Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Exp Eye Res. 2015 Dec 1;145:88–92. doi: 10.1016/j.exer.2015.11.012

Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice

Neda Afsharkhamseh 1, Asadolah Movahedan 1, Sanaz Gidfar 1, Michael Huvard 1, Lisa Wasielewski 1, Behrad Y Milani 1, Medi Eslani 1, Ali R Djalilian 1
PMCID: PMC4979604  NIHMSID: NIHMS807972  PMID: 26607808

Abstract

We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency.

A clear cornea is essential for normal vision. The corneal epithelium, the most superficial layer of the cornea, protects the cornea against pathogen invasion and is essential for maintaining the integrity and clarity of the cornea. The corneal epithelium is a stratified squamous epithelium that is endlessly renewed throughout life. The epithelial stem cells that maintain the corneal epithelium are primarily located at the corneal limbus in the basal layer of the epithelium (Ahmad et al., 2006; Dua et al., 2003). These stem cells are constantly self-renewing, repairing, and regenerating the corneal epithelium (Chang et al., 2008). After an injury to the epithelium, the remaining epithelial cells flatten, spread and move across the defect. The cells in the basal layer with proliferative capacity undergo further proliferation to restore cell numbers and cell mass. The newly regenerated epithelium is anchored firmly to the underlying tissue and the new basement membrane (Dua et al., 1994). This process is mediated by growth factors, adhesion proteins, proteases, cytokines, and other factors provided in part by the limbal blood supply, tear film, stromal and immune cells (Freire et al., 2014). Interactive cross-talk between surrounding cells, the extracellular matrix, and soluble signals are critical for epithelial homeostasis and the ocular surface wound healing response (Dua et al., 2003; Gipson, 1989).

Limbal Stem Cell Deficiency (LSCD) is a pathologic state that results from failure of stem cells to renew the corneal epithelium (Hatch and Dana, 2009; Sejpal et al., 2013). Inherited forms of LSCD, for example, are seen in aniridia (Mayer et al., 2003) and ectodermal dysplasia (Di Iorio et al., 2012) while acquired LSCD can arise following chemical injuries, Stevens Johnson syndrome and long-term contact lens wear (Chan and Holland, 2013; Huang and Tseng, 1991; Puangsricharern and Tseng, 1995). Because stem cells and/or their niche are lost or dysfunctional, corneal epithelial regeneration is impaired and corneal surface becomes repopulated by conjunctival epithelial cells (“conjunctivalization”), a finding that is considered the hallmark of LSCD (Dua et al., 2003). Conjunctivalization has clinical implications for patients. Most significantly, it causes superficial corneal neovascularization with recurrent/persistent epithelial defects, chronic ocular surface inflammation, scarring and loss of vision (Hatch and Dana, 2009; Puangsricharern and Tseng, 1995).

Understanding the pathophysiologic mechanisms of LSCD is critical to the development of novel therapies for this challenging condition. Animal models of LSCD are necessary both for studying the disease process at a cellular and molecular level and for testing the efficacy of treatments. Some of the earliest experimental models of LSCD were based on chemical destruction of the limbal area, particularly with alkaline agents (Luengo Gimeno et al., 2007; Phan et al., 1991). Alkali substances are known to cause severe ocular damage and inflammation with compromising stroma and resulting in corneal ulceration, hyphema, hypopyon, and even corneal perforation (Ma et al., 2006). Other models involve surgical removal of the limbal tissue (Chen and Tseng, 1991), cauterization (Majo et al., 2008) and more recently, using benzalkonium chloride to simulate chronic injury to the ocular surface (Lin et al., 2013). In this study, our goal was to evaluate and validate a reproducible mouse model of LSCD using mechanical and thermal injury in a controlled manner, thereby avoiding extensive damage to the corneal stroma and other anterior chamber structures.

