the hypothalamic suprachiasmatic nucleus , the primary circadian oscillator in humans , is the site for the master circadian clock [ 1 , 2 ] . light phase shifts the endogenous oscillator in the hypothalamic suprachiasmatic nucleus , synchronizing it with the day - night cycle [ 1 , 2 ] . some years ago , a study of human eyes culminated in the breakthrough discovery of intrinsically photosensitive retinal ganglion cells ( iprgcs ) , which constitute a subtype of retinal ganglion cells ( rgcs ) that express melanopsin , a photopigment that is most sensitive to short - wavelength blue light ( 480  nm ) [ 3 , 4 ] .
in addition to being intrinsically light - sensitive [ 3 , 57 ] , iprgcs are associated with circadian , neuroendocrine , and neurobehavioral functions as well as having influence on some image - forming functions [ 69 ] .
glaucoma , an optic neuropathy characterized by the progressive loss of rgcs , is associated with morphological changes in the optic nerve and the retinal nerve fiber layer ( rnfl ) .
several investigators have explored whether light transmission to the hypothalamic suprachiasmatic nucleus or the postillumination pupillary light response ( plr ) is compromised as a result of iprgc damage in glaucoma [ 1114 ] .
the activity of iprgcs has been evaluated in patients with glaucoma using either the light - induced reduction of nocturnal pineal melatonin secretion or the plr test as a functional indicator of melanopsin - based phototransduction [ 1214 ] .
most of these previous studies have shown significant reductions in iprgc function in affected eyes compared with the eyes of healthy subjects [ 1114 ] .
in addition , previous studies using the plr test have demonstrated significant correlations between decreased iprgc function and functional [ 11 , 13 ] and structural   damage in patients with glaucoma .
few studies have tried to find an association between abnormal iprgc function and the severity of sleep disorders in glaucoma patients [ 16 , 17 ] .
wang et al . used the self - rated pittsburgh sleep quality index ( psqi ) to evaluate whether glaucoma affects sleep quality .
these authors showed that the prevalence of sleep disorders was higher among patients with glaucoma than among healthy participants .
although these studies indicate that patients with glaucoma exhibit significantly altered sleep patterns that are also correlated with the plr test , the relationships between objective polysomnographic measures and self - reported diurnal symptoms ( e.g. , excessive daytime sleepiness ) have yet to be established .
the degree of daytime sleepiness can be assessed using the epworth sleepiness scale ( ess ) , which can be helpful in diagnosing sleep disorders .
the ess score has never been determined in a glaucomatous population , nor has it been associated with the objective polysomnography measures that are considered as the gold - standard test for assessing sleep disorders .
the study uses the plr ( as a parameter for iprgc dysfunction ) and correlated these measurements with ess and polysomnography to evaluated correlations between sleep disorder and iprgc function .
the protocol for this cross - sectional study followed the tenets of the declaration of helsinki and was approved by the institutional review board of the federal university of so paulo ( cep 262.470 ) . written informed consent
thirty of these participants had primary open - angle glaucoma , and 10 others were included for the control group .
the inclusion and exclusion criteria , as well as the ophthalmological assessments , have been previously used and are described in detail as follows [ 15 , 17 ] .
all subjects underwent a complete ophthalmological examination , including a medical history review , best - corrected visual acuity measurement , slit - lamp biomicroscopy , gonioscopy , intraocular pressure ( iop ) measurement , dilated fundoscopic examination using a 78-diopter ( d ) lens , refraction , and standard automated perimetry ( sap ) using the swedish interactive threshold algorithm ( sita standard 24 - 2 ; carl zeiss meditec , inc . ,
subjects were excluded if they were younger than 40 years old or older than 80 years old ; had a best - corrected visual acuity worse than 0.2 logmar ( logarithm of the minimum angle of resolution ) ; had a previous history of ophthalmic surgery ; had lens opacity greater than 0.5 ( cortical opacity , nuclear opalescence , posterior subcapsular opacity , or changes in nuclear color ) according to the lens opacity classification system iii ( locs iii ) ; had any corneal , retinal , or orbital diseases ; presented with an absolute spherical refractive error > 5  d or an absolute cylindrical error > 3  d ; or were using alpha - adrenergic agonist eye drop medication or any systemic medication that might affect plr .
