Adjunctive bevacizumab therapy in an equine corneal stromal invasive squamous cell carcinoma with a 53-months follow-up.

A 17-year-old Appaloosa mare was referred for evaluation of presumed refractory keratitis of the left eye. Gross examination revealed ocular discomfort and corneal neovascularization with a nasal focal opacification affecting approximately 40% of the corneal surface. On ophthalmic examination, extensive subepithelial to mid-stromal vascular branching accompanied by a homogeneous white, dense opacification, which affected up to 80% of the total corneal thickness, were apparent. Signs of concurrent uveitis were absent. Deep-stromal lamellar keratectomy with a conjunctival pedicle graft was performed under general anesthesia. Histopathology confirmed a poorly differentiated corneal stromal invasive squamous cell carcinoma (SI-SCC) with neoplastic cell extension to the surgical margins. Postoperatively, 4 topical mitomycin C 0.04% chemotherapy cycles combined with oral firocoxib therapy were initiated. Seven months after surgery, regrowth of the SI-SCC was clinically suspected. A total volume of 1 ml bevacizumab 2.5% was administered in the standing sedated horse via 3 mid-stromal corneal injections. Four weeks later, intrastromal bevacizumab injections (ISBIs) were repeated, however, this time the solution was injected directly into the main corneal vessel branches.Seven weeks after the second ISBIs, the left eye was comfortable and significant remission of corneal vascularization and opacity was recognized. No recurrence has been noted for a follow-up period of more than 53 months.Equine SI-SCC usually has a very poor prognosis for globe maintenance. To the authors' knowledge this is the first report of well-tolerated intrastromal antivascular endothelial growth factor adjunctive therapy with bevazicumab 2.5% and SI-SCC resolution after a multimodal treatment approach.

Bevacizumab Efficiently Inhibits VEGF-Associated Cellular Processes in Equine Umbilical Vein Endothelial Cells: An In Vitro Characterization.

Anti-VEGF agents were found to have clinical implications for the successful treatment of vascular-driven diseases in humans. In this study, a detailed biological characterization of bevacizumab in a variety of in vitro assays was carried out to determine the effect of bevacizumab on equine umbilical vein endothelial cells (EqUVEC). EqUVECs were harvested from umbilical cords of clinically healthy horses and exposed to different concentrations (1, 2, 4, 6, 8 mg/mL) of bevacizumab (Avastin®). Assays concerning the drug's safety (cell viability and proliferation assay) and efficacy (cell tube formation assay, cell migration assay, and Vascular endothelial growth factor (VEGF) expression) were carried out reflecting multiple cellular processes. Bevacizumab significantly decreased VEGF expression at all concentrations over a 72 h period. No cytotoxic effect of bevacizumab on EqUVECs was observed at concentrations of 4 mg/mL bevacizumab or lower. Incubated endothelial cells showed delayed tube formation and bevacizumab efficiently inhibited cell migration in a dose-dependent manner. Bevacizumab potently inhibits VEGF-induced cellular processes and could be a promising therapeutic approach in vascular-driven diseases in horses.

[Ophthalmic findings caused by Encephalitozoon cuniculi (strain III) in a dog]. / Ophthalmologische Befunde durch Enzephalitozoon cuniculi (Stamm III) bei einem Hund.

A 2-year-old female mixed-breed canine patient from Namibia presented originally with chronic uveitis. A serum antibody titer and a PCR test performed on the aqueous humor were positive for encephalitozoon cuniculi. The left eye showed an immature anterior focal cortical cataract in the periphery with suspected lens capsule rupture and signs of chronic uveitis. An incipient anterior focal cortical cataract was also perceivable in the patient's right eye. Despite local treatment as well as systemic administration of carprofen, prednisolone, and fenbendazol recurrent uveitis occurred. The patient then underwent bilateral extracapsular lensextraction via phacoemulsification. A PCR test of the lens material was positive for encephalitozoon cuniculi strain III. Recurring uveitis and secondary glaucoma 10 months post-op resulted in permanent blindness of the left eye. The patient then continued to receive local anti-inflammatory treatment. The last recheck examination of both eyes, 31 month post-op, revealed no signs of uveitis. This is the first case reported of a cataract in a canine patient caused by encephalitozoon cuniculi strain III.

