institutional animal care and use committee approval was obtained and adherence to the arvo statement for the use of animals in ophthalmic and vision research was observed . experimental anterior uveitis was induced in both eyes of six dutch - belted rabbits utilizing a previously published protocol with the following modifications . one hundred milligrams of heat - killed m. tuberculosis h37ra ( tb ) antigen ( difco laboratories , detroit , mi , usa ) was ultrasonicated in 5 ml mineral oil , usp ( sigma - aldrich corp . , st . five hundred microliters was administered subcutaneously via two dorsal injection sites ( each site received 250 l ) to minimize local tissue reaction . one hundred milligrams of heat - killed tb antigen was ultrasonicated in 10 ml balanced salt solution ( alcon , fort worth , tx , usa ) for 30 to 45 seconds until homogenous . the excess cell debris was separated from the mixture by centrifugation at 2500 g for 5 minutes . the supernatant containing the tb antigen was diluted with additional balanced salt solution ( bss ) to obtain concentrations between 1 g/l and 0.5 g/l , in order to induce a range of inflammatory responses . general anesthesia was induced with intramuscular ketamine ( 45 mg / kg ) and xylazine ( 6 mg / kg ) , and subcutaneous buprenorphine ( 0.03 mg / kg ) was given for postoperative analgesia . once the rabbit was anesthetized , one drop each of 0.5% proparacaine hydrochloride ophthalmic solution , usp ( akorn , lake forest , il , usa ) and 1% atropine sulfate ophthalmic solution , usp ( bausch&lomb , tampa , fl , usa ) were given to each eye for pain control and pupil dilation . spectral - domain optical coherence tomography and slit - lamp examination were performed on each rabbit to confirm the absence of cells at baseline . twenty microliters of prepared tb antigen solution was injected into the anterior chamber of each eye . bss was used to wash away excess betadine after the injection followed by application of bacitracin zinc and polymyxin b sulfate ophthalmic ointment usp ( bausch&lomb ) . seven days after the intracameral injections of tb antigen , the rabbits were ready for oct imaging . ketamine ( 45 mg / kg ) and xylazine ( 6 mg / kg ) were administered intramuscularly for anesthesia . the rabbit was positioned upright in a custom holder , and the upper eyelid was temporarily retracted using tape . the eyes were hydrated at all times with polyethylene glycol eye drops ( systane ultra , alcon ) . a custom - built sd - oct system was used to capture scans of the anterior chamber at 74 khz line - rate . the spectral - domain optical coherence tomography used a broadband superluminescent diode light source centered at 820 nm with 150 nm bandwidth ( superlum ) , which provided 1.98 m axial resolution in air . lateral resolution was limited by the anterior segment imaging probe optics to approximately 21 m . the system had a measured 6 db fall off at 1.1 mm . a 12-mm scan was taken for central cornea thickness measurement , followed by 1 mm  1 mm scans superior and inferior to the center of the cornea , for determining cell density values ( cells/l ) . the optical coherence tomography system was calibrated using a ruler prior to collecting data , and the volumes were corrected accordingly . at each location , a 500 ( a - scans / b - scan )  500 ( b - scans / volume ) scan density was used , with a second scan performed with each horizontal raster in triplicate ( three - frames / b - scan ) . image frames were manually counted for hyperreflective specks , which were used for cell density calculations . if a speck was detected in the same location in consecutive scans , it was only counted once to prevent oversampling errors . a haag - streit slit - lamp was used to examine and clinically grade each eye immediately after imaging , using the sun grading scale for cells within a 1 mm  1 mm slit - beam field of view . the standardization of uveitis nomenclature grading scale is as follows : 0.5 + refers to 1 to 5 cells , 1 + for 6 to 15 cells , 2 + for 16 to 25 cells , 3 + for 26 to 50 cells , and 4 + for more than 50 cells . in 54 out of 62 examinations , other observations were also noted , including cornea clarity , and the presence of iris inflammatory nodules , fibrin , and cell clumps . the graders were masked to each other and also to the results of the oct cell counts and aqueous sampling . immediately following slit lamp grading , isoflurane anesthetic ( 2%4% ) was