Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism.

Retinitis pigmentosa (RP) is a heterogeneous inherited retinal disorder. Mutations in KIZ cause autosomal recessive (AR) RP. We aimed to characterize the genotype, expression pattern, and phenotype in a large cohort of KIZ cases. Sanger and whole exome sequencing were used to identify the KIZ variants. Medical records were reviewed and analyzed. Thirty-one patients with biallelic KIZ mutations were identified: 28 homozygous for c.226C>T (p.R76*), 2 compound heterozygous for p.R76* and c.3G>A (p.M1?), and one homozygous for c.247C>T (p.R83*). c.226C>T is a founder mutation among patients of Jewish descent. The clinical parameters were less severe in KIZ compared to DHDDS and FAM161A cases. RT-PCR analysis in fibroblast cells revealed the presence of four different transcripts in both WT and mutant samples with a lower percentage of the WT transcript in patients. Sequence analysis identified an exonic sequence enhancer (ESE) that includes the c.226 position which is affected by the mutation. KIZ mutations are an uncommon cause of IRD worldwide but are not rare among Ashkenazi Jews. Our data indicate that p.R76* affect an ESE which in turn results in the pronounced skipping of exon 3. Therefore, RNA-based therapies might show low efficacy since the mutant transcripts are spliced.

Technical note: a nose ring sensor system to monitor dairy cow cardiovascular and respiratory metrics.

Monitoring cardiovascular and respiratory measurements corresponds to the precision livestock farming (PLF) objective to continuously monitor and assess dairy cows' welfare and health. Changes in heart rate, breathing rate, and oxygen saturation (SpO2) are valuable metrics in human and veterinary medicine to assess stress, pain, illness, and detect critical conditions. The common way to measure heart rate is either manually or with a stethoscope. Under research conditions, heart rate is usually measured with a sports watch chest belt. Breathing rate is obtained by counting the cow's flank movements which is a time-consuming and labor-intensive method that requires training and is prone to human error. No devices are available on the market that enable practical and easy pulse oximetry in farm animals. This study presents a wireless nose ring sensor system (NoRS) composed of thermal and photoplothysmography sensors that attach to the nostrils of four Holstein dairy cows. The NoRS's thermocouple measured the cow's nasal cavity air temperature; an optic sensor measured the IR (660 nm) and RED (660 nm) signals reflected from the cow's nasal septum. Breathing was calculated from the thermocouple signal's center frequency with a fast Fourier transformation or the signal peak count (i.e., oscillations). The breathing rate was compared to breathing observed by concurrently counting the flank movements. Heart rate and SpO2 were measured by integrated pulse oximetry and heart rate monitor module (MAX30101 TinyCircuit) assembled on the NoRS circuit. Heart rate was also measured with FFT and by counting the number of peaks from the optic sensor's raw IR and RED signals. These measures were compared to an off-the-shelf hand-held pulse oximeter's heart rate and SpO2 readings during the same time. The comparisons revealed highly significant correlations for the heart rate readings where the strength of the correlation was sensitive to the method. The correlation between breathing rate and the veterinarian's visual observations was low, albeit significant. Thus, inhale-exhale cycle counting constitutes a more precise approach than flank movement counts. The hand-held device's 96% SpO2 is compatible with near-saturation values expected in healthy cows. The mean NoRS SpO2 reading was 3% less. After further piloting under field conditions, the NoRS will require no animal restraining to automatically and continuously record cows' breathing rate, heart rate, and SpO2.

Monitoring cardiovascular and respiratory measurements responds to the precision livestock farming objective to continuously monitor and assess dairy cows' welfare and health. Changes in heart rate, breathing rate, and oxygen saturation are valuable metrics in human and veterinary medicine that are used to assess stress, pain, illness, and detect critical conditions. This article describes a wireless nose ring sensor system (NoRS) developed to read heart rate, breathing rate, and oxygen saturation from the cow's nostrils and tested on four Holstein dairy cows. These measures were compared to heart rate and oxygen saturation readings obtained from an off-the-shelf hand-held pulse oximeter and a veterinarian's concurrent count of flank movements. The comparison revealed highly significant correlations between the heart rate readings and a low, albeit significant correlation for breathing rate. The mean NoRS oxygen saturation reading was 3% less than the hand-held device. Although commonly used techniques for detecting vital parameters such as heart rate, breathing rate, and oxygen saturation only provide information about the time of examination, the NoRS is a wearable device that can monitor cardiovascular and respiratory measurements remotely and over time.

