retinitis pigmentosa ( rp ) is a genetically heterogeneous hereditary disorder characterized by night blindness and progressive concentric visual field restriction , which may lead to severe central vision impairment due to degeneration and loss of photoreceptors and retinal pigment epithelium . on the other hand
, haemodynamic studies have demonstrated that rp patients show ocular blood flow disturbances , not only in retina and choroid but also in the retrobulbar vessels [ 24 ] , and this finding may potentially contribute to the retinal damage .
moreover , increased endothelin-1 ( et-1 ) plasma levels have been described repeatedly in rp [ 57 ] , and this increase might play a key role in determining vasoconstriction and ischemia , reducing ocular blood flow [ 8 , 9 ] , and leading to worsening of the abiotrophic process , as previously reported by our group in early stage rp .
finally , alterations of the blood - ocular barrier and signs of intraocular inflammation , including cystoid macular edema , have been demonstrated in rp patients , by clinical examination and by fluorophotometric studies [ 1113 ] , and notably , kchle and associates showed that rp eyes have increased aqueous flare values compared with controls that is strictly associated with cystoid macular edema . in an attempt to establish better whether a relationship between ocular inflammation and haemodynamic alterations may exist and whether rp subjects may show some abnormalities in the choroid , we investigated the status of the blood - ocular barrier in early stage rp subjects by using the laser flare - cell meter and evaluated subfoveal choroidal thickness ( sct ) by using spectral domain optical coherence tomography ( sd - oct ) .
the current study was an observational , case - control , single - center study performed in accordance with the tenets of the declaration of helsinki , was conducted between october 2012 and june 2014 at the s. orsola - malpighi hospital in bologna , and was reviewed and approved by the institutional and ethical committee of university of bologna .
each patient signed an informed consent form , before being enrolled in the study , after a full explanation of the aim of the study and of the procedures .
24 patients aged 25 and 42 years , affected by early stage rp , were enrolled from a cohort of 58  rp subjects followed up by our center and compared with 24 age- and sex - matched healthy controls , aged between 28 and 45 years .
the diagnostic criteria for the early stage of rp we followed up are those described by hamel in 2006 : night blindness , peripheral visual field defects , normal or subnormal visual acuity , color vision and life habits , modest attenuation of retinal arterioles , normal or fairly pale optic disc , absent or rare peripheral bone spicule - shaped pigment deposits , and a decrease in maximum electroretinogram ( erg ) amplitude .
thus , we selected only young rp patients with preserved visual function , nyctalopia , peripheral visual field defects , and decreased but not extinct erg and excluded those who had systemic diseases , such as systemic hypertension , diabetes , and cardiovascular disease , were taking any medications , had cystoid macular edema demonstrated with sd - oct and advanced posterior subcapsular cataract , or were syndromic rp patients .
all participants underwent a complete ophthalmological evaluation , including visual acuity , goldmann applanation tonometry , and slit - lamp examination of anterior and posterior segment . a visual field test , an erg , and assessment of choroidal thickness , aqueous flare , and et-1 plasma levels were also performed .
visual field test was performed by using standard automated perimetry with the humphrey 740 field analyzer 30.2 full threshold program ( humphrey instruments inc .
, san leandro , ca , usa ) , and both mean defect ( md ) and pattern standard deviation ( psd ) were obtained using humphrey statpac software and were expressed in decibels ( db ) .
electroretinogram ( erg ) was recorded by using retimax plus advanced ( cso ophthalmic , florence , italy ) .
after pupil dilation with 1% tropicamide and topical anesthesia of the cornea with 0.4% oxybuprocaine hydrochloride , hk - loop erg electrodes , a ground electrode , and the reference electrodes were applied , respectively , to the ocular surface , to the forehead , and to the temporal region .
ergs were recorded from both eyes simultaneously , according to international society for clinical electrophysiology of vision ( iscev ) standards , by using a standard flash strength of 2.5  cdsm .
band - pass filter was between 0.3  hz and 300  hz with an amplification of 5  k while artifactual signals were automatically removed .
the amplitudes of b - wave and a - wave were measured from the baseline and were expressed in microvolts ( v ) .
sd - oct is a noninvasive , objective technique that allows in vivo cross - sectional high resolution visualization of retina and choroid with a fast scanning speed ; however , to provide many details of the structure of the choroid , a new technique called enhanced depth imaging ( edi ) has been developed in the recent years and described elsewhere [ 18 , 19 ] .