in addition , none of our patients reported any travel or shift work during the examination period . only patients with an open angle at gonioscopy
the participants were considered as having glaucoma if they had at least 3 repeatable , consecutive , abnormal visual field test results , which were defined as a pattern standard deviation outside the 95% confidence interval for the normal range or a glaucoma hemifield test result outside the normal limits and a corresponding alteration in the appearance of the optic disc .
patients were also considered as having glaucoma if they showed signs of glaucomatous optic neuropathy at clinical examination by a glaucoma expert , confirmed by stereophotography .
glaucomatous damage to the optic disc nerve was defined as the presence of rnfl defects or localized or diffuse neuroretinal rim loss .
the control participants showed normal results at ophthalmological examination , an iop of < 21  mmhg , normal visual field test results , and absence of glaucomatous optic neuropathy based on fundoscopic and stereoscopic optic disc photograph evaluations .
the entire cohort ( i.e. , the 30 patients with primary open - angle glaucoma and the 10 controls ) belongs to the same sample of a study that was published elsewhere [ 15 , 17 ] .
no additional participant was recruited to augment that previously published sample [ 15 , 17 ] .
sleep disorders were evaluated using the ess , which has been widely used as a subjective measurement of daytime sleepiness .
this test measures the probability of falling asleep across eight daily situations ; each question is scored as 0 ( e.g. , would never doze ) , 1 ( slight change of dozing ) , 2 ( moderate chance of dozing ) , or 3 ( high chance of dozing ) , for a total possible score of 24 . the clinically normal score of this scale ranges from 2 to 10 .
a score higher than 10 indicates a borderline or increased level of chronic sleepiness , and a score higher than 15 is defined as excessive daytime sleepiness .
measurement of the plr was based on a method developed by park et al . ;
our previous studies [ 15 , 17 ] and other research groups have used this technique . to preferentially stimulate iprgc function , we used 470  nm ( blue ) flashes with luminance of 250  cd / m ( 14.1 log photons / cm / s melanopic excitation )   of 1  s in duration . alternatively , to preferentially stimulate different retinal photoreceptors ( e.g. , cones and rods ) without directly stimulating the iprgcs , we used 640  nm ( red ) flashes of 1  s in duration with luminance of 250  cd / m ( 10.7 log photons / cm / s melanopic excitation ) .  
table 1 summarizes full equations for calculating illuminance values for iprgc , cones , and rods functions . for each stimulus ,
a red flash was presented first , followed by a blue flash 60  s after the offset of the red flash .
the interval between stimuli was used to allow the pupil size to return to baseline before the presentation of the subsequent stimulus .
the patients were dark - adapted for 10  min ; then , alternating 1  s red and blue flashes were presented at luminance of 250  cd / m ( 10.7 and 14.1 log photons / cm / s melanopic excitation , resp . ) .
stimuli were generated by corresponding light - emitting diodes ( leds ) in a ganzfeld system ( retiport ; roland consult , brandenburg , germany ) , and responses were recorded using an eye - tracking camera system with an infrared led ( ir333-a ; arrington research , scottsdale , az , usa ) .
the peak wavelength of the infrared leds was 940  nm [ 20 , 22 , 23 ] .
pupil size was directly measured ; the largest diameter was considered as the primary measure .
the peak plr amplitude was calculated as the maximal pupillary constriction and expressed relative to the baseline value ( peak plr amplitude = maximal constriction diameter / baseline diameter ) .
in addition , the sustained plr was expressed as the pupil diameter 6  s after the flash offset relative to baseline .
