Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane.

OBJECTIVES: This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies. PROCEDURES: A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression. RESULTS: Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins. CONCLUSIONS: This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

Preliminary evaluation of safety and migration of immune activated mesenchymal stromal cells administered by subconjunctival injection for equine recurrent uveitis.

Introduction: Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. Methods: Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 µg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. Results: No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 µg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. Discussion: Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

Retrobulbar embryonal tumor with multilayered rosettes in a golden retriever dog.

Although rare, embryonal tumors (previously called primitive neuroectodermal tumors) should be considered in the differential diagnosis of retrobulbar tumors in dogs regardless of the age of the patient, and ancillary tests are required for definitive diagnosis.

Evaluation of pigment epithelium-derived factor concentration in equine amniotic membrane homogenate and its in-vitro vascular endothelial growth factor inhibition effect in tears of dogs with vascularized ulcerative keratitis.

Background: Corneal neovascularization can result from many pathological processes affecting the ocular surface leading to disturbances and opacifications that reduce corneal clarity and may impact vision. In veterinary medicine, the use of topical corticosteroid is contraindicated in the presence of ulcerative keratitis, and there is sparse research regarding safe medical alternatives to inhibit corneal neovascularization in dogs to improve visual outcome. Aim: To investigate the pigment epithelium-derived factor (PEDF) concentration in equine amniotic membrane homogenate (EAMH) and its in-vitro vascular endothelial growth factor (VEGF) inhibition in tears of dogs with vascularized ulcerative keratitis. Methods: Homogenates from 10 equine amniotic membranes (AM) were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of equine PEDF and VEGF. Forty tear samples were collected from both eyes of dogs diagnosed with vascularized ulcerative keratitis, and 50 samples from healthy dogs. Samples from affected eyes were allocated to G1 - affected undiluted tears; G2 - affected tears diluted with phosphate-buffer solution; G3 - affected tears treated with low-concentrated EAMH; and G4 - affected tears treated with high-concentrated EAMH. Tears from the unaffected contralateral eyes were composed in G5, while G6 was composed by tears from healthy dogs (control). The presence and levels of VEGF were evaluated in all groups by Western blot and ELISA. Results: The PEDF:VEGF ratio in EAMH was 110:1. An increase in VEGF levels was observed in tears from eyes with vascularized corneal ulcers (G1) as well as in contralateral tears (G5), compared to normal dogs (G6). High-concentrated EAMH provided a greater decrease in VEGF levels in-vitro compared to low-concentrated EAMH. Conclusion: EAMHs exhibited high concentrations of PEDF in comparison to VEGF and were able to partially decrease VEGF levels in tears of dogs with vascularized ulcers, in-vitro. Our results suggest that VEGF concentration is elevated in tears of dogs with active vascularized ulcerative keratitis in both affected and contralateral eyes compared to that of healthy dogs.

Osmolarity of basal and reflex tears of normal dogs.

OBJECTIVE: To compare basal and reflex tear osmolarity in healthy dogs and to evaluate for correlation among tear film osmolarity, tear production, and tear fern pattern. ANIMALS STUDIED: The population consisted of 22 healthy adult dogs. PROCEDURES: Reflex tear osmolarity was measured in both eyes using the I-PEN®  VET osmometer 30 minutes following the Schirmer tear test (STT-1) measurement. Subsequently, two minutes following topical anesthetic application, the lacrimal lake and conjunctival fornices were dried, and 3 minutes later, basal tear osmolarity was measured. Tears were extracted from the dye-free STT-1 strip by centrifugation, placed on a glass slide, and the ferning pattern was determined by light microscopy. Comparisons between basal and reflex tears were performed with one-way ANOVA. Correlations between tear osmolarity, STT-1, and tear ferning were verified by Pearson's correlation coefficient. RESULTS: No statistical difference was found between right and left eyes for STT-1, tear fern pattern, and osmolarity of reflex or basal tears (P < .05). There was no statistical difference between osmolarity of basal and reflex tears among individuals (P < .05). No correlation was detected between tear fern pattern and osmolarity of reflex tears. A weak positive correlation was detected between STT-1 values and osmolarity of reflex, but not basal tears. CONCLUSIONS: The osmolarity of basal tears did not differ from that of reflex tears. A positive weak correlation exists between tear production and osmolarity of reflex tears. No correlation was detected between tear osmolarity measured by handheld osmometer and tear ferning.