administered via inhalation to continue sedation . proparacaine drops were given for analgesia , atropine drops for dilation , and 5% betadine solution was used for sterilization of ocular surface , intended injection site , and surrounding eye - lid tissue . approximately 0.15 ml fluid was drawn from the anterior chamber through the superior peripheral cornea , using a 0.3-ml syringe and 31-gauge needle ( bd , franklin lakes , nj , usa ) bd # 328438 . the needle was passed through the cornea approximately 1 to 2 mm from the limbus with the tip of the needle aiming toward the center of the eye with the bevel down . care was taken not to disturb the anterior lens capsule and the needle was kept parallel to the iris plane . fluid was withdrawn slowly into the syringe , the needle removed from the eye , and a sterile swab placed over the wound to stop the egress of fluid . the eye was flushed with bss then bacitracin / polymyxin b antibiotic ophthalmic ointment was applied . the fluid within the syringe was immediately transferred to a nageotte ( hausser scientific , horsham , pa , usa ) or neubauer ( hausser scientific ) hemocytometer at the time of collection . for each imaging session , a total of four sd - oct scans were obtained ( superior / inferior , high / low density ) and the mean was used for total sd - oct counts . for analysis of superior and inferior scans , the average of the low and high density scan was used . since the goal was to evaluate associations between systems , and different sessions within each eye reflected different levels of inflammation , each session was treated independently in the statistical analysis . analysis was performed using microsoft excel , and a p value less than 0.05 was considered statistically significant . one hundred milligrams of heat - killed m. tuberculosis h37ra ( tb ) antigen ( difco laboratories , detroit , mi , usa ) was ultrasonicated in 5 ml mineral oil , usp ( sigma - aldrich corp . , st . five hundred microliters was administered subcutaneously via two dorsal injection sites ( each site received 250 l ) to minimize local tissue reaction . one hundred milligrams of heat - killed tb antigen was ultrasonicated in 10 ml balanced salt solution ( alcon , fort worth , tx , usa ) for 30 to 45 seconds until homogenous . the excess cell debris was separated from the mixture by centrifugation at 2500 g for 5 minutes . the supernatant containing the tb antigen was diluted with additional balanced salt solution ( bss ) to obtain concentrations between 1 g/l and 0.5 g/l , in order to induce a range of inflammatory responses . general anesthesia was induced with intramuscular ketamine ( 45 mg / kg ) and xylazine ( 6 mg / kg ) , and subcutaneous buprenorphine ( 0.03 mg / kg ) was given for postoperative analgesia . once the rabbit was anesthetized , one drop each of 0.5% proparacaine hydrochloride ophthalmic solution , usp ( akorn , lake forest , il , usa ) and 1% atropine sulfate ophthalmic solution , usp ( bausch&lomb , tampa , fl , usa ) were given to each eye for pain control and pupil dilation . spectral - domain optical coherence tomography and slit - lamp examination were performed on each rabbit to confirm the absence of cells at baseline . twenty microliters of prepared tb antigen solution was injected into the anterior chamber of each eye . bss was used to wash away excess betadine after the injection followed by application of bacitracin zinc and polymyxin b sulfate ophthalmic ointment usp ( bausch&lomb ) . seven days after the intracameral injections of tb antigen , the rabbits were ready for oct imaging . ketamine ( 45 mg / kg ) and xylazine ( 6 mg / kg ) were administered intramuscularly for anesthesia . the rabbit was positioned upright in a custom holder , and the upper eyelid was temporarily retracted using tape . the eyes were hydrated at all times with polyethylene glycol eye drops ( systane ultra , alcon ) . a custom - built sd - oct system was used to capture scans of the anterior chamber at 74 khz line - rate . the spectral - domain optical coherence tomography used a broadband superluminescent diode light source centered at 820 nm with 150 nm bandwidth ( superlum ) , which provided 1.98 m axial resolution in air . lateral resolution was limited by the anterior segment imaging probe optics to approximately 21 m . the system had a measured 6 db fall off at 1.1 mm . a 12-mm scan was taken for central cornea thickness measurement , followed by 1 mm  1 mm scans superior