were performed on day 7. For the remaining 4 horses, upper respiratory tract endoscopy was repeated at 1 month postoperatively, with no subsequent postmortem exam. RESULTS: No perioperative complications occurred, and the DLPS was effectively delivered in all horses under standing sedation. The left-to-right quotient ratio at day 7 postoperatively could be altered from a resting position of 0.76 (± 0.06) to a maximum of 0.97 (± 0.06; P < .05). The degree of arytenoid abduction could not be significantly altered after 1 month of device implantation, suspected to be due to peri-implant fibrosis. No coughing nor tracheal contamination was observed at all time points or during inflation. CLINICAL RELEVANCE: The ability to alter the degree of abduction at 7 days postoperatively with the DLPS may be beneficial in selective cases.

Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing.

BACKGROUND: Supporting limb laminitis (SLL) is a complication of severe orthopedic disease in horses and is often life-limiting, yet the pathophysiology remains obscure. HYPOTHESIS/OBJECTIVES: To investigate the role of digital lamellar inflammatory signaling in the pathophysiology of SLL using a model of unilateral weight bearing, hypothesizing that there would be evidence of lamellar inflammation in limbs subjected to the model. ANIMALS: Thirteen healthy adult Standardbred horses were used for this study (11 geldings, 2 mares; mean age 6.5 ± 2.5 years; mean body weight 458.3 ± 32.8 kg). METHODS: Randomized controlled experimental study. A steel shoe with a custom insert was applied to a randomly selected front foot of 7 horses; 6 horses were unshod and served as controls. After 92 hours, all horses were humanely euthanized, and digital lamellar samples were collected. Lamellar protein and mRNA were isolated and used to perform western blot and PCR. RESULTS: Lamellar concentrations of IL-6 mRNA were higher in SL tissue than IL HIND tissue (median [25%-75%] normalized copy number 191 [111-3060] and 48 [25-74], respectively; P=.003), and lamellar concentrations of COX-2 mRNA were higher in SL tissue than CON tissue (normalized copy number 400 [168-634] and 125 [74-178], respectively; P=.007). Lamellar concentrations of IL-1B, IL-10, and COX-1 mRNA were not significantly different between groups. The concentrations of phosphorylated (activated) STAT1 and STAT3 proteins were higher in SL (0.5 [0.35-0.87] and 1.35 [1.1-1.7], respectively) compared to CON (0.24 [0.09-0.37] and 0.31 [0.16-037]) and UL HIND (0.27 [0.19-0.37] and 0.38 [0.24-0.5]); P=0.01 and P<0.001. CONCLUSIONS AND CLINICAL IMPORTANCE: Lamellar inflammatory signaling was higher in tissue from horses subjected to prolonged unilateral weight-bearing, suggesting that these pathways could be relevant to the pathophysiology of SLL.

Effect of sirolimus on insulin dynamics in horses.

BACKGROUND: Sirolimus, a mechanistic target of rapamycin inhibitor, suppresses insulin production in other species and has therapeutic potential for hyperinsulinemia in horses. HYPOTHESIS/OBJECTIVE: Determine the pharmacokinetics (PKs) of sirolimus and evaluate its effect on insulin dynamics in healthy and insulin dysregulation (ID) horses. ANIMALS: Eight Standardbred geldings. METHODS: A PK study was performed followed by a placebo-controlled, randomized, crossover study. Blood sirolimus concentrations were measured by liquid chromatography-mass-spectrometry. PK indices were estimated by fitting a 2-compartment model using nonlinear least squares regression. An oral glucose test (OGT) was conducted before and 4, 24, 72, and 144 hours after administration of sirolimus or placebo. Effects of time, treatment and animal on blood glucose and insulin concentrations were analyzed using mixed-effects linear regression. Sirolimus was then administered to 4 horses with dexamethasone-induced ID and an OGT was performed at baseline, after ID induction and after 7 days of treatment. RESULTS: Median (range) maximum sirolimus concentration was 277.0 (247.5-316.06) ng/mL at 5 (5-10) min and half-life was 3552 (3248-4767) min. Mean (range) oral bioavailability was 9.5 (6.8-12.4)%. Sirolimus had a significant effect on insulin concentration 24 hours after a single dose: median (interquartile range) insulin at 60 min (5.0 [3.7-7.0] µIU/mL) was 37 (-5 to 54)% less than placebo (8.7 [5.8-13.7] µIU/mL, P = .03); and at 120 min (10.2 [8.4-12.2] µIU/mL) was 28 (-15 to 53)% less than placebo (14.9 [8.4-24.8] µIU/mL, P = .02). There was minimal effect on glucose concentration. Insulin responses decreased toward baseline in ID horses after 7 days of treatment. CONCLUSION AND CLINICAL IMPORTANCE: Sirolimus decreased the insulinemic response to glucose and warrants further investigation.

