the vis - oct system enables the quantification of both retinal so2 and retinal blood flow in vivo . using a supercontinuum laser as a light source ( center wavelength : 568.5 nm ; bandwidth : 107 nm ; superk nkt photonics , birkerd , denmark ) , we built a free - space spectral - domain ( sd)-oct ( fig .
1a ) with the theoretical axial resolution being 1.3 m in air . to quantify retinal so2 ,
we raster - scanned 256  256 a - lines onto the mouse retina to acquire a retinal fundus image , covering a 1.05  1.05 mm area centered at the optic disc ( fig .
by analyzing the reflected light spectrum from each retinal vessel , we determined the corresponding so2 value ( fig .
2 ) . to quantify the retinal blood flow , we measured the phase shifts , vessel size , and doppler angles ( fig .
, 16 pairs of small - big rings were scanned onto a mouse retina centered at the optic disc .
the radius was 0.31 mm for the big scanning ring , and 0.21 mm for the small scanning ring , with 4096 a - lines in each ring . during the experiments
, we first performed the raster - scanning , and then the dual - ring scanning protocol .
the a - line rate was 25 khz for both scanning protocols . experimental setup and scanning protocols .
( b ) raster - scanning protocol to acquire the retinal fundus image and measure retinal oxygen saturation ( so2 ) .
l1 to l5 are lenses ; bs , beam splitter ; dc , dispersion compensation .
( b ) sample b - scan from the red dashed line in ( a ) .
( c ) short - time fourier transforms with different center frequencies ( green , light green , and red ) performed on the spectral interferogram ( blue line ) .
( d ) sub - band b - scans of the vessel in ( b ) denoted by the red dashed circle .
( c ) sample phase - shift b - scan image from the small scanning ring .
( h ) total blood flow rate in the retinal arterial and venous vascular system .
scale bar : 200 m . the procedure for retinal oximetry with vis - oct was reported in great detail previously .
2a ) , and reconstructed the cross - sectional b - scan using the full band of the spectral interferogram . from the cross - sectional b - scan
2b ) . as illustrated in figure 2c , short - time fourier transforms ( stft ) , with different band center frequencies ( depicted as the gaussian - shaped colored curves ) ,
were performed on the spectral interferogram ( blue line ) to reconstruct sub - band b - scans .
examples of the obtained sub - band b - scans , for the circled vessel in figure 2b , are shown in figure 2d .
the bandwidth for each of the stfts was 15 nm , which reduced the axial resolution of each sub - band b - scan to 9.5 m .
although the axial resolution was reduced , it is still sufficient to resolve the major murine retinal vessels , which have vessel diameters of 40 m or more .
using the already determined vessel coordinates , we extracted the oct signal from the bottom of the vessel across all the sub - band b - scans , as highlighted by the arrows in figure 2d .
examples of the obtained oct signal spectra , for an artery and a vein , are shown in figure 2e . to obtain the so2 from the oct signal spectra ,
we needed to model the reflectance and attenuation of the oct signal by using the optical attenuation coefficient of oxygenated hemoglobin ( hbo2 ) and deoxygenated hemoglobin ( hb ) as follows : 

  here i0 is the incident intensity on the retina , and r0 is the reflectance of the reference arm .
the spectral behavior of r was as scattering under first - order born approximation , which is given by the power law function : r( ) = a. here , a and  are constants .
the exponential term in equation 1 is attenuation according to beer - lambert 's law , where n is the mean refractive index of the blood ( 1.35 ) , and hbo2 and hb denote the attenuation coefficients .
the backscattered light experiences two - way ( round - trip ) attenuation through the blood vessel diameter , d [ m ] , hence the factor of 2 in the exponential expression . the wavelength - dependent attenuation coefficients ( hbo2 and hb ) incorporate both the absorption coefficient ( a ) and scattering coefficients ( s ) of the whole blood ( figs .
our previous investigation suggested the relationship between the attenuation coefficient , absorption coefficient , and scattering coefficient as follows : 

  to obtain so2 , the oct signal spectra for each vessel was fit to a modified form of equation 1 ( see appendix ) using least - squares ( ls ) fitting .
this retinal oximetry technique using vis - oct has been validated in silico with monte carlo simulations , in vitro with artificially oxygenated bovine blood and a commercial blood gas analyzer , and in vivo with systemic hypoxia experiments and a commercial pulse oximeter .
doppler sd - oct can measure only the projected flow along the probing beam . to achieve the absolute flow measurements , the doppler angle , which is the angle between the probing beam and blood flow ,
we used the dual - ring scanning protocol to simultaneously extract the projected flow and doppler angle . 

