tear film ( tf ) constantly protects the exposed surface of the eye , the cornea , and the conjunctiva from environmental stresses including desiccation , temperature change , physical injury , and infections . by providing optimal concentrations of electrolytes , proteins , mucin , and lipids , dry eye disease ( ded ) is a multifactorial dysfunction of the tf , resulting in symptoms of discomfort , visual disturbance , and even loss of vision due to damage to the ocular surface . ded is generally acknowledged to be , in large part , due to reduced secretion or increased evaporation of the tear fluid , resulting in subsequent increase in osmolarity and inflammation at the ocular surface . since ded represents a diverse group of conditions that manifest as inadequate ocular surface lubrication , restoration of a sufficient tear volume remains the mainstay of current dry eye ( de ) treatment . although lacrimal gland ( lg ) is considered the main source of tears , increasing evidence suggests that under certain conditions conjunctival epithelium has the capacity to be the primary source of tf . removal of the main lg of squirrel monkeys does not lead to keratoconjunctivitis sicca ( kcs ) . in humans , up to 86% of patients with epiphora who underwent palpebral dacryoadenectomy ( pda ) did not develop de , and in up to 50% of such patients the epiphora persisted [ 5 , 6 ] . although accessory lgs were believed to be mostly responsible in these cases , the conjunctiva certainly plays a role as a compensatory tissue . the human conjunctiva occupying 17 times more surface area than the cornea has the potential to be the primary modulator of tear volume and component . we are interested in understanding the physiology of conjunctival epithelium so as to maximize its fluid secretion capacity as an alternative to ded treatment . a rabbit model with intact conjunctiva and equal de phenotype bilaterally is ideal in such research . we created a de model in rabbits by surgical excision of the nictitating membrane ( nm ) , harderian gland ( hg ) , and main lg . although de associated ocular surface phenotype and inflammatory biomarkers elevated in the immediate postoperative period , they gradually decreased over 4-month duration to near preoperative level without therapeutic intervention . these findings suggest that the rabbit ocular surface can potentially compensate for the loss of these seemingly vital ocular surface structures , including the main lg . the results also indicate that , in acute de condition ( as created in our experiment ) , ocular surface injury and inflammation can be mostly reverted . to gain further insight into the exact mechanisms of conjunctiva mediated tear compensation , the present study further explored methods of conjunctival characterization in this mixed mechanism rabbit de model . male new zealand white rabbits ( n = 8 , 16 eyes , harlan sprague dawley , indianapolis , in , usa ) weighing 2.02.5  kg were used for this study . the rabbits were reared under standard laboratory conditions ( 22  2c , 40%  5% relative humidity , and a 12-hour light - dark cycle ) with free access to food and water throughout the experiment . the study was conducted in compliance with the tenets of the declaration of helsinki and arvo statement for the use of animals in ophthalmic and visual research . the protocol was approved by the university of arizona ( tucson , az , usa ) institutional animal care and use committee ( protocol # 14 - 511 ) . the surgical protocol for resection of main lg , hg , and nm was published previously   which was modified from established procedures [ 9 , 10 ] . the rabbits were assessed before excision ( be ) and after excision ( ae ) . to minimize slit lamp finding artifact from other tests , the evaluations were carried out in two days in the following sequence of each eye . the first day begins with corneal fluorescein test , followed immediately by rose bengal staining and cic . on the second day , schirmer tests , without ( schirmer i test , sit ) and with anesthesia ( schirmer ii test , siit ) , were performed separately in the morning and afternoon . the eyes of all rabbits were examined under a slit lamp microscope ( gr-54 , gilras llc , miami , fl ) by the same ophthalmologist ( yn ) following protocol described previously . both sit and siit were carried out in our study . one drop of 0.5% proparacaine hydrochloride ( bausch and lomb , tampa , fl , usa ) was placed and the excess fluid was blotted away with soft paper tissue , prior to the insertion of the filter paper strips ( alcon laboratories , inc . , fort worth , tx , usa ) in the lower lateral one - third of conjunctival