the absolute values of the peak and sustained responses to blue and red flashes at 250  cd / m for a similar cohort were reported in our previous studies [ 15 , 17 ] . to obtain sleep parameter data ,
polysomnographic recordings were performed over a full night in a temperature - controlled and sound - attenuated room ( embla_s7000 , embla systems inc . ,
the following parameters were included in the polysomnographic assessment : total sleep time ( tst ; the amount of actual sleep time over a sleep period ) ; sleep efficiency ( tst per sleep period ) ; sleep latency ( i.e. , the amount of time before starting the effective sleep period ) ; rem sleep latency ( i.e. , the amount of time before starting rem sleep ) ; and the s1 to s3 and rem sleep stages ( as a percentage of tst ) .
arousal was defined as a  3  s increase in the electroencephalographic frequency preceded by 10  s of stable sleep .
the following data were collected for the arousal assessment : total arousal duration after falling asleep ( in minutes ) , total number of arousals ( n ) , and arousal index ( number of events per hour ) . the apnea - hypopnea index ( ahi , event count per hour of tst ) was calculated as the total number of episodes of apnea and hypopnea per hour of electroencephalographically confirmed sleep .
apnea was defined as the cessation of airflow for at least 10  s combined with the effort to breathe .
hypopnea was classified as a minimum 30% reduction in thoracoabdominal movement or airflow compared with baseline lasting at least 10  s together with 4% oxygen desaturation .
our previous study reported different polysomnographic parameters from a similar cohort [ 15 , 17 ] .
a board - certified physician who was unaware of the study design ( and was therefore blind to patients ' ophthalmic examination results ) analyzed the polysomnographic tests .
the polysomnographic tests were performed within 6 months of the administration of the ess questionnaire , the ophthalmological exams , and the plr tests .
both eyes of each participant were included in this analysis . to correct for the bias introduced by the expected correlation between the two eyes of each participant , a generalized estimating equation ( gee )
was used to adjust for intereye correlations . after adjusting for within - patient intereye correlations ,
the gee was used to examine the relationship between the ess score and the plr across the entire cohort . in addition
, a linear regression was used to determine the association between the ess score and the polysomnographic parameters for both groups . to obtain the correlation coefficients regarding the association between the ess score and the plr as well as the polysomnographic parameters ,
a linear regression was performed . whenever both eyes were eligible , the right eye was arbitrarily selected for this specific analysis .
statistical analyses were performed using commercially available stata software ( version 13 , statacorp lp , college station , tx , usa ) . the  level ( type i error )
this study examined both eyes of 40 subjects : 30 with glaucoma and 10 healthy ( control group ) .
the mean ( standard deviation ) ages of the control and glaucoma groups were 56.10  8.08 ( range = 45 to 74 ) and 62.00  9.39 ( range = 42 to 75 ) years ,
respectively ( p = 0.084 ) . the demographic and clinical characteristics of the participants are summarized in table 2 .
relevant variation was present with regard to the average md in the glaucomatous eyes , ranging from 32.28 to 0.53  db .
the mean ess score of the glaucoma patients was significantly higher than that of the control group ( 13.10  5.14 and 9.10  3.73 , resp .
, nineteen glaucoma patients ( 63.33% ) and three control patients ( 30.00% ) reported an ess score > 10 , which is considered as borderline or indicative of an increased level of chronic sleepiness ; however , this difference was not significant ( p = 0.234 ; fisher 's exact test ) .
the ess score was greater than 15 for 10 of the participants in the glaucoma group ( 33.33% ) , suggesting a high level of daytime sleepiness ; however , none of the participants in the control group had an ess score greater than 15 .  
figure 1 illustrates the distribution of the ess scores among the controls and glaucoma patients .
a significant association was found between the ess score and glaucoma severity based on sap md ( p < 0.001 ) ; specifically , higher ess scores were related to worse damage ( figure 2(a ) ) . 