figure 3a shows the geometry of the dual - ring scanning protocol . using this geometry , equations for the doppler angle 
, we see that the doppler angle requires the three - dimensional vessel axis coordinates , hence the requirement for two circular rings in the dual - ring method .
figure 3b shows an example of a mouse fundus with the scanning paths of the dual rings .
figure 3d shows the calculated doppler angles of all the vessels in this example retina . after acquiring the doppler angles , we calculated the absolute retinal blood flow velocity v ( m / s ) as follows : 

  where fsample ( khz ) is the a - line rate ; 0 ( nm ) is the center wavelength of the sd - oct ;  is the phase shift ( rad ) ( fig .
3c ) between adjacent a - lines after bulk motion correction ; n is the refractive index of the sample ( n = 1.35 ) ; and  [ degree ] is the doppler angle .
3c ) in experiments ; to correct this , the axial blood flow direction is necessary .
the flow direction is recognizable from the phase shifts near the vessel border , where low flow velocity prevents phase - wrapping . assuming the actual phase  < 0
, we corrected the phase - wrapping as follows : 

  for the actual phase that is  > 0 , we corrected the phase - wrapping as 

  in this study , we had 16 pairs of big - small ring scanning , providing 32 continuous b - scans .
we tested the measured velocity stability by averaging the velocity results across different cumulative b - scans and comparing the mean velocity difference between adjacent cumulative b - scans .
we considered the velocity stable if the velocity difference was smaller than 0.1 mm / s between adjacent cumulative b - scans . after averaging results across 20 b - scans ( fig .
the velocities for the sample retina are shown in figure 3f , where the arterial flow has positive velocities running from the optic disc to the peripheral retina , and the venous flow has negative velocities draining blood from the peripheral retina to the optic disk .
we measured the vessel diameter dia ( m ) in the axial direction in the oct b - scan structural , the actual vessel diameter dia_r ( m ) was : 

  where  ( radians ) is the doppler angle .
the vessel cross - sectional area z ( m ) was : 

  with the absolute velocity and vessel size , the corresponding blood flow in the ith vessel was determined : 

  where vi is the average velocity and ai is the vessel cross - sectional area .
we calculated the total blood flow ( f [ l / min ] ) by summing all blood flow in the retinal arterial / vein vascular system , as shown in equation 9 : 

  where n is the total number of retinal arteries or veins .
we calculated the irmro2 as follows : 

  where wo2 and whbo2 are the molecular weights of o2 and oxygenated hemoglobin , which were 32 and 68,000 ( g / mol ) , respectively .
[ hbt ] is the hemoglobin concentration in the mouse , which was measured to be 150 (
g / l ) ; so2a and so2v are the mean so2 values in the artery and vein vascular system , respectively .
we can convert ng / min to l / min as : 