fornix and eyelids closed by gentle force for 5  mins . the filter paper discs were peeled off and immediately placed in either 500  l trizol solution ( invitrogen , ca , usa ) for rna isolation or 100  l of radio immunoprecipitation assay ( ripa ) buffer ( teknova , ca , usa ) for protein isolation . total rna was isolated from the cic specimens in trizol solution according to manufacturer 's instructions ( invitrogen , ca , usa ) . rna concentrations were estimated by nanodrop nd-1000 spectrophotometer ( nanodrop technologies , wilmington de , usa ) in 1  l volume . purity of the rna was assessed by the ratio of absorbance at 260/280  nm . a ratio of 1.9 to 2 was considered to be good quality rna specimen and used for further experiments . the first strand of cdna was synthesized with quantitect reverse transcription kit ( qiagen , valencia , ca , usa ) using 500  ng total rna according to the manufacturer 's instructions . the rt - qpcr reactions were set using sybr green pcr master mix ( applied biosystems , foster city , ca , usa ) according to manufacturer 's instructions . the primer sequences for muc5ac were as follows : muc5ac - f : ccccaacgtcaagaacaact and muc5ac - r : tcaaacaggcagttcgagtg . the rt - qpcr was performed on steponeplus real - time pcr system ( applied biosystems ) with the following cycling conditions : 15  min 95c , 40 cycles of 15  sec 95c , and 30  sec 60c . a melting curve analysis was performed at the end of each pcr by gradually increasing the temperature from 60 to 95c while recording the fluorescence . a single peak at the melting temperature of the pcr product confirmed primer specificity . to compare between different runs , a fixed fluorescence threshold for derivation of ct value for all runs relative quantification of muc5ac expression in rabbit cic specimens was performed be and 1 month ae . the fold change in muc5ac expression was relative to the internal housekeeping gene , -actin ( endogenous control ) . mean fold change in muc5ac expression was calculated using 2 method , where ct = ( ctgene  ctactin)after  excision  ( ctgene  ctactin)before  excision . difference between ct for muc5ac and -actin mrna in each specimen was used to calculate level of target mrna relative to that of -actin mrna in the same specimen . total cell lysate proteins were isolated from cic in radio immunoprecipitation assay ( ripa ) buffer with 1x halt protease and phosphatase inhibitor single use inhibitor cocktail ( thermoscientific , rockford , il , usa ) by incubating on ice for 30  min . protein concentration was determined by pierce bca protein assay kit ( thermo fisher scientific , ny ) . hercules , ca , usa ) containing -mercaptoethanol and heated at 95c for 10  min . the primary rabbit monoclonal antibodies to il-1 , tnf- , and muc5ac ( abcam , cambridge , ma , usa ) were used at a dilution of 1 : 200 . to test the effects of epithelial sodium channel ( enac ) blockers on tear secretion , amiloride and benzamil were administered topically to the right eyes ( n = 8) of the operated rabbits 2 months ae . a 0.1% of amiloride and benzamil   ( both from sigma - aldrich , inc . louis , mo , usa ) were prepared in sterile buffered saline solution ( bss ) and tested in separate experiments . the right eyes were allocated to enac blockers and left eyes to bss as control . a 50  l of enac blocker eye drops or bss was instilled into the lower conjunctival sac by a micropipette at the beginning of the experiments . siit was performed before and at 5  min , 15  min , 30  min , 60 , and 90  min after application of amiloride or benzamil . potential difference ( pd ) is generated by electrogenic cl secretion and na reabsorption across superficial cell apical membrane of the corneal and conjunctival epithelia . pd measurement is a sensitive modality in detecting transepithelial electrolyte conductance at the ocular surface . therefore , to help delineate the underlying physiological change which contributes to the increased output of tears by the rabbit conjunctiva ae , open - circuit pd was measured with a method modified from a previously established protocol in mice . briefly , the rabbits were anesthetized with 100  mg / kg ketamine and 10  mg / kg xylazine ( sigma - aldrich , mo , usa ) and placed on a heating pad in a stereotaxic device with conjunctival and corneal tissues exposed and faced upwards . two different solutions were perfused in series over the ocular surface at a rate of 10  ml / min using a pinch valve perfusion system ( ps-8h ; bioscience tools , san diego , ca , usa ) and peristaltic pump ( 13 - 876 - 1 ; fisher scientific , pittsburgh , pa , usa ) with 1/16 inner - diameter plastic tubing . first , phosphate - buffered saline ( 1x pbs ) was perfused for 5  min to establish a stable baseline , and then 100  m amiloride ( sigma - aldrich ) prepared in 1x pbs was perfused . a low powered wall vacuum attached to 1/16 i d tubing was placed next to the fluid bolus covering the ocular surface to keep the volume constant and avoid fluid runoff . the pds were measured with a high - impedance digital voltmeter , isomilivolt meter ( world precision instruments , sarasota , fl , usa ) with two ag / agcl electrodes connected through a 1  m kcl agar bridge . one probe was placed in contact with the ocular fluid , and the other was placed subcutaneously in the rabbit 's mid - back . the pds were measured on operated rabbit eyes ( n = 4 ) 5 months ae and compared with normal rabbit eyes ( n = 4 ) as controls . data in figures are presented as mean standard error method , the bars representing standard errors . statistical significance between two groups ( be and ae ) was evaluated using unpaired 2-tailed t - test . a probability of p equal to 0.05 was considered significant ( where applicable , p < 0.05 , p < 0.01 , and p < 0.001 ) . the spearman correlation analysis was employed to determine the correlation between every pair of the tests performed , be and ae . in addition , massive hemorrhage tends to occur while excising the hg from between the medial rectus muscle and the anterior orbital wall . less hemorrhage and improved visibility of the surgical field ensured complete ablation of the hg ( figure 1 ) . however , a 5  mm palpebral conjunctival incision was made in their study to extract lobes of the main lg . in our experience , such a small incision would not permit adequate access to all lobes of the lg , especially the intraorbital lobe , which is deeply embedded beneath the lateral orbital rim and separated by a dense membranous connective tissue from the superficial temporal lobe . no additional conjunctival incision was necessary in our procedure and hence the entire conjunctival surface is preserved . the skin incision only needed to cover the lateral two - thirds of the orbital rim in order to have a good exposure to adequately remove the infraorbital , temporal , and intraorbital lobes of the lg . a rabbit model with intact conjunctiva and equal de phenotype bilaterally is ideal for our research to comparatively assess modalities that potentially stimulate conjunctival fluid secretions . as compared to be , both fluorescein and rose bengal staining increased on the cornea and conjunctiva ( figure 2 ) 1 month ae . < 0.0001 in both ) demonstrated the presence of de phenotype at the ocular surface . for all tests conducted , there were no significant differences found as a function of left versus right eye . in our study , large variations were noted in both schirmer tests among eyes tested either be or ae . with both sit ( p = 0.104 ) and siit ( p = 0.478 ) , no significant reduction in tear production was seen 1 month ae ( figure 3 ) . there was , however , significant difference between the sit and siit ( p < 0.0001 ) either be or ae , with tear secretion being lower under topical anesthesia . the protein levels of ded associated inflammatory cytokines ( tnf- and il-1 ) increased 1 month ae ( figure 4 ) which corroborated with the mrna levels of the inflammatory cytokines as reported previously . increase of conjunctival epithelium encoded goblet cell - specific muc5ac at mrna and protein levels were observed 1 month ae ( figure 4 ) . the two enac blockers did not increase tear secretion in our rabbit de model as measured by siit ( figure 5 ) . the pd measurements for the 10 seconds before the perfusion system was switched from pbs to amiloride channel were 272  6  mv for rabbit eyes in the operated group ( n = 4 ) and 159  3  mv for the control group ( n = 4 ) . after the ocular surface was perfused with amiloride , the 10-second average pd reached 133  4  mv in the operated eyes and 90  4  mv in the control eyes . the magnitude of depolarization was statistically larger ( p < 0.05 ) in the operated eyes than in the control eyes ( figure 6 ) . using spearman correlation analysis , higher siit scores are closely associated with lower rose bengal test scores ( negatively correlated , correlation coefficient = 0.57 , p = 0.02 ) . additionally , rt - qpcr of il-1 and tnf- were significantly correlated ( correlation coefficient = 0.72 , p = 0.02 ) . the changes of inflammatory biomarkers did not correlate with that of the clinical tests ( fluorescein staining , rose bengal staining , and schirmer tests ) . in our study , as expected , the rabbits showed increased fluorescein and rose bengal staining of