The effect of cyanoacrylate on knot elongation in three sutures used for prosthetic laryngoplasty in the horse.

OBJECTIVE: This study investigated the elongation following cyclic loading on square knots of 5 USP multifilament long-chain ultra-high molecular weight polyethylene core (UHMWPE), 2 mm woven UHMWPE tape, and 5 USP braided polyester, with and without cyanoacrylate glue. STUDY DESIGN: Experimental study. SAMPLE POPULATION: n = 4. METHODS: Three conditions (suture without knot, suture with knot, suture with knot + cyanoacrylate) were evaluated for each suture material on a mechanical test stand by measuring the increased length of the construct after cycling from 25 to 50N for 1000 repetitions at 20 mm/second. Knot elongation was determined by subtracting the length of the control suture from the suture with knot or suture with knot + cyanoacrylate. The data were analyzed with a linear regression model with robust estimation of variance. Post-hoc analysis determined the model adjusted differences (square knot vs. cyanoacrylate) as a difference from control. t-tests were conducted to identify the significant findings. RESULTS: Total elongation of polyester (6.2-7.8 mm) was greater than multifilament UHMWPE (3.4-6.4 mm) and UHMWPE tape (2-3.7 mm) for all conditions. Polyester had the lowest knot elongation (1.6 mm) and the addition of cyanoacrylate decreased knot elongation for polyester by 1 mm. CONCLUSIONS: Polyester had the most total construct elongation followed by multifilament UHMWPE and UHMWPE tape. Polyester showed the least knot elongation and cyanoacrylate decreased this knot elongation. CLINICAL SIGNIFICANCE: Total construct and knot elongation should be considered as contributing factors to loss of arytenoid abduction following prosthetic laryngoplasty when using polyester, multifilament UHMWPE, or UHMWPE tape. Addition of cyanoacrylate to polyester knots should be explored to limit elongation.

Treatment of primary hyperparathyroidism in a Miniature Horse using chemical ablation of abnormal parathyroid tissue localized by 3-phase computed tomography.

A 15-year-old Miniature Horse mare with persistently increased plasma calcium (total and ionized) and serum parathyroid hormone concentrations was presented for suspected primary hyperparathyroidism. Ultrasonography of the thyroid region identified an enlarged heterogeneous mass axial to the right thyroid lobe suggestive of an enlarged parathyroid gland, which was further confirmed using sestamibi nuclear scintigraphy and 3-phase computed tomography. Percutaneous ultrasound-guided ethanol ablation of the mass, a method not previously described in the horse, was performed under general anesthesia resulting in rapid normalization of plasma ionized calcium and serum parathyroid hormone concentrations. Ablation of abnormal parathyroid gland tissue may be a suitable alternative to surgical resection in certain cases of primary hyperparathyroidism in the horse.

Plasma amino acid concentrations during experimental hyperinsulinemia in 2 laminitis models.

