research on rhesus macaques ( macaca mulatta ) followed the guidelines of the arvo statement for the use of animals in ophthalmic and vision research , complied with the national institutes of health ( nih ) guide for the care and use of laboratory animals , and were approved by the university of california - davis institutional animal care and use committee .
the cnprc is accredited by the association for the accreditation and assessment of laboratory animal care ( aaalac ) international .
adult animals were selected from the rhesus macaque colony at cnprc that were undergoing routine semiannual physical examinations .
the animals were sedated with intramuscular injection of ketamine hydrochloride , midazolam , and dexmedetomidine , followed by pupillary dilation with tropicamide .
all subjects underwent complete ophthalmic examination including slit - lamp biomicroscopy , dilated fundus biomicroscopy , streak retinoscopy , a - scan biometry , and rebound tonometry , as well as spectral - domain oct ( sd - oct ) of the macula in edi mode .
animal eyes that showed any retinal or choroidal lesions on exam , or demonstrated refractive error greater than 6 diopters ( d ) spherical equivalent based on streak retinoscopy , were excluded from this study .
research in humans was approved by the institutional review board of the university of california - davis and was performed in accordance with the tenets of the declaration of helsinki .
age - matched human subjects were selected retrospectively from a database search of patients seen in the vitreoretinal service at the university of california - davis eye center who had a normal ophthalmologic examination and underwent edi - oct macular imaging .
these subjects underwent imaging for eyes that were evaluated for possible ocular pathology but in which no abnormalities were found ( n = 9 ) ; or were contralateral eyes in patients with unilateral pathologies , such as epiretinal membrane or vitreomacular traction ( n = 7 ) , retinal tear or retinal detachment ( n = 6 ) , posterior vitreous detachment ( n = 5 ) , choroidal nevus ( n = 1 ) , white without pressure ( n = 1 ) , or open globe injury ( n = 1 ) .
eyes that had any retinal or choroidal diseases , history of vitreoretinal surgery , or myopia greater than 6 d were excluded .
effort was made to age - match human subjects to rhesus macaques ( approximately 3 human years for every year in macaques ) , and to select a similar proportion of patients who identified themselves as white or black race .
for both rhesus macaques and humans , only one eye from each subject was selected for analysis .
if both eyes qualified based on inclusion and exclusion criteria , the right eye was selected for patients with an even - numbered birth month , and the left eye selected for an odd - numbered birth month .
demographic data , including age and sex , were collected for all subjects . for macaques , cycloplegic refraction ( d ) , intraocular pressure ( mm hg ) measured by rebound
tonometry , and axial length ( mm ) on a - scan biometry were also recorded . for human subjects , race ,
lens status ( phakic , pseudophakic , or aphakic ) , best - corrected visual acuity ( bcva ; logmar ) , manifest refraction ( d ) , and intraocular pressure ( mm hg ) measured by applanation tonometry were collected . enhanced - depth imaging 
oct imaging for both humans and rhesus macaques was performed using the spectralis sd - oct device ( heidelberg engineering , heidelberg , germany ) .
oct was performed using a single 30 horizontal line scan with 1536 a - scans per b - scan , centered on the fovea , in high - resolution edi mode . up
to 100 scans ( range , 50100 ) were averaged for each b - scan , using the heidelberg eye tracking automatic real - time ( art ) software .
all imaging on rhesus macaques ( n = 25 ) was performed by the first author ( gy ) at cnprc . for nonhuman primate imaging
, the head and chin rests of the imaging device were removed and replaced with a custom metal bar to allow the animal 's head to rest comfortably .
animals were monitored by a trained technician and a cnprc veterinarian ( lg ) at all times .
the mean age of the animals was 20.3  5.9 years ( range , 1030 years ) , with 14 females and 11 male primates .
study eyes included 14 right eyes ( 56% ) and 11 left eyes ( 44% ) .
mean cycloplegic refraction was + 0.3  2.0 d spherical equivalent , mean intraocular pressure was 18.0  4.1 mm hg , and mean axial length was 19.7  1.4 mm . for humans ( n = 30 ) ,
edi - oct was performed by trained ophthalmic photographers at the uc davis eye center .
the mean age of the subjects was 62.8  17.3 ( range , 3188 years ) , with 15 males and 15 female subjects .
eighteen were self - reported as white or caucasian , while 12 were black or african american .
of the study eyes , 17 were right eyes ( 56.7% ) and 13 were left eyes ( 43.3% ) ; 27 were phakic ( 90% ) , and the remainder were pseudophakic .
mean logmar bcva was 0.11  0.13 ( snellen equivalent 20/25.8 ) , mean refractive error was + 0.19  2.0 d , and mean intraocular pressure was 15.1  3.4 mm hg .
images were exported from heidelberg explorer software ( version 1.8.6.0 , heidelberg engineering ) to imagej software ( version 1.49v ; http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) to generate vertical reflectivity profiles at the foveal center . for each eye ,
reflectivity values were averaged over 10 adjacent a - scans , centered at the fovea , and presented on a scale from 0 to 1.00 grayscale units after normalization to the peak of the hyperreflective band corresponding to the rpe / bruch 's membrane , which was assigned to a value of 1.0 .