we chose the horizontal scan running directly through the center of the fovea of both eyes of each patient by using sd - oct ( heidelberg engineering gmbh , heidelberg , germany ) with the edi technique and choroidal thickness was measured manually and was defined as the vertical distance from the hyperreflective line of the retinal pigment epithelium - bruch 's membrane complex to the innermost hyperreflective line of the choroidal scleral interface , exactly below the foveola .
the values were expressed in micrometers ( m ) ( figure 1 ) . for et-1 measurements
the plasma samples were drawn from the antecubital vein and collected in a container with edta , cooled , and stored in ice .
after centrifugation , the extraction was performed using a sep - column containing c-18 ( peninsula laboratories , belmont , ca , usa ) and et-1 concentration was determined by using a commercial radioimmunoassay ( ria ) kit ( rik-6901 peninsula laboratories , belmont , ca , usa ) ; after that samples and standards were firstly incubated with rabbit anti - et-1 serum for 24 hours at 4c .
a second 24-hour incubation was performed after the addition of an iodinated tracer [ i]-et-1 ( peninsula laboratories , belmont , ca , usa ) .
free and bound radioligands were separated with centrifugation and radioactivity in the precipitate was counted with an automatic gamma - counter ( packard industries , boonton , nj , usa ) .
examination of the aqueous humor by slit - lamp biomicroscopy is the primary method by which ophthalmologists evaluate the severity of anterior segment inflammation , by counting the number of cells and estimating  flare ,  that is , the amount of protein in the aqueous humor .
obviously , the ability to quantify inflammation relies on the experience of the examiner and this may result in substantial interobserver variability .
for this reason , in the late 1980s , a laser flare - cell meter , an automated objective and noninvasive technique that enables rapid and reproducible measurement of cells and flare in the aqueous humor [ 20 , 21 ] , by providing information about the status of the blood - aqueous barrier ( bab ) , and is able to identify small changes in aqueous humor proteins during the course of a disease that are not apparent clinically by slit - lamp biomicroscopy , was developed .
the device we used to assess intraocular inflammation was the laser flare - cell meter fc-500 ( kowa company , ltd , tokyo , japan ) , which uses a diode laser that is projected into the anterior chamber to scan a measurement window of 0.3  0.5  mm over 0.5 seconds , and the amount of backscattered light by protein particles in the aqueous humor , which is proportional to the concentration and size of proteins , is detected by a photomultiplier and processed by a computer .
the average of signals coming from above and below the measurement window ( background signals ) is subtracted from the signal obtained from inside the scanned window to provide a laser flare photometry measurement .
the accuracy and reproducibility of the method have been shown in several studies by different groups [ 20 , 22 , 23 ] , the coefficient of variation is less than 10% , and measurements are independent of the examiner using the instrument .
seven measurements for each eye were obtained and averaged , and those with artifacts were eliminated .
all data were expressed as the mean  standard deviation ( sd ) and only 1 eye , for each subject , was randomly selected for statistical analysis .
the statistical analysis was performed with medcalc 10.9.1 statistical program ( medcalc software , ostend , belgium ) , to assess the differences between rp patients and controls by using the wilcoxon rank - sum test .
spearman 's correlation test was used to evaluate the relationship between choroidal thickness and aqueous flare and the association between et-1 plasma levels and both choroidal thickness and subclinical ocular inflammation .
a total of 48 eyes from 24  rp patients ( male - to - female ratio , 14  :  10 ; mean age , 33.8  7.3 years ) and 24 healthy subjects ( male - to - female ratio , 12  :  12 ; mean age , 36  6.8 years ) were examined in this study ( table 1 ) . as regards rp patients , the limits of visual field test parameters we considered for inclusion in the study were md > 5  db and psd > 2.5  db , whereas the limits of erg were b - wave amplitude < 50  v and a - wave amplitude < 35  v ( in our lab , normal range for a - wave and b - wave was 56.8772.02 and 35.3740.78  v , resp . ) .
clinically , no participants showed anterior chamber inflammatory reaction ( cells and flare ) or abnormalities in the lens and vitreous .
mild to modest attenuation of retinal arterioles , normal or fairly pale optic disc , and absent or rare peripheral bone spicule - shaped pigment deposits were also observed by slit - lamp biomicroscopic examination .
age , best - corrected visual acuity , and intraocular pressure did not differ significantly between rp patients and healthy controls ( p > 0.05 ) , whereas there was a highly significant difference with regard to visual field parameters , md ( p < 0.006 ) and psd ( p
< 0.001 ) and erg ; indeed , rp had peripheral visual field defects and decreased b - wave and a - wave amplitude compared to controls ( p < 0.002 and p < 0.019 , resp . ) .
