v12a128-liang pgmkr

Molecular Vision 2007; 13:1169-80 <http://www.molvis.org/molvis/v13/a128/>Received 4 April 2007 | Accepted 1 July 2007 | Published 17 July 2007

Although less common than Gram-positive (GP) bacte-rial infections, Gram-negative (GN) bacterial infections oftenlead to more serious ocular surface inflammation with a highrisk of visual impairment and even blindness [1,2]. GN bacte-rial infections are characterized by a rapidly progressing, sup-purative stromal infiltration with marked mucopurulent exu-dates [3]. GN bacteria such as Pseudomonas aeruginosa (P.aeruginosa) are the most common bacteria isolated from con-tact lenses, which remain important causes of highly destruc-tive keratitis and neovascularization [3,4]. Moreover, the suc-cessful treatment of GN bacteria is being challenged by in-creasing antibiotic resistance [5].

In addition to direct infectious diseases induced by GNspecies, lipopolysaccharide (LPS or endotoxin), a highly con-served molecular pattern of GN bacteria, has also been sug-gested to play a direct role in severe ocular surface patholo-gies [3] such as delayed corneal wound healing, complica-tions after corneal surgery [6,7], or aggravation of certain in-fectious situations [8]. LPS induces the release of a number ofproinflammatory cytokines, i.e. interleukin(IL)-1, IL-6, andtumor necrosis factor (TNF)α [9]. LPS also activates the in-

nate immune system via CD14 and toll-like receptors (TLRs)[10]. Therefore, it is an important environmental agent thatinduces and aggravates ocular bacteria-related inflammatoryreactions and is directly involved in keratitis, conjunctivitis,and uveitis. LPS also interferes with wound healing, whichcould cause severe concerns after refractive surgery. In a rab-bit laser in situ keratomileusis (LASIK) model, LPS was ca-pable of reproducing diffuse lamellar keratitis(DLK)-like in-flammation by stimulating production of IL-8 when appliedbeneath the corneal flaps [6]. Conversely, endotoxin blockerssuch as polymyxin were effective in decreasing DLK inci-dence [7]. LPS-related TNF production was shown to inhibitgrowth factors and decrease collagen production, thus impair-ing the wound healing process in skin wounds [11].

Human cornea, conjunctiva, and uvea were shown to ex-press LPS receptor proteins such as CD14 and TLR4 [12,13].Corneal inflammatory response triggered by LPS is charac-terized by neutrophil (polymorphonuclear leukocytes, PMNs)and macrophage recruitment [14,15]. Recently, within an acuteinflammation model of subconjunctival injection of LPS inrabbits using an in vivo confocal microscopy (IVCM) tech-nique, we demonstrated the time sequence of inflammatoryinfiltration [16]. Co-injection of neutralizing anti-TNF-α inthe conjunctiva could significantly reduce LPS-induced in-flammation and apoptosis in the epithelium and substantiapropria.

©2007 Molecular Vision

LPS-stimulated inflammation and apoptosis in corneal injurymodels

Hong Liang,1,2 Françoise Brignole-Baudouin,2,3 Antoine Labbé,1,2 Aude Pauly,2 Jean-Michel Warnet,1,3 ChristopheBaudouin1,2

1Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital and Ambroise Paré Hospital, APHP, Univer-sity of Versailles, Paris, France; 2INSERM, UMR S 872, Cordeliers, University Paris Descartes, France; 3Department of Toxicol-ogy, Faculty of Biological and Pharmacological Sciences, University Paris Descartes, France

Purpose: To evaluate and compare the proinflammatory and apoptotic effects of lipopolysaccharide (LPS) in three rabbitcorneal injury models using a new in vivo confocal microscope (IVCM) and immunohistological techniques.Methods: Adult male New Zealand albino rabbits were used in this study. Three corneal models were tested: cornealincision, corneal epithelium scraping, and corneal suture. Ten rabbits were used in each model and these three groups weresubdivided into two subgroups: with or without LPS instillation (with saline used as control) for eight days. Rabbitcorneas were analyzed in vivo by using the Rostock Cornea Module (RCM) of the Heidelberg Retina Tomograph (HRT)-II. Immunohistology was used to evaluate inflammatory, proliferating, and apoptotic cells in the different injury modelsfollowing saline or LPS instillations.Results: Clinically, LPS induced earlier and higher levels of inflammation and corneal neovascularization in eyes sub-jected to scraping and suturing compared to saline. The RCM/HRT successfully presented high-quality images allowinganalysis of all pathological corneal layers. Compared to groups receiving saline, LPS caused earlier and greater surfaceand stromal inflammatory infiltration as well as neovascularization. Immunohistology was correlated with in vivo find-ings and confirmed these results by showing greater infiltration of KI 67+ proliferating cells, TUNEL+ apoptotic cells, andTNF-α+, TNFR1+, TLR4/MD2+, ICAM-1+, RLA-DR+, CD11b+, and CD11c+ inflammatory cells, in eyes receiving LPScompared to those receiving saline.Conclusions: These results indicate that in various models of corneal injury, LPS is a potent proinflammatory stimulusand its exposure has major effects on determinants of inflammation, angiogenesis, and apoptosis.

