Expression of Antimicrobial Peptide Genes in the Canine Amniotic Membrane.

The human amniotic membrane has been successfully used in human ocular reconstruction. Several studies have demonstrated its properties, including antimicrobial features. As a result of the restricted availability of human amniotic membrane for veterinary use, canine amniotic membrane has become an attractive alternative. Clinical studies of the application of canine amniotic membrane in animals and the understanding of its biological properties are limited. This study aimed to determine the expression of peptide genes of natural antimicrobials in canine amniotic membrane. Expressions of canine ß-defensin 1, 102, and 103, and canine Elafin were determined in healthy puppies by real-time quantitative polymerase chain reaction. Canine ß-defensin 1, 103, and Elafin were expressed in all samples, possibly suggesting a role in the innate immune system of normal canine amniotic membrane. Further investigations of protein expression and localization are recommended.

Biological Compositions of Canine Amniotic Membrane and Its Extracts and the Investigation of Corneal Wound Healing Efficacy In Vitro.

The usage of canine amniotic membrane (cAM) is mainly of interest in veterinary ophthalmology. Topical formulations of cAM could deliver the beneficial properties of cAM without the need for surgical intervention. The present study aimed to investigate biological compositions of cAM and its extracts, including their corneal wound healing efficacy. In this study, canine amniotic membrane extract (cAME) and lyophilized canine amniotic membrane extract (cAMX) were developed. Bioactive molecules related to corneal wound healing, including hepatocyte growth factor, tissue inhibitor of metalloproteinase-1 and -2, Thrombospondin-1 and Interleukin-1 receptor antagonist were studied at both gene and protein expression levels. Cell viability and wound healing assays were investigated for the possibility of cAME and cAMX as topical applications. The results demonstrated that all of the relevant genes and proteins were detected in cAM, cAME and cAMX. Both cAME and cAMX showed wound healing properties in vitro and cAME at 1.0 mg/mL concentration appeared to have the best healing efficacy. In conclusion, cAME and cAMX generated for topical use provided promising results in the healing of corneal defects.

Comparative study of healing time of canine non-infectious deep ulcerative keratitis between medical therapy alone and combined treatment with medical therapy and a nictitating membrane flap: A retrospective study.

Background: Canine non-infectious deep ulcerative keratitis is considered a severe ocular disorder that possibly can progress to perforation. Immediate treatment should be directed to stimulate corneal wound healing, control infection, and minimize self-trauma while eliminating the underlying causes. Aim: This retrospective study was aimed to compare the difference in non-infectious deep corneal wound healing time between cases treated with medical therapy alone and those treated with medical therapy combined with a nictitating membrane flap. Methods: The medical records at the Ophthalmology Clinic, Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University between January 2018 and March 2020 were retrospectively reviewed. Sixty-six eyes (from 65 dogs) diagnosed with non-infectious deep ulcerative keratitis from the medical treatment group (n = 34) and the combined treatment group (n = 32) were included. The combined treatment group was prescribed the same conservative medical administrations plus a surgical nictitating membrane flap for 14 days. Results: Healing time was defined as the duration of time from the day that the dog had been diagnosed with deep ulcerative keratitis by a fluorescein staining test to the day that the corneal fluorescein stain was negative. Overall, the mean age of dogs with deep ulcerative keratitis was 10.49 ± 4.7 years. The disease was commonly evident in females more than males. Shih Tzu was the most prevalent dog breed. The corneal healing time between dogs receiving medical therapy alone and those receiving combined treatment was not statistically significant (p = 0.386). Healing times were not significantly different between sex and breed (p = 0.41). The median corneal healing time for dogs older than 10 years in the combined treatments group (29.5 days; ranging from 20 to 46 days) was longer than for those receiving medical therapy alone (21 days; ranging from 9.5 to 30.5 days). Conclusion: Supportive therapy including a nictitating membrane flap is suggested in dogs prone to deep corneal ulcers not involving infection. Even though the healing time is not statistically significant, a nictitating membrane flap acts as a tissue bandage to reduce friction over the cornea, and it also alleviates the healing process by moistening the ocular surface.

Characterization of Cryopreserved Canine Amniotic Membrane.