Tear production, intraocular pressure, and central corneal thickness in white-tailed deer (Odocoileus virginianus).

OBJECTIVE: To evaluate the tear production, intraocular pressure, and central corneal thickness in healthy white-tailed deer (Odocoileus virginianus) and assess for seasonal variability. ANIMALS STUDIED: Nineteen healthy fawns (3-10 days old) and 8 healthy adult white-tailed deer (164-280 days old) were studied. PROCEDURES: All fawns were manually restrained for the study, while adult animals were immobilized with an intramuscular injection of combined xylazine (2.2 mg/kg), tiletamine (1 mg/kg), and zolazepam (1 mg/kg). Tear production (STT-1) was evaluated in both eyes of all fawns. Intraocular pressure (applanation and rebound tonometry) and central corneal thickness (ultrasound pachymetry) were determined in both eyes of all fawns and adult deer. RESULTS: Mean ± SD tear production in fawns was 12.32 ± 4.46 mm/min. Mean ± SD IOP values in the fawns were 16.21 ± 4.97 mm Hg (TonoPen® ) and 14.05 ± 5.03 mm Hg (TonoVet® ). In adult deer, mean ± SD IOP values were 15.57 ± 2.88 mm Hg with TonoPen® and 12.87 ± 2.57 with TonoVet® , and TonoPen® significantly overestimated the IOP, compared to TonoVet® (P < 0.005). The IOP was not significantly different between young and adult animals (P = 0.22). A significant difference (P < 0.005) in CCT values was observed between fawns (528.42 ± 27.65 µm) and adult deer (747.55 ± 41.60 µm). CONCLUSIONS: This study reports normal values of tear production in healthy fawns, and intraocular pressure and central corneal thickness in healthy young and adult white-tailed deer.

Ophthalmic abnormalities secondary to periocular or ocular snakebite (pit vipers) in dogs--11 cases (2012-2014).

OBJECTIVE: To describe ophthalmic abnormalities secondary to periocular and ocular snakebite in dogs. ANIMAL STUDIED: Retrospective review of medical records from dogs presenting to the Small Animal Hospital at University of Florida following snakebites to the face (2012-2014). Two groups were identified: periocular bites (PB) and ocular bites (OB). RESULTS: Records from eleven dogs matched the search criteria and were included in the study (PB=9, 81.8%; OB=2, 18.2%). Both OB cases involved the cornea. Facial edema, blepharospasm, chemosis, and conjunctival hyperemia occurred in all cases (100%). Hemorrhage from the eyelids occurred in eight cases (72.7%; PB=7, OB=1). Subconjunctival hemorrhage occurred in seven cases (63.6%; PB=6, OB=1). Third eyelid laceration and nictitans gland prolapse occurred in 1 case each (9%; PB=1). Lagophthalmia was present in three cases (27.3%; PB=3), with secondary corneal ulcer in two cases (18.2%; PB=2). Corneal ulcer due to direct corneal bite occurred in two cases (18.2%-partial thickness with melting (1) and full thickness (1) ). Uveitis was present in 6 cases (54.5%; PB=4, OB=2), with flare and miosis in 4 cases (36.4%; PB=2, OB=2). Hyphema, fibrin in anterior chamber, and cataract occurred in one case (9%; OB=1). Vision loss occurred in two cases (18.2%; PB=2), secondary to retinal degeneration (PB=1) and amaurosis (PB=1). Mean follow-up time was 7 weeks (range: 3 days-11 months). Most clinical signs had resolved by last examination. CONCLUSIONS: Periocular symptoms were more commonly observed than ocular alterations, regardless of bite location. Appropriate supportive therapy should be instituted according to clinical signs.

