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

Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia (“cryotherapy”), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10\ua0g/kg, n\ua0=\ua014 horses). Lamellae were harvested at 24\ua0h post-oligofructose administration (DEV, n\ua0=\ua07) or at the onset of OG1 laminitis (OG1, n\ua0=\ua07). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n\ua0=\ua010 (40× fields/digit for MAC387 and 20\ua0x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p\ua