Detection of Equine Papillomaviruses and Gamma-Herpesviruses in Equine Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) seriously compromises the health and welfare of affected horses. Although robust evidence points to equine papillomavirus type 2 (EcPV2) causing genital lesions, the etiopathogenesis of equine SCC is still poorly understood. We screened a series of SCCs from the head-and-neck (HN), (peri-)ocular and genital region, and site-matched controls for the presence of EcPV2-5 and herpesvirus DNA using type-specific EcPV PCR, and consensus nested herpesvirus PCR followed by sequencing. EcPV2 DNA was detected in 45.5% of HN lesions, 8.3% of (peri-)ocular SCCs, and 100% of genital tumors, whilst control samples from tumor-free horses except one tested EcPV-negative. Two HNSCCs harbored EcPV5, and an ocular lesion EcPV4 DNA. Herpesvirus DNA was detected in 63.6%, 66.6%, 47.2%, and 14.2% of horses with HN, ocular, penile, and vulvar SCCs, respectively, and mainly identified as equine herpesvirus 2 (EHV2), 5 (EHV5) or asinine herpesvirus 5 (AsHV5) DNA. In the tumor-free control group, 9.6% of oral secretions, 46.6% of ocular swabs, 47% of penile samples, and 14.2% of vaginal swabs scored positive for these herpesvirus types. This work further highlights the role of EcPV2 as an oncovirus and is the first to provide information on the prevalence of (gamma-)herpesviruses in equine SCCs.

Morphology and volume of Meibomian glands ex vivo pre and post partial tarsal plate excision, cryotherapy and laser therapy in the dog using microCT.

OBJECTIVE: To determine the morphology and volume of Meibomian glands (MG) of dogs with microCT before and after partial tarsal plate excision (PTPE), cryotherapy, and laser therapy. PROCEDURE: MicroCT scans were made of 12 upper lids (ULs) and lower lids (LLs) of 12 dogs. After undergoing PTPE, 10 ULs and LLs were scanned again, and one UL and one LL was scanned after laser therapy and one UL and one LL after cryotherapy. RESULTS: The length of the area containing MGs did not change pre- and post-PTPE, and cryo- or laser therapy. The mean number of MGs in the ULs and LLs was 30.50 and 29.42, respectively, and did not change during the procedures. The average length of one individual MG was 2.60 mm. The mean volume of MGs in the 10 ULs and LLs pre-PTPE was 21.45 and 17.2 mm3 , respectively, and 12.84 and 11.25 mm3 in the UL and LL after PTPE, respectively. The mean volume of MGs decreased from 29.78 mm3 precryotherapy to 28.91 mm3 post-treatment and in the lower eyelid from 22.87 to 22.4 mm3 after cryotherapy. The mean volume of MGs in the UL and LL before laser therapy was 8.95 and 6.78 mm3 , respectively, and after 9.25 and 6.38 mm3 , respectively. CONCLUSION: MicroCT is a valuable tool to determine the morphology and the volume of MGs and to demonstrate changes that occur after PTPE, laser-, and cryotherapy. There is no need for additional preparation, such as staining, of the specimen prior to scanning.

Feline intralenticular Encephalitozoon cuniculi: three cases from California.

Case series summary: Three domestic shorthair cats from California presented to veterinary ophthalmologists with immature cataracts. Other presenting clinical signs included corneal edema, anisocoria, anterior uveitis, elevated intraocular pressure, blepharospasm and/or lethargy. All patients were immunocompromised due to concurrent diseases and/or immunomodulatory drugs. Diagnostics included serial comprehensive ophthalmic examinations with tonometry, ocular ultrasound, electroretinogram and testing for other causes of feline uveitis. Testing for Encephalitozoon cuniculi included serology, histopathology and/or PCR of aqueous humor, lens material or paraffin-embedded whole eye. Treatments included antiparasitic medication, anti-inflammatory medication and supportive care in all three cases. Surgical treatment included enucleation (one case), bilateral phacoemulsification and unilateral intraocular lens placement (one case) and bilateral phacoemulsification with bilateral endolaser ciliary body ablation and bilateral intraocular lens implantation (one case). Both cats for which serologic testing for E cuniculi was performed were positive (1:64-1:4096). In all cats, diagnosis of intraocular E cuniculi was based on at least one of the following: lens histopathology or PCR of aqueous humor, lens material or paraffin-embedded ocular tissue. The clinical visual outcome was best in the patient undergoing phacoemulsification at the earliest stage of the cataract. Relevance and novel information: Encephalitozoon cuniculi should be considered as a differential cause of cataracts and uveitis in cats in California, the rest of the USA and likely worldwide.