table 3 shows the associations between the ess scores and the polysomnographic parameters for the entire cohort .
a significant inverse correlation was observed between the ess score and sleep efficiency ( p = 0.002 ) , indicating that higher ess scores were associated with lower sleep efficiencies .
a significant association was also found between the ess score and arousal duration after falling asleep ( p < 0.001 ) ; specifically , higher ess scores were related to longer arousal durations after falling asleep .
the ess score was also associated with the number of arousals and the arousal index ( p = 0.039 and p = 0.013 , resp . ) .  
figure 2(b ) illustrates the association between the ess score and the number of arousals at polysomnography for the entire cohort .
age was included in the multivariate model but did not exert a significant effect ( p > 0.05 for all polysomnographic parameters ) .
a significant inverse correlation was observed between the ess score and the peak response to the blue flash with a luminance of 250  cd / m ( p = 0.017 , r = 0.080 ) .
furthermore , a significant correlation was found between the ess score and the sustained response to the blue flash with luminance of 250  cd / m ( p = 0.009 , r = 0.068 ) .
a higher ess score was associated with lower peak and sustained responses to the blue flash at 250  cd / m .  
figure 2(c ) shows the association between the ess score and the sustained response to the blue flash with luminance of 250  cd / m for the entire cohort .
an inverse association was also found between the ess score and the sustained response to the red flash at 250  cd / m ( 10.7 log photons / cm / s ) ( p = 0.010 ; r = 0.080 ) ; specifically , a higher ess score was associated with a lower sustained response to the red flash with luminance of 250  cd / m ( 10.7 log photons / cm / s ) .
however , no correlation was observed between the ess score and the peak response to the red flash at 250  cd / m ( 10.7 log photons / cm / s ; p = 0.199 ; r = 0.021 ) .
in addition , age was examined in the multivariate model ; however , it was found not to exert a significant effect on the associations between the ess score and either the peak or sustained response to the blue flash with luminance of 250  cd / m for either the control or glaucoma group ( p = 0.403 and p = 0.290 , resp . ) .
in addition , age did not significantly affect the associations between the ess score and either the peak or sustained response to the red flash with luminance of 250  cd / m for either the control or glaucoma group ( p = 0.223 and p = 0.375 , resp . ) .
this study used the ess to evaluate sleep disorders , specifically daytime sleepiness , in glaucoma patients .
in addition , we addressed the relationship between the ess score and both polysomnographic measures and plr in glaucoma patients . increased daytime sleepiness , as measured by the ess ,
was associated with arousal duration after falling asleep , sleep efficiency , number of arousals , and the arousal index at polysomnography .
in addition , increased ess scores were associated with reduced peak and sustained plrs to a blue flash with luminance of 250  cd / m . to the best of our knowledge
, this study is the first to identify the associations between daytime sleepiness and polysomnography and plr parameters .
sleep disorders ( evaluated using ess and polysomnography ) and plr might be related to certain types of iprgcs .
the results of this study are in agreement with those of previous studies showing that glaucoma is associated with a decrease in sleep quality [ 16 , 30 , 31 ] .
wang et al . used the psqi to investigate whether glaucoma affects sleep quality and to evaluate the differences between disparate types of glaucoma ( i.e. , primary open - angle glaucoma and primary closed - angle glaucoma ) .
these authors found that the prevalence of sleep disorders was higher among patients with glaucoma and that the patients with primary closed - angle glaucoma had a greater incidence of sleep disorders than those with primary open - angle glaucoma . using the ess
the ess is a self - report questionnaire that is considered as the gold standard for assessing daytime sleepiness ; however , to date , the ess score has never been used to investigate glaucoma patients .
we hypothesized that ocular damage in patients with glaucoma affects all types of rgcs ( including iprgcs ) and that this damage is linked to the dysregulation of the circadian system , thereby leading to decreased sleep quality and resulting in excessive daytime sleepiness .
