The authors thank Ruth Zelkha, MS, for her generous assistance in imaging. This research was supported by Clinical Scientist Development Program Award K12EY021475 to M.E., grant R01 EY024349-01A1 to A.R.D., core grant EY01792 from the 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.

All procedures performed on animals comply with the Association for Research in Vision and Ophthalmology statements for animal use in vision research. Fourteen four- to six-month-old male/female C57BL/6J wild type mice were used: ten mice for the injury model and four as control animals (unwounded corneas).
1. Animal Preparation
<ol>
	<li>Anesthetize a mouse with an intraperitoneal injection of a 100 mg/kg ketamine and 5 mg/kg xylazine mixture. After the anesthesia has taken effect, pinch the toe to confirm the anesthesia depth; the animal should not react.</li>
	<li>Inspect the eye under a slit lamp before conducting the experiments to verify the absence of any previous corneal injury.</li>
	<li>Instill a drop of 1% tetracaine hydrochloride onto the eye. After 60 - 90 sec, test the corneal reflex to confirm the absence of sensation. Proparacaine hydrochloride 0.5% can be used instead.</li>
	<li>When the loss of corneal sensation is certain, gently place a drop of 5% povidone-iodine on the eye and on the hair around the eye. Wipe off the drop after 1 min or spread it on the hair around the eye to prevent the hair from interfering with the tool tip.</li>
</ol>
2. Abrading the Corneal Epithelium
<ol>
	<li>Wear surgical gloves and a gown. Prepare the required tools: a sterile rotating burr and a pair of sterile tweezers. For better control, use bent tweezers. Place the tools on a sterile sheet during the procedure.</li>
	<li>Using the bent tweezers, stabilize the eye by grabbing the lids from the nasal side and gently proptosing the eye. Avoid using too much pressure.</li>
	<li>Under a surgical microscope, shave 360&#176; around the limbal area twice using the sterile rotating burr. Go around the limbus with a speed of 5 - 6 sec per round. 
	NOTE: The burr has one fixed speed. However, for it to rotate at the established speed, the &#34;end nut&#34; should be completely tightened.</li>
	<li>Remove the whole corneal epithelium from limbus to limbus with the rotating burr. For better results, move the tool in a circular fashion and hold it somewhat parallel to the corneal surface to work with the lateral side of its tip. Take care not to injure the stroma by not re-brushing the areas where the epithelium has already been removed. Do not apply any pressure on the eye. Clean the brush tip as required.</li>
	<li>Instill a drop of sterile phosphate-buffered saline on the cornea and wipe away any detached epithelial sheets with a soft cleaning tissue.</li>
	<li>Shave the remaining epithelium, if any.</li>
	<li>Pay attention to the mouse tail during the surgery. 
	NOTE: If any movement occurs, the anesthesia depth has faded and further injection is required. One-third to two-thirds of the primary injected volume should be sufficient. Do not overdose the animal with the anesthetic medication.</li>
	<li>Sterilize all the tools before using them on another eye.</li>
</ol>
3. Confirm Complete Epithelial Removal
<ol>
	<li>Apply a drop of 1 mg/ml fluorescein sodium onto the cornea. Clean the cornea after 30 sec and examine it under a microscope with a cobalt blue filter to verify the complete removal of the epithelium. 
	NOTE: Corneal areas with epithelial defects are visualized in green.</li>
</ol>
4. Post-operation Care
<ol>
	<li>Put the animal on a heating blanket and keep it in a safe place (e.g., in its cage) while under recovery. Do not keep an unconscious animal in the company of conscious ones.</li>
	<li>Apply 1% erythromycin ophthalmic ointment. This will also prevent eye dryness. Continue the application the antibiotic daily for a week.</li>
	<li>Inject 0.1 mg/kg buprenorphine subcutaneously after the procedure and again after 12 hr. Continue the injection twice per day for two days.</li>
	<li>Observe the animal for 30 - 45 min, until complete recovery from anesthesia. 
	NOTE: Mild movement of the respiratory muscles on the ventral surface indicates animal wellness while under recovery.</li>
</ol>