Poly(N-Isopropylacrylamide)-co-Acrylamide Hydrogels for the Controlled Release of Bromelain from Agroindustrial Residues of Ananas comosus.

This works reports the purification of bromelain extracted from Ananas comosus industrial residues by ethanol purification, its partial characterization from the crude extract as well as the ethanol purified enzyme, and its application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels. Bromelain was recovered within the 30-70 % ethanol fraction, which achieved a purification factor of 3.12-fold, and yielded more than 90 % of its initial activity. The resulting purified bromelain contained more than 360 U · mg(-1), with a maximum working temperature of 60 °C and pH of 8.0. Poly(N-isopropylacrylamide)-co-acrylamide hydrogels presented a swelling rate of 125 %, which was capable of loading 56 % of bromelain from the solution, and was able to release up to 91 % of the retained bromelain. Ethanol precipitation is suitable for bromelain recovery and application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels based on its processing time and the applied ethanol prices.

Effects of antiproteolytic agents on corneal epithelial viability and matrix metalloproteinase-2 and metalloproteinase-9 activity in alkali-burned corneas of rats.

OBJECTIVE: To evaluate the effects of agents on corneal re-epithelization and metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) activities in corneas of rats submitted to ulceration. ANIMALS STUDIED: Ninety eight healthy rats. PROCEDURES: Corneal ulcers were created using 1N NaOH in their left eye. Eyes were treated every 6 h with 1% ethylenediaminetetraacetic acid (EDTA), 3% chondroitin sulfate (CS), 10% N-acetylcysteine NAc and saline (S) at 6-h intervals. Corneas were stained with fluorescein and photographed at the same time points. Following 20 h and 40-42 h of corneal injury, corneas were processed for scanning electron microscopy (SEM) to quantify microvilli density, and MMPs activities were analyzed using zymography. RESULTS: The percentage of wound area and the time in hours for corneal re-epithelization did not differ significantly among treatment groups (P > 0.05). In first and the second moments, latent MMP-2 was significantly elevated in the eyes treated with NAC and CS (P < 0.001). Active MMP-2 did not change significantly among treatment groups in the first moment (P > 0.05); significantly higher activity was observed in the second moment in the eyes treated with CS (P <0.001). In the second moment, latent MMP-9 decreased significantly in eyes treated with EDTA and S (P < 0.01). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05). CONCLUSION: Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.

Efectos cardiorespiratorios y requerimientos de isofluorano en conejos sometidos a cirugía oftalmológica tratados con tramadol pre, intra y postoperatorio / Cardiorespiratory effects and isoflurane requirements in rabbits subjected to ophthalmologic surgery treated with tramadol pre-, intra- or postoperatively

Para evaluar los efectos cardiorespiratorios y los requerimientos de isofluorano en conejos sometidos a cirugía oftálmica tratados en diferentes momentos con tramadol, fue realizado un estudio prospectivo experimental de tipo ciego en 24 conejos distribuidos aleatoriamente en tres grupos de ocho individuos cada uno: grupo tramadol preoperatorio (Tpre), grupo tramadol transoperatorio (Ttrans) y grupo tramadol postoperatorio (Tpos). Cada animal recibió tres inyecciones intramusculares de igual volumen en los periodos pre, intra y postoperatorio, conteniendo tramadol (4mg/kg) o NaCl 0,9%, de acuerdo al momento indicado para cada grupo. La inducción y mantenimiento anestésico fueron realizados con isofluorano en FiO2 1,0. Una vez estabilizado el plano anestésico, fue iniciado el procedimiento quirúrgico. Se evaluaron: frecuencia cardíaca (FC), presión arterial media (PAM), frecuencia respiratoria (f), presión de CO2 al final de la expiración (EtCO2), saturación parcial de oxihemoglobina (SpO2) y concentración de isofluorano al final de la expiración (EtISO). Las variables fueron medidas después de la estabilización anestésica (m1) y cada 10 min desde el inicio de la cirugía, hasta el final del experimento (m10 a m40). La FC, SpO2, f , EtCO2 y EtISO fueron similares entre grupos durante todo el tiempo. La PAM fue significativamente mayor (p<0,01) durante m1 comparada con el resto del tiempo y el grupo Tpre mostró valores inferiores (p=0,04) de este parámetro durante m1 con relación a los demás grupos. El clorhidrato de tramadol promueve la reducción transitoria de la presión arterial en conejos anestesiados con isofluorano y sometidos a cirugía oftálmica. La administración preoperatoria del fármaco no disminuye el requerimiento anestésico y resulta en hipotensión más pronunciada. Adicionalmente, la anestesia con isofluorano, asociada al reflejo óculo-cardiaco, generan hipotensión arterial, fenómeno que debe tenerse en cuenta durante cirugías oftalmológicas.

