Brow rhytidectomy with or without Stades-like procedure for correction of pseudoptosis and trichiasis-entropion of the upper eyelid in Chow Chow and Shar-Pei dogs: A retrospective study of 27 dogs (2019-2022).

PURPOSE: Heavy brows in Chow Chow and Shar-Pei dogs can be associated with pseudoptosis and trichiasis-entropion of the upper eyelids, causing vision impairment and ocular discomfort. This study describes the outcomes of brow rhytidectomy ± Stades-like procedure to address these anatomical abnormalities. METHODS: Retrospective medical records review (2019-2022). RESULTS: Twenty-seven dogs (n = 15 Chow Chow, n = 12 Shar-Peis) were included, aged 1.9 ± 1.5 years (0.5-6.5 years). Presenting complaints included recurrent episodes of ocular inflammation (n = 13, 48.1%), blepharospasm (n = 10, 37.0%), corneal ulcers (n = 8, 29.6%), entropion (n = 6, 22.2%), and impaired vision (n = 4, 14.8%). Brow skin was removed as follows: (i) First incision parallel and 10-15 mm dorsal to upper lid, slightly extending beyond medial/lateral canthi; (ii) second incision forming an arc with its apex 15-35 mm dorsal to the first incision; and (iii) standard two or three layers closure. In 22/27 dogs, a Stades-like procedure was performed by removing a 2-4 mm strip of skin above eyelid margin and leaving it to heal by secondary intention (no sutures). At last recheck (follow-up 354.5 ± 187.8 days), 19/27 dogs (70.4%) had an adequate eyelid conformation, 7/27 dogs (25.9%) were considered under-corrected, and 1/27 dogs (3.7%) was over-corrected. Most owners (81.5%) were satisfied with the surgical results. Most cases (96.3%) had no recurrence of the clinical signs during the entire follow-up period. CONCLUSIONS: Brow rhytidectomy ± Stades-like procedure provide good cosmetic and clinical outcomes in Chow Chow and Shar-Pei dogs, although under correction may occur in severely affected dogs and with advancing time.

Standard and modified glabellar flap for medial canthus reconstruction following mass removal in three dogs and two cats.

PURPOSE: To describe the surgical technique and clinical outcomes of the glabellar flap and its modification for the reconstruction of the medial canthus following resection of tumors in three dogs and two cats. METHODS: Three dogs (7-, 7-, and 12.5-year-old mixed breeds) and two cats (10- and 14-year-old Domestic shorthair) presented with a 7-13 mm tumor affecting the eyelid and/or conjunctiva in the medial canthal region. Following en bloc mass excision, an inverted V-shaped skin incision was made in the glabellar region (i.e., the area between the eyebrows in humans). The apex of the inverted V-shaped flap was rotated in three cases, whereas a horizontal sliding movement was performed in the other two cases to better cover the surgical wound. The surgical flap was then trimmed to fit the surgical wound and sutured in place in two layers (subcutaneous and cutaneous). RESULTS: Tumors were diagnosed as mast cell tumors (n = 3), amelanotic conjunctival melanoma (n = 1), and apocrine ductal adenoma (n = 1). No recurrence was noted in a follow-up time of 146 ± 84 days. Satisfactory cosmetic outcome with normal eyelids closure was achieved in all cases. Mild trichiasis was present in all patients and mild epiphora was noted in 2/5 patients, but there were no associated clinical signs such as discomfort or keratitis. CONCLUSIONS: The glabellar flap was easy to perform and provided a good outcome in terms of cosmetic, eyelid function, and corneal health. Postoperative complications from trichiasis appear to be minimized by the presence of the third eyelid in this region.

Prevalence and characteristics of ocular diseases in Sphynx cats: A retrospective assessment (2012-2021) and comparison with non-Sphynx cats.

PURPOSE: To describe the prevalence of ocular diseases in Sphynx cats, and compare disease characteristics with other feline breeds. METHODS: Medical records of Sphynx cats presented to an ophthalmology referral center between 1/2012 and 4/2021 were examined. Cats of other breeds examined during the same period served as controls. RESULTS: One hundred ten Sphynx cats were examined during the study period, diagnosed with corneal sequestrum (n = 42 cats), lower eyelid entropion (n = 25), presumed herpetic keratoconjunctivitis (n = 19), conjunctivitis (n = 7), corneal ulcers (n = 6), nasolacrimal duct atresia/obstruction (n = 6), corneal dystrophy (n = 3), eosinophilic keratitis (n = 2), uveitis (n = 2), non-ulcerative keratitis (n = 1), and cataract (n = 1). Corneal sequestrum was significantly more common in Sphynx versus non-Sphynx cats (odds ratio = 8.0, 95% CI = 5.3-12.2, p < .001), and age of diagnosis was significantly younger in Sphynx cats (2.0 ± 1.5 years vs. 5.9 ± 4.0 years, p < .001). Corneal sequestrum recurrence was noted in 19.4% Sphynx eyes and 6.4% non-Sphynx eyes undergoing surgery (p = .015). Lower eyelid entropion-most often bilateral (80%)-was significantly more common in Sphynx versus non-Sphynx cats (odds ratio = 4.1, 95% CI = 2.3-7.1, p < .001), and age of diagnosis was significantly younger in Sphynx cats (0.9 ± 1.4 years vs. 3.5 ± 3.3 years, p < .005). CONCLUSIONS: Corneal sequestrum and entropion were overrepresented and diagnosed at an earlier age in Sphynx cats when compared with the general feline population. Given the high prevalence, early age of onset, and relatively high recurrence of corneal sequestrum in Sphynx cats, further studies are warranted to better understand etiopathogenesis and preferred therapies.

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.

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.

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.

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.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.

Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia.

Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

Flicker cone function in normal and day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation.

PURPOSE: Recently we reported on day blindness in sheep caused by a mutation in the CNGA3 gene, thus making affected sheep a naturally occurring large animal model for therapeutic intervention in CNGA3 achromatopsia patients. The purpose of this study was to characterize flicker cone function in normal and day blind sheep, with the aim of generating a normative data base for ongoing gene therapy studies. METHODS: Electoretinographic (ERG) cone responses were evoked with full-field conditions in 10 normal, 6 heterozygous carriers and 36 day blind sheep. Following light adaptation (10 min, 30 cd/m(2)), responses were recorded at four increasing light intensities (1, 2.5, 5 and 10 cd s/m(2)). At each of these intensities, a single photopic flash response followed by 8 cone flicker responses (10-80 Hz) was recorded. Results were used to generate a normative data base for the three groups. Differences between day blind and normal control animals were tested in two age-matched groups (n = 10 per group). RESULTS: The normal sheep cone ERG wave is bipartite in nature, with critical flicker fusion frequency (CFF) >80 Hz. In all four flash intensities, the single photopic flash a-wave and b-wave amplitudes were significantly lower (p < 0.005), and implicit times significantly delayed (p < 0.0001), in day blind animals. In all four flash intensities, CFF values were significantly lower (p < 0.0001) in day blind sheep. CONCLUSIONS: Cone function is severely depressed in day blind sheep. Our results will provide a normative data base for ongoing gene therapy studies.

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.

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.

Functional and Structural Evaluation of Sildenafil in a Rat Model of Acute Retinal Ischemia/Reperfusion Injury.

PURPOSE: Retinal ischemia is a common cause of visual impairment and blindness. Sildenafil, a PDE5 inhibitor which inhibits cGMP degradation and in turn prolongs the effect of nitric oxide, has been shown to be protective in a number of ischemia/reperfusion (I/R) injuries, as well as in neuronal damage. We hypothesized that treatment with sildenafil might be neuroprotective in a model of acute retinal I/R injury. METHODS: Anterior chamber cannulation was performed to induce unilateral I/R injury in 38 Lewis rats. Animals received intraperitoneal injections of sildenafil (0.5 and 1 mg/kg once a day, for a period of 7 and 18 days, respectively), or saline. Electroretinography recordings, retinal ganglion cell (RGC) counts following retrograde labeling with fluorogold, histopathology, and immunohistochemistry (IHC) using antibodies against PDE5, NOS2, caspase-3, caspase-9, and Bcl-2 were conducted. RESULTS: No significant differences in electroretinography, RGC counts, or retinal morphometry were observed between experimental eyes of sildenafil- and saline-treated animals. A tendency toward less necrosis in histopathology, and a slight trend toward lower PDE5, NOS2, and caspase-9 and higher Bcl-2 IHC scores were evident in experimental retinas of sildenafil-treated animals. CONCLUSIONS: Electroretinography, RGC counts, and retinal morphometry failed to show any neuroprotective effect of sildenafil in acute retinal I/R injury in rats. A slight positive effect of sildenafil was qualitatively indicated by histopathology and IHC.

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.

Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep.

Purpose: Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods: Impaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA. Results: Behavioral assessment revealed day blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams. Conclusions: The c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.

Pam3CSK4/TLR2 signaling elicits neutrophil recruitment and restricts invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine mastitis model.

Mastitis-inflammation of the mammary gland is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria, and Escherichia coli is frequently implicated. Intramammary challenge with bacterial LPS is sufficient to elicit the disease. However, using toll-like receptor (TLR) 4-deficient mice, we previously found that mammary pathogenic E. coli is still able to elicit neutrophil recruitment, indicating the presence of bacterial virulence factors other than LPS. To date, no specific virulence factors have been identified in E. coli isolates associated with mastitis, and other microbe-associated molecular patterns (MAMPs), such as bacterial lipoproteins, are prime candidates. The synthetic analog of bacterial lipopeptides, Pam3CSK4, is recognized by TLR2 and mimics the proinflammatory properties of triacylated lipoproteins of Gram-negative bacteria. The aim of the present work was to determine the role of bacterial lipoproteins recognized by TLR2 on mammary cells as virulence factors in the mammary gland. Using the murine mastitis model, we previously showed that following intramammary LPS challenge, neutrophil recruitment is strictly dependent on alveolar macrophages. Thus, the role of alveolar macrophages in the response to intramammary bacterial lipoprotein challenge was also studied. Here, Pam3CSK4 infusion induced mastitis in wild-type mice, but not in TLR2-deficient mice. The wild-type phenotype was not restored by adoptive transfer of TLR2-expressing macrophages into the alveolar milk space of TLR2-deficient mice, indicating that cells other than alveolar macrophages are essential for Pam3CSK4/TLR2 signaling. In contrast to the Pam3CSK4 treatment, infection with E. coli P4 resulted in inflammation, even in the absence of TLR2 signaling, indicating that lipoproteins are sufficient, but not essential virulence factors in the pathogenesis of the intact bacteria. However, in the absence of TLR2, the infecting E. coli P4 invaded the alveolar epithelial cells and formed intracellular bacterial communities, indicating that intact lipoprotein/TLR2 signaling is essential to restricting bacterial invasion.

ApoE4 induces synaptic and ERG impairments in the retina of young targeted replacement apoE4 mice.

The vertebrate retina, which is part of the central nervous system, is a window into the brain. The present study investigated the extent to which the retina can be used as a model for studying the pathological effects of apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD). Immunohistochemical studies of retinas from young (4 months old) apoE4-targeted replacement mice and from corresponding mice which express the AD benign apoE3 allele, revealed that the density of the perikarya of the different classes of retinal neurons was not affected by apoE4. In contrast, the synaptic density of the retinal synaptic layers, which was assessed immunohistochemically and by immunoblot experiments, was significantly lower in the apoE4 than in the apoE3 mice. This was associated with reduced levels of the presynaptic vesicular glutamatergic transporter, VGluT1, but not of either the GABAergic vesicular transporter, VGaT, or the cholinergic vesicular transporter, VAChT, suggesting that the glutamatergic nerve terminals are preferentially affected by apoE4. In contrast, the post synaptic scaffold proteins PSD-95 and Gephyrin, which reside in excitatory and inhibitory synapses, respectively, were both elevated, and their ratio was not affected by apoE4. Electroretinogram (ERG) recordings revealed significant attenuation of mixed rod-cone responses in dark-adapted eyes of apoE4 mice. These findings suggest that the reduced ERG response in the apoE4 mice may be related to the observed decrease in the retinal nerve terminals and that the retina could be used as a novel model for non-invasive monitoring of the effects of apoE4 on the CNS.