Amniotic membrane is an effective corneal reconstruction material in veterinary surgery. Cryopreserved amniotic membrane is widely used in practice. Properties of cryopreserved canine amniotic membranes are currently not well studied. This study aimed to compare three properties between canine amniotic membranes cryopreserved for 7 days and 30 days, including tensile strength, transparency, and cell viability. After their respective cryopreservation time, stress-strain curves of the cryopreserved membranes' tensile strength were assessed using a universal testing machine. Both groups produced J-shaped stress-strain curves with statistically comparable parameters, including maximum stress, strain, and Young's modulus. The percentage of cell viability was observed by trypan blue staining under a light microscope. Membrane transparency was tested with a spectrophotometer. Transparency tests showed high levels of light transmission and low haze, with no statistical difference between groups. Cell viability was statistically lower in the 30-day cryopreserved group. Tensile strength and transparency of cryopreserved CAM were not significantly impeded for up to 30 days. For CAM to be used as an alternative corneal transplant material in veterinary and regenerative medicine, further research on cell biology, biomechanical properties of the membrane, and cell viability should be conducted.

Comparison of intraocular pressure in healthy brachycephalic and nonbrachycephalic cats using the Icare® TONOVET Plus rebound tonometer.

OBJECTIVE: To compare intraocular pressure using the Icare® TONOVET Plus rebound tonometer in healthy brachycephalic and nonbrachycephalic cats. ANIMALS STUDIED: Both eyes of 78 healthy cats were investigated in this study. Cats were divided into two groups: brachycephalic (n = 39) and nonbrachycephalic (n = 39). PROCEDURES: Nose position and muzzle ratio were photographically recorded and analyzed. Physical and ophthalmic examinations were performed. Intraocular pressure was measured using the Icare® TONOVET Plus rebound tonometry instrument. Quantitative mean values were statistically compared using an unpaired t-test at a significance level of p < .05. RESULTS: Mean values of the nose position and muzzle ratio were significantly lower in the brachycephalic group (20.14 ± 5.43%, 9.61 ± 3.29%) compared with the nonbrachycephalic group (29.21 ± 4.30%, 13.97 ± 6.01%). The mean intraocular pressure for brachycephalic cats (15.76 ± 0.50 mmHg) was significantly lower (p < .001) than for nonbrachycephalic cats (18.77 ± 0.49 mmHg). CONCLUSIONS: Intraocular pressure was significantly lower in brachycephalic cats using the Icare® TONOVET Plus rebound tonometer. Intraocular pressure values obtained in this study could be used as a guideline for measurements obtained using this tonometry device in healthy brachycephalic and nonbrachycephalic cats.

Comparison of computed tomographic ocular biometry in brachycephalic and non-brachycephalic cats.

BACKGROUND AND AIM: Ocular biometry has been used to evaluate ocular parameters; however, several factors need to be considered. In humans, age and sex have been shown to affect ocular biometry. The main factor that affects feline ocular biometry is the head circumference. At present, several reports have revealed that canine ocular biometry differs among dog breeds. However, there are no reports on normal ocular biometry in cats using computed tomography (CT). Therefore, this study aimed to explore feline ocular parameters between brachycephalic (B) and non-brachycephalic (NB) cats using CT and to evaluate the influence of age or sex of cats on ocular biometry. MATERIALS AND METHODS: Twenty-four normal cats were divided into two groups: B (n=12) and NB (n=12). Each group had an equal number of designated males and females. CT was performed under mechanical restraint without general anesthesia and intravenous contrast enhancement. Ocular biometry, dimensions of the internal structure, including attenuation numbers and extra-ocular structures, were evaluated and compared. RESULTS: B-cats had a significantly wider globe width (GW) than NB-cats (p<0.05). In addition, globe length (GL) and GW were significantly correlated with the age of the cats. Significant correlation between GL and age was observed in all cats (r=0.4867; p<0.05), NB-cats (r=0.8692; p<0.05), and B-cats (r=0.4367; p<0.05), whereas the correlation between GW and age was observed in B-cats only (r=0.7251; p<0.05). For extra-ocular structures, NB-cats had significantly greater orbital depth than B-cats (p<0.05), and orbital diameter was significantly correlated with age in all cats and B-cats (p<0.05). CONCLUSION: CT can be used for ocular biometric evaluation in cats with different skull types. GW was wider in B-cats, whereas the orbital depth was greater in NB-cats. Moreover, GW, GL, and orbital diameter were affected by the age of the cats. This information will be useful for further ocular diagnosis and treatment, especially in prosthetic surgical procedures.

Effect of glycerol concentrations and temperatures on epidermal growth factor protein expression in preserved canine amniotic membrane.

Amniotic membrane has been widely applied as a biological graft in both medical and veterinary practice. In ophthalmology, epidermal growth factor (EGF) in human amniotic membrane (HAM) promotes corneal epithelial cell proliferation and migration, thus it facilitates corneal wound healing. In dogs, with limited cryopreserved HAM availability, different cold glycerol preserving protocols have been developed for the storage canine amniotic membrane (CAM). This study aimed to study protein expression of EGF in CAM preserved with different concentrations of glycerol and storage temperatures, using enzyme-linked immunosorbent assay. CAM preserved in 50% glycerol and 99.5% glycerol and kept at 4 and - 20 °C for 7-30 days were compared. We found that preserving membrane with 50% glycerol at - 20 °C has significantly higher EGF protein expression compared with that at 4 °C (p < 0.05). There was a trend that the storage in 50% glycerol achieved higher EGF protein expression than 99.5% glycerol at both 4 °C and - 20 °C. In conclusion, 50% glycerol at - 20 °C was the best condition to preserve CAM in our study. Therefore, there is likely an alternative method to maintain level of EGF protein expression in preserved CAM.

Characterization of a canine model of autosomal recessive retinitis pigmentosa due to a PDE6A mutation.

PURPOSE: To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. METHODS: Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry, and assay for retinal PDE6 levels and enzymatic activity. RESULTS: The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments stopped, and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells, which appeared to be reduced in number relatively early in the disease process, other inner retinal cells were preserved in the early stages of the disease, although there was marked and early activation of Müller glia. Western blot analysis showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is essential for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. CONCLUSIONS: This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa, and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face of a rapid loss of rod cells.

Assessment of the dark-adaptation time required for recovery of electroretinographic responses in dogs after fundus photography and indirect ophthalmoscopy.

OBJECTIVE: To investigate the duration of dark-adaptation time required for recovery of electroretinographic responses after fundus photography or indirect ophthalmoscopy in dogs. ANIMALS: 6 dogs. PROCEDURE: Initially, scotopic-intensity series of electroretinograms (ERGs) were recorded after 20 minutes of dark adaptation. The fundus of the left eye of each dog was photographed (n = 10) or examined via indirect ophthalmoscopy for 5 minutes with moderate- (117 candela [cd]/m2) or bright-intensity (1,693 cd/m2) light; ERGs were repeated after a further 20 or 60 minutes of dark adaptation (6 procedures/dog). RESULTS: Following 20 minutes of dark adaptation after fundus photography, the b- and a-wave amplitudes were reduced in response to brighter stimuli, compared with pretest ERGs; after 60 minutes of dark adaptation, ERG amplitudes had recovered. Following 20 minutes of dark adaptation after indirect ophthalmoscopy (moderate-intensity light), significantly lower b-wave amplitudes were recorded in response to 2 of the brighter flash stimuli, compared with pretest ERGs; after 60 minutes of dark adaptation, ERG amplitudes had recovered. Following 20 minutes of dark adaptation after indirect ophthalmoscopy (bright-intensity light), all ERG amplitudes were significantly decreased and implicit times were significantly decreased at several flash intensities, compared with pretest ERGs; after 60 minutes of dark adaptation, ERG amplitudes and implicit times had returned to initial values, except for b-wave amplitudes recorded in response to dimmer stimuli. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that at least 60 minutes of dark adaptation should be allowed before ERGs are performed in dogs after fundus photography or indirect ophthalmoscopy.

Influence of recording electrode type and reference electrode position on the canine electroretinogram.

Electroretinography is commonly used to assess the functional integrity of the retina. There are many external variables that can influence the electroretinographic waveforms recorded, and it is important to be aware of these so as not to misinterpret their effects as abnormalities in retinal function. In this study we examined the effect of three different recording electrodes on the ERGs recorded from normal dogs. A bipolar Burian-Allen lens, a monopolar Dawson Trick Litzkow (DTL) fiber electrode, and a monopolar ERG-Jet lens electrode were compared. The effect of altering the distance of the reference electrode from the eye was also examined; using the ERG-Jet lens electrode, the ERG was recorded with the reference electrode placed over the zygomatic arch at 1, 3 and 5 cm caudal to the lateral canthus. The ERGs recorded with the bipolar Burian-Allen lens had significantly lower amplitudes, higher a-wave thresholds and a shallower initial a-wave slope, than those recorded by the two monopolar electrodes. Positioning the reference electrode further from the eye resulted in significantly higher amplitudes. Naka-Rushton fitting and calculation of retinal sensitivity (K) gave significantly different results between the Burian-Allen lens and ERG-Jet lens electrode with the reference electrode 5 cm from the lateral canthus. These results demonstrate that recording electrode type and distance of the reference electrode from the eye significantly affect the ERG tracings of the dog, and may alter the assessment of retinal function that can therefore be derived. Results obtained using these three different types of electrodes cannot be directly compared.