Effects of diluted povidone iodine on viability and migration of canine corneal epithelial and stromal cells in tissue culture.

BACKGROUND/OBJECTIVE: The purpose of this study was to establish a reliable and reproducible method to isolate and cultivate canine corneal epithelial and stromal cells (cCECs and cCSCs). The cells were subsequently used for in vitro testing of topically applied diluted povidone iodine (PVI). METHODS: Corneas of dogs, euthanized for reasons unrelated to this study, were used to collect primary cCECs and cCSCs. Corneas were enzymatically digested and explants obtained using biopsy punches. Epithelial and stromal explants were separately taken into culture. Cell proliferation and migration was evaluated after incubation of cCECs and cCSCs with PVI in different concentrations (1, 2, or 5%) and with different exposure times (1, 3, or 10 min). RESULTS: Solely incubation of 4 mm diameter corneal epithelial explants in a 48-well culture plate in full medium led to sufficient growth of cCECs. Up to four passages were achieved with a cell density of 10,000-20,000 cells/cm2 after dissociation of cells in trypsin for 8 min. Cell detachment and passaging for cCSCs were possible with almost every cornea and explant. Canine CSCs were less sensitive to PVI in all concentrations and over time than cCECs. Epithelial and stromal cell proliferation and migration decreased with increasing exposure times and PVI concentrations across all groups. CONCLUSIONS: The described method is a straightforward and sound way to isolate and cultivate cCECs and cCSCs in vitro. Basic information on cCEC and cCSC migration and proliferation after incubation with PVI, was gathered. The results may provide a step towards an optimal preoperative antisepsis protocol for ophthalmic surgery in future.

Immunohistochemical staining of immunoglobulin G in healthy equine, canine, and feline corneas.

OBJECTIVE: Establishing an immunohistochemical approach for semi-quantitative assessment of the presence of immunoglobulin G (IgG) in equine, canine, and feline corneas. PROCEDURES: Healthy corneas of horses, dogs, and cats, euthanized because of a fatal disease or an unrecoverable trauma unrelated to and without a history of ophthalmic disease were formalin-fixed, paraffin-embedded, and determined to be pathomorphologically healthy by light microscopy. Automated immunohistochemistry was performed using primary antibodies against IgG, biotin-conjugated secondary antibodies, and streptavidin-peroxidase, as well as diaminobenzidine for visualization. After counterstaining with hematoxylin, epithelium, stroma, Descemet´s membrane (DM), and endothelium were semi-quantitatively scored for the presence of IgG on a 4-grade scale (0 = no, 1 = faint, 2 = medium, 3 = strong staining) by light microscopy. RESULTS: Corneal specimens of 20 horses (40 eyes) with a median age of 15.5 years (range 2-31 years), 12 dogs (21 eyes) with a median age of 10.0 years (range 4-16), and 13 cats (24 eyes) with a median age of 10.0 years (range 2-18) were included in the study. Different sexes and breeds were represented. In all corneas (100%), significant medium signal intensity in the stroma was observed. Variable immunosignal was obtained in epithelium, DM, and endothelium. CONCLUSION: This method reproducibly allows for the detection of IgG in healthy equine, canine, and feline corneas, particularly stroma. Semi-quantitative results evidence medium presence of IgG in the corneal stroma. Further research is needed to evaluate IgG presence in diseased corneas.

Topical bevacizumab for the treatment of corneal vascularization in dogs: A case series.