in addition , rp patients showed significantly higher et-1 plasma levels and aqueous flare than controls , 2.143  0.258 versus 1.219  0.236  pg / ml ( p < 0.002 ) and 10.51  3.97 versus 5.66  1.29  pc / ms ( p < 0.0001 ) , respectively , but also a significant reduction in choroidal thickness : 226.75  76.37 versus 303.9  39.87  m ( p < 0.03 ) ( table 1 ) .
furthermore , spearman 's correlation test highlighted that the increase of et-1 plasma levels in rp was related with the decrease of choroidal thickness ( r = 0.702 ; p < 0.023 ; figure 2 ) and the increase of intraocular inflammation , represented by aqueous flare ( r = 0.580 ; p < 0.007 ; figure 3 ) , whereas no statistically significant correlation between aqueous flare and choroidal thickness ( r = 0.308 ; p = 0.124 ) was reported .
retinitis pigmentosa is a group of inherited disorders characterized by progressive peripheral visual field loss , abnormal erg responses and variable clinical presentation , severity , age of onset , and progression and may lead to central vision loss because it diffusely involves photoreceptors and retinal pigment epithelium ( rpe ) . to the best of our knowledge
, no data have been published concerning the relationship between intraocular inflammation and et-1 plasma levels in rp patients .
our results demonstrate that subjects affected by early stage rp with preserved central visual acuity have an 86% increase in aqueous flare values , a 34% decrease in choroidal thickness , and statistically significant higher et-1 plasmatic levels compared with healthy controls .
the increase in aqueous flare reflects a disruption of the bab , which allows leakage of serum proteins , as well as inflammatory molecules and cells , into the anterior segment , by causing a change in aqueous protein composition and concentration . by means of the noninvasive laser flare - cell meter that may provide an objective assessment of the status of the bab
, we showed that rp leads to a breakdown of the bab that causes a local anterior subclinical inflammation that is not apparent clinically by slit - lamp biomicroscopy .
this finding is in agreement with previous studies ; indeed , fluorophotometric studies reported increased amount of fluorescein leakage into the vitreous of eyes with rp , whereas kchle and associates   demonstrated that subjects affected by rp have higher aqueous flare values compared with healthy controls .
finally , yoshida and coworkers   showed that aqueous flare is increased in rp patients and negatively correlates with visual function in phakic eyes .
the exact mechanism by which ocular inflammation occurs in rp patients is not clear , but two reasons may be postulated : firstly , most dystrophic and degenerative diseases are accompanied by low - grade inflammation ; it is well known that increased retinal lipofuscin fluorophores in rp may determine damage , disturbed polarity , death of rpe , and apoptosis of photoreceptors . in response to this stimulation , rpe synthesizes and releases a large variety of inflammatory molecules such as cytokines and chemokines , which , in turn , promote the recruitment of inflammatory cells that leak into the vitreous and may reach the aqueous , as there is no barrier separating the posterior from the anterior segment [ 27 , 28 ] , with a resulting enhanced aqueous flare .
secondly , as blood retinal barrier breakdown occurs both in retinal vessels and in rpe [ 29 , 30 ] , even bab may be affected , leading to increased amount of proteins into the aqueous .
the thinnest choroidal thickness values we measured in rp patients compared with controls ( p = 0.03 ) , by using edi sd - oct , are in agreement with those already demonstrated by dhoot et al .   with the same objective technique .
it is possible that rpe and photoreceptor degeneration may result in choroidal thinning due to choriocapillaris atrophy [ 32 , 33 ] ; in animal models , it has been demonstrated that these cells are necessary for choroidal maintenance and thickness by producing several factors , such as vascular endothelial growth factor .
however , we reported choroidal thinning in patients with preserved visual acuity , suggesting that , probably , abnormalities in choroidal circulation and flow are the primum movens of photoreceptor degeneration . indeed , both retinal and choroidal blood flow have previously been shown to be reduced in patients with rp , by using color doppler imaging and laser doppler flowmetry [ 24 , 35 , 36 ] , and fluorescein angiography studies further support that choroidal circulation and volume are reduced and disturbed in rp [ 3739 ] . finally , these patients showed increased et-1 plasma levels , and this finding was well related with the decrease of choroidal thickness and the increase of intraocular subclinical inflammation .
such an increase in et-1 plasma levels in rp patients has been described in previous studies [ 57 ] , but not confirmed by all researchers
. different disease heterogeneity , stages of rp , race , sample size , and the method used for the et-1 determination may justify contrasting results and different values reported in published data . currently , the exact mechanism why et-1 levels are increased is not completely elucidated , but possible explanations may be formulated .