<ol><li>Afsharkhamseh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stability+of+limbal+stem+cell+deficiency+after+mechanical+and+thermal+injuries+in+mice%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.</a> Exp. Eye Res. 145, 88-92 (2015).</li>
<li>Dorà, N. J., Hill, R. E., Collinson, J. M., West, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+tracing+in+the+adult+mouse+corneal+epithelium+supports+the+limbal+epithelial+stem+cell+hypothesis+with+intermittent+periods+of+stem+cell+quiescence%5BTitle%5D)+AND+%22Stem+Cell+Res%22%5BJournal%5D)">Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence.</a> Stem Cell Res. 15 (3), 665-677 (2015).</li>
<li>Ahmad, S., Osei-Bempong, C., Dana, R., Jurkunas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+culture+and+transplantation+of+human+limbal+stem+cells%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">The culture and transplantation of human limbal stem cells.</a> J. Cell Physiol. 225 (1), 15-19 (2010).</li>
<li>Kolli, S., Ahmad, S., Lako, M., Figueiredo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+clinical+implementation+of+corneal+epithelial+stem+cell+therapy+for+treatment+of+unilateral+limbal+stem+cell+deficiency%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.</a> Stem Cells. 28 (3), 597-610 (2010).</li>
<li>Ahmad, S., Figueiredo, F., Lako, M. Corneal epithelial stem cells: characterization, culture and transplantation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAhmad%2C+S+%22Regen.+Med%22">Regen. Med</a>. 1 (1), 29-44 (2006).</li>
<li>He, H., Yiu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell-based+therapy+for+treating+limbal+stem+cells+deficiency%3A+A+review+of+different+strategies%5BTitle%5D)+AND+%22Saudi+J.+Ophthalmol%22%5BJournal%5D)">Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies.</a> Saudi J. Ophthalmol. 28 (3), 188-194 (2014).</li>
<li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Removal+of+the+basement+membrane+enhances+corneal+wound+healing%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Removal of the basement membrane enhances corneal wound healing.</a> Exp Eye Res. 93 (6), 927-936 (2011).</li>
<li>Stepp, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wounding+the+cornea+to+learn+how+it+heals%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">Wounding the cornea to learn how it heals.</a> Exp. Eye Res. 121, 178-193 (2014).</li>
<li>Amirjamshidi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbal+fibroblast+conditioned+media%3A+a+non-invasive+treatment+for+limbal+stem+cell+deficiency%5BTitle%5D)+AND+%22Mol.+Vis%22%5BJournal%5D)">Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency.</a> Mol. Vis. 17, 658-666 (2011).</li>
<li>Li, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+AlgerBrush+II+rotating+burr+as+a+tool+for+inducing+ocular+surface+failure+in+the+New+Zealand+White+rabbit%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.</a> Exp. Eye Res. 147, 1-11 (2016).</li>
<li>Ti, S. E., Anderson, D., Touhami, A., Kim, C., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+affecting+outcome+following+transplantation+of+ex+vivo+expanded+limbal+epithelium+on+amniotic+membrane+for+total+limbal+deficiency+in+rabbits%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.</a> Invest. Ophthalmol. Vis. Sci. 43 (8), 2584-2592 (2002).</li>
<li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstruction+of+chemically+burned+rat+corneal+surface+by+bone+marrow-derived+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.</a> Stem Cells. 24 (2), 315-321 (2006).</li>
<li>Bu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+activated+omental+cells+on+rat+limbal+corneal+alkali+injury%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">Effects of activated omental cells on rat limbal corneal alkali injury.</a> Exp. Eye Res. 121, 143-146 (2014).</li>
<li>Luengo Gimeno, F., Lavigne, V., Gatto, S., Croxatto, J. O., Correa, L., Gallo, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+corneal+stem-cell+transplantation+in+rabbits+with+severe+ocular+alkali+burns%5BTitle%5D)+AND+%22J.+Cataract+Refract.+Surg%22%5BJournal%5D)">Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns.</a> J. Cataract Refract. Surg. 33 (11), 1958-1965 (2007).</li>
<li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+of+limbal+stem+cell+deficiency+induced+by+topical+medication+with+the+preservative+benzalkonium+chloride%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">A mouse model of limbal stem cell deficiency induced by topical medication with the preservative benzalkonium chloride.</a> Invest. Ophthalmol. Vis. Sci. 54 (9), 6314-6325 (2013).</li>
<li>Huang, A. J., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corneal+epithelial+wound+healing+in+the+absence+of+limbal+epithelium%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Corneal epithelial wound healing in the absence of limbal epithelium.</a> Invest. Ophthalmol. Vis. Sci. 32 (1), 96-105 (1991).</li>
<li>Liu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cornea-specific+expression+of+K12+keratin+during+mouse+development%5BTitle%5D)+AND+%22Curr.+Eye+Res%22%5BJournal%5D)">Cornea-specific expression of K12 keratin during mouse development.</a> Curr. Eye Res. 12 (11), 963-974 (1993).</li>
<li>Nakatsu, M. N., González, S., Mei, H., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+limbal+mesenchymal+cells+support+the+growth+of+human+corneal+epithelial+stem%2Fprogenitor+cells%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells.</a> Invest. Ophthalmol. Vis. Sci. 55 (10), 6953-6959 (2014).</li>
<li>Pajoohesh-Ganji, A., Pal-Ghosh, S., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corneal+goblet+cells+and+their+niche%3A+implications+for+corneal+stem+cell+deficiency%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Corneal goblet cells and their niche: implications for corneal stem cell deficiency.</a> Stem Cells. 30 (9), 2032-2043 (2012).</li>
<li>McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., Burgess, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13, 1400-1412 (2014).</li>
</ol>