and inferior to the center of the cornea , for determining cell density values ( cells/l ) . the optical coherence tomography system was calibrated using a ruler prior to collecting data , and the volumes were corrected accordingly . at each location , a 500 ( a - scans / b - scan )  500 ( b - scans / volume ) scan density was used , with a second scan performed with each horizontal raster in triplicate ( three - frames / b - scan ) . image frames were manually counted for hyperreflective specks , which were used for cell density calculations . if a speck was detected in the same location in consecutive scans , it was only counted once to prevent oversampling errors . a haag - streit slit - lamp was used to examine and clinically grade each eye immediately after imaging , using the sun grading scale for cells within a 1 mm  1 mm slit - beam field of view . the standardization of uveitis nomenclature grading scale is as follows : 0.5 + refers to 1 to 5 cells , 1 + for 6 to 15 cells , 2 + for 16 to 25 cells , 3 + for 26 to 50 cells , and 4 + for more than 50 cells . in 54 out of 62 examinations , grading was performed by two trained nonclinicians . in 8 out of 62 examinations , other observations were also noted , including cornea clarity , and the presence of iris inflammatory nodules , fibrin , and cell clumps . the graders were masked to each other and also to the results of the oct cell counts and aqueous sampling . immediately following slit lamp grading , isoflurane anesthetic ( 2%4% ) was administered via inhalation to continue sedation . proparacaine drops were given for analgesia , atropine drops for dilation , and 5% betadine solution was used for sterilization of ocular surface , intended injection site , and surrounding eye - lid tissue . approximately 0.15 ml fluid was drawn from the anterior chamber through the superior peripheral cornea , using a 0.3-ml syringe and 31-gauge needle ( bd , franklin lakes , nj , usa ) bd # 328438 . the needle was passed through the cornea approximately 1 to 2 mm from the limbus with the tip of the needle aiming toward the center of the eye with the bevel down . care was taken not to disturb the anterior lens capsule and the needle was kept parallel to the iris plane . fluid was withdrawn slowly into the syringe , the needle removed from the eye , and a sterile swab placed over the wound to stop the egress of fluid . the eye was flushed with bss then bacitracin / polymyxin b antibiotic ophthalmic ointment was applied . the fluid within the syringe was immediately transferred to a nageotte ( hausser scientific , horsham , pa , usa ) or neubauer ( hausser scientific ) hemocytometer at the time of collection . for each imaging session , a total of four sd - oct scans were obtained ( superior / inferior , high / low density ) and the mean was used for total sd - oct counts . for analysis of superior and inferior scans , the average of the low and high density scan was used . since the goal was to evaluate associations between systems , and different sessions within each eye reflected different levels of inflammation , each session was treated independently in the statistical analysis . analysis was performed using microsoft excel , and a p value less than 0.05 was considered statistically significant . the tb model produced exuberant anterior segment inflammation , often with associated corneal thickening , corneal haze , and inflammatory cell clumps on the surface of the iris and anterior lens capsule ( table 1 ) . over a course of 2 to 3 weeks , inflammatory levels decreased and rabbits were imaged over multiple time points in order to obtain a range of inflammatory grades . a total of six rabbits and 12 eyes were imaged over 62 different sessions ( range per eye : four to six sessions ) . figure 1 shows examples of hyperreflective specks seen on sd - oct imaging in inflamed rabbit eyes compared with a control eye . a shapiro - wilk test for normality was performed for sd - oct counts , hemocytometer counts ( aqueous sampling ) , and sun grades ( table 2 ) . all of the data were found to be nonnormal , and thus spearman rank correlation was used for our analysis . the weighted  between the two graders was 0.76 ( 95% confidence interval : 0.660.87 ) , which shows good agreement between the graders . frequency of severe ocular inflammatory findings examples of sd - oct b - scan image in an inflamed eye ( a ) , control eye ( b ) , and inflamed eyes with thickened corneas ( c , d ) . . the thick cornea ( c , d ) attenuates the cell signal compared with a. the signal attenuation is greatest with increasing depth of penetration ( d ) . comparison between aqueous sampling , sd - oct , and sun grading systems figure 2a shows a scatterplot of aqueous sampling versus sd - oct cell counts . these eyes also had associated central corneal thickness ( cct ) of 470 m or greater . fifty - two of the 62 ( 84% ) samples had cct below 470 m . the average corneal thickness of our complete data set was 412  57 ( mean  sd ) m , and for samples below 470 m it was 392  34 m . overall , sd - oct correlated well with aqueous sampling with a spearman coefficient (  ) of 0.53 ( p < 0.001 ) . this improved to  = 0.65 ( p < 0.001 ) when cct was limited to below 470 m . standardization of uveitis nomenclature grading of the same eyes correlated poorly with aqueous sampling ( fig . the overall  was 0.02 ( p = 0.88 ) and this did not significantly improve when cct was below 470 m (  = 0.06 , p = 0.65 ) . similarly , sd - oct correlated poorly with sun grading in our study (  = 0.13 , p = 0.34 ; fig . ( a ) several outliers with very high aqueous cell counts are seen toward the bottom right of the plot . ( b ) when the population is limited to cct below 470 m , the outliers are excluded . standardization of uveitis nomenclature grading does not correlate with aqueous sampling or sd - oct cell density estimates . although anterior chamber cells are mobile , they appear to settle with gravity ( i.e. , formation of a hypopyon ) . we wanted to test if regional differences could be detected with sd - oct by imaging each eye in a location superior to the central cornea and also inferior to the central cornea . no significant difference in sd - oct values between these two locations were detected by wilcoxon signed rank test ( p = 0.50 ) , even after limiting the analysis to cct below 470 m ( p = 0.44 ) . anterior chamber inflammation is currently measured and graded by clinical examination using slit - lamp biomicroscopy and the sun scale . this approach is considered the gold - standard for clinical diagnosis , where aqueous sampling can not be easily obtained . this study investigates an alternative method for grading inflammatory cells within the anterior chamber using sd - oct , a technology already being used in clinical settings . spectral - domain optical coherence tomography allows for the noninvasive capture of cross - sectional image frames where cells can be visualized as reflective spots . our data show that sd - oct counts correlated with aqueous cell counts better than the correlation between sun grading and aqueous cell counts . spectral - domain optical coherence tomography has known limitations , however . the axial resolution of sd - oct is approximately 5 m , and the transverse resolution is approximately 20 m . cells smaller than 5 m may be undetectable and two small cells separated by less than 20 m may appear as one hyperreflective speck . fortunately , rabbit lymphocytes , and heterophils are approximately 10 to 15 m in diameter and monocytes are even larger . the optical coherence tomography signal is also limited by optical penetration depth , as well as signal roll - off , and thus the entire anterior chamber can not be imaged at once . signal roll - off limits the ability of sd - oct to distinguish cells from background with increasing axial depth . if the distribution of cells between anterior and posterior regions is different , then sd - oct may not provide a completely accurate estimate of anterior chamber cellular density . our study also suggests corneal thickening attenuates the oct signal and results in erroneous cellular density estimates . scans with cct  470 m resulted in sd - oct cell counts that poorly correlated with aqueous sampling including outliers with very high aqueous sampling cell counts , but low sd - oct counts ( fig . this is consistent with previous reports where corneal edema resulted in low sd - oct cell counts compared with eyes with clear corneas and high grades of inflammation . in contrast to previous reports comparing sun clinical grading to sd - oct grading , we found no correlation between sun grading and sd - oct grading in our study . the current study describes a uveitis model in rabbit instead of clinical uveitis in human patients . to our knowledge , sun grading of rabbit uveitis in the tb model has yet to be validated . standardization of uveitis nomenclature grading of