Lipid analysis of flat-mounted eye tissue by imaging mass spectrometry with identification of contaminants in preservation.

Matrix-assisted laser desorption/ionization imaging mass spectrometry was used to analyze donor eye tissue specimens for phospholipid content to evaluate lipid distribution. Phosphatidylcholines and sphingomyelins were detected in the positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. During this study, unknown ion signals in the lower m/z region (less than m/z 400) were detected, mainly in the far periphery of human flat-mounted tissue but not in age-matched rhesus monkey tissue prepared in a similar manner. The unknown ion signals occurred at m/z 304, 332, 360, and 388. These ions were subjected to tandem mass spectrometry directly from the tissue sample, and exact mass measurements of extracts were prepared for further identification. These ions were identified as alkyl dimethylbenzylammonium surfactants (benzalkonium chlorides (BACs)). The classification of these species was verified by comparing an eye tissue extract to an over-the-counter eye-care product containing BACs.

Aqueous humor dynamics in inbred rhesus monkeys with naturally occurring ocular hypertension.

This study evaluates aqueous humor dynamics in rhesus monkeys from the University of Florida inbred colony with ocular normotension and naturally occurring ocular hypertension. Eight monkeys with untreated intraocular pressures (IOPs) of less than 18 mmHg in one eye (ONT group) and seven with untreated IOPs of greater than or equal to 18 mmHg in one eye (OHT group) were included in the study. Assessments included central cornea thickness by ultrasound pachymetry, IOP by tonometry, aqueous flow and outflow facility by fluorophotometry, and uveoscleral outflow by mathematical calculation. Animals were sedated with ketamine for all measurements. Values from the two eyes of each animal were averaged, with the exception of one animal that had only one good eye. Comparisons between groups were made by Student's two-tailed unpaired t-tests. Compared to the ONT group, the OHT group had higher IOPs at all times measured (4:00 PM the day before the study, 21.2 ± 6.5 versus 14.4 ± 1.5 mmHg, p = 0.01; 9:00 AM the day of the study, 20.7 ± 6.6 versus 14.8 ± 1.2 mmHg, p = 0.03; 11:00 AM the day of the study, 16.0 ± 1.6 versus 13.3 ± 2.9 mmHg, p = 0.05) and lower aqueous flow (2.12 ± 0.40 versus 4.54 ± 1.11 µl/min, p = 0.0001), outflow facility (0.17 ± 0.10 versus 0.33 ± 0.07 µl/min/mmHg, p = 0.01) and uveoscleral outflow (p < 0.05). The elevated IOP in inbred Florida rhesus monkeys is a result of significantly reduced outflow facility and uveoscleral outflow. These animals also have slower aqueous flow than the ONT animals which does not contribute to the higher IOP.

Lipid geographical analysis of the primate macula by imaging mass spectrometry.

Imaging mass spectrometry (IMS) provides a unique method to probe for chemical distributions within tissue sections with high chemical specificity. The direct analysis of tissue sections by mass spectrometry, which is the field of IMS, is relatively young, 10 year old; however, the techniques for mass spectrometric analysis are well known. Critical aspects of IMS then are the preparation of tissue specimens for insertion into a vacuum chamber and the interpretation of results with respect to disease studies. Here, we describe the methodologies for geographic localization of phospholipids in flat-mounted eye segments from rhesus monkey using IMS with matrix-assisted laser desorption/ionization (MALDI) and tandem mass spectrometry.

Eye damage control by reduced blue illumination.

The aim of this study was to demonstrate that a blue light and ultraviolet cut-off filter (blue filter) could reduce short-wavelength retina/RPE damage threshold by a continuous spectrum source. Sixteen normal eyes of two rhesus monkeys and six cynomolgus monkeys were subjected to macular irradiation of 20, 24, 27.4, 30, 35, 45, 50 and 60 J/cm(2) energy densities. The values of energy density were measured before the blue filter. Lesions were measured before and at 2 and 30 days after irradiation of a 2.8 mm diameter region within the macular arcade. Measures were fundoscopy, fluorescein angiography and long wavelength scanning by the Heidelberg Retinal Tomograph (HRT) unit. The lesions, which were produced, were scored and compared to irradiant energy density of the blue LED (NSPB500S, Nichia, Tokushima, Japan). The exposure at the 20 J/cm(2) produced no detectable result at 2 or 30 days. Exposure at 35 J/cm(2) showed definite lesion production without blue filter. With the filter added there was one indication of minor change. At 60 J/cm(2) there was extensive heavy, enduring damage without the filter and with the filter damage was present but was significantly attenuated. These results strongly support the conclusion that the blue filter attenuation reduces the frequency of damage by exposure. This experimental system is a useful model for normal human eye aging and continuous spectrum environment irradiance.

The effect of blue light exposure in an ocular melanoma animal model.

BACKGROUND: Uveal melanoma (UM) cell lines, when exposed to blue light in vitro, show a significant increase in proliferation. In order to determine if similar effects could be seen in vivo, we investigated the effect of blue light exposure in a xenograft animal model of UM. METHODS: Twenty New Zealand albino rabbits were injected with 1.0 x 10(6) human UM cells (92.1) in the suprachoroidal space of the right eye. Animals were equally divided into two groups; the experimental group was exposed to blue light, while the control group was protected from blue light exposure. The eyes were enucleated after sacrifice and the proliferation rates of the re-cultured tumor cells were assessed using a Sulforhodamine-B assay. Cells were re-cultured for 1 passage only in order to maintain any in vivo cellular changes. Furthermore, Proliferating Cell Nuclear Antigen (PCNA) protein expression was used to ascertain differences in cellular proliferation between both groups in formalin-fixed, paraffin-embedded eyes (FFPE). RESULTS: Blue light exposure led to a statistically significant increase in proliferation for cell lines derived from intraocular tumors (p < 0.01). PCNA expression was significantly higher in the FFPE blue light treated group when compared to controls (p = 0.0096). CONCLUSION: There is an increasing amount of data suggesting that blue light exposure may influence the progression of UM. Our results support this notion and warrant further studies to evaluate the ability of blue light filtering lenses to slow disease progression in UM patients.

Association of HTRA1 and ARMS2 gene variation with drusen formation in rhesus macaques.

Age-related macular degeneration (AMD) is a clinically heterogeneous disease and constitutes the major cause of visual impairment in the elderly population worldwide. Rhesus macaques also possess a macula and, like humans, develop drusen-associated, age-related macular pathologies. Susceptibility to AMD in humans and drusen formation in rhesus macaques both have been shown to be associated with variation in the HTRA1 and ARMS2 genes. To corroborate these results we genotyped a cohort of 116 rhesus macaques with and without macular drusen. Like in humans, markers in the two genes showed a significant association with drusen formation at the genotype level. Joint haplotype analysis revealed, however, that the disease association observed in rhesus macaques was entirely attributable to a promoter polymorphism (-558G>T) of the HTRA1 gene.

Maculas, monkeys, models, AMD and aging.

Age related macular degeneration (AMD) signs may be found reliably in monkeys (Macaca mulatta) bred selectively in Florida after 14 generations of inbreeding in a closed colony at the University of Puerto Rico. Progression, ultrastructure and functional losses are parallel to those found in humans.

IMPG1 gene variation in rhesus macular drusen.

Drusen is a hallmark of human age-related maculopathy. Rhesus macaques (Macaca mulatta) represent a natural model of age-related maculopathy with drusen. We have already mapped the macular drusen susceptibility locus in rhesus macaques to the homolog of human chromosome 6q14-15 and shown that a particular IMPG1 gene SNP haplotype was apparently associated with drusen formation in the rhesus macaques maintained by the Caribbean Primate Research Center (CPRC), Puerto Rico, USA. The aim of the present study was to verify this finding in the macaques kept at the German Primate Research Center (DPZ), Germany. The study group comprised 64 animals (34 affected, 30 unaffected). These monkeys were genotyped for all known variations in the IMPG1 gene and haplotype analysis was performed. A total absence of the previously identified risk haplotype of the IMPG1 gene, and a much lower drusen prevalence in comparison to the CPRC group, was observed in the DPZ samples. This prompted a re-analysis of the original disease association in the CPRC, which revealed that the implied risk haplotype was in fact a sequencing artifact. Taken together, the data highlight that additional factors, other than IMPG1 variation, must play a role in drusen pathogenesis in rhesus macaques.

Propofol modulates inner retina function in Beagles.

PURPOSE: To further understand a common veterinary anesthetic, propofol (2,6- di-isopropylphenol) and effects of infusion rates on the retinal neurons in Beagle dogs. METHODS: Standard full-field blue xenon-flash stimulation elicited responses of dark-adapted eyes, which were recorded from dogs before and after a propofol infusion rate increase. RESULTS: Electroretinogram b-waves increased significantly after the infusion rate increase and decreased with decline (P < 0.0001). Also, a weak significance (P = 0.041) for a-wave peak amplitude increase was found after infusion rate increase. The initial part (first 18 ms) of the leading edge of the a-wave remained unchanged. No significant differences in times to a- and b-wave peaks were found. CONCLUSION: Enhanced b-wave response and decline is due to sensitivity of postreceptoral cells, possibly interplexiform and amacrine cells, to propofol concentration.

Drusen and lipid-filled retinal pigment epithelium cells in a rhesus macula.

A middle-aged rhesus monkey with detailed clinical history exhibited progression of a macular abnormality with a variety of clinical drusen and pigment changes typical of the Cayo Santiago phenotype. Numerous frozen sections of one sample of the macular retina/retinal pigment epithelium (RPE)/choroid showed a single classical druse but extensive single and clustered lipid-filled RPE cells. The monkey exhibited functional outer retinal decline and an insignificant number of 'window defects' as found among signs of relatively benign human macular aging. The clinical and histologically defined results agree if lipid-filled RPE cells are included among the clinically apparent signs of drusen.

Repeat sample intraocular pressure variance in induced and naturally ocular hypertensive monkeys.

PURPOSE: To compare repeat-sample means variance of laser induced ocular hypertension (OH) in rhesus monkeys with the repeat-sample mean variance of natural OH in age-range matched monkeys of similar and dissimilar pedigrees. MATERIALS & METHODS: Multiple monocular, retrospective, intraocular pressure (IOP) measures were recorded repeatedly during a short sampling interval (SSI, 1-5 months) and a long sampling interval (LSI, 6-36 months). There were 5-13 eyes in each SSI and LSI subgroup. Each interval contained subgroups from the Florida with natural hypertension (NHT), induced hypertension (IHT1) Florida monkeys, unrelated (Strasbourg, France) induced hypertensives (IHT2), and Florida age-range matched controls (C). Repeat-sample individual variance means and related IOPs were analyzed by a parametric analysis of variance (ANOV) and results compared to non-parametric Kruskal-Wallis ANOV. RESULTS: As designed, all group intraocular pressure distributions were significantly different (P < or = 0.009) except for the two (Florida/Strasbourg) induced OH groups. A parametric 2 x 4 design ANOV for mean variance showed large significant effects due to treatment group and sampling interval. Similar results were produced by the nonparametric ANOV. Induced OH sample variance (LSI) was 43x the natural OH sample variance-mean. The same relationship for the SSI was 12x. CONCLUSION: Laser induced ocular hypertension in rhesus monkeys produces large IOP repeat-sample variance mean results compared to controls and natural OH.

Mapping of a macular drusen susceptibility locus in rhesus macaques to the homologue of human chromosome 6q14-15.

Rhesus macaques (Macaca mulatta) are a natural model for retinal drusen formation. The present study aimed at clarifying whether chromosomal regions homologous to candidate genes for drusen formation and progression in humans are also associated with a drusen phenotype in rhesus macaques. Some 42 genetic markers from seven chromosomal regions implicated in macular degeneration syndromes in humans were tested for whether they identified homologous, polymorphic sequences in rhesus DNA. This was found to be the case for seven markers, all of which were subsequently screened for the presence of potentially disease-predisposing alleles in 52 randomly chosen adult animals from the Cayo Santiago population of rhesus macaques (Caribbean Primate Research Center, PR, USA). The high drusen prevalence expected in the Cayo Santiago colony was confirmed in our sample in that 38 animals were found to have drusen (73%). Logistic regression analysis revealed that some alleles of the rhesus homologue of anonymous human marker D6S1036 were consistently over-represented among affected animals. Of two candidate genes located in the respective region, allelic variation in one (IMPG1) showed strong association with drusen formation. We conclude that one or more genes located at the rhesus homologue of human 6q14-15 are likely to play a role in retinal drusen formation, a finding that represents a first step towards the identification of genetic factors implicated in macular drusen formation in rhesus macaques. This is an important tool for the separation of genetic and environmental factors which must occur before satisfactory management methods can be developed.

Pattern electroretinography in a rat model of ocular hypertension: functional evidence for early detection of inner retinal damage.

With the increasing use of the rat as an animal model for glaucoma and for the evaluation of neuroprotective treatments, there is a need for a sensitive test of retinal ganglion cell (RGC) function in this species. The aims of this study were to detect functional abnormalities of the inner retina in a rat model of high intraocular pressure (IOP) using the pattern electroretinogram (PERG), and to correlate them with morphometric analysis of RGC survival and the functional integrity of the inner retina. Unilateral ocular hypertension was induced in 17 Lewis rats through laser photocoagulation. Pattern ERGs were recorded prior to lasering and 3 weeks later, using a series of shifting patterns of decreasing spatial frequency projected directly onto the animals' fundus. IOP was measured at the same intervals, and the number of surviving RGCs estimated. Low amplitude PERG signals could be recorded in response to a narrow grating of 0.368 cycles per degree (cpd), and increased with stimulus size. Lasering caused mean (+/-s.d.) IOP to increase significantly from 18.3+/-4.5 (baseline) to 29.8+/-8.8 mmHg within 3 weeks (p<0.0001). At this time, PERG amplitudes were significantly reduced (p<0.05), declining an average of 45% compared to the normotensive, control eyes. No outer retinal damage was observed, but the mean number of RGCs decreased significantly (p<0.001), from 2 525.0+/-372.4 to 1 542.8+/-333.8 cells per mm2. This decrease in RGC number was significantly (p=0.03) correlated the decrease in PERG amplitude. The correlation between functional integrity of the inner retina and the rat PERG was further demonstrated by intravitreal tetrodotoxin injections, which temporarily abolished the PERG but did not affect outer retinal activity, reflected in the flash ERG. The evidence for early functional deficits, combined with tonometry and documentation of correlated ganglion cells loss, confirms the sensitivity of this diagnostic tool and the validity and importance of this animal model in glaucoma research.

Fundus pigment distribution in rhesus monkeys.

There is growing evidence for an interaction among fundus pigmentation, character, geography and the function and structure of the outer retina. We examined three inbred groups of rhesus macaques (132 eyes) including all ages. One was a smaller (18 eyes) group. Coat colors were variations of brown-tan however, the smaller group had lighter 'golden' coats and colors. Fundus images were classified for pigmentation and its geographic distribution. In golden-coated animals there was bias toward nasal fundus hypopigmentation with the optic disk as a watershed demarcation zone, which extended in the superior-inferior direction. Temporal fundus hypopigmentation did not occur in the absence of nasal hypopigmentation. More common, darker coated samples showed a characteristic diffuse fundus pigmentation. There was no evidence for albinism or large variations in macular pigmentation. Rhesus monkeys can exhibit geographically controlled genetic development of fundus pigmentation. Hypopigmentation provides for access to the choroidal infrastructure.

Functional and structural analysis of the visual system in the rhesus monkey model of optic nerve head ischemia.

PURPOSE: A redistribution of neurochemicals has been identified in the visual cortex of monkeys with laser-induced glaucoma. Examined were functional, structural, and neurochemical changes to the retina, optic nerve, and central visual system in a nonhuman primate model of optic nerve head (ONH) ischemia caused by sustained unilateral administration of endothelin (ET)-1 to the optic nerve. METHOD: ET-1 or sham control solution was delivered by osmotic minipump to the retrolaminar region of one optic nerve of rhesus monkeys (Macaca mulatta) for 1.5 years. ONH topography and blood flow velocity were serially studied with scanning laser tomography and laser Doppler flowmetry, respectively. Retinal and cortical electrophysiologic measurements from pattern-derived stimuli were obtained quarterly. Immunohistochemistry was used to identify the distribution of calbindin (CB) and c-Fos labeled neurons in the visual cortex areas V1 and V2, and lateral geniculate nucleus (LGN). Retinal ganglion cell counts and optic nerve axon density were determined by light microscopy. RESULTS: No significant changes in retinal and ONH morphology, ONH blood flow velocity, and retinal and cortical pattern-derived functional activity were detected. Measurement of CB-positive cell density in V1 and V2 showed a significant decrease in CB labeling to the contralateral side of the ET-1-treated eye (P < 0.04). CB-positive cells were present in the magnocellular layers of the LGN with no differences noticed between the ET-1- and sham-treated eyes. c-Fos-labeled neurons were found in striate area V1 and extrastriateV2 of both groups. No c-Fos labeling was observed in the LGN. CONCLUSIONS: Administering ET-1 to the orbital optic nerve alters neuronal metabolic activity in the visual cortex in rhesus monkeys. Metabolic activity reductions in the visual cortex precede the ability to detect functional and structural alterations in the retina, ONH, and visual cortex in this animal model.

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.

Technical issues in electrodiagnostic recording.

Electroretinogram (ERG) and visual-evoked potentials (VEPs) are used in veterinary ophthalmology to assess the functional integrity of the retina and the central visual pathway. The interpretation of altered electrophysiologic potentials in diseases can be of great diagnostic value, although it is important to be aware of technical factors and the limitations of these techniques which may result in over-interpretation and misinterpretation of the recordings, such that they are similar to those found in disease-related electrophysiologic changes. The recorded potentials represent the differences in voltage between the active and reference electrodes. The ground electrode serves as zero. A differential or instrumentation amplifier selectively amplifies signals of interest while rejecting noise. Differences between inputs are amplified, whereas common signals are rejected in a process called common mode rejection (CMR). In order for CMR to be most effective in reducing noise, the electrode impedances should be balanced. Filters are part of the differential amplifier as they remove unwanted noise of a certain frequency. The frequency bandwidth, or passband, is the range of frequencies between low- and high-frequency filter settings that are not filtered out. Major sources of noise that cause interpretation artifacts are power lines, amplifier noise, physiologic activity, electrochemical electrode noise and circular grounds. Noise reduction is achieved with high amplifier input impedance, balanced electrode impedances, CMR, filters and signal averaging. Maintaining electrodes in good condition, achieving proper contact between electrode and animal, and keeping electrode leads short aid in achieving noise reduction.