Changes in Epithelial and Stromal Corneal Stiffness Occur with Age

bioengineering

Article Changes in Epithelial and Stromal Corneal Stiﬀness Occur with Age and Obesity

Peiluo Xu 1, Anne Londregan 2 , Celeste Rich 3 and Vickery Trinkaus-Randall 3,4,*

1 Department of Biomedical Engineering, Boston University, Boston, MA 02115, USA; [email protected] 2 Department of Biochemistry/Molecular Biology, Boston University, Boston, MA 02215, USA; [email protected] 3 Department of Biochemistry, Boston University School of Medicine, Boston, MA 02115, USA; [email protected] 4 Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02115, USA * Correspondence: [email protected]

Received: 14 January 2020; Accepted: 4 February 2020; Published: 7 February 2020

Abstract: The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and compare them to 15-week pre-Type 2 diabetic obese mouse. Nanoindentation was performed on corneal epithelium in situ and then the epithelium was abraded, and the procedure repeated on the basement membrane and stroma. Confocal imaging was performed to examine the localization of proteins. Stiffness was found to be age and obesity dependent. Young’s modulus was greater in the epithelium from 15-week mice compared to 8-week mice. At 15 weeks, the epithelium of the control was significantly greater than that of the obese mice. There was a difference in the localization of Crb3 and PKCζ in the apical epithelium and a lack of lamellipodial extensions in the obese mouse. In the pre-Type 2 diabetic obese mouse there was a difference in the stiffness slope and after injury localization of fibronectin was negligible. These indicate that age and environmental changes incurred by diet alter the integrity of the tissue with age rendering it stiffer. The corneas from the pre-Type 2 diabetic obese mice were significantly softer and this may be a result of changes both in proteins on the apical surface indicating a lack of integrity and a decrease in fibronectin.

Keywords: basement membrane; confocal imaging; nanoindenter; pre-Type 2 diabetic

1. Introduction One area of great interest is understanding if the changes that occur to tissues and cells with disease or age reflect physical changes. The result of mechanical forces can be seen in endothelial vasculature [1], bone remodeling [2], and cell-cell communication between vascular endothelial cells on substrata [3]. In addition, when epithelial cells migrate they generally move as a sheet of cells to heal a wound rather than discrete cells and we hypothesize that the sheet generates unique forces on the underlying basement membrane depending on the stiffness of the substrate [4]. The motility of the sheet is enabled by protrusion of lamellipodia at the leading edge of the wound and requires a continual assembly and disassembly of focal adhesions that occur during extension, retraction, maturation of nascent adhesions [5]. The cornea provides a superb tissue to study changes in stiffness in epithelium, basement membrane proteins and stromal matrix proteins as it is accessible, and its properties can be readily examined. This tissue is constantly subjected to a wide range of mechanical stimuli. Response to the basement membrane is critical as changes in the composition of the basement membrane have been shown to mediate cell signaling associated with cell adhesion and migration [6,7]. Others have

Bioengineering 2020, 7, 14; doi:10.3390/bioengineering7010014 www.mdpi.com/journal/bioengineering Bioengineering 2020, 7, 14 2 of 11 demonstrated that the degree of force generated by cells depends on substrate stiffness [8]. However, most of these studies were performed on single cells [9–11] or on monolayer cultures [12,13]. More recently Onochie et al., demonstrated that the leading-edge dynamics of migrating corneal epithelium were significantly different on a substrate of 8kPa compared to a stiffer substrate. In addition, there was a significantly greater phosphorylation of focal adhesion kinase proteins when cells were cultured and wounded on stiffer substrata [4]. The unwounded corneal epithelium consists of 5–7 layers of cells and the basal cells adhere to the basement membrane through structures called hemidesmosomes that are comprised of several large proteins that contact collagen anchoring fibrils in the anterior stroma. The stroma is comprised of an array of collagens and proteoglycans that permit the light to pass unaltered to the retina. Upon injury fibronectin is released and present transiently [14,15]. In addition, investigators have demonstrated that the matrix proteins change in the basement membrane zone of diabetic corneas with a decrease in laminin and an increase in heparan sulfate proteoglycans [16]. Here we investigate if the change in stiffness of corneal epithelium, basement membrane zone and stroma are altered either with age or in an obese mouse that is a model for a Type 2 pre-diabetes (DiO; diet induced obesity). Previously, investigators demonstrated that the DiO mice had impaired skin and corneal wound healing and a change in the regulation of an ionotropic receptor, P2X7 [17,18]. In our current study we found a significant increase in epithelial stiffness with age and an age matched decrease in the DiO epithelium. In contrast, there is a greater percent change in stiffness in the corneal stroma (anterior to posterior) in the 8-week mice compared to the 15-week mice. Furthermore, there is little if any change in stiffness in the stroma in the 15-week DiO mice. To understand these changes, we examined the polarity protein, Crumbs3, that is associated with tight junctions in the epithelium. Together our results demonstrate that age and environment impact the integrity of the cornea.