reflectivity profiles from different eyes were aligned along the posterior border of this hyperreflective rpe / bruch 's membrane band for averaging .
visualization of the choriocapillaris layer and the csj was qualitatively graded as percent of the linear distance over which the layer boundaries were visible , and classified as clear ( 75% visibility ) , intermediate ( 25% and < 75% visibility ) , or unclear ( < 25% visibility ) by two experienced graders ( gy and vv ) who were masked to species and subject demographic .
choriocapillary and choroidal thicknesses were measured after semiautomated segmentation of the central 3 mm of the macula ( 1.5 mm nasal1.5 mm temporal to the fovea ) using duke optical coherence tomography retinal analysis program ( doctrap , version 62.0 ; durham , nc , usa ) , a custom image segmentation software designed using matlab ( mathworks , natick , ma , usa ) . for thickness measurements , the inner boundary was automatically determined by doctrap as the outer border of the hyperreflective rpe / bruch 's complex ; and two masked graders manually traced the outer border of the hyporeflective choriocapillaris layer to measure the choriocapillary thickness , and the outer border of the choroid stroma , corresponding to the csj , to measure the choroidal thickness .
all thickness measurements were determined from the mean of the two graders ' measurements , and reproducibility was determined by measuring intergrader reliability using intraclass correlations ( icc ) by the bland - altman method .
histologic sections from rhesus macaque eyes ( n = 6 , mean age 19.2  7.9 years ) were taken from age - matched animals at cnprc undergoing necropsy for nonocular health issues .
eyes were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde for 24 to 48 hours , transferred to 4% formalin , then embedded in paraffin and stained with standard hematoxylin - eosin .
histologic sections from age - matched adult postmortem human eyes ( n = 10 , mean age 52.8  23.5 years ) were selected from an archive of cadaveric eyes with no known ocular diseases at the duke university medical center ( courtesy of allan proia ) . all sections also underwent standard fixation in formalin , paraffin embedding , and staining with hematoxylin - eosin . among the eyes analyzed ,
five were from white subjects ( mean age 63.2  18.9 years , range , 3571 years , three males / two females ) , and five were from black subjects ( mean age 42.4  24.7 years , range , 2583 years , three males / two females ) .
to quantify the cell density , size , and pigmentation of choroidal melanocytes in histologic sections , three 100  100-m representative regions of choroid stroma were selected for each study eye from images of histologic sections captured at 20 using an eclipse te 200 ( nikon , tokyo , japan ) microscope .
we then performed automated color - based segmentation using the lab color space and k - means clustering in matlab ( mathworks ) to isolate the pigmented melanocytes .
the number of pigmented cells in each field was manually counted to determine melanocyte density ( cells per 10,000 m ) .
average melanocyte size was calculated by dividing the total segmented area of melanin pigment by the cell number in each field .
finally , uveal pigmentation was evaluated by measuring the average luminosity ( l - component of the lab color space ) of the segmented areas from each image as an estimate of optical density ( supplementary fig .
the relationship of choriocapillaris or csj visibility to species or race was determined using the kruskal - wallis test .
multivariate ordinal logistic regression was used to determine the independent association of age , race , and refractive error with choriocapillaris or csj visibility .
choriocapillary and choroidal thickness measurements , as well as differences in cell density , size , and pigmentation of melanocytes , were compared using student 's t - tests .
all statistical analyses were performed using spss statistics ( version 22 ; ibm , armonk , ny , usa ) .
research on rhesus macaques ( macaca mulatta ) followed the guidelines of the arvo statement for the use of animals in ophthalmic and vision research , complied with the national institutes of health ( nih ) guide for the care and use of laboratory animals , and were approved by the university of california - davis institutional animal care and use committee .
the cnprc is accredited by the association for the accreditation and assessment of laboratory animal care ( aaalac ) international .
adult animals were selected from the rhesus macaque colony at cnprc that were undergoing routine semiannual physical examinations .
the animals were sedated with intramuscular injection of ketamine hydrochloride , midazolam , and dexmedetomidine , followed by pupillary dilation with tropicamide .
all subjects underwent complete ophthalmic examination including slit - lamp biomicroscopy , dilated fundus biomicroscopy , streak retinoscopy , a - scan biometry , and rebound tonometry , as well as spectral - domain oct ( sd - oct ) of the macula in edi mode .
animal eyes that showed any retinal or choroidal lesions on exam , or demonstrated refractive error greater than 6 diopters ( d ) spherical equivalent based on streak retinoscopy , were excluded from this study .
research in humans was approved by the institutional review board of the university of california - davis and was performed in accordance with the tenets of the declaration of helsinki .
age - matched human subjects were selected retrospectively from a database search of patients seen in the vitreoretinal service at the university of california - davis eye center who had a normal ophthalmologic examination and underwent edi - oct macular imaging .
these subjects underwent imaging for eyes that were evaluated for possible ocular pathology but in which no abnormalities were found ( n = 9 ) ; or were contralateral eyes in patients with unilateral pathologies , such as epiretinal membrane or vitreomacular traction ( n = 7 ) , retinal tear or retinal detachment ( n = 6 ) , posterior vitreous detachment ( n = 5 ) , choroidal nevus ( n = 1 ) , white without pressure ( n = 1 ) , or open globe injury ( n = 1 ) .
