The authors thank Ya-Lan Chien and Chia-Ying Lee for excellent technical assistance, and OnLine English company for the linguistic edition. This study was funded in part by Chang Gung Medical Research Project (Grant No. CMRPG3L1491).

The research was performed in compliance with institutional guidelines at the beginning of the protocol section. All protocols and procedures were carried out according to the Declaration of Helsinki and were reviewed and approved by the Chang Gung Medical Foundation Institutional Review Board. All volunteers were informed of the nature of this study and signed an informed consent form prior to their inclusion. The consumables required for the entire experimental procedure are presented in Figure 1 and Figure 2, as well as in the Table of Materials.
1. Preparation of the materials needed to produce mACS eye drops
<ol>
	<li>Prepare 250 mL of 10% glycerol solution and keep a 21 G butterfly-winged infusion set, a 3 mL syringe without the needle, and six 10 mL vacutainer tubes containing heparin 158 USP units ready (Figure 1).</li>
	<li>Connect the 21 G blood collection needle to the 3 mL syringe and withdraw 3 mL of 10% glycerol solution into the prepared syringe. 
	NOTE: All materials must be sterilized before the needle is inserted.</li>
	<li>Distribute the 10% glycerol solution into the vacutainer tubes in sequence, with approximately 0.5 mL of 10% glycerol solution in each (Figure 3A). 
	&#8203;NOTE: Because of the negative pressure in the test tube, the needle must come out immediately after it goes in to evenly distribute 3 mL of glycerol solution to the six test tubes.</li>
	<li>Sterilize the patient&#39;s skin with 75% alcohol sterile cotton swabs. Puncture the superficial vein of the patient&#39;s upper limbs with the 18 G blood collection needle. Draw 60-70 mL of venous blood from the superficial vein in total.</li>
</ol>
2. Preparation for mACS eye drops
<ol>
	<li>Sequentially inject 10 mL of the drawn venous blood into each of the six vacutainer tubes (Figure 3B). 
	NOTE: This step relies on the negative pressure of the vacuum to fill the tubes. To avoid blood cell disruption and hemolysis, do not apply any positive pressure.</li>
	<li>Place the six vacutainer tubes in the incubator with a constant temperature of 37 &#176;C for 4 h (Figure 3C). 
	NOTE: The hypoxic status is maintained and stabilized by the remaining negative pressure in a sealed tube that received glycerol.</li>
	<li>Remove the tubes from the incubator after 4 h and centrifuge them at 3,500 &#215; g for 10 min at room temperature.</li>
	<li>At this point, prepare the materials for mACS extraction, including the sterilized eye drop bottles, a 3 mL syringe with the needle, a 0.22 &#181;m filter, an 18 G needle, and a pair of sterile gloves (Figure 2). 
	NOTE: The operating table should be wiped with 75% alcohol to ensure an aseptic environment. The supernatant, after centrifugation, is already a semi-finished product of mACS.</li>
	<li>Place the six tubes on the tube rack and open the caps after complete centrifugation (Figure 3D). 
	NOTE: Sterility is not required in this step.</li>