the finding in our study supported these hypotheses , showing that patients with glaucoma have higher daytime sleepiness associated with worse parameters in polysomnography .
the excessive daytime sleepiness observed among glaucoma patients was likely because of the increased number or total duration of arousals after falling asleep , decreased sleep efficiency , and briefer tst .
daytime sleepiness ( as measured using the ess ) was also associated with different polysomnographic parameters such as sleep efficiency , arousal duration after falling asleep , arousals , and the arousal index .
a previous study performed by our group demonstrated that patients with glaucoma have significantly lower average tsts and sleep efficiencies as well as longer arousal durations after falling asleep .
daytime sleepiness was also associated with plr , a well - known indicator of the afferent input from the retina and the optic nerve .
briefly , the plr shows a transient phase of pupillary constriction , which is attributed to rod and cone input to the iprgcs , followed by sustained pupil constriction , which is primarily driven by the melanopsin response in the iprgcs [ 20 , 37 ] .
previous studies have shown an inverse correlation between sustained pupillary constriction and glaucoma severity [ 11 , 13 , 15 ] .
for example , gracitelli et al . showed that the sustained response to the blue flash at 250  cd / m ( which preferentially stimulates iprgcs [ 3 , 4 ] ) was reduced in patients with advanced glaucoma ( i.e. , patients with sap md worse than 12  db ) .
the present study adds new information by showing that , in addition to the decreased sustained pupillary response in glaucoma patients , the degree of daytime sleepiness is associated with a reduced sustained response to a blue flash at 250  cd / m .
one possible explanation for this finding is that iprgcs are among the cells in the total rgc population that are damaged in glaucoma patients ; thus , they affect several functions of iprgcs , including the plr and circadian rhythms .
importantly , although a significant association was observed between daytime sleepiness and the plr in this study , the correlation coefficient was not substantial ( r = 0.080 for the correlation between the ess score and peak plr and r = 0.068 for the correlation between the ess score and the sustained plr ) .
for example , the subjectivity of all self - report questionnaires might affect the final score .
showed that self - reported questionnaire of sleepiness can often be inaccurate when viewed alongside quantitative measures of alertness and sometimes is often viewed as a common and natural aspect of aging that can lead to a misinterpretation of the results [ 38 , 39 ] .
in addition , daytime sleepiness , in glaucoma group , was associated with a sustained plr to the red flash .
the peak and sustained plrs to a red flash are well known to be correlated with the number and function of the rods and cones , not iprgcs .
one hypothesis explaining this finding is that rod and cones photoreceptors might also be affected in patients with chronic or advanced glaucoma , resulting in decreased signaling from these cells [ 4042 ] .
however , this topic remains controversial in the literature [ 4043 ] , and additional studies must be conducted to clarify the actual connection between the functions of iprgcs and rod and cones photoreceptors .
another interesting issue is the controversy concerning the relative preservation of iprgcs among the diversity of rgcs [ 21 , 4447 ] .
showed that although the conventional rgc number was decreased in rats with increased iop , the number of iprgcs did not change .
this result suggests that iprgcs were resistant to the deleterious effects caused by increased iop .
however , another study in mice demonstrated that iprgcs appeared to be resistant to damage resulting from iop elevation at an early age ( approximately 5 months ) but became vulnerable at a later age ( approximately 11 months ) .
the authors of that study suggested that the magnitude of iop elevation required to damage iprgcs must be greater than that required to induce rgc damage [ 48 , 49 ] .
in addition , drouyer et al . and de zavala et al . showed that glaucomatous rats and other rodents exhibited a delayed phase angle with respect to darkness [ 46 , 47 ] .
these results are in agreement with ours , suggesting that glaucoma induces a circadian system dysfunction .
one explanation for the differences between certain animal models and studies of patients with glaucoma is the endpoint evaluated , that is , morphological versusfunctional endpoints .