  where v is the volume of gas ; ng is the number of moles of gas ; r is 8.3145 ( jmolek ) ; t is the blood temperature ( 311.15 k ) ; and p is the standard atmospheric pressure ( 101,325 pa ) .
statistic results were obtained from seven akita/+ , tsp1 dr mice and seven tsp1 control mice , respectively . retinal vessel sample preparation and ec , pc quantification can be found in our previous article . in this study ,
all retinal vessel preparations , ec and pc number counting were performed on 13-week - old mice .
we fixed mouse eyes in 4% paraformaldehyde for 24 hours , bisected equatorially , and removed the entire retina .
the retinas were washed overnight in distilled water and incubated in 3% trypsin ( trypsin 1:250 ; difco , bd diagnostic systems , sparks , md , usa ) prepared in 0.1 m tris , 0.1 m maleic acid , 0.2 m naf for approximately 1.0 to 1.5 hours at 37c .
there was 0.2 m sodium fluoride in the digestion buffer to inhibit dnases from contaminating the crude trypsin preparation .
after digestion , the retinal vessels were flattened by four radial cuts and mounted on glass slides for periodic acid - schiff and hematoxylin staining .
the nuclei of ecs are oval or elongated in shape , laid within the vessel wall , and extended along the axis of the capillary ; in comparison , pericyte nuclei are typically small , spherical in shape , and stain densely .
generally , the pericyte nuclei is characterized by a protuberant position on the capillary wall .
we determined the number of ecs and pcs by counting nuclei under the microscope with 400 magnification , where the mounting reticle ( 10  10 m ) was placed in one of the viewing oculars to facilitate counting .
we included only retinal capillaries in the mid - zone for the cell count , and measured the number of ecs and pcs in four reticles from the four quadrants of each retina .
the mean value of ecs and pcs for each retina was determined by averaging the values from the four reticles .
briefly , we counted acellular capillaries in five field areas in the middle of the retina samples ( 200 magnification ) .
acellular capillaries were defined as capillary - sized vessel tubes without nuclei anywhere along their length .
tubes with a diameter < 20% of the diameter of adjacent capillaries were identified as strands and were not counted .
we weighed each mouse before imaging , and anesthetized them by intraperitoneal injection with a cocktail of ketamine ( 87 mg / kg body weight ) and xylazine ( 13 mg / kg body weight ) . during the experiment
, we applied 0.5% tetracaine hydrochloride ophthalmic solution to paralyze the iris sphincter muscle and 1% tropicamide ophthalmic solution to dilate the pupil .
fort worth , tx , usa ) were added every minute to the mouse cornea to prevent dehydration and cataract formation .
the blood so2 were measured to be approximately 98 and the heart rate was approximately 320 beats per minute ( pulse oximetry , model 8500av ; nonin medical , inc . ,
all experimental procedures were in compliance with the arvo statement for the use of animals in ophthalmic and vision research , and the laboratory animal protocol approved by the institutional animal care and use committee at northwestern university .
blood glucose levels were measured with a commercial available glucometer ( glucometer elite ; bayer , inc . ,
whippany , nj , usa ) by using a drop of tail blood collected from mice models .
the vis - oct system enables the quantification of both retinal so2 and retinal blood flow in vivo . using a supercontinuum laser as a light source ( center wavelength : 568.5 nm ; bandwidth : 107 nm ; superk nkt photonics , birkerd , denmark ) , we built a free - space spectral - domain ( sd)-oct ( fig .
1a ) with the theoretical axial resolution being 1.3 m in air . to quantify retinal so2 ,
we raster - scanned 256  256 a - lines onto the mouse retina to acquire a retinal fundus image , covering a 1.05  1.05 mm area centered at the optic disc ( fig .
by analyzing the reflected light spectrum from each retinal vessel , we determined the corresponding so2 value ( fig .
2 ) . to quantify the retinal blood flow , we measured the phase shifts , vessel size , and doppler angles ( fig .
, 16 pairs of small - big rings were scanned onto a mouse retina centered at the optic disc .
the radius was 0.31 mm for the big scanning ring , and 0.21 mm for the small scanning ring , with 4096 a - lines in each ring . during the experiments
, we first performed the raster - scanning , and then the dual - ring scanning protocol .
the a - line rate was 25 khz for both scanning protocols . experimental setup and scanning protocols .
( b ) raster - scanning protocol to acquire the retinal fundus image and measure retinal oxygen saturation ( so2 ) .
l1 to l5 are lenses ; bs , beam splitter ; dc , dispersion compensation .
( b ) sample b - scan from the red dashed line in ( a ) .
( c ) short - time fourier transforms with different center frequencies ( green , light green , and red ) performed on the spectral interferogram ( blue line ) .
( d ) sub - band b - scans of the vessel in ( b ) denoted by the red dashed circle .
( c ) sample phase - shift b - scan image from the small scanning ring .
( h ) total blood flow rate in the retinal arterial and venous vascular system .
the procedure for retinal oximetry with vis - oct was reported in great detail previously .
2a ) , and reconstructed the cross - sectional b - scan using the full band of the spectral interferogram . from the cross - sectional b - scan
2b ) . as illustrated in figure 2c , short - time fourier transforms ( stft ) , with different band center frequencies ( depicted as the gaussian - shaped colored curves ) , were performed on the spectral interferogram ( blue line ) to reconstruct sub - band b - scans .
examples of the obtained sub - band b - scans , for the circled vessel in figure 2b , are shown in figure 2d .
the bandwidth for each of the stfts was 15 nm , which reduced the axial resolution of each sub - band b - scan to 9.5 m .
although the axial resolution was reduced , it is still sufficient to resolve the major murine retinal vessels , which have vessel diameters of 40 m or more .
using the already determined vessel coordinates , we extracted the oct signal from the bottom of the vessel across all the sub - band b - scans , as highlighted by the arrows in figure 2d .
examples of the obtained oct signal spectra , for an artery and a vein , are shown in figure 2e . to obtain the so2 from the oct signal spectra , we needed to model the reflectance and attenuation of the oct signal by using the optical attenuation coefficient of oxygenated hemoglobin ( hbo2 ) and deoxygenated hemoglobin ( hb ) as follows : 

  here i0 is the incident intensity on the retina , and r0 is the reflectance of the reference arm .
the spectral behavior of r was as scattering under first - order born approximation , which is given by the power law function : r( ) = a. here , a and  are constants .
the exponential term in equation 1 is attenuation according to beer - lambert 's law , where n is the mean refractive index of the blood ( 1.35 ) , and hbo2 and hb denote the attenuation coefficients .
the backscattered light experiences two - way ( round - trip ) attenuation through the blood vessel diameter , d [ m ] , hence the factor of 2 in the exponential expression . the wavelength - dependent attenuation coefficients ( hbo2 and hb ) incorporate both the absorption coefficient ( a ) and scattering coefficients ( s ) of the whole blood ( figs .
our previous investigation suggested the relationship between the attenuation coefficient , absorption coefficient , and scattering coefficient as follows : 