	<li>Put on sterile gloves and draw out the mACS one by one using a 3 mL syringe with an 18 G needle. 
	NOTE: Be careful not to draw the lower blood cell layer during this step (Figure 3E).</li>
	<li>Pull out the needle, connect it to the 0.22 &#181;m filter, and connect the 23 G, 1.5 in blood collection needle with the original 3 mL syringe to the outlet below (Figure 3F).</li>
	<li>Push the needle gently through the 0.22 &#181;m filter into the prepared sterile eye drop bottles (Figure 3G).</li>
	<li>Repeat the above steps until all the mACS has been filtered and stored in eye drop bottles (Figure 3H).</li>
	<li>Store the mACS eye drops at 4 &#176;C for immediate use; store at -20 &#176;C for long-term preservation. 
	&#8203;NOTE: Do not keep them for more than 2 weeks at 4 &#176;C or for longer than 3 months at -20 &#176;C9,19.</li>
</ol>
3. Ex vivo wound healing model of murine corneal epithelium
NOTE: The following ex vivo animal model was based on prior experience from Hung et al. on mechanical injuries of the corneal epithelium20. The following steps should be performed under the microscope to create a well-circumscribed and consistent corneal epithelial wound.
<ol>
	<li>Anesthetize the C57BL/6 mice with 3%-4% isoflurane. Indent the skin biopsy punch over the murine central cornea, leaving a shallow circle on the epithelium as a uniform wound margin. 
	NOTE: Be gentle to avoid eyeball rupture.</li>
	<li>Debride the corneal epithelium within the confirmed area down to the Bowman&#39;s layer, with a corneal rust ring remover equipped with a 0.5 mm burr.</li>
	<li>To establish the ex vivo animal model of the mechanical corneal wound, harvest the eyeball and prepare the culture well.
	<ol>
		<li>Euthanize the mice first; then, gently press the superior and inferior orbital rims of the mice, introduce the tip of the forceps over the retrobulbar space, and out the eyeball and hold it by the forceps.</li>
		<li>Cut the optic nerve and periorbital soft tissue with corneal scissors to isolate the eyeball perfectly.</li>
		<li>Prepare a 96-well plate with melted wax inside it. Quickly create a round hole using the tips of forceps and then wait for solidification.</li>
	</ol>
	</li>
	<li>Prepare the media to be tested: 0.5% mACS, 0.5% AS for comparison, normal saline as a negative control, and Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). 
	NOTE: mACS is obtained using the protocol mentioned above.</li>
	<li>For the ex vivo culture, place the harvested eyeball onto the prepared 96-well plate. Add 200 &#181;L of each medium into the 96-well plate.</li>
	<li>Place the 96-well culture plate into the incubator at 37 &#176;C with 5% CO2. Ensure that the culture media is changed every 24 h.</li>
	<li>To confirm the sequential wound healing effect, monitor the epithelial wound area under the microscope every 8 h by fluorescein staining.
	<ol>
		<li>Dissolve the fluorescein on the fluorescein paper with normal saline.</li>
		<li>Drop the fluorescein dye onto the murine central cornea, then observe and document it under a microscope. A typical result is shown in Figure 4. 
		NOTE: One drop (approximately 0.05 mL) of fluorescein dye is enough for the observation.</li>
	</ol>
	</li>
</ol>