the primary focus of human studies has typically been iprgc function , whereas the number and morphology of these cells have usually been considered in animal studies . the resistance mechanism that underlies
although this question persists , our study analyzed two different functions involving iprgcs in the same group of patients , and we found a strong association between them .
thus , our results strongly suggest that both non - image - forming functions of iprgcs were affected .
recently , chen et al . demonstrated that there is not one specific type of iprgc ; rather , different subpopulations of iprgcs exist with similar properties that innervate disparate brain regions to execute specific functions , such as the plr and circadian photoentrainment . in this study , pupil constriction , circadian oscillation ( wheel running activity ) , the adjustment of the circadian clock to different light stimuli
( i.e. , circadian photoentrainment ,  jet - lag  paradigms , phase shifting , and skeleton photoperiod ) , and the direct effect of constant and ultradian light on activity were measured .
the results showed that iprgcs comprise functionally distinct subpopulations that differentially express a specific transcription factor .
iprgcs that do not express this transcription factor innervate the suprachiasmatic nucleus of the hypothalamus , whereas iprgcs that express this transcription factor innervate other known brain targets such as the olivary pretectal nucleus .
thus , the iprgcs that express this transcription factor are associated with the plr but not with circadian photoentrainment .
because we did not evaluate this issue in the present study , future investigations should be conducted to elucidate the subpopulations of iprgcs to determine how they are involved in non - image - forming functions .
the primary clinical finding of this study was that glaucoma patients had increased daytime sleepiness compared with healthy participants , and these results were associated with worse polysomnographic parameters and lower sustained plrs to a blue flash with luminance of 250  cd / m .
therefore , circadian rhythm regulation , which is linked with iprgcs ' functions , should be evaluated in certain patients with glaucoma .
excessive daytime sleepiness is well known to affect quality of life , daytime function , and mortality ; furthermore , previous studies of geriatric populations have revealed that excessive daytime sleepiness is associated with age , disability , dementia , and vision and hearing impairments .
although future studies with greater numbers of patients should be undertaken to better understand the role of iprgcs in sleep disorders , the present study did show strong agreement between the subjective and objective measures of sleep quality among patients with glaucoma .
second , sleep patterns are most likely affected by factors other than the hypothesized iprgc loss associated with glaucoma .
however , the subjective and objective measures of sleep quality ( i.e. , the ess questionnaire and the polysomnographic parameters ) strongly suggested that the damage to iprgcs associated with glaucoma results in certain types of sleep disorders .
furthermore , regarding the primary endpoint of this study , we relied on self - reported histories of daytime sleepiness ; it is possible that inaccuracies were present in patient recollection and reporting .
however , we also used polysomnographic parameters ( i.e. , the gold standard test of sleep quality ) , and these parameters were in agreement with the ess scores .
in addition , because patient age is associated with a decrease in sleep quality and can affect plr , this variable might have affected our final results [ 16 , 51 ] .
although the influence of age - related factors could not be completely excluded , our study did not reveal any significant differences in age between the two groups , and the multivariate analysis found that age was not a significant covariate .
in addition , previous studies have shown that the postillumination response did not significantly decrease with age   and that only pupil diameter was reduced among the elderly population . in conclusion , the results of this study demonstrated that patients with glaucoma had increased daytime sleepiness relative to healthy controls as measured by a self - report questionnaire . in addition , daytime sleepiness was associated with a reduced sustained pupillary response and polysomnographic parameters .
these iprgc functions might be impaired in patients with glaucoma , thereby affecting their quality of life .
 patients with glaucoma showed to have higher daytime sleepiness measured by epworth sleepiness scale . in addition , this symptom was associated with pupillary reflex and polysomnography parameters . 
 these iprgc functions might be impaired in patients with glaucoma , leading to worse quality of life . 
1. Introduction
2. Methods
3. Results
4. Discussion