OBJECTIVE: To evaluate the effect and safety of topical anti-human vascular endothelial growth factor bevacizumab in dogs with persistent corneal vascularization. ANIMALS STUDIED: Prospective case series of 15 adult dogs (20 eyes). PROCEDURES: Dogs received 0.25% bevacizumab eye drops BID for 28 days. Follow-ups were scheduled 28 days and 6-7 months after treatment start. Macroscopic findings were scored for conjunctival hyperemia, chemosis, ocular discharge, corneal edema, vascularization, and pigmentation. Vascularized area was assessed by analyzing photographs using an imaging software. RESULTS: The treatment response was variable. Some cases showed a marked reduction in vascularized area and edema, while other eyes had subtle signs of improvement. Vascularization score decreased from 1.5 to 1.1 and vascularized area was reduced by 48.8% after 28 days. A thinning of vessels, consolidation of areal bleedings into fine vascular networks, decrease in distal vessel branching, and a change from blurry vascularized beds into demarcated thin vessels were observed. One dog developed a SCCED 6 months after the last bevacizumab administration. Two dogs died 4 and 4.5 months after the last bevacizumab administration, aged 16 and 12 years, respectively. In all events, a causal relationship is unlikely but cannot be ruled out with certainty. CONCLUSIONS: Our findings suggest that topical 0.25% bevacizumab may be an effective treatment option for corneal vascularization in dogs. Further long-term placebo-controlled studies with larger patient cohorts are recommended to provide scientific evidence of efficacy and to investigate dosage, safety, possible use as a single treatment, and routes of administration.

Labial and buccal minor salivary glands of the dog - location, three-dimensional arrangement and histology.

OBJECTIVE: Transplantation of minor salivary glands (MSGs) to the conjunctiva is a treatment option for patients suffering from dry eye disease. As there is not enough information about labial and buccal MSGs in dogs, the aim of this study was to provide evidence of the presence of these glands and to investigate their spatial arrangement and excretory ducts. METHODS: The oral mucosa of the lower lip of 4 dogs and the whole lower jaw of 1 dog were used for histological and microCT analysis. Presence, number, volumes and the tissue depth of MSGs were assessed. RESULTS: Histological analysis showed that compact tubulo-acinar glands were located in the submucosal connective tissue. MicroCT images revealed that 9 to 21 MSGs were arranged in a single row at the level of the dental alveolae. The volume of the MSGs increased from rostral to caudal and the total volume of glandular tissue per animal ranged from 35.01 mm3 to 549.43 mm3 . The mean tissue depth of MSGs ranged from 0.57 mm to 1.37 mm (upper surface of glands) and between 1.43 mm and 3.09 mm (lower surface of the glands). Excretory ducts left the dorsal part of the glands and ran in dorso-rostral direction. CONCLUSIONS: The location, number and volume of the labial and buccal MSGs in the dog could be detected and described using microCT scans and histology. The present results can provide valuable information for future transplantation of labial MSGs as therapeutic measure against keratoconjunctivitis sicca.

Diagnosis and successful treatment of an Austrian dog infected with Trypanosoma congolense forest type.

Canine African Trypanosomosis (CAT) is a rarely described disease with frequently lethal outcome. A 5-year-old female mongrel dog weighing 22 kg was presented in Austria due to unilateral uveitis, pancytopenia, and anorexia 4 months after return from a trip through Western Africa. Trypanosoma spp. flagellates were detected in a blood smear and identified as Trypanosoma congolense forest type by PCR. Initial treatment with imidocarb and miltefosine led to clinical improvement but only isometamidium chloride hydrochloride applied intramuscularly led to complete eradication of the pathogen from the dog's blood 4 months later.

Equine non-healing corneal ulcers: a retrospective evaluation of 57 cases (2001-2017).

BACKGROUND AND OBJECTIVES: Non-healing corneal ulcers (NHCU) are a common problem in equine practice and several treatment options are available with different success and healing times. The aim of this retrospective study was to evaluate and to compare treatment protocols, clinical courses, corneal healing time and outcomes of NHCU. METHODS: From December 2001 to December 2017, a total of 57 horses with NHCU were presented at the Vetmeduni Vienna. Recorded data included affected eye, signalment, clinical symptoms, season of diagnosis, treatment protocols, complications and corneal healing rate. RESULTS: Sixty-three eyes were diagnosed with a NHCU. Follow-up information was available for 48/63 eyes. For those treated medically mean corneal healing time was 15.7 days (± SD 12.0). Medical treatment included topical antibiotics, antimycotics, cycloplegics, and systemic anti-inflammatory drugs. Twelve eyes received treatment with a poly-carboxymethylglucose-sulfate regenerating agent (Cacicol®; Thea Pharma GmbH, Wien, Austria). Other common additional treatments included debridement with an iodine drenched cotton tip (48 eyes; 76.2 %) and diamond burr debridement (30 eyes; 47.6 %). A bandage contact lens (BCL) was used for 10 eyes. Each eye received at least one additional treatment, although none of them led to a statistically significant alteration in healing time. Only usage of a BCL significantly increased healing time when compared to not using a BCL (p = 0.035). When all treatments failed, superficial keratectomy with placement of a conjunctival flap was performed. Secondary complications included stromal cellular infiltration, keratomycosis, keratomalacia, and corneal abscess formation. CONCLUSIONS: Results correlated with those previously described and thus demonstrated the difficulty and complexity of this disease. Further research is needed to determine an optimal treatment protocol for non-healing ulcers in horses. CLINICAL RELEVANCE: Since NHCUs are a commonly encountered problem in equine practice a reliable treatment protocol is required. This study reflects the problems with those ulcers and provides several protocols for possible treatments.