anterior chamber cell in our model may be less accurate than in humans , resulting in sun grades that do not accurately reflect anterior segment inflammation . the rabbit model produced severe inflammation including 42% of eyes with inflammatory cell clumps or fibrin in the anterior chamber ( table 1 ) . the average corneal thickness of new zealand white rabbits is 350 m and 362  15.5 m for dutch - belted rabbits . in our study , 65% ( 40 out of 62 samples ) were 2 standard deviations above the average cct ( data not shown ) . this increased corneal thickness may have limited the ability of our graders from accurately assigning sun grades . our study also used two nonclinicians to grade the majority of the slit lamp examinations ( 87% ) as opposed to grading by trained uveitis specialists . our results suggest sun grading accuracy is highly dependent on observer level of expertise and quality of the sample . standardization of uveitis nomenclature grading requires a dynamic measurement of cells moving in and out of a defined volume through a slit beam , whereas sd - oct cell counts are taken from static optical sections through a defined volume . this fundamental difference could also help explain why no correlation was found between sun grading and sd - oct cell counts or aqueous taps . the lack of a 1:1 correlation between sd - oct counts and aqueous cell counts may be due to several possibilities . spectral - domain optical coherence tomography may underestimate the actual cell count due to the sd - oct limitations outlined above . another possibility , however , is oversampling of the  free floating aqueous cell population in our experimental model by inadvertently collecting cell clumps stuck to the iris and lens . although we made every effort not to disturb these clusters of inflammatory cells , it is possible that in some samples , collection errors might have resulted in higher than usual cell counts . by using an animal model , we were able to determine the aqueous cell density by directly counting the cells withdrawn in an aqueous sample . we found sd - oct to correlate well with aqueous cell density when corneal thickening was limited . spectral - domain optical coherence tomography is currently being used in many ophthalmic offices and transition to anterior segment scans could be rapidly and easily adapted . development of automated cell counting algorithms could further improve the accuracy and precision of sd - oct cell counts and allow for the rapid evaluation of anterior segment inflammation . standardization of uveitis nomenclature grading in humans is well validated and remains the gold standard for monitoring anterior uveitis . however , the use of sd - oct may become a useful adjunctive test for the evaluation of patients with anterior uveitis .
purposeto determine the feasibility and accuracy of spectral - domain optical coherence tomography ( sd - oct ) based grading of anterior chamber cell , using aqueous sampling as a standard , in a rabbit model of anterior uveitis.methodsadult dutch - belted rabbits were preimmunized with m. tuberculosis ( tb ) h37ra antigen , 1 week prior to induction of anterior uveitis with an intracameral injection of tb antigen . 
 the anterior chamber was imaged with sd - oct , followed by a slit lamp examination . 
 two independent , trained graders recorded their estimate of anterior chamber cell count using the standardization of uveitis nomenclature ( sun ) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer . using the aqueous cell density as a standard , correlation with sd - oct counts 
 were compared to those with sun scores.resultsoverall , sd - oct correlated well with the hemocytometer counts ( spearman coefficient = 0.53 , p < 0.001 ) compared with sun grading and hemocytometer counts ( spearman coefficient = 0.02 , p = 0.88 ) . 
 the correlation improved to 0.65 ( p < 0.001 ) when we excluded eyes with corneal thickness  470 m . 
 eyes with corneal thickness  470 m exhibited the greatest degree of ocular inflammation and corneal opacity.conclusionsin our rabbit model , sd - oct grading of anterior chamber cell correlated significantly better with aqueous cell counts , compared to traditional slit lamp grading . 
 spectral - domain optical coherence tomography grading of anterior chamber cell may be a good alternative to sun grading . 
 although sun grading remains the clinical gold standard , alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response .