All animal investigations were carried out in accordance with recommendations of Association for Research in Vision and Ophthalmology (ARVO). Experiments were performed on 84 eyes of four to six month-old male and female C57BL/6J mice. Eyes were clinically normal under slit lamp examination before conducting the experiments. Subjects were anesthetized by intraperitoneal injection of ketamine (100 mg/Kg) and xylazine (5 mg/Kg) mixture. A drop of proparacaine 0.5% was instilled before and during the procedures. Three methods were conducted to generate LSCD: mechanical alone - using a blunt spatula or Alger Brush- and mechanical alongside with heat injury.

We have previously described a model of partial LSCD (Amirjamshidi et al., 2011), which was a modified mouse model described by Pal-Ghosh et al. (Pal-Ghosh et al., 2008). The model involved scraping the entire corneal epithelium from limbus to limbus using a blunt metal spatula with a 0.3 mm tip. The main advantages of that model are that it limits the injury to the epithelium and is able to produce a state of partial limbal stem cell deficiency. However, its main disadvantages are that the degree of limbal stem cell deficiency is variable since it is difficult to standardize the injury. In the current study, the blunt spatula method is compared with two others using AlgerBrush II rust ring remover (0.5 mm burr, Rumex international Co, Clearwater, FL) alone or followed by heat injury (16 eyes for each group). Unwounded corneas served as control.

Method one

Using Algerbrush II, the limbal epithelium was shaved twice after which, the entire corneal epithelium was removed once from limbus to limbus. Care was taken to avoid injuring the stroma and conjunctiva (Video 1). Brush tip was cleaned as required.

Method two

Limbal and corneal epithelium were removed with AlgerBrush II as described above in method one, followed by controlled thermal injury to the limbus. To exert a precise thermal injury, we designed an adjustable electric cautery that could yield a constant and measurable amount of heat at its fine tip. We used an ophthalmic cautery device (Medtronic Low Temp Cautery, Jacksonville, FL) connected to an adjustable electrical power supply. A 1.5 cm single solid copper wire (gauge 16, GLT, Solon, Ohio) was bent on itself and was tied to the tip of the cautery on the other end to create a very fine blunt tip (Video 2, epithelial removal not shown). The optimum temperature at the copper tip to create the desired damage without perforation was found to be 50 ± 1 °C. The temperature was measured indirectly by measuring the temperature of 150 µL water heated with the cautery tip (the water was heated for at least 30 minutes and the temperature was recorded when the thermometer showed a constant value for five minutes). Limbal injury was induced by touching the instrument to the limbal area for two to three seconds -just long enough to cause a slight impression on the eyeball (Video 2).

All corneas were examined by Nikon FS-2 photo-slit lamp under bright field and cobalt blue filter three minutes after applying 1 mg/ml fluorescein sodium (late fluorescein staining) on days zero, 30 (data not shown), 60 and 90 after the procedures. Fluorescein staining on day zero confirmed complete epithelial removal throughout the cornea and limbus (Fig. 1A). Histologic examination was used to confirm that the injuries did not violate the stroma (Fig. 1B), although keratocyte loss in the anterior stroma was seen as expected following an epithelial debridement (Wilson et al., 1996, Fig. 1B).

Figure 1.

Figure 1

Open in a new tab

Slit lamp examination after total corneal epithelial removal with a blunt spatula, AlgerBrush alone or followed by thermal injury to the limbus. Unwounded cornea as control. Corneal neovascularization and late fluorescein staining at days 60 and 90 (A). H&E staining confirms that following epithelial removal the stroma remains intact (B, scale bar: 100 µm). DAPI staining shows keratocyte death in the stroma after epithelial removal (B, scale bar: 20 µm). CD31 wholemount immunostaining demonstrating neovascularization three months after the injury (C, scale bar: 200 µm)

Platelet endothelial cell adhesion molecule-1 or CD31 was used as an endothelial cell marker. To confirm and visualize corneal vascularization, wholemount immunostaining for CD31 (anti-CD31 antibody, Biolegend inc., San Diego, CA, 1:50) was carried out on four corneas of each group three months following the injury (Fig. 1C).

Corneal epithelial phenotype was evaluated by immunostaining for cytokeratin (CK) 12, an intermediate filament specific to normal corneal epithelial cells (Liu et al., 1993) and CK8, a marker of simple epithelia (Pajoohesh-Ganji et al., 2012). Following euthanasia at three months, eyes (16 in each group) were enucleated and corneas were prepared for wholemount staining similar to what described previously (Amirjamshidi et al., 2011). Double indirect immunostaining of corneal wholemounts for CK12 (goat polyclonal anti-CK12, Santa Cruz, CA, 1:100) and CK8 (rat monoclonal anti-CK8 TROMA-I, Iowa City, IA, 1:50) were carried out (Fig. 2A). Cytokeratin-12 and CK8 immunofluorescence densities were separately measured in the central corneal of wholemount images using Image-J software (version 1.47, NIH) with correction for the background (Burgess et al., 2010).

Figure 2.

Figure 2

Open in a new tab

CK12 immunostaining of corneal wholemounts at three months. Epithelial removal using Algerbrush with or without heat injury results in near total loss of CK12 expression on the cornea compared to spatula treatment. DAPI staining on wholemounts confirms the absence of corneal erosions (A, scale bar: 200 µm). CK12 immunostaining on frozen sections of unwounded cornea and all injury groups at three months. Alger and thermal injury models have less CK12 than spatula group (A, DAPI: blue, scale bar: 50 µm). CK8 positive cells are seen more in Alger and thermal treated eyes than spatula group, compared to no staining in unwounded cornea (A, Scale bar: 200 µm). Muc-5AC immunostaining at two months: all injury models demonstrate goblet cells on the cornea (A, scale bar: 200 µm).

Significantly reduced CK12 to CK8 staining ratio in all groups compared to unwounded corneas (B, P<0.0001, error bars represent standard deviation). The Alger and Alger+heat groups also have lower CK12 to CK8 ratio compared to the blunt spatula group (B, P-value of <0.0001).

Shaving the corneal epithelium with AlgerBrush followed by heat injury, results in more significant stromal opacity compared to using AlgerBrush alone or spatula (C, *: P-value<0.0001).

Normal uninjured corneas demonstrated CK12 throughout the corneal epithelium. In both Algerbrush and combined AlgerBrush and thermal injury groups CK12 was almost totally absent at three months, while in the blunt spatula group variable amounts of CK12 staining was noted in the cornea (Fig. 2A). DAPI staining of wholemounts verified no erosion on corneal surface. Positive CK8 staining was observed in Algerbrush and thermal injury groups at three months, while spatula scraped corneas showed less CK8 staining and unwounded corneas devoid of any (Fig. 2A). Cytokaratin 12/8 immunostaining of frozen sections (four eyes in each group) also revealed the same results (Fig. 2A, data for CK8 not shown).

The ratio of CK12 to CK8 densities for each eye was calculated and the averages were reported for each group. The mean ratio of CK12 to CK8 staining was found to be significantly less in Algerbrush and heat treated corneas compared to unwounded eyes (P<0.0001, Fig. 2B) and spatula group (P<0.0001, Fig. 2B). There was no significant difference in the ratios between Alger alone and Alger plus heat methods (P= 0.1507).

To additionally confirm LSCD, immunostaining for MUC5AC (goblet cell-specific mucin) (Pajoohesh-Ganji et al., 2012) was performed on four wholemount samples from each group at two months (mouse anti-MUC5AC, Thermo Scientific, Fremont, CA, 1:200, M.O.M. kit, Vector Laboratories, Burlingame, CA). No goblet cells were observed in unwounded corneas, while other three groups showed variable amounts of mucin staining, confirming the presence of goblet cells and LSCD (Fig. 2A).

Corneal opacity was scored on slit lamp images taken three months after injury using a previously published system (Yoeruek et al., 2008); 0: completely clear; 1: slightly hazy, iris and pupil easily visible; 2: somewhat opaque, but iris and pupil still detectable; 3: strong opacity, pupil hardly detectable, and 4: completely opaque, pupil not seen. Average scores in the groups were charted (P< 0.0001, Fig. 2C). Adding thermal injury to the burr shaving, produced a more severe phenotype of limbal stem cell deficiency with greater stromal opacity.

Using chemical agents to induce LSCD can lead to variable phenotypes ranging from mild injury to severe ocular damage, hemorrhage and perforation, often involving corneal stroma and conjunctiva (Ma et al., 2006). Therefore, chemical destruction may not be a suitable way to specifically destroy limbal stem cells particularly in small animals such as mice. In this study, we examined the stability of LSCD after mechanical and thermal controlled injuries to the corneal epithelium and limbus. We aimed to identify a protocol that is simple, uniform, and reproducible in our hands. Previously, we reported the use of a blunt spatula to scrape the corneal epithelium as a model of limbal stem cell deficiency (Amirjamshidi et al., 2011). As mentioned in the discussion of that paper, that model is more consistent with a partial limbal stem cell deficiency, given that CK12 expression may be still maintained or restored in some cases (Amirjamshidi et al., 2011; Dua et al., 2005; Pal-Ghosh et al., 2004). Furthermore, we found scraping the entire epithelium with a spatula to be difficult to perform in a reproducible manner with some operators being more delicate and others being more aggressive.

As reported by others before, the AlgerBrush II (0.5mm burr) is an easy tool for shaving corneal epithelium (Meyer-Blazejewska et al., 2011). This device is commonly used in emergency departments to remove the rust ring in patients with a metallic corneal foreign body. The use of a burr is advantageous because it requires less mechanical force by the operator and the epithelium can be removed without placing excessive pressure on the cornea. Removing the epithelium with a rotating burr is also different from a spatula in that it leads to the removal of the basement membrane while epithelial debridement with a blunt knife often maintains the basement membrane (Pal-Ghosh et al., 2011). The use of a burr however, does require the operator to be aware of the force and the speed at which they move the tip, since applying persistent pressure to one spot could potentially lead to removal of the stromal tissue. As with previous reports, our histologic examination confirmed that the underlying stroma remained intact following removal of the epithelium with a burr that was moved constantly with minimal force.

These current results confirm our past experience that mechanical debridement with a blunt spatula can lead to a more variable phenotype. This likely reflects the fact that injury may be less uniform especially in the limbal area. In our mechanical injury model with a burr we shaved the limbal region two additional times to achieve a more complete removal of the limbal epithelial/niche cells. We further hypothesized that thermal injury may be a more effective means to focally destroy the limbal epithelium and niche. As the 3-month results indicated, both methods were nearly equally effective and mechanical injury alone can lead to a stable model with less stromal scarring. The thermal injury model in particular led to a more significant stromal wound healing response and scarring. This may be useful for studies focused on the pathophysiology of stromal responses after severe ocular surface injury.

The use of a burr for inducing limbal stem cell deficiency in mice has been reported in some studies before (Ksander et al., 2014; Meyer-Blazejewska et al., 2011). While the first study (Meyer-Blazejewska et al., 2011) actually reported only short term results, the more recent one (Ksander et al., 2014), provided longer follow-up up to 13 months. In both studies, the details of the procedure were not provided and the reproducibility of the model was not specifically mentioned. The current study provides more specific details about the procedure while validating the stability of the limbal stem cell deficiency for at least three months. In particular, since both previous studies assessed the outcome by immunostaining of cross sections, the current study highlights the use of wholemount staining for assessing the expression of keratin 12 in the entire cornea.

Specifically, given the focal nature of the limbal area, using cross sections can potentially miss small pockets of remaining limbal stem cells and corneal type epithelium. As shown in Fig. 2A, in both injury models, we occasionally noted very small pockets of corneal type epithelium on the cornea. This is in fact analogous to clinical scenarios where patients rarely present with a complete loss of K12 expression and mosaic expression patterns for limbal and corneal keratins are common. Wholemount staining does have one limitation experimentally in that it uses the entire tissue and therefore it does not allow to repeat staining for the same or other antigens which can be done in cross section by cutting additional sections of the tissue block.

