Immunological and pathological investigations in equine experimental uveitis

Background

The pathogenic mechanism of equine recurrent uveitis (ERU) is still poorly defined and many variations between experimental animal models and spontaneous disease exist.

Objectives

The aim of our study was to investigate if Th17 cell-mediated response plays role in the pathogenesis of the used experimental model in horses and to reveal its pathological findings.

Methods

Experimental uveitis was induced in 6 healthy horses. The concentrations of retinal autoantigen CRALBP and IL-17 were measured using ELISA in aqueous humor and vitreous body of the 12 inflamed eyes as well as in 12 control non-inflamed eyes taken from 6 horses in slaughter house. After centrifugation of the two eye media, smears were prepared and cytological investigation was performed. Tissue specimens were taken from all eye globes and were submitted to histopathological investigation.

Results

CRALBP and IL-17 concentrations were significantly elevated in eye media of horses with experimental uveitis in comparison with controls. Cytological and histopathological findings corresponded to the changes characteristic of chronic immune-mediated inflammation with mononuclear cell infiltration of uvea, choroid, retina, and eye media as well as severe retinal destruction.

Conclusions

Our study demonstrated the involvement of the retinal autoantigen CRALBP as well as IL-17 in the pathogenesis of experimental uveitis in horses. These findings suggests that this experimental uveitis in horses may serve as a suitable animal model for investigation of IL-17- mediated immune response during spontaneous autoimmune uveitis in horses as well as in humans.

     

Marked hyperphosphatasemia associated with an acute leukemia in a Great Dane

This is the report of a 5‐year‐old male neutered Great Dane with an extreme leukocytosis (544.9 × 10⁹ cells/L; RI 5.2–13.9 × 10⁹ cells/L) characterized by highly atypical round cells. Cellular morphologic features such as cytoplasmic membrane blebs, a high nuclear‐to‐cytoplasmic ratio, and nuclear indentations and irregularities and large nucleoli, as well as immunocytochemistry for CD3 and CD79, myeloperoxidase cytochemistry, and clonality testing were not conclusive for myeloid or lymphoid origin. Marked alkaline hyperphosphatasemia was present at the first visit (2783.0 U/L; RI 6–80.0 U/L), followed by a 5‐fold increase (14,000 U/L) a week later, identified as being mostly contributed by the bone‐ALP isoform (11,062 U/L; RI 0–30 U/L). In addition, the atypical leukocytes were strongly positive for cytoplasmic ALP activity. In vitro lysis of a heparin blood sample resulted in a 1.7‐fold increase of ALP activity, supporting the origin of the hyperphosphatasemia at least in part from the leukemic cell population. To the authors’ knowledge, this is a unique case of alkaline hyperphosphatasemia, due at least to a leukemic cell population producing a bone‐ALP isoform, regardless of the exact nature of the leukemia.     

Ultrastructural Characterization of Fresh and Vitrified In Vitro‐ and In Vivo‐Produced Sheep Embryos

The lower results in cryopreservation of in vitro‐produced (IVP) sheep embryos, when compared to the in vivo derived, limits its use. Four groups of blastocyst (BL) were evaluated: fresh IVP (n = 3), fresh in vivo derived (n = 3), warmed IVP cryopreserved in open pulled straws (OPS, n = 3) and warmed in vivo derived cryopreserved in OPS (n = 3). Ultrastructural observation of processed fresh embryos showed a reduced number of microvilli and mitochondria in the IVP ones, as well as a lower number of mature mitochondria, that can be associated with deficient metabolism in IVP embryos, possibly involved in the lower resistance to cryopreservation. Both in vivo‐derived and IVP embryos had a large number of vesicles, with light and dense content. In embryos vitrified by OPS, major changes were observed mainly in IVP embryos with small changes in grade 2 (fair) and high changes in grade 3 (bad) semithin scoring. The main changes associated with cryopreservation included disruption of cellular membranes and poor intracellular preservation, with loss of microvilli and the presence of cellular debris. In conclusion, ultrastructural evaluation of IVP blastocysts cryopreserved in OPS was herein described for the first time, reporting more severe cellular damage in these embryos when compared to those produced in vivo. This is probably associated with a lower cryotolerance that can be related to their lipid content and metabolism.     

Effect of dietary Schizochytrium microalga oil on selected markers of low‐grade inflammation in rats

The objective of the present study was to evaluate a potential of Schizochytrium microalga oil to alleviate possible negative effects of high‐fat‐high‐energy diets. Forty adult male rats (Wistar Albino) were fed 7 weeks the diet containing beef tallow + evaporated sweetened milk (diet T) intended to cause mild obesity and low‐grade systemic inflammation. Consequently, the animals were divided into four groups by 10 animals each and fed either the T‐diet (control) or the diet containing 6% of safflower oil (S), 6% of fish oil (F) and 6% of Schizochytrium microalga oil (A), respectively, for another 7 weeks. The A‐diet decreased (p < 0.05) live weight to 86% and glycaemia to 85% of control, respectively; an effect of the S‐ and F‐diet on these markers was insignificant (p > 0.05). In comparison with control, higher (p < 0.05) deposition of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in the epididymal adipose tissue (EAT) of the A‐rats correlated with increased (p < 0.05) plasma adiponectin concentration, but it was without the effect (p > 0.05) on cellular adiponectin content in the EAT. Higher (p < 0.05) EPA+DHA deposition in the liver of the A‐rats correlated with higher expression (149% of control; p < 0.05) of the gene coding for peroxisome proliferator‐activated receptor gamma, and with lower expression (82% and 66%; p < 0.05) of the genes coding for adiponectin receptors AdipoR1 and AdipoR2; no relationship to the expression of receptor GPR120 was found. The A‐diet did not affect amount of the nuclear fraction of the nuclear factor kappa B in the liver, but increased plasma level of anti‐inflammatory cytokine TGF‐β1 (p < 0.05). The presented data agree with results of other in vivo rodent and human studies, but not with literature data regarding in vitro experiments: it can be concluded that the effects of dietary oils on inflammatory markers need further investigation.     

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.