The authors declare that they have nothing to disclose.

This article describes a reproducible and relatively simple technique to create a mouse model of LSCD. There are several important aspects of this model that are worth noting. First, unlike models that use chemicals11, 12, the injury primarily involves the surface epithelium (and basement membrane), with minimal damage to the underlying corneal stroma or to other intraocular structures. Thus, there is only limited inflammation and scarring, both of which can complicate the procedure and make it more difficult to assess the outcome of any interventions aimed at the limbal stem cells. An interesting recent study by Li et al.10, which compares the use of the rotating burr with different combinations of surgical and chemical treatments, also found the burr to be safer and more effective in creating LSCD in rabbits. Second, the model seems to be stable for at least up to three months1. Creating this model involves the use of the rotating burr, an instrument that is familiar to clinicians who work with the cornea, as well as to those who do mouse wounding studies. Its advantage over using a blunt spatula is that the injury is more uniform and reproducible1.
To better destroy the limbal stem cells, the limbal area was treated twice. Based on experience working on mouse eyes, treatment of more than two rounds often abrades limbal blood vessels and causes bleeding. In case the technique is used in other animals, this step might be modified depending on the corneal epithelial thickness. In this study, a motorized tool with a 0.5-mm burr tip was utilized; burr tips in other shapes and sizes are also available and can be used instead, depending on corneal specifications and on eye size. Li et al.10 reported the 2.5-mm burr to be suitable to consistently remove rabbit corneal and limbal epithelia.
To obtain the optimal result, limbal and corneal epithelia were shaved in a circular fashion and the lateral side of the burr was used. To prevent the animal hair from interfering with the tool tip, the povidone-iodine drop was spread on the hair around the eye, instead of being wiped off. The tool should not be operated statically in one spot, as this may cause perforation. Treating the limbus at a very slow pace can result in bleeding; a speed of almost 6 sec per round is suitable for a C57BL/6J mouse eye. It is critical to avoid re-brushing the bare stroma, as this will induce more inflammation and stroma opacification. Pressure should not be applied to the eye while abrading the epithelium. After the procedure, the cornea was stained with fluorescein dye to make sure of complete epithelial removal. It is important to prevent eye dryness during animal recovery. Limitations are that this technique cannot remove 100% of the stem cells. It is also somewhat operator-dependent and requires practice to give consistent results.
When studying the corneal epithelial phenotype, whole mount staining is the best way to assess the outcome of this model. This is because it allows the whole corneal epithelium to be evaluated at once, whereas cross sections would only give information about each particular section.
This controllable technique can induce LSCD in mice, and probably in other animals as well. It provides a suitable model of LSCD to study novel treatments and to discover wider aspects of the disease pathophysiology. With some alterations, it can be useful in studying corneal wound healing, angiogenesis, and lymphangiogenesis as well.