In this study, we used the absence of CK12 staining and the presence of goblet cells as the hallmarks of limbal stem cell deficiency. Recent studies have shed light on the origin of goblet cells in the cornea following injuries close to the limbal area (Pajoohesh-Ganji et al., 2012; Pajoohesh-Ganji et al., 2015). In particular, they have identified compound niches within the mouse limbal area that give rise to corneal goblet cells after large corneal debridement wounds that remove 70% of the epithelium. They have proposed that goblet cells in the cornea in the setting of “corneal stem cell deficiency” may actually be derived from the limbus and not necessarily from the conjunctiva. More studies are needed to investigate this finding in clinical limbal stem cell deficiency.

In conclusion, this study validates the use of mechanical burr for shaving the entire corneal and limbal epithelium in mice with or without additional heat injury to the limbus, as a controllable and consistent method for destroying limbal stem cells and niche. These injury models produce a durable phenotype that mimics clinical LSCD. By combining mechanical and thermal injuries, the model may be tailored to simulate severe stromal wound healing response.

Supplementary Material

1

Video 1: Creating a mechanical injury model of LSCD; using AlgerBrush II with 0.5 mm burr to shave the limbal epithelium twice and the entire corneal epithelium once. Care was taken not to damage the stroma.

Download video file (7.7MB, avi)
2

Video 2: Inducing thermal injury to the limbal area with a modified low temperature ophthalmic cautery (49–51 °C). Removal of the epithelium with AlgerBrush (done prior to starting the cautery treatment) is not shown in the video. Also complete 360 degrees of cautery treatment is not shown.

Download video file (11.8MB, avi)

Acknowledgments

The authors thank Ruth Zelkha, MS, for her generous technical assistance in imaging.

This research was supported by R01 EY024349-01A1 to A.R.D. and core grant EY01792 from National Eye Institute, NIH and an unrestricted grant from Research to Prevent Blindness. A.R.D. is the recipient of a Career Development Award from Research to Prevent Blindness.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Disclosure: None to report.