  to obtain so2 , the oct signal spectra for each vessel was fit to a modified form of equation 1 ( see appendix ) using least - squares ( ls ) fitting .
this retinal oximetry technique using vis - oct has been validated in silico with monte carlo simulations , in vitro with artificially oxygenated bovine blood and a commercial blood gas analyzer , and in vivo with systemic hypoxia experiments and a commercial pulse oximeter .
doppler sd - oct can measure only the projected flow along the probing beam . to achieve the absolute flow measurements , the doppler angle , which is the angle between the probing beam and blood flow ,
we used the dual - ring scanning protocol to simultaneously extract the projected flow and doppler angle . 

figure 3a shows the geometry of the dual - ring scanning protocol . using this geometry , equations for the doppler angle 
, we see that the doppler angle requires the three - dimensional vessel axis coordinates , hence the requirement for two circular rings in the dual - ring method .
figure 3b shows an example of a mouse fundus with the scanning paths of the dual rings .
figure 3d shows the calculated doppler angles of all the vessels in this example retina . after acquiring the doppler angles , we calculated the absolute retinal blood flow velocity v ( m / s ) as follows : 

  where fsample ( khz ) is the a - line rate ; 0 ( nm ) is the center wavelength of the sd - oct ;  is the phase shift ( rad ) ( fig .
3c ) between adjacent a - lines after bulk motion correction ; n is the refractive index of the sample ( n = 1.35 ) ; and  [ degree ] is the doppler angle .
3c ) in experiments ; to correct this , the axial blood flow direction is necessary .
the flow direction is recognizable from the phase shifts near the vessel border , where low flow velocity prevents phase - wrapping . assuming the actual phase  < 0
, we corrected the phase - wrapping as follows : 

  for the actual phase that is  > 0 , we corrected the phase - wrapping as 

  in this study , we had 16 pairs of big - small ring scanning , providing 32 continuous b - scans .
we tested the measured velocity stability by averaging the velocity results across different cumulative b - scans and comparing the mean velocity difference between adjacent cumulative b - scans .
we considered the velocity stable if the velocity difference was smaller than 0.1 mm / s between adjacent cumulative b - scans . after averaging results across 20 b - scans ( fig .
the velocities for the sample retina are shown in figure 3f , where the arterial flow has positive velocities running from the optic disc to the peripheral retina , and the venous flow has negative velocities draining blood from the peripheral retina to the optic disk .
we measured the vessel diameter dia ( m ) in the axial direction in the oct b - scan structural , the actual vessel diameter dia_r ( m ) was : 

  where  ( radians ) is the doppler angle .
the vessel cross - sectional area z ( m ) was : 

  with the absolute velocity and vessel size , the corresponding blood flow in the ith vessel was determined : 

  where vi is the average velocity and ai is the vessel cross - sectional area .
we calculated the total blood flow ( f [ l / min ] ) by summing all blood flow in the retinal arterial / vein vascular system , as shown in equation 9 : 

  where n is the total number of retinal arteries or veins .
we calculated the irmro2 as follows : 

  where wo2 and whbo2 are the molecular weights of o2 and oxygenated hemoglobin , which were 32 and 68,000 ( g / mol ) , respectively .
[ hbt ] is the hemoglobin concentration in the mouse , which was measured to be 150 (
g / l ) ; so2a and so2v are the mean so2 values in the artery and vein vascular system , respectively .
we can convert ng / min to l / min as : 

  where v is the volume of gas ; ng is the number of moles of gas ; r is 8.3145 ( jmolek ) ; t is the blood temperature ( 311.15 k ) ; and p is the standard atmospheric pressure ( 101,325 pa ) .
statistic results were obtained from seven akita/+ , tsp1 dr mice and seven tsp1 control mice , respectively .
retinal vessel sample preparation and ec , pc quantification can be found in our previous article . in this study , all retinal vessel preparations , ec and pc number counting were performed on 13-week - old mice .
we fixed mouse eyes in 4% paraformaldehyde for 24 hours , bisected equatorially , and removed the entire retina .
the retinas were washed overnight in distilled water and incubated in 3% trypsin ( trypsin 1:250 ; difco , bd diagnostic systems , sparks , md , usa ) prepared in 0.1 m tris , 0.1 m maleic acid , 0.2 m naf for approximately 1.0 to 1.5 hours at 37c .
there was 0.2 m sodium fluoride in the digestion buffer to inhibit dnases from contaminating the crude trypsin preparation .
after digestion , the retinal vessels were flattened by four radial cuts and mounted on glass slides for periodic acid - schiff and hematoxylin staining .
the nuclei of ecs are oval or elongated in shape , laid within the vessel wall , and extended along the axis of the capillary ; in comparison , pericyte nuclei are typically small , spherical in shape , and stain densely .
generally , the pericyte nuclei is characterized by a protuberant position on the capillary wall .
we determined the number of ecs and pcs by counting nuclei under the microscope with 400 magnification , where the mounting reticle ( 10  10 m ) was placed in one of the viewing oculars to facilitate counting .
we included only retinal capillaries in the mid - zone for the cell count , and measured the number of ecs and pcs in four reticles from the four quadrants of each retina .
the mean value of ecs and pcs for each retina was determined by averaging the values from the four reticles .
briefly , we counted acellular capillaries in five field areas in the middle of the retina samples ( 200 magnification ) .
acellular capillaries were defined as capillary - sized vessel tubes without nuclei anywhere along their length .
tubes with a diameter < 20% of the diameter of adjacent capillaries were identified as strands and were not counted .
we weighed each mouse before imaging , and anesthetized them by intraperitoneal injection with a cocktail of ketamine ( 87 mg / kg body weight ) and xylazine ( 13 mg / kg body weight ) . during the experiment
, we applied 0.5% tetracaine hydrochloride ophthalmic solution to paralyze the iris sphincter muscle and 1% tropicamide ophthalmic solution to dilate the pupil .
commercial artificial tears ( systane ; alcon laboratories , inc . , fort worth , tx , usa ) were added every minute to the mouse cornea to prevent dehydration and cataract formation .
the blood so2 were measured to be approximately 98 and the heart rate was approximately 320 beats per minute ( pulse oximetry , model 8500av ; nonin medical , inc . ,
all experimental procedures were in compliance with the arvo statement for the use of animals in ophthalmic and vision research , and the laboratory animal protocol approved by the institutional animal care and use committee at northwestern university .
blood glucose levels were measured with a commercial available glucometer ( glucometer elite ; bayer , inc . ,
whippany , nj , usa ) by using a drop of tail blood collected from mice models .
we monitored the body weight and blood glucose levels for both diabetic and control nondiabetic mice from 5 weeks to 13 weeks of age , as shown in figure 4 . during the imaging window ,
we also noted that at 13 weeks of age , the weights of the diabetic mice were slightly less than the control mice ( 25.1  1.0 vs. 28.5  2.0 g ) ( fig .
4b ) were observed in the diabetic mice at 5 weeks of age compared with normal blood glucose levels in the control mice ( 205  7 vs. 132  6.5 mg / dl ) .
after the final imaging at 13 weeks of age , we examined histology of the retinal microvasculature in both the diabetic and control mice .
we found no retinal acellular capillaries or pc loss in the diabetic mice ( figs .
4c , 4d ) ; both the diabetic and control mice had similar retinal ec density and pc density ( fig .
comparison of body weights , blood glucose levels , and ec and pc densities between diabetic and control groups . ( a ) body weight comparison between diabetic and control mice from 5 to 13 weeks of age .
( b ) comparison of blood glucose between diabetic and control mice from 4 to 13 weeks of age .
( c ) sample retinal vessels from diabetic mouse group at 13 weeks of age .
( d ) sample retinal vessels from control mouse group at 13 weeks of age . ( e )
we used vis - oct to quantify the retinal so2 , blood flow rate , and irmro2 simultaneously ( fig .
1 ) . to resolve the retinal so2 , we first extracted the light reflectance spectrum from each vessel , and then fitted the extracted light reflectance spectrum with the known absorption / scattering spectra of both oxygenated and deoxygenated hemoglobin based on a modified beer - lambert law ( fig .
to calculate retinal blood flow rate , we first extracted flow - induced phase shifts , then calculated the doppler angle for each retinal vessel and measured the corresponding vessel diameter . finally , with these measurements , the absolute flow rate was quantified ( see fig .
longitudinal retinal imaging for both the diabetic and control groups was performed every other week , from 5 to 13 weeks of age . at each imaging time point
, we measured seven physiological parameters , including blood velocity , vessel diameter , blood flow rate , arterial so2 , venous so2 , artery - vein so2 difference , and irmro2 , as shown in figures 5b through 5h . by comparing results between the diabetic group and control group
first , we did not find significant differences in blood velocity or arterial so2 between the diabetic mice and control mice ( p  0.128 for velocity , and  0.09 for arterial so2 ; see supplementary table s1 ) ( figs .
5b , 5d ) . these two parameters , blood velocity and arterial so2 , were also stable ( less than 4% variation ) over the course of the experiment within both the diabetic mice and control mice .
