Calprotectin in Myeloid and Epithelial Cells of Laminae from Horses with Black Walnut Extract-Induced Laminitis

Background: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin.
Hypothesis: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis.
Animals: Twenty adult horses.
Methods: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed.
Results: Laminar epidermal CP signal was significantly increased ( P = .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups ( P = .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group.
Conclusions and Clinical Importance: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.     

Immunohistochemistry Diagnosis of an Ovarian Dysgerminoma in One Bitch

An ovarian enlargement (diameter, 8 cm) was identified and surgically excised from a 5-year-old female dog. Microscopic examination of the multinodular neoplasm revealed sheets of polygonal neoplastic cells with large nuclei, frequent mitosis, necrosis and haemorrhage. Immunohistochemically, the neoplastic cells were positive for vimentin and alkaline phosphatase but were negative for CD3, CD79a, cytokeratin, alpha-fetoprotein, inhibin-α and S-100. The histopathological diagnosis of the mass was unilateral ovarian dysgerminoma.     

Cryotherapy provides transient analgesia in an induced lameness model in horses

The analgesic effect of cryotherapy in an induced lameness model was evaluated. Lameness was induced with solar pressure from a custom-made shoe in a 10-horse, cross-over study. The degree of lameness was recorded with a commercial non-invasive inertial sensor. The distal limbs were maintained in an ice and water slurry (cryotherapy) or at ambient temperature (control) for 1 hour. Lameness was assessed serially over the following hour. Lameness at each time point was compared to the baseline induced lameness, within and between groups. Lameness had improved significantly in all horses 5 minutes after treatment but remained improved 10 minutes after treatment for the cryotherapy group only. Fifteen minutes after treatment, lameness in the cryotherapy group was improved relative to the control. Cryotherapy produced moderate, transient analgesia. Additional research is required to determine if altering the method, duration, or temperature of cryotherapy, as well as the targeted pathology and anatomy, alters the analgesic effect.     

Expression and Activity of Collagenases in the Digital Laminae of Horses with Carbohydrate Overload‐Induced Acute Laminitis

Background

Matrix metalloproteinases (MMP) are hypothesized to degrade structurally important components of the laminar extracellular matrix (ECM) in horses with laminitis.

Objective

To compare levels of expression of stromelysin‐1 (MMP‐3), collagenases (MMP‐1, ‐13), and membrane type‐MMPs (MMP‐14, ‐15, ‐16), and the distribution of their ECM substrates, in laminae of healthy horses and horses with carbohydrate overload laminitis.

Animals

Twenty‐five adult horses.

Methods

Gene and protein expression were determined in extracts of laminae using real‐time quantitative polymerase chain reaction and Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis. Distribution of MMP‐13 and ECM components was determined using indirect immunofluorescent microscopy of nonfixed frozen sections. ECM morphology was assessed by hematoxylin and eosin staining.

Results

Of the genes studied, only those encoding MMP‐1 and ‐13 were upregulated in CHO‐induced laminitis; MMP‐1 at Obel grade (OG)1 lameness and MMP‐13 at OG3 lameness. Laminar MMP‐1 was present as 52 kDa proenzyme only. MMP‐13 was present as pro‐ (61 kDa) and processed (48 kDa) enzyme. MMP‐13 localized to the basal epithelium of the secondary epidermal laminae and its increased expression were accompanied by the appearance in secondary dermal laminae (SDL) of multiple foci that were devoid of collagen I, fibronectin, chondroitin and keratan sulfate glycosaminoglycans, and eosin‐staining material.

Conclusions and Clinical Relevance

MMP‐13 is upregulated in laminae of horses with CHO‐induced OG3 lameness and, by degrading components of the ECM, may contribute to the formation of ECM‐free lesions (gaps or tears) that appear in the SDL with OG3 lameness.     

Hormonal Response of Female Goats to Active Immunization against a Recombinant Human Inhibin α-subunit, and establishment of an Enzyme-linked Immunosorbent Assay for Caprine Follicle-stimulating Hormone

The effect of selective immunosuppression of endogenous inhibin in goats on FSH, LH, progesterone and estradiol-17β profiles was studied during the breeding and nonbreeding seasons. Eighteen adult female Boer goats were immunized against the recombinant human inhibin α-subunit (hINH-α). With the exception of estradiol, which was determined by radio-immunoassay (RIA), all plasma hormone concentrations were determined by ELISA. The ELISA for FSH presented in this paper was established in the authors' laboratory, based on an existing RIA. Mean basal concentrations of FSH were not affected by immunosuppression of endogenous inhibin, nor was there a difference in the amplitude of the pre-ovulatory FSH surge. Immunization against inhibin appears to eliminate the slight secondary rise of FSH occurring 12–20 h after the major surge associated with ovulation. The LH profiles of the immunized goats were characterized by lower basal concentrations both before and after the pre-ovulatory LH surge which itself was reduced by 50% in immunized does. By contrast, concentrations of circulating estradiol were significantly elevated after inhibin-immunization. Progesterone profiles were not affected. Extending immunization into the anoestrous season by a booster injection of hINH-α, implicating oestrus induction with a progestagen and eCG, produced no discernible differences in FSH and LH profiles in comparison with nonimmunized control goats. The findings suggest that in goats, paracrine factors may play a more significant role in controlling follicular activity than a feedback mechanism acting via the pituitary.     

Malignant neoplasia in four alpacas.

Malignant neoplasia in 4 alpacas was characterized by acute onset of clinical signs and rapidly deteriorating condition. Postmortem examination revealed metastatic or multicentric neoplasia in the abdominal organs of alpacas 1, 3, and 4 and an extensive thoracic mass in alpaca 2. Immunohistochemical stains supported a diagnosis of B-cell lymphosarcoma in alpacas 1-3 and a neuroendocrine neoplasm in alpaca 4.