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression.

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or ß-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Is it necessary to wait several minutes between applications of different topical ophthalmic solutions? A preliminary study with tropicamide eye drops in healthy dogs.

OBJECTIVE: To evaluate the efficacy of topical tropicamide when placed at different time intervals before or after a saline drop. ANIMALS STUDIED: Eight healthy Labrador and golden retriever dogs. PROCEDURES: The effect of 1% tropicamide on pupillary diameter (PD) was measured over 240 min when administered alone (control) and then 1 and 5 min prior to, or following, application of a saline drop, with 1-week washout between each of the five trials. Data were analyzed using repeated-measures ANOVA and Tukey post hoc test. RESULTS: Only 6/110 pairwise comparisons among the 5 trials were statistically significant (p ≤ .035), with post-hoc analysis showing no significant differences (p ≥ .14) between the overall means of all trials. In all five trials, maximal PD was reached 30 min after tropicamide application and maintained until 210 min for 180 min (p = .0005). CONCLUSIONS: Our results suggest that waiting 1 min between applications of different ophthalmic solutions may be sufficient for maximal drug effect. Care should be taken when extrapolating these results to other species and different ophthalmic formulations.

Treatment with chloroquine is retinotoxic in captive African penguins (Speniscus demersus). Attenuation and recovery of electroretinographic responses.

PURPOSE: To evaluate the effect of prophylactic anti-malarial chloroquine treatment, and its cessation, on electroretinographic (ERG) responses of captive African penguins. METHODS: A brief ERG protocol ("QuickRetCheck") was recorded under mesopic conditions with manual restraint and no sedation or pupil dilation. Birds were recorded on two separate occasions, first while being treated with a daily chloroquine dose of 10 mg/kg for 12 days (n = 15, treatment group) and second after 4 months without chloroquine treatment (n = 6, off-treatment group). Three birds were recorded on both occasions. Three other birds from the flock that died were studied histopathologically. RESULTS: Scotopic responses were unmeasurable in either recording and therefore were not analyzed. Mean a- and b-wave amplitudes of the mixed rod-cone responses to standard (3 cd·s/m2 ) and high (10 cd·s/m2 ) intensity flashes were higher in the off-treatment group. No difference in implicit times was observed. Sex, age, and number of previous chloroquine treatments did not affect ERG responses. Histopathology revealed Plasmodium spp.in the lungs, liver, and brain, but not in the eyes, of the necropsied birds, and there were no signs of retinitis or retinopathy. CONCLUSIONS: Daily chloroquine treatment was associated with attenuated ERG responses in penguins, which improve following cessation of treatment. Further work is warranted to establish a chloroquine dose that is efficacious yet has minimal adverse effects. Our results suggest that ERG responses of captive penguins undergoing ERG for any indication (such as prior to cataract surgery), must be evaluated in light of the birds' anti-malaria treatment status.

Ocular anatomy and correlation with histopathologic findings in two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) diagnosed with inflammatory phakitis and retinitis.

