Mechanisms of Retinal Damage after Ocular Alkali Burns
2017; Elsevier BV; Volume: 187; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2017.02.005
ISSN1525-2191
AutoresEleftherios I. Paschalis, Chengxin Zhou, Fengyang Lei, Nathan L. Scott, Vassiliki Kapoulea, Marie-Claude Robert, Demetrios G. Vavvas, Reza Dana, James Chodosh, Claes H. Dohlman,
Tópico(s)Traumatic Brain Injury and Neurovascular Disturbances
ResumoAlkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection. Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection. Alkali burns may cause significant corneal scarring and blindness even if promptly treated.1McCulley J.P. Chemical injuries.in: Smolin G. Thoft R.A. The Cornea: Scientific Foundations and Clinical Practice. ed 2. Lippincott Williams & Wilkins, Philadelphia, PA1987: 527-542Google Scholar, 2Wagoner M.D. Chemical injuries of the eye: current concepts in pathophysiology and therapy.Surv Ophthalmol. 1997; 41: 275-313Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 3Schrage N.F. Langefeld S. Zschocke J. Kuckelkorn R. Redbrake C. Reim M. Eye burns: an emergency and continuing problem.Burns. 2000; 26: 689-699Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 4Pfister R.R. Chemical trauma.in: Foster C.S. Azar D.T. Dohlman C.H. Smolin and Thoft's The Cornea: Scientific Foundations and Clinical Practice. ed 4. Lippincott Williams & Wilkins, Philadelphia, PA2005: 781-796Google Scholar Standard corneal transplantation can temporarily restore corneal clarity, but long-term results have been disappointing.5Abel R. Binder P.S. Polack F.M. Kaufman H.E. The results of penetrating keratoplasty after chemical burns.Trans Am Acad Ophthalmol Otolaryngol. 1975; 79: 84-95Google Scholar Burn-induced loss of limbal epithelial stem cells that regenerate corneal epithelium complicates surface healing.6Fagerholm P. Lisha G. Corneal stem cell grafting after chemical injury.Acta Ophthalmol Scand. 1999; 77: 165-169Crossref PubMed Scopus (14) Google Scholar, 7Tuft S.J. Shortt A.J. Surgical rehabilitation following severe ocular burns.Eye (Lond). 2009; 23: 1966-1971Crossref PubMed Scopus (59) Google Scholar Implantation of an artificial cornea can restore transparency. In many patients, the clear ocular media reveals the presence of optic nerve pallor and cupping, characteristic of retinal degeneration and severe glaucoma.8Cade F. Grosskreutz C.L. Tauber A. Dohlman C.H. Glaucoma in eyes with severe chemical burn, before and after keratoprosthesis.Cornea. 2011; 30: 1322-1327Crossref PubMed Scopus (56) Google Scholar The mechanism of the damage to the posterior eye is not clear, but previous reports postulated that the alkali diffuses posteriorly and directly damages the retina.9Miyamoto F. Sotozono C. Ikeda T. Kinoshita S. Retinal cytokine response in mouse alkali-burned eye.Ophthalmic Res. 1998; 30: 168-171Crossref PubMed Scopus (21) Google Scholar Inflammatory intraocular pressure (IOP) elevation has also been implicated.10Lin M.P. Ekşioğlu Ü. Mudumbai R.C. Slabaugh M.A. Chen P.P. Glaucoma in patients with ocular chemical burns.Am J Ophthalmol. 2012; 154: 481Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar However, in a recent study on corneal alkali burns in rabbits (in vivo) and porcine eyes (ex vivo), direct pH determination performed in the vitreous revealed an unchanged, normal pH up to 6 hours after the burn. This casts doubt on the possibility of a direct effect of the alkali on the retina. In addition, tumor necrosis factor (TNF)-α expression was shown to acutely increase in the retinas of mice, followed by retinal ganglion cell (RGC) apoptosis 24 hours after the burn.11Cade F. Paschalis E.I. Regatieri C.V. Vavvas D.G. Dana R. Dohlman C.H. Alkali burn to the eye: protection using TNF-α inhibition.Cornea. 2014; 33: 382-389Crossref PubMed Scopus (51) Google Scholar This suggests that the damage to the retina may be mediated by immunological processes, such inflammatory cytokines, rather than by direct pH change. These findings also raise the possibility of using targeted immunomodulatory therapy, such as antibody against TNF-α, for neuroprotection. Indeed, antibody against TNF-α given soon after burn was shown to provide significant protection to both the cornea and retina.11Cade F. Paschalis E.I. Regatieri C.V. Vavvas D.G. Dana R. Dohlman C.H. Alkali burn to the eye: protection using TNF-α inhibition.Cornea. 