eyes that had any retinal or choroidal diseases , history of vitreoretinal surgery , or myopia greater than 6 d were excluded .
effort was made to age - match human subjects to rhesus macaques ( approximately 3 human years for every year in macaques ) , and to select a similar proportion of patients who identified themselves as white or black race .
for both rhesus macaques and humans , only one eye from each subject was selected for analysis .
if both eyes qualified based on inclusion and exclusion criteria , the right eye was selected for patients with an even - numbered birth month , and the left eye selected for an odd - numbered birth month .
demographic data , including age and sex , were collected for all subjects . for macaques , cycloplegic refraction ( d ) , intraocular pressure ( mm hg ) measured by rebound
tonometry , and axial length ( mm ) on a - scan biometry were also recorded . for human subjects , race ,
lens status ( phakic , pseudophakic , or aphakic ) , best - corrected visual acuity ( bcva ; logmar ) , manifest refraction ( d ) , and intraocular pressure ( mm hg ) measured by applanation tonometry were collected .
enhanced - depth imaging  oct imaging for both humans and rhesus macaques was performed using the spectralis sd - oct device ( heidelberg engineering , heidelberg , germany ) .
oct was performed using a single 30 horizontal line scan with 1536 a - scans per b - scan , centered on the fovea , in high - resolution edi mode . up
to 100 scans ( range , 50100 ) were averaged for each b - scan , using the heidelberg eye tracking automatic real - time ( art ) software .
all imaging on rhesus macaques ( n = 25 ) was performed by the first author ( gy ) at cnprc . for nonhuman primate imaging
, the head and chin rests of the imaging device were removed and replaced with a custom metal bar to allow the animal 's head to rest comfortably .
animals were monitored by a trained technician and a cnprc veterinarian ( lg ) at all times .
the mean age of the animals was 20.3  5.9 years ( range , 1030 years ) , with 14 females and 11 male primates .
study eyes included 14 right eyes ( 56% ) and 11 left eyes ( 44% ) .
mean cycloplegic refraction was + 0.3  2.0 d spherical equivalent , mean intraocular pressure was 18.0  4.1 mm hg , and mean axial length was 19.7  1.4 mm .
for humans ( n = 30 ) , edi - oct was performed by trained ophthalmic photographers at the uc davis eye center . the mean age of the subjects was 62.8  17.3 ( range , 3188 years ) , with 15 males and 15 female subjects .
eighteen were self - reported as white or caucasian , while 12 were black or african american .
of the study eyes , 17 were right eyes ( 56.7% ) and 13 were left eyes ( 43.3% ) ; 27 were phakic ( 90% ) , and the remainder were pseudophakic .
mean logmar bcva was 0.11  0.13 ( snellen equivalent 20/25.8 ) , mean refractive error was + 0.19  2.0 d , and mean intraocular pressure was 15.1  3.4 mm hg .
images were exported from heidelberg explorer software ( version 1.8.6.0 , heidelberg engineering ) to imagej software ( version 1.49v ; http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) to generate vertical reflectivity profiles at the foveal center . for each eye ,
reflectivity values were averaged over 10 adjacent a - scans , centered at the fovea , and presented on a scale from 0 to 1.00 grayscale units after normalization to the peak of the hyperreflective band corresponding to the rpe / bruch 's membrane , which was assigned to a value of 1.0 .
reflectivity profiles from different eyes were aligned along the posterior border of this hyperreflective rpe / bruch 's membrane band for averaging .
visualization of the choriocapillaris layer and the csj was qualitatively graded as percent of the linear distance over which the layer boundaries were visible , and classified as clear ( 75% visibility ) , intermediate ( 25% and < 75% visibility ) , or unclear ( < 25% visibility ) by two experienced graders ( gy and vv ) who were masked to species and subject demographic .
choriocapillary and choroidal thicknesses were measured after semiautomated segmentation of the central 3 mm of the macula ( 1.5 mm nasal1.5 mm temporal to the fovea ) using duke optical coherence tomography retinal analysis program ( doctrap , version 62.0 ; durham , nc , usa ) , a custom image segmentation software designed using matlab ( mathworks , natick , ma , usa ) . for thickness measurements , the inner boundary was automatically determined by doctrap as the outer border of the hyperreflective rpe / bruch 's complex ; and two masked graders manually traced the outer border of the hyporeflective choriocapillaris layer to measure the choriocapillary thickness , and the outer border of the choroid stroma , corresponding to the csj , to measure the choroidal thickness .
all thickness measurements were determined from the mean of the two graders ' measurements , and reproducibility was determined by measuring intergrader reliability using intraclass correlations ( icc ) by the bland - altman method .
histologic sections from rhesus macaque eyes ( n = 6 , mean age 19.2  7.9 years ) were taken from age - matched animals at cnprc undergoing necropsy for nonocular health issues .
eyes were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde for 24 to 48 hours , transferred to 4% formalin , then embedded in paraffin and stained with standard hematoxylin - eosin .