BACKGROUND: Endocrinopathic laminitis develops in association with insulin dysregulation, but the role of insulin in the pathogenesis remains unclear. Hyperinsulinemia can cause hypoaminoacidemia, which is associated with integumentary lesions in other species and therefore warrants investigation as a potential mechanism in laminitis. OBJECTIVE: Evaluate plasma amino acid concentrations in the euglycemic-hyperinsulinemic clamp (EHC) and prolonged glucose infusion (PGI) laminitis models. ANIMALS: Sixteen Standardbred horses. METHODS: Prospective experimental study. Plasma amino acid concentrations were measured in samples collected every 6 hours from horses that underwent a 48-hour EHC (n = 8) or 66-hour PGI (n = 8) after a 24- or 6-hour baseline period in EHC and PGI groups, respectively. RESULTS: Fifteen of the 20 measured amino acid concentrations decreased over time in both EHC and PGI horses (P < 0.001). The median percentage change from baseline for these amino acids was: histidine (EHC: 41.5%; PGI: 43.9%), glutamine (EHC: 51.8%; PGI: 35.3%), arginine (EHC: 51.4%; PGI: 41%), glutamic acid (EHC: 52.4%; PGI: 31.7%), threonine (EHC: 62.8%; PGI: 25.2%), alanine (EHC: 48.9%; PGI: 19.5%), proline (EHC: 56.2%; PGI: 30.3%), cystine (EHC: 34.9%; PGI: 31.2%), lysine (EHC: 46.4%; PGI: 27.8%), tyrosine (EHC: 27.5%; PGI: 16.9%), methionine (EHC: 69.3%; PGI: 50.8%), valine (EHC: 50.8%; PGI: 34.4%), isoleucine (EHC: 60.8%; PGI: 38.7%), leucine (EHC: 48.2%; PGI: 36.6%), and phenylalanine (EHC: 16.6%; PGI: 12.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: Hypoaminoacidemia develops in EHC and PGI laminitis models. The role of hypoaminoacidemia in the development of hyperinsulinemia-associated laminitis warrants further investigation.

Lamellar perfusion and energy metabolism in a preferential weight bearing model.

BACKGROUND: Supporting limb laminitis (SLL) is suspected to be caused by lamellar ischaemia as a consequence of increased mechanical load. OBJECTIVES: Examine the effects of prolonged preferential weight bearing (PWB) on lamellar perfusion and metabolism. STUDY DESIGN: In vivo experiment. METHODS: Microdialysis probes were inserted in the lamellar and sublamellar dermis of one forelimb in 13 Standardbred horses. In six horses, a platform shoe (contralateral forelimb) was used to induce increased load on the microdialysis-instrumented forelimb (PWB). The remaining seven horses were controls (CON). All horses were housed in stocks with limb weight distribution logged continuously for 92 hours. Microdialysate was collected and analysed every 4 hours for glucose, lactate, pyruvate, and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Median [IQR] load on the microdialysis-instrumented limb was equivalent to 38.7% bwt. [37.3-40.3] in PWB and 27.3% bwt. [26.6-28] in CON. Limb offloading frequency increased in CON (P < .001) but not PWB (P = .2). Lamellar microdialysate glucose decreased in PWB (P < .001) and CON (P = .004), however, the rate of decrease was higher in PWB (P = .007). Lamellar L:P increased in PWB (P < .001) and peaked at 196 [79-656], whereas L:P did not change over time in CON (P = .6) and peaked at 42 [41-49]. Lamellar urea clearance decreased in PWB (P < .001) but not in CON (P = .3). Sublamellar L:P and urea clearance did not change over time in either group (P > .05). MAIN LIMITATIONS: The PWB model may not be representative of naturally occurring SLL. CONCLUSIONS: Evidence of lamellar ischaemia (increased L:P and decreased urea clearance) was detected exclusively in the lamellar dermis of PWB feet subjected to persistently increased load. Lamellar ischaemia is a consequence of increased mechanical load and likely contributes to the development of SLL.

Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. HYPOTHESIS/OBJECTIVES: To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. ANIMALS: Eight Standardbred horses. METHODS: Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL-11, cyclooxygenase 1 and 2, tumour necrosis factor-alpha [TNF-α], E-selectin, and intercellular adhesion molecule-1 [ICAM-1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3). RESULTS: Compared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL-6 (P = .008), IL-1ß (P = .008), IL-11 (P = .008), and cyclooxygenase-2 (P = .008) were decreased in CDH. Cyclooxygenase-1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP-1 (P = .05), MCP-2 (P = .46), TNF-α (P = .05), E-selectin (P = .15), and ICAM-1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P-STAT3 [S727] P = .19; P-STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P-STAT1 [S727], P = .25; P-STAT1 [Y701], P = .64). CONCLUSIONS AND CLINICAL IMPORTANCE: These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

Evaluation of the effect of laser salpingopharyngostomy on the guttural pouch environment in horses.