the ocular surface 1 month ae , characteristic of de phenotype . interestingly , no significant reduction was found in tear secretion by schirmer tests as compared to be . possible explanations as to why no significant reduction in tear secretion was seen after resection of the lg , hg , and nm have been extensively discussed in a separate publication . it has been assumed that accessory lgs are responsible for the remaining tear secretion capacity in the absence of the main lg [ 6 , 15 ] . however , increasing evidence supports the notion that the conjunctiva can be an important contributor [ 5 , 8 , 16 , 17 ] . the accessory lgs are embedded in the conjunctiva , and hence the surface area of conjunctiva is substantially larger than the sum of secreting acinar cell surface area of the accessory lgs . it is not unreasonable to assume that conjunctiva contributes substantial amount to the tear volume in the absence of main lg . significant difference between sit and siit scores in our rabbit model suggests that sensory regulation of the ocular surface plays an important role . since accessory lgs have similar functions   and innervations as the main lg , they are assumed to be under identical reflex control . although a local transcellular osmotic mechanism is believed to govern the fluid and electrolyte transport   fluid secretion by the conjunctiva can also be stimulated . the presence of parasympathetic nerves in rat conjunctiva   and increased conjunctiva cl and fluid secretion by sympathomimetic agonists [ 22 , 23 ] suggests that neural influence of conjunctiva secretion can not be ruled out . and if so , local anesthesia of the secretory nerve terminals could also suppress the secretion output of the conjunctival epithelium . differences between scores of sit and siit in our study could reflect , at least to a large extent , the basal level tear secretion from accessory lgs and the conjunctiva , whereas it is very difficult , if not impossible , to determine the proportion of contributions from accessory lgs or conjunctiva to the remaining tear secretion capacity . tear breakup time and corneal / conjunctiva staining are extremely difficult to evaluate objectively , especially in small animals . correlations between clinical symptoms , signs of de , and diagnostic test results have been disappointing as well [ 2529 ] . in our study , poor correlation among the clinical tests ( fluorescein staining , rose bengal staining , and schirmer tests ) is consistent with previous studies . molecular biomarker based diagnostics , on the other hand , can offer a standardized , objective , and precise measurement of the status of ocular diseases   and should be used as adjuncts when possible . ded associated ocular surface inflammation is caused by increased level of inflammatory cytokines ( il-1 , il-6 , tnf- , and il-17 ) in tear fluid , corneal / conjunctival epithelia , and increased infiltration of dendritic and t - cells in conjunctiva . in our studies , removal of main lg , hg , and nm led to inflammatory responses at the ocular surface as depicted by increased mrna   and protein levels of tnf- and il-1. rabbits with sham surgeries did not show significant increase in biomarker mrna and protein ( data not shown ) , suggesting that persistent elevation of these markers 1 month ae is not a direct result of surgical procedure itself . although there was no significant change in tear production at 1 month ae , biomarker evaluations confirmed the increased inflammation which corroborated with the presence of de phenotype at the ocular surface . our data is consistent with solomon et al . who demonstrated that de is associated with increased production of proinflammatory cytokines ( il-1 and tnf- ) in conjunctiva . to the best of our knowledge , overexpression of goblet cell - specific muc5ac in response to acute de condition created by surgery is a novel finding in our study . in association with the persistent normal level of tear secretion , muc5ac overproduction likely contributed to the spontaneous recovery of ocular de phenotype with time in our rabbit de model . noted reduced conjunctival goblet cell density in their rabbit de model after cauterizing the lg excretory duct and surgically removing the nm and hg , whereas with mucin - specific staining , we were not able to discern any changes in the number or morphology of goblet cells in cic specimens be and ae . the exact mechanisms of goblet cell mucin regulations in our rabbit de model await further investigation . we isolated both total rna and protein from cic specimens , a rapid , convenient , and minimally invasive technique to collect one to three layers of cells from bulbar conjunctival surface . the cic has been widely performed on subjects to confirm a variety of ocular surface diseases and monitor changes at conjunctival surface . total rna and protein isolated from cic specimen detected subtle changes in mrna and protein levels of the ded associated cytokines ( tnf- and il-1 ) and muc5ac . biomarkers provided objective and quantitative data that significantly enhanced the characterization of rabbit ocular surface pathology . one cic specimen per eye at a specific time point offered sufficient high quality total rna and protein for analyzing several genes without sacrificing the animals . shi and candia concluded that the electrogenic na reabsorption across rabbit conjunctiva was amiloride - insensitive , indicating the important roles played by na dependent cotransporters such as those carrying glucose and amino acids in series with the basolaterally located na - k pump . recently demonstrated increased tear secretion as measured by schirmer test after the application of amiloride at the rabbit ocular surface . even using more potent enac inhibitor , benzamil , no significant increase in tear production was seen in the present study . we concluded that schirmer test , given its large variation between measurements , may not be sensitive enough to detect subtle change in tear production . therefore , we further assessed the baseline ocular pd and its response to the application of amiloride in rabbit eyes with and without surgery . significantly higher ( more negative ) pd in the operated rabbit eyes was noted in comparison to eyes without surgery . since electrogenic cl secretion and na reabsorption across superficial cell apical membrane of the corneal and conjunctival epithelia contribute to the pd , the ocular surface tissues must have reached a new equilibrium of higher cl secretion and/or na reabsorption . higher magnitude of pd depolarization in the operated eyes in response to the application of amiloride indicates the presence of an elevated amiloride - sensitive na conductance ( reabsorption ) across the epithelia . although amiloride - insensitive higher na reabsorption mechanism could not be measured in the study , it presumably exists . likewise , a higher cl conductance ( secretion ) most probably is present as well . our pd measurements demonstrate electrophysiological support of higher tear output across the ocular surface in rabbit eyes without lg , hg , and nm . to summarize , in this rabbit ded model , although schirmer tests were unchanged be and ae , analysis of biomarkers corroborated with the clinical examination findings and confirmed the development of de condition . assessing ded pertinent biomarkers enhanced the results obtained from standard clinical tests and is a valuable addition to the tools of ocular surface evaluation . it was interesting to note the elevated muc5ac expression in the acute de condition created by surgery but its mechanism requires further investigation . no measurable increased tear secretion was detected with schirmer test with topical application of amiloride in rabbit eyes ae . however , the open - circuit pd measurement provided a sensitive modality to detect the underlying electrophysiological changes at the rabbit ocular surface ae .
purpose . to assess the conjunctival functionality in a rabbit dry eye ( de ) model . methods . nictitating membrane , lacrimal and harderian glands 
 were surgically excised from male new zealand white rabbits using minimally invasive surgery . 
 fluorescein / rose bengal staining of ocular surface ( os ) and schirmer test were done before ( be ) and after excision ( ae ) . 
 the expression of interleukin- ( il- ) 1 , tumor necrosis factor- ( tnf- )  , and muc5ac proteins were estimated by immunoblotting from conjunctival impression cytology specimens . 
 muc5ac mrna was quantified as well . 
 the effect of epithelial sodium channel ( enac ) blockers on tear production and potential differences ( pd ) of os were assessed under anesthesia in rabbits with and without surgery . 
 results . 
 increase in corneal and conjunctival staining was observed 1 month ae compared to be . 
 schirmer tests failed to show decrease in tear production . 
 elevated il-1 , and tnf- , 1 month ae indicated inflammation . 
 muc5ac expression was elevated 1 month ae . 
 enac blockers did not improve tear production in rabbit eyes ae but characteristic changes in pd were observed in rabbits with surgery 
 . conclusions . 
 de biomarkers are important tools for os assessment and muc5ac expression is elevated in rabbit de . 
 pd measurement revealed significant electrophysiological changes in rabbits with surgery .