histologic sections from age - matched adult postmortem human eyes ( n = 10 , mean age 52.8  23.5 years ) were selected from an archive of cadaveric eyes with no known ocular diseases at the duke university medical center ( courtesy of allan proia ) . all sections also underwent standard fixation in formalin , paraffin embedding , and staining with hematoxylin - eosin . among the eyes analyzed ,
five were from white subjects ( mean age 63.2  18.9 years , range , 3571 years , three males / two females ) , and five were from black subjects ( mean age 42.4  24.7 years , range , 2583 years , three males / two females ) .
to quantify the cell density , size , and pigmentation of choroidal melanocytes in histologic sections , three 100  100-m representative regions of choroid stroma were selected for each study eye from images of histologic sections captured at 20 using an eclipse te 200 ( nikon , tokyo , japan ) microscope .
we then performed automated color - based segmentation using the lab color space and k - means clustering in matlab ( mathworks ) to isolate the pigmented melanocytes .
the number of pigmented cells in each field was manually counted to determine melanocyte density ( cells per 10,000 m ) .
average melanocyte size was calculated by dividing the total segmented area of melanin pigment by the cell number in each field .
finally , uveal pigmentation was evaluated by measuring the average luminosity ( l - component of the lab color space ) of the segmented areas from each image as an estimate of optical density ( supplementary fig .
the relationship of choriocapillaris or csj visibility to species or race was determined using the kruskal - wallis test .
multivariate ordinal logistic regression was used to determine the independent association of age , race , and refractive error with choriocapillaris or csj visibility .
choriocapillary and choroidal thickness measurements , as well as differences in cell density , size , and pigmentation of melanocytes , were compared using student 's t - tests .
all statistical analyses were performed using spss statistics ( version 22 ; ibm , armonk , ny , usa ) .
enhanced - depth  oct images of rhesus macaque and human eyes showed similar appearance of the retinal layers at the foveal region .
rhesus macaque eyes uniformly showed a distinct hyporeflective choriocapillaris layer immediately posterior to the rpe / bruch 's complex , and no distinct csj was seen in any of the animals ( figs .
by contrast , images of human eyes showed variable appearance of the choriocapillaris , and many showed a well - delineated csj ( figs . 1b , 1d ) .
all macaques showed 75% visibility of the choriocapillaris , unlike humans , where few normal eyes showed 75% visibility of this layer ( p < 0.001 ) ( table 1 ) .
in contrast , few macaques showed 25% visibility of the csj , while half of human subjects showed 25% visibility of this boundary ( p = 0.003 ) ( table 1 ) .
these results show that choroidal morphology on edi - oct is different between rhesus macaques and humans , with better visualization of the choriocapillaris in macaques and greater visualization of the csj in humans .
comparison of representative edi - oct images from normal rhesus macaque eyes ( a , c ) and human eyes ( b , d ) . the choriocapillaris layer ( arrows ) is well demarcated in rhesus macaques , but not human eyes , while the choroidal  scleral junction ( arrowheads ) is better delineated in some human eyes ( 33% ) in comparison to macaques .
visibility of choroidal morphology in rhesus macaques and humans to further compare the differential appearance of the choroidal layers in humans and macaques on edi - oct , we obtained vertical reflectivity profiles of the choroid at the fovea from all the images .
all rhesus macaque eyes showed a narrow band of hyporeflectivity just posterior to the peak intensity of the rpe band , and delineated posteriorly by another peak of reflectivity that gradually tapers toward the sclera with no clearly demarcated vessel lumen or csj ( fig .
2a ) . compared with histologic sections of age - matched animals , the hyporeflective band corresponds well with the location of the choriocapillaris .
the signal sensitivity roll - off posterior to the choriocapillaris corresponds to the presence of large , darkly pigmented uveal melanocytes that are densely distributed between the medium- to large - caliber vessels , but not in the choriocapillaris layer .
this heavy melanin pigmentation appears to result in obscuration of the csj in the macaque choroid .
reflectivity profiles of representative edi - oct images of a rhesus macaque ( a ) and human eye ( b ) , measured from 10 adjacent a - scans centered at the fovea ( magnified from area of dashed box ) . in macaques , a hyperreflective band corresponds to the rpe layer , while the adjacent hyporeflective band corresponds to the choriocapillaris ( cc ) on age - matched histologic sections .
the gradual signal attenuation corresponds to large , darkly pigmented melanocytes that are densely distributed across the choroid stroma up to the posterior border of the choriocapillaris , and the choroidal  scleral junction ( csj ) is poorly demarcated . in humans ,
no distinct choriocapillaris band can be seen posterior to the hyperreflective rpe band , and the variable reflectivities of the choroidal vessels correspond to the small , lightly pigmented melanocytes that are loosely distributed mostly in the posterior choroid and suprachoroidal layer .
plots of mean reflectivities of rhesus macaques ( c ) and human subjects ( d ) measured from the posterior border of the rpe / bruch 's complex , which shows a clear inflection point corresponding to the choriocapillaris in macaques but not humans .
mean reflectivities are measured in arbitrary grayscale units scaled from 0 to 1.00 , normalized to the peak of the rpe / bruch 's membrane , which is assigned to a value of 1.00 . by contrast , human eyes showed variable reflectivity levels between the rpe and sclera with no single distinct hyporeflective choriocapillaris , with some eyes ( 33% ) showing a well - delineated reflectivity drop at the choroidal  scleral interface ( fig .
