Intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report.

Cecal fluid from two adult horses was assayed by the limulus amebocyte lysate system for endotoxin before and after carbohydrate overload of the gastrointestinal tract. There were increases in cecal fluid endotoxin concentrations at the 3-, 6-, and 12-hour samplings when compared with base-line values. Concomitant cecal fluid lactate concentrations and pH values increased and decreased, respectively. Both horses subsequently developed clinical signs of acute laminitis.     

In vitro evaluation of intraluminal factors that may alter intestinal permeability in ponies with carbohydrate-induced laminitis

OBJECTIVES: To study the in vitro effects of cecal contents incubated with corn starch on colonic permeability in horses. ANIMALS: 4 healthy adult ponies. PROCEDURE: Mucosal specimens were obtained from the right ventral colon and mounted in Ussing chambers. Changes in short circuit current, conductance, and large-molecule permeability in response to addition of cecal contents and cecal contents incubated with corn starch were evaluated for 120 minutes. RESULTS: Incubation of cecal contents with corn starch for 8 hours resulted in a decrease in cecal content pH and an increase in lactic acid concentration. These changes were similar to those reported in vivo for ponies given corn starch. Exposure of colonic mucosa to cecal contents incubated with corn starch resulted in an increase in tissue conductance and permeability of technetium Tc 99m pentetate, compared with mucosa exposed to cecal contents alone. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro exposure of colonic mucosa to cecal contents incubated with starch resulted in increased paracellular permeability. Fermentation of excessive amounts of carbohydrate in the intestinal lumen of horses may directly induce increased intestinal permeability associated with carbohydrate-induced laminitis.     

Submural histopathologic changes attributable to peracute laminitis in horses

OBJECTIVE: To describe submural histopathologic changes attributable to peracute laminitis in horses. ANIMALS: 20 adult horses. PROCEDURE: A concurrent-control design was used to compare laminar lesions in 10 horses subjected to carbohydrate-induced laminitis with laminar characteristics of 10 sex- and aged-matched control horses with normal feet. Horses in the treatment group were administered an overload of carbohydrate. Tissues were obtained by biopsy 4 to 8 hours after onset of lameness or 72 hours after administration of the carbohydrate overload when lameness did not develop. Sections were stained with H&E, Masson's trichrome, and periodic acid-Schiff stains. Histopathologic changes were analyzed to detect differences between groups and to correlate epidermal changes with severity and duration of lameness. RESULTS: Analysis indicated that dermal and epidermal lesions were evident despite lack of visible separation of the epidermal basement membrane, can be found in horses without detectable lameness, and were nonspecific and progressive following onset of lameness. Furthermore, severity and location of lesions were associated with severity and duration of lameness. CONCLUSION AND CLINICAL RELEVANCE: These observations are consistent with the concept that separation of the laminar epithelial basement membrane is a delayed step in the pathogenesis of acute laminitis, digital vascular hypoperfusion is an underlying cause for laminitis, and the potential for repeated episodes of subclinical laminitis may underlie the development of structural and mechanical changes consistent with chronic laminitis despite lack of clinical signs of acute laminitis.     

Production of amines in equine cecal contents in an in vitro model of carbohydrate overload