PURPOSE: Review octopus ocular anatomy and describe the histopathologic findings in three octopuses diagnosed with phakitis and retinitis. ANIMALS: Two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) with a history of ophthalmic disease. METHODS: A literature search was performed for the ocular anatomy section. Both eyes from all three octopuses, and two control eyes, were submitted for histopathologic evaluation. Hematoxylin and eosin stain was used for standard histopathologic evaluation; GMS stain was used to screen for fungi, gram stain for bacteria; and Fite's acid fast stain for acid fast bacteria. RESULTS: Anatomically, the anterior chamber of the octopus has direct contact with ambient water due to an opening in the dorsal aspect of a pseudocornea. The octopus lens is divided into anterior and posterior segments. The anterior half is exposed to the environment through the opening into the anterior chamber. Neither part of the lens has a lens capsule. The retina is everted, unlike the inverted vertebrate retina, and consists of just two layers. Histopathology revealed inflammatory phakitis and retinitis of varying severity in all six eyes of the study animals. No intraocular infectious organisms were recognized but one common octopus eye had clusters of coccidian parasites, identified as Aggregata sp., in extraocular tissues and blood vessels. CONCLUSION: We describe inflammatory phakitis and retinitis in two species of octopuses. The underlying cause for the severe intraocular response may be direct intraocular infection, water quality, an ocular manifestation of a systemic disease, or natural senescence.

Does the order of intraocular pressure measurement affect tonometry results?

PURPOSE: To determine whether the order of intraocular pressure (IOP) measurement affects readings, regardless of which eye is measured first. METHODS: Intraocular pressure was measured in 31 and 41 dogs using applanation and rebound tonometry, respectively. Initially, IOP was measured in the first (randomly chosen) eye (reading A), followed by measurement in the fellow eye (reading B), and a third (repeated) measurement in the first eye (reading C). After 15 minutes, measurements were repeated in reverse order (readings D - F). RESULTS: Applanation tonometry revealed significant differences between readings A & B (15.6 ± 2.3 and 14.8 ± 2.7 mm Hg, respectively, p = .02), A & C (15.6 ± 2.3 and 14.5 ± 2.4 mm Hg, respectively, p = .002), D & E (14.5 ± 2.3 and 13.7 ± 2.1 mm Hg, respectively, p = .02), D & F (14.5 ± 2.3 and 13.9 ± 1.9 mm Hg, respectively, p = .05), and A & E (15.6 ± 2.3 and 13.7 ± 2.1 mm Hg, respectively, p = .001). Rebound tonometry yielded similar results, with additional differences between B & C (19.1 ± 3.0 and 18.2 ± 2.4 mm Hg, respectively, p = .002) and E & F (18.7 ± 3.3 and 18.2 ± 3.3 mm Hg, respectively, p = .02). CONCLUSIONS: Intraocular pressure measured in the first eye, whether right or left, is higher than in the fellow eye. Repeated tonometry in the same visit could result in a significant IOP decrease, though the magnitude may not be clinically appreciable.

The future of retinal gene therapy: evolving from subretinal to intravitreal vector delivery.

Inherited retinal degenerations are a leading and untreatbale cause of blindness, and as such they are targets for gene therapy. Numerous gene therapy treatments have progressed from laboratory research to clinical trails, and a pioneering gene therapy received the first ever FDA approval for treating patients. However, currently retinal gene therapy mostly involves subretinal injection of the therapeutic agent, which treats a limited area, entails retinal detachment and other potential complications, and requires general anesthesia with consequent risks, costs and prolonged recovery. Therefore there is great impetus to develop safer, less invasive and cheapter methods of gene delivery. A promising method is intravitreal injection, that does not cause retinal detachment, can lead to pan-retinal transduction and can be performed under local anesthesia in out-patient clinics. Intravitreally-injected vectors face several obstacles. First, the vector is diluted by the vitreous and has to overcome a long diffusion distance to the target cells. Second, the vector is exposed to the host's immune response, risking neutralization by pre-existing antibodies and triggering a stronger immune response to the injection. Third, the vector has to cross the inner limiting membrane which is both a physical and a biological barrier as it contains binding sites that could cause the vector's sequestration. Finally, in the target cell the vector is prone to proteasome degradation before delivering the transgene to the nucleus. Strategies to overcome these obstacles include modifications of the viral capsid, through rational design or directed evolution, which allow resistance to the immune system, enhancement of penetration through the inner limiting membrane or reduced degradation by intracellular proteasomes. Furthermore, physical and chemical manipulations of the inner limiting membrane and vitreous aim to improve vector penetration. Finally, compact non-viral vectors that can overcome the immunological, physical and anatomical and barriers have been developed. This paper reviews ongoing efforts to develop novel, safe and efficacious methods for intravitreal delivery of therapeutic genes for inherited retinal degenerations. To date, the most promising results are achieved in rodents with robust, pan-retinal transduction following intravitreal delivery. Trials in larger animal models demonstrate transduction mostly of inner retinal layers. Despite ongoing efforts, currently no intravitreally-injected vector has demonstrated outer retinal transduction efficacy comparable to that of subretinal delivery. Further work is warranted to test promising new viral and non-viral vectors on large animal models of inherited retinal degenerations. Positive results will pave the way to development of the next generation of treatments for inherited retinal degeneration.