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All authors declare that there is no conflict of interest.

In this study, a protocol for the preparation of mACS is described and the benefit of mACS eye drops in the wound healing of animal models is further shown. The crucial modification of this mACS protocol is the addition of approximately 0.5 mL of 10% glycerol solution in each test tube, which creates suitable hypoxic conditions during the 4 h incubation at 37 &#176;C. This setting provides the AS with proper stress and prompt cells to secrete the necessary growth factors that help wound healing. The 0.22 &#181;m filter c...

The therapeutic effects of autologous serum (AS) in dry eye diseases were first reported in the 1980s by Fox et al.1. It is believed that both the lubricating property and the essential epitheliotropic biochemical components in AS, mimicking natural tears, benefit the proliferation of corneal epithelial cells. Over the past decades, several studies have been performed on this basis. Trophic components include epidermal growth factor (EGF), vitamin A, transforming growth factor &#946; (TGF- &#946;), and other cytokines. Interestingly, the serum is rich in TGF- &#946; and vitamin A, which are believed to play a pivotal role in epidermal proliferation2,3,4,5. In addition, when treating patients with ocular surface diseases, several studies have showed some advantages of AS eye drops in patient-reported outcomes, other objective dry eye parameters6,7, and microscopic findings such as cell density8. Meta-analysis studies revealed that there might be some benefits in improving patient&#39;s syndromes with AS eye drops treatment, but long-term results and observations are still lacking9,10.
Unlike AS, platelet-rich plasma (PRP) is derived from adding an anticoagulant during preparation, with further differential centrifugation and chemical activation of the platelets. Compared with AS, numerous chemicals and growth factors, such as TGF- &#946;, vascular endothelial growth factor (VEGF), and EGF, are present in PRP. It has also been applied to ocular surface diseases with clinical benefits in symptom relief11.
The cross-link between epithelial defects and inflammation is complex. Notably, immunopathophysiology is another important issue in ocular surface diseases. Pro-inflammatory cytokines, such as IL-1&#946; and IFN-&#947;, are believed to be pivotal mediators in inflammatory cascades12. New avenues of treatment are thus opened based on understanding the immune mechanism. Strategies to stop this inflammatory process, including the production of interleukin-1 receptor antagonist (IL-1Ra) and other anti-inflammatory cytokines, may also play an important role in ocular surface diseases13,14,15.
Since 1998, Orthokine, a commercialized autologous conditioned serum (ACS), has been used clinically in orthopedic patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), and spinal disorders13. Compared with AS and PRP, treatment with chemically coated glass beads and hypoxic incubation to activate monocytes are the specific features of ACS16. Theoretically, more anti-inflammatory factors can be secreted by adding survival stress to the cells, resulting in a higher concentration of essential immune-modulating components, including IL-1Ra. The improved therapeutic benefits of ACS in OA, compared with AS, have also been reported17. Ocular surface diseases share similar immune backgrounds with orthopedic inflammatory diseases in some respects. Therefore, based on the successful results of human blood-derived therapy in the orthopedic field, ACS might have advantages over conventional treatments in clinical practice by epitheliotropic and immune-modulating properties. Although ACS has been widely used in orthopedic inflammatory diseases, its clinical applications in ophthalmology still need to be explored, which may be hindered by its high cost, lack of literature support, and lack of standardization of the preparation process, resulting in diverse performance.
In this video article, a novel, cost-effective, and convenient method was demonstrated to generate the modified ACS (mACS), or plasma rich in growth factors (PRGF), producing an eye drop solution with a comparable practical value to commercialized ACSs. The key ideas of adding anticoagulants and triggering the blood cells to secrete anti-inflammatory cytokines by stressed incubation were retained, but unlike the chemically-induced methods, such as those based on CrSO4-coated glass beads and commercial kits, the critical stress status is physically induced by hypoxic incubation in this method. Moreover, glycerol was added to provide extra benefits, including an increase in the stability of the membrane of blood cells, maintenance of a proper osmotic extracellular fluid pressure18, and an appropriate source of nutrients in hypoxic conditions that avoid&#160;overstressing the cells.