BACKGROUND: Laser salpingopharyngostomy has been used as an adjunct to primary medical therapy allowing for topical debridement, topical treatment or to potentially alter the guttural pouch environment in horses with guttural pouch disease. OBJECTIVES: To evaluate the effect of a laser salpingopharyngostomy on the guttural pouch environment in healthy horses. STUDY DESIGN: Experimental study. METHODS: In six adult Standardbred female horses, under standing sedation a stab incision was made ventral to the sternocephalicus tendon and a customised trocar inserted into the medial guttural pouch compartment. Baseline (day 0) temperature, humidity, oxygen (O2 ) and carbon dioxide (CO2 ) levels were recorded. A laser salpingopharyngostomy was subsequently made into the right guttural pouch. The guttural pouch environment was re-sampled as above on days 7 and 14 to detect differences in the local environment of the guttural pouch caused by the laser salpingopharyngostomy. RESULTS: The guttural pouch environment was altered by the laser salpingopharyngostomy. Baseline recorded values within the guttural pouch were highly constant within each horse. Post laser salpingopharyngostomy there was considerable variation within the guttural pouch between horse and during the period of sampling. Specifically, CO2 levels post laser salpingopharyngostomy appeared to be linked directly to the horse's respiratory pattern with peaks and troughs recorded post laser salpingopharyngostomy. The variation in CO2 was significantly different at days 7 and 14 post laser salpingopharyngostomy compared with baseline levels (day 7 = 6 mm Hg, day 14 = 8.83 mm Hg). Oxygen levels were also more varied during sampling compared with baseline (day 7 = 3.4%, day 14 = 1.83%). There was no change in humidity or temperature during the study. MAIN LIMITATIONS: This study was conducted on horses with no evidence of guttural pouch abnormalities or disease. CONCLUSIONS: Laser salpingopharyngostomy alters the guttural pouch environment and this may be useful in the treatment of guttural pouch disease where this alteration is desirable.

Lamellar energy metabolism and perfusion in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Hyperinsulinaemia is associated with the development of endocrinopathic laminitis; however, the mechanisms remain unclear. OBJECTIVES: Evaluate the effects of hyperinsulinaemia on lamellar energy metabolism and perfusion during laminitis development. STUDY DESIGN: In vivo experiment. METHODS: Eight Standardbred horses were instrumented with a microdialysis probe in the lamellae of a forelimb. A 24 hours baseline period (BASELINE) was followed by 48 hours of a continuous euglycaemic hyperinsulinaemic clamp (EHC) from 24 to 72 hours (CLAMP). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate-to-pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Archived microdialysis samples from six identically instrumented Standardbred horses served as controls (CON). Variables were compared over time and between EHC and CON horses using a mixed-effects linear regression model. RESULTS: Glucose concentration decreased during the CLAMP period in CON and EHC horses (P < .001), but there was no difference between CON and EHC (P > .9). Lactate concentration increased during the CLAMP period in CON and EHC horses (P < .001), however, the rate of increase was significantly higher in EHC horses relative to CON (P = .014). There was a relative increase in pyruvate concentration in EHC horses compared with CON during the CLAMP period (P = .03). L:P increased significantly in CON horses during the CLAMP period (P < .001) but not in EHC (P = .1). Urea clearance did not change in CON (P = .9) or EHC (P = .05) during the CLAMP, but did increase in EHC relative to CON (P = .02). MAIN LIMITATIONS: The effects of microdialysis probe implantation on perfusion and metabolism remain unclear. The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: Laminitis developed without evidence of lamellar hypoperfusion or energy stress. Therapies to improve perfusion are unlikely to affect the initial development of endocrinopathic laminitis.

The effect of continuous digital hypothermia on lamellar energy metabolism and perfusion during laminitis development in two experimental models.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp (EHC) and oligofructose (OF) laminitis models, but the mechanisms remain unclear. OBJECTIVES: To evaluate the effects of CDH on lamellar energy metabolism and perfusion in healthy horses and during EHC and OF laminitis models. STUDY DESIGN: In vivo experiment. METHODS: Archived samples were used from Standardbred geldings that received no treatment (CON) (n = 8) or underwent EHC (n = 8) or OF (n = 6) laminitis models. Both forelimbs were instrumented with a lamellar microdialysis system, and one forelimb was cooled (CDH) with the other maintained at ambient temperature (AMB). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Glucose did not change in CDH limbs relative to AMB in CON (P = .3), EHC (P = .3) or OF (P = .6) groups. There was a decrease in lactate (P < .001) and pyruvate (P < .01) in CDH limbs relative to AMB in all groups. L:P decreased in CON CDH relative to CON AMB (P < .001) but was not different in EHC (P = .6) and OF (P = .07) groups. Urea clearance decreased in CDH limbs relative to AMB in CON (P = .002) and EHC (P < .001), but not in OF (P = .4). MAIN LIMITATIONS: The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: CDH caused a marked decrease in lamellar glucose metabolism (CON, EHC and OF) and perfusion (CON and EHC) without affecting lamellar glucose concentration. Although cellular energy failure is not a primary pathophysiological event in EHC and OF laminitis models, CDH may act by limiting energy supply to pathologic cellular processes whilst preserving those critical to lamellar homoeostasis.