Clinical utility, dose determination, and safety of ocular contrast-enhanced ultrasonography in horses: A pilot study.

OBJECTIVE: To determine efficacy of contrast-enhanced ultrasonography (CEUS) using different sulfur hexafluoride (SF6) doses to assess blood flow and perfusion in equine eyes and to evaluate safety of SF6 in horses. PROCEDURES: Ocular B-mode and contrast-enhanced ultrasonography were performed bilaterally in nine sedated university-owned horses. Intravenous SonoVue® bolus injections of 5, 10, 15, 20, 25, and 30 mL were administered for 2/18, 5/18, 6/18, 3/18, 1/18, and 1/18 eyes, respectively. Doses were increased based on ascending bodyweight. Each eye within one horse was examined utilizing a different dose. Qualitative blood flow and quantitative perfusion were analyzed. Heart and respiratory rates were monitored nonsedated, sedated, and during first and second minutes of CEUS. RESULTS: Qualitative contrast enhancement (CE) was visible in 7/9 animals. Quantitative CE was measurable bilaterally in four horses, unilaterally in three individuals, and not detected in two animals. In all horses with unilateral CE, the positive eye received the higher dose. Fifteen mL dose resulted in significantly shorter time to peak than 10 mL (P < .05). Peak intensity, maximum signal increase, and corresponding area under the curve were significantly higher for 15 and 20 mL doses compared with 10 mL (P < .05). Uveal and retinal tissues were enhanced frequently. Twenty-five and 30 mL doses revealed no CE. Only sedation reduced heart rates significantly (P < .05). Clinically relevant changes in respiratory rates or adverse reactions following SF6 application were not observed. CONCLUSIONS: Contrast enhancement was in most instances dose-dependent. Fifteen mL appeared appropriate to assess equine ocular perfusion. The reliability in horses remains questionable; however, CEUS was well-tolerated.

In-vitro binding analysis of anti-human vascular endothelial growth factor antibodies bevacizumab and aflibercept with canine, feline, and equine vascular endothelial growth factor.

PURPOSE: Promising results have been described for antibodies binding vascular endothelial growth factor (VEGF) in patients with corneal neovascularization. Whether veterinary patients would also benefit from this therapeutic approach has not been investigated yet. We examined binding properties of anti-human VEGF antibodies bevacizumab (Avastin®) and aflibercept (Zaltrap®) for canine, feline, and equine VEGF. METHODS: Human, equine, feline, and canine VEGF were analyzed for sequence similarity using the "Basic Local Alignment Search Tool" (BLAST). Western-blot analysis and ELISA were used to assess binding properties. RESULTS: BLAST analysis revealed a sequence homology of canine, feline, and equine VEGF to human VEGF-A of 93%, 92%, and 89%, respectively. Western-blot analysis showed immunoreactivity of bevacizumab with human, canine, and feline VEGF, but not with equine VEGF. Aflibercept recognized VEGF of all tested species. ELISA data indicated that bevacizumab and aflibercept bind canine VEGF in a dose-dependent manner. Feline VEGF was bound by bevacizumab and aflibercept in a dose-independent manner. ELISA study further confirmed the lack of bevacizumab binding to equine VEGF, and yielded also a dose-independent binding by aflibercept. CONCLUSIONS: Bevacizumab and aflibercept turned out to bind VEGF with species-specific differences. Further studies are required to investigate their efficacy and safety under clinical conditions.

Quantitative, noninvasive assessment of intra- and extraocular perfusion by contrast-enhanced ultrasonography and its clinical applicability in healthy dogs.