References

  • 1.Ahmad S, Figueiredo F, Lako M. Corneal epithelial stem cells: Characterization, culture and transplantation. Regen. Med. 2006;1(1):29–44. doi: 10.2217/17460751.1.1.29. [DOI] [PubMed] [Google Scholar]
  • 2.Amirjamshidi H, Milani BY, Sagha HM, Movahedan A, Shafiq MA, Lavker RM, Yue BY, Djalilian AR. Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency. Mol. Vis. 2011;17:658–666. [PMC free article] [PubMed] [Google Scholar]
  • 3.Burgess A, Vigneron S, Brioudes E, Labbé JC, Lorca T, Castro A. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc. Natl. Acad. Sci. USA. 2010;107(28):12564–12569. doi: 10.1073/pnas.0914191107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chang CY, Green CR, McGhee CN, Sherwin T. Acute wound healing in the human central corneal epithelium appears to be independent of limbal stem cell influence. Invest. Ophthalmol. Vis. Sci. 2008;49:5279–5286. doi: 10.1167/iovs.07-1260. [DOI] [PubMed] [Google Scholar]
  • 5.Chen JJ, Tseng SC. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Invest. Ophthalmol. Vis. Sci. 1991;32:2219–2233. [PubMed] [Google Scholar]
  • 6.Di Iorio E, Kaye SB, Ponzin D, Barbaro V, Ferrari S, Böhm E, Nardiello P, Castaldo G, McGrath JA, Willoughby CE. Limbal stem cell deficiency and ocular phenotype in ectrodactyly-ectodermal dysplasia-clefting syndrome caused by p63 mutations. Ophthalmology. 2012;119(1):74–83. doi: 10.1016/j.ophtha.2011.06.044. [DOI] [PubMed] [Google Scholar]
  • 7.Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J Ophthalmol. 1994;78:401–408. doi: 10.1136/bjo.78.5.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Dua HS, Joseph A, Shanmuganathan VA, Jones RE. Stem cell differentiation and the effects of deficiency. Eye. 2003;17:877–885. doi: 10.1038/sj.eye.6700573. [DOI] [PubMed] [Google Scholar]
  • 9.Dua HS, Shanmuganathan VA, Powell-Richards AO, Tighe PJ, Joseph A. Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. BrJOphthalmol. 2005;89:529–532. doi: 10.1136/bjo.2004.049742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Freire V, Andollo N, Etxebarria J, Hernaez-Moya R, Duran JA, Morales MC. Corneal wound healing promoted by 3 blood derivatives: An in vitro and in vivo comparative study. Cornea. 2014;33(6):614–620. doi: 10.1097/ICO.0000000000000109. [DOI] [PubMed] [Google Scholar]
  • 11.Gipson IK. The epithelial basement membrane zone of the limbus. Eye. 1989;3:132–140. doi: 10.1038/eye.1989.21. [DOI] [PubMed] [Google Scholar]
  • 12.Hatch KM, Dana R. The structure and function of the limbal stem cell and the disease states associated with limbal stem cell deficiency. Int. Ophthalmol. Clin. 2009;49:43–52. doi: 10.1097/IIO.0b013e3181924e54. [DOI] [PubMed] [Google Scholar]
  • 13.Huang AJ, Tseng SC. Corneal epithelial wound healing in the absence of limbal epithelium. Invest. Ophthalmol. Vis. Sci. 1991;32:96–105. [PubMed] [Google Scholar]
  • 14.Ksander BR, Kolovou PE, Wilson BJ, Saab KR, Guo Q, Ma J, McGuire SP, Gregory MS, Vincent WJ, Perez VL, Cruz-Guilloty F, Kao WW, Call MK, Tucker BA, Zhan Q, Murphy GF, Lathrop KL, Alt C, Mortensen LJ, Lin CP, Zieske JD, Frank MH, Frank NY. ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature. 2014;511(7509):353–357. doi: 10.1038/nature13426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Lin Z, He H, Zhou T, Liu X, Wang Y, He H, Wu H, Liu Z. A mouse model of limbal stem cell deficiency induced by topical medication with the preservative benzalkonium chloride. Invest. Ophthalmol. Vis. Sci. 2013;54:6314–6325. doi: 10.1167/iovs.12-10725. [DOI] [PubMed] [Google Scholar]
  • 16.Liu CY, Zhu G, Westerhausen-Larson A, Converse R, Kao CW, Sun TT, Kao WW. Cornea-specific expression of K12 keratin during mouse development. Curr Eye Res. 1993;12(11):963–974. doi: 10.3109/02713689309029222. [DOI] [PubMed] [Google Scholar]
  • 17.Luengo Gimeno F, Lavigne V, Gatto S, Croxatto JO, Correa L, Gallo JE. Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns. J. Cataract Refract Surg. 2007;33:1958–1965. doi: 10.1016/j.jcrs.2007.07.020. [DOI] [PubMed] [Google Scholar]
  • 18.Ma Y, Xu Y, Xiao Z, Yang W, Zhang C, Song E, Du Y, Li L. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells. 2006;24(2):315–321. doi: 10.1634/stemcells.2005-0046. [DOI] [PubMed] [Google Scholar]