Are There Shared Mechanisms in the Pathophysiology of Different Clinical Forms of Laminitis and What Are the Implications for Prevention and Treatment?

Laminitis is a consequence of primary disease processes elsewhere in the body. The key pathophysiologic events are insulin dysregulation in endocrinopathic laminitis, ischemia in supporting limb laminitis, and inflammation in sepsis-related laminitis. These apparently disparate mechanisms converge to cause lamellar attachment failure through epithelial cell adhesion loss and stretch, possibly mediated by common growth factor signaling pathways. Tissue damage through mechanical distraction, inflammation, pain, and a proliferative epithelial healing response are features of acute laminitis regardless of the cause. Preventive and treatment strategies based on knowledge of these unique and common mechanistic events are likely to improve clinical outcomes.

In vitro comparison of 3 techniques of prosthesis attachment to the muscular process of the equine arytenoid cartilage.

OBJECTIVE: To determine the degree and ease of arytenoid abduction achieved with abaxial placement of a FASTak II suture anchor compared to 2 suture patterns with different directions of insertion-caudomedial to craniolateral and medial to lateral. STUDY DESIGN: Ex vivo experimental. STUDY POPULATION: Cadaveric larynges from 10 Standardbred racehorses. METHODS: Each larynx was sequentially instrumented with all 3 arytenoid suture attachment in random order: (1) abaxial placement of a FASTak II suture, (2) caudomedial to craniolateral suture, and (3) medial to lateral suture placement. Each construct was abducted at 5N increments from 0 to 25N and the left to right quotient angle ratio (LRQ) measured from digital pictures acquired at each sequential increment. RESULTS: Arytenoid abduction (higher LRQ) was greater with FASTak II construct than either of the suture patterns. The largest difference occurred at 5N. Approximately 50% less force was required to achieve an LRQ of 1.0 with the FASTak II anchor compared to the suture patterns. No difference was detected between the 2 suture constructs throughout the study. CONCLUSION: Use of the FASTak II suture anchor improved arytenoid abduction compared to 2 suture patterns and minimized the suture loads required to achieve maximum arytenoid abduction. CLINICAL RELEVANCE: Use of the FASTak II anchor may decrease the suture load required to achieve arytenoid abduction in clinical cases. This may reduce the load placed on the laryngoplasty, thereby, minimizing postoperative loss of abduction.

The effect of hypothermia on influx of leukocytes in the digital lamellae of horses with oligofructose-induced laminitis.

Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia ("cryotherapy"), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10g/kg, n=14 horses). Lamellae were harvested at 24h post-oligofructose administration (DEV, n=7) or at the onset of OG1 laminitis (OG1, n=7). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n=10 (40× fields/digit for MAC387 and 20x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p<0.05. MAC387-positive cells were present in low numbers in the lamellar tissue and were decreased in the hypothermic limbs (vs. AMB limbs, p<0.05) in the OG1 group; no change in CD163-positive cell numbers was noted across the conditions of the model. This study demonstrated that hypothermia of the distal limbs instituted early in the disease process in the horse at risk of SRL significantly attenuates the increase of MAC387-positive leukocytes in the digital lamellae, but has minimal effect on increases in lamellar concentrations of the major leukocyte cell type present in that tissue, CD163-positive mononuclear cells.

Distribution of technetium-99m PEG-liposomes during oligofructose-induced laminitis development in horses.