OBJECTIVE: To assess quantitative perfusion of intra- and extraocular regions of interest (ROIs) in conscious, healthy dogs utilizing contrast-enhanced ultrasonography (CEUS); to compare varying enhancement with the first and second bolus injection and in the right and left eye; and to determine the most appropriate examination time. PROCEDURES: Gray scale ultrasonography and contrast harmonic imaging using sulfur hexafluoride were performed randomly assigned in both eyes in 10 university-owned beagles. Perfusion parameters including slope time, time to peak (TTP), peak intensity (PI), and area under the curve (AUC) were measured at individually drawn ROIs (retrobulbar cone = ROI 1, choroid-retina complex = ROI 2, medial = ROI 3, and lateral anterior uvea = ROI 4). RESULTS: Time-intensity curve parameters revealed no significant differences in eyes examined by the first or second bolus injection (P > 0.05) or in the right or left eye (P > 0.05). Pooled data from all eyes were analyzed. Peak intensity of ROI 2 was significantly higher compared to all other ROIs (P < 0.001). Area under the curve at ROI 2 was significantly higher compared to all other ROIs (P < 0.05), and AUC at ROI 1 was significantly higher than at ROI 4 (P < 0.05). No significant differences in TTP were observed between different ROIs (P > 0.05). Ratios relative to different ROI sizes showed fastest enhancement in the retrobulbar cone and most intense perfusion in the anterior uveal regions. The first minute after contrast injection provided the highest diagnostic value. CONCLUSION: Quantitative perfusion in nondiseased canine eyes revealed consistent parameters. Application of standardized CEUS protocols may be a promising diagnostic tool to differentiate ocular lesions.

Morphological and immunohistochemical characteristics of the equine corneal epithelium.

OBJECTIVE: The morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation-, differentiation-, stem cell-associated markers and cell junction proteins were assessed. METHODS: Corneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E-cadherin, ß-catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium. RESULTS: The height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea-specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E-cadherin, ß-catenin, and Cx43 revealed a similar apico-lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface. CONCLUSIONS: This study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.

Serum vascular endothelial growth factor changes and safety after topical anti-human VEGF antibody bevacizumab in healthy dogs.

OBJECTIVE: To evaluate ocular and general safety of topical anti-human VEGF bevacizumab and the effect on serum vascular endothelial growth factor (VEGF) values in healthy dogs. PROCEDURES: Nine university-owned beagles received 0.05 mL of 0.25% bevacizumab eyedrops (Avastin® , Roche) in one eye and 0.05 mL of 0.9% saline solution in the other eye as a control, administered at 12 hours intervals over a period of 28 days. Continuous monitoring for vital parameters and ocular examinations were conducted. Complete blood counts including hematology and coagulation parameters were performed before trial start as well as 24 hours, 7 days, and 28 days after trial start. Measurements of serum VEGF values were obtained using an ELISA-based approach at days 0, 7, and 28. The experiment was designed as a masked placebo-controlled study. RESULTS: No clinical signs of ocular toxicity or systemic incompatibility were noted in any dog at any time point of the study. No signs of pain were present in any dog at any time point. All blood count values remained in normal clinical ranges without relevant variation. There was no significant change in mean serum VEGF values between day 0 and day 7 and between day 0 and day 28. CONCLUSIONS: The results indicate that topical bevacizumab treatment is safe in healthy dogs. However, further studies are needed to assess safety and efficacy in diseased dogs with naturally occurring corneal neovascularization.

Circulating cell-free DNA does not harbour a diagnostic benefit in cats with feline diffuse iris melanomas.

