Proteomic analysis of equine amniotic membrane: characterization of proteins.

INTRODUCTION: Human amniotic membrane (AM) has been used as a biomaterial for surgical wound skin and ocular surface reconstruction for several years. Currently, equine AM has been used for corneal reconstruction in several animal species, and appears to have the same properties as human AM. Despite the observed positive healing abilities of this tissue in horses with ulcerative keratitis the proteins of equine AM have not been described. OBJECTIVE: To identify proteins known to be associated with corneal healing from frozen equine AM. PROCEDURES: Placentas were acquired from healthy live foal births from a local Thoroughbred breeding farm. The amnion was removed from the chorion by blunt dissection, washed with phosphate-buffered saline (PBS), and treated with 0.05% trypsin and 0.02% ethylene diaminetetraacetic acid in PBS. Amnion was attached to nitrocellulose paper (epithelial side up), and cut into 4 × 4 cm pieces. The sheets were frozen at -80 °C. The protein samples were solubilized, and analyzed by 2D gel electrophoresis and shotgun proteomics. RESULTS: A reference identification map of the equine AM proteins was produced and 149 different proteins were identified. From gel-based proteomics, 49 spots were excised and 43 proteins identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Shotgun proteomics identified 116 proteins with an overlap of 10 proteins in both analyses. CONCLUSIONS: We have described a reference map for equine AM proteins that may provide a background to explain the positive results found in horses with ulcerative keratopathies using this biomaterial.

IOP change undergoing anesthesia in rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

The objective of the study was to examine changes of intraocular pressure (IOP) undergoing anesthesia in Rhesus Macaques (Macaca mulatta) with Laser-induced Ocular Hypertension. Twenty male rhesus macaques (hypertensive glaucoma eye in OD; normal eye in OS) between 6 and 20 years of age were used for the study. The monkeys were anesthetized with ketamine hydrochloride (10 mg/kg intramuscularly) and 1% isoflurane, and then IOP in both eyes was measured by a single investigator using a calibrated Tonopen(TM) applanation tonometer (Mentor, Norwell, MA, U.S.A.). The mean IOP with ketamine anesthesia was 36.70 ± 12.04 (right eye: OD) and 15.88 ± 2.84 (left eye: OS). The mean IOP with isoflurane anesthesia was 19.98 ± 6.67 (right eye: OD) and 15.32 ± 2.15 (left eye: OS). Undergoing isoflurane anesthesia, the IOP of OD was significantly decreased. Conclusively, careful examination of IOP is required to prevent unexpected contraindication on glaucoma patient with isoflurane anesthesia.

Long-term effect of retinal ganglion cell axotomy on the histomorphometry of other cells in the porcine retina.

PURPOSE: To determine the effect of retinal ganglion cell axotomy on the thickness of inner plexiform, inner nuclear, and outer plexiform layers, as well as the densities of short- and middle-to-long-wavelength cones, in the porcine retina. METHODS: Unilateral retinal ganglion cell axotomy was performed in seven domestic pigs by either surgical optic nerve section or peripapillary argon laser photocoagulation. Damage to the retinal vasculature was ruled out with fluorescein angiography. Histologic examination of the retinal tissue was performed nine months later. Cone densities were determined immunohistochemically with the anti-visual pigment antibodies COS-1 and OS-2. Image analysis of semithin retinal cross sections was used to measure the thickness of the retinal layers. The effect of axotomy was quantified by optic nerve axon counts and estimations of retinal ganglion cell counts. The data were compared between the eyes with axotomy and the contralateral normal eye using the nonparametric Wilcoxon rank sum test. RESULTS: Treatment of the peripapillary retina with the argon laser resulted in a median decrease in axon counts and retinal ganglion cell density estimates of 31%. No optic nerve axons and cells resembling retinal ganglion cells were found in the eyes with transected optic nerves. There was no significant difference in either the thickness of any retinal layers or cone densities between axotomized and normal control eyes. CONCLUSION: No signs of retrograde transsynaptic degeneration were observed in porcine retinas nine months after retinal ganglion cell axotomy.

Passive attenuation of cortical pattern evoked potentials with increasing body weight in young male rhesus macaques.

The purpose of our study was to determine changes in amplitudes and implicit times of retinal and cortical pattern evoked potentials with increasing body weight in young, growing rhesus macaques (Macaca mulatta). Retinal and cortical pattern evoked potentials were recorded from 29 male rhesus macaques between 3 and 7 years of age. Thirteen animals were reexamined after 11 months. Computed tomography (CT) was performed on two animals to measure the distance between the location of the skin electrode and the surface of the striate cortex. Spearman correlation coefficients were calculated to describe the relationship between body weights and either root mean square (rms) amplitudes or implicit times. For 13 animals rms amplitudes and implicit times were compared with the Wilcoxon matched pairs signed rank test for recordings taken 11 months apart. Highly significant correlations between increases in body weights and decreases in cortical rms amplitudes were noted in 29 monkeys (p < 0.0005). No significant changes were found in the cortical rms amplitudes in thirteen monkeys over 11 months. Computed tomography showed a large increase of soft tissue thickness over the skull and striate cortex with increased body weight. The decreased amplitude in cortical evoked potentials with weight gain associated with aging can be explained by the increased distance between skin electrode and striate cortex due to soft tissue thickening (passive attenuation).

Flash electroretinography in standing horses using the DTL microfiber electrode.

PURPOSE: The goal of our study was the evaluation of a practical method for the recording of flash electroretinograms (ERGs) in sedated, standing horses with the DTL microfiber electrode. METHODS: The horses were sedated intravenously with detomidine hydrochloride (0.015 mg/kg). The pupil was dilated and the auriculopalpebral nerve was blocked. The ERGs were recorded with the active electrode on the cornea (DTL), the reference electrode near the lateral canthus, and the ground electrode over the occipital bone. The light intensities of the white strobe light were 0.03 cd x s/m2 (scotopic) and 3 cd x s/m2 (scotopic and photopic). Photopic and scotopic single flash and flicker responses to Ganzfeld stimulation were recorded. During the 20-min dark adaptation period the retina was stimulated every 5 min with the 0.03 cd x s/m2 single flash. RESULTS: The median b-wave amplitudes and implicit times were 38 microV and 33 ms (photopic cone-dominated response), 43 microV and 63 ms (5-min dark adaptation), 72 microV and 89 ms (10 min), 147 microV and 103 ms (15 min), 188 microV and 109 ms (20 min, 0.03 cd x s/m2, rod response), and 186 microV and 77 ms (20 min, 3 cd x s/m2, maximal combined rod-cone response). A steady increase in amplitude and implicit time was noted during dark adaptation. No oscillatory potentials could be isolated. CONCLUSIONS: The use of detomidine hydrochloride sedation and the DTL microfiber electrode allowed the recording of good quality ERGs. This protocol should permit the detection of functional problems in the retina without the risk involved with general anesthesia.