Liposomes are phospholipid nanoparticles used for targeted drug delivery. This study aimed to determine whether intravenous liposomes accumulate in lamellar tissue during laminitis development in horses so as to assess their potential for targeted lamellar drug delivery. Polyethylene-glycol (PEG) coated liposomes were prepared according to the film hydration method and labelled using (99m)Tc-hexamethyl-propylene-amine-oxime. Six horses received 10 g/kg oligofructose via nasogastric tube to induce laminitis, and four control horses received water via nasogastric tube. All horses received 300 µmol (99m)Tc-PEG-liposomes (5.5 GBq) plus 5.5 µmol/kg PEG-liposomes by slow intravenous infusion. Scintigraphic imaging was performed at 0, 6 and 12 h post-infusion. Technetium-99m liposome uptake was measured in regions of interest over the hoof, fetlock and metacarpus. At the study end-point horses were euthanased, tissue samples collected and tissue liposome levels were calculated as the percentage of the injected dose of (99m)Tc-liposomes per kilogram of tissue. Data were analysed non-parametrically. All horses receiving oligofructose developed clinical and histological signs of laminitis. Technetium-99m liposome uptake in the hoof increased with time in laminitis horses (P = 0.04), but decreased with time in control horses (P = 0.01). Technetium-99m liposome levels in lamellar tissue from laminitis horses were 3.2-fold higher than controls (P = 0.02) and were also higher in laminitis vs. control skin, muscle, jejunum, colon, and kidney (P < 0.05). Liposomes accumulated in lamellar tissue during oligofructose-induced laminitis development and demonstrated potential for targeted lamellar drug delivery in acute laminitis. This study provides further evidence that lamellar inflammation occurs during laminitis development. Liposome accumulation also occurred in the skin, muscle, jejunum, colon and kidneys, suggesting systemic inflammation in this model.

Intraosseous infusion of the distal phalanx compared to systemic intravenous infusion for marimastat delivery to equine lamellar tissue.

No validated laminitis drug therapy exists, yet pharmaceutical agents with potential for laminitis prevention have been identified. Many of these are impractical for systemic administration but may be effective if administered locally. This study compared intraosseous infusion of the distal phalanx (IOIDP) with systemic intravenous constant rate infusion (CRI) to determine which was more effective for lamellar marimastat delivery. Ultrafiltration probes were placed in both forefeet of five horses to collect lamellar interstitial fluid as lamellar ultrafiltrate (LUF). Marimastat solution (3.5 mg/mL) containing lidocaine (20 mg/mL) was infused by IOIDP at 0.15 mL/min for 12 h. After a 12 h wash-out, marimastat (3.5 mg/mL) and lidocaine were infused by constant rate infusion (CRI) at 0.15 mL/min for 12 h. LUF, plasma and lamellar tissue marimastat concentrations were quantified using UPLC-MS. Zymography was used to establish the inhibitory concentrations of marimastat for equine lamellar matrix metalloproteinases (MMPs). Data were analysed non-parametrically. There was no difference between the steady-state marimastat concentration in lamellar ultrafiltrate (LUF[M]) during IOIDP (139[88-497] ng/mL) and CRI (136[93-157] ng/mL). During IOIDP, there was no difference between marimastat concentrations in the treated foot (139[88-497] ng/mL), the untreated foot (91[63-154] ng/mL) and plasma (101[93-118] ng/mL). LUF[M] after IOIDP and CRI were >IC50 of lamellar MMP-2 and 9, but below the concentration considered necessary for in vivo laminitis prevention. Lamellar drug delivery during IOIDP was inconsistent and did not achieve higher lamellar marimastat concentrations than CRI. Modification or refinement of the IOIDP technique is necessary if it is to be consistently effective.

Ultrafiltration of equine digital lamellar tissue.

There are no experimentally validated pharmacological means of preventing laminitis; however, locally acting pharmaceutical agents with the potential to prevent laminitis have been identified. Demonstrating therapeutic drug concentrations in lamellar tissue is essential for evaluating the efficacy of these agents. The aim of this study was to develop an experimental technique for repeatedly sampling lamellar interstitial fluid. A technique for placing ultrafiltration probes was developed in vitro using 15 cadaver limbs. Subsequently, lamellar ultrafiltration probes were placed in one forelimb in six living horses. Interstitial fluid was collected continuously from the probes as ultrafiltrate for 4 (n = 4) or 14 days (n = 2). The rate of ultrafiltrate collection was calculated every 12 h. Biochemical analyses were performed on ultrafiltrate collected on night 1 (12-24 h post-implantation) and night 4 (84-96 h post-implantation). Sections surrounding the probe and control tissue from the contralateral limb were harvested, stained with H&E and Masson's trichrome and scored based on the tissue response to the probe. Ultrafiltration probes were placed in the lamellar tissue in all six horses. Ultrafiltrate was collected from these probes at 55 (30-63) µL/h (median [interquartile range]). Fluid production decreased significantly with time from night 3 onwards (P < 0.05). There was no significant change in the constituents of the ultrafiltrate between nights 1 and 4 (P > 0.05). The technique was well tolerated. This study demonstrates that ultrafiltration can be used to sample equine digital lamellar interstitial fluid, and has potential for measuring lamellar drug levels.

