Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Determination of morphological, biometric and biochemical susceptibilities in healthy Eurasier dogs with suspected inherited glaucoma.

In both humans and dogs, the primary risk factor for glaucoma is high intraocular pressure (IOP), which may be caused by iridocorneal angle (ICA) abnormalities. Oxidative stress has also been implicated in retinal ganglion cell damage associated with glaucoma. A suspected inherited form of glaucoma was recently identified in Eurasier dogs (EDs), a breed for which pedigrees are readily available. Because of difficulties in assessing ICA morphology in dogs with advanced glaucoma, we selected a cohort of apparently healthy dogsfor the investigation of ICA morphological status, IOP and plasma concentrations of oxidative stress biomarkers. We aimed to establish correlations between these factors, to identify predictive markers of glaucoma in this dog breed. A cohort of 28 subjects, volunteered for inclusion by their owners, was selected by veterinary surgeons. These dogs were assigned to four groups: young males, young females (1-3 years old), adult males and adult females (4-8 years old). Ocular examination included ophthalmoscopy, tonometry, gonioscopy, biometry and ultrasound biomicroscopy (UBM), and the evaluation of oxidative stress biomarkers consisting of measurements of plasma glutathione peroxidase (GP) activity and taurine and metabolic precursor (methionine and cysteine) concentrations in plasma. The prevalence of pectinate ligament abnormalities was significantly higher in adult EDs than in young dogs. Moreover, in adult females, high IOP was significantly correlated with a short axial globe length, and a particularly large distance between Schwalbe's line and the anterior lens capsule. GP activity levels were significantly lower in EDs than in a randomized control group of dogs, and plasma taurine concentrations were higher. Hence, ICA abnormalities were associated with weaker antioxidant defenses in EDs, potentially counteracted by higher plasma taurine concentrations. This study suggests that EDs may constitute an appropriate canine model for the development of glaucoma. This cohort will be used as a sentinel for longitudinal monitoring.

Use of bovine pericardium (Tutopatch®) graft for surgical repair of deep melting corneal ulcers in dogs and corneal sequestra in cats.

OBJECTIVE: To evaluate the efficacy of bovine pericardium (BP) graft in the treatment of deep melting corneal ulcers in three dogs and corneal sequestra in three cats. PROCEDURE: Three dogs with keratomalacia affecting the deep third of the stroma and three cats with corneal sequestrum were evaluated and underwent surgery. Following keratectomy, BP material was placed into the keratectomy bed and sutured to the recipient cornea with 9/0 polyglactin suture material. Postoperative treatment with topical and systemic antibiotics, systemic nonsteroidal anti-inflammatory agents, and topical atropine was prescribed. Follow-up examinations were carried out 1, 2 weeks, 1 and 2 months after the surgery and consisted of a complete ophthalmic examination. Optical coherence tomography (OCT) was performed 1 and 2 months after the surgery in one dog and in one cat. RESULTS: At 1 week, corneal neovascularization was present around the BP graft in all cases. Four weeks after the BP graft, in two dogs and in all cats, the vascularization was regressing and the graft was integrated into the cornea, which was regaining transparency. Topical treatment with anti-inflammatory agents was then prescribed for 2 weeks. Two months after the surgery, 5 of 6 corneas in two dogs and three cats had healed with focal corneal scarring. The remaining dog had progression of the keratomalacia involving the deep BP graft that required additional surgery, but became blind. CONCLUSION: Bovine pericardium graft offers a promising option for surgical reconstruction of the cornea following keratectomy for the management of corneal ulcers and sequestra.

Adjunctive cryotherapy for pigmentary keratitis in dogs: a study of 16 corneas.

OBJECTIVE: To assess whether soft cryotherapy (dimethylether, isobutene, and propane) can remove pigmentation of the cornea that has accumulated under different conditions when conventional therapy has been unsuccessful. ANIMALS STUDIED: Nine dogs with unilateral or bilateral corneal pigmentation (16 eyes) were included based on progressive corneal pigmentation that was unresponsive to long-term etiological treatment. The dogs had keratoconjunctivitis sicca or chronic superficial keratitis. PROCEDURES: A cryogen of 95% dimethylether, 3% isobutane, and 2% propane was applied to the pigmented areas of each cornea under anesthesia. Initial corneal pigmentation and changes were documented over the entire study period using a grading scheme and clinical photographs. RESULTS: Most of the pigment deposits were gone by 5-15 days after cryosurgery. Postoperatively, the dogs showed some corneal edema and corneo-conjunctival inflammation, and three dogs had superficial corneal ulcers; these symptoms had resolved by 1 month after the procedure. Follow-up for more than 90 days was available in five dogs (nine corneas), and we observed total or partial repigmentation when the underlying disease was not controlled. A new cryotherapy procedure was successfully performed in two of these dogs. CONCLUSIONS: Given the sensitivity to cold of melanocytes, cryotherapy is a viable adjunctive treatment for refractory severe corneal pigmentation. Etiological treatment remains necessary to prevent pigmentation from rapidly reappearing. Only a few dogs were followed for more than 90 days; further study is necessary to evaluate the long-term safety and efficacy of soft cryotherapy.