2014; 33: 382-389Crossref PubMed Scopus (51) Google Scholar The promise of neuroprotection in human eyes after alkali burn prompted the present study. The goal of this study is to elucidate the biophysical and biological processes that lead to retina damage after alkali trauma and to explore the protective role of targeted immunomodulation with an anti–TNF-α agent as a novel adjunct therapy. All animal-based procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the NIH Guide for the Care and Use of Laboratory Animals.12Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.ed 8. National Academies Press, Washington, DC2011Crossref Google Scholar This study was approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. C57BL/6 and TNFRSF1A1B knockout mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice between the ages of 6 and 12 weeks were used for this study. This model of alkali chemical burn is based on our previous study.11Cade F. Paschalis E.I. Regatieri C.V. Vavvas D.G. Dana R. Dohlman C.H. Alkali burn to the eye: protection using TNF-α inhibition.Cornea. 2014; 33: 382-389Crossref PubMed Scopus (51) Google Scholar In brief, mice were anesthetized using 60 mg/kg ketamine and 6 mg/kg xylazine, and deep anesthesia was confirmed by a toe pinch test. Proparacaine hydrocloride USP 0.5% (Bausch and Lomb, Tampa, FL) eye drop was applied to the cornea for 1 minute and carefully dried with a Weck-Cel (Beaver Visitec International, Inc., Waltham, MA). A 2-mm-diameter filter paper was soaked into 1 mol/L sodium hydroxide solution for 10 seconds, dried from excess sodium hydroxide by a single touch on a paper towel, and applied onto the mouse cornea for 20 seconds. Complete adherence of the filter paper on the corneal surface was ensured by gentle push of the perimeter using forceps. After the filter paper was removed, prompt irrigation with sterile saline for 10 seconds was applied using a 50-mL syringe with a 25-gauge needle. The mouse was then placed on a heating pad, positioned laterally, and irrigated for another 15 minutes at low pressure using sterile saline. Buprenorphine hydrochloride (0.05 mg/kg; Buprenex Injectable; Reckitt Benckiser Healthcare Ltd, Hull, UK) was administered s.c. for pain management. A single drop of topical Polytrim antibiotic was administered after the irrigation (polymyxin B/trimethoprim; Bausch & Lomb Inc., Bridgewater, NJ). Mice were kept on a heating pad until fully awake. New Zealand white rabbits, weighing 4 to 5 kg, were obtained from Charles River (Woburn, MA). Rabbits were placed on a heating pad, and anesthesia was administered using i.m. ketamine, 20 mg/kg, followed by i.p. injection of urethane, 1300 mg/kg (Sigma Aldrich, St. Louis, MO) diluted in 2-mL sterile water for injection.13Moore L.R. Chang S.F. Greenstein E.T. Urethane-acepromazine: a novel method of administering parenteral anesthesia in the rabbit.Methods Find Exp Clin Pharmacol. 1987; 9: 711-715PubMed Google Scholar Additional administration of urethane, 400 mg/kg, was provided every 5 hours to maintain anesthesia for 24 hours. Heart rate and temperature were continuously monitored. Alkali burn to the cornea was performed by placing an 8-mm trephine on the corneal surface and filling the trephine with 1 mL 2 mol/L sodium hydroxide for 40 seconds. The alkali was carefully absorbed from inside the trephine using a Weck-cel sponge, and the trephine was filled with 3 mL of sterile saline, which was then aspirated. The eye was then irrigated with saline solution for 20 seconds, followed by slow irrigation for 15 minutes, as described above. At the end of the experiment, an epidermal fentanyl patch was placed (12 μg/hour) for 3 days to reduce discomfort. Rabbits received twice-daily topical antibiotic ointment (erythromycin). Rabbits were euthanized at the completion of the experiment with 40 mg/kg ketamine and 10 mg/kg xylazine, followed by 100 mg/kg Fatal Plus IV injection (sodium pentobarbital). In vivo pH measurements were performed using two different pH probes: fiberoptic pH-1-micro with an outer diameter of 140 μm (PreSens, Regensburg, Germany) and microelectrode pH-50 with an outer diameter of 50 μm (Unisense, Aarhus, Denmark). Two different probes were used to validate the results. Briefly, the fiberoptic pH technology is based on dual-lifetime referencing technique. A fiberoptic probe is inserted in the eye and a coupled photo-emitting diode performs simultaneous excitation of a pair of luminophores, one reporting the pH and the other acting as reference.14Boniello C. Mayr T. Bolivar J.M. Nidetzky B. Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts.BMC Biotechnol. 2012; 12: 11Crossref PubMed Scopus (37) Google Scholar The phase difference between the two excitations represents the pH of the sample and is independent of light intensity or wavelength interference. The probe has a linear response time of approximately 30 seconds at a pH range between 5.5 and 8.5 at 5°C to 50°C. At neutral pH, the probe has resolution ±0.001, accuracy ±0.05, and drift <0.05. The electrode pH sensor is based on selective diffusion of protons through pH-sensitive glass, and measures the potentials between the electrolyte and a reference electrode. The response time of the electrode is 0.98 in all cases. In vivo oxygen measurements were performed using a 50-μm diameter glass-electrode oxygen sensor (Unisense, Aarhus, Denmark). The oxygen probe measures the diffusion of oxygen through a silicone membrane to an oxygen-reducing cathode. The reducing cathode is polarized against an internal silver/silver chloride anode, and the potential difference between the anode and cathode represents the oxygen partial pressure. The probe has linear response between 0 and 1 Atm pO2, with negligible oxygen consumption (10 to 16 mol/second) and response time <1 second. Before implantation, the probe was thoroughly prepolarized with −0.8 V for 24 hours and calibrated at maximum oxygen saturation (0.9% saline at 37°C with air agitation), and zero oxygen (2% sodium ascorbate in 0.1 mol/L sodium hydroxide solution). The probe was connected to a portable computer and measurements were acquired at 1 Hz (Nyquist-Shannon sampling theorem). In vivo oxidation reduction (redox) measurements were performed using a 50-μm-diameter platinum glass-electrode redox sensor (Unisense, Aarhus, Denmark). An open-ended silver-silver chloride reference electrode was used. Briefly, redox electrode sensors measure the electric potential relative to the reference electrode, which shows the tendency of the solution to release (oxidation) or take up (reduction) electrons. Both electrodes were connected to a high-impedance millivolt meter and calibrated in a 4 and 7 pH/quinhydrone buffer (equal molarity mixture of hydrochinone and quinone). Measurements were acquired by a computer. The sensor can detect electric potential changes of 0.1 mV and has a response time 24 hours) was achieved with urethane i.p. injection. The upper lid was excised to expose the posterior segment of the globe, and the conjunctiva was surgically dissected to reveal the sclera. A full-thickness scleral tunnel was generated using a custom-made beveled glass needle (approximately 10 μm in diameter) that was marked with dye (Accu-line Products Inc.). The probes were introduced into the vitreous via the tunnel, and measurements were performed and logged in a computer. The probes were introduced into the vitreous cavity via the tunnel, and pH, oxygen, and redox measurements were performed and logged in a computer. The sensors were inserted into the suprachoroidal space through a small scleral incision 4 mm posterior to the limbus, and advanced posteriorly close to the optic nerve. A stereotaxic device (Leica 39463001) was used to control the position of the probe. Alkali burn was performed as described above, and measurements were performed every 20 seconds for 25 hours. A temperature probe, inserted into the rectum of the rabbit, was used to monitor body temperature. Anesthetized rabbits were euthanized after 25 hours. Intraocular pressure measurements were performed in anesthetized rabbits and mice using a custom-made intracameral pressure transducer connected to a 33-gauge needle. The device was designed using a differential microelectromechanical pressure sensor 40 PC (Honeywell, Freeport, IL) connected to a 14-bit, 48 kilo samples