histologic sections of age - matched human choroid can be clearly distinguished from rhesus macaques by the smaller size and lighter pigmentation of the melanocytes ( table 2 ) .
the melanocytes are also less densely distributed and are distributed more posteriorly within the choroid stroma and in the suprachoroidal layer ( fig .
the marked difference in melanocyte pigmentation is in contrast to the rpe pigmentation , which is similar between the two species ( figs .
hence , melanin pigmentation in uveal melanocytes rather than rpe cells appears to contribute to the differential visualization of the choriocapillaris and csj in human and macaque eyes on edi - oct .
characteristics of choroidal melanocytes in rhesus macaques and humans we averaged the choroidal reflectivity profiles from edi - oct images across all macaque animals ( n = 25 ) and from all human subjects ( n = 30 ) to compare differences in reflectivity patterns , and found that only the nonhuman primates exhibit a consistent clear hyporeflective band corresponding to the choriocapillaris ( figs .
the mean location of the hyporeflective band is 12.5  6.7 m posterior to the posterior border of the rpe / bruch 's complex , which corresponds to the location of the choriocapillaris on histology . using a semiautomated graph  based oct image segmentation software previously described , we measured the thickness of the choriocapillaris and choroid along the central 3-mm segment around the fovea .
the mean choriocapillary thickness was similar between rhesus macaques and humans ( 19.3  3.4 vs. 19.8  3.4 m ) , with no significant difference between the two species ( p = 0.615 ) .
however , the mean choroidal thickness measured was significantly lower in macaques compared with humans ( 191.2  43.0 vs. 266.8  78.0 m ; p < 0.001 ) . in both species
, while the choriocapillary thickness remains relatively constant across the central macula , the choroid is thickest at the fovea and slightly thinner in the nasal macula region ( fig .
3 ) . representative edi - oct images with semiautomatic segmentation of the choriocapillaris and choroid stroma in rhesus macaque ( a ) and human eyes ( b ) .
the posterior border of the rpe / bruch 's complex is semiautomatically determined ( red line ) , while the outer borders of the hyporeflective choriocapillaris ( green line ) and choroid stroma ( blue line ) are manually traced .
plots of mean choroidal thickness ( ct ) and choriocapillary thickness ( cct ) measured from edi - oct images of rhesus macaque ( c ) and human eyes ( d ) .
while cct is similar between the two species , the measured ct is significantly thinner in macaques than in humans .
the overall intergrader reliability of choroidal thickness ( icc = 0.760 ) and choriocapillary thickness ( icc = 0.607 ) measurements was good .
consistent with the difference in choroidal morphology on edi - oct between the two species , the reliability of choriocapillary thickness measurements was greater than that of choroidal thickness measurements in macaque eyes ( icc = 0.661 vs. 0.297 ) , while the opposite was true in human eyes , where measurements of choroidal thickness were more reproducible than choriocapillary thickness ( icc = 0.776 vs. 0.590 ) .
the lower reliability of choroidal thickness measurements in macaques is likely due to the poor visualization of the csj , and likely contributes to an underestimate of the actual choroidal thickness in these animals .
given the differential oct appearance of the choroid between humans and macaques , which appears to be attributed to the marked difference in the distribution and pigmentation of choroidal melanocytes , we decided to assess whether differences in race may affect uveal pigmentation on histology and choroidal morphology on edi - oct . while the distribution and pigmentation of melanocytes were variable among different individuals , eyes from white subjects ( figs .
c ) have smaller and less darkly pigmented melanocytes than those from black individuals ( figs .
f ) ( table 3 ) , as reported in prior studies . otherwise , in all human eyes , uveal melanocytes were not only smaller and less pigmented than those in rhesus macaques ( table 2 ) , but also less densely packed and more posteriorly distributed , with the majority of the cells located in haller 's layer and the suprachoroidal layer ( figs .
representative histology images of human choroid from white subjects ( a  c ) and black subjects ( d  f ) .
a ) a 35-year - old white female , ( b ) a 71-year - old white male , ( c ) a 54-year - old white male , ( d ) a 29-year - old black male , ( e ) a 49-year - old black male , and ( f ) a 25-year - old black female . characteristics of choroidal melanocytes in humans of different races consistent with our hypothesis that uveal pigmentation affects choroidal morphology on edi - oct , masked grading of images of human eyes showed greater choriocapillaris visibility in black subjects ( p = 0.017 ) and higher visibility of the csj among white individuals ( p = 0.004 ) ( table 4 ) . to determine
if race is independently associated with these choroidal morphologic parameters , we performed a multivariate logistic regression including age and refractive error , which are both known determinants of choroidal thickness . among these factors
, our data showed that choriocapillaris visibility was independently associated with race alone ( p = 0.016 ) , while csj visibility was associated with both race ( p = 0.004 ) and age ( p = 0.026 ) in our study population ( table 5 ) .
there were no significant racial differences in both the mean choriocapillary thickness ( 19.0  3.2 m in whites vs. 21.0  3.3 m in blacks ; p = 0.574 ) and mean choroidal thickness ( 240.0  66.2 m in whites vs. 306.9.8  79.5 m in blacks ; p = 0.377 ) .
thus , racial differences have a greater impact on the visibility than the vascular dimensions of the choroid .
