Digital hypothermia inhibits early lamellar inflammatory signalling in the oligofructose laminitis model

Reasons for performing study: The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown. Objectives: To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis. Methods: Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON‐RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time‐quantitative PCR analysis of mRNA concentrations of cytokines (IL‐6, IL‐1β, IL‐10), chemokines (CXCL1, CXCL6, CXCL8/IL‐8, MCP‐1, MCP‐2), cell adhesion molecules (ICAM‐1, E‐selectin), COX‐2 and 3 housekeeping genes. Data were analysed (NON‐RX vs. CRYO, NON‐RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests. Results: Compared with CON, the OG1 NON‐RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL‐10, IL‐1β and MCP‐1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON‐RX. Within the OG1 group, CRYO limbs (compared with NON‐RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP‐1 and IL‐10). Within the DEV group, mRNA concentrations of CXCL‐1, ICAM‐1, IL‐1β, CXCL8 and MCP‐2 were decreased (P<0.05) and the anti‐inflammatory cytokine IL‐10 was increased (compared with NON‐RX limbs; P<0.05). Conclusions: Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX‐2 and endothelial adhesion molecules. Potential relevance: This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.     

Vascular perfusion in horses with chronic laminitis

Vascular perfusion casts were used to define and characterise the macroscopic perfusion defects present in the distal digit of 11 horses affected by chronic laminitis. Five clinically normal horses were used as controls. Based on clinical history and clinical status, horses with chronic laminitis were classified as being potentially treatable or clinically refractory. Eleven macroscopic vascular defects were noted in the casts from horses with laminitis. Four types of lesions were identified in the submural laminar circulation, 3 in the coronary bed and 4 were associated with the solar circulation. Multiple defects were present and a definite trend was noted for the perfusion defects to be worse in the casts of clinically refractory subjects than in those considered treatable. This information suggests that evaluation of circulatory status should add significantly to the ability to separate treatable from clinically refractory patients. Results also indicated that ventral displacement of the third phalanx (sinkers) and compression of the solar vasculature are more prevalent than is presently thought.     

Equine laminitis model: Cryotherapy reduces the severity of lesions evaluated seven days after induction with oligofructose

Reasons for performing study: A previous preliminary study demonstrated the potential of distal limb cryotherapy (DLC) for preventing laminitis. Clinically, DLC must be effective for periods longer than 48 h and the preventive effect must extend beyond its discontinuation. Objectives: To evaluate the effect of DLC, applied during the developmental phase of induced laminitis, on the severity of clinical laminitis and lamellar histopathology 7 days after dosing. Methods: Eighteen normal Standardbred horses were divided into 3 groups of 6. Continuous cryotherapy was applied for 72 h to the distal limbs of the first group. The second and third groups were administered laminitis inducing doses of oligofructose and 72 h of cryotherapy applied (immediately after dosing) to the second group. After clinical assessment all horses were subjected to euthanasia 7 days after dosing and hoof lamellar tissues were harvested and analysed. Results: In the laminitis induced horses clinical lameness and laminitis histopathology was significantly reduced in horses that underwent 72 h of DLC compared with untreated controls. Cryotherapy alone produced no significant lameness or other ill effect. Conclusions: Continuous, medium‐ to long‐term (72 h) cryotherapy applied to the distal limbs of horses safely and effectively ameliorates the clinical signs and pathology of acute laminitis. Potential relevance: Pre‐emptive distal limb cryotherapy is a practical method of ameliorating laminitis in ill horses at risk of developing the disease.     

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.     

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.     

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.     

Equine laminitis: Induced by 48 h hyperinsulinaemia in Standardbred horses

Reasons for performing study: Hyperinsulinaemia is known to induce laminitis experimentally in healthy ponies with no history of the condition. Horses are more insulin sensitive than ponies and whether prolonged hyperinsulinaemia and euglycaemia would have a similar laminitogenic effect requires study. Objectives: To determine if laminitis results when the prolonged euglycaemic hyperinsulinaemic clamp technique (p‐EHC) is applied to clinically normal Standardbred horses, and to monitor hoof wall temperature seeking an association between vascular activity and laminitis development. Methods: Eight young, clinically normal Standardbred horses were assigned into 4 pairs and within each pair, one was assigned randomly to either treatment (n = 4) or control (n = 4) groups. Treated horses received continuous infusions of insulin and glucose until clinical signs of laminitis developed, at which point the horses were subjected to euthanasia. Control horses received an equivalent volume of a balanced electrolyte infusion for the same period. Hoof wall surface temperature (HWST) was monitored continuously throughout the experimental period. Results: All horses in the treatment group were calculated to have normal insulin sensitivity. All treated horses, and none in the control group, developed laminitis (P = 0.01). Pronounced digital pulses were a feature of the treatment group, while insignificant digital pulses occurred in control horses. HWST was higher and less variable in treated horses once hyperinsulinaemia was established. Conclusions: Healthy Standardbred horses subjected to prolonged hyperinsulinaemia develop laminitis within 48 h, demonstrating that laminitis in horses can be triggered by insulin. Potential relevance: Insulin resistance and the associated hyperinsulinaemia place horses and ponies at risk of developing laminitis. This study demonstrates a need for prompt management of the persistent hyperinsulinaemia seen in some endocrinopathies.     

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.     

Evaluation using hoof wall strain gauges of a therapeutic shoe and a hoof cast with a heel wedge as potential supportive therapy for horses with laminitis

OBJECTIVE: To evaluate using strain gauges, a hoof cast with heel wedge, and a therapeutic shoe with unsupported toe for their effectiveness in redistribution of load from the dorsal hoof wall. STUDY DESIGN: In vitro biomechanical study. SAMPLE POPULATION: Twenty forelimb specimens. METHODS: Rosette strain gauges were placed on the dorsal and lateral hoof wall of 20 normal shaped hooves. Limbs were loaded vertically using a tensile testing machine with a 1 Hz sinusoidally cycling load up to 3000 N during 15 seconds. Mean values of principal strain and direction at 2500 N load were calculated for 3 experimental conditions (unshod, therapeutic shoe with unsupported toe, and hoof cast with heel elevation) and tested by ANOVA (P<.05). RESULTS: Vertical limb loading in an unshod hoof leads to a biaxial compression of the dorsal wall with high longitudinal compression (epsilon2 = -1515 microm/m). Principal strain at the dorsal wall (epsilon2) was decreased by 23% with the therapeutic shoe and by 59% with the hoof cast. On the lateral hoof wall principal strain was unchanged with the shoe, but increased by 34% with the cast. CONCLUSIONS: Strain measurements indicate unloading of the dorsal hoof wall by both methods with the cast being more effective than the shoe. CLINICAL RELEVANCE: The hoof cast with wedge offers substantial unloading of the dorsal wall, but increases load on the quarter. Therefore a hoof cast would likely be most helpful in acute laminitis when palmar structures can still bear load. The therapeutic shoe offers rehabilitation and regrowth of the dorsal wall without increased load on the quarter wall.     

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.