the generation and preservation of the microenvironment of the tear film is guaranteed by the biological interplay between the ocular surface together with the related glands and local neural interconnections , that represent the core players for the protection of the transparency of the cornea and the health of the ocular surface . in particular , the tear layer can be considered a reservoir of soluble factors with biological effects involved in the maintenance of the physiology of surface epithelial cells including protective and antimicrobial activities , nourishing functions , and contribution to local wound healing / anti - inflammatory responses [ 24 ] . in normal conditions , it has been demonstrated that tears of healthy subjects are characterized by a so - called  immune tone  generated by local levels of cytokines and chemokines released by different cell types , such as immune cells and epithelial cells [ 5 , 6 ] . therefore , any alteration of the tear film may have direct consequences on the ocular surface integrity leading to pathological condition such as dry eye [ 7 , 8 ] . however , at the same time , any changes on the composition of the conjunctival sac fluid might have a potential as clinical biomarker to monitor the onset - evolution of ocular surface diseases [ 911 ] and the response to pharmacological and/or surgical interventions [ 1214 ] . photorefractive keratectomy ( prk ) is one of the most commonly used surgical techniques to correct refractive errors through a laser - induction ablation of the corneal epithelium , which is able to induce corneal stromal remodelling with changes in corneal refraction . this procedure is followed by the release of several factors including cytokines , growth factors , and matrix metalloproteases that are involved in both corneal wound healing process and possible postoperative ( inflammatory ) complications . in this light , prk might be considered a model of corneal regeneration for studies aiming at improving the comprehension of the complex biological processes underlying the maintenance of local corneal microenvironment and the wound healing process . therefore , the objective of this study was to monitor the local cytokines / chemokines levels in patients undergoing prk including a wide inflammatory / immune mediators ' profile . by this experimental approach , we aimed ( i ) to increase the understanding of the physiopathological response after prk in a long term postoperative period ; ( ii ) to identify molecular mediators involved in the wound healing process after prk ; and ( iii ) to assess a methodological approach for the identification of molecular mediators that could act as laboratory biomarkers for prognostic and monitoring purposes in the context of ocular surface diseases . the subjects involved in the study included 25 patients enrolled by the ophthalmology section at the university - hospital of ferrara . all patients underwent complete preoperatively ophthalmic examination before receiving myopic prk treatment by using the 200  hz allegretto laser platform ( wavelight laser technologie ag , erlangen , germany ) . in particular , preoperative ( 1 day before prk ) , as well as postoperative ( 2 , 5 , and 30 days after prk ) , follow - up examinations included analyses of uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest refraction , corneal topography , and complications . before and after prk , at the time of patients ' clinical assessment , conjunctival sac fluid samples were collected by using standard strips of schirmer test as previously described . briefly , for each subject a volume corresponding to three notches of the strip was collected . strips were then transferred in 400  l of 0.9% nacl solution at 4c for 36 hours , to allow the release of the conjunctival sac fluid proteins from the strip in the solution . as control , conjunctival sac fluid samples were also collected from the normal contralateral eye of the 52% of enrolled patients . aliquots of conjunctival sac fluid solutions were stored at 80c and thawed only once before analyses . forearm blood samples were collected from healthy subjects in the presence of sodium citrate and immediately centrifuged for plasma isolation that were stored at 80c in single - use aliquots . written informed consents were obtained from each patient and all the procedures that followed were in accordance with the declaration of helsinki and were approved by the institutional review board ( university hospital of ferrara ) . the biological samples were frozen and thawed only once before performing the milliplex map human cytokine / chemokine panel ( merck millipore , billerica , ma ) , a bead - based multiplex immunoassay , which allows the simultaneous quantification of the following 29 human cytokines : egf , il-1 , il-1 receptor antagonist ( ra ) , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12(p40 ) , il-12(p70 ) , il-13 , il-15 , 1l-17a , eotaxin , g - csf , gm - csf , ifn-2 , ifn- , cxcl10 , mcp-1 , mip-1 , mip-1 , tnf- , tnf- , and vegf . samples were analysed in duplicate following the manufacturer 's recommended protocols and the results were read on a magpix instrument equipped with the milliplex - analyst software using a five - parameter nonlinear regression formula to compute sample concentrations from the standard curves , as previously described . the identified proteins were analysed by using the ipa software ( http://www.ingenuity.com/ ; ingenuity systems , redwood city , ca , usa ) in order to identify biological functions and molecular pathways associating the proteins , as well as predict protein - protein interaction networks determined by the ingenuity knowledge base . briefly , the ingenuity pathway knowledge base identifies protein networks that are ranked in relation to the biological functions assigned to the network . the score indicates the probability that eligible proteins are in the network by random chance . in other words , high - score values indicate high reliability of protein association ( a score > 2 is considered to be significant ) . the most highly scored networks identified were then graphically visualized showing the major molecular relationship between proteins . data were analysed by spss statistical software and calculated as median , mean  standard deviation ( sd ) for each group of data obtained from samples analysis . differences between values were evaluated by using a pairwise sign - rank wilcoxon 's test and a p value < 0.05 was considered statistically significant . the cohort of patients enrolled for this study consisted of 64% of females with age ranging between 24 and 48 years and a mean age of 36.1 years and of 36% of males characterized by age ranging between 22 and 54 years with a mean age of 36.7 years . after prk treatment the postoperative course was clinically monitored : no complications such as keratitis , infections and/or delay on corneal epithelium regeneration were reported and all patients showed full correction of the refractive errors and restoration of a proper visual quality , with the exception of one patient showing hypermetropy at the last clinical follow - up assessment performed 30 days after surgical intervention . in a preliminary experiment , we have analysed the levels of expression of 29 cytokines / chemokines in the conjunctival sac fluid samples collected from the normal contralateral eyes of patients ( see figure 1(a ) and supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/942948 ) . in particular , as summarized in figure 1(a ) , among the 29 cytokines / chemokines analysed by multiplex assay , only il-3 and il-17a were undetectable in all samples while the following 27 cytokines / chemokines were detectable at different levels ( up to over 1000  pg / ml ) : egf , il-1ra , mcp-1 , ip-10/cxcl10 , eotaxin , gm - csf , ifn- , il-10 , il-12(p40 ) , il-12(p70 ) , il-15 , il-1 , il-1 , il-2 , il-4 , il-5 , il-6 , mip-1 , mip-1 , tnf- , tnf- , ifn-2 , g - csf , il-13 , il-7 , il-8 , and vegf . notably , the expression profile and the levels assessed in the conjunctival sac fluids were significantly different from those documented in serum samples of healthy subjects ( figure 1(b ) and supplementary table 1 ) . in fact , a higher number of soluble factors were undetectable at the serum level : il-10 , il-12(p40 ) , il-15 , il-13 , il-17a , il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , and tnf- ( figure 1(b ) ) . moreover , when clustered for ranges of expression levels , a limited number of cytokines / chemokines were detected at serum levels higher than 50  pg / ml and no soluble factor showed systemic levels over 1000  pg / ml ( figures 1(a)-1(b ) ) . the patients enrolled in the study underwent prk surgery and the levels of the same 29 cytokines / chemokines were evaluated in serial samples of conjunctival sac fluid collected one day before and at different time points ( days 2 , 5 , and 30 ) after prk . the levels of the cytokines and chemokines assessed before and after the prk surgery are reported in table 1 . in order to identify clusters of cytokines / chemokines that might potentially be linked and interact at the conjunctival microenvironment level , we have firstly analysed the kinetics of release of the different cytokines / chemokines ( table 1 ) . then , in order to understand the potential association among the cytokines / chemokines having in common the same kinetic profile , the data were analysed using ingenuity pathway analysis ( ipa ) software ( http://www.ingenuity.com/ ) which identifies relevant interactions and biological mechanisms among proteins ( supplementary table 2 ) . based on these analyses , we have identified two different kinetics of modulation , referred to as profile i and profile ii . in particular , as detailed in table 1 , the majority of the cytokines / chemokines analysed , including egf , ip-10/cxcl10 , eotaxin , gm - csf , ifn- , il-10 , il-12(p40 ) , il-12(p70 ) , il-15 , g - csf , il-1 , il-2 , il-4 , il-5 , mip-1 , mip-1 , tnf- , tnf- , ifn-2 , il-1ra , il-13 , il-7 , and vegf , showed a dramatic decrease at day 2 after prk , followed by a rapid recover at day 5 and thereafter ( profile i ) . the kinetics of modulation of egf , g - csf , and il-1ra are shown in figure 2(a ) as representative of profile i. within the 23 cytokines / chemokines of profile i , two networks were found with score > 2 and involving 12 and 10 proteins , respectively ( figure 2(b ) ) . of interest , both of these networks involve a high number of focus molecules mainly related to immune cell trafficking , cellular growth / proliferation , and inflammatory responses ( supplementary table 2 ) . on the other hand , il-6 , il-8 , and mcp-1 showed a different profile of expression ( profile ii ) with respect to the majority of the cytokines / chemokines analysed , characterized by an increase at day 2 after prk ( table 1 and figure 3(a ) ) . these proteins are functionally involved in a network related to inflammatory processes ( figure 3(b ) ) . anyhow , among the panel of 29 cytokines / chemokines assessed in the present study , the peculiar kinetic of expression / modulation of mcp-1 characterized by a significant ( p < 0.05 ) and persistent increase at all times ( up to 30 days ) after prk ( figure 3(a ) ) is certainly of note . moreover , analysis of patient 's matched samples shows that the levels of mcp-1 measured at the last clinical follow - up assessment performed 30 days after prk were significantly ( p < 0.05 ) different from the levels of mcp-1 detected in paired normal contralateral eyes ( figure 3(c ) ) . the first observation derived from our study is that the profile of expression of cytokines / chemokines at the conjunctival sac fluid level does not match the systemic profile for the great majority of the cytokines / chemokines analysed in line with previous observations . in this respect , we are aware that the procedures for the measurements of the cytokines and chemokines in tears show some difficulties and that the procedure employed to measure soluble cytokines / chemokines in conjunctival sac fluid might underestimate the real concentrations ( since a percentage of the proteins could remain trapped in schirmer strips ) . in spite of these potential technical limitations , the comparative analysis of the concentrations of the same cytokines / chemokines between serum samples and tears shows a more complex pattern of expression and higher levels of cytokines / chemokines in the microenvironment of the anterior surface of the eye , with respect to the systemic circulation . this observation is likely the results of the synthesis / release from different cellular source , such as epithelial cells and immune cells , which might contribute to generate the observed differences between serum and tears . moreover , we report for the first time the pattern of expression / modulation of a wide panel of cytokines / chemokines in the conjunctival sac fluid of patients before and after prk treatment . as observed in a previous study focused on the analysis of a single cytokine ( trail ) , involved in migration [ 20 , 21 ] and inflammatory   processes , we found that the large majority of the cytokines / chemokines investigated in this study were characterized by a drastic decrease at day 2 after surgery , followed by a recover thereafter . interestingly , only three cytokines ( mcp-1 , il-6 , and il-8 ) did not show any significant decrease after surgery , with mcp-1 exhibiting the most remarkable pattern . indeed , the mcp-1 levels in the conjunctival sac fluid showed a pronounced and significant increase already at day 2 , peaking at day 5 after surgery and maintaining high levels up to day 30 after surgery . this pattern is particularly noteworthy , as mcp-1 has been involved in wound healing processes [ 2327 ] . therefore , it is likely that mcp-1 elevation plays a significant role in the process of reepithelialization of the cornea . in conclusion , in the context of prk , our data contribute to identify the key inflammatory / immune mediators involved in the local physiopathological responses to the surgical procedure and allowed to identify mcp-1 as a key molecular mediator involved in the wound healing process after prk . moreover , the present study , based on the application of a multiplex - array approach in the prk model of corneal regeneration , certainly contributes to the improvements in the development of biomarker profiles for other ocular surface diseases requiring monitoring of the pathology development as well as monitoring after surgery .
photorefractive keratectomy ( prk ) represents a therapeutic option to remodel corneal stroma and to compensate refractive errors , which involves inflammatory and/or regenerative processes . in this context 
 , the modulation of cytokines / chemokines in the conjunctival sac fluid and their role in the maintenance of the corneal microenvironment during the healing process upon refractive procedures has not been deeply investigated . in this study , 
 serial samples of conjunctival sac fluid of patients ( n = 25 ) undergoing prk were harvested before and at different time points after surgery . 
 the levels of 29 cytokines / chemokines / growth factors involved in inflammatory / immune processes were measured with a multiplex array system . 
 the results have firstly highlighted the different pattern of cytokine expression between the microenvironment at the anterior surface of the eye and the systemic circulation . 
 more importantly , the kinetic of modulation of cytokines / chemokines at the conjunctival level following prk revealed that while the majority of cytokines / chemokines showed a significant decrease , mcp-1 emerged in light of its pronounced and significant increase soon after prk and during the follow - up . 
 this methodological approach has highlighted the role of mcp-1 in the healing process following prk and has shown a potential for the identification of expression / modulation of soluble factors for biomarker profiling in ocular surface diseases .