visibility of choroidal morphology in humans of different races factors affecting choroidal morphology on edi - oct in humans
enhanced - depth  oct images of rhesus macaque and human eyes showed similar appearance of the retinal layers at the foveal region .
rhesus macaque eyes uniformly showed a distinct hyporeflective choriocapillaris layer immediately posterior to the rpe / bruch 's complex , and no distinct csj was seen in any of the animals ( figs .
by contrast , images of human eyes showed variable appearance of the choriocapillaris , and many showed a well - delineated csj ( figs . 1b , 1d ) .
all macaques showed 75% visibility of the choriocapillaris , unlike humans , where few normal eyes showed 75% visibility of this layer ( p < 0.001 ) ( table 1 ) .
in contrast , few macaques showed 25% visibility of the csj , while half of human subjects showed 25% visibility of this boundary ( p = 0.003 ) ( table 1 ) .
these results show that choroidal morphology on edi - oct is different between rhesus macaques and humans , with better visualization of the choriocapillaris in macaques and greater visualization of the csj in humans .
comparison of representative edi - oct images from normal rhesus macaque eyes ( a , c ) and human eyes ( b , d ) . the choriocapillaris layer ( arrows ) is well demarcated in rhesus macaques , but not human eyes , while the choroidal  scleral junction ( arrowheads ) is better delineated in some human eyes ( 33% ) in comparison to macaques .
to further compare the differential appearance of the choroidal layers in humans and macaques on edi - oct , we obtained vertical reflectivity profiles of the choroid at the fovea from all the images .
all rhesus macaque eyes showed a narrow band of hyporeflectivity just posterior to the peak intensity of the rpe band , and delineated posteriorly by another peak of reflectivity that gradually tapers toward the sclera with no clearly demarcated vessel lumen or csj ( fig .
compared with histologic sections of age - matched animals , the hyporeflective band corresponds well with the location of the choriocapillaris .
the signal sensitivity roll - off posterior to the choriocapillaris corresponds to the presence of large , darkly pigmented uveal melanocytes that are densely distributed between the medium- to large - caliber vessels , but not in the choriocapillaris layer .
this heavy melanin pigmentation appears to result in obscuration of the csj in the macaque choroid .
reflectivity profiles of representative edi - oct images of a rhesus macaque ( a ) and human eye ( b ) , measured from 10 adjacent a - scans centered at the fovea ( magnified from area of dashed box ) . in macaques , a hyperreflective band corresponds to the rpe layer , while the adjacent hyporeflective band corresponds to the choriocapillaris ( cc ) on age - matched histologic sections .
the gradual signal attenuation corresponds to large , darkly pigmented melanocytes that are densely distributed across the choroid stroma up to the posterior border of the choriocapillaris , and the choroidal  scleral junction ( csj ) is poorly demarcated . in humans ,
no distinct choriocapillaris band can be seen posterior to the hyperreflective rpe band , and the variable reflectivities of the choroidal vessels correspond to the small , lightly pigmented melanocytes that are loosely distributed mostly in the posterior choroid and suprachoroidal layer .
plots of mean reflectivities of rhesus macaques ( c ) and human subjects ( d ) measured from the posterior border of the rpe / bruch 's complex , which shows a clear inflection point corresponding to the choriocapillaris in macaques but not humans .
mean reflectivities are measured in arbitrary grayscale units scaled from 0 to 1.00 , normalized to the peak of the rpe / bruch 's membrane , which is assigned to a value of 1.00 . by contrast
, human eyes showed variable reflectivity levels between the rpe and sclera with no single distinct hyporeflective choriocapillaris , with some eyes ( 33% ) showing a well - delineated reflectivity drop at the choroidal 
histologic sections of age - matched human choroid can be clearly distinguished from rhesus macaques by the smaller size and lighter pigmentation of the melanocytes ( table 2 ) .
the melanocytes are also less densely distributed and are distributed more posteriorly within the choroid stroma and in the suprachoroidal layer ( fig .
the marked difference in melanocyte pigmentation is in contrast to the rpe pigmentation , which is similar between the two species ( figs .
hence , melanin pigmentation in uveal melanocytes rather than rpe cells appears to contribute to the differential visualization of the choriocapillaris and csj in human and macaque eyes on edi - oct .
characteristics of choroidal melanocytes in rhesus macaques and humans we averaged the choroidal reflectivity profiles from edi - oct images across all macaque animals ( n = 25 ) and from all human subjects ( n = 30 ) to compare differences in reflectivity patterns , and found that only the nonhuman primates exhibit a consistent clear hyporeflective band corresponding to the choriocapillaris ( figs .
the mean location of the hyporeflective band is 12.5  6.7 m posterior to the posterior border of the rpe / bruch 's complex , which corresponds to the location of the choriocapillaris on histology .
using a semiautomated graph  based oct image segmentation software previously described , we measured the thickness of the choriocapillaris and choroid along the central 3-mm segment around the fovea .
the mean choriocapillary thickness was similar between rhesus macaques and humans ( 19.3  3.4 vs. 19.8  3.4 m ) , with no significant difference between the two species ( p = 0.615 ) .
however , the mean choroidal thickness measured was significantly lower in macaques compared with humans ( 191.2  43.0 vs. 266.8  78.0 m ; p < 0.001 ) . in both species
, while the choriocapillary thickness remains relatively constant across the central macula , the choroid is thickest at the fovea and slightly thinner in the nasal macula region ( fig .
