<meta charset="utf8"></head><body>用于毒性检测和伤口愈合的离体猪角膜培养系统

<ol>
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<table><tbody><tr><td>1.7 mL tubes</td><td>Axygen</td><td>AXYMCT175SP</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Thermo Scientific</td><td>R0491</td><td></td></tr><tr><td>Bromfenac (Prolensa) 0.09%&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td>Canon</td><td>PowerShot A620</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>D-glucose&amp;nbsp;</td><td>Sigma</td><td>50-99-7</td><td></td></tr><tr><td>Dissecting microscope&amp;nbsp;</td><td>Zeiss</td><td>Stemi 2000c</td><td></td></tr><tr><td>Forceps</td><td>FisherScientific</td><td>10-316A</td><td></td></tr><tr><td>Hemostat</td><td>FisherScientific</td><td>13-812-14</td><td></td></tr><tr><td>Ketorolac (Acular) 0.45%</td><td></td><td>Kresge Clinic</td><td></td></tr><tr><td>Kimwipes</td><td>Kimtech</td><td>34155</td><td></td></tr><tr><td>LL-37</td><td>Tocris</td><td>5213/1</td><td></td></tr><tr><td>Minimum essential medium (MEM)&amp;nbsp;</td><td>Gibco</td><td>A1048901</td><td></td></tr><tr><td>Nepafenac (Ilevro) 0.1%&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Pig eyes&amp;nbsp;</td><td>Bernthal Packing Inc.</td><td></td><td></td></tr><tr><td>Pipet tips</td><td>VWR</td><td>76322-164</td><td></td></tr><tr><td>Porcine corneas</td><td>Bernthal Packing , Inc. Frankenmuth, MI&amp;nbsp;</td><td></td><td></td></tr><tr><td>Povidone-Iodine&amp;nbsp;</td><td>Betadine</td><td></td><td></td></tr><tr><td>Q-Tips cotton swabs</td><td>Q-Tips</td><td></td><td></td></tr><tr><td>Razor blade</td><td>Electron Microscopy Sciences</td><td>72002-01</td><td></td></tr><tr><td>Razor blade holder&amp;nbsp;</td><td>Stotz</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Bard-Parker</td><td>377112</td><td></td></tr><tr><td>Scalpel Handle</td><td>Bard-Parker</td><td>#3</td><td></td></tr><tr><td>Scissors</td><td>FisherScientific</td><td>08-951-20</td><td></td></tr><tr><td>Silicon mold</td><td></td><td></td><td></td></tr><tr><td>Tissue culter enclosure</td><td>Labconco</td><td>5100000</td><td></td></tr><tr><td>Trephine</td><td>Acu.Punch</td><td>3813775</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>VWR</td><td>1235</td><td></td></tr></tbody></table>

evaluating therapeutic and chemical toxicity using organ-cultured porcine corneas and epithelial wound healing

<meta charset="utf8"></head><body>作者聚焦：探索糖尿病患者的角膜先天免疫和伤口愈合延迟

使用器官培养的猪角膜评估治疗和化学毒性以及上皮伤口愈合

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular ...

evaluating-therapeutic-chemical-toxicity-using-organ-cultured-porcine

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Research

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

JoVE Journal

Medicine

6.9K 次观看.  Wayne State University School of Medicine. 我们正在研究角膜先天免疫和糖尿病伤口愈合。具体来说，我们旨在了解为什么糖尿病患者更容易受到细菌感染，以及导致与低血糖相关的伤口愈合延迟的原因。我们已经确定 IL-1 β 和 IL-1 受体拮抗剂以及 TGF β-1 和 TGF β-3 之间的平衡对于角膜伤口愈合很重要。这可能会导致一种新的糖尿病护理治疗方法有毒。我们的模型可能会使糖尿病患者的?...

视频: 作者聚焦：探索糖尿病患者的角膜先天免疫和伤口愈合延迟

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

科研

免疫与感染

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.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

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 ...

医学

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

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.

创面愈合研究的前活体角膜器官培养模型

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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.

角膜上皮机械和化学损伤的离体和体内动物模型

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

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

生物学

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Source: Okurowska, K., et al. <a href="https://www.jove.com/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp (2020).
This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection.

猪离体角膜细菌性角膜炎模型:一种在角膜上皮细胞中建立细菌感染的技术

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp (2020).
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JoVE Encyclopedia of Experiments

Large Animal Models

Porcine

Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

Source: Holzhey, A. et al., <a href="https://www.jove.com/v/60542">Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss.</a> J. Vis. Exp. (2020)
This video demonstrates a non-invasive laser-assisted procedure to selectively damage the corneal endothelial cells in the porcine eye. The developed ex vivo model of endothelial cell damage can be used to study diseases of the corneal endothelium.

猪角膜内皮细胞的激光辅助选择性消融:一种诱导离体研究聚焦猪角膜内皮损伤的技术

Source: Holzhey, A. et al., Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss. J. Vis. Exp. (2020)
This ...