2. Materials and Methods

2.1. Chemicals Anti-fibronectin monoclonal mouse antibodies (clone FN-3E2, Lot# 104M4800V) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhodamine phalloidin (Lot# 1842723) was purchased from Invitrogen by Thermo Fisher Scientific (Waltham, MA, USA). Alexa Fluor 488 goat anti-mouse was obtained from Invitrogen by Thermo Fisher Scientific (Waltham, MA, USA). VectaSHIELD with 40,6-diamidino-2-phenylindole (DAPI) was purchased from Vector Labs (Burlingame, CA, USA).

2.2. Tissue Preparation The research protocol conformed to the standards of the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic Care and Vision Research and the Boston University Institutional Animal Care and Use Committee (IACUC). Control and DiO mice were obtained from Jackson Laboratory (The Jackson Laboratory; Bar Harbor, ME, USA). Control mice were maintained on the diet, D1425OB (10 kcal% fat, 3.8 kcal/g), while the DiO mice were fed a high fat diet, D12492 for 15 weeks (60 kcal% fat, 5.2 kcal/g). The mice exhibited obesity (40.8 g versus 32.4 g at 16 weeks), an elevated HbA1c, mild hyperglycemia, significantly elevated triglycerides, impaired glucose tolerance, and blood glucose for DiO that remained elevated at 120 min. In addition, no difference in bone mineral density in DiO mice was detected. Prior to delivery, body weight, blood glucose and other measurements were performed as described [1–9]. All eyes were examined under a microscope prior to experiments to make sure there were no ocular injuries prior to debridement. Experiments were performed ex vivo and all animals were sacrificed prior to measurements or injury. To examine localization of proteins, a debridement wound was made with a dull laser blade following demarcation with a 1.5 mm-diameter trephine in the central cornea [19]. After wounding, the eyes were incubated in Dulbecco’s Modified Eagles medium Bioengineering 2020, 7, 14 3 of 11

(DMEM) at 37 ◦C and 5% CO2 for diﬀerent time points. After incubation, the eyes were ﬁxed with 4% paraformaldehyde for 30 min at room temperature. The tissue was then dissected, leaving an intact scleral rim, and cut into radial wedges.

2.3. Nanoindentation Corneal epithelium and stromal stiffnesses were measured with a Piumananoindenter system (Optics11, Amsterdam, the Netherlands). To perform the measurements the head was immobilized. The eye was immersed in phosphate buffered saline (PBS) and the probe was lowered onto the center of the cornea. The probe had a tip radius of 26 µm and stiffness of 4.4 N/m. Epithelium measurements were taken through 10 µm indentations. To examine the basement membrane and stroma, the epithelium was abraded. Measurements were taken using various indentation depths, ranging from 1 µm to 17 µm. The Young’s modulus of the sample was calculated using the built-in PIUMA software based on the Hertzian contact mechanics model, assuming cornea tissue is perfectly incompressible with a Poisson ratio of 0.5. The loading curves were fit to the following equation by the software:

4 p 3 F = E R h 2 3 e f f i · where F represents the applied force, Ee f f represents the eﬀective Young’s modulus, Ri represents the spherical tip radius, and h represents indentation depth. The bulk Young’s modulus we used for analysis was generated using the following equation:

E = E (1 ν2) e f f − where ν represents Poisson’s ratio of the measured material.

2.4. Immunohistochemistry Tissues were permeabilized with 0.1% v/v Triton X-100 in PBS and blocked with 4% bovine serum albumin (BSA) solution in PBS for 1 h for indirect immunofluorescence. Samples were incubated in anti-fibronectin solution (1:200, Sigma-Aldrich, St. Louis, MO, USA), or anti-laminin-5 (γ2 chain) (1:200, MilliporeSigma, Burlington, MA, USA) at 4 ◦C overnight, washed with PBS, and incubated in Alexa-Fluor-conjugated secondary antibodies (1:200; Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) for 1 hr. Samples were washed with PBS, then incubated in rhodamine phalloidin (1:50, Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) for 20 min.

2.5. Confocal Microscopy Tissues were mounted using VectaSHIELD antifade mounting medium with DAPI during image acquisition. Tissues were placed apical side down on a glass coverslip bottom petri dish and ﬂattened by placing another glass coverslip on top of the tissue. Tissues were imaged using a 40 oil immersion × objective. The gain and laser intensity were set according to a secondary control sample and remained constant throughout the experiment. The pinhole was maintained at 1 airy unit for all sample images. The slice interval for all z-stack images was 1 µm.

2.6. Statistical Analysis Values were presented as the mean standard error of the mean (SEM). Statistical signiﬁcance ± was determined by the Wilcoxon rank-sum test (also known as the Mann-Whitney U Test) using the MATLAB function. BioengineeringBioengineering 20202020,, 77,, x 14 FOR PEER REVIEW 44 of of 11 11

3. Results 3. Results The corneal epithelium is typically a very stable structure due to tight junctions that are located in theThe apical corneal epithelium epithelium that isprevent typically growth a very fact stableors structureand other due molecules to tight junctionsfrom penetrating, that are located binding in tothe their apical receptors, epithelium and thatactivating prevent signaling growth cascades factors and [14]. other molecules from penetrating, binding to their receptors, and activating signaling cascades [14]. 3.1. Stiffness is Age and Obesity Dependent 3.1. Stiffness Is Age and Obesity Dependent In the following experiments we compared mice of 8 and 15 weeks with a naturally occurring In the following experiments we compared mice of 8 and 15 weeks with a naturally occurring murine obesity model (pre-Type 2 diabetic mice) to examine changes in stiffness in epithelium and murine obesity model (pre-Type 2 diabetic mice) to examine changes in stiffness in epithelium and the the stroma. Both eyes from five 8-week control mice, five 15-week control mice, and 2 DiO stroma. Both eyes from five 8-week control mice, five 15-week control mice, and 2 DiO 15-week-old 15-week-old mice were used. Previously, we demonstrated that corneal epithelial wound repair is mice were used. Previously, we demonstrated that corneal epithelial wound repair is impaired in impaired in corneas from the obese mice [19]. These results stimulated us to examine changes in the corneas from the obese mice [19]. These results stimulated us to examine changes in the cornea that cornea that might be underlying causes of the changes in the cell migration and wound repair. In might be underlying causes of the changes in the cell migration and wound repair. In Figure1 we Figure 1 we examined the stiffness of epithelium, basement membrane, and stroma in intact eyes. examined the stiffness of epithelium, basement membrane, and stroma in intact eyes. Young’s modulus Young’s modulus was calculated and the intact corneal epithelium from a 15-week C57Bl6 mouse was calculated and the intact corneal epithelium from a 15-week C57Bl6 mouse was significantly was significantly (Wilcoxon rank-sum test, *** p < 0.01) greater than the epithelium from an 8-week (Wilcoxon rank-sum test, *** p < 0.01) greater than the epithelium from an 8-week mouse. At the mouse. At the equivalent age, the epithelium was significantly (Wilcoxon rank-sum test, *** p < 0. 01) equivalent age, the epithelium was significantly (Wilcoxon rank-sum test, *** p < 0. 01) greater than an greater than an age matched DiO mouse cornea. Here the background of the mice are similar and the age matched DiO mouse cornea. Here the background of the mice are similar and the DiO mouse is DiO mouse is fed a high fat diet for 15 weeks. fed a high fat diet for 15 weeks.

Figure 1. Corneal epithelium stiffness (mean standard deviation) for 8-week control, assuming cornea ± Figuretissue is1. perfectly Corneal incompressibleepithelium stiffness with a(mean Poisson ± ratiostandard of 0.5. deviation) for 8-week control, assuming cornea tissue is perfectly incompressible with a Poisson ratio of 0.5. 3.2. Localization of Polarity Proteins 3.2. Localization of Polarity Proteins Since there was a major difference in stiffness in the control and DiO epithelium, we examined localizationSince there of a was polarity a major protein, difference Crumbs3 in stiffness (Crb3), whichin the control is associated and DiO with epithelium, epithelial tightwe examined junctions localizationand recruits of tight a polarity junction protein, proteins, Crumbs3 such as(Crb3), ZO-1, which to tight is associated junction structures. with epithelial Crumbs3 tight junctions has been andshown recruits to maintain tight junction apical-basal proteins, polarity, such apical as ZO-1 stability,, to tight cell adhesion, junction andstructures. epithelial Crumbs3 integrity has [20 been] and shownis required to maintain for airway apical-basal differentiation. polarity, We apical hypothesized stability, that cell we adhesion, would find andchanges epithelial in integrity Crb3. Images [20] andare presentedis required in for 2 ways airway (Figure differentiation.2). We hypothesized that we would find changes in Crb3. Images are presented in 2 ways (Figure 2). BioengineeringBioengineering 20202020,, 77,, x 14 FOR PEER REVIEW 55 of of 11 11

Figure 2. Localization of Crb3 and PKCζ in unwounded control and DiO corneal epithelium. An apical Figureand a basal2. Localization image are displayed of Crb3 and for eachPKC condition.ζ in unwounded Tissue was control stained and for DiO Crb3 corneal and PKC epithelium.ζ and counter An apicalstained and with a basal rhodamine image phalloidinare displayed and for DAPI. each Apicalcondition. cells Tissue at z-plane was 5stained out of 25for slices, Crb3 and imaged PKC atζ 1andµm counterinterval, stained are shown with (i –rhodaminep). Both the phalloidin control (j, kand) and DA DiOPI. (nApical,o) tissue cells show at z-plane no staining 5 out for of either 25 slices, Crb3 imagedor PKC ζat(Control 1μm interval, apical-( area). shown DAPI ( andi–p). actin, Both (theb). control Crb3, (c ().j,k PKC) andζ,( DiOd) merged; (n,o) tissue DiO show apical-( no estaining). DAPI forand either actin, Crb3 (f). Crb3, or PKC (g).ζ PKC(Controlζ,(h )apical-( merged;a). Control DAPI and basal-( actin,i). DAPI(b). Crb3, and actin,(c). PKC (j).ζ Crb3,, (d) merged; (k). PKC DiOζ,(l). apical-(merged;e). DiO DAPI basal-( andm actin,). DAPI (f). andCrb3, actin, (g). ( nPKC). Crb3,ζ, (h ()o merged;). PKCζ,( Controlp) merged). basal-(i). DAPI and actin, (j). Crb3, (k). PKCζ, (l). merged; DiO basal-(m). DAPI and actin, (n). Crb3, (o). PKCζ, (p) merged). Tissue was stained for Crb3 and PKCζ and counter stained with rhodamine phalloidin and DAPI andTissue an image was is stained shown for of theCrb3 most and apical PKCζ image and counter and of astained basal image.with rhodamine Both Crb3 phalloidin and PKC ζandare DAPIpresent and in an apical image images is shown but localization of the most is apical different. image The and control of a basal tissue image. shows Both punctate Crb3 staining and PKC ofζ Crb3 are present(Figure 2inb) apical and PKC imagesζ (Figure but localization2c). The DiO is tissue different. shows The di ffcontroluse staining tissue forshows both punctate Crb3 (Figure staining2f) and of Crb3PKC ζ(Figure(Figure 2b)2g). and In addition,PKCζ (Figure there 2c). is no The staining DiO tissue of either shows in thediffuse basal staining layer. To for examine both Crb3 changes (Figure in 2f)localization and PKC afterζ (Figure injury 2g). we firstIn addition, examined there F-actin is no at thestaini edgeng ofof theeither wound in the (Figure basal3) layer. and demonstrated To examine changesthat the morphologyin localization of after the cells injury is diwefferent. first examin We thened examined F-actin at thethe localizationedge of the ofwound Crb3 and(Figure performed 3) and demonstratedmaximal projection that the images morphology and made of orthogonalthe cells is sections different. to examineWe then apicalexamined polarity the inlocalization cross section of Crb3(Figure and4). performed maximal projection images and made orthogonal sections to examine apical polarity in cross section (Figure 4). Bioengineering 2020, 7, x FOR PEER REVIEW 6 of 11 Bioengineering 2020, 7, 14 6 of 11 Bioengineering 2020, 7, x FOR PEER REVIEW 6 of 11

Figure 3. Wound healing is reduced in DiO tissue stained for Crb3 and PKCζ. A tiled image was taken comprised of 20 slices taken at 0.5 μm intervals; shown is z-plane 4 stained only for actin. The z-stackFigure 3.used3. WoundWound as a representative healing healing is is reduced reduced image in in DiOat DiOthe tissue woun tissue stainedd stainededge for and Crb3for distal Crb3 and to PKCand the ζPKC wound. A tiledζ. A edge imagetiled is image wasindicated taken was bycomprisedtaken the comprisedwhite of box. 20 slices Tissueof 20 takenslices shown attaken 0.5 wasµ atm also 0.5 intervals; stainedμm intervals; shownfor Crb3 shown is and z-plane PKC is z-plane 4ζ stained. The 4wound stained onlyfor edge only actin. is forindicated The actin. z-stack The by theusedz-stack white as used a representativedotted as a line.representative (a) imageIn control atimage the tissue wound at the apical woun edge cellsd and edge can distal beand identified to distal the wound to distalthe wound edge to the is edge indicatedwound is indicated by by their the ζ largerwhiteby the box.polygonalwhite Tissue box. Tissueshape shown shownwhile was also thewas stained cellsalso stainedalong for Crb3 thfore Crb3 andwound PKC and edge PKC. The ζare. woundThe smaller wound edge and edge is indicatedmore is indicated uniform, by the by indicatingwhitethe white dotted dottedthat line. they line. (a are) In ( abasal control) In control cells tissue which tissue apical have apical cells collectively cancells be can identiﬁed migratedbe identified distal over distal tothe the wound to wound the area.wound by their(b )by In larger theirDiO tissue,polygonallarger apicalpolygonal shape cells while areshape also the while apparent cells alongthe distalcells the woundalongto the wound,th edgee wound are but smaller alongedge andtheare morewoundsmaller uniform, edge and there more indicating is uniform,reduced that b basaltheyindicating arecell basal migration. that cells they which are basal have cells collectively which have migrated collectively over the migrated wound area. over (the) In wound DiO tissue, area. apical(b) In cellsDiO are also apparent distal to the wound, but along the wound edge there is reduced basal cell migration. tissue, apical cells are also apparent distal to the wound, but along the wound edge there is reduced basal cell migration.

FigureFigure 4.4. ChangeChange in in Crb3 Crb3 after after epithelial epithelial injury. injury. Maximal Maximal projection projection images wereimages taken, were and taken, orthogonal and orthogonalimages made. images The side made. bars The show side cross-sections bars show with cross-sections an apical-basal with view an ofapical-basal Crb3 (green), view DAPI of (blue)Crb3 and actin (red). Asterisk denotes wound. (green),Figure DAPI4. Change (blue) in and Crb3 actin after (red). epithelial Asterisk denotesinjury. Maximalwound. projection images were taken, and orthogonal images made. The side bars show cross-sections with an apical-basal view of Crb3 (green), DAPI (blue) and actin (red). Asterisk denotes wound. Bioengineering 2020, 7, x FOR PEER REVIEW 7 of 11

Bioengineering 2020, 7, x FOR PEER REVIEW 7 of 11 BioengineeringWhen 2020the ,control7, 14 tissue was treated with PNGase we found the major form of Crb3 at 38kDa.7 of 11 Likewise,When when the controlthe DiO tissue tissue was was treated treated with with PNGase PNGase we the found major the form major of Crb3form shiftedof Crb3 to at 27kDa, 38kDa. indicating that Crb3 is more highly glycosylated. The major 38kDa form is also present (Figure 5). Likewise,When when the control the DiO tissue tissue was was treated treated with with PNGase PNGa wese the found major the form major of form Crb3 of shifted Crb3 atto 38kDa.27kDa, Likewise,indicating when that Crb3 the DiO is more tissue highly was glycosylat treated withed. PNGaseThe major the 38kDa major form form is of also Crb3 present shifted (Figure to 27kDa, 5). indicating that Crb3 is more highly glycosylated. The major 38kDa form is also present (Figure5).

Figure 5. Glycosylation of Crb3 in control and DiO corneal epithelium. Protein extracts treated with Figure 5. Glycosylation of Crb3 in control and DiO corneal epithelium. Protein extracts treated with PNGase and then were run on a 12% SDS-PAGE gel. The protein was transferred overnight to a PNGaseFigure 5 and. Glycosylation then were runof Crb3 ona in 12% control SDS-PAGE and DiO gel. corn Theeal proteinepithelium. was Protein transferred extracts overnight treated towith a nitrocellulose membrane for Western blotting. The blot was then probed for Crb3. The associated line nitrocellulosePNGase and membranethen were forrun Western on a 12% blotting. SDS-PAGE The blot ge wasl. The then protein probed was for transferred Crb3. The associatedovernight lineto a graphs are indicative of the relative densities within the lane. (a) Control; (b) DiO. graphsnitrocellulose are indicative membrane of the for relative Western densities blotting. within The bl theot lane.was then (a) Control; probed (forb) DiO.Crb3. The associated line graphs are indicative of the relative densities within the lane. (a) Control; (b) DiO. 3.3.3.3. StiStiffnessffness of Corneal BasementBasement Membrane andand StromaStroma 3.3. WeStiffnessWe abradedabraded of Corneal thethe corneas,corneas, Baseme nt removedremoved Membrane thethe and epithelium,epithelium Stroma , andand indentedindented at didifferentfferent depthsdepths toto examineexamine thethe didifferencesWefferences abraded inin cornealthecorneal corneas, basementbasement removed membranemembrane the epithelium andand stromalstromal, and indented stistiffness.ffness. at We different havehave shown showndepths previously previouslyto examine usingusingthe differences electronelectron microscopymicroscopy in corneal thatbasement thethe basementbasement membrane membranememb andrane stromal isis intactintact stiffness. [[21].21]. Data We waswashave collectedcollected shown previously fromfrom fivefive controlcontrolusing electron 8-week8-week mice, microscopymice, five five control controlthat 15-weekthe 15-weekbasement mice, mice, andmemb twoandrane DiO two is mice.intact DiO Results [21].mice. Data haveResults was shown collectedhave that shown basement from that five membranebasementcontrol 8-week membrane and stromal mice, and stifive stromalffness control shows stiffness 15-week an increasingshows mice, an increasingand trend two as indentationDiO trend mice. as indentation depthResults increases have depth shown inincreases control that miceinbasement control andis micemembrane similar and among is andsimilar stromal diff amongerent stiffness individuals different shows individuals (Figure an increasing6). However,(Figure trend 6). this asHowever, indentation trend is this not depthtrend as obvious isincreases not inas DiOobviousin control mice in and DiOmice has mice and greater andis similar has variance greater among between variance different individuals. between individuals individuals. (Figure 6). However, this trend is not as obvious in DiO mice and has greater variance between individuals.

Figure 6. Corneal stromal stiffness. Young’s modulus (Pa) was determined throughout the depth of the Figure 6. Corneal stromal stiffness. Young’s modulus (Pa) was determined throughout the depth of stroma in control 8- and 15-week corneas and compared to 15-week DiO corneas. (a) 8-week control theFigure stroma 6. Corneal in control stromal 8- and stiffness. 15-week Young’s corneas modulus and compared (Pa) was to determined 15-week DiO throughout corneas. the(a) depth8-week of (R2 = 0.5527) (b) 15-week control (R2 = 0.4534) (c) 15-week DiO (R2 = 0.311 for DiO mouse 1; R2 = control (R2 = 0.5527) (b) 15-week control (R2 = 0.4534) (c) 15-week DiO (R2 = 0.311 for DiO mouse 1; R2 0.3744the stroma for DiO in mousecontrol 2). 8- Both and eyes15-week were corneas measured and for compared each mouse. to 15-week DiO corneas. (a) 8-week =control 0.3744 for(R2 DiO= 0.5527) mouse (b )2). 15-week Both eyes control were (R measured2 = 0.4534) for (c) each15-week mouse. DiO (R2 = 0.311 for DiO mouse 1; R2 3.4. Changes= 0.3744 in for Fibronectin DiO mouse 2). Both eyes were measured for each mouse. 3.4. Changes in Fibronectin We examined for changes in fibronectin as it is known to be transiently localized in the stroma 3.4. Changes in Fibronectin and woundWe examined edge of for control changes corneas. in fibronectin In the control as it cornea is known we foundto be transiently that fibronectin localized was presentin the stroma at the woundand woundWe edge examined edge (arrows). of forcontrol Inchanges addition, corneas. in fibronectin we In detectedthe control as fibronectinit iscornea known we alongto found be transiently the that stromal fibronectin localized nerves was in in the presentthe control stroma at corneastheand wound wound (arrowheads) edge edge (arrows).of control where itIn corneas. is addition, presumably In thewe secretedcontroldetected cornea by fibronectin the we nerves found (Figurealong that the7 fibronectina). stromal The stromal wasnerves branchpresent in the is at detectedcontrolthe wound corneas in the edge orthogonal (arrowheads) (arrows). image. In where addition, In contrast,it is presumably we in detected the DiO secreted corneafibronectin by fibronectin the along nerves isthe di(Figure ffstromaluse (arrow) 7a). nerves The and stromal in does the notbranchcontrol appear is corneas detected in an organized(arrowheads) in the orthogonal manner where along it image. is thepresumably wound In contrast, edge secreted (Figurein the by7 DiOtheb). Innerves cornea addition, (Figure fibronectin the 7a). staining The is diffusestromal along branch is detected in the orthogonal image. In contrast, in the DiO cornea fibronectin is diffuse Bioengineering 2020, 7, x FOR PEER REVIEW 8 of 11 Bioengineering 2020, 7, 14 8 of 11 (arrow) and does not appear in an organized manner along the wound edge (Figure 7b). In addition, the staining along the stromal nerve is negligible (arrowheads). As the stromal nerves are present in thethe 15-week stromal nerveDiO mouse is negligible cornea (arrowheads).[19], we hypothesized As the stromalthat the nervessecretion are is present defective. in theFibronectin 15-week was DiO notmouse detected cornea in [ 19the], control we hypothesized unwounded that cornea the secretion (Figure is 7c) defective. as has been Fibronectin described was previously not detected [15,22]. in the control unwounded cornea (Figure7c) as has been described previously [15,22].

Figure 7. Corneas stained for fibronectin and counter stained with rhodamine phalloidin. 40 µm of 2 × 2 tile z-sections were taken and presented as maximum intensity with orthogonal sections. (a) 15-week Figure 7. Corneas stained for fibronectin and counter stained with rhodamine phalloidin. 40 μm of 2 unwounded control (b) 15-week unwounded DiO (c) 15-week control 20 h after wounding (d) 15-week × 2 tile z-sections were taken and presented as maximum intensity with orthogonal sections. (a) DiO 20 h after wounding. 15-week unwounded control (b) 15-week unwounded DiO (c) 15-week control 20 h after wounding 4. Discussion(d) 15-week DiO 20 h after wounding. The cornea is unique because it serves an essential function as the strongest refracting surface of 4. Discussion the eye while also maintaining an impermeable barrier between the eye and the external environment. We haveThe cornea shown is that unique changes because in sti it ffservesness occuran essentia withl age function as well as asthe on stronges obeset mice refracting (DiO) surface that are of a themodel eye of while pre-Type also II diabetes.maintaining Previously, an impermeabl we demonstratede barrier that between corneas ofthe DiO eye mice and heal the significantly external environment.slower than corneas We have from shown control that C57BL6 changes mice in stiffnes [19]. Ours occur data with in this age manuscript as well as on support obese a mice report (DiO) that thatpre-diabetics are a model have of anpre-Type increased II di incidenceabetes. Previously, of corneal surface we demonstrated disorders compared that corneas to controls of DiO (20.67%mice heal vs. significantly3.33%, respectively; slower pthan< 0.05) corneas [23]. from control C57BL6 mice [19]. Our data in this manuscript supportWe demonstrateda report that thatpre-diabetics the stiffness have of epithelium an increased was ageincidence dependent of corneal and diet surface dependent disorders for age comparedmatched animals. to controls As (20.67% the epithelium vs. 3.33%, of therespectively; DiO animals p < 0.05) was not[23]. as stiff we reflected on the changes in integrityWe demonstrated of the epithelium that the and stiffness stained of forepitheli polarityum was proteins age dependent that are typically and diet seen dependent in the apical for ageepithelium. matched Weanimals. examined As the unwounded epithelium andof the wounded DiO animals corneas was as not planar as stiff polarity we reflected can be initiated on the changesby several in integrity different of cues, the includingepithelium changes and stained in growth for polarity factors proteins and/or that the extracellularare typically matrixseen in [the16]. apicalThe cues epithelium. are thought We to examined be converted unwounded into directional and migration,wounded whichcorneas requires as planar the reorganizationpolarity can be of Bioengineering 2020, 7, 14 9 of 11 the cellular components by signaling pathways. These proteins generally become localized to the front of the migrating cells resulting in cytoskeletal changes with membrane protrusions at the leading edge and directional movement [24]. These changes were observed in the control and were modified in the DiO corneas. Previously, in other tissue Crb3 was found to localize to the apical surface in both differentiated secretory and differentiated ciliated cells [20]. Differences in stiffness were also detected in the basement membrane and stroma with a change in the percent change of stiffness through the stroma. All the measurements were made in the central cornea. The reason for the change in stiffness is not well understood [25]. showed that with age connective tissue is characterized by an accumulation of Advanced Glycation End-Products (AGEs) and that states that diabetics are impacted more by their accumulation. This is important as it can alter the glycation of proteins such as collagen that are abundant in the stroma. Their study on tendons in vitro demonstrated that AGEs reduce the viscoelasticity of the tissue. The data on the accumulation of AGEs over time and their impact on collagen fibrils and the spacing of proteoglycans is not known. In a recent review, McKay et al., stated that collagen cross-linking provides the strength for maintaining the integrity of the cornea and that aging and diabetes are associated with an increase in collagen cross-linking [26]. However, the review then stated that while corneal hysteresis and corneal resistance factor were elevated in the diabetic population there were inconsistencies and they proposed it was due to the heterogeneity of the human patient population. Consistency within a diabetic population is extremely difficult to control for several reasons including severity and length of the disease. For this reason, study of several different mouse diabetic models over time will be interesting to resolve this question. McKay did report increased stromal thickness with none to moderate increase in stiffness depending on the studies. In our studies we found that there was an increase in stromal thickness in the DiO cornea Kneer et al. (2018). Other investigators hypothesized that the increase was associated with diabetes, and found instead that there was no association of lysyl oxidase (LOX) with diabetes or obesity in their cohort [27]. While we measured stiffness, we did not measure LOX and its expression needs to be examined in the DiO mouse at different times of diet. In fact, their in vitro studies showed a greater association of elevated LOX with hypoxia. Furthermore, Mankus et al. (2012) demonstrated in the P2X7 knock out mouse that LOX was elevated as was Type III collagen and was not indicative of increased cross-linking [28]. However, in the DiO mouse and in diabetic human corneas, P2X7 mRNA is significantly elevated 7–10 fold [19,28]. The functional meaning of this elevated expression is not yet understood. In our current studies collagen fibril measurements were not performed, and future studies will further examine the extracellular matrix of the stroma. It is possible that there is a change in the sulfation of proteoglycans as this could also affect the stiffness. Our studies propose that the impaired wound healing in the DiO cornea could reflect the difference in stiffness or a change in the matrix molecules such as fibronectin. In the control the fibronectin was detected along the stromal nerves and at the leading edge of control compared to the DiO corneas. A reduction in fibronectin was detected in corneas subjected to hypoxia, where healing was delayed compared to control [15,22,29]. It is even possible that the lack of compliancy reflects the decrease in fibronectin as in vitro studies have shown that stretching of fibronectin causes it to unfold leading to exposure of cryptic binding sites [30]. The usage of the nanoindenter on mice of the same background at different ages and environmental stresses provides a controlled study for a field that has much conflicting data because the human model is so complex. Additional studies over time and on Type 2 diabetic models of obese and non-obese mice will be conducted to further test our results. Identification of specific factors that alter the integrity of the cornea will be used in treating and preventing changes. In summary, study of the physical features of the cornea such as stiffness in situ provides insight into the mechanisms of wound repair in control and diseased conditions.

Author Contributions: Conceptualization, V.T.-R. and C.R.; methodology, P.X. and A.L.; software, P.X.; validation, P.X., A.L., C.R., and V.T.-R. formal analysis, P.X., V.T.-R.; investigation, P.X., A.L., C.R. and V.T.-R.; resources, V.T.-R., P.X.; writing—Original draft preparation, P.X., A.L. and V.T.-R.; writing—Review and editing, P.X., A.L., Bioengineering 2020, 7, 14 10 of 11

C.R. and V.T.-R.; visualization, P.X., A.L., C.R. and V.T.-R.; supervision, V.T.-R.; project administration, V.T.-R.; funding acquisition, V.T.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Institutes of Health Grants, grants R01EYO6000 and R21EY029097, and New England Corneal Transplant Fund, and a departmental grant from Massachusetts Lions Eye Research (V.T.-R.). Peiluo Xu was funded in part by the Boston University Undergraduate Research Opportunities Program. Acknowledgments: We acknowledge Xin Brown for her assistance and usage of the Nanoindenter and YoonJoo Lee and James Zieske for their discussions on the apical proteins. We acknowledge the Confocal Imaging Core. This work is in honor of James Zieske who contributed extensively to corneal research and to his many discussions on the role of corneal stiffness. Conflicts of Interest: The authors declare no conflict of interest.

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