<table><tbody><tr><td>&amp;nbsp;96-well culture plate</td><td>Merck KGaA, Germany</td><td>CLS3997</td><td></td></tr><tr><td>Barraquer lid speculum</td><td>katena</td><td>K1-5355</td><td>15 mm</td></tr><tr><td>Barraquer needle holder</td><td>Katena</td><td>K6-3310</td><td>without lock&amp;nbsp;</td></tr><tr><td>Barron Vacuum Punch 8.0 mm</td><td>katena</td><td>K20-2108</td><td>for cutting filter paper</td></tr><tr><td>BD 10.0 mL vacutainer tubes containing heparin 158 USP units</td><td>Becton,Dickinson and Company, US</td><td>367880</td><td>At least 6 tubes, necessary to collect blood for subsequent experiments and to avoid blood agglutination</td></tr><tr><td>BD 21 G butterfly-winged infusion set</td><td>Becton,Dickinson and Company, US</td><td>367281</td><td>For even distribution of glycerol solution</td></tr><tr><td>C57BL/6 mice&amp;nbsp;</td><td>National Laboratory Animal Center</td><td>RMRC11005</td><td>for mouse model</td></tr><tr><td>Castroviejo forceps 0.12 mm</td><td>katena&amp;nbsp;</td><td>K5-2500</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Germany</td><td>5811000428</td><td>3,500 x g for 10 min</td></tr><tr><td>Cheng Yi 10.0 mL sterilized eye dropper bottle</td><td>Cheng Yi Chemical, Taiwan</td><td>CP405141</td><td>Must be sterile and as the storage container for the final product</td></tr><tr><td>Corneal rust ring remover with 0.5 mm burr</td><td>Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX</td><td>CHI-675</td><td>for debridement of the corneal epithelium</td></tr><tr><td>Dulbecco&#039;s modified minimal essential medium</td><td>Merck KGaA, Germany</td><td>D6429</td><td></td></tr><tr><td>Filter paper&amp;nbsp;</td><td>Toyo Roshi Kaisha,Ltd.</td><td>1.11</td><td></td></tr><tr><td>Fluorescein sodium ophthalmic strips U.S.P</td><td>OPTITECH</td><td>OPTFL100</td><td>staining for corneal epithelial defect&amp;nbsp;</td></tr><tr><td>Incubator</td><td>Firstek, Taiwan</td><td>S300S</td><td>37 &amp;deg;C for 4 h</td></tr><tr><td>Kanam sterile gloves</td><td>Kanam Latex Industries, India</td><td>EN455</td><td>For aseptic operation</td></tr><tr><td>Merck 0.22 &amp;micro;m filter</td><td>Merck KGaA, Germany</td><td>PR05359</td><td>At least 2 filters for mACS filtration</td></tr><tr><td>Nang Kuang 250 mL 10% glycerol solution</td><td>Nang Kuang Pharmaceutical, Taiwan</td><td>19496</td><td>To offer suitable membrane stabilization effect and extracellular osmotic pressure for blood cells</td></tr><tr><td>Normal saline</td><td>TAIWAN BIOTECH CO., LTD.</td><td>100-120-1101</td><td></td></tr><tr><td>Skin biopsy punch 2mm</td><td>STIEFEL</td><td>22650</td><td></td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss Meditec, Dublin, CA</td><td>SV11</td><td>microscope for surgery</td></tr><tr><td>Terumo 18 G needle</td><td>Terumo, Taiwan</td><td>SMACF0120-18BX</td><td>3.0 mL syringe with 18 G needle to extract the supernatant after centrifugation</td></tr><tr><td>Terumo 20.0 mL syringe</td><td>Terumo, Taiwan</td><td>MDSS20ES</td><td>Could be used to collect serum after initial centrifugation and use it for secondary centrifugation.</td></tr><tr><td>Terumo 3.0 mL syringe with the 23 G needle</td><td>Terumo, Taiwan</td><td>MDSS03S2325</td><td>3.0 mL syringe is used to extract the supernatant after centrifugation. Then connect the filter and the 23 G needle for injection into the eye drop bottles.</td></tr><tr><td>Westcott Tenotomy Scissors Medium</td><td>katena</td><td>K4-3004</td><td></td></tr></tbody></table>

production of modified autologous conditioned serum and ex vivo assessment of its healing potential in murine corneal epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Figure 1 and Figure 2 show the materials needed for the experiment, and Figure 3 displays the sequential steps and the successful mid-products during the preparation of mACS. First, 0.5 mL of 10% glycerol solution was added into each 10 mL sterile test tube (Figure 3A). Then, 60-70 mL of venous blood was obtained from the patient, and 10 mL of blood was injected into each tube (Fig...

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Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

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Research

JoVE Journal

Medicine

1.8K Views.  Chang Gung Memorial Hospital Linkou. Dit proces presenteert een innovatief bloedafgeleide, mACS genaamd, dat geen complexe technische verwerking vereist. Het versnelt ook de wondgenezing in grotere mate dan conventioneel autoloog serum. Naast het bieden van een gestandaardiseerd voorbereidingsproces, kan deze techniek ook de kosten van serumoogdruppels verlagen, waardoor de toegang van patiënten tot een meer betaalbare behandelingsoptie wordt verbeterd.Deze techniek kan worden toegepast op gebieden die het bevorderen van...

Video: Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

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 ...

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

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 ...

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.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

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