3 ) . representative edi - oct images with semiautomatic segmentation of the choriocapillaris and choroid stroma in rhesus macaque ( a ) and human eyes ( b ) .
the posterior border of the rpe / bruch 's complex is semiautomatically determined ( red line ) , while the outer borders of the hyporeflective choriocapillaris ( green line ) and choroid stroma ( blue line ) are manually traced . plots of mean choroidal thickness ( ct ) and choriocapillary thickness ( cct ) measured from edi - oct images of rhesus macaque ( c ) and human eyes ( d ) . while cct is similar between the two species , the measured ct is significantly thinner in macaques than in humans .
the overall intergrader reliability of choroidal thickness ( icc = 0.760 ) and choriocapillary thickness ( icc = 0.607 ) measurements was good .
consistent with the difference in choroidal morphology on edi - oct between the two species , the reliability of choriocapillary thickness measurements was greater than that of choroidal thickness measurements in macaque eyes ( icc = 0.661 vs. 0.297 ) , while the opposite was true in human eyes , where measurements of choroidal thickness were more reproducible than choriocapillary thickness ( icc = 0.776 vs. 0.590 ) .
the lower reliability of choroidal thickness measurements in macaques is likely due to the poor visualization of the csj , and likely contributes to an underestimate of the actual choroidal thickness in these animals .
given the differential oct appearance of the choroid between humans and macaques , which appears to be attributed to the marked difference in the distribution and pigmentation of choroidal melanocytes , we decided to assess whether differences in race may affect uveal pigmentation on histology and choroidal morphology on edi - oct . while the distribution and pigmentation of melanocytes were variable among different individuals , eyes from white subjects ( figs .
c ) have smaller and less darkly pigmented melanocytes than those from black individuals ( figs .
f ) ( table 3 ) , as reported in prior studies . otherwise , in all human eyes , uveal melanocytes were not only smaller and less pigmented than those in rhesus macaques ( table 2 ) , but also less densely packed and more posteriorly distributed , with the majority of the cells located in haller 's layer and the suprachoroidal layer ( figs .
representative histology images of human choroid from white subjects ( a  c ) and black subjects ( d  f ) .
histologic sections are taken from ( a ) a 35-year - old white female , ( b ) a 71-year - old white male , ( c ) a 54-year - old white male , ( d ) a 29-year - old black male , ( e ) a 49-year - old black male , and ( f ) a 25-year - old black female . characteristics of choroidal melanocytes in humans of different races consistent with our hypothesis that uveal pigmentation affects choroidal morphology on edi - oct , masked grading of images of human eyes showed greater choriocapillaris visibility in black subjects ( p = 0.017 ) and higher visibility of the csj among white individuals ( p = 0.004 ) ( table 4 ) . to determine if race is independently associated with these choroidal morphologic parameters , we performed a multivariate logistic regression including age and refractive error , which are both known determinants of choroidal thickness . among these factors
, our data showed that choriocapillaris visibility was independently associated with race alone ( p = 0.016 ) , while csj visibility was associated with both race ( p = 0.004 ) and age ( p = 0.026 ) in our study population ( table 5 ) .
there were no significant racial differences in both the mean choriocapillary thickness ( 19.0  3.2 m in whites vs. 21.0  3.3 m in blacks ; p = 0.574 ) and mean choroidal thickness ( 240.0  66.2 m in whites vs. 306.9.8  79.5 m in blacks ; p = 0.377 ) .
thus , racial differences have a greater impact on the visibility than the vascular dimensions of the choroid .
visibility of choroidal morphology in humans of different races factors affecting choroidal morphology on edi - oct in humans
together , our results demonstrate that the cross - sectional visualization of choroidal structures including the choriocapillaris and csj on edi - oct may be affected by pigmented uveal melanocytes in the choroid of humans and rhesus macaques .
additionally , racial differences in pigmentation among human subjects may affect the morphologic appearance of the choroid on edi - oct .
these results have important implications in human studies , where the ability to define a well - delineated csj for reliable choroidal thickness measurements may be affected by the subject 's race .
nonhuman primates are the gold standard among animal models of human macular diseases such as amd because they possess a macula  a feature absent in most other mammals .
rhesus macaques exhibit amd - like drusen with a prevalence of up to 50% , and even higher in the free - ranging cayo santiago colony in puerto rico .
humans and macaques also share genetic polymorphisms in genes such as htra1 ( high temperature requirement factor a1 ) and arms2 ( age - related maculopathy susceptibility 2 ) that are associated with drusen formation . yet , while oct imaging has been widely adopted in human clinical practice , the in vivo characterization of the retina and choroid using oct in rhesus macaques has not been pursued at length . early work by toth et al . demonstrated the feasibility of using oct in primates , while a few later studies employed oct in macaques to visualize laser - induced choroidal neovascularization .
the ocular anatomy of rhesus macaques has remarkable resemblance to human eyes , allowing ocular imaging technologies in clinical use to be readily adapted for primate research .