Acute laminitis can be induced experimentally in horses by the administration of carbohydrate, resulting in fermentation within the cecum and ischemia-reperfusion of the digits. The products of fermentation that trigger acute laminitis are as yet unknown; however, compounds such as amines might play a role due to their potential vasoactive properties. The objectives of this study were to quantify the amines present in equine cecal contents and to use a model of carbohydrate overload in vitro to test the hypothesis that carbohydrate fermentation is associated with increased amine production. Cecal contents from each horse were divided into aliquots and incubated anaerobically with either cornstarch or inulin (a form of fructan carbohydrate; both 1 g/100 mL). The pH was measured and samples were taken at the same time for amine measurement by HPLC at 2-h intervals over a 24-h period. In a second set of experiments, the effects of the antibiotic virginiamycin (1 mg/100 mL), calcium (CaPO4; 0.3 g/100 mL), and plant steroidal saponin (Yucca schidigera extract; 0.1 g/100 mL) were examined on pH and amine concentrations in cecal contents incubated with starch or inulin. Both starch and inulin caused significant time-dependent falls in pH, from 6.7 +/- 0.1 at 0 h to 5.2 +/- 0.1 (starch) and 5.0 +/- 0.1 (inulin) at 24 h. Fermentation of carbohydrate was also associated with increased production of phenylethylamine and isoamylamine (two- to threefold increases) as well as putrescine and cadaverine (1.5- to twofold increases). Virginiamycin inhibited the fall in pH and increases in production of phenylethylamine and isoamylamine, while calcium phosphate moderated the changes in pH only. Yucca schidigera extract was without effect. These data show that fermentation of carbohydrate by equine cecal microbiota may lead to increased production of amines.     

Circulatory and blood gas changes accompanying the development and treatment of induced laminitis

A peripheral vasodilatory agent, isoxsuprine hydro-chloride, was evaluated in a controlled study for its efficacy in the treatment of acute equine laminitis. Eight healthy, adulthorses of variable age and sex were used in the trial. Acute laminitis was induced in 5 of the horses by oral carbohydrate overload. Intravenous isoxsuprine therapy (1.8 mg/kg) was initiated in 3 of the horses receiving carbohydrate overload at first sign of clinical lameness and repeated at 12-hour intervals. Intravenous saline placebos were administered on a similar schedule to 2 control horses which also received a carbohydrate overload. The remaining 3 horses served as further controls. Local and systemic responses to induction of laminitis and isoxsuprine administration were assessed by subjective evaluation of clinical lameness in a double blind trial; nuclear scintigraphy and radiography of the distal forelimbs; and assessment of physical, hematological and biochemical parameters.

Pronounced tachycardia, hypotension and sweating accompanied the intravenous infusion of isoxsuprine. The 3 horses treated with isoxsuprine following the induction of laminitis showed a more rapid improvement in soundness than horses receiving saline placebos. No horse developed rotation of the third phalanx in response to the diet Nuclear scintigraphy indicated that blood perfusion patterns within the hoof of laminitic horses altered with isoxsuprine therapy, but an overall increase or decrease in perfusion was not apparent. Alterations in serum enzyme and electrolyte profiles with the onset of laminitis generally concurred with findings previously reported for this model of the disease. No change in coagulation profiles accompanied the onset of laminitis or isoxsuprine administration. Blood gas analysis indicated an increase in median palmar vein oxygen partial pressure (PO₂) levels with onset of laminitis. A concurrent decrease in the median palmar arteriovenous oxygen partial pressure difference (AVO₂) was significant at the P<0.01 level. There was no difference in median palmar vein PO₂ values between thoseanimals receiving isoxsuprine and those receiving saline placebo therapy. Results of the trialindicated that isoxsuprine may be beneficial in the treatment of acute laminitis. Further controlled studies are appropriate.     

Intestinal bacterial overgrowth includes potential pathogens in the carbohydrate overload models of equine acute laminitis

Carbohydrate overload models of equine acute laminitis are used to study the development of lameness. It is hypothesized that a diet-induced shift in cecal bacterial communities contributes to the development of the pro-inflammatory state that progresses to laminar failure. It is proposed that vasoactive amines, protease activators and endotoxin, all bacterial derived bioactive metabolites, play a role in disease development. Questions regarding the oral bioavailability of many of the bacterial derived bioactive metabolites remain. This study evaluates the possibility that a carbohydrate-induced overgrowth of potentially pathogenic cecal bacteria occurs and that bacterial translocation contributes toward the development of the pro-inflammatory state. Two groups of mixed-breed horses were used, those with laminitis induced by cornstarch (n=6) or oligofructan (n=6) and non-laminitic controls (n=8). Cecal fluid and tissue homogenates of extra-intestinal sites including the laminae were used to enumerate Gram-negative and -positive bacteria. Horses that developed Obel grade2 lameness, revealed a significant overgrowth of potentially pathogenic Gram-positive and Gram-negative intestinal bacteria within the cecal fluid. Although colonization of extra-intestinal sites with potentially pathogenic bacteria was not detected, results of this study indicate that cecal/colonic lymphadenopathy and eosinophilia develop in horses progressing to lameness. It is hypothesized that the pro-inflammatory state in carbohydrate overload models of equine acute laminitis is driven by an immune response to the rapid overgrowth of Gram-positive and Gram-negative cecal bacterial communities in the gut. Further equine research is indicated to study the immunological response, involving the lymphatic system that develops in the model.     

Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses

Reasons for performing study: Insulin resistance (IR) is a risk factor for pasture‐associated laminitis in equids and alimentary carbohydrate overload may trigger laminitis. Whether glucose metabolism responses to carbohydrate overload are more pronounced in insulin‐resistant horses requires further study. Hypothesis: Horses pretreated with endotoxin to alter insulin sensitivity differ significantly in their glucose and insulin responses to carbohydrate overload. Methods: Horses (n = 24) were divided into 3 groups. A lipopolysaccharide (LPS; n = 8) group that received endotoxin as an 8 h 7.5 ng/kg bwt/h i.v. continuous rate infusion, an oligofructose (OF; n = 8) group that received an infusion of saline followed by 5 g/kg bwt OF via nasogastric intubation, and a LPS/OF (n = 8) group that received LPS followed 16 h later by OF. Glucose and insulin dynamics were evaluated at ‐24 h and 48 h using the frequently sampled i.v. glucose tolerance test and minimal model analysis. Physical examinations and haematology were performed and the severity of laminitis assessed. Results: Horses receiving LPS developed leucopenia and both LPS and OF induced clinical signs consistent with systemic inflammation. Insulin sensitivity significantly decreased (P<0.001) over time, but responses did not differ significantly among groups. Time (P<0.001) and treatment × time (P = 0.038) effects were detected for the acute insulin response to glucose, with mean values significantly increasing in LPS and LPS/OF groups, but not the OF group. Five horses in the LPS/OF group developed clinical laminitis compared with 0 and 2 horses in the LPS and OF groups, respectively. Conclusions: Endotoxaemia and carbohydrate overload reduce insulin sensitivity in horses. Endotoxin pretreatment does not affect the alterations in glucose metabolism induced by carbohydrate overload. Potential relevance: Insulin sensitivity decreases after carbohydrate overload in horses, which may be relevant to the development of pasture‐associated laminitis.     

Inflammatory Cytokine Gene Expression in Blood During the Development of Oligofructose-Induced Laminitis in Horses

Systemic inflammation is a risk factor for laminitis in horses and precedes the onset of lameness in experimental models. We therefore hypothesized that whole-blood inflammatory cytokine expression would increase during the development of laminitis in a carbohydrate overload model. Blood samples were obtained from 14 horses undergoing laminitis induction with 10 g/kg oligofructose as part of another study. Samples were collected at 0, 8, 12, 16, 20, and 24 hours, and lameness evaluations were performed every 4 hours. Expression levels of interleukin-1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor-α were measured in whole blood by using real-time PCR. IL-1β, IL-8, and IL-10 expression increased above baseline from 8 to 24 hours (P < .001), and IL-6 expression increased at 16 and 20 hours (P = .005). Expression of tumor necrosis factor-α did not change over time. All horses developed clinical laminitis between 12 and 24 hours. Increased mean IL-1β, IL-8, and IL-10 expression detected at 8 hours therefore preceded the onset of lameness. We conclude that peripheral leukocyte cytokine expression increases as systemic inflammation develops in an alimentary carbohydrate overload model of laminitis, and this precedes detection of lameness. Results support current recommendations to control the systemic inflammatory response in order to lower the risk of laminitis in horses.     

Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitis

In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean +/- s.e. 218.04 +/- 18.59 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.     

Illumina sequencing of the V4 hypervariable region 16S rRNA gene reveals extensive changes in bacterial communities in the cecum following carbohydrate oral infusion and development of early-stage acute laminitis in the horse

In the equine carbohydrate overload model of acute laminitis, disease progression is associated with changes in bacteria found in the cecum. To date, research has focused on changes in specific Gram-positive bacteria in this portion of the intestinal tract. Metagenomic methods are now available making it possible to interrogate microbial communities using animal protocols that sufficiently power a study. In this study, the microbiota in cecal fluid collected from control, non-laminitic horses (n = 8) and from horses with early-stage acute laminitis induced with either oligofructan (n = 6) or cornstarch (n = 6) were profiled. The microbiota were identified based on sequencing the V4 hypervariable region of the 16S rRNA gene. The results of the study show that the relative abundance of Lactobacillus sp. and Streptococcus sp. increased significantly (p < 0.05) following OF and CS infusion. Other significant changes included an increase (p < 0.05) in relative abundance of Veillonella sp. and Serratia sp., two potentially pathogenic, Gram-negative bacteria. Significant decreases in the relative abundance of presumptive normal flora were detected as well. Although changes in cecal microbiota described in this communication are from a pilot study, it is hypothesized that an overgrowth of pathogenic Gram-negative bacteria develops and contributes to enterocolitis, pyrexia and lameness in the carbohydrate overload model of acute laminitis.     

Palmar digital vessel relaxation in healthy horses and in horses given carbohydrate

OBJECTIVE: To compare in vitro smooth muscle relaxation of palmar digital vessels from healthy horses with those from horses in the prodromal stage of experimentally (carbohydrate) induced laminitis. ANIMALS: 16 adult horses. PROCEDURE: Segments of palmar digital vessels were obtained from 5 healthy horses and 6 horses given carbohydrate. Vascular rings from the palmar digital artery and vein were suspended in individual organ baths containing buffer solution and indomethacin; isometric tension was recorded, and contraction and relaxation were compared. Smooth muscle contraction in response to cumulative addition of phenylephrine was recorded in the absence and presence of 1 microM NG-nitro-L-arginine methyl ester (L -NAME). After wash out, vascular rings were preconstricted with phenylephrine (0.3 microM), and cumulative endothelium-dependent (acetylcholine-induced) and independent (nitroprusside-induced) smooth muscle relaxations were recorded in the absence or presence of L -NAME. RESULTS: Phenylephrine increased vascular smooth muscle tone in ring preparations of palmar digital arteries and veins. Addition of acetylcholine or nitroprusside induced relaxation of palmar digital artery and vein ring preparations. Use of L-NAME (1 microM) significantly reduced maximal relaxation induced by acetylcholine, but not by nitroprusside. Maximal relaxation induced by acetylcholine, but not by nitroprusside, was reduced in vascular rings prepared from carbohydrate-overloaded horses. CONCLUSION AND CLINICAL RELEVANCE: Reduced endothelium-dependent relaxation of palmar digital vessels may have a role in the pathophysiology of acute laminitis after carbohydrate overload in horses.     

Effectiveness of a unique dihydropyridine (BAYTG 1000) for prevention of laminitis in horses

