Kara R. Gornik

Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.

A partial gene deletion of SLC45A2 causes oculocutaneous albinism in doberman pinscher dogs

The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (p<0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; TYR, OCA2, TYRP1 and SLC45A2. SLC45A2 was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of SLC45A2 (chr4∶77,062,968-77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable SLC45A2 transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model.

Prevalence of uveal cysts and pigmentary uveitis in Golden Retrievers in three Midwestern states.

OBJECTIVE To determine the prevalence of uveal cysts and pigmentary uveitis (PU) in Golden Retrievers in 3 Midwestern states. DESIGN Prospective cross-sectional study. ANIMALS 164 American Kennel Club-registered Golden Retrievers in the states of Illinois, Indiana, and Michigan. PROCEDURES For all dogs, biomicroscopic and binocular indirect ophthalmoscopic examinations of both eyes were performed after pupillary dilation. A finding of pigment deposition in a radial pattern or in zones on the anterior aspect of the lens capsule of 1 or both eyes was required for a diagnosis of PU. RESULTS Eighty of the 328 (24.4%) eyes and 57 of the 164 (34.8%) dogs had visible uveal cysts. Of those 80 eyes with cysts, 41 (51.3%) had a single cyst located nasally and posterior to the iris, 33 (41.3%) had multiple uveal cysts, and 6 (75%) had a single, free-floating cyst. A diagnosis of PU was made for 9 (5.5%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE Prevalences of uveal cysts (34.3%) and PU (5.5%) in the examined Golden Retrievers were both higher than prevalences reported previously (5.4% for uveal cysts and 1.5% for PU) in the Canine Eye Registry Foundations 2009 All-Breeds Report. Study findings have indicated that PU is not a rare condition and should be considered as a differential diagnosis for Golden Retrievers with ocular disease.

Unilateral choristoma of the nictitating membrane in a horse

CASE DESCRIPTION A 2-year-old Morgan mare was evaluated because of a corneal ulceration. CLINICAL FINDINGS An irregular, deep stromal corneal ulcer in an area of malacia was noted in the left eye. Hypopyon was present in the ventral portion of the anterior chamber with moderate aqueous flare. The nictitating membrane of the left eye had hairs originating from its leading edge that contacted the corneal surface. TREATMENT AND OUTCOME General anesthesia was induced, and a bulbar pedicle conjunctival graft was performed. The conjunctiva at the leading edge of the nictitating membrane, including the aberrant hair follicles, was excised. Microscopically, a nonkeratinized stratified squamous epithelium, sebaceous glands, and hair shafts were present, confirming a choristoma of pilosebaceous origin at the leading edge of the nictitating membrane. Six weeks after surgery, the horse had no signs of discomfort, with no regrowth of the hairs; no loss of vision was evident. CLINICAL RELEVANCE Ocular choristomas develop secondary to defective fetal cellular differentiation and are rarely reported in the equine literature. The choristoma in this horse contained ectopic hair follicles with hair growth as well as sebaceous glands. This finding emphasizes the importance of a thorough adnexal examination in horses with corneal disease.

Ophthalmic variables in rehabilitated juvenile Kemp's ridley sea turtles (Lepidochelys kempii)

OBJECTIVE To determine central corneal thickness (total corneal thickness [TCT], epithelial thickness [ET], and stromal thickness [ST]), anterior chamber depth (ACD), and intraocular pressure (IOP) in Kemps ridley sea turtles (Lepidochelys kempii). DESIGN Prospective cross-sectional study. ANIMALS 25 healthy rehabilitated juvenile Kemps ridley sea turtles. PROCEDURES; Body weight and straight-line standard carapace length (SCL) were recorded. All turtles underwent a complete anterior segment ophthalmic examination. Central TCT, ET, ST, and ACD were determined by use of a spectral-domain optical coherence tomography device. Intraocular pressure was determined with a rebound tonometer; the horse setting was used to measure IOP in all 25 turtles, and the undefined setting was also used to measure IOP in 20 turtles. For each variable, 3 measurements were obtained bilaterally. The mean was calculated for each eye and used for analysis purposes. RESULTS The mean ± SD body weight and SCL were 3.85 ± 1.05 kg (8.47 ± 2.31 lb) and 29 ± 3 cm, respectively. The mean ± SD TCT, ET, ST, and ACD were 288 ± 23 μm, 100 ± 6 μm, 190 ± 19 μm, and 581 ± 128 μm, respectively. Mean ± SD IOP was 6.5 ± 1.0 mm Hg when measured with the horse setting and 3.8 ± 1.1 mm Hg when measured with the undefined setting. CONCLUSIONS AND CLINICAL RELEVANCE Results provided preliminary reference ranges for objective assessment of ophthalmic variables in healthy juvenile Kemps ridley sea turtles.

Baseline Corneal Sensitivity and Duration of Action of Proparacaine in Rehabilitated Juvenile Kemp's Ridley Sea Turtles (Lepidochelys kempii)

Abstract Corneal sensitivity and the duration of action of a topical anesthetic drop, proparacaine, were evaluated prospectively. Baseline corneal touch threshold (CTT) was determined using a Cochet-Bonnet esthesiometer in 25 healthy, rehabilitated Kemps ridley sea turtles (Lepidochelys kempii). Corneal touch threshold was defined as the filament length required to elicit a blink in at least three out of five applications. The onset and duration of action of proparacaine was determined in nine of the Kemps ridley sea turtles. One drop of proparacaine was applied to each eye and the CTT was subsequently measured at 1, 5, 15, 30, and 45 min. Results demonstrated that the mean CTT for the Kemps ridley sea turtles was 5.7 cm, with a median of 6 cm. Additionally, proparacaine had an onset of action within 1 min after application (17/18 eyes). The duration of action of proparacaine exceeded the 45 min time limit in all eyes. This study shows that Kemps ridley sea turtles have an exceptionally sensitive cornea. This study also establishes that proparacaine, a topical anesthetic, is effective in Kemps ridley sea turtles, with an onset of action within 1 min and a duration of action greater than 45 min following application.

Orbital and Subcutaneous Emphysema Following Enucleation and Respiratory Distress in a Japanese Chin.

A 7 yr old, neutered male Japanese chin presented to the Cummings School of Veterinary Medicine at Tufts University (CSVMTU) for evaluation of chronic unilateral orbital swelling that worsened following an episode of respiratory distress. The left eye had been enucleated 5 yr previously. Intermittent mild-to-moderate left orbital swelling had been noted by the owner since the initial surgery. Examination demonstrated a moderate-to-severe, soft, fluctuant swelling involving the left orbit with erythema of the overlying skin. Crepitus was noted over the occipital tuberosity. Computed tomography revealed a large volume of gas involving the left orbit. The gas extended caudally within the subcutaneous tissues to both hemimandibles, dorsal to the cranium, and partially surrounded the cranial neck. The presence of a mucosa-lined, air-filled space with a patent nasolacrimal duct was noted on orbital exploration. The lining was removed and the duct closed. Histopathology confirmed the presence of an epithelial lining. No recurrence of the swelling was observed on examination 8 wk after surgery. This is the first report documenting acute worsening of orbital swelling following an episode of respiratory distress. This case highlights the importance of addressing the nasolacrimal duct while performing an enculeation in a brachycephalic dog.