per second data acquisition NI USB-6009 (National Instruments, Austin, TX), controlled by a proprietary software algorithm operating in Labview 2011 (National Instruments) environment. A special algorithm was designed to compensate for minute aqueous humor volume displacement during in vivo pressure measurements in mice. The device was assembled using microfluidic components (IDEX Health & Science, Oak Harbor, WA) with minimum dead volume. Before measurements, the remaining dead volume of the syringe was prefilled with sterile water, thus minimizing air compressibility only within the microelectromechanical cavity, which was compensated by the software algorithm. To perform measurements, the needle was inserted into the anterior chamber of the eye through a temporal clear corneal puncture, adjacent to the limbus, and the needle was advanced approximately 500 μm toward the center of the chamber. The contralateral eye served as internal control, and naive eyes served as physiological pressure reference. Infliximab (Remicade) lyophilized powder was reconstituted to final concentration of 10 mg/kg of Remicade using 10 mL of 0.9% sodium chloride injection, USP. A 26-gauge butterfly catheter was inserted and secured with tape in the marginal ear vein, and the drug was slowly infused over 60 minutes, starting immediately after ocular irrigation (15 minutes). After alkali burn, eyes were enucleated at indicated time points and processed using OCT (Tissue Tek; Sakura, Leiden, the Netherlands) compound. Multiple sagittal sections of approximately 10 μm in thickness were obtained from the center and the periphery of the globe. Sectioned tissues were transferred to positive charged glass slides Superfrost Plus 75 × 25 mm, 1 mm thickness (VWR, Radnor, PA) and stored at −80°C for further processing. After alkali burn, eyes were enucleated at indicated time points and fixed in 4% paraformaldehyde solution for 1 hour at 4°C. The cornea and retina tissue were carefully isolated using microsurgical techniques and washed three times for 5 minutes in phosphate-buffered saline solution (PBS) at 4°C. The tissues were then blocked using 5% bovine serum albumin and permeabilized using 0.3% Triton-X for 1 hour at 4°C. The specific antibody was added in blocking solution, incubated overnight at 4°C, and then washed three times for 10 minutes with 0.1% Triton-X in PBS. Tissues were transferred from the tube to positive charged glass slides Superfrost Plus 75 × 25 mm, 1 mm thickness using a wide pipette tip with the concave face upwards. Four relaxing incisions from the center to the periphery were made to generate four flat tissue quadrants. VECTRASHIELD mounting medium (H-1000; Vector Laboratories, Burlingame, CA) was placed over the tissue, followed by a coverslip. After alkali burn, eyes were enucleated and cryosectioned. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) labeling was performed using a Roche TUNEL kit (12156792910; Roche, Basel, Switzerland), as previously reported.15Paschalis E.I. Chodosh J. Spurr-Michaud S. Cruzat A. Tauber A. Behlau I. Gipson I. Dohlman C.H. In vitro and in vivo assessment of titanium surface modification for coloring the backplate of the Boston keratoprosthesis.Invest Ophthalmol Vis Sci. 2013; 54: 3863-3873Crossref PubMed Scopus (29) Google Scholar Mounting medium with DAPI (UltraCruz; sc-24941; Santa Cruz Biotechnology, Dallas, TX) was placed over the tissue, followed by a coverslip. Images were taken with an epifluorescent microscope (Zeiss Axio Imager M2; Zeiss, Oberkochen, Germany), using the tile technique. DAPI signal (blue) was overlayed with Texas red (TUNEL+ cells) and quantified using ImageJ software version 1.43 or above (NIH, Bethesda, MD; http://imagej.nih.gov/ij) to measure the number of TUNEL+ cells overlapping with DAPI in the areas of interest. All experiments were performed in triplicate. After alkali burn, eyes were enucleated at indicated time points and processed for frozen section, as described above. Sectioned tissues were fixated on the glass slides using 4% paraformaldehyde for 15 minutes at room temperature. The slides were then washed three times for 5 minutes in PBS and dried for paraffin boundary marking using paraffin pen. Tissues were blocked with 5% bovine serum albumin (BSA) and incubated at 4°C overnight using 1:100 dilution of monoclonal anti-mouse fluorescein isothiocyanate conjugated antibody (NBP1-51502; Novus Biologicals, Littleton, CO). Tissues were washed three times for 5 minutes in PBS at room temperature and mounted using either VECTASHIELD or mounting medium with DAPI (UltraCruz; sc-24941; Santa Cruz Biotechnology), and covered with a coverslip. Tissues were blocked with 5% BSA in PBS + 0.1 Tween-20 for 30 minutes at room temperature. Overnight incubation at 4°C was performed using rabbit monoclonal cleaved caspase-3 primary antibody (9664; Cell Signaling, Danvers, MA) at 1:300 dilution. Tissues were washed three times for 5 minutes in PBS, followed by incubation with preabsorbed goat anti-rabbit secondary antibody (DyLight 488; ab96895; Abcam, Cambridge, MA) at room temperature for 1 hour. Tissues were washed three times for 5 minutes in PBS at room temperature and mounted using medium with DAPI (UltraCruz) and covered using a coverslip. Tissues were blocked with 5% BSA in PBS + 0.1 Tween-20 for 30 minutes at room temperature. Incubation at 4°C for 3 days was performed using goat polyclonal Brn3a (C-20) primary antibody (sc-31984Santa Cruz Biotechnology Inc.) at 1:50 dilution. Tissues were washed three times for 5 minutes in PBS, followed by donkey anti-goat secondary antibody (Alexa Fluor 488; ab150129; Abcam) incubation at room temperature for 1 hour. Tissues were washed three times for 5 minutes in PBS at room temperature, mounted using either VECTASHIELD or mounting medium with DAPI (UltraCruz), and covered using a coverslip. Tissues were blocked with 5% BSA in PBS + 0.1 Tween-20 for 30 minutes at room temperature. Overnight incubation at 4°C was performed using conjugated mouse monoclonal antibody (NL557; R&D Systems, Minneapolis, MN) at 1:100 dilution. Tissues were washed three times for 5 minutes in PBS at room temperature, mounted using either VECTASHIELD or mounting medium with DAPI (UltraCruz), and covered using a coverslip. Optic nerve axon degeneration was evaluated in explanted rabbit eyes using paraphenylenediamine staining. Optic nerves were dissected from the explanted eyes, fixed in Karnovsky fixative solution for 24 hours at 4°C, then processed and embedded in acrylic resin. Cross sections (1 μm thick) were obtained and stained with 1% paraphenylenediamine in absolute methanol. Each section was mounted onto glass slides and imaged with a bright field microscope (Nikon eclipse E800 DIC; Tokyo, Japan) using a 100× objective lens. Tile images of the whole nerve section were produced, and axon degeneration was analyzed using ImageJ software, according to previous protocols.16Madigan M.C. Sadun A.A. Rao N.S. Dugel P.U. Tenhula W.N. Gill P.S. Tumor necrosis factor-alpha (TNF-alpha)-induced optic neuropathy in rabbits.Neurol Res. 1996; 18: 176-184Crossref PubMed Scopus (58) Google Scholar, 17Sadun A.A. Win P.H. Ross-Cisneros F.N. Walker S.O. Carelli V. Leber's hereditary optic neuropathy differentially affects smaller axons in the optic nerve.Trans Am Ophthalmol Soc. 2000; 98: 223-232PubMed Google Scholar, 18Kimura K. Morita Y. Orita T. Haruta J. Takeji Y. Sonoda K.-H. Protection of human corneal epithelial cells from TNF-α-induced disruption of barrier function by rebamipide.Invest Ophthalmol Vis Sci. 2013; 54: 2572-2760Crossref PubMed Scopus (65) Google Scholar After alkali burn, eyes were enucleated and fresh retinas were dissected under a surgical microscope. Harvested tissues were placed in cryovials, frozen in liquid nitrogen, and stored at −80°C for further processing. When all tissue samples were collected, frozen tissues were suspended in buffer mixture of protease/phosphatase inhibitors and homogenized using a rotating cone. Samples were centrifuged at 11 × g for 15 minutes, and supernatant was collected and recentrifuged. A BSA assay was performed to quantify protein concentration, and all samples were normalized to 30 μg/mL using SDS buffer. Gels were prepared using acrylamide agarose 10% or purchased from Bio-Rad Laboratories (Hercules, CA). Cellulose transfer membrane was used to transfer the proteins from the gel to the methyl cellulose paper, followed by primary and secondary antibody staining. TNF-α expression was quantified using ImageJ software on whole eye tissue cross sections stained with DAPI and anti–TNF-α monoclonal antibody. Different time points were selected for analysis. For each time point, three different tissue sections from three different mice (n = 3) were used. The area of interest (cornea or retina) was marked using the freehand selection tool from ImageJ software and according to DAPI boundaries. The enclosed area was measured (mm2), and the image was decomposed to red, green, and blue channels. The green channel that demonstrated expression of green fluorescent protein of TNF-α antibody was selected and converted to binary, and the positive pixels (bright pixels) were quantified by the ImageJ software. The outcome was normalized according to the total sampled area, and the resulting number was plotted. Quantification of retinal TUNEL+ cells was performed on whole eye tissue cross sections stained with DAPI and TUNEL assay using ImageJ software. Different retinal layers were quantified (ganglion cell layer; inner nuclear/plexiform layer; outer nuclear/plexiform layer). For each layer, two different tissue sections from three different mice (n = 3) were used. For each retinal layer, the percentage of TUNEL expression was calculated as the ratio of TUNEL/DAPI (area %) using particle analysis of ImageJ software. Data were presented as means ± SD. After burn, eyes were enucleated at indicated time points. Under the surgical dissecting microscope, the cornea, iris, lens, retina, and scleral tissues of each eye were isolated and placed in separate vials containing tissue lysis and protease inhibitor buffer. The tissues were homogenized using ultrasonication. The vials were maintained in ice for temperature control. Vials were centrifuged, and the supernatant was collected and stored at −20°C for further processing. TNF-R1 and TNF-R2 protein levels were quantified using enzyme-linked immunosorbent assay (R&D Systems), as described by the manufacturer. After alkali burn, eyes were harvested at indicated time points. Corneal and retinal tissues were surgically dissected, placed in cryovials, and rapidly frozen in liquid nitrogen. RNA isolation was performed using Uneasy mini kit (74106; Qiagen, Valencia, CA) for retinas and Direct-zol RNA MiniPrep (R2052; Zymo Research Corp., Irvine, CA) for corneas. RNA quantification was performed using a nanodrop spectrophotometer (Nanodrop 2000; Thermo Scientific, Waltham, MA), and each sample was normalized before cDNA synthesis. cDNA synthesis was performed using superscript III (18080-044; Invitrogen, Carlsbad, CA). One microliter of cDNA was used in each real-time PCR. TaqMan primers were used to assess the expression levels of the following genes: TNFA (Mm99999068_m1; Life Technologies, Woburn, MA), TNFR1 (Mm01182929_m1; Life Technologies), TNFR2 (Mm00441889_m1; Life Technologies), FAS (Mm01204974_m1; Life Technologies), FASLG (Mm00438864_m1; Life Technologies), IL1B (MmMm00434228_m; Life Technologies), INFG (Mm01168134_m1; Life Technologies), and MMP9 (Mm00442991_m1; Life Technologies). Each sample was run in triplicate, and three samples were used at each time point. The average cycle threshold (CT) value was used in the analysis. CT values were normalized using β-actin probe, and ranges were normalized based on naive control tissue. ΔΔCT algorithm was used to compare mRNA levels. Eyes were harvested at indicated time points. Corneas and retinas were isolated and processed for flow cytometry using collagenase type I and papain dissociation system (Worthington, Lakewood, NJ). Thy1.1-PE antibody (BioLegend, San Diego, CA) was used to identify RGCs, whereas immune cell activation was assessed using antibodies against CD45 and major histocompatibility complex (MHC)-II. Cells were analyzed on a BD LSR II cytometer (BD Biosciences, San Jose, CA) using FlowJo software version 10.2 (Tree Star, Ashland, OR). TNF-α blockade was performed using infliximab, a Federal Drug Administration–approved chimeric (mouse-human) monoclonal antibody (Remicade; Janssen Biotech, Inc., Horsham, PA). Administration in mice was performed by i.p. injection of 6.25 mg/kg antibody reconstituted in normal salin
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