second , we did not find significant differences in vessel diameter or blood flow rate between the diabetic mice and control mice ( p  0.128 for diameter , and  0.26 for flow rate ; see supplementary table s1 ) . however , we observed a moderate increase in vessel diameter ( 4 [ m ] in absolute value or 10% in relative percentage increase ) and blood flow rate ( 0.2 [ l / min ] in absolute value or 13% in relative percentage increase ) in the diabetic mice over the course of experiments .
such increases were not present in the control mice ( less than 3% in relative percentage change ) ( figs . 5c , 5f ) .
third , between the diabetic mice and control mice , we observed significant differences in venous so2 and artery - vein so2 difference from 9 weeks of age ( p = 0.006 ; see supplementary table s1 ) , and significant differences in irmro2 from 11 weeks of age ( p = 0.001 ; see supplementary table s1 ) .
across the course of experiments in diabetic mice , we also found significant changes in the venous so2 ( 9.6 [ % ] in absolute decrease or 16% in relative percentage decrease ) , artery - vein so2 difference ( 9.0 [ % ] in absolute increase or 27.9% in relative percentage increase ) and irmro2 ( 54.45 [ nl / min ] in absolute increase or 41.3% in relative percentage increase ) . in comparison , during the course of experiments in the control mice , we found stable values among venous so2 , artery - vein so2 difference , and irmro2 ( less than 5% in relative percentage change ) ( figs .
5e , 5 g , 5h ) . longitudinal monitoring retinal flow , so2 , and irmro2 for both dr mice and control mice from 5 to 13 weeks of age .
( b ) longitudinal monitoring results of mean retinal arterial blood velocity . ( c )
we monitored the body weight and blood glucose levels for both diabetic and control nondiabetic mice from 5 weeks to 13 weeks of age , as shown in figure 4 . during the imaging window ,
we also noted that at 13 weeks of age , the weights of the diabetic mice were slightly less than the control mice ( 25.1  1.0 vs. 28.5  2.0 g ) ( fig .
4b ) were observed in the diabetic mice at 5 weeks of age compared with normal blood glucose levels in the control mice ( 205  7 vs. 132  6.5 mg / dl ) .
after the final imaging at 13 weeks of age , we examined histology of the retinal microvasculature in both the diabetic and control mice .
we found no retinal acellular capillaries or pc loss in the diabetic mice ( figs .
4c , 4d ) ; both the diabetic and control mice had similar retinal ec density and pc density ( fig .
comparison of body weights , blood glucose levels , and ec and pc densities between diabetic and control groups . ( a ) body weight comparison between diabetic and control mice from 5 to 13 weeks of age .
( b ) comparison of blood glucose between diabetic and control mice from 4 to 13 weeks of age .
( c ) sample retinal vessels from diabetic mouse group at 13 weeks of age .
( d ) sample retinal vessels from control mouse group at 13 weeks of age . ( e )
we used vis - oct to quantify the retinal so2 , blood flow rate , and irmro2 simultaneously ( fig .
1 ) . to resolve the retinal so2 , we first extracted the light reflectance spectrum from each vessel , and then fitted the extracted light reflectance spectrum with the known absorption / scattering spectra of both oxygenated and deoxygenated hemoglobin based on a modified beer - lambert law ( fig .
to calculate retinal blood flow rate , we first extracted flow - induced phase shifts , then calculated the doppler angle for each retinal vessel and measured the corresponding vessel diameter . finally , with these measurements , the absolute flow rate was quantified ( see fig .
longitudinal retinal imaging for both the diabetic and control groups was performed every other week , from 5 to 13 weeks of age . at each imaging time point
, we measured seven physiological parameters , including blood velocity , vessel diameter , blood flow rate , arterial so2 , venous so2 , artery - vein so2 difference , and irmro2 , as shown in figures 5b through 5h . by comparing results between the diabetic group and control group
first , we did not find significant differences in blood velocity or arterial so2 between the diabetic mice and control mice ( p  0.128 for velocity , and  0.09 for arterial so2 ; see supplementary table s1 ) ( figs .
these two parameters , blood velocity and arterial so2 , were also stable ( less than 4% variation ) over the course of the experiment within both the diabetic mice and control mice .
second , we did not find significant differences in vessel diameter or blood flow rate between the diabetic mice and control mice ( p  0.128 for diameter , and  0.26 for flow rate ; see supplementary table s1 ) . however , we observed a moderate increase in vessel diameter ( 4 [ m ] in absolute value or 10% in relative percentage increase ) and blood flow rate ( 0.2 [ l / min ] in absolute value or 13% in relative percentage increase ) in the diabetic mice over the course of experiments .
such increases were not present in the control mice ( less than 3% in relative percentage change ) ( figs . 5c , 5f ) .
third , between the diabetic mice and control mice , we observed significant differences in venous so2 and artery - vein so2 difference from 9 weeks of age ( p = 0.006 ; see supplementary table s1 ) , and significant differences in irmro2 from 11 weeks of age ( p = 0.001 ; see supplementary table s1 ) . across the course of experiments in diabetic mice , we also found significant changes in the venous so2 ( 9.6 [ % ] in absolute decrease or 16% in relative percentage decrease ) , artery - vein so2 difference ( 9.0 [ % ] in absolute increase or 27.9% in relative percentage increase ) and irmro2 ( 54.45 [ nl / min ] in absolute increase or 41.3% in relative percentage increase )
during the course of experiments in the control mice , we found stable values among venous so2 , artery - vein so2 difference , and irmro2 ( less than 5% in relative percentage change ) ( figs . 5e , 5 g , 5h ) .
longitudinal monitoring retinal flow , so2 , and irmro2 for both dr mice and control mice from 5 to 13 weeks of age .
( b ) longitudinal monitoring results of mean retinal arterial blood velocity . ( c )
in this study , we found increased irmro2 at early diabetes when there were no detectable anatomical retinal vascular complications .
