Quantitative assessment of increased sensitivity of chronic laminitic horses to hoof tester evoked pain

Reasons for performing study: To evaluate quantitative sensory testing (QST) of the feet of laminitic horses using a power‐assisted hoof tester. Hypothesis: Hoof Compression Thresholds (HCTs) can be measured reliably and are consistently lower in horses with chronic laminitis than in normal horses. Methods: HCTs of chronic laminitic (n = 7) and normal horses (n = 7) were repeatedly measured using a hydraulically powered and feedback controlled hoof tester. Data from 2 tests, at 3 sites in both forefeet, during 3 sessions were collected and statistically analysed using linear mixed models. Results: The mean ± s.e. HCT for the laminitic horses was 29.6 ± 3.5 kg/cm² and for horses in the normal group was 59.8 ± 4.3 kg/cm². Residual variance was the largest of the error components and was greater (P<0.001) for the normal horses; none of the other components significantly differed between the 2 groups. Averaging of HCTs from each foot could produce a test with intraclass correlation coefficients of 0.83 for the normal group and 0.87 for the laminitic group, with an estimated sensitivity of 0.94 and a specificity of 0.93. This test would permit detection with 80% power and 95% confidence of a reduction of over 40% in the difference in mean HCTs between laminitic and normal horses following effective treatment provided that the experimental groups are of 9 or more horses. Conclusions: HCTs can be safely and reliably measured experimentally using this hoof tester. The level of variability found indicates that, under these conditions, treatments may need to produce at least a 40% improvement to be detected. Simplification of the hoof tester, training of the horse and repeated testing may permit the method to be used clinically to detect changes in the HCTs of individual laminitic horses but these potential improvements will require further investigation. Potential relevance: Measurement of HCTs can provide an additional means for assessing the effectiveness of treatments for alleviation of chronic equine laminitis.     

Glucose transport in the equine hoof

Reasons for performing study: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin‐responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. Objectives: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. Methods: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2‐deoxy‐D‐[2,6‐³H]glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin‐induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. Results: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin‐independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. Conclusions: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1‐mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. Potential relevance: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.     

Effects of a supplement containing chromium and magnesium on morphometric measurements, resting glucose, insulin concentrations and insulin sensitivity in laminitic obese horses

Reasons for performing study: Obesity and insulin resistance are risk factors for laminitis in equids and supplements containing chromium and magnesium might improve insulin sensitivity. Hypothesis: A supplement containing chromium, magnesium and other nutraceuticals would alter morphometric measurements, blood variables, and insulin sensitivity in laminitic obese horses. Methods: Twelve previously laminitic obese (body condition score ≥ 7/9) horses were randomly allocated to treatment (n = 6) and control (n = 6) groups and 2 obese horses with clinical laminitis were included in the treatment group. Treated animals received 56 g supplement with 0.25 kg oats once daily for 16 weeks. The supplement contained chromium (5 mg/day as yeast), magnesium (8.8 g/day as oxide/proteinate), and other nutraceuticals. Insulin‐modified frequently sampled i.v. glucose tolerance tests were performed with hay provided at 0, 8 and 16 weeks, and insulin sensitivity was estimated by minimal model analysis. Physical measurements were collected at the same points. Horses were not exercised. Results: Hyperinsulinaemia (>30 µu/ml) was detected in 12 of 14 horses prior to treatment. Glucose and insulin data from one mare with clinical laminitis were excluded because of persistent pain. Mean ± s.d. insulin sensitivity was 0.64 ± 0.62 × 10^?4 l/min/mu prior to treatment for the remaining 13 horses. Time and treatment × time effects were not significant for any of the variables examined, with the exception of resting insulin concentrations, which significantly increased over time (P = 0.018). Health status remained the same. Conclusions: The supplement containing chromium and magnesium evaluated in this study did not alter morphometric measurements, blood variables, resting insulin concentrations or insulin sensitivity in laminitic obese horses. Potential relevance: Additional research is required to determine the appropriate use of chromium and magnesium supplements in horses.     

Use of stem cells in equine musculoskeletal disorders

Stem cell therapies for musculoskeletal disorders are becoming commonplace in the horse. In order to decipher the many options available for stem cell therapy and interpret results of accumulating experimental and clinical data, practitioners should have a basic understanding of stem cells.     

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.     

Isolation of equine bone marrow‐derived mesenchymal stem cells: a comparison between three protocols

Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 ⁶ for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.     

Venographic evaluation of the circumflex vessels and lamellar circumflex junction in laminitic horses

Venograms provide information about areas of vascular compression or damage within the hoof. Key areas of the venogram to evaluate include the circumflex vessels, papillae and lamellar circumflex junction. This article describes and illustrates the appearance of the circumflex vessels, papillae and lamellar circumflex junction in sound and laminitic horses.     

The effect of hoof angle variations on dorsal lamellar load in the equine hoof

Reasons for performing study: In the treatment of laminitis it is believed that reducing tension in the deep digital flexor tendon by raising the palmar angle of the hoof can reduce the load on the dorsal lamellae, allowing them to heal or prevent further damage. Objective: To determine the effect of alterations in hoof angle on the load in the dorsal laminar junction. Methods: Biomechanical finite element models of equine hooves were created with palmar angles of the distal phalanx varying from 0–15°. Tissue material relations accounting for anisotropy and the effect of moisture were used. Loading conditions simulating the stages in the stance where the vertical ground reaction force, midstance joint moment and breakover joint moment were maximal, were applied to the models. The loads were adjusted to account for the reduction in joint moment caused by increasing the palmar angle. Models were compared using the stored elastic energy, an indication of load, which was sampled in the dorsal laminar junction. Results: For all loading cases, increasing the palmar angle increased the stored elastic energy in the dorsal laminar junction. The stored elastic energy near the proximal laminar junction border for a palmar angle of 15° was between 1.3 and 3.8 times that for a palmar angle of 0°. Stored elastic energy at the distal laminar junction border was small in all cases. For the breakover case, stored elastic energy at the proximal border also increased with increasing palmar angle. Conclusions and potential relevance: The models in this study predict that raising the palmar angle increases the load on the dorsal laminar junction. Therefore, hoof care interventions that raise the palmar angle in order to reduce the dorsal lamellae load may not achieve this outcome. See also correspondence by Redden See also correspondence by Curtis