OBJECTIVE: To determine whether a unique dihydropyridine (BAYTG 1000) would be beneficial in preventing laminitis in horses. ANIMALS: 16 clinically normal adult horses. PROCEDURE: 8 pairs of horses were used in a controlled double-blind study, using sex- and age-matched horses randomly assigned to treatment or control groups. Horses were subjected to carbohydrate overload to induce laminitis. Treated horses were administered BAY TG 1000 (30 mg/kg, PO, q 24 h) for 3 days. Hoof wall surface temperature (HWST) and lameness were recorded at 4-hour intervals. The HWST was adjusted on the basis of time of onset of lameness and evaluated, using a repeated-measures ANOVA. Lameness 8 hours after onset and clinical status 72 hours after onset of lameness were evaluated, using Mann-Whitney procedures. RESULTS: Analysis revealed that BAYTG 1000 did not decrease the incidence of lameness but significantly ameliorated prodromal hypothermia, lessened the severity of lameness 8 hours after onset of lameness, and improved the clinical status of horses 72 hours after onset of lameness. CONCLUSION AND CLINICAL RELEVANCE: Results support the conclusion that BAYTG 1000 was protective when used in prevention of laminitis. The drug decreased severity and improved clinical status (recovery) of induced lameness, which was interpreted to mean that the drug's actions were on mechanisms important but secondary to primary causal mechanisms of laminitis. Therefore, drugs that enhance digital perfusion via alteration of rheologic activity may have potential use in the prevention and management of laminitis in horses.     

In vitro reactivity of digital arteries and veins to vasoconstrictive mediators in healthy horses and in horses with early laminitis

The in vitro reactivity of vasoconstrictive mediators that are implicated in acute laminitis was determined in palmar and plantar digital arteries and veins obtained from healthy horses and in palmar digital vessels of horses with early laminitis (Obel grade I). To obtain baseline reactivity data, 3 experiments were conducted, using healthy horses: (1) the reactivity of palmar and plantar digital arteries and veins to angiotensin II, norepinephrine, and 5-hydroxytryptamine (serotonin) were compared; (2) the direct effects of bacterial endotoxin on vascular reactivity were assessed; and (3) the reactivity of palmar digital arteries and veins to angiotensin II, norepinephrine, prostaglandin F2 alpha (PGF2 alpha), serotonin, and a thromboxane-endoperoxide analog (U46619) were determined. The vascular reactivity of these same 5 vasoconstrictors then was determined in horses with early laminitis and was compared with data from healthy (control) horses. Obel grade-I laminitis was experimentally induced in horses, using carbohydrate overload. Dose responses were conducted for each agent at concentrations between 10(-8)M and 10(-4)M. The potency of a drug was defined as the mean effective concentration necessary to induce 50% of maximal contraction (EC50). There were no differences in EC50 concentrations and in maximal contractions between forelimb and hind limb arteries and veins for angiotensin II, norepinephrine, and serotonin. Incubation with endotoxin had no effect on the reactivity of arteries and veins to angiotensin II, norepinephrine, and serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokeratins of the matrices of the chestnut (torus carpeus) and periople in horses with acute laminitis

OBJECTIVES: To determine whether there is a change in the expression of cytokeratins in the epidermal cells of the non-weight-bearing parts of the limb in horses with acute laminitis and thus determine whether the morphologic changes that develop in the periople and chestnut (torus carpeus) of horses early in acute laminitis are caused by inhibition of keratinocyte differentiation. ANIMALS: 8 horses with acute laminitis. PROCEDURE: Tissue specimens were obtained from the chestnuts of all 8 horses and from the stratum externum of the hoof wall of 3 horses. Tissue specimens were obtained within 48 hours of the first clinical signs of laminitis. The cytokeratins were characterized by 1- and 2-dimensional gel electrophoresis, and the tissue distribution of the cytokeratins was studied by immunohistochemical staining. RESULTS: The biochemical findings indicated that the epidermal cells of tissues from horses affected by laminitis contained the same set of cytokeratins as corresponding tissues from clinically normal horses. Immunohistochemistry on sections from specimens of horses with laminitis versus clinically normal horses indicated a difference in the expression of cytokeratin in the basal cells in the matrix of the stratum externum of the hoof wall and in the matrix of the chestnut of horses with laminitis in which the most severe morphologic changes were observed. CONCLUSIONS: Inhibition of keratinocyte differentiation, as observed by immunohistochemical changes, in cells in parts of the chestnut and periople may indirectly indicate that the observed epidermal changes in horses with laminitis are primary and are unaffected by weight-bearing.     