The corneal epithelium is required to maintain the clarity and integrity of the cornea. It is constantly renewed throughout life by the epithelial stem cells residing in the limbus-a narrow zone at the junction of the cornea and the conjunctiva. These self-renewing limbal stem cells play a crucial role in regenerating the corneal epithelium, both normally and after injury. Partial or complete depletion of these stem cells will result in recurrent corneal erosions, pain, corneal scarring and neovascularization, appearance of goblet cells, and if left untreated, corneal blindness. This condition is known as limbal stem cell deficiency (LSCD) and can be idiopathic; hereditary; or acquired due to chemical or thermal injuries, long-term contact lens wear, and chronic inflammation1-6.
Research on LSCD requires a suitable animal model that not only mimics the disease in humans, but is reproducible and sustainable, with the least amount of injury to other corneal and ocular structures. This model is necessary to assess treatments and to clarify the disease mechanisms at molecular and cellular levels. As described before1, with the use of a rotating burr, one can easily develop a mouse model of LSCD that features the aforementioned advantages and persists for at least three months. The goal of this study is to present a simple, reproducible, and sustainable mouse model of LSCD.
The rotating burr is a handy tool that evenly removes the epithelium without injuring the underlying stroma1. It has been used to induce central corneal erosion7, 8 in wound healing studies. The hereby-presented technique to create LSCD in a mouse has not been reported before. Previously introduced methods of scraping the epithelium with a blunt spatula result in a less uniform injury-particularly at the limbus-and a more variable phenotype1, 9, with more restoration of normal corneal epithelium1. Unlike the blunt scraper, the rotating burr removes the epithelial basement membrane as well7, 8, 10. Other reported methods to sever stem cells involve the use of chemicals, such as sodium hydroxide, n-heptanol, and benzalkonium chloride, which not only may induce unwanted injury to underlying eye structures, but also may lead to significant inflammation and subsequent corneal opacification or epithelial squamous metaplasia11-15. The rotating burr is not associated with these severe complications. Surgically removing the limbal epithelium, alone or with the use of chemicals10, 11, 16, is more difficult to perform and is not the best option in animals with small eyes and a thin limbal epithelia (like mice). In addition, surprisingly, limbectomy may still leave some of the limbal epithelium behind10.
The below-described technique will result in LSCD with neovascularization and conjunctivalization that mimics the presentation of complete LSCD in patients and lasts for at least three months1. It is suitable for those who aim to study LSCD or wound healing pathophysiology, immunology, and potential treatments in mice. Possibly, with some modifications, this procedure can be performed in rats or rabbits10. As the rotating burr efficiently removes the epithelium, it can be utilized in any research pertaining to corneal epithelial abrasion/wounding and in any related treatment or molecular biology studies.

<table><tbody><tr><td>Rotating burr: AlgerBrush II rust ring remover&amp;nbsp;</td><td>Rumex International Co, Clearwater, FL</td><td>16-141</td><td>0.5 mm burr. Also available from other companies.</td></tr><tr><td>Surgical microscope</td><td>Wild Heerbrugg, Switzerland</td><td>Wild M691</td><td></td></tr><tr><td>Digital camera</td><td>Nikon, Thailand</td><td></td><td></td></tr><tr><td>Nikon FS-2 slit lamp</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Polyclonal anti-CK12 antibody</td><td>Santa Cruz Blotechnology, CA,</td><td>SC-17101</td><td>1:100 concentration</td></tr><tr><td>Monoclonal anti-CK8 antibody</td><td>TROMA-I-s, Iowa City, IA</td><td>AB_531826</td><td>1:50 concentration</td></tr></tbody></table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

Within a month after performing this technique, 100% of the corneas developed superficial neovascularization (Figure 1A. Photos were taken with a camera connected to a slit lamp under a bright field and cobalt blue filter). In 18/20 (90%) of the eyes, neovascularization involved the whole corneal surface (Figure 1A). In 2/20 (10%), neovascularization was observed in three of four corneal quadrants, but it still reached the corneal center.
To validate the development of LSCD, whole mount immunostaining was performed1. This step is not a part of the protocol and can be altered depending on goals and interests. Three variations were considered as indicators of LSCD: absence of cytokeratin (CK) 12, an intermediate filament specific to normal corneal epithelial cells17, 18; appearance of CK8, a simple epithelia marker20 that is not present on the normal cornea; and presence of goblet cells on the corneal surface. To evaluate the corneal epithelial phenotype, double immunostaining for CK12 and CK8 was performed on whole mount samples three months after the injury, as described previously1, 9. Goblet cell existence was assessed by MUC5AC-goblet cell-specific mucin-immunostaining1, 15, 19 45 days following epithelial removal. The appearance of goblet cells reduces in time. For the best results, it is important to perform the staining less than two months after the injury.
Normal, uninjured corneas express CK12-a specific marker of mature corneal epithelial cells17, 18-throughout the corneal surface, but they are devoid of any CK8 or goblet cells (MUC5AC) on the corneal part of the whole mount samples (Figure 1B). In comparison to normal eyes, CK12 expression is almost absent on injury model corneas, while they significantly show positive staining for CK8 and goblet cells (Figure 1B). These observations indicate the development of LSCD.
If interested in quantifying the immunostaining results, use Image-J to separately quantify20 immunostaining densities on the original, unaltered images taken by the microscope under the same settings. Briefly, select the corneal part of the whole mount sample, measure the integrated density and area for that region, and calculate the corrected density for the background20. The ratio of CK12 to CK8 densities were previously presented on a greater number of samples1. The reduced ratio in comparison to controls (normal corneas) and the appearance of goblet cells on the corneal surface demonstrate the development of LSCD.
<img alt="Figure 1" src="/files/ftp_upload/54658/54658fig1.jpg" /> 
Figure 1: Corneal Phenotype After Creating LSCD in Mice. Slit lamp examination shows that all corneas develop some degree of neovascularization by one month (A, Scale bar: 0.5 mm). A normal cornea expresses CK12 all over the cornea but is devoid of any CK8 or goblet cells (MUC5AC). CK12 is absent in the LSCD model, while CK8 and goblet cells appear on the cornea (B, Scale bar: 200 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54658/54658fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

a-simple-mechanical-procedure-to-create-limbal-stem-cell-deficiency

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K Views.  University of Illinois at Chicago.  Het volgende artikel biedt een eenvoudige, reproduceerbare techniek om effectief een duurzame muismodel van limbale stamcellen deficiëntie (LSCD). Dit diermodel is bruikbaar bij het testen en vergelijken van de werkzaamheid van behandelingen voor limbale stamcellen ziekten. 

Video: A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Developmental Biology

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Neuroscience

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

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