as such , our comparative analysis of choroidal morphology in humans and macaques establishes a critical basis for future translational research involving choroidal imaging in these animals . in this study
, we found that the distribution and pigmentation of uveal melanocytes are major determinants of choroidal morphology on edi - oct . in macaques ,
the melanocytes are bigger , more darkly pigmented , and densely dispersed across the choroid stroma up to the posterior border of the choriocapillaris , while in humans they are smaller , less pigmented , and more loosely distributed in the posterior choroid , as described previously .
this difference accounts for the more distinctly hyporeflective choriocapillaris band seen in macaques and better delineation of the csj in humans on edi - oct .
interestingly , the retinal layers and rpe appear similar on oct , and the rpe pigmentation is also similar on histology between the two species .
thus , the melanin pigment in the uvea may be a more important contributor to choroidal morphology on oct imaging than rpe pigment .
oct imaging of patients with ocular albinism shows better visualization of choroidal morphology without edi due to their reduced pigmentation . compared to amelanotic nevi , pigmented choroidal nevi on edi - oct appear as a highly reflective band with posterior shadowing .
patients with choroidal melanocytosis also show an apparent increase in choroidal thickness and perivascular stromal tissue . in animal studies ,
comparisons of oct reflectivities from pigmented and albino mice showed differential appearance of the choroid and sclera due to the signal attenuation from choroidal melanin in pigmented mice .
light scattered by tissues containing melanin is also depolarized , as shown in comparisons between pigmented and albino rats , supporting the use of polarization - sensitive oct technology for imaging pigmented structures in the eye .
we note in our study that race may be an important factor in determining choroidal morphology on edi - oct , presumably due to differences in choroidal pigmentation
. however , a person 's self - identified race is a poor surrogate for pigmentation , and skin color may not correlate well with uveal pigmentation .
other ocular features such as iris color may be a better marker for uveal pigmentation and oct morphology
. nevertheless , both race and iris color are factors associated with amd risk , and a better understanding of melanin pigmentation in the choroid may provide important insights into the pathophysiology of these retinal diseases .
importantly , the reduced visibility of the csj and lower reliability of choroidal thickness measurements in blacks should be taken into account in clinical studies involving multiethnic populations .
the limitations of our study include the small sample size and the retrospective selection of age - matched human subjects for comparative analyses .
many of the normal human eyes were contralateral eyes of subjects with unilateral pathologies that are not known to affect the macular choroid or fellow eye , but an association can not be completely excluded .
also , the variable appearance of the choriocapillaris in humans may reduce segmentation reliability , while the poor visualization of the csj in macaques may lead to an artifactually lower choroidal thickness measurement .
future research using oct systems such as swept - source oct devices with longer - wavelength light sources to better visualize deeper ocular structures may help circumvent such limitations . while newer technologies such as oct angiography provide better en face visualization of the choriocapillaris ,
edi - oct remains more widely accessible by taking advantage of the existing generation of sd - oct devices .
however , despite some attempts at segmentation of the choriocapillaris or csj , none have yet to become widely accepted owing to their variable and often ambiguous appearance in human subjects .
our study of choroidal morphology in rhesus macaques showed a very clearly defined choriocapillaris and poor visualization of the csj , likely due to the abundance and darker pigmentation of uveal melanocytes in these animals .
future studies on the relationship between melanin pigment and ocular imaging may improve the development of segmentation algorithms and interpretation of oct findings in retinal and choroidal diseases .
 purposeto compare cross - sectional choroidal morphology in rhesus macaque and human eyes using enhanced - depth imaging optical coherence tomography ( edi - oct ) and histologic analysis.methodsenhanced-depth imaging  
 oct images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal  scleral junction ( csj ) visibility in the central macula based on oct reflectivity profiles , and compared with age - matched histologic sections . 
 semiautomated segmentation of the choriocapillaris and csj was used to measure choriocapillary and choroidal thickness , respectively . 
 multivariate regression was performed to determine the association of age , refractive error , and race with choriocapillaris and csj visibility.resultsrhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on edi - oct , while the csj can not be visualized . 
 in contrast , humans show variable reflectivities of the choriocapillaris , with a distinct csj seen in many subjects . 
 histologic sections demonstrate large , darkly pigmented melanocytes that are densely distributed in the macaque choroid , while melanocytes in humans are smaller , less pigmented , and variably distributed . 
 optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density , size , and pigmentation of choroidal melanocytes . 
 mean choriocapillary thickness was similar between the two species ( 19.3  3.4 vs. 19.8  3.4 m , p = 0.615 ) , but choroidal thickness may be lower in macaques than in humans ( 191.2  43.0 vs. 266.8  78.0 m , p < 0.001 ) . 
 racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and csj on edi-oct.conclusionspigmented uveal melanocytes affect choroidal morphology on edi - oct in rhesus macaque and human eyes . 
 racial differences in pigmentation may affect choriocapillaris and csj visibility , and may influence the accuracy of choroidal thickness measurements . 
Methods
Subject and Eye Selection
Enhanced-Depth Imaging OCT
OCT Image Analysis
Histologic Analysis
Statistical Analysis
Results
Qualitative Comparison of Choroidal Morphology on EDI-OCT
Comparison of Reflectivity Profiles and Histology
Choriocapillary and Choroidal Thicknesses
Effect of Race on Choroidal Morphology
Discussion
Supplementary Material