first , retinal and choroidal circulations together provide retinal tissue oxygen and nutrients , with choroidal circulation nourishing the outer retina and retinal circulation supporting inner retina .
reduced choroidal blood flow rate was reported in early - stage diabetic mice ( 1-month - old akita/+ , tsp1 mice ) , when no retinal structural changes were observable .
decreased choroidal flow can lower the choroidal oxygen supply for photoreceptors ; however , to maintain the normal function of photoreceptors , enough oxygen is necessary . to compensate for decreased supply from the choroid in early - stage diabetes , photoreceptors have to extract more oxygen from the retinal circulation . in this view
, the increased irmro2 would reflect increased extraction to supply the outer retina , rather than an increase in the utilization of oxygen in the inner retina .
second , high blood glucose in early diabetes prompts mitochondria to generate more reactive oxygen species ( ros ) . in the presence of high ros ,
mitochondrial uncoupling is activated to reduce ros production ; however , increased mitochondrial uncoupling will contribute to increased oxygen consumption .
our results also imply that the increased irmro2 at early diabetes is mainly from the decreased retinal venous so2 levels associated with the increased retinal artery - vein so2 differences , instead of the variation of retinal blood flow rate
. this may be reasonable for early diabetes in which no retinal microvasculature complications were observable .
in early diabetes , the unchanged blood flow can be explained by the hagen - poiseuille law . according to the hagen - poiseuille law
, blood flow rate is determined by the pressure drop across the vessel and the vessel diameter .
for one thing , the pressure drop across inner retinal vasculature depends on the systemic arterial and venous pressures as well as the intermediate resistance of the capillary network .
the systemic arterial and venous pressures are likely the same between early - stage diabetic mice and control mice .
histologic examination in our experiments found no microvascular changes in diabetic mice , which may indicate normal retinal intermediate resistance .
in such a scenario , the pressure drop across the inner retina should be normal for diabetic mice .
thus , the retinal blood flow rate was expected to be normal in early diabetes , as we observed in the longitudinal monitoring experiments . with unchanged blood flow rate ,
the elevated inner retinal oxygen metabolism in diabetic mice is due to the decrease in venous so2 and increase in the artery - vein so2 difference .
previous studies have reported retinal tissue hypoxia in dr , which , however , are different from the increased irmro2 we observed .
dr is a dynamic disease , in which retinal oxygen metabolism may change with dr development . in this study
, we focused on early diabetes when there are no observable microvascular complications . in comparison
, the reported studies targeted dr , when retinal capillary dysfunctions are already present ; nonperfused retinal capillaries in dr can cause hypoxia .
for another , reported studies applied multi - wavelength fundus photography or hyperspectral photography to measure retinal so2 in dr . because of lack of depth resolution , performance of both fundus photography and hyperspectral photography are sensitive to light scattering in retinal tissue and variation of local retinal geometrical parameters , including retinal vessel diameter , which usually generate biased so2 results . in comparison , as a depth - resolved imaging technique , vis - oct can precisely extract light absorption spectra from retinal blood vessels and accurately quantify so2 .
first , there is a possibility that the volume of the retina in diabetic mice could have increased , resulting in the measured increase of irmro2 . quantifying the whole retinal volume was not feasible with our current techniques and is a limitation to all present in vivo oxygen consumption studies .
moreover , several groups have studied the retinal thickness of akita/+ mice , as a surrogate to the cell count , and found no significant changes in the thickness .
second , because we focused on measuring the functional state of the retina with vis - oct , we did not acquire oct angiograms as part of our imaging protocol . optical coherence tomography angiography has been recently reported using vis - oct to study the capillary network in rodents .
structural changes in the vasculature would be useful information to obtain , in addition to the measured functional parameters that we collected .
however , it has been shown that the akita/+ mouse does not develop retinal vascular changes until at least 6 months .
future studies with vis - oct will likely correlate longitudinal changes in vascular structure with functional oxygenation parameters .
last , a concern with vis - oct is that the illumination beam , being visible , might activate retinal neurons and , through neurovascular coupling , alter the measured so2 or blood flow rate .
studies of retinal flicker in rodents suggest that this is unlikely , because the peak response for blood flow due to whole - eye retinal flicker in rodents is at 10 hz . in vis - oct ,
a - lines are collected at 25 khz , which is several orders of magnitude higher than in flicker studies . moreover ,
the vis - oct beam is focused by the lens of the rodent eye to a small spot on the retina , which is much smaller than the whole - eye illumination used in flicker experiments . despite these facts ,
further studies are required to further investigate this issue . in conclusion , we have applied vis - oct to measure irmro2 in akita/+ , tsp1 mice and controls .
this is the first attempt to measure irmro2 in a diabetic mouse model using oct .
we found that irmro2 in diabetic mice was elevated from weeks 5 to 13 as compared with the controls .
the elevation was primarily due to a measured decrease in venous so2 , because arterial so2 and total blood flow rate remained insignificantly changed . during this same study period
, we also found that pc and ec densities were the same between diabetic mice and control mice , which shows that the changes in irmro2 occurred before vascular alterations . using vis - oct , future studies in human dr
by substituting equation 2 in equation 1 and taking the natural log , equation 1 can be rewritten as follows : 