Naturally-occurring myopia and loss of cone function in a sheep model of achromatopsia.

Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.

Evaluation of Photoreceptor Transduction Efficacy of Capsid-Modified Adeno-Associated Viral Vectors Following Intravitreal and Subretinal Delivery in Sheep.

Gene augmentation therapy based on subretinal delivery of adeno-associated viral (AAV) vectors is proving to be highly efficient in treating several inherited retinal degenerations. However, due to potential complications and drawbacks posed by subretinal injections, there is a great impetus to find alternative methods of delivering the desired genetic inserts to the retina. One such method is an intravitreal delivery of the vector. Our aim was to evaluate the efficacy of two capsid-modified vectors that are less susceptible to cellular degradation, AAV8 (doubleY-F) and AAV2 (quadY-F+T-V), as well as a third, chimeric vector AAV[max], to transduce photoreceptor cells following intravitreal injection in sheep. We further tested whether saturation of inner limiting membrane (ILM) viral binding sites using a nonmodified vector, before the intravitreal injection, would enhance the efficacy of photoreceptor transduction. Only AAV[max] resulted in moderate photoreceptor transduction following intravitreal injection. Intravitreal injection of the two other vectors did not result in photoreceptor transduction nor did the saturation of the ILM before the intravitreal injection. However, two of the vectors efficiently transduced photoreceptor cells following subretinal injection in positive control eyes. Previous trials with the same vectors in both murine and canine models resulted in robust and moderate transduction efficacy, respectively, of photoreceptors following intravitreal delivery, demonstrating the importance of utilizing as many animal models as possible when evaluating new strategies for retinal gene therapy. The successful photoreceptor transduction of AAV[max] injected intravitreally makes it a potential candidate for intravitreal delivery, but further trials are warranted to determine whether the transduction efficacy is sufficient for a clinical outcome.

Contrary to popular belief, chinchillas do not have a pure rod retina.

PURPOSE: To establish whether there is cone contribution to retinal function and structure in chinchillas (Chinchilla lanigera), in view of the prevailing notion that this species possesses a pure rod retina. METHODS: Photopic electroretinography (ERG) responses to high-intensity flashes (10 and 25 cd*s/m2 ) were recorded unilaterally in six pigmented chinchillas following 10 minutes of light adaptation (30 cd/m2 ). Retinas of two animals were studied histologically, and immunohistochemistry (IHC) was conducted to detect the presence of short and medium/long wavelength cone photoreceptors. RESULTS: ERG recordings revealed photopic responses, albeit of low amplitudes. Histopathology demonstrated presumptive cone inner segments in the photoreceptor layer. Presence of cone photoreceptors was confirmed by IHC. Cone density was higher in the central retina, and red/green cones outnumbered blue cones. CONCLUSIONS: Our results provide convincing evidence for the presence of functioning cone photoreceptors in the chinchilla retina, disproving the established belief that the species has a pure rod retina.

Consecutive unilateral recording of the two eyes affects dark-adapted ERG responses, when compared to simultaneous bilateral recording.

PURPOSE: Our aim was to compare the electroretinographic (ERG) responses of two eyes obtained by consecutive unilateral recordings to those obtained by a simultaneous bilateral recording in sheep. METHODS: Eight sheep underwent two full-field ERG recordings, using two recording strategies of the standard ISCEV protocol: consecutive unilateral recordings of one eye after the other, and simultaneous bilateral recording of both eyes. The order of recording strategy within an animal (unilateral/bilateral), eye recording sequence in the unilateral session (OD/OS), and amplifier channel assignment for each eye were all randomized. To test whether duration of dark adaptation and/or anesthesia affect the results, the ISCEV protocol was recorded bilaterally in six additional eyes following 38 min of patched dark adaptation, as was done for the second eye recorded in the consecutive unilateral recordings. RESULTS: The second recorded eye in the unilateral session had significantly higher scotopic b-wave amplitudes compared to the first recorded eye and to the bilaterally recorded eyes. A-wave amplitudes of the dark-adapted mixed rod-cone responses to a high-intensity flash were also significantly higher in the second eye compared to the first eye recorded unilaterally and to the bilaterally recorded eyes. Light-adapted responses were unaffected by the recording strategy. When the ISCEV protocol was recorded after 38 min of dark adaptation, the scotopic responses were higher than in the first eyes, and similar to those of the second eyes recorded unilaterally, suggesting that indeed the longer duration of anesthesia and dark adaptation are responsible for the increased scotopic responses of the second eye. CONCLUSIONS: Consecutive unilateral ERG recordings of two eyes result in higher amplitudes of the dark-adapted responses of the eye recorded second, compared to the eye recorded first and to bilaterally recorded eyes. The differences in scotopic responses can be attributed to different duration of dark adaptation and/or anesthesia of the two consecutively recorded eyes. Photopic responses are not affected. Therefore, simultaneous bilateral ERG responses should be recorded when possible, especially for evaluation of scotopic responses.

Can an in vivo imaging system be used to determine localization and biodistribution of AAV5-mediated gene expression following subretinal and intravitreal delivery in mice?

Recombinant adeno associated viruses (AAV) are the most commonly used vectors in animal model studies of gene therapy for retinal diseases. The ability of a vector to localize and remain in the target tissue, and in this manner to avoid off-target effects beyond the site of delivery, is critical to the efficacy and safety of the treatment. The in vivo imaging system (IVIS) is a non-invasive imaging tool used for detection and quantification of bioluminescence activity in rodents. Our aim was to investigate whether IVIS can detect localization and biodistribution of AAV5 vector in mice following subretinal (SR) and intravitreal (IVT) injections. AAV5 carrying firefly luciferase DNA under control of the ubiquitous cytomegalovirus (CMV) promoter was injected unilaterally IVT or SR (in the central or peripheral retina) of forty-one mice. Luciferase activity was tracked for up to 60 weeks in the longest surviving animals, using repeated (up to 12 times) IVIS bioluminescence imaging. Luciferase presence was also confirmed immunohistochemically (IHC) and by PCR in representative animals. In the SR group, IVIS readings demonstrated luciferase activity in all (32/32) eyes, and luciferase presence was confirmed by IHC (4/4 eyes) and PCR (12/12 eyes). In the IVT group, IVIS readings demonstrated luciferase activity in 7/9 eyes, and luciferase presence was confirmed by PCR in 5/5 eyes and by IHC (2/2 eyes). In two SR-injected animals (one each from the central and peripheral injection sites), PCR detected luciferase presence in the ipsilateral optic nerves, a finding that was not detected by IVIS or IHC. Our results show that when evaluating SR delivery, IVIS has a sensitivity and specificity of 100% compared with the gold standard PCR. When evaluating IVT delivery, IVIS has a sensitivity of 78% and specificity of 100%. These finding confirm the ability of IVIS to detect in-vivo localized expression of AAV following SR delivery in the retina up to 60 weeks post-treatment, using repeated imaging for longitudinal evaluation, without fading of the biological signal, thereby replacing the need for post mortem processing in order to confirm vector expression. However, IVIS is probably not sensitive enough, compared with genome detection, to demonstrate biodistribution to the optic nerve, as it could not detect luciferase activity in ipsilateral optic nerves following SR delivery in mice.

Six Years and Counting: Restoration of Photopic Retinal Function and Visual Behavior Following Gene Augmentation Therapy in a Sheep Model of CNGA3 Achromatopsia.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values (p = 0.0025 and p < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment (p < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model.

The retina of the collared peccary (Pecari tajacu): structure and function.

OBJECTIVE: To study retinal morphology and function in the collared peccary, an ungulate species distantly related to the domestic pig. ANIMAL STUDIES: Twenty captive peccaries anesthetized for routine health examinations. Procedures No abnormalities were noted on a complete ophthalmic examination. Fundi were examined ophthalmoscopically and photographed. The eyes of an individual that died of unrelated, nonocular reasons were studied histologically and by immunohistochemistry. Scotopic, mixed rod-cone, and photopic electroretinography (ERG) responses were recorded using the 'QuickRetCheck' (n = 6) and 'Dog diagnostic' (n = 5) protocols of the Handheld Multispecies ERG (HMsERG). RESULTS: The fundus of the peccary is atapetal, with varying amounts of pigmentation seen ophthalmoscopically, and histologically in the retinal pigment epithelium (RPE) and choroid. The retina is holangiotic with dichotomously branching vessels. These cross, and apparently loop on, the optic disk surface, but no venous circle was seen. Immunohistochemistry suggests a high concentration of cone photoreceptors with red/green cones being more abundant than blue cones. Rod ERG responses were very low with no evident dark adaptation. Mixed rod-cone and cone ERG response amplitudes were low compared to those of domestic pigs, but quite similar to those of minipigs. CONCLUSIONS: To the best of our knowledge, this study describes the collared peccary's retinal features for the first time. A comparison of our findings with data from other ungulate species shows some similarities between the peccary and pig retinas. Further studies are warranted to determine whether the peccary can be used alongside the pig as an animal model in retinal studies.

Electroretinography is a prognostic indicator for postoperative vision in dogs undergoing retinal reattachment surgery.

PURPOSE: To determine whether pre-operative electroretinography (ERG) predicts postoperative vision in dogs undergoing retinal reattachment surgery (RRS). METHODS: This 18-month prospective study recorded signalment, duration, cause, and extent of retinal detachment and pre-operative vision status. Rod and mixed rod-cone ERG responses were recorded prior to RRS. Referring veterinary ophthalmologists assessed vision 2 months postoperatively. RESULTS: Thirty dogs (40 affected eyes) aged 4 months to 12.1 years were included. The detachment extent was 150° -320° in 15 of 40 eyes, 360° in 24 of 40 eyes, and not recorded in one eye. Most dogs had a genetic predisposition for retinal detachment. Eight eyes of seven dogs had previous cataract surgery. Mean estimated duration of detachment prior to surgery was 24.5 ± 19.6 days. Pre-operatively, 34 of 40 eyes were blind, two of 40 eyes were sighted, and four of 40 eyes had severely diminished vision. Compared to normative ERG values in our clinics, pre-operative ERGs were classified as "normal" in five of 40 eyes, "attenuated" in seven of 40 eyes, and "flat" in 28 of 40 eyes. Following RRS, the retina was fully reattached in all operated eyes. Two-month postoperatively, 30 of 40 eyes had "normal" vision as defined by referring veterinary ophthalmologists, six of 40 eyes had "limited" or "diminished" vision and four of 40 eyes were blind. Normal vision was regained in 12 of 12 (100%) of eyes with normal or attenuated pre-operative ERG's, but only in 18 of 28 (64%) of eyes with flat pre-operative ERG 's (Linear-by-linear test, P = 0.029). CONCLUSIONS: A recordable pre-operative ERG, even if attenuated, is associated with return of vision in canine RRS patients, and is a favorable prognostic indicator.

Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 µL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 µL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/or toxic effects.