To evaluate the cardiorespiratory effects and isoflurane requirements of tramadol applied at different times during ophthalmic surgery, a blind prospective study was made in 24 rabbits, randomly divided into three groups of eight subjects each: pre-operatory tramadol group (Tpre), trans-operatory tramadol group (Ttrans) and post-operatory tramadol group (Tpos), which received three intramuscular injections of equal volume, in the pre, intra and post-operative period, containing tramadol hydrochloride (4 mg/kg) or NaCl 0,9%, according to each group. Anesthetic induction and maintenance was made with isoflurane diluted FiO2 1.0. Once the anesthetic plane was stabilized, surgical procedure was started. Were evaluated heart rate (HR), medium arterial presseure (MAP), respiratory rate (f), end tidal CO2 (EtCO2), Oxihemoglobin saturation (SpO2) and end tidal isoflurane (EtISO). Variables were measured after anesthesia stabilization (m1) and every 10 min after onset of surgery, until the end of experimental protocol (m10 to m40). HR, SpO2, f, EtCO2 and EtISO were similar among groups at all times. MAP were significantly higher (p<0.01) in m1 than in other moments and Tpre group showed lower (p=0.04) values than the other groups at this moment. Tramadol hydrochloride promotes transient reduction of blood pressure in isoflurane anesthetized rabbits subjected to ophthalmic surgery. The preoperative administration of the drug does not reduce the anesthetic requirement and results in a more pronounced hypotension. Additionally, isoflurane anesthesia, associated to oculocardiac reflex generates a hypotension phenomenon that must be taken in to considertation during ophthalmologic surgeries.

Light microscopic evaluation and scanning electron microscopic analysis of horse eyes following deep anterior lamellar keratectomy.

OBJECTIVE To describe the technique of deep anterior lamellar keratoplasty (DALK) with Descemet's membrane (DM) exposure in horse eyes. Also, to compare the efficacy and safety of viscodissection and big-bubble techniques for DALK. ANIMALS STUDIED Thirty-four ex vivo horse eyes. PROCEDURE Deep anterior lamellar keratoplasty was performed in 34 ex vivo horse eyes. Two groups (Group V--viscodissection--2% sodium hyaluronate; Group A--air--big-bubble) of 17 eyes were studied. Other than the substance used, the surgical technique was similar for both groups. Nonperforated eyes were submitted for light microscopic histologic evaluation and scanning electron microscopic (SEM) analysis. RESULTS Group V--Perforations occurred in 18% of the eyes during surgery. Light microscopy revealed exposure of DM in 28% of the eyes with mean thickness of the remaining stroma being 70.4 µm. Group A--Perforations occurred in 42% of the eyes. Light microscopy revealed exposure of DM in 60% of the eyes with mean thickness of the remaining stroma being 23.3 µm. No significant differences in safety, efficacy and thickness of the remaining stroma (including all eyes or excluding those with DM exposure) were observed. SEM of the surgical site revealed a more even surface in those eyes with DM exposure compared to eyes with thicker remaining stroma in both groups. CONCLUSIONs We describe two DALK techniques (viscodissection and big-bubble) for use in horses. No significant differences in safety, efficacy and thickness of the remaining stroma were observed. However, a nonsignificant trend toward the big-bubble technique being more efficacious but less safe was observed.