  using this modified equation a1 , an ls fit can be performed to fit the oct signal spectrum from a vessel , resulting in values for ln(ar0 ) , so2 , and .
the doppler angle  can be calculated by inverting the cosine in the equation : 

  which results from the geometry shown in figure 3a .
the  denotes the vector dot product ;   and   are the direction of blood flow and the probing light , respectively .
the   and   were determined by the coordinates of e , s , and n. the e and s represent the center points of the sample vessel within the small and big circular b - scan images ; n signifies the nodal point of the eye .
coordinates of e , s , and n were derived from vessel centers within the circular b - scan images , as shown in figure 3b . for simplicity
3a ) were ( 1 , d1 ) and ( 2 , d2 ) within the inner and outer circular b - scans ; we also assumed the mouse eyeball diameter was h ( the reported value is approximately 3 mm ) , which was the length of no .
the diameters of the big ring and the small ring on the retina were r1 and r2 .
the coordinates of e , s , and n were estimated as follows : 



  with these equations , the doppler angles were calculated .
 purposeto investigate inner retinal oxygen metabolic rate ( irmro2 ) during early stages of type 1 diabetes in a transgenic mouse model.methodsin current study , we involved seven diabetic mice ( akita/+ , tsp1/ ) and seven control mice ( tsp1/ ) , and applied visible - light optical coherence tomography ( vis - oct ) to image functional parameters including retinal blood flow rate , oxygen saturation ( so2 ) and the irmro2 value longitudinally from 5 weeks of age to 13 weeks of age . 
 after imaging at 13 weeks of age , we analyzed the imaging results , and examined histology of mouse retina.resultsbetween diabetic mice and the control group , we observed significant differences in venous so2 from 9 weeks of age ( p = 0.006 ) , and significant increment in irmro2 from 11 weeks of age ( p = 0.001 ) in diabetic mice compared with control group . 
 we did not find significant differences in retinal blood flow rate as well as arterial so2 during imaging between diabetic and control mice . 
 histologic examination of diabetic and control mice at 13 weeks of age also revealed no anatomical retinal alternations.conclusionsin diabetic retinopathy , complications in retinal oxygen metabolism may occur before changes of retinal anatomical structure . 
Methods
Visible-Light OCT System and Scanning Protocol
Quantification of Retinal sO
Quantification of Retinal Blood Flow Rate
Calculation of IRMRO
Retinal Vessel Preparation and Endothelial Cell (EC) Density and PC Density Quantification
Assessment of Acellular Capillaries
Animal Anesthetic Procedure
Blood Glucose Measurements
Results
Histologic Examination Finds No Microvascular Structural Complications in Early-Stage Diabetic Mice
Longitudinal Imaging Reveals Increased Retinal Oxygen Metabolism in Early Diabetic Mice
Discussion
Supplementary Material
Calculating sO
Calculating the Doppler Angle