Alfaxalone compared with ketamine for induction of anaesthesia in horses following xylazine and guaifenesin.

OBJECTIVE: To compare anaesthesia induced with either alfaxalone or ketamine in horses following premedication with xylazine and guaifenesin. STUDY DESIGN: Randomized blinded cross-over experimental study. ANIMALS: Six adult horses, five Standardbreds and one Thoroughbred; two mares and four geldings. METHODS: Each horse received, on separate occasions, induction of anaesthesia with either ketamine 2.2 mg kg(-1) or alfaxalone 1 mg kg(-1) . Premedication was with xylazine 0.5 mg kg(-1) and guaifenesin 35 mg kg(-1) . Incidence of tremors/shaking after induction, recovery and ataxia on recovery were scored. Time to recovery was recorded. Partial pressure of arterial blood oxygen (PaO(2) ) and carbon dioxide (PaO(2) ), arterial blood pressures, heart rate (HR) and respiratory rates were recorded before premedication and at intervals during anaesthesia. Data were analyzed using Wilcoxon matched pairs signed rank test and are expressed as median (range). RESULTS: There was no difference in the quality of recovery or in ataxia scores. Horses receiving alfaxalone exhibited a higher incidence of tremors/shaking on induction compared with those receiving ketamine (five and one of six horses respectively). Horses recovered to standing similarly [28 (24-47) minutes for alfaxalone; 22 (18-35) for ketamine] but took longer to recover adequately to return to the paddock after alfaxalone [44 (38-67) minutes] compared with ketamine [35 (30-47)]. There was no statistical difference between treatments in effect on HR, PaO(2) or PaCO(2) although for both regimens, PaO(2) decreased with respect to before premedication values. There was no difference between treatments in effect on blood pressure. CONCLUSIONS AND CLINICAL RELEVANCE: Both alfaxalone and ketamine were effective at inducing anaesthesia, although at induction there were more muscle tremors after alfaxalone. As there were no differences between treatments in relation to cardiopulmonary responses or quality of recovery, and only minor differences in recovery times, both agents appear suitable for this purpose following the premedication regimen used in this study.

Measurement of digital laminar and venous temperatures as a means of comparing three methods of topically applied cold treatment for digits of horses.

OBJECTIVE: To compare effects of 3 methods of topically applied cold treatment (cryotherapy) on digital laminar and venous temperatures in horses. ANIMALS: 9 healthy adult Thoroughbreds. PROCEDURES: Thermocouples were placed in palmar digital veins and digital laminae of both forelimbs of horses. Three methods of cryotherapy were applied to the distal aspects of the limbs: wader boot (63-cm-tall vinyl boot filled with ice and water [ice slurry]), ice bag (5-L fluid bag filled with ice slurry), and a gel pack boot (boot containing frozen gel packs). Gel packs and ice slurries were replenished every hour during cryotherapy. The forelimb that received the first treatment was randomly assigned; thereafter, control and treated forelimbs were alternated for each treatment. For each treatment, temperatures were recorded every minute during 15-minute pretreatment, 2-hour treatment, and ≥ 30 minute rewarming periods. Once temperatures had returned to within 3°C below pretreatment values, the experiment was repeated in a similar manner for other cryotherapy methods. RESULTS: Digital venous temperatures were similar to laminar temperatures during each treatment. Ice bag and wader boot treatments caused similar cooling of digits. Gel boot treatment did not cause substantial cooling of digits. CONCLUSIONS AND CLINICAL RELEVANCE: Ice bag treatment caused laminar and digital venous cooling equivalent to that of wader boot treatment. Cryotherapy by use of 5-L fluid bags with an ice slurry may be a readily available, practical, and efficient method for prevention of laminitis in horses. Digital laminar and venous temperatures were similar in forelimbs of horses before and during cryotherapy.