Ultrassonografia em modo A e B na avaliação pré-operatória da lente e do segmento posterior de olhos de cães com catarata / A and B mode ultrasonography in preoperative evaluation of lens and posterior segment of dogs eyes with cataract

Ultrasonography of the lens and posterior segment is an indispensable step in the preoperative evaluation of dogs with cataracts, since ophthalmoscopy is not feasible when there is opacification of the lens. This study evaluated the echographic conditions of cataractous lens and fundus of the eye in dogs affected by cataracts. The study was conducted in 30 dogs (56 eyes), 10 males and 20 females, with different types of cataracts at different stages of development. Echography in A and B modes, simultaneously, was carried out for the examination of the lens and posterior segment. The examinations revealed anterior cortical, posterior cortical and nuclear cataract in 12 eyes (21.4 percent), anterior cortical, posterior cortical, nuclear and posterior capsular in 23 eyes (41 percent), anterior cortical, posterior cortical and posterior capsular cataract in one eye (1.7 percent), anterior cortical and nuclear cataract in one eye (1.7 percent), anterior cortical, nuclear and posterior capsular cataract in five eyes (8.9 percent), and anterior cortical cataract in seven eyes (12.5 percent). Abnormal ultrasonographic alterations were observed in the posterior segment in 26 eyes evaluated (46.4 percent). Vitreal degeneration was detected in 12 eyes (21.4 percent), images of vitreal exudate or hemorrhage in seven eyes (12.5 percent), persistence of hyaloid artery in four eyes (7.1 percent) and lens subluxation in three eyes (5.3 percent). The results obtained reiterate the importance of ultrasonography in canine patients presented for cataract surgery given that alterations of the posterior segment are difficult to identify in a clinical examination when the lens is opacified.

A ultrassonografia do segmento posterior do bulbo do olho é etapa indispensável na avaliação de cães com catarata que serão submetidos à facectomia, uma vez que a oftalmoscopia não é factível quando há opacificação da lente, notadamente nas cataratas maduras. Este estudo avaliou as condições ecográficas da lente cataratogênica e do fundo de olho de cães cursando com catarata. Foram avaliados 30 cães (56 olhos), sendo 10 machos e 20 fêmeas, cursando com catarata em diferentes estágios de desenvolvimento. Realizou-se a ecografia em modo A e B simultaneamente. Os exames revelaram catarata cortical anterior, posterior e nuclear em 12 olhos (21,4 por cento), catarata cortical anterior, posterior, nuclear e capsular posterior em 23 olhos (41 por cento), catarata cortical anterior, posterior e capsular posterior em um olho (1,7 por cento), catarata cortical anterior e nuclear em um olho (1,7 por cento), catarata cortical anterior, nuclear e capsular posterior em cinco olhos (8,9 por cento) e catarata cortical anterior em sete olhos (12,5 por cento). Alterações no segmento posterior foram observadas, à ultrassonografia, em 26 olhos avaliados (46,4 por cento). Degeneração vítrea foi detectada em 12 olhos (21,4 por cento), imagens compatíveis com exsudato ou hemorragia em sete olhos (12,5 por cento), persistência da artéria hialóide em quatro olhos (7,1 por cento) e subluxação da lente em três olhos (5,3 por cento). Os resultados obtidos reiteram a importância da ultrassonografia em cães candidatos à cirurgia de catarata, uma vez que o exame do segmento posterior do bulbo do olho é de difícil execução, devido à opacificação da lente.

Ophthalmic patterns of captive brown brocket deer (Mazama gouazoubira).

Captive brown brocket deer (Mazama gouazoubira) were manually restrained to assess tear production by the Schirmer tear test I to measure intraocular pressure by applanation tonometry, to examine ocular conjunctival epithelial cells via cytologic and histologic samples, and to survey ocular conjunctival microflora by microbiologic culture. The mean value for the Schirmer tear test I was 8.9 +/- 1.8 mm/min, and the mean intraocular pressure was 15.3 +/- 3.1 mm Hg. Conjunctival epithelium contained stratified pavimentous layers of cells, and the microflora consisted of predominantly gram-positive bacteria.