OBJECTIVES: Feline diffuse iris melanoma (FDIM) is the most common malignant primary intraocular tumour in cats, with reported metastases rates between 19% and 63%. Currently, the only available diagnostic tool for a tentative diagnosis is histopathological examination of the enucleated eye. Therefore, the veterinary ophthalmologist is often faced with the dilemma of whether to enucleate an oftentimes visual eye or to continue monitoring, with the risk of metastases developing. In the past, cell-free DNA (cfDNA) gained more attention in human medicine, especially in the field of oncology. Prior studies have shown the use of cfDNA as diagnostic or prognostic markers in canine and human cancer patients. Therefore, the aim of this study was to investigate cfDNA concentration and integrity in cats with FDIMs compared with cats with benign iris naevi and without ocular abnormalities. METHODS: cfDNA from plasma of cats with iris melanoma (n = 34), iris naevus (n = 30) and without ocular abnormalities (n = 32) were extracted. Primer and probes for feline amyloid beta precursor protein ( APP) and beta actin ( ACTB) were designed for amplicons of various lengths and quantitative PCRs of extracted cfDNA were performed to measure cfDNA concentration and integrity of the plasma samples. Differences of cfDNA concentrations and integrity levels between the three groups (iris melanoma, iris naevi and controls) were analysed using the Mann-Whitney U-test. RESULTS: cfDNA concentration and integrity analysis revealed no significant differences between the cats with iris melanoma, iris naevus or the control group ( P >0.01). Cats with metastases showed similar cfDNA concentration and integrity to cats without metastases. CONCLUSIONS AND RELEVANCE: cfDNA concentration and integrity seem to be insufficient as a diagnostic or prognostic marker in cats with FDIMs.

[Untersuchung des Tränenfilms und histologische Untersuchung der Augenlider von Möpsen]. / Tear film examinations and eyelid histology of pugs.

OBJECTIVES: To determine if qualitative tear film and histological changes of the eyelid margins in pugs compared to other brachycephalic dogs could be potential contributing factors to the high prevalence of corneal diseases in this breed. METHODS: Ophthalmic examin ation (including tear film break-up time [TFBUT] and meibometry) was undertaken on three groups (pugs with and without ophthalmologic abnormalities as well as on other brachycephalic breeds with history of ophthalmologic abnormalities). Histology of eyelid tissue obtained during medial canthoplasty was performed, using hematoxylin-eosin and oil-red-O-staining. RESULTS: Seventy-eight pugs and 11 brachycephalic dogs were included. Mean ages were 3.54 and 5.5 years respectively. STT 1 values below 15 mm/min were found in 12 of 150 eyes of pugs and in three of 18 eyes of other brachycephalic dogs. Tear film break-up time values below 20 seconds were determined in 118 of 126 eyes of pugs, and in eight of 18 eyes of other brachycephalic dogs. Meibometry values over 100 MU were only identified in 20 of 147 eyes of pugs and 12 of 20 eyes in other brachycephalic dogs. Eyelid tissue of 21 pugs and 11 brachycephalic dogs was obtained. All pugs had a higher number of inflammatory cells in eyelid tissue than other brachycephalic breeds. CONCLUSIONS: Young pugs are often presented with STT 1 values within the reference range and low TFBUT and meibometry values. As other brachycephalic breeds are often presented with STT 1 values within reference ranges as well as low TFBUT values, the only marked difference between pugs and other brachycephalic breeds are low meibometry values and the higher number of inflammatory cells in the medial canthal lid margins.

Density and distribution of feline conjunctival goblet cells.

Objectives The objective of this study was to examine the density and distribution of goblet cells (GCs) in the feline conjunctiva and to investigate a potential effect of age and sex on GC density (GCD). Methods Thirty-nine eyes of 21 cats euthanased for reasons unrelated to this study were used. Fixed upper and lower eyelid and bulbar conjunctiva were divided into nasal and temporal regions. The third eyelid was excised and investigated separately. Samples were embedded in paraffin wax; sections were stained with periodic acid-Schiff reaction and analysed with light microscopy. To determine the topographic distribution of GCs, each region was subdivided into the marginal, palpebral and bulbar zone. In each zone 200 epithelial cells, including GCs, were counted. Goblet cell index was defined as a percentage of the epithelial cells. Results The palpebral zone of both eyelids contained significantly ( P <0.001) more GCs (27.5-32.0%) than the marginal or bulbar areas. The highest GCD was found in the nasal palpebral zone of the upper eyelid (32.0%). Marginal and bulbar sites contained fewer numbers of GCs (2.6-10.0%). The lowest GCD was detected in the nasal bulbar zone of the lower eyelid (2.6%). Overall the nasal region contained significantly ( P = 0.036) more GCs than the temporal region, but there was no significant difference in GCD between the upper and lower eyelids. Correlation analysis did not show any effect of age or sex on GC counts. Conclusions and relevance GCD in the palpebral zones and on the anterior surface of the third eyelid was highest; the lowest density was found in the bulbar zones of the lower eyelid and in the marginal zones of both eyelids. Overall, higher GCD was found in the cat than in other species. Age and sex have no effect on GCD.