  • 19.Majo F, Rochat A, Nicolas M, Jacoude GA, Barrandon Y. Oligopotent stem cells are distributed throughout the entire mammalian ocular surface. Nature. 2008;456:250–254. doi: 10.1038/nature07406. [DOI] [PubMed] [Google Scholar]
  • 20.Mayer KL, Nordlund ML, Schwartz GS, Holland EJ. Keratopathy in congenital aniridia. Ocul. Surf. 2003;1(2):74–79. doi: 10.1016/s1542-0124(12)70130-1. [DOI] [PubMed] [Google Scholar]
  • 21.Meyer-Blazejewska EA, Call MK, Yamanaka O, Liu H, Schlötzer-Schrehardt U, Kruse FE, Kao WW. From Hair to Cornea: Towards the Therapeutic Use of Hair Follicle- Derived Stem Cells in the Treatment of Limbal Stem Cell Deficiency. Stem Cells. 2011;29:57–66. doi: 10.1002/stem.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Movahedan A, Afsharkhamseh N, Sagha HM, Shah JR, Milani BY, Milani FY, Logothetis HD, Chan CC, Djalilian AR. Loss of Notch1 disrupts the barrier repair in the corneal epithelium. PLoS One. 2013;8(7):e69113. doi: 10.1371/journal.pone.0069113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Pajoohesh-Ganji A, Pal-Ghosh S, Tadvalkar G, Stepp MA. Corneal Goblet Cells and their Niche: Implications for Corneal Stem Cell Deficiency. Stem Cells. 2012;30(9):2032–2043. doi: 10.1002/stem.1176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pajoohesh-Ganji A, Pal-Ghosh S, Tadvalkar G, Stepp MA. K14+ compound niches are present on the mouse cornea early after birth and expand after debridement wounds. Dev Dyn. 2015 doi: 10.1002/dvdy.24365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Pal-Ghosh S, Pajoohesh-Ganji A, Tadvalkar G, Stepp MA. A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease. Invest. Ophthalmol. Vis. Sci. 2004;45(6):1775–1788. doi: 10.1167/iovs.03-1194. [DOI] [PubMed] [Google Scholar]
  • 26.Pal-Ghosh S, Pajoohesh-Ganji A, Tadvalkar G, Stepp MA. Removal of the basement membrane enhances corneal wound healing. Exp. Eye Res. 2011;93(6):927–936. doi: 10.1016/j.exer.2011.10.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Pal-Ghosh S, Tadvalkar G, Jurjus RA, Zieske JD, Stepp MA. BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds. Exp. Eye Res. 2008;87:478–486. doi: 10.1016/j.exer.2008.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Phan TM, Foster CS, Shaw CD, Zagachin LM, Colvin RB. Topical fibronectin in an alkali burn model of corneal ulceration in rabbits. Arch. Ophthalmol. 1991;109(3):414–419. doi: 10.1001/archopht.1991.01080030116051. [DOI] [PubMed] [Google Scholar]
  • 29.Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology. 1995;102:1476–1485. doi: 10.1016/s0161-6420(95)30842-1. [DOI] [PubMed] [Google Scholar]
  • 30.Sejpal K, Bakhtiari P, Deng SX. Presentation, diagnosis and management of limbal stem cell deficiency. Middle East Afr J Ophthalmol. 2013;20(1):5–10. doi: 10.4103/0974-9233.106381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Singh R, Joseph A, Umapathy T, Tint NL, Dua HS. Impression cytology of the ocular surface. BrJOphthalmol. 2005;89(12):1655–1659. doi: 10.1136/bjo.2005.073916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wilson SE, He YG, Weng J, Li Q, McDowall AW, Vital M, Chwang EL. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res. 1996;62(4):325–327. doi: 10.1006/exer.1996.0038. [DOI] [PubMed] [Google Scholar]
  • 33.Yoeruek E, Ziemssen F, Henke-Fahle S, Tatar O, Tura A, Grisanti S, Bartz-Schmidt KU, Szurman P. Tübingen Bevacizumab Study Group. Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn. Acta. Ophthalmol. 2008;86(3):322–328. doi: 10.1111/j.1600-0420.2007.01049.x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Video 1: Creating a mechanical injury model of LSCD; using AlgerBrush II with 0.5 mm burr to shave the limbal epithelium twice and the entire corneal epithelium once. Care was taken not to damage the stroma.

Download video file (7.7MB, avi)
2

Video 2: Inducing thermal injury to the limbal area with a modified low temperature ophthalmic cautery (49–51 °C). Removal of the epithelium with AlgerBrush (done prior to starting the cautery treatment) is not shown in the video. Also complete 360 degrees of cautery treatment is not shown.

Download video file (11.8MB, avi)

RESOURCES