OBJECTIVE:To estimate the prevalence of ocular disease and obtain normative ocular data for free-living hummingbirds. ANIMALS STUDIED:Two hundred and sixty-three free-living, adult Hummingbirds from coastal and inland central California were studied, including Anna's (Calypte anna, n = 186) and Black-chinned (Archilochus alexandri; n = 77) hummingbirds. PROCEDURES:Slit lamp biomicroscopy and indirect ophthalmoscopy were performed on all individuals. Rebound tonometry, measurement of horizontal palpebral fissure length, and streak retinoscopy were performed on select individuals. Five conscious Anna's Hummingbirds underwent ocular imaging including fundus photography, digital slit lamp photography, and anterior segment and retinal optical coherence tomography. RESULTS:The prevalence of ocular disease in this population was 2.28%. Ocular imaging revealed a thin cornea, shallow anterior chamber, large lens, and a single central, deep convexiclivate fovea. Mean ± SD intraocular pressure was 11.21 ± 2.23 mm Hg. Mean ± SD eyelid length was 2.59 ± 0.19 mm. All eyes were emmetropic or mildly hyperopic with a mean (range) ± SD refractive error of +0.32 (-0.25 to +1) ± 0.33 diopters. CONCLUSIONS:Consistent with previous reports, these data suggest that hummingbirds have visual characteristics found in predatory and prey species, as well as a low prevalence of spontaneous ocular disease. This work provides a set of reference values and clinical findings that can be used in the future research on hummingbird vision and ocular disease. It also provides representative diagnostic images of normal birds and demonstrates that advanced ocular imaging can be performed on manually restrained hummingbirds without pharmacologic dilation.

Purpose

Non-human primates (NHPs) are useful models for human retinal disease. Chromatic pupillometry has been proposed as a noninvasive method of identifying inherited retinal diseases (IRDs) in humans; however, standard protocols employ time-consuming dark adaptation. We utilized shortened and standard dark-adaptation protocols to compare pupillary light reflex characteristics following chromatic stimulation in rhesus macaques with achromatopsia to wild-type (WT) controls with normal retinal function.

Methods

Nine rhesus macaques homozygous for the p.R656Q mutation (PDE6C HOMs) and nine WT controls were evaluated using chromatic pupillometry following 1-minute versus standard 20-minute dark adaptations. The following outcomes were measured and compared between groups: pupil constriction latency, peak constriction, pupil constriction time, and constriction velocity.

Results

Pupil constriction latency was significantly longer in PDE6C HOMs with red-light (P = 0.0002) and blue-light (P = 0.04) stimulation versus WT controls. Peak constriction was significantly less in PDE6C HOMs with all light stimulation compared to WT controls (P < 0.0001). Pupil constriction time was significantly shorter in PDE6C HOMs versus WT controls with red-light (P = 0.04) and white-light (P = 0.003) stimulation. Pupil constriction velocity was significantly slower in PDE6C HOMs versus WT controls with red-light (P < 0.0001), blue-light (P < 0.0001), and white-light (P = 0.0002) stimulation. Dark adaptation time only significantly affected peak (P = 0.008) and time of pupil constriction (P = 0.02) following blue-light stimulation.

Conclusions

Chromatic pupillometry following 1- and 20-minute dark adaptation is an effective tool for screening NHPs for achromatopsia.

Translational relevance

Rapid identification of NHPs with IRDs will provide animal research models to advance research and treatment of achromatopia in humans.

Purpose

To perform in vivo evaluation of the structural morphology and vascular plexuses of the neurosensory retina and choroid across vertebrate species using swept-source optical coherence tomography (SS-OCT) and SS-OCT angiography (SS-OCTA) imaging.

Methods

A custom-built SS-OCT system with an incorporated flexible imaging arm was used to acquire the three-dimensional (3D) retinal OCT and vascular OCTA data of five different vertebrates: a mouse (C57BL/6J), a rat (Long Evans), a gray short-tailed opossum (Monodelphis domestica), a white sturgeon (Acipenser transmontanus), and a great horned owl (Bubo virginianus).

Results

In vivo structural morphology of the retina and choroid, as well as en face OCTA images of retinal and choroidal vasculature of all species were generated. The retinal morphology and vascular plexuses were similar between rat and mouse, whereas distinct choroidal and paired superficial vessels were observed in the opossum retina. The retinal and vascular structure of the sturgeon, as well as the pecten oculi and overlying the avascular and choroidal vasculature in the owl retina are reported in vivo.

Conclusions

A high-quality two-dimensional and 3D in vivo visualization of the retinal structures and en face visualization of the retina and choroidal vascular plexus of vertebrates was possible. Our studies affirm that SS-OCT and SS-OCTA are viable methods for evaluating the in vivo retinal and choroidal structure across terrestrial, aquatic, and aerial vertebrates.

Translational relevance

In vivo characterization of retinal morphology and vasculature plexus of multiple species using SS-OCT and SS-OCTA imaging can increase the pool of species available as models of human retinal diseases.

Purpose

Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model.

Methods

C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina.

Results

In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis.

Conclusions

Intravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.

Purpose

To define the normal range of central corneal thickness (CCT) and corneal endothelial cell density (ECD) in rhesus macaques (Macaca mulatta) and the effects of age, body weight, sex, and intraocular pressure (IOP) on these parameters.

Methods

Ophthalmic examinations were performed on 144 rhesus macaques without anterior segment pathology. The CCT was measured via ultrasound pachymetry (USP) and specular microscopy, and the ECD was semiautomatically and manually counted using specular microscopy. Rebound tonometry was used to measure IOP. Linear regression and mixed-effects linear regression models were used to evaluate the effects of age, body weight, sex, and IOP on CCT and ECD.

Results

We included 98 females and 46 males with an age range of 0.2 to 29.4 years. The mean CCT by USP and specular microscopy were 483 ± 39 and 463 ± 33 µm, respectively, and were statistically different (P < 0.001). The ECDs were 2717 ± 423 and 2747 ± 438 cells/mm2 by semiautomated and manual analysis, respectively. Corneal endothelial degeneration was identified in one aged rhesus macaque.

Conclusions

The mean USP and specular microscopy CCT values differed significantly, whereas the semiautomatic and manual ECD did not. The CCT was associated with the IOP and sex, whereas the ECD was associated with body weight and age (P < 0.05). As in humans, corneal disease in rhesus macaques is uncommon.

Translational relevance

Establishing reference values is fundamental to use rhesus macaques as a model for corneal disease or to identify toxicity in studies of ocular drugs or devices.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.