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

Abstract

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 (10g/kg, n=14 horses). Lamellae were harvested at 24h post-oligofructose administration (DEV, n=7) or at the onset of OG1 laminitis (OG1, n=7). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n=10 (40× fields/digit for MAC387 and 20x 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<0.05. MAC387-positive cells were present in low numbers in the lamellar tissue and were decreased in the hypothermic limbs (vs. AMB limbs, p<0.05) in the OG1 group; no change in CD163-positive cell numbers was noted across the conditions of the model. This study demonstrated that hypothermia of the distal limbs instituted early in the disease process in the horse at risk of SRL significantly attenuates the increase of MAC387-positive leukocytes in the digital lamellae, but has minimal effect on increases in lamellar concentrations of the major leukocyte cell type present in that tissue, CD163-positive mononuclear cells.

Effect of Continuous Digital Hypothermia on Lamellar Inflammatory Signaling When Applied at a Clinically‐Relevant Timepoint in the Oligofructose Laminitis Model

“…OG4 time point] versus mean: 3.1 cells/40× field in untreated/ambient lamellae in the previous study [approx. OG1 time point]) …”

“…However, a more recent study in which CDH was initiated at the onset of clinical lameness (OG2) did not find the same degree of inhibition of lamellar inflammation with CDH despite it still conferring a dramatic protective effect upon the lamellae . Similarly, a recent study investigating the effects of CDH (instituted immediately after administration of enteral OF) on leukocyte infiltration in the OF SRL model reported only minor changes in lamellar leukocyte numbers, indicating that leukocyte infiltration may have less of a role in lamellar tissue damage than previously thought …”

Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light‐breed horses

“…Leukocyte emigration into digital lamellar tissue was assessed with MAC387/calprotectin and CD163 immunohistochemical staining using the avidin‐biotin complex (ABC) method as previously described . Briefly, 5‐μm tissue sections were prepared from formalin‐fixed, paraffin‐embedded lamellar tissue and affixed to slides.…”

Cellulitis: Any change?

“…Indeed, in terms of the care of cellulitis cases, in the light of cryotherapy research in laminitis studies (Godman et al . ; Dern et al . ), cryotherapy becomes a logical choice for the possibility of reducing inflammation and enzymatic activity, which may reduce bacterial protein synthesis.…”

“…Superantigens nonspecifically activate T lymphocytes and cause widespread pro-inflammatory cytokine release, often leading to toxic shock syndrome and necrotising lesions. Indeed, in terms of the care of cellulitis cases, in the light of cryotherapy research in laminitis studies (Godman et al 2016;Dern et al 2018), cryotherapy becomes a logical choice for the possibility of reducing inflammation and The elaboration of highly pathogenic exotoxins from both Staphylococcal and Streptococcal organisms is well entrenched in our collective veterinary psyche. However, the sole reliance on antimicrobial drugs to combat such infections may also be well entrenched.…”