Serial alterations in digital hemodynamics and endothelin-1 immunoreactivity, platelet-neutrophil aggregation, and concentrations of nitric oxide, insulin, and glucose in blood obtained from horses following carbohydrate overload

OBJECTIVE: To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. ANIMALS: 20 adult horses without abnormalities of the digit. PROCEDURES: Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. RESULTS: Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.     

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.     

Effects of an endothelin receptor antagonist and nitroglycerin on digital vascular function in horses during the prodromal stages of carbohydrate overload-induced laminitis

OBJECTIVE: To evaluate changes in digital vascular function in horses with carbohydrate overload (CHO)-induced laminitis and determine the effects of an endothelin (ET) receptor antagonist and nitroglycerin on laminitis-associated vascular dysfunction. ANIMALS: 20 adult horses without abnormalities of the digit. PROCEDURES: Hemodynamic variables were recorded before (baseline) and hourly after all horses were administered a CHO ration via nasogastric tube. In 4 groups of 5 horses each, saline (0.9% NaCl) solution or ET receptor antagonist (10(5)M in digital blood) was administered into the digital arterial circulation according to 1 of 2 schedules. During anesthesia, blood flow; arterial, venous, and capillary pressures; and total, precapillary, and postcapillary resistances were measured in an isolated perfused digit of each horse. In all groups, nitroglycerin was infused (10(5)M in digital blood), and digital microvascular assessments were repeated. RESULTS: The CHO caused a significant decrease in right atrial pressure by 14 hours that was not affected by administration of saline solution or ET receptor antagonist. In isolated digits of anesthetized horses, CHO resulted in a significant decrease in digital blood flow associated with a significant increase in total and postcapillary resistances. Treatment with the ET receptor antagonist and nitroglycerin caused a significant decrease in total resistance. Postcapillary resistance was significantly decreased following treatment with the ET receptor antagonist but was not altered by treatment with nitroglycerin. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with an ET receptor antagonist and nitroglycerin resulted in significant improvement in vascular resistance in isolated perfused digits of anesthetized horses with CHO-induced laminitis.     

Equine laminitis: cryotherapy reduces the severity of the acute lesion

REASONS FOR PERFORMING STUDY: The hypometabolic and vasoconstrictive effects of cryotherapy could prevent the development of laminitis. OBJECTIVES: To use distal limb cryotherapy to prevent laminitis induced by alimentary carbohydrate overload. METHODS: Laminitis was induced in 6 Standardbred horses that had one front limb continuously cooled in an ice/water mixture. Lameness evaluation, blinded lamellar histological grading and analysis for lamellar matrix metalloproteinase-2 (MMP-2) mRNA expression were used to evaluate the severity of laminitis. RESULTS: Cryotherapy was well tolerated and effective in cooling the feet. In each horse no lameness was observed in the treated limbs. Laminitis histology scores in the treated limbs were significantly less than those of the corresponding untreated forelimbs (P < 0.05). Laminitis histology scores in the treated limbs were also significantly less than those of the untreated limbs (fore- and hind) as a group (P < 0.05). Expression of MMP-2 mRNA in the iced feet was significantly (P < 0.05) less than that detected in the untreated feet. CONCLUSIONS: Cryotherapy, when applied to one foot, markedly reduced the severity of acute laminitis in this study. We propose that vasoconstriction (preventing delivery of haematogenous trigger factors) and hypometabolism (reduction in lamellar MMP activity) were the primary therapeutic mechanisms. POTENTIAL RELEVANCE: Although further research is needed, we suggest cryotherapy as a potentially effective prophylactic strategy in horses at risk of developing acute laminitis.     

Assessment of apoptosis in epidermal lamellar cells in clinically normal horses and those with laminitis

OBJECTIVE: To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis. SAMPLE POPULATION: Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis). PROCEDURE: Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis. RESULTS: The number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUNEL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14. CONCLUSIONS AND CLINICAL RELEVANCE: The large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated.