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Quantification of Optic Nerve Axon Loss Associated With a Relative Afferent Pupillary Defect in the Monkey

Quantification of Optic Nerve Axon Loss Associated With a Relative Afferent Pupillary Defect in... ObjectiveTo quantify the amount of optic nerve axonal loss associated with the presence of a mild relative afferent pupillary defect (RAPD) in an experimental monkey model.MethodsThe right macula of 5 rhesus monkeys (Macaca mulatta) was treated with concentrically enlarging diode laser burns until an RAPD was detected using a transilluminator light and measured with neutral density filters. Intervals between treatments were 3 to 7 days over a period of 2 months. Pupillary responses to light stimulation were recorded with a monocular infrared television pupillometer. Two months after detection of an RAPD, 5 treated and 4 control monkeys underwent euthanasia and enucleation. Histopathologic analysis and quantification of optic nerve axon counts using an image analysis system were performed.ResultsNo RAPD was observed despite an estimated ganglion cell loss of up to 26%. A 0.6 log unit RAPD was present in 5 monkeys when the laser scar incorporated the entire macula within the temporal vascular arcades. One eye had progressive vitreomacular traction with worsening of the RAPD to 1.8 log units without further laser treatment. Histopathologic evaluation disclosed complete loss of the normal retinal architecture within the macula. The average fiber loss for the 4 treated eyes with 0.6 log unit RAPDs compared with fellow eyes was 53.3% (95% confidence interval [CI], 45.0%-61.6%). The average difference in axon counts between untreated pairs of optic nerves was 12.8% (95% CI, 10.0%-15.6%). Optic nerve axon loss between pairs of experimental and control eyes was statistically significant (P<.001).ConclusionIn rhesus monkeys, an RAPD develops after an approximate unilateral loss between 25% and 50% of retinal ganglion cells.Clinical RelevanceOwing to redundancy in the anterior visual pathways, unilateral retinal ganglion cell loss may occur prior to the observation of an RAPD. The presence of an RAPD measuring 0.6 log units implies that significant retinal ganglion cell injury has occurred.ASSESSMENT OF the pupillary reaction to light is one of the few tests of visual function that does not require a subjective patient response. Detection of abnormalities in the pupillary light reflex is performed by alternately illuminating each eye while comparing the velocity and amplitude of the pupillary responses.Asymmetry in this response is referred to as a relative afferent pupillary defect (RAPD) and indicates either unilateral or bilateral asymmetric disease of the anterior visual system.An RAPD can be quantified by sequentially placing optical filters of increasing density in front of the normal eye as a light source alternately illuminates each eye.These filters logarithmically reduce the light input into the normal eye until the pupillary responses are symmetric. Using this technique, the severity of an RAPD can be quantified as the density of the filter required to balance the response of each eye, ranging from 0.3 to 3.0 log units. While an RAPD measuring 0.3 to 0.6 log units might clinically be considered to be within the mild spectrum of disease, it represents a 50% to 87% decrement in light input, respectively.Although the severity of an RAPD does not correlate with reduction in visual acuity, it does correlate with the visual field lossand the anatomic extent of retinal disease.Despite the fundamental clinical importance of the RAPD in assessment of visual function, it is not known how much optic nerve damage is present when one observes an RAPD. The present study quantified the amount of axon loss associated with the presence of a 0.6 log unit RAPD in an experimental animal model (rhesus monkey) of retinal nerve fiber loss produced by unilateral retinal laser photocoagulation.MATERIALS AND METHODSEXPERIMENTAL MODEL AND PUPIL RECORDINGRhesus monkeys (Macaca mulatta) were chosen for use in this study because their eyes closely resemble the human eye in foveal structure, pigmentation, and the pupillary response to light. They were anesthetized and handled for treatment, photography, pupillography, and euthanasia in accordance with standards established by the Association for Research in Vision and Ophthalmology (Rockville, Md) resolution on Use of Animals in Research and US Department of Defense guidelines. Based on 3 prior unpublished observations (Dr Quigley, unpublished data, 1990), the number of axons between monkey eyes can vary by 10%. We estimated the need to treat between 4 and 5 monkeys to detect at least a 20% difference in axonal counts. Prior to laser treatment, all animals underwent eye examination with a hand light, clinical assessment of the pupillary response, measurement of intraocular pressures, dilated fundus examination, and fundus photography using a Kowa fundus camera (Kowa, Torrance, Calif). All monkeys participating in the study had normal findings on eye examination prior to inclusion. Sedation consisted of intramuscular injection of ketamine (10 mg/kg) using a 25-gauge needle. The monkeys were then placed in primate restraint chairs as a means of stabilizing the head.Following baseline pupillometric recording from all animals, the right eye was dilated with 2.5% phenylephrine and 1% tropicamide. The treatment protocol was initiated with an approximately 800-µm laser lesion centered on the nasal aspect of the foveal depression in the papillomacular bundle of the right eye. The laser consisted of an Oculight Diode Laser (Iris Medical Instruments Inc, Mountain View, Calif) administered through an indirect ophthalmoscope delivery system. The laser emission was approximately 810 nm in the infrared part of the optical spectrum. The spot size was 500 µm with duration ranging from 400 to 600 milliseconds and power ranging from 340 to 810 mW. To ensure damage to the ganglion cell and nerve fiber layers, treatments were repeated and intense. Between 2 and 5 days following each treatment, the eyes were examined clinically for the presence of an RAPD. If an RAPD was not present, the eyes were dilated and further laser emissions were administered to the prior treatment area followed by concentric expansion around the lesion. Once the nasal aspect of the lesion reached the optic nerve, the lesion was expanded temporally.Fundus photography was performed periodically. Images were digitized and a photomontage created (Adobe Photoshop 4.0; Adobe Systems Inc, San Jose, Calif). A map of retinal ganglion cell isodensities from monkey eyes published by Perry and Coweywas projected onto each retinal photomontage (Figure 1). This template was referenced to the horizontal diameter of the optic nerve determined in the pupil/optic nerve head histopathologic sections. The area of scarring within each isodensity contour line was outlined and measured using computer software that allows one to capture, display, analyze, and measure images (Scion Image; Scion Corp, Frederick, Md). This area was multiplied by the ganglion cell density for that isocontour. The total estimated ganglion cell loss was the sum of the estimated ganglion cell loss for each area.Figure 1.Photomontage of the lesion prior to (A) and after (B) the development of a relative afferent pupillar defect (RAPD) in monkey 89-135/R99-41 with overlay of a template of retinal ganglion cell density isocontours by Perry and Cowey.Prior to the development of an RAPD, the size of the lesion was 5.82 mm2with a predicted percent ganglion cell loss of 8.9% to 11.1% (Table 2). After the development of an RAPD, the size of the lesion was 37.66 mm2with a predicted percent ganglion cell loss of 29.8% to 37.2%. The actual percent ganglion cell loss was 43.2%.Clinical testing for the presence of an RAPD was performed under ketamine sedation in darkness using a transilluminator light (Welch-Allyn, Skaneateles Falls, NY), illuminating each eye separately while assessing the pupillary response for asymmetry. The transilluminator used a 3.5 V halogen bulb. Following this, the light was alternated back and forth at varying intervals between 1 and 3 seconds. If asymmetry in the pupillary responses was observed, neutral density filters of increasing density were placed in front of the untreated eye while the light was once again alternated back and forth. The density of the filter was recorded when the pupillary responses were symmetric. Pupil recordings were performed in treated monkeys with a monocular infrared television pupillometer (Eye Scan Inc, Burlington, Mass). The source of light photostimulation was a handheld mini-Ganzfeld photostimulator (LKC Technologies Inc, Gaithersburg, Md). The photostimulator provided a square-wave stimulation of 13 candela (cd)/mm2as a 2-second pulse or continuous mode, the signal for which was output to the pupillometer.The recording protocol consisted of illuminating 1 eye while recording the response from the fellow eye in the darkened room. The mini-Ganzfeld photostimulator was used to stimulate an eye for 2 seconds at 3- to 5-second intervals. The photostimulator output signaled to the computer the onset and cessation of the stimulus. Each trial consisted of 6 to 9 stimulations. After a trial, several minutes were allowed to elapse in darkness before conducting another trial. Each eye underwent 3 to 6 trials. The constriction amplitudes were averaged for each eye at examinations performed at the initial observation of an RAPD and 2 weeks later.EUTHANASIA, HISTOPATHOLOGIC ANALYSIS, AND AXON COUNTINGFor euthanasia, monkeys were administered intramuscular ketamine (10 mg/kg) in the caudal thigh muscle using a 25-gauge needle, followed by intravenous pentobarbital sodium via the lateral saphenous vein using a 23-gauge needle. Perfusion fixation was performed after exsanguination through an incision in the femoral artery. An abdominal incision was performed with isolation of the descending aorta approximately 3 inches prior to the bifurcation. A 14-gauge cannula was inserted into the aorta, and a preplaced 4-0 silk suture was tied. After clearing the line with heparinized isotonic sodium chloride solution, infusion with 1% procaine/isotonic sodium chloride solution was performed until the blood began to clear. Then, infusion with approximately 2 liters of 4% paraformaldehyde/2% glutformaldehyde in 0.1mM phosphate buffer (pH 7.4) was performed. The eyes and optic nerves were harvested along with other organs.Following enucleation, globes were placed in fixative after slits were made in the pars plana. The globes were opened by removal of the superior and inferior caps. Pupil/optic nerve sections incorporating the area of laser treatment were prepared and stained with hematoxylin-eosin. Cross sections of the optic nerves were also obtained. A 1-mm thick section of optic nerve was obtained within 3 mm of the posterior surface of the globe and the superior and nasal quadrants marked with single and double razor blade cuts, respectively. These sections were rinsed in cacodylate buffer (pH 7.4), postfixed in 2% osmium tetroxide in cacodylate buffer, dehydrated in alcohol, and embedded in epoxy resin. Cross sections 1-µm thick were cut with an ultramicrotome, mounted on glass slides, and stained with toluidine blue, which allows one to distinguish residual, degenerating myelin bundles from normal ones. Neural bundle areas, from normal profiles, were measured by planimetry on enlarged photographs of each nerve cross section, and each nerve was divided into 16 segments of approximately equal area. Four random 50 × 50 µm areas from each of 16 segments were examined using an image analysis system (Zeiss VIDAS; Carl Zeiss Inc, Thornwood, NY) to determine absolute axon number and fiber diameter. To determine fiber diameter, an algorithm was used by which each myelinated nerve fiber had its geometric center identified and 32 radii drawn to the inner edge of the myelin sheath. The smallest radius was multiplied by 2 to determine the smallest diameter. Fiber diameters were sorted into bins separated by 0.1 µm. Optic nerve axon counts were performed on all specimens and the difference between eyes determined. The difference in axon counts between pairs of eyes from treated monkeys was compared with the difference in axon counts between pairs of eyes from untreated animals using the 2-tailed ttest with unequal variance.RESULTSFive monkeys received concentrically enlarging diode laser treatments to the macula until an RAPD was detected (Table 1). The amount of treatment varied from 390 to 810 mW delivered between 6 and 11 treatment sessions over the course of 3 months. The number of spots per session varied from 1 to 798, with the total number of spots varying from 2262 to 3378 spots per eye. Four eyes experienced hemorrhaging during laser treatment. Two eyes developed small choroidal hemorrhages that resolved. Two eyes had choroidal hemorrhages with extension into the vitreous following laser-induced breaks in the Bruch membrane. The vitreous hemorrhages cleared over 2 weeks. In 1 eye, vitreomacular traction became apparent on subsequent examinations.Table 1. Summary of Treatment*MonkeyNo. of Treatment SessionsTotal Spot No. (Range of Spots per Treatment)Power, mWComplicationsTime From Observation of RAPD to Euthanasia, dTime From Last Treatment (Within Previously Treated Area) to Euthanasia, dSize of RAPD in Log Units at Euthanasia89-133/R99-40112932 (37-783)400-700None60300.689-140/R99-4282262 (37-798)390-810Choroidal hemorrhage with extension into the vitreous, clearing over 10 days46320.689-135/R99-41113378 (1-680)390-700Small choroidal hemorrhage60300.689-126/R99-2762660 (153-652)400-870Small choroidal hemorrhage60300.689-142/R99-43102800 (37-787)400-800Break in Bruch membrane with vitreous hemorrhage, clearing over 2 weeks62621.8*After a relative afferent pupillar defect (RAPD) was observed, further treatment was only applied to the previously treated area.Prior to each successive treatment, the monkeys were examined for the presence of an RAPD. When the treatment area, based on the retinal photomontage, measured an average of 9.43 mm2(3 monkeys: range, 5.82-14.65 mm2) at the nasal aspect of the foveal depression within the maculopapillary bundle, the pupils of treated and untreated monkeys were examined by an observer (M.L.R.) who was masked to treatment status of the monkey and which eye was treated (Table 2). The observer did not detect an RAPD in any of the monkeys at this stage. Estimation of the retinal ganglion cell death at this time, based on the optic nerve axon counts of untreated eyes in the present study and the projection of monkey retinal ganglion cell isodensity mapsonto the retinal photomontage, resulted in a mean estimated percent ganglion cell loss of 16.5% (range, 11.1%-26.0%) (Table 2).Table 2. Estimate of Retinal Area Lesioned at an Interval Prior to Relative Afferent Pupillary Defect (RAPD) in 3 Monkeys and After Development of RAPD in 5 MonkeysMonkey (Size of RAPD)Optic Nerve Head Area, mmLesioned Area, mm2*Ganglion Cells Killed, No.†Ganglion Cell Death, %‡Ganglion Cell Death, %§89-133/R99-40 (no RAPD)2.0414.65290 60120.826.089-133/R99-40 (0.6 log RAPD)1.9047.90494 44735.344.189-140/R99-42 (no RAPD)1.657.83138 0959.912.389-140/R99-42 (0.6 log RAPD)2.0728.36347 23824.831.089-135/R99-41 (no RAPD)1.945.82124 5218.911.189-135/R99-41 (0.6 log RAPD)2.0237.66413 49829.636.989-126/R99-27 (0.6 log RAPD)2.0447.74416 92729.837.289-142/R99-43 (1.8 log RAPD)2.2835.22429 72330.738.3*Lesioned areas were determined after construction of retinal photomontages encompassing the lesion, using the maximal horizontal diameter of the optic nerve as measured in the pupil/optic nerve histopathologic sections.†The number of retinal ganglion cells (RGC) killed was predicted by projection of the RGC isodensity map of Perry and Coweyonto each photomontage. The percentage of RGC death was computed using either the total RGC count of 1 million predicted by Perry and Cowey(‡) and the mean total RGC count of 1 122 012 of all untreated eyes in this study (§).When the lasered area incorporated the entire area within the arcades, a 0.6 log unit RAPD was detected in the treated eye of 5 monkeys (Table 1). All monkeys were euthanized 2 months after the detection of an RAPD. In 4 monkeys, to destroy any possible remaining ganglion cells within the laser scar, a final laser treatment session was applied only within the previously treated area 1 month prior to euthanasia. At the time of euthanasia, the size of the RAPD remained 0.6 log units in all 4 animals. The treated eye of the fifth monkey, which had experienced a vitreous hemorrhage, had no further photocoagulation. This eye developed worsening vitreomacular traction, and a 1.8 log unit RAPD was detected prior to euthanasia.Prior to euthanasia, the lesioned area in all the monkeys incorporated the entire macula within the temporal arcades (Figure 1). Based on reconstruction of the retinal photomontage, the lesions measured an average of 39.38 mm2in 5 monkeys (range, 28.36-47.90 mm2) (Table 2).Pupillography was performed on 5 lesioned monkeys after initial observation of an RAPD and again 2 weeks later. Technically consistent recordings were obtained after stimulation with the mini-Ganzfeld photostimulator. The amplitude of pupillary constriction was decreased an average of 30% in treated eyes in comparison with untreated eyes (Table 3; Figure 2).Table 3. Results of Pupillography With Mini-Ganzfeld Stimulus Performed After Initial Observation of an RAPD (Examination 1) and 2 Weeks Later (Examination 2)*Monkey (Size of RAPD)ExaminationDecrease in Pupillary Constriction Amplitude in Treated Eye vs Untreated Eye, %Average Decrease in Pupillary Constriction Amplitude in Treated Eye vs Untreated Eye, %89-133/R99-40 (0.6 log RAPD)127.136.1245.189-140/R99-42 (0.6 log RAPD)139.428.4217.389-135/R99-41 (0.6 log RAPD)1−1.310.9223.089-126/R99-27 (0.6 log RAPD)139.744.9250.089-142/R99-43 (1.8 log RAPD)137.128.8220.5Overall decrease in pupillary constriction amplitude in treated eye vs untreated eye, %. . .. . .29.8*An overall decrement in the pupillary constriction amplitude of approximately 30% was observed in treated eyes in comparison with untreated eyes. RAPD indicates relative afferent pupillary defect.Figure 2.Pupil recording from monkey 89-140/R99-42 (examination 1) after detection of a 0.6 log unit relative afferent pupillary defect in the right eye on clinical examination. The tracing on the left is performed during stimulation of the left eye for 2 seconds (horizontal bar) with a mini-Ganzfeld stimulus while recording from the right eye. The tracing on the right is performed while stimulating the right eye and recording from the left eye.Following enucleation, histopathologic evaluation of treated eyes disclosed complete loss of the normal retinal architecture within the macula (Figure 3A). The retina was replaced by avascular tissue composed of glial cells and pigmented macrophages. A thin preretinal membrane was present overlying the scar. For an area extending approximately 18mm on either side of the scar, there was loss of the photoreceptor layer with some remaining cells in the inner nuclear and ganglion cell layers. The retinal pigment epithelium was absent in the area of the scar. The underlying choroid was thickened with pigmented cells. The temporal aspect of the optic nerve was atrophic with thinning of the temporal nerve fiber layer. Examination of the optic nerve cross sections disclosed a C-shaped area of atrophy with vacuolization of the nerve fiber bundles and gliosis (Figure 3C). Optic nerve cross sections stained with toluidine blue demonstrated some residual degenerating myelin bundles (Figure 4).Figure 3.Histopathologic analysis of the retina and optic nerve from monkey 89-126/R99-27 (hematoxylin-eosin). A, Within the macula, there was complete loss of the normal retinal architecture (original magnification ×25). The retina was replaced by avascular tissue composed of glial cells and pigmented macrophages (arrows). A focal, thin preretinal membrane was present overlying the scar (arrowheads). The retinal pigment epithelium was absent in the area of the scar. The underlying choroid was thickened with pigmented cells (asterisks). B, The temporal aspect of the optic nerve was atrophic (arrowheads) with thinning of the temporal nerve fiber layer (original magnification ×10). For an area extending approximately 18mm on either side of the scar, there was loss of the photoreceptor layer with some remaining cells in the inner nuclear and ganglion cell layers. C, Microscopic examination of right optic nerve cross sections (original magnification ×10) disclosed a C-shaped area of atrophy (between arrowheads) temporally with vacuolization of the nerve fiber bundles and gliosis.Figure 4.Right optic nerve (monkey 89-140). A, Injured area demonstrates mostly degenerating myelin profiles (arrow) and glial cells (asterisk). Some residual normal myelinated axons (arrowhead) are present, particularly at the margin of injury, as in this area. B, Normal area demonstrates well-formed myelinated axons (arrowhead) (toluidine blue, original magnification ×400).Quantification of axon loss between eyes in experimental monkeys and control monkeys demonstrated a significant axon loss in treated eyes (Table 4). The mean ± SD fiber loss for the 4 treated eyes with 0.6 log unit RAPDs in comparison with fellow eyes was 53.3% ± 8.0% (95% CI, 45.0%-61.6%). Compared with the difference between pairs of untreated eyes of 4 control monkeys, this loss significantly exceeded the normal intereye difference in axon counts of 12.8% ± 2.8% (95% CI, 10.0%-15.6%) (P<.001, 2-tailed ttest). Axon loss was greatest in the temporal sector of the optic nerve (Table 5; Figure 3). Estimation of the retinal ganglion cell death prior to euthanasia based on projection of monkey retinal ganglion cell isodensity maps of Perry and Coweyonto the retinal photomontage resulted ina mean estimated percent ganglion cell loss of 30.0% (range, 24.8%-35.3%) (Table 2), lower than what was observed for axon counts.Table 4. Total Retinal Ganglion Cell Axonal Counts and Mean Axonal Diameters in Treated and Untreated Monkeys*MonkeySize of RAPD, Log UnitsAxon No., Right EyeAxon No., Left EyeAxon No., % Difference†Mean Axon Diameter, Right EyeMean Axon Diameter, Left EyeAxon Diameter, % DifferenceTreated89-133/R99-400.6 RAPD425 476‡1 022 55958.40.8750‡1.020214.289-140/R99-420.6 RAPD520 349‡1 328 17560.80.9553‡1.01425.889-135/R99-410.6 RAPD530 113‡932 75543.20.9072‡0.97436.989-126/R99-270.6 RAPD557 400‡1 135 35750.91.0229‡1.0176−0.589-142/R99-431.8 RAPD405 515‡1 142 46764.50.9881‡0.9249−6.8Mean (SD). . .. . .. . .55.6 (8.5). . .. . .3.9 (8.0)95% CI. . .. . .. . .47.3-63.9. . .. . .. . .UntreatedB652/R98-638no RAPD1 083 4481 281 41015.40.98330.94574.047R/R98-639no RAPD1 031 3941 212 71415.01.07021.08030.9E473/R98-640no RAPD1 113 4481 005 38710.71.00741.09768.284-456/R99-26no RAPD1 088 6121 208 4419.91.04321.08103.5Mean (SD). . .. . .. . .12.8 (2.8). . .. . .4.2 (3.0)95% CI. . .. . .. . .10.0-15.6. . .. . .. . .Pvalue§. . .. . .. . .<.001. . .. . ..95*RAPD indicates relative afferent pupillary defect; CI, confidence interval.†The mean (SD) percent difference in axon No. for the 4 pairs of eyes with a 0.6 log unit RAPD was 53.3% (8.0); 95% CI, 45.0%-61.6%; and P<.001 (compared with untreated eyes, 2-tailed ttest, unequal variance).‡Laser-treated eye.§Compared with untreated eyes, 2-tailed ttest, unequal variance.Table 5. Retinal Ganglion Cell Axonal Counts in Various Sectors of the Optic Nerves in Treated and Untreated MonkeysMonkeySize of RAPD, Log UnitsAxonal CountEyeCentralPeripheralSuperior QuadrantInferior QuadrantNasal QuadrantTemporal QuadrantTreated89-133/R99-400.6 RAPDRight*166 166263 071217 36740 860145 62033 553Left498 699519 029243 440251 996221 894300 26589-140/R99-420.6 RAPDRight*268 310243 76545 788220 245188 48657 632Left582 453749 357351 746325 628248 185411 00189-135/R99-410.6 RAPDRight*250 394279 341140 317124 606237 70849 487Left460 147463 978219 411258 037210 427245 56489-126/R99-270.6 RAPDRight*279 238273 65567 088226 110203 84567 596Left496 505641 356260 055298 164253 429318 90289-142/R99-431.8 RAPDRight*215 935183 231143 98467 995184 1429016Left505 979635 122247 860268 192260 501364 838Percent difference, mean (SD)52.48 (10.44)53.47 (11.76)51.98 (35.00)48.01 (26.50)16.25 (20.44)83.36 (4.82)UntreatedB652/R98-638No RAPDRight522 110551 304222 481273 201220 230369 652Left610 212666 492273 980226 606453 996339 11447R/R98-639No RAPDRight492 325539 435276 278230 304241 579281 535Left603 919605 627282 049288 259275 213365 950E473/R98-640No RAPDRight597 356503 888262 258302 903230 321306 800Left485 012515 760262 907232 557185 545329 24484-456/R99-26No RAPDRight579 071497 998261 735279 887307 794222 491Left623 640584 327296 179286 899283 806339 708Percent difference, mean (SD)4.22 (18.85)11.32 (6.55)8.17 (8.66)−7.06 (22.71)7.78 (32.72)13.84 (19.00)Pvalue.004<.001.05.01.67<.001*Treated eye.Comparison of mean axonal diameters between pairs of eyes in treated monkeys and pairs of eyes in untreated monkeys demonstrated no significant difference (Table 4). The distribution of the diameters for treated and untreated eyes was similar (Figure 5).Figure 5.Graph of axonal diameters of treated right and untreated left eyes.COMMENTThe principal finding of this study is that an RAPD, measuring 0.6 log units, developed after an approximate loss of between 25% and 50% of retinal ganglion cells. To determine the amount of retinal ganglion cell loss using the present model, full-thickness destruction of the retina within a circumscribed area had to be achieved followed by an adequate interval of time for ascending atrophy to take place. Complete destruction of all retinal layers was achieved by repeated laser treatments and confirmed by histopathologic examination. The area of injury was essentially confined to the treatment area as only a thin rim of outer retinal injury, with some remaining inner nuclear and ganglion cell layers was present. The monkeys underwent euthanasia 2 months after the detection of an RAPD. To ensure complete destruction within the scar, the previously treated area was retreated 4 weeks prior to euthanasia. In the squirrel monkey, Andersonobserved that atrophy occurs in the distal axon segment following retinal photocoagulation between 2 and 4 weeks after injury. Four weeks following retinal laser treatment, most of the axon debris had cleared without the appearance of phagocytes.In the present study, the prior heavy treatment within the laser scar over the preceding months, the lack of a change in the RAPD prior to euthanasia, and the ability to distinguish residual degenerating neural bundle profiles from normal profiles in axon counting suggest that theinterval between treatment and euthanasia was adequate and did not result in an underestimation of the extent of damage. Thus, the optic nerve axon counts accurately reflect the extent of terminal ganglion cell injury at the time of euthanasia.Ganglion cell injury beyond the area of treatment may have occurred from either damage to the nerve fibers passing through the treated area or from secondary injury.Although damage to axon processes passing though the treatment area with subsequent descending atrophy may have occurred, this was minimized by expanding the lesion temporally rather than superiorly and inferiorly. In addition, one would expect injury of axons passing through the lesion to be reflected in both the pupillary responses and axon counts. Secondary retinal ganglion cell damage, possibly mediated through glutamate excitotoxicity outside the laser treatment area, may also have occurred. It is difficult to estimate the role this may have played. If the effect was progressive following injury, it was not large enough to alter the size of the RAPD over 8 weeks. If secondary injury took place simultaneous to primary death of the retinal ganglion cells that were photocoagulated, its effect would be expected to be observed in both the pupillary responses and in the axon counts.Overlay of the retinal ganglion cell templates on the retinal photographs resulted in an underestimation of axon loss in comparison with postmortem axon counts. This may be owing to loss of retinal ganglion cells outside the treatment area as discussed or underestimation of the density of retinal ganglion cells in these templates.This model, in which ganglion cell loss was associated with outer retinal damage, differs from most clinical optic neuropathies in which damage is generally isolated to the retinal ganglion cell and nerve fiber layers. The particular advantage of the present model is that it allowed ganglion cell damage to take place in a controlled, graded manner followed by pupil examination. For the present model to be compared with clinically encountered optic neuropathies, it is assumed that photoreceptors within a scotoma do not contribute significant pupillomotor input through lateral transmission of impulses to regions outside the scotoma.In humans, RAPDs of 0.3 log units can easily be detected, and defects as small as 0.1 log units can be measured using cross-polarizing filters. Good correlation has been demonstrated between intraobserver clinical measurements as well as between clinical measurementand automated infrared pupillometry.In the present study, pupillary responses were assessed both clinically and using infrared pupillometry. Assessment of the pupillary responses in this study was performed while these monkeys were sedated with ketamine. Although this may have influenced the pupillary response, we would expect the response of each eye to be equally affected; hence, this would not impair our ability to identify a difference.Regarding clinical assessment, a 0.6 log unit deficit was the smallest RAPD that could be reliably detected in any of the monkeys. The development of a 0.6 log unit RAPD in the setting of significant injury may reflect a threshold effect with regard to ganglion cell loss and pupillary responses, or it may be secondary to technical factors. In humans, the ability to detect subtle abnormalities with the alternating flashlight test is highly dependent on timing. We suspect that the responses of the monkey pupil are different from human responses in that they have a shorter latency and a more rapid recovery phase. Thus, the most sensitive testing paradigm for detecting an RAPD in humans may be different from that used in monkeys.In humans, the RAPD correlates with the anatomic extent of retinal damage in macular degenerationand retinal detachment.In patients with macular degeneration, an RAPD is observed most often with disciform scars larger than 6 disc diameters.In one study of RAPDs in retinal detachments, each peripheral quadrant of detachment contributed to 0.35 log units of defect, whereas detachment of the macula caused an additional 0.68 log units.In another study of retinal detachments, abnormal pupillary responses were uncommon in peripheral detachments and occurred in approximately half of the detachments involving the macula.Although the depth of an RAPD does not correlate with visual acuity, it does correlate with the extent of visual field loss.The correlation between the RAPD and static threshold as measured by perimetryis consistent with the observation that the light reflex is only 0.2 log units above the threshold for light perceptionand closer to the threshold for light perception with larger areas of stimulation.Visual fields have been used to estimate the amount of ganglion cell loss in association with RAPDs by superimposing templates of human ganglion cell densitiesover visual fields. A linear correlation was observed, which predicted that a 0.6 log unit RAPD would be associated with an estimated ganglion cell loss of between 6% and 18%. However, the authors acknowledged that these results should be interpreted with caution because the study was unable to account for the presence of relative scotomas and did not take into account the observation of other investigators that the relationship between ganglion cell loss and visual field loss has been observed to be nonlinear.Bilateral quantification of optic nerve axon loss in humans with RAPDs has been performed. In these cases, the axon loss was severe and bilateral, but asymmetric. For example, in a case of bilateral anterior ischemic optic neuropathy, the axon counts were reduced to 4% of normal in one eye and 28% of normal in the other eye.In 3 cases with compressive lesions of the anterior visual pathways and RAPDs, the reduction in optic nerve axons, compared with normal optic nerves, was 30% and 70%, 9% and 32%, and 0% and 3% (right and left eyes).Thus, in the setting of bilateral optic neuropathies, an RAPD may be present with an estimated retinal ganglion cell reduction by as much as 57% in one eye compared with the other. Although RAPDs were present in these cases, the severity of the RAPDs was not quantified.Although our study does not demonstrate the retinal ganglion cell loss that is sufficient to produce the minimum clinically detectable RAPD of 0.3 log units, it does demonstrate that an RAPD developed when retinal ganglion cell loss was between approximately 25% and 50%. This would be consistent with a threshold effect regarding estimated ganglion cell loss and central visual function. A threshold effect may be observed owing to an exponential relationship between ganglion cell loss and visual function or a limit beyond which this relationship is linear. A nonlinear relationship has been observed with regard to visual acuityand visual fields.Taken together, these studies imply that disease may affect retinal ganglion cells prior to a significant decrease in visual acuity, a mild abnormality on visual field testing, or a mild RAPD.A threshold effect with regard to retinal ganglion cell damage and the RAPD may be explained by redundancy in the anterior visual pathways. The neuroanatomic substrate for this redundancy may be overlapping receptive fields.The relationship between visual function and retinal ganglion cell loss may be influenced by receptive field size and overlap as well as by the pattern of retinal ganglion cell loss, whether it is focal and complete, as in the present study, or diffuse and partial. The receptive field size and overlap varies with the ratio of photoreceptors to retinal ganglion cells, which varies with location (central vs peripheral) and ganglion cell type. The pattern of retinal ganglion cell loss also depends on the pathologic process. Thus, the estimation of retinal ganglion cell loss in association with an RAPD, as determined in the present study, may differ in comparison to another model, such as glaucoma, which results in a different pattern of retinal ganglion cell loss.These data challenge the hypothesis that a mild asymmetry of 53% of crossed fibers compared with 47% of uncrossed fibersunderlies the RAPD seen in optic tract injury.Although asymmetry of fiber crossing at the chiasm is the most plausible explanation for an RAPD with optic tract lesions, as many as 4 other possible explanations are possible: (1) the amount of crossing may be greater than previously estimated, (2) the asymmetry may be greater for pupillomotor fibers,(3) a functional asymmetry may be present that is not represented in anatomical studies, or (4) additional physiologic processes such as inhibition may be playing a role. Nevertheless, optic tract lesions can be associated with RAPDs of 0.3 log units, and a threshold effect might allow for an RAPD to be present in the setting of small amounts of asymmetric injury to the anterior visual pathways.We observed that an RAPD developed when retinal ganglion cell loss was between approximately 25% and 50% in rhesus monkeys. To the extent that this may be extrapolated to clinically encountered optic neuropathies, it implies that unilateral retinal ganglion cell damage may occur prior to the development of an RAPD. Furthermore, the presence of a 0.6 log unit RAPD implies significant injury that may have prognostic significance for a patient's ability to recover from future insults.PLevatinPupillary escape in disease of the retina and optic nerve.Arch Ophthalmol.1959;62:768-779.TACoxPupillography of a relative afferent pupillary defect.Am J Ophthalmol.1986;101:320-324.HSThompsonJJCorbettTACoxHow to measure the relative afferent pupillary defect.Surv Ophthalmol.1981;26:39-42.HSThompsonPMontagueTACoxJJCorbettThe relationship between visual acuity, pupillary defect, and visual field loss.Am J Ophthalmol.1982;93:681-688.LNJohnsonRAHillMJBartholomewCorrelation of afferent pupillary defect with visual field loss on automated perimetry.Ophthalmology.1988;95:1649-1655.RHKardonCLHaupertHSThompsonThe relationship between static perimetry and the relative afferent pupillary defect.Am J Ophthalmol.1993;115:351-356.JABovinoTCBurtonMeasurement of the relative afferent pupillary defect in retinal detachment.Am J Ophthalmol.1980;90:19-21.JCFolkHSThompsonSGFarmerTWO'GormanRFDreyerRelative afferent pupillary defect in eyes with retinal detachment.Ophthalmic Surg.1987;18:757-759.DANewsomeRCMiltonJDGassAfferent pupillary defect in macular degeneration.Am J Ophthalmol.1981;92:396-402.GDFrischPDShawalukDOAdamsRemote nerve fibre bundle alterations in the retina as caused by argon laser photocoagulation.Nature.1974;248:433-435.VHPerryACoweyThe ganglion cell and cone distributions in the monkey's retina: implications for central magnification factors.Vision Res.1985;25:1795-1810.DRAndersonAscending and descending optic atrophy produced experimentally in squirrel monkeys.Am J Ophthalmol.1973;76:693-711.EYolesMSchwartzDegeneration of spared axons following partial white matter lesion: implications for optic neuropathies.Exp Neurol.1998;153:1-7.MJCroweJCBresnahanSLShumanJNMastersMSBeattieApoptosis and delayed degeneration after spinal cord injury in rats and monkeys.Nat Med.1997;3:73-76.IDusartMESchwabSecondary cell death and inflammatory reaction after dorsal hemisection of the rat spinal cord.Eur J Neurosci.1994;6:712-724.RTBartusEYChenGLynchJHKordowerCortical ablation induces spreading calcium-dependent, secondary pathogenesis which can be inhibited by calpain.Exp Neurol.1999;155:315-326.RABellPMWaggonerWMBoydREAkersCEYeeClinical grading of relative afferent pupillary defects.Arch Ophthalmol.1993;111:938-942.WDLagrèzeRHKardonCorrelation of relative afferent pupillary defect and estimated retinal ganglion cell loss.Graefes Arch Clin Exp Ophthalmol.1998;236:401-404.Not AvailableThe light reflex.In: Loewenfeld IE. The Light Reflex in the Pupil. Boston, Mass: Butterworth-Heinemann; 1999:122-135.EAlexandridisSpatial and temporal summation of pupillomotor contraction upon light stimulation in man.Albrecht Von Graefes Arch Klin Exp Ophthalmol.1970;180:12-19.CACurcioKAAllenTopography of ganglion cells in human retina.J Comp Neurol.1990;300:5-25.HAQuigleyGRDunkelbergerWRGreenRetinal ganglion cell atrophy correlated with automated perimetry in human eyes with glaucoma.Am J Ophthalmol.1989;107:453-464.RSHarwerthLCarter-DawsonFShenELSmith IIIMLCrawfordGanglion cell losses underlying visual field defects from experimental glaucoma.Invest Ophthalmol Vis Sci.1999;40:2242-2250.LAKerrigan-BaumrindHAQuigleyMEPeaseDFKerriganRSMitchellNumber of ganglion cells in glaucoma eyes compared with threshold visual field tests in the same persons.Invest Ophthalmol Vis Sci.2000;41:741-748.HAQuigleyNRMillerWRGreenThe pattern of optic nerve fiber loss in anterior ischemic optic neuropathy.Am J Ophthalmol.1985;100:769-776.PSLevinSANewmanHAQuigleyNRMillerA clinicopathologic study of optic neuropathies associated with intracranial mass lesions with quantification of remaining axons.Am J Ophthalmol.1983;95:295-306.LFrisénHAQuigleyVisual acuity in optic atrophy: a quantitative clinicopathological analysis.Graefes Arch Clin Exp Ophthalmol.1984;222:71-74.LFrisénThe neurology of visual acuity.Brain.1980;103:639-670.LFrisénMFrisénA simple relationship between the probability distribution of visual acuity and the density of retinal output channels.Acta Ophthalmol (Copenh).1976;54:437-444.MMeisterMultineuronal codes in retinal signaling.Proc Natl Acad Sci U S A.1996;93:609-614.SHDeVriesCorrelated firing in rabbit retinal ganglion cells.J Neurophysiol.1999;81:908-920.CKupferLChumbleyJ deCDownerQuantitative histology of optic nerve, optic tract and lateral geniculate nucleus of man.J Anat.1967;101:393-401.RABellHSThompsonRelative afferent pupillary defect in optic tract hemianopias.Am J Ophthalmol.1978;85:538-540.PJSavinoMParisNJSchatzLSOrrJJCorbettOptic tract syndrome: a review of 21 patients.Arch Ophthalmol.1978;96:656-663.SANewmanNRMillerOptic tract syndrome: neuro-ophthalmologic considerations.Arch Ophthalmol.1983;101:1241-1250.PSO'ConnorDKasdonTJTrediciDJIvanMarcus Gunn pupil in experimental tract lesions.Ophthalmology.1982;89:160-164.DEliottETCunningham JrNRMillerFourth nerve paresis and ipsilateral relative afferent pupillary defect without significant visual sensory disturbance.J Clin Neuroophthalmol.1991;11:169-172.Accepted for publication February 23, 2001.This work was supported by a departmental grant, Uniformed Services University of Health Sciences, Bethesda, Md (Dr Kerrison); EY02120, National Eye Institute, Bethesda (Dr Quigley); and EY01765, National Eye Institute (Core Facility Grant, Wilmer Institute).We thank Anthony C. Kouzis, PhD, of the Wilmer Eye Institute for statistical consultation.Corresponding author and reprints: John B. Kerrison, MD, Wilford Hall Medical Center, 2200 Bergquist Dr, Suite 1, Lackland AFB, TX 78236 (e-mail: jkerrison@yahoo.com). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA Ophthalmology American Medical Association

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American Medical Association
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Copyright 2001 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
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2168-6165
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2168-6173
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10.1001/archopht.119.9.1333
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Abstract

ObjectiveTo quantify the amount of optic nerve axonal loss associated with the presence of a mild relative afferent pupillary defect (RAPD) in an experimental monkey model.MethodsThe right macula of 5 rhesus monkeys (Macaca mulatta) was treated with concentrically enlarging diode laser burns until an RAPD was detected using a transilluminator light and measured with neutral density filters. Intervals between treatments were 3 to 7 days over a period of 2 months. Pupillary responses to light stimulation were recorded with a monocular infrared television pupillometer. Two months after detection of an RAPD, 5 treated and 4 control monkeys underwent euthanasia and enucleation. Histopathologic analysis and quantification of optic nerve axon counts using an image analysis system were performed.ResultsNo RAPD was observed despite an estimated ganglion cell loss of up to 26%. A 0.6 log unit RAPD was present in 5 monkeys when the laser scar incorporated the entire macula within the temporal vascular arcades. One eye had progressive vitreomacular traction with worsening of the RAPD to 1.8 log units without further laser treatment. Histopathologic evaluation disclosed complete loss of the normal retinal architecture within the macula. The average fiber loss for the 4 treated eyes with 0.6 log unit RAPDs compared with fellow eyes was 53.3% (95% confidence interval [CI], 45.0%-61.6%). The average difference in axon counts between untreated pairs of optic nerves was 12.8% (95% CI, 10.0%-15.6%). Optic nerve axon loss between pairs of experimental and control eyes was statistically significant (P<.001).ConclusionIn rhesus monkeys, an RAPD develops after an approximate unilateral loss between 25% and 50% of retinal ganglion cells.Clinical RelevanceOwing to redundancy in the anterior visual pathways, unilateral retinal ganglion cell loss may occur prior to the observation of an RAPD. The presence of an RAPD measuring 0.6 log units implies that significant retinal ganglion cell injury has occurred.ASSESSMENT OF the pupillary reaction to light is one of the few tests of visual function that does not require a subjective patient response. Detection of abnormalities in the pupillary light reflex is performed by alternately illuminating each eye while comparing the velocity and amplitude of the pupillary responses.Asymmetry in this response is referred to as a relative afferent pupillary defect (RAPD) and indicates either unilateral or bilateral asymmetric disease of the anterior visual system.An RAPD can be quantified by sequentially placing optical filters of increasing density in front of the normal eye as a light source alternately illuminates each eye.These filters logarithmically reduce the light input into the normal eye until the pupillary responses are symmetric. Using this technique, the severity of an RAPD can be quantified as the density of the filter required to balance the response of each eye, ranging from 0.3 to 3.0 log units. While an RAPD measuring 0.3 to 0.6 log units might clinically be considered to be within the mild spectrum of disease, it represents a 50% to 87% decrement in light input, respectively.Although the severity of an RAPD does not correlate with reduction in visual acuity, it does correlate with the visual field lossand the anatomic extent of retinal disease.Despite the fundamental clinical importance of the RAPD in assessment of visual function, it is not known how much optic nerve damage is present when one observes an RAPD. The present study quantified the amount of axon loss associated with the presence of a 0.6 log unit RAPD in an experimental animal model (rhesus monkey) of retinal nerve fiber loss produced by unilateral retinal laser photocoagulation.MATERIALS AND METHODSEXPERIMENTAL MODEL AND PUPIL RECORDINGRhesus monkeys (Macaca mulatta) were chosen for use in this study because their eyes closely resemble the human eye in foveal structure, pigmentation, and the pupillary response to light. They were anesthetized and handled for treatment, photography, pupillography, and euthanasia in accordance with standards established by the Association for Research in Vision and Ophthalmology (Rockville, Md) resolution on Use of Animals in Research and US Department of Defense guidelines. Based on 3 prior unpublished observations (Dr Quigley, unpublished data, 1990), the number of axons between monkey eyes can vary by 10%. We estimated the need to treat between 4 and 5 monkeys to detect at least a 20% difference in axonal counts. Prior to laser treatment, all animals underwent eye examination with a hand light, clinical assessment of the pupillary response, measurement of intraocular pressures, dilated fundus examination, and fundus photography using a Kowa fundus camera (Kowa, Torrance, Calif). All monkeys participating in the study had normal findings on eye examination prior to inclusion. Sedation consisted of intramuscular injection of ketamine (10 mg/kg) using a 25-gauge needle. The monkeys were then placed in primate restraint chairs as a means of stabilizing the head.Following baseline pupillometric recording from all animals, the right eye was dilated with 2.5% phenylephrine and 1% tropicamide. The treatment protocol was initiated with an approximately 800-µm laser lesion centered on the nasal aspect of the foveal depression in the papillomacular bundle of the right eye. The laser consisted of an Oculight Diode Laser (Iris Medical Instruments Inc, Mountain View, Calif) administered through an indirect ophthalmoscope delivery system. The laser emission was approximately 810 nm in the infrared part of the optical spectrum. The spot size was 500 µm with duration ranging from 400 to 600 milliseconds and power ranging from 340 to 810 mW. To ensure damage to the ganglion cell and nerve fiber layers, treatments were repeated and intense. Between 2 and 5 days following each treatment, the eyes were examined clinically for the presence of an RAPD. If an RAPD was not present, the eyes were dilated and further laser emissions were administered to the prior treatment area followed by concentric expansion around the lesion. Once the nasal aspect of the lesion reached the optic nerve, the lesion was expanded temporally.Fundus photography was performed periodically. Images were digitized and a photomontage created (Adobe Photoshop 4.0; Adobe Systems Inc, San Jose, Calif). A map of retinal ganglion cell isodensities from monkey eyes published by Perry and Coweywas projected onto each retinal photomontage (Figure 1). This template was referenced to the horizontal diameter of the optic nerve determined in the pupil/optic nerve head histopathologic sections. The area of scarring within each isodensity contour line was outlined and measured using computer software that allows one to capture, display, analyze, and measure images (Scion Image; Scion Corp, Frederick, Md). This area was multiplied by the ganglion cell density for that isocontour. The total estimated ganglion cell loss was the sum of the estimated ganglion cell loss for each area.Figure 1.Photomontage of the lesion prior to (A) and after (B) the development of a relative afferent pupillar defect (RAPD) in monkey 89-135/R99-41 with overlay of a template of retinal ganglion cell density isocontours by Perry and Cowey.Prior to the development of an RAPD, the size of the lesion was 5.82 mm2with a predicted percent ganglion cell loss of 8.9% to 11.1% (Table 2). After the development of an RAPD, the size of the lesion was 37.66 mm2with a predicted percent ganglion cell loss of 29.8% to 37.2%. The actual percent ganglion cell loss was 43.2%.Clinical testing for the presence of an RAPD was performed under ketamine sedation in darkness using a transilluminator light (Welch-Allyn, Skaneateles Falls, NY), illuminating each eye separately while assessing the pupillary response for asymmetry. The transilluminator used a 3.5 V halogen bulb. Following this, the light was alternated back and forth at varying intervals between 1 and 3 seconds. If asymmetry in the pupillary responses was observed, neutral density filters of increasing density were placed in front of the untreated eye while the light was once again alternated back and forth. The density of the filter was recorded when the pupillary responses were symmetric. Pupil recordings were performed in treated monkeys with a monocular infrared television pupillometer (Eye Scan Inc, Burlington, Mass). The source of light photostimulation was a handheld mini-Ganzfeld photostimulator (LKC Technologies Inc, Gaithersburg, Md). The photostimulator provided a square-wave stimulation of 13 candela (cd)/mm2as a 2-second pulse or continuous mode, the signal for which was output to the pupillometer.The recording protocol consisted of illuminating 1 eye while recording the response from the fellow eye in the darkened room. The mini-Ganzfeld photostimulator was used to stimulate an eye for 2 seconds at 3- to 5-second intervals. The photostimulator output signaled to the computer the onset and cessation of the stimulus. Each trial consisted of 6 to 9 stimulations. After a trial, several minutes were allowed to elapse in darkness before conducting another trial. Each eye underwent 3 to 6 trials. The constriction amplitudes were averaged for each eye at examinations performed at the initial observation of an RAPD and 2 weeks later.EUTHANASIA, HISTOPATHOLOGIC ANALYSIS, AND AXON COUNTINGFor euthanasia, monkeys were administered intramuscular ketamine (10 mg/kg) in the caudal thigh muscle using a 25-gauge needle, followed by intravenous pentobarbital sodium via the lateral saphenous vein using a 23-gauge needle. Perfusion fixation was performed after exsanguination through an incision in the femoral artery. An abdominal incision was performed with isolation of the descending aorta approximately 3 inches prior to the bifurcation. A 14-gauge cannula was inserted into the aorta, and a preplaced 4-0 silk suture was tied. After clearing the line with heparinized isotonic sodium chloride solution, infusion with 1% procaine/isotonic sodium chloride solution was performed until the blood began to clear. Then, infusion with approximately 2 liters of 4% paraformaldehyde/2% glutformaldehyde in 0.1mM phosphate buffer (pH 7.4) was performed. The eyes and optic nerves were harvested along with other organs.Following enucleation, globes were placed in fixative after slits were made in the pars plana. The globes were opened by removal of the superior and inferior caps. Pupil/optic nerve sections incorporating the area of laser treatment were prepared and stained with hematoxylin-eosin. Cross sections of the optic nerves were also obtained. A 1-mm thick section of optic nerve was obtained within 3 mm of the posterior surface of the globe and the superior and nasal quadrants marked with single and double razor blade cuts, respectively. These sections were rinsed in cacodylate buffer (pH 7.4), postfixed in 2% osmium tetroxide in cacodylate buffer, dehydrated in alcohol, and embedded in epoxy resin. Cross sections 1-µm thick were cut with an ultramicrotome, mounted on glass slides, and stained with toluidine blue, which allows one to distinguish residual, degenerating myelin bundles from normal ones. Neural bundle areas, from normal profiles, were measured by planimetry on enlarged photographs of each nerve cross section, and each nerve was divided into 16 segments of approximately equal area. Four random 50 × 50 µm areas from each of 16 segments were examined using an image analysis system (Zeiss VIDAS; Carl Zeiss Inc, Thornwood, NY) to determine absolute axon number and fiber diameter. To determine fiber diameter, an algorithm was used by which each myelinated nerve fiber had its geometric center identified and 32 radii drawn to the inner edge of the myelin sheath. The smallest radius was multiplied by 2 to determine the smallest diameter. Fiber diameters were sorted into bins separated by 0.1 µm. Optic nerve axon counts were performed on all specimens and the difference between eyes determined. The difference in axon counts between pairs of eyes from treated monkeys was compared with the difference in axon counts between pairs of eyes from untreated animals using the 2-tailed ttest with unequal variance.RESULTSFive monkeys received concentrically enlarging diode laser treatments to the macula until an RAPD was detected (Table 1). The amount of treatment varied from 390 to 810 mW delivered between 6 and 11 treatment sessions over the course of 3 months. The number of spots per session varied from 1 to 798, with the total number of spots varying from 2262 to 3378 spots per eye. Four eyes experienced hemorrhaging during laser treatment. Two eyes developed small choroidal hemorrhages that resolved. Two eyes had choroidal hemorrhages with extension into the vitreous following laser-induced breaks in the Bruch membrane. The vitreous hemorrhages cleared over 2 weeks. In 1 eye, vitreomacular traction became apparent on subsequent examinations.Table 1. Summary of Treatment*MonkeyNo. of Treatment SessionsTotal Spot No. (Range of Spots per Treatment)Power, mWComplicationsTime From Observation of RAPD to Euthanasia, dTime From Last Treatment (Within Previously Treated Area) to Euthanasia, dSize of RAPD in Log Units at Euthanasia89-133/R99-40112932 (37-783)400-700None60300.689-140/R99-4282262 (37-798)390-810Choroidal hemorrhage with extension into the vitreous, clearing over 10 days46320.689-135/R99-41113378 (1-680)390-700Small choroidal hemorrhage60300.689-126/R99-2762660 (153-652)400-870Small choroidal hemorrhage60300.689-142/R99-43102800 (37-787)400-800Break in Bruch membrane with vitreous hemorrhage, clearing over 2 weeks62621.8*After a relative afferent pupillar defect (RAPD) was observed, further treatment was only applied to the previously treated area.Prior to each successive treatment, the monkeys were examined for the presence of an RAPD. When the treatment area, based on the retinal photomontage, measured an average of 9.43 mm2(3 monkeys: range, 5.82-14.65 mm2) at the nasal aspect of the foveal depression within the maculopapillary bundle, the pupils of treated and untreated monkeys were examined by an observer (M.L.R.) who was masked to treatment status of the monkey and which eye was treated (Table 2). The observer did not detect an RAPD in any of the monkeys at this stage. Estimation of the retinal ganglion cell death at this time, based on the optic nerve axon counts of untreated eyes in the present study and the projection of monkey retinal ganglion cell isodensity mapsonto the retinal photomontage, resulted in a mean estimated percent ganglion cell loss of 16.5% (range, 11.1%-26.0%) (Table 2).Table 2. Estimate of Retinal Area Lesioned at an Interval Prior to Relative Afferent Pupillary Defect (RAPD) in 3 Monkeys and After Development of RAPD in 5 MonkeysMonkey (Size of RAPD)Optic Nerve Head Area, mmLesioned Area, mm2*Ganglion Cells Killed, No.†Ganglion Cell Death, %‡Ganglion Cell Death, %§89-133/R99-40 (no RAPD)2.0414.65290 60120.826.089-133/R99-40 (0.6 log RAPD)1.9047.90494 44735.344.189-140/R99-42 (no RAPD)1.657.83138 0959.912.389-140/R99-42 (0.6 log RAPD)2.0728.36347 23824.831.089-135/R99-41 (no RAPD)1.945.82124 5218.911.189-135/R99-41 (0.6 log RAPD)2.0237.66413 49829.636.989-126/R99-27 (0.6 log RAPD)2.0447.74416 92729.837.289-142/R99-43 (1.8 log RAPD)2.2835.22429 72330.738.3*Lesioned areas were determined after construction of retinal photomontages encompassing the lesion, using the maximal horizontal diameter of the optic nerve as measured in the pupil/optic nerve histopathologic sections.†The number of retinal ganglion cells (RGC) killed was predicted by projection of the RGC isodensity map of Perry and Coweyonto each photomontage. The percentage of RGC death was computed using either the total RGC count of 1 million predicted by Perry and Cowey(‡) and the mean total RGC count of 1 122 012 of all untreated eyes in this study (§).When the lasered area incorporated the entire area within the arcades, a 0.6 log unit RAPD was detected in the treated eye of 5 monkeys (Table 1). All monkeys were euthanized 2 months after the detection of an RAPD. In 4 monkeys, to destroy any possible remaining ganglion cells within the laser scar, a final laser treatment session was applied only within the previously treated area 1 month prior to euthanasia. At the time of euthanasia, the size of the RAPD remained 0.6 log units in all 4 animals. The treated eye of the fifth monkey, which had experienced a vitreous hemorrhage, had no further photocoagulation. This eye developed worsening vitreomacular traction, and a 1.8 log unit RAPD was detected prior to euthanasia.Prior to euthanasia, the lesioned area in all the monkeys incorporated the entire macula within the temporal arcades (Figure 1). Based on reconstruction of the retinal photomontage, the lesions measured an average of 39.38 mm2in 5 monkeys (range, 28.36-47.90 mm2) (Table 2).Pupillography was performed on 5 lesioned monkeys after initial observation of an RAPD and again 2 weeks later. Technically consistent recordings were obtained after stimulation with the mini-Ganzfeld photostimulator. The amplitude of pupillary constriction was decreased an average of 30% in treated eyes in comparison with untreated eyes (Table 3; Figure 2).Table 3. Results of Pupillography With Mini-Ganzfeld Stimulus Performed After Initial Observation of an RAPD (Examination 1) and 2 Weeks Later (Examination 2)*Monkey (Size of RAPD)ExaminationDecrease in Pupillary Constriction Amplitude in Treated Eye vs Untreated Eye, %Average Decrease in Pupillary Constriction Amplitude in Treated Eye vs Untreated Eye, %89-133/R99-40 (0.6 log RAPD)127.136.1245.189-140/R99-42 (0.6 log RAPD)139.428.4217.389-135/R99-41 (0.6 log RAPD)1−1.310.9223.089-126/R99-27 (0.6 log RAPD)139.744.9250.089-142/R99-43 (1.8 log RAPD)137.128.8220.5Overall decrease in pupillary constriction amplitude in treated eye vs untreated eye, %. . .. . .29.8*An overall decrement in the pupillary constriction amplitude of approximately 30% was observed in treated eyes in comparison with untreated eyes. RAPD indicates relative afferent pupillary defect.Figure 2.Pupil recording from monkey 89-140/R99-42 (examination 1) after detection of a 0.6 log unit relative afferent pupillary defect in the right eye on clinical examination. The tracing on the left is performed during stimulation of the left eye for 2 seconds (horizontal bar) with a mini-Ganzfeld stimulus while recording from the right eye. The tracing on the right is performed while stimulating the right eye and recording from the left eye.Following enucleation, histopathologic evaluation of treated eyes disclosed complete loss of the normal retinal architecture within the macula (Figure 3A). The retina was replaced by avascular tissue composed of glial cells and pigmented macrophages. A thin preretinal membrane was present overlying the scar. For an area extending approximately 18mm on either side of the scar, there was loss of the photoreceptor layer with some remaining cells in the inner nuclear and ganglion cell layers. The retinal pigment epithelium was absent in the area of the scar. The underlying choroid was thickened with pigmented cells. The temporal aspect of the optic nerve was atrophic with thinning of the temporal nerve fiber layer. Examination of the optic nerve cross sections disclosed a C-shaped area of atrophy with vacuolization of the nerve fiber bundles and gliosis (Figure 3C). Optic nerve cross sections stained with toluidine blue demonstrated some residual degenerating myelin bundles (Figure 4).Figure 3.Histopathologic analysis of the retina and optic nerve from monkey 89-126/R99-27 (hematoxylin-eosin). A, Within the macula, there was complete loss of the normal retinal architecture (original magnification ×25). The retina was replaced by avascular tissue composed of glial cells and pigmented macrophages (arrows). A focal, thin preretinal membrane was present overlying the scar (arrowheads). The retinal pigment epithelium was absent in the area of the scar. The underlying choroid was thickened with pigmented cells (asterisks). B, The temporal aspect of the optic nerve was atrophic (arrowheads) with thinning of the temporal nerve fiber layer (original magnification ×10). For an area extending approximately 18mm on either side of the scar, there was loss of the photoreceptor layer with some remaining cells in the inner nuclear and ganglion cell layers. C, Microscopic examination of right optic nerve cross sections (original magnification ×10) disclosed a C-shaped area of atrophy (between arrowheads) temporally with vacuolization of the nerve fiber bundles and gliosis.Figure 4.Right optic nerve (monkey 89-140). A, Injured area demonstrates mostly degenerating myelin profiles (arrow) and glial cells (asterisk). Some residual normal myelinated axons (arrowhead) are present, particularly at the margin of injury, as in this area. B, Normal area demonstrates well-formed myelinated axons (arrowhead) (toluidine blue, original magnification ×400).Quantification of axon loss between eyes in experimental monkeys and control monkeys demonstrated a significant axon loss in treated eyes (Table 4). The mean ± SD fiber loss for the 4 treated eyes with 0.6 log unit RAPDs in comparison with fellow eyes was 53.3% ± 8.0% (95% CI, 45.0%-61.6%). Compared with the difference between pairs of untreated eyes of 4 control monkeys, this loss significantly exceeded the normal intereye difference in axon counts of 12.8% ± 2.8% (95% CI, 10.0%-15.6%) (P<.001, 2-tailed ttest). Axon loss was greatest in the temporal sector of the optic nerve (Table 5; Figure 3). Estimation of the retinal ganglion cell death prior to euthanasia based on projection of monkey retinal ganglion cell isodensity maps of Perry and Coweyonto the retinal photomontage resulted ina mean estimated percent ganglion cell loss of 30.0% (range, 24.8%-35.3%) (Table 2), lower than what was observed for axon counts.Table 4. Total Retinal Ganglion Cell Axonal Counts and Mean Axonal Diameters in Treated and Untreated Monkeys*MonkeySize of RAPD, Log UnitsAxon No., Right EyeAxon No., Left EyeAxon No., % Difference†Mean Axon Diameter, Right EyeMean Axon Diameter, Left EyeAxon Diameter, % DifferenceTreated89-133/R99-400.6 RAPD425 476‡1 022 55958.40.8750‡1.020214.289-140/R99-420.6 RAPD520 349‡1 328 17560.80.9553‡1.01425.889-135/R99-410.6 RAPD530 113‡932 75543.20.9072‡0.97436.989-126/R99-270.6 RAPD557 400‡1 135 35750.91.0229‡1.0176−0.589-142/R99-431.8 RAPD405 515‡1 142 46764.50.9881‡0.9249−6.8Mean (SD). . .. . .. . .55.6 (8.5). . .. . .3.9 (8.0)95% CI. . .. . .. . .47.3-63.9. . .. . .. . .UntreatedB652/R98-638no RAPD1 083 4481 281 41015.40.98330.94574.047R/R98-639no RAPD1 031 3941 212 71415.01.07021.08030.9E473/R98-640no RAPD1 113 4481 005 38710.71.00741.09768.284-456/R99-26no RAPD1 088 6121 208 4419.91.04321.08103.5Mean (SD). . .. . .. . .12.8 (2.8). . .. . .4.2 (3.0)95% CI. . .. . .. . .10.0-15.6. . .. . .. . .Pvalue§. . .. . .. . .<.001. . .. . ..95*RAPD indicates relative afferent pupillary defect; CI, confidence interval.†The mean (SD) percent difference in axon No. for the 4 pairs of eyes with a 0.6 log unit RAPD was 53.3% (8.0); 95% CI, 45.0%-61.6%; and P<.001 (compared with untreated eyes, 2-tailed ttest, unequal variance).‡Laser-treated eye.§Compared with untreated eyes, 2-tailed ttest, unequal variance.Table 5. Retinal Ganglion Cell Axonal Counts in Various Sectors of the Optic Nerves in Treated and Untreated MonkeysMonkeySize of RAPD, Log UnitsAxonal CountEyeCentralPeripheralSuperior QuadrantInferior QuadrantNasal QuadrantTemporal QuadrantTreated89-133/R99-400.6 RAPDRight*166 166263 071217 36740 860145 62033 553Left498 699519 029243 440251 996221 894300 26589-140/R99-420.6 RAPDRight*268 310243 76545 788220 245188 48657 632Left582 453749 357351 746325 628248 185411 00189-135/R99-410.6 RAPDRight*250 394279 341140 317124 606237 70849 487Left460 147463 978219 411258 037210 427245 56489-126/R99-270.6 RAPDRight*279 238273 65567 088226 110203 84567 596Left496 505641 356260 055298 164253 429318 90289-142/R99-431.8 RAPDRight*215 935183 231143 98467 995184 1429016Left505 979635 122247 860268 192260 501364 838Percent difference, mean (SD)52.48 (10.44)53.47 (11.76)51.98 (35.00)48.01 (26.50)16.25 (20.44)83.36 (4.82)UntreatedB652/R98-638No RAPDRight522 110551 304222 481273 201220 230369 652Left610 212666 492273 980226 606453 996339 11447R/R98-639No RAPDRight492 325539 435276 278230 304241 579281 535Left603 919605 627282 049288 259275 213365 950E473/R98-640No RAPDRight597 356503 888262 258302 903230 321306 800Left485 012515 760262 907232 557185 545329 24484-456/R99-26No RAPDRight579 071497 998261 735279 887307 794222 491Left623 640584 327296 179286 899283 806339 708Percent difference, mean (SD)4.22 (18.85)11.32 (6.55)8.17 (8.66)−7.06 (22.71)7.78 (32.72)13.84 (19.00)Pvalue.004<.001.05.01.67<.001*Treated eye.Comparison of mean axonal diameters between pairs of eyes in treated monkeys and pairs of eyes in untreated monkeys demonstrated no significant difference (Table 4). The distribution of the diameters for treated and untreated eyes was similar (Figure 5).Figure 5.Graph of axonal diameters of treated right and untreated left eyes.COMMENTThe principal finding of this study is that an RAPD, measuring 0.6 log units, developed after an approximate loss of between 25% and 50% of retinal ganglion cells. To determine the amount of retinal ganglion cell loss using the present model, full-thickness destruction of the retina within a circumscribed area had to be achieved followed by an adequate interval of time for ascending atrophy to take place. Complete destruction of all retinal layers was achieved by repeated laser treatments and confirmed by histopathologic examination. The area of injury was essentially confined to the treatment area as only a thin rim of outer retinal injury, with some remaining inner nuclear and ganglion cell layers was present. The monkeys underwent euthanasia 2 months after the detection of an RAPD. To ensure complete destruction within the scar, the previously treated area was retreated 4 weeks prior to euthanasia. In the squirrel monkey, Andersonobserved that atrophy occurs in the distal axon segment following retinal photocoagulation between 2 and 4 weeks after injury. Four weeks following retinal laser treatment, most of the axon debris had cleared without the appearance of phagocytes.In the present study, the prior heavy treatment within the laser scar over the preceding months, the lack of a change in the RAPD prior to euthanasia, and the ability to distinguish residual degenerating neural bundle profiles from normal profiles in axon counting suggest that theinterval between treatment and euthanasia was adequate and did not result in an underestimation of the extent of damage. Thus, the optic nerve axon counts accurately reflect the extent of terminal ganglion cell injury at the time of euthanasia.Ganglion cell injury beyond the area of treatment may have occurred from either damage to the nerve fibers passing through the treated area or from secondary injury.Although damage to axon processes passing though the treatment area with subsequent descending atrophy may have occurred, this was minimized by expanding the lesion temporally rather than superiorly and inferiorly. In addition, one would expect injury of axons passing through the lesion to be reflected in both the pupillary responses and axon counts. Secondary retinal ganglion cell damage, possibly mediated through glutamate excitotoxicity outside the laser treatment area, may also have occurred. It is difficult to estimate the role this may have played. If the effect was progressive following injury, it was not large enough to alter the size of the RAPD over 8 weeks. If secondary injury took place simultaneous to primary death of the retinal ganglion cells that were photocoagulated, its effect would be expected to be observed in both the pupillary responses and in the axon counts.Overlay of the retinal ganglion cell templates on the retinal photographs resulted in an underestimation of axon loss in comparison with postmortem axon counts. This may be owing to loss of retinal ganglion cells outside the treatment area as discussed or underestimation of the density of retinal ganglion cells in these templates.This model, in which ganglion cell loss was associated with outer retinal damage, differs from most clinical optic neuropathies in which damage is generally isolated to the retinal ganglion cell and nerve fiber layers. The particular advantage of the present model is that it allowed ganglion cell damage to take place in a controlled, graded manner followed by pupil examination. For the present model to be compared with clinically encountered optic neuropathies, it is assumed that photoreceptors within a scotoma do not contribute significant pupillomotor input through lateral transmission of impulses to regions outside the scotoma.In humans, RAPDs of 0.3 log units can easily be detected, and defects as small as 0.1 log units can be measured using cross-polarizing filters. Good correlation has been demonstrated between intraobserver clinical measurements as well as between clinical measurementand automated infrared pupillometry.In the present study, pupillary responses were assessed both clinically and using infrared pupillometry. Assessment of the pupillary responses in this study was performed while these monkeys were sedated with ketamine. Although this may have influenced the pupillary response, we would expect the response of each eye to be equally affected; hence, this would not impair our ability to identify a difference.Regarding clinical assessment, a 0.6 log unit deficit was the smallest RAPD that could be reliably detected in any of the monkeys. The development of a 0.6 log unit RAPD in the setting of significant injury may reflect a threshold effect with regard to ganglion cell loss and pupillary responses, or it may be secondary to technical factors. In humans, the ability to detect subtle abnormalities with the alternating flashlight test is highly dependent on timing. We suspect that the responses of the monkey pupil are different from human responses in that they have a shorter latency and a more rapid recovery phase. Thus, the most sensitive testing paradigm for detecting an RAPD in humans may be different from that used in monkeys.In humans, the RAPD correlates with the anatomic extent of retinal damage in macular degenerationand retinal detachment.In patients with macular degeneration, an RAPD is observed most often with disciform scars larger than 6 disc diameters.In one study of RAPDs in retinal detachments, each peripheral quadrant of detachment contributed to 0.35 log units of defect, whereas detachment of the macula caused an additional 0.68 log units.In another study of retinal detachments, abnormal pupillary responses were uncommon in peripheral detachments and occurred in approximately half of the detachments involving the macula.Although the depth of an RAPD does not correlate with visual acuity, it does correlate with the extent of visual field loss.The correlation between the RAPD and static threshold as measured by perimetryis consistent with the observation that the light reflex is only 0.2 log units above the threshold for light perceptionand closer to the threshold for light perception with larger areas of stimulation.Visual fields have been used to estimate the amount of ganglion cell loss in association with RAPDs by superimposing templates of human ganglion cell densitiesover visual fields. A linear correlation was observed, which predicted that a 0.6 log unit RAPD would be associated with an estimated ganglion cell loss of between 6% and 18%. However, the authors acknowledged that these results should be interpreted with caution because the study was unable to account for the presence of relative scotomas and did not take into account the observation of other investigators that the relationship between ganglion cell loss and visual field loss has been observed to be nonlinear.Bilateral quantification of optic nerve axon loss in humans with RAPDs has been performed. In these cases, the axon loss was severe and bilateral, but asymmetric. For example, in a case of bilateral anterior ischemic optic neuropathy, the axon counts were reduced to 4% of normal in one eye and 28% of normal in the other eye.In 3 cases with compressive lesions of the anterior visual pathways and RAPDs, the reduction in optic nerve axons, compared with normal optic nerves, was 30% and 70%, 9% and 32%, and 0% and 3% (right and left eyes).Thus, in the setting of bilateral optic neuropathies, an RAPD may be present with an estimated retinal ganglion cell reduction by as much as 57% in one eye compared with the other. Although RAPDs were present in these cases, the severity of the RAPDs was not quantified.Although our study does not demonstrate the retinal ganglion cell loss that is sufficient to produce the minimum clinically detectable RAPD of 0.3 log units, it does demonstrate that an RAPD developed when retinal ganglion cell loss was between approximately 25% and 50%. This would be consistent with a threshold effect regarding estimated ganglion cell loss and central visual function. A threshold effect may be observed owing to an exponential relationship between ganglion cell loss and visual function or a limit beyond which this relationship is linear. A nonlinear relationship has been observed with regard to visual acuityand visual fields.Taken together, these studies imply that disease may affect retinal ganglion cells prior to a significant decrease in visual acuity, a mild abnormality on visual field testing, or a mild RAPD.A threshold effect with regard to retinal ganglion cell damage and the RAPD may be explained by redundancy in the anterior visual pathways. The neuroanatomic substrate for this redundancy may be overlapping receptive fields.The relationship between visual function and retinal ganglion cell loss may be influenced by receptive field size and overlap as well as by the pattern of retinal ganglion cell loss, whether it is focal and complete, as in the present study, or diffuse and partial. The receptive field size and overlap varies with the ratio of photoreceptors to retinal ganglion cells, which varies with location (central vs peripheral) and ganglion cell type. The pattern of retinal ganglion cell loss also depends on the pathologic process. Thus, the estimation of retinal ganglion cell loss in association with an RAPD, as determined in the present study, may differ in comparison to another model, such as glaucoma, which results in a different pattern of retinal ganglion cell loss.These data challenge the hypothesis that a mild asymmetry of 53% of crossed fibers compared with 47% of uncrossed fibersunderlies the RAPD seen in optic tract injury.Although asymmetry of fiber crossing at the chiasm is the most plausible explanation for an RAPD with optic tract lesions, as many as 4 other possible explanations are possible: (1) the amount of crossing may be greater than previously estimated, (2) the asymmetry may be greater for pupillomotor fibers,(3) a functional asymmetry may be present that is not represented in anatomical studies, or (4) additional physiologic processes such as inhibition may be playing a role. 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Kouzis, PhD, of the Wilmer Eye Institute for statistical consultation.Corresponding author and reprints: John B. Kerrison, MD, Wilford Hall Medical Center, 2200 Bergquist Dr, Suite 1, Lackland AFB, TX 78236 (e-mail: jkerrison@yahoo.com).

Journal

JAMA OphthalmologyAmerican Medical Association

Published: Sep 1, 2001

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