Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture.

The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with ßIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

OBJECTIVES: We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. RESULTS: Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. CONCLUSIONS: Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

Solitary (primary) uveal T-cell lymphoma in a horse.

A 22-year-old Australian stockhorse gelding was presented with anterior uveitis in the right eye which was nonresponsive to anti-inflammatory therapy. Clinical examination revealed corneal edema and vascularization, marked hypopyon, and thickening of the dorsal iris, which was confirmed by ultrasonography. Hematologic and biochemical analyses, abdominal and thoracic ultrasonography, and abdominocentesis with cytologic and biochemical analysis revealed no significant abnormalities. Cytological examination of an aqueous humor sample revealed a population of predominantly large lymphoblasts with high nuclear-to-cytoplasmic ratio, round or irregular nuclei, clumped nuclear chromatin, multiple large prominent nucleoli, and a small volume of basophilic cytoplasm. The cytologic diagnosis was intraocular lymphoma. Biopsy of the right submandibular lymph node revealed no evidence of neoplastic invasion. Euthanasia and a complete necropsy were performed and revealed no evidence of neoplasia in any tissue other than the right eye, which had an extensive, well-defined infiltrate of neoplastic lymphocytes expanding the ciliary body and iris, infiltrating the ciliary epithelium, and extending into the pars plana and peripheral choroid. Immunohistochemistry confirmed that neoplastic cells expressed the T-cell marker CD3. To the authors' knowledge, this is the first description of primary, solitary uveal T-cell lymphoma in a horse. Although apparently rare, lymphoma should be considered in horses with uveitis, even when inflammation is unilateral and in the absence of extraocular signs of neoplasia. Aqueocentesis and cytological examination provided an antemortem diagnosis in this case and should be considered as a diagnostic tool for investigation of uveal thickening and hypopyon.

Distensible superficial venous orbital malformations involving the lower eyelid in two horses.

CASE DESCRIPTION: 2 horses were examined because of vascular masses involving the lower eyelid. CLINICAL FINDINGS: Both horses had a unilateral, fluctuant mass involving the lower eyelid. For horse 1, the mass had been present since birth and had slowly increased in size over time. The mass also changed in size in response to various environmental stimuli, alterations in the position of the horse's head, and digital obstruction of superficial vessels adjacent to the mass. Horse 2 was brought to the hospital for euthanasia, and no historical or antemor-tem data were available. A combination of contrast angiography, Doppler ultrasonography, surgical exploration, and blood gas analysis (horse 1) and postmortem and histologic examination (horse 2) were used to determine that the masses consisted of non-neoplastic distended venous channels with anastomoses to the inferior lateral palpebral and angularis oculi veins (both horses) as well as the facial vein (horse 2). Histologic examination (horse 2) revealed large, endothelial cell-lined, blood-filled spaces within the deep dermis consistent with a distensible superficial venous orbital malformation. TREATMENT AND OUTCOME: Horse 1 underwent surgical exploration and ligation of the vascular malformation. Six months after surgery, the mass was markedly reduced in size, and size of the mass was static regardless of head position or environmental stimuli. CLINICAL RELEVANCE: Thorough preoperative planning with Doppler ultrasonography, contrast angiography, and blood gas analysis is recommended when attempting surgical correction of these malformations in horses. Surgical ligation can result in a successful cosmetic and functional outcome.

Third metacarpal condylar fatigue fractures in equine athletes occur within previously modelled subchondral bone.

Bone modelling and remodelling reduce the risk of fatigue fractures; the former by adapting bone to its loading circumstances, the latter by replacing fatigued bone. Remodelling transiently increases porosity because of the normal delay in onset of the formation phase of the remodelling sequence. Protracted intense loading suppresses remodelling leaving modelling as the only means of maintaining bone strength. We therefore hypothesized that race horses with fatigue fractures of the distal third metacarpal bone (MC3) will have reduced porosity associated with suppressed remodelling while continued adaptive modelling will result in higher volume fraction (BV/TV) at this site. Using high resolution peripheral quantitative computed tomography (HR-pQCT), we measured the distal aspect of the MC3 obtained at postmortem from 13 thoroughbred race horses with condylar fractures of the MC3 (cases), 8 horses without fractures (training controls), 14 horses with a fracture at another site (fractured controls) and 9 horses resting from training (resting controls). Porosity of the subchondral bone of MC3 was lower in cases than resting controls (12±1.4% vs. 18±1.6%, P=0.017) although areas of focal porosity were observed adjacent to fractures in 6/13 horses. BV/TV of the distal metacarpal epiphysis tended to be higher in horses with condylar fractures (0.79±0.015) than training controls (0.74±0.019, P=0.070), but also higher in controls with a fracture elsewhere (0.79±0.014) than the training controls (0.74±0.019, P=0.040). BV/TV was higher in horses over three years of age than those aged two or three years (0.79±0.01 vs. 0.74±0.01, P=0.016). All metacarpal condylar fractures occurred within focal areas of high BV/TV. We infer that intense training in equine athletes suppresses remodelling of third metacarpal subchondral bone limiting damage repair while modelling increases regional bone volume in an attempt to minimise local stresses but may fail to offset bone fragility.