whether et-1 is mainly produced by vascular endothelial cells , also several kinds of cells may synthetize and secrete et-1 when they are under stress conditions , such as hypoxia and oxidative stress , and interestingly , it has been widely suggested that oxidative stress is a typical finding in rp and possibly contributes to its pathogenesis .
on the other hand , some authors have hypothesised a primary vascular dysregulation syndrome [ 42 , 43 ] that might be the cause for the observed findings in blood flow reduction and increase in et-1 and could explain all the signs and symptoms both in eye and in the body of rp patients .
indeed , cellini and coworkers demonstrated , by using laser doppler flowmetry , that blood flow abnormality in rp patients was not confined to the eye but also occurred in the peripheric circulatory system .
thus , the observed increase of et-1 plasma level might be secondary to vascular dysfunction and/or subclinical systemic inflammation .
notably , we reported a negative association between choroidal thickness and et-1 ( r = 0.702 ; p < 0.023 ) and hypothesize that the reduction of choroidal thickness may be determined by the vasoconstrictive effect of et-1 , which may lead to vasospasms and unstable ocular blood supply , by altering the regulation of choroidal perfusion with a resulting relative ischemia .
there was also a significant positive correlation between aqueous flare and et-1 plasmatic levels ( p < 0.007 ) in rp patients .
it is interesting to notice that rp subjects have an imbalance of the antioxidant - oxidant status in the peripheral blood   that leads to an increase in free radicals , chronic oxidative stress , and subclinical inflammation which may stimulate cells to secrete more et-1 , as in a vicious circle ; on the other hand , increased et-1 may , in turn , lead to vascular dysregulation and hypoxic stress with resulting activation of inflammatory pathways .
independent of the cause of the et-1 increase in the plasma , this increase has consequences on ocular blood impairment , and the thinned choroid reported in our patients may be a manifestation of this decreased flow . in addition
, choroidal thickness did not correlate with the extent of subclinical inflammation of the anterior chamber ; this means that bab breakdown does not directly influence the thinning of the choroid .
firstly , the sample size was small because participants were accurately selected among young early stage rp patients , to limit confounding factors , such as age , systemic diseases , and different stages of rp , which could affect plasmatic et-1 levels .
finally , we have no data on the systemic inflammatory status of the affected patients to strengthen our results .
larger evaluation of rp patients to investigate the correlation between vascular dysregulation and inflammation will help shed further light on the key role of these elements in rp .
to our knowledge , this is the first study that demonstrates a correlation between ocular inflammation and et-1 plasma levels in early stage rp patients .
this means that increased et-1 plasma levels and subclinical inflammation may interact to play a key role in the impairment of choroidal blood flow , supply , and thickness which lead to an increase in free radicals and chronic oxidative stress .
although our results require further confirmation and investigation , this study might provide reasonable data about the possibility to use antagonists of et-1 and anti - inflammatory molecules together with antioxidants with the purpose of improving the ocular blood flow to ameliorate microvascular function and reduce the progression of the disease .
 
 purpose . to assess endothelin-1 ( et-1 ) plasma levels , choroidal thickness , and aqueous flare in patients with early stage retinitis pigmentosa ( rp ) and to search for possible correlations 
 . methods . 
 we compared 24 rp patients with 24 healthy controls . 
 choroidal thickness and aqueous flare were measured , respectively , by using a spectral domain optical coherence tomography and a laser flare - cell meter , whereas plasma samples were obtained from each patient to evaluate et-1 plasma levels . 
 results . 
 notably , rp subjects showed significantly increased et-1 plasma levels and reduced choroidal thickness compared with controls : 2.143  0.258 versus 1.219  0.236  pg / ml , p < 0.002 , and 226.75  76.37 versus 303.9  39.87  m , p < 0.03 , respectively . 
 higher aqueous flare values were also demonstrated in rp compared to controls : in detail , 10.51  3.97 versus 5.66  1.29 photon counts / ms , p < 0.0001 . 
 spearman 's correlation test highlighted that the increase of et-1 plasma levels was related with the decrease of choroidal thickness ( r = 0.702 ; p < 0.023 ) and the increase of aqueous flare ( r = 0.580 ; p < 0.007 ) . conclusions . 
 early stage rp patients show a breakdown of blood - ocular barrier and increased et-1 plasma levels and these findings may contribute to the reduction of choroidal thickness . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions