Ocular Phenotype of Relaxin Gene Knockout (Rln-/-) Mice

ABSTRACT Purpose: To test if relaxin deficiency affects ocular structure and function we investigated expression of relaxin (Rln) and RXFP receptors (Rxfp1, Rxfp2), and compared ocular phenotypes in relaxin gene knockout (Rln−/−) and wild type (Rln+/+) mice. Materials and Methods: Rln, Rxfp1 and Rxfp2 mRNA expression was detected in ocular tissues of Rln+/+ mice using RT-PCR. The eyes of 11 Rln−/− and 5 Rln+/+ male mice were investigated. Corneal and retinal thickness was assessed using optical coherence tomography. Intraocular pressure was measured using a rebound tonometer. Retinal, choroidal and sclera morphology and thickness were evaluated histologically. Eyes were collected and fixed for immunofluorescence staining or used for RNA extraction to evaluate mRNA expression using real-time PCR. Results: Rln mRNA was expressed only in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid. Rxfp2 was only present in the cornea. None of these genes were expressed in the lacrimal gland, eyelid or lens. Intraocular pressure was higher and central cornea of Rln−/− mice was significantly thicker and had significantly larger endothelial cells and a lower endothelial cell density than Rln+/+ mice. Immunohistochemistry demonstrated no significant difference in AQP3 and AQP5 staining in the cornea or other regions between wildtype and Rln−/− mice. mRNA expression of Aqp4 was significantly higher in Rln−/− than in Rln+/+ corneas, whereas Col1a2, Mmp9, Timp1 and Timp2 were significantly decreased. Expression of Aqp1, Aqp4, Aqp5, Vim and Tjp1 was significantly decreased in Rln−/− compared to Rln+/+ uvea. No significant differences in these genes were detected in the retina. Retinal, choroidal and scleral thicknesses were not different and morphology appeared normal. Conclusion: The findings indicate that loss of Rln affects expression of several genes in the uvea and cornea and results in thicker corneas with altered endothelial cells. Many of the gene changes suggest alterations in extracellular matrix and fluid transport between cells.


Introduction
The relaxin peptide family, including relaxin and insulin-like peptide 3 (INSL3) are endogenous peptides that are structurally related to insulin.Humans and higher primates have two relaxin (RLN1 and RLN2) genes, whereas other mammals only have RLN1. 1 The circulating relaxin peptide (referred to herein as relaxin, RLN), originally detected during pregnancy, is encoded by RLN2 in humans, and by RLN1 in other mammals, including rodents. 2 Many animal studies have characterized the biological action of relaxin as a pregnancy hormone.Specifically, it is well known that RLN contributes to some of the key maternal cardiovascular and renal adaptations during pregnancy. 3,4For example, RLN can influence the expression of aquaporins, water channels that regulate the hydration of tissue not just those found in the cervix or kidney but also in the eye. 5espite structural similarities between RLN and INSL3, they bind to and exert their biological function via different receptors.Specifically, RLN is the cognate ligand for the relaxin family peptide receptor, RXFP1, which is a G-protein coupled receptor.1][12] In male Rln gene knockout (Rln −/− ) mice, there is endothelial dysfunction and reduced volume compliance in mesenteric arteries when compared to their wildtype (Rln +/+ ) counterparts. 13In addition to the effects on vasculature, the RLN-RXFP1 system also has well-established antifibrotic effects in several organs such as the kidney, heart, liver and lungs by regulating extracellular matrix (e.g.5][16] In contrast to the RLN-RXFP1 system, less is known about the biological action of INSL3, which is the cognate ligand for RXFP2.RXFP2 is known to be expressed in the ovary, testis, and gubernaculum, and has specific roles in modulating testicular descent and gonadal function. 17ore recently, RLN2 and RXPF1 have been detected in the eye and whole eyelid in humans. 18Specifically, RLN2 and RXFP1 mRNA transcripts are present in lacrimal gland, eyelid, conjunctiva, cornea, nasolacrimal ducts and primary corneal fibroblasts, suggesting a functional role for the RLN-RXFP1 system in the eye.Indeed, in vitro studies using recombinant RLN2 demonstrated accelerated wound healing in corneal and conjunctival epithelial cell lines via increased proliferation and faster migration, involving regulation of matrix metalloproteinase (MMP) and tissue inhibitors of MMPs (TIMP) expression.In particular, the gelatinases MMP2 and MMP9 as well as their inhibitors TIMP1 and TIMP2 were found to be regulated by RLN2.In a mouse model, topical application of recombinant RLN2 resulted in improved wound closure after alkali burns. 18Other evidence that the RLN-RXFP1 system modulates ocular physiology comes from a preliminary study showing that relaxin injected intramuscularly decreased the intraocular pressure (IOP) in six humans. 19However, this result has not been recapitulated in laboratory models, as recombinant RLN2 treatment failed to change intraocular pressure or dilate retinal vessels in rat eyes. 20Therefore, despite the wide distribution of RXFP1 within the eye, these data highlight potential region-specific (e.g.retina, cornea and uvea) differences in the function of relaxin within the eye.In addition to the detection of RLN2 and RXFP1 in the eyes, RLN2 was detected in human tears, suggesting that it is produced endogenously and it may regulate ocular physiology in a paracrine or autocrine manner. 18o date, the role of endogenous RLN2 in the eyes and the possibility that RLN2 is secreted to regulate the ocular milieu has not been investigated.
We sought to examine ocular phenotypes in Rln −/− mice to determine the endogenous role of RLN in corneal and ocular structure and function.The specific aim of this study was to investigate if Rln −/− leads to region-specific ocular phenotypes in the retina, cornea and uvea.

Animals
The present study used the original Rln −/− mouse backcrossed on a C57BL/6J background to the F14 generation. 21eterozygous Rln ± mice were mated to generate male Rln −/− and Rln +/+ littermates as controls.The eyes of eleven male Rln −/− (age: 10.9 ± 0.6 months) and five male Rln +/+ mice (age: 10.7 ± 1.4 months) were investigated.The average body weight was 39.4 ± 1.8 g in the Rln −/− mice and 42.0 ± 2.8 g in the Rln +/+ mice group (p = .435).Genotypes were confirmed by PCR analysis of genomic DNA from ear clips as previously described. 22All mice were housed on a 12:12-h day/night cycle at a room temperature of 20 ± 2°C in the School of BioSciences Animal Facility (The University of Melbourne) and were given ad libitum access to standard rodent chow (Barastock, VIC, Australia) and water.
The anterior segment of each eye was imaged using spectral-domain OCT (VHR, Bioptigen Inc., Durham, NC) to evaluate any structural changes of the cornea in vivo as described previously. 23Colour images and spectral-domain OCT cross-sectional images of the retina through the center of the optic nerve and fundus were acquired using the Micron III small animal imaging platform (Phoenix Research Labs, Pleasanton, CA, USA).Intraocular pressure was measured in each eye using a rebound tonometer (Tonolab, iCare, Helsinki, Finland), and the average of 10 readings for each eye was used for analysis.Following OCT and IOP measurement, animals were killed via cervical dislocation and eyes were removed for histological analysis or tissues were dissected and stored in RNAlater for qPCR analysis.

Measurement of corneal thickness and anterior chamber depth
Once anaesthetized, a drop of sterile saline was applied to the mouse eye to prevent corneal drying.Mice were placed on the animal imaging mount and rodent alignment stage attached to the Bioptigen SD-OCT imaging device and aligned with a noncontact 18 mm Telecentric lens (Bioptigen, Inc., Durham, NC).Volumes of 4 mm x 4 mm (100 B-scans equally separated vertically, 1000 A-scans/B-scan) were captured for calculating central corneal thickness. 23All scans were exported as TIFF files and imported into Image J software (v1.47;US National Institutes of Health) for analysis.Total central corneal thickness was measured at the point where a vertical line was orthogonal to the anterior corneal curvature.Final values represent the average thickness across eleven individual frames, each spaced 40 microns apart.Anterior chamber depth was measured at the point where a vertical line was orthogonal to the posterior corneal curvature and lens anterior surface.Final values represent the average measure of five central individual frames, spaced 40 microns apart.

Measurement of retinal layer and vessel thickness
Spectral-domain optical coherence tomography (OCT) was utilized to quantify retinal thickness (Image-Guided 830 nm OCT, Phoenix Research Laboratories, Pleasanton, CA).
A circle pattern was used to scan (1024-A-scans per b-scan, 10 repeats; axial resolution 4 µm, lateral resolution 8 µm) a cross section of the retina centered on the optic nerve.Retinal thickness was measured from the vitreal-retinal interface to Bruch's membrane.Segmentation was undertaken manually by a masked grader.Thickness measurements represent the average for regions at a distance of 36 µm away from the centre of the optic nerve.Average retinal thicknesses from right and left eyes were used for calculations (Rln −/− n = 11 animals, Rln +/+ n = 5 animals).Colour images of the fundus were used to measure the retinal vessel diameter at a distance of 36 µm from the optic disc.Average retinal vessel diameter from right and left eyes were used for calculations (Rln −/− n = 10 animals, Rln +/+ n = 5 animals).

Histological assays
Whole eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 1 h at room temperature.For quantitative analysis of endothelial cell density (ECD), corneas were dissected as flat mounts, washed with phosphatebuffered saline (PBS), incubated with 20 mM EDTA for 1 h at 37°C then blocked in PB containing 3% BSA, 0.3% triton X-100 and 5% goat serum.TJP1 immunofluorescence was used to visualize cell borders of endothelial cells for cell size measurements.Corneas were incubated overnight at 4°C with an antibody to tight junction protein 1B (TJP1; rabbit, HPA001636, Sigma-Aldrich), washed in PBS and subsequently incubated with Alexa488 conjugated goat-anti-rabbit antibody (MoBiTec GmbH, Göttingen, Germany, 1:200 in PBS) for 1 h at room temperature.Flat mounts were counterstained with 4′,6-diamidine-2-phenylindole (DAPI; 1:1000, Roche Applied Science, Mannheim, Germany), washed and cover slipped epithelial side down.Images were acquired using a Keyence BZ9000 microscope (Keyence GmbH, Neu-Isenburg, Germany) at three different central positions per cornea at 60x magnification.Endothelial cell densities were manually counted by a masked observer using FIJI software.Endothelial cell size was manually measured by a masked observer using imageJ software.Cell borders were marked with the freehand tool and area measured with preset scales.For epithelial thickness, corneal tissue was stained with sirius red and central epithelial thickness from the surface of the epithelium to the basal epithelial layer was manually measured in three positions by a masked observer using ImageJ software (Figure 3g).
For aquaporin localization, eyes were embedded in paraffin, sectioned (6 μm) and dewaxed.Sections were treated with trypsin (Sigma-Aldrich, 5 mg/ml in PBS) for 10 min at 37°C for permeabilization.Heat-induced epitope recovery was carried out in citrate buffer (pH 6, 1.8 mM citric acid, 8.2 mM sodium citrate) in a microwave at 600 W. Sections were pretreated with 1% powdered milk solution in PBS-Tween (Carl Roth GmbH & Co, Karlsruhe, Germany) for 1 h at room temperature to prevent nonspecific binding.Sections were incubated in primary antibody AQP3 (1:50, Santa Cruz sc-9885), and AQP5 (1:100, Santa Cruz sc-9890) in PBS with 2% [w/v] bovine serum albumin (Merck, Darmstadt, Germany) and 0.2% [v/v] Triton X100) overnight at 4°C, rinsed in PBS three times for 10 min each time, followed by secondary antibody treatment (Alexa488 conjugated rabbitanti-goat antibody, MoBiTec GmbH, Göttingen, Germany, 1:100 in PBS).Nuclear counterstaining was done with DAPI.Two negative control sections were used in each case: one was incubated with the secondary antibody only, and the other with the primary antibody only.The slides were examined with a Keyence BZ9000 microscope (Keyence GmbH, Neu-Isenburg, Germany).
For retinal layer morphological evaluation and quantification of retinal, choroidal and scleral thickness, haematoxylin and eosin-stained slides were examined with a Keyence BZ9000 microscope (Keyence GmbH, Neu-Isenburg, Germany) and images of the posterior retina close to the optic nerve of each eye were taken.Retinal layers, choroid and sclera were manually measured in five positions in each section by a masked observer using ImageJ software.

RNA preparation and cDNA synthesis for conventional RT-PCR
Samples of retina, choroid and sclera, cornea, lacrimal gland, whole eyelid and lens were crushed in a Wig-L-Bug® grinder/ mixer (Sigma-Aldrich) under liquid nitrogen.Tissues from the right and left eyes of one mouse were pooled.Total RNA was isolated using TRI-Reagent (Ambion, Mulgrave, VIC, Australia) according to the manufacturer´s instructions.Five hundred nanograms of total RNA was used for each cDNA reaction.The cDNA was generated with 50 ng/µl (20 pmol) oligo-dT15 primer (Biosearch Technologies, Novato, CA) and 0.8 µl Superscript RNase III reverse transcriptase (100 U; Invitrogen, Mount Waverly, VIC, Australia) for 60 min at 37°C.Reverse transcriptase-polymerase chain reaction (RT-PCR) RT-PCR amplifications were carried out with 1 µl cDNA in a final volume of 24 µl containing 12.5 µl Go Taq Green, 9.5 µl PCR buffer, and 1 µl forward/reverse primer mix (10 µM, synthesized at Biosearch Technologies, Novato, CA).To exclude genomic amplifications, PCR was performed with specific primers for Rln, Rxfp1 and Rxfp2 as well as Gapdh, which all spanned at least one intron.Details for primers are listed in supplemental Table 1.After 2 min of heat denaturation at 85°C, the PCR cycle consisted of 40 cycles of 94°C for 1 min, 60°C for 1 min; and 72°C for 1 min, followed by a final elongation step of 72°C for 10 min.The PCR reactions (10 µl) were loaded onto a 1% agarose gel, and the amplicons were visualized via fluorescence.Control reactions, in which the reverse transcriptase was omitted, did not result in observable amplificons.Uterus served as a positive control as it expresses Rln, Rxfp1 and Rxfp2. 24A preparation and cDNA synthesis for real-time PCR For each sample, tissues from two mice (WT or Rln −/− ) were pooled.For uvea preparation, the retina was removed after the globe was cut along the equator.Choroid attached to the sclera was scraped off and ciliary body and iris were dissected from the other half to collect uveal tissue.Samples were mechanically lysed in 350 µl RLT buffer, containing βmercaptoethanol and guanidine-isothiocyanatae (QIAGEN Group, Hilden, Germany) in an innuSPEED Lysis Tube placed in a SpeedMill plus homogenizer (both Analytik Jena AG, Jena, Germany).Total RNA was purified with RNeasy MinElute® spin columns using RNeasy Plus Micro Kit (QIAGEN Group) according to the manufacturer's protocol.High-quality RNA was eluted in 14 µl RNase-free water.Reverse transcription of 500 ng RNA from each sample to first-strand cDNA was performed by Transcriptor First-Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer's protocol.Fluorescence visualization of reference gene β-actin PCR amplification product on agarose gel-assessed integrity and stability of the cDNA used for real-time PCR assays.

Real-time PCR assays
Real-time PCR reactions were conducted with custom-made human RealTime ready Custom Panels (supplemental Table 2 for details) from Roche.Amplifications were performed with gene-specific primers and a Universal ProbeLibrary (UPL) probe on a LightCycler® 480.As per the manufacturer's instructions, reactions were carried out in a final volume of 20 μl with 0.4 μl of each cDNA.All 96-well plates were run at 95°C for 10 min, followed by 55 cycles at 95°C for 10 s, 60°C for 30 s and 72°C for 1 s.The cycle threshold (C t ) parameter was defined by second derivative maximum analysis with LightCyler480 software v1.5.To standardize mRNA concentration, transcript levels of Gapdh and Rn18s as reference genes were determined in parallel in each sample.Gapdh showed no regulation and was therefore chosen for calculations.Relative transcript levels were corrected by normalization with the Gapdh C t levels and relative changes in gene expression were calculated using the delta-delta C t method. 25

Statistical analysis
After evaluating that the measurements were normally distributed using the Kolmogorov-Smirnov test, statistical significance was evaluated with independent t-tests when homogeneity of variances was given.Otherwise, we decided to use the nonparametric Mann Whitney U test.All data were analyzed with GraphPad Prism (version 5 GraphPad Software, Inc).A p value less than 0.05 was considered statistically significant.
Rln and Rxfp receptor gene expression in wild-type Rln +/+ mice In Rln +/+ mice, Rln mRNA expression was only detected in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid (Figure 2).The retina expressed only the Rxfp1 splice variant 1 amplicon of 450 bp, whereas cornea and sclera/choroid expressed both Rxfp1 transcripts (splice variant 1: 450 bp, splice variant 3: 320 bp) that can be detected in the uterus.Rxfp2 was only detected in the cornea.None of these genes were expressed in the lacrimal glands, whole eyelids or lenses.

Corneal gene expression
In Rln −/− mice, relative expression of corneal Aqp4 was significantly increased 3.1 ± 0.83-fold (p = .030)compared to wild-type mice (Figure 4a).By contrast, expression of Aqp1, Aqp3 and Aqp5 in whole corneas were not different between Rln −/− and Rln +/+ mice.Immunofluorescence in sagittal sections of whole eyes showed that AQP3 and AQP5 were localized in the corneal epithelium (Figure 4b) of both Rln −/ − and Rln +/+ mice and there was no demonstrable difference in distribution or level of staining for either of these proteins in the cornea or other ocular regions.Negative controls did not show any detectable staining.

Discussion
The aim of the study was to determine the homeostatic role of RLN in corneal and ocular structure and function.The key findings are that there were tissue-specific changes to ocular structure and gene expression associated with the extracellular matrix (ECM) and aquaporins in Rln −/− mice (see also summary Table 1).Specifically, there was down-regulation of ECM-associated genes, but upregulation of Aqp4 expression in the cornea of Rln −/− mice.This was accompanied by increased corneal thickness, reduced corneal endothelial cell density and endothelial cell hypertrophy.In contrast to the cornea, Rln −/− mice showed down-regulation of Vim and Aqp1, Aqp4 and Aqp5 in the uvea.Interestingly, Rln −/− mice showed few difference in the expression of retinal genes that were analysed.There was no impact on retinal thickness or intraocular pressure in Rln −/− mouse eyes (Figure 3).
The pregnancy hormone RLN2 acts via activation of the receptor, RXFP1. 26Besides the detection of RXFP1 in RT-PCR analyses of Rln1, Rxfp1, Rxfp2, and GAPDH in WT mice (n = 3).Rln mRNA was only detected in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid.Rxfp2 was only present in the cornea.None of these genes were expressed in lacrimal glands, eyelids or lenses.Key: (1) retina, (2) choroid and sclera, (3) cornea, (4) lacrimal gland, ( 5) eyelid, (6) lens, ( 7) uterus served as a positive control, (8) water negative control, (9) reverse transcriptase negative control.reproductive tissues such as ovary, uterus, placenta, mammary gland, prostate, and testis, RXFP1 mRNA and protein are also located in the heart, kidney, lung, liver, blood cells and also in areas of brain including cortex, organum vasculosum of the lamina terminalis, and subfornical organ. 2 Furthermore, the RLN2 hormone is present in human tears and is expressed in tissues of the anterior eye including conjunctiva and cornea as well as the lacrimal gland, meibomian gland and nasolacrimal duct. 18However, in our study, Rln1 mRNA was expressed in the mouse retina but not in any other anatomical regions within the eyes.Furthermore, the retina expressed an Rxfp1 transcript of 450 bp suggesting a paracrine action of RLN in the mouse retina.Interestingly, the cornea and sclera/choroid expressed both Rxfp1 transcripts (450 bp and 344 bp) that can also be detected in the uterus, which served as a positive control.From our experiments, Rxfp2 was only present in the cornea.Although RLN has been shown to activate RXFP2 at a much lower affinity in vitro; there is no evidence that RLN activates RXFP2 in vivo. 2 Therefore, it is unlikely that RLN mediates effects by activating RXFP2.Surprisingly, none of these genes were expressed in the murine lacrimal gland, eyelid or lens and thus RLN is not produced by or has effects on these tissues.IWhile the retina is a potential source of RLN1, it is also possible that RLN1 is transported via the bloodstream to the eye.This may explain how RLN1 is present in tears as is the case in humans. 18s the cornea expresses Rxfp1, a potential influence through the activation of this receptor by RLN, might be expected in mice.In humans, RLN has been shown to promote cell migration and proliferation of corneal epithelial cells in vitro. 18Cell culture experiments show an influence of RLN2 on matrix metalloproteinase (MMP) expression.In immortalized conjunctival epithelial cells Mmp2, Mmp9, Timp1 and Timp2 are upregulated by RLN2, while in immortalized corneal cells especially Timp1 and Timp2 levels were increased by RLN2.Wound healing also involves degradation of damaged extracellular matrix and reorganization which in part is regulated by MMPs and TIMPs.Additionally, we demonstrated in an earlier study that RLN improved ocular surface wound healing in a mouse model of corneal injury. 18In the present study, we demonstrate a role for RLN in modulating corneal thickness and endothelial cell density and size implicating a role in corneal morphology and physiology.The changes in corneal thickness most likely arise in the stroma as the epithelium was unchanged.The cornea of Rln −/− mice was approximately 10 µm thicker than Rln +/+ mice as measured by OCT.Since endothelial cells are very thin it is unlikely that they would contribute a change of that magnitude.
Aquaporins are membrane-integrated water channels that regulate the influx of water molecules. 27In the eye, aquaporins contribute to the maintenance of corneal and lens transparency by mediating flow of water across epithelial and endothelial barriers. 28They are also involved in corneal epithelial repair following injury, intraocular pressure (IOP) regulation, retinal signal transduction and retinal swelling following injury. 29In this study, we observed that the whole cornea of Rln −/− mice was thicker than of Rln +/+ mice with no difference in corneal epithelial thickness.This might be due to our observation that endothelial cell density was lower, which could result in a reduced functional capacity to regulate corneal hydration, and thus minor corneal stromal edema.Given that Rln −/− mice have lower Aqp3 and Apq5 expression and that these genes can  be increased by RLN supplementation, 30 we also examined the expression of other aquaporins in Rln −/− and Rln +/+ eyes.In the cornea of Rln −/− mice, the expression of Aqp4 transcripts was upregulated, compared to Rln +/+ corneas.AQP4 has been detected in corneal endothelium and might also be an aquaporin regulating corneal water homeostasis. 31In the cornea, water homeostasis is maintained by AQP1, AQP3 and AQP5. 28QP1 is expressed in corneal endothelium, 29 whereas AQP3 and AQP5 are expressed by corneal epithelium. 29,32AQP1 and AQP5 are responsible for corneal transparency and have been linked to corneal barrier function. 28In the mouse cervix, RLN2 regulates AQP3 and AQP5 expression. 30However, mRNA expression of Aqp1, Aqp3 and Aqp5 showed no difference between corneas from wildtype and knockout mice.Expression of Aqp1, Aqp4 and Aqp5 was reduced in Rln −/− uvea compared to Rln +/+ uvea.Previous studies have reported the expression of AQP1 and AQP4 in the non-pigmented ciliary epithelium. 28owever, in the present study, microdissection of the ciliary epithelium was not possible, and technical limitations were encountered when immunostaining with AQP1 and AQP4 antibodies, preventing visualization of AQP1 and AQP4 protein expression in frozen sections.
A further proposed role for aquaporins in the eye is the regulation of IOP.In mice lacking Aqp1 and/or Aqp4, aqueous fluid production and IOP were decreased compared with wildtype mice. 33However, the Rln −/− mice showed no dramatic changes in IOP, thus it is likely that the changes in aquaporin expression observed in this study in the cornea and uvea have had little impact on IOP.It is worth noting that whilst IOP was measured under the same conditions in both strains of mice, values derived under anesthesia underestimate true IOP.By contrast, a preliminary study from 1963 demonstrated that intramuscular injection of RLN resulted in reduced IOP in six participants. 195][36][37][38][39] However, others have reported that there was little difference. 40Discrepancies in these outcomes are likely to be related to the timing of measurement, as RLN blood levels are highest during the first trimester of pregnancy. 41,42Whether the topical application of RLN influences IOP remains to be demonstrated.
The corneal stroma is comprised of flattened layers of mostly type 1 collagen.Corneal thickness can be influenced by changes in expression of genes encoding collagen proteins.Surprisingly, Col1a2 mRNA was reduced in Rln −/− mice, suggesting reduced extracellular matrix production, which would be expected to result in thinner corneas.MMPs are a family of enzymes that can degrade extracellular matrix proteins, including collagens, and together with TIMPs play an important role in tissue remodeling and wound healing at the ocular surface, as well as in dry eye disease, corneal neo-vascularization, the development of corneal ulcers, or pterygia. 43For example, MMP2 is expressed in healthy cornea and is up-regulated during wound healing, 44 suggesting an important role for MMP2 during physiological extracellular matrix turnover and wound healing of the cornea. 45In this study, Mmp9, Timp1 and Timp2 mRNA expression was decreased in Rln −/− corneas.As Mmp9 and Timp1 are modulators of extracellular tissue composition and a decreased expression might lead to increased extracellular matrix and therefore thicker corneas.This is consistent with other studies showing that Rln −/− mice have reduced ECM and MMP expression and/or activity in other tissues. 46,47Further, biochemical investigations and ultrastructural analyses of collagenous architecture would be required to confirm whether aberrant Mmp9 and Timp1 gene expression relates directly to the observed corneal thickness changes in Rln −/− mice.
Although Rln as well as the receptors Rxfp1 and Rxfp2 were expressed in the mouse retina, retinal layer thickness and expression of aquaporins, Tjp1, collagens as well as Mmps and Timps remained unchanged in Rln −/− mice compared to wild-type mice.The role of Rln in the retina remains unclear and needs further investigation.

Conclusions
In conclusion, we found subtle changes in expression of aquaporins and genes associated with ECM remodeling in Rln1 −/− mice, along with a significantly thicker cornea, increase endothelial cell size and decrease corneal endothelial cell count in eyes of Rln −/− mice.We also found that the expression patterns of Rln, Rxfp1 and Rxfp2 differed among the various regions within the eye.Interestingly, Rln was only detected in the retina, suggesting that it may be the source of RLN within the eye and potentially regulates the neighboring tissues, which express various receptor combinations.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by Deutsche Forschungsgemeinschaft (DFG) [HA 6344/2-1] and Boehringer Ingelheim travel grants.This project was partially funded by Novartis Pharma, as a condition of the Australian Research Council Linkage Grant that funded this research.
All animal experiments were approved by the Faculty of Science, University of Melbourne Animal Experimental Ethics Committee (AEC #0911478.1)and conducted in accordance with the Australian Code of Practice the National Health and Medical Research Council (https://nhmrc.gov.au/about-us/publications/australian-code-responsible-con duct-research-2007) and the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Ophthalmic and Vision Research.