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.     

Laminar leukocyte accumulation in horses with carbohydrate overload-induced laminitis

Background: While there is evidence of laminar leukocyte infiltration in black walnut extract (BWE)‐induced laminitis, there is no such evidence for carbohydrate overload (CHO) laminitis. Objective: To assess presence of leukocytes and signs of epidermal stress/injury in the laminar tissue from horses with CHO‐induced laminitis. Animals: Twenty‐four adult horses. Methods: Immunohistochemistry for myeloid cell markers calprotectin (CP) and monocyte‐specific marker (CD163) was performed on laminar sections obtained from 2 groups of horses in the CHO model: the developmental time point (DTP) group (n = 6) and the onset of lameness (LAM) group (n = 6), and a control (CON) group (n = 8). Results: DTP was characterized by an increase in CP⁺ leukocytes (7.8‐fold increase versus CON, P < .001), and LAM time point was characterized by a more marked increase in laminar CP⁺ (108.5‐fold, P < .001) and mild increase in CD163⁺ (1.9‐fold, P= .007) cell counts. Increased CP epidermal signal (indicating epidermal stress or injury) occurred consistently at the LAM time point, although histological evidence of basement membrane (BM) detachment was minor, only being present in 3/6 horses. Conclusions and Clinical Relevance: Maximal laminar leukocyte infiltration and epithelial stress occurred at the onset of lameness in the CHO model showing a different temporal pattern from the BWE model, where maximal leukocyte infiltration clearly precedes epithelial stress. Leukocyte infiltration before major histological changes in the CHO model indicates that leukocyte infiltration can be a cause of and not a reaction to BM degradation and structural failure.     

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.     

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.     

Ultrastructural study of the equine cecum during onset of laminitis

The morphologic and pathologic changes which occurred within the cecal mucosa of 4 horses during the onset of laminitis were determined from cecal biopsy materials obtained via a cecal fistula; the laminitis was induced with carbohydrate overload. The cecal epithelial mucosa specimens were obtained at 0 (base line), 24, 32, 48, and 72 hours after horses were given carbohydrate overload, and these were fixed and subsequently photographed. Changes in the cecal epithelium were examined by transmission electron and scanning electron microscopies. These histopathologic changes indicated that the mucosal barrier was substantially damaged by the carbohydrate overload.     

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.     

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

REASONS FOR PERFORMING STUDY: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. OBJECTIVES: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. METHODS: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. RESULTS: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. CONCLUSIONS: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. POTENTIAL RELEVANCE: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.     

Voluntary limb-load distribution in horses with acute and chronic laminitis

OBJECTIVES: To compare limb-load distribution between horses with and without acute or chronic laminitis. ANIMALS: 10 horses with carbohydrate-induced acute laminitis, 20 horses with naturally occurring chronic laminitis, and 20 horses without foot abnormalities (controls). PROCEDURES: Limb-load distribution was determined, using a custom-designed system that allowed simultaneous quantification of the mean percentage of body weight voluntarily placed on each limb (ie, mean limb load) and the SD of the mean load over a 5-minute period (ie, load distribution profile [LDP]). Load distribution profile was used as an index of frequency of load redistribution. RESULTS: Mean loads on fore- and hind limbs in control horses were 58 and 42%, respectively, and loads were equally and normally distributed between left and right limbs. In addition, forelimb LDP was greater, compared with hind limbs, and was affected by head and neck movement. In comparison, limb-load distribution in horses with chronic laminitis was characterized by an increase in the preferential loading of a forelimb, a decrease in total forelimb load, and an increase in LDP that was correlated with severity of lameness. In horses with carbohydrate-induced acute laminitis, mean limb loads after onset of lameness were not different from those prior to lameness; however, LDP was significantly decreased after onset of lameness. CONCLUSION AND CLINICAL RELEVANCE: Quantification of limb-load distribution may be an applicable screening method for detecting acute laminitis, grading severity of lameness, and monitoring rehabilitation of horses with chronic laminitis.     

Equine laminitis: ultrastructural lesions detected 24-30 hours after induction with oligofructose

REASONS FOR PERFORMING STUDY: The pathology of equine laminitis has been well-documented 48 h after dosing with oligofructose when clinical lameness and lamellar disintegration is well advanced. Further analysis of the earliest lesions, by collecting lamellar samples at the first sign of foot lameness after oligofructose dosing is required in order to increase understanding of the disease. OBJECTIVES: To investigate lamellar epidermal hemidesmosome damage and basement membrane dysadhesion by transmission electron microscopy (TEM). METHODS: Eight clinically normal, mature Standardbred horses were divided randomly into 2 groups of 4. The treatment group were dosed with oligofructose (10 g/kg bwt) and subjected to euthanasia when shifting weight from one foot to other commenced and at the first sign of lameness during walking and turning. This occurred at 24 h in 3 horses and 30 h in one. The sham treatment control group were dosed with water and subjected to euthanasia after 48 h. Lamellar tissues of the front feet were harvested and processed for ultrastructural study using TEM. RESULTS: Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased. CONCLUSIONS: Laminitis lesions are detectable 24 h after oligofructose administration. POTENTIAL RELEVANCE: Hindgut events occurring in the first 24 h after dosing have begun the destruction of the hoof lamellar interface. Prevention and treatment strategies should precede lameness if they are to be efficacious.     

Hindlimb laminar inflammatory response is similar to that present in forelimbs after carbohydrate overload in horses

Reasons for performing study: A significant proinflammatory response is known to occur in the forelimb lamina after carbohydrate administration. As the hindlimbs are often less affected by laminitis compared with the forelimbs, we assessed hindlimb inflammatory response in the early stages of carbohydrate‐induced laminitis to determine whether differences in the response existed. Objective: To determine whether a similar proinflammatory response occurs in the hindlimb laminae to that previously reported for the forelimb. Methods: Archived laminar samples from 12 horses administered 17.6 g of starch (85% corn starch, 15% wood flour)/kg bwt via nasogastric tube that were anaesthetised either after developing a temperature >38.9°C (DEV; n = 6) or at the onset of Obel grade 1 lameness (OG1; n = 6) were used in addition to 6 control horses (CON) that were anaesthetised 24 h after administration of water. Real‐time quantitative polymerase chain reaction for selected proinflammatory mediators and MAC387 immunohistochemistry were performed. The data were analysed nonparametrically to compare groups. Results: Increases in laminar MAC387‐positive leucocytes and laminar messenger ribonucleic acid (mRNA) concentrations (P<0.05) for interleukin‐1β, interleukin‐6, cyclo‐oxygenase‐2, chemokine (C‐X‐C motif)ligand (CXCL)1 and CXCL8 were present in both fore‐ and hindlimb laminae from horses with OG1 lameness. Both CXCL1 and CXCL8 were also increased in forelimb and hindlimb laminae in the DEV horses. Conclusions: Administration of carbohydrate resulted in a similar inflammatory response in the hindlimb laminae to that previously reported for the forelimb laminae. These findings suggest that other factors, such as weightbearing, may play an important role in the development of laminitis after a systemic inflammatory condition develops. Potential relevance: Evidence of inflammation in the hindlimb laminae suggests that the hindfeet should be addressed in the septic horse at risk for laminitis; however, laminitis is often less severe in the hindlimbs due to other factors, such as weightbearing and hoof angle.     

Calprotectin in Myeloid and Epithelial Cells of Laminae from Horses with Black Walnut Extract-Induced Laminitis

Background: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin.
Hypothesis: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis.
Animals: Twenty adult horses.
Methods: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed.
Results: Laminar epidermal CP signal was significantly increased ( P = .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups ( P = .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group.
Conclusions and Clinical Importance: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.     

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis

Reasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis‐related laminitis than the black walnut extract (BWE) model. Objectives: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature >38.9°C (DEV group, n = 8) or at onset of Obel grade 1 lameness (OG1 group, n = 8). Control horses (CON group, n = 8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time‐quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. Results: Increased mRNA concentrations (P<0.05) for IL‐1β, IL‐6, IL‐12p35, COX‐2, E‐selectin and ICAM‐1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL‐2, IL‐4, IL‐10, TNF‐α, IFN‐γ or COX‐1 at either the DEV or OG1 time points. Conclusions: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. Potential relevance: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.     

Laminar microvascular flow, measured by means of laser Doppler flowmetry, during the prodromal stages of black walnut-induced laminitis in horses

OBJECTIVE: To measure changes in laminar microvascular blood flow (LMBF) over time in healthy horses and horses in the prodromal stage of black walnut-induced laminitis and to determine the effects of glyceryl trinitrate application on LMBF in horses with acute laminitis. ANIMALS: 10 healthy adult horses. PROCEDURE: Laser Doppler flowmetry was used to measure LMBF Baseline measurements were obtained, horses were given deionized water via a nasogastric tube, and measurements were obtained hourly for 12 hours. Twenty-four hours later, baseline measurements were again obtained, and horses were given black walnut extract. Measurements were obtained hourly for 12 hours or until development of Obel grade-3 laminitis. At this time, 5 horses were treated with phenylbutazone, and the other 5 were treated with phenylbutazone and glyceryl trinitrate, and measurements were obtained hourly for an additional 12 hours. RESULTS: LMBF was significantly decreased 1 and 2 hours after administration of the black walnut extract but then returned to near-baseline values for the next 6 hours. Eight hours after extract administration, there was a second significant decrease in LMBF that persisted until the end of the study. Glyceryl trinitrate had no effect on LMBF. Clinical signs of laminitis developed 8 to 12 hours after extract administration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that in horses with black walnut-induced laminitis, there is an early decrease in LMBF followed by reperfusion prior to onset of clinical signs. Treatment with glyceryl trinitrate after development of clinical signs of laminitis did not have a significant effect on LMBF.     

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.     

Histopathology of insulin-induced laminitis in ponies

Reasons for performing study: Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. Objectives: To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin‐induced laminitis. Methods: Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid‐dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. Results: No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. Conclusions: Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. Potential relevance: Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.     

Presence of mononuclear cells in normal and affected laminae from the black walnut extract model of laminitis

Reasons for performing study: There is increasing evidence of involvement of inflammatory cells in acute laminitis. Objective: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)‐induced laminitis. Methods: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. Results: Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3‐ and CD20‐positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163‐positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P = 0.0016) in laminar CD163‐positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. Conclusions: Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163‐positive macrophages in SDL. Potential relevance: Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.     

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.     

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.     

Equine laminitis: Ultrastructural lesions detected in ponies following hyperinsulinaemia

Reasons for performing study: Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. Objectives: To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. Methods: Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. Results: Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/μm of BM was decreased in all insulin treated ponies. Conclusions: Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. Potential relevance: The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.