Correspondence to: Christophe Baudouin, MD, PhD, Department ofOphthalmology III, Quinze-Vingts National Ophthalmology Hospi-tal, 28 Rue de Charenton, 75012, Paris, France; Phone: (33) 1 40 0213 04; FAX: (33) 1 40 02 13 99; email: [email protected]

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©2007 Molecular VisionMolecular Vision 2007; 13:1169-80 <http://www.molvis.org/molvis/v13/a128/>

After the study of LPS in conjunctiva, we wished to con-tinue our research by seeking LPS potentiation ofneovascularization, inflammation, apoptosis, and cicatrizationin various models involving the cornea. We studied the ef-fects of LPS in three types of corneal injuries in rabbits: deeptransversal incision, epithelium scraping, and corneal suture.In the literature, the incision models were used to mimic ra-dial keratotomy or simple corneal incision while scraping wasused as a photorefractive keratectomy (PRK) model and su-ture placements were considered to mimic corneal allograftsuture and a model for inducing corneal neovascularization.The effects of LPS on cornea were previously studied follow-ing injection into the corneal stroma [14,15]. Here, we usedtopical instillation of LPS, considering that direct contact be-tween LPS and the wounded tissue would be more similar tothe environmental stimulus. We used LPS derived from Es-cherichia coli (E. coli) because on corneal fibroblasts, it had agreater effect than P. aeruginosa. Probably because LPS fromP. aeruginosa has a smaller lipid A component [17-19]. LPSwas instilled directly onto the ocular surface in these threetypes of corneal wounds and saline instillation was used ascontrol. We quantified in vivo the inflammatory processesusing IVCM and completed with standard immunohistologyto confirm and analyze the inflammatory and apoptotic ef-fects of LPS in corneal injuries.

METHODSAnimals: Adult, male, 10-month-old New Zealand albino rab-bits weighing 2.5-3 kg with ocular surface integrity observedby a slit lamp biomicroscope were used. All animals weretreated according to the Association for Research in Visionand Ophthalmology Resolution on the Human Use of Ani-mals in Vision Research under the supervision of a health au-thority-accredited staff member for animal care and manage-ment. Considering the damage to the eyes of rabbits, espe-cially those in LPS-instilled scraping and suture models, weused just one eye of each rabbit to do the experiments. Beforeall experiments, rabbits were anesthetized by subcutaneousinjection of ketamine (35 mg/kg, Imalgéne 500; Merial, Lyon,France) and xylazine (5 mg/kg; Bayer, Puteaux, France). Atotal of 30 rabbits were divided into six groups. In each group,five rabbits were used for IVCM observation at 30 min (M30),1 h, 4 h (H1 and H4), and from day(D)1 to D9; three rabbitswere then observed from D10 to D30. Two rabbits from eachtreatment group were sacrificed for immunohistological pro-cedures at D9, a time point chosen for optimally observingneovascularization according to a previous preliminary studyin three rabbits (data not shown). The scores were assessed bytwo ophthalmologists in a masked manner. The final scorewas the average of these two scores.

Corneal models and saline or lipopolysaccharide instil-lation: Three corneal models consisting of corneal incision,corneal epithelium scraping, and corneal suture were repro-duced in one eye of 30 rabbits (ten for each type of injury). Asimple linear incision was made in the cornea by a cut using asterile 25-G needle from the limbus and directed toward the

center of the cornea. Incision length (7 mm) was measuredwith a caliper and depth (midstroma) was controlled under anoperating microscope. Epithelial scraping was performed us-ing a mechanical scraping of the corneal epithelium with asterile blade under an operating microscope. The superior cor-neal epithelium was removed, leaving an untreated area 0.5mm along the limbus. In the corneal suture model, three 10-0nylon nonabsorbable monofilament sutures were placed in therabbit cornea at midstromal depth, parallel to the limbus, andapproximately 5 mm along the limbus at the two, ten, andtwelve o’clock positions.

The three corneal injury models were divided into twosubgroups: with LPS instillation (from Escherichia coli, SigmaAldrich, St. Louis, MO) and endotoxin-free saline as control.We administered 25 µl of endotoxin-free saline or 25 µl ofLPS solution at a concentration of 50 µg/ml [6] onto the rabbit’socular surface immediately after corneal surgery and then ev-ery day from D1 to D8 at the end of IVCM examination. Sa-line and LPS instillations were then stopped between D9 andD30 in the three remaining animals in each group in order toobserve corneal recovery.

Clinical findings and Draize test: Using slit lamp mi-croscopy, the eyes were examined for irritation and scoredaccording to a weighted scale for grading the severity of ocu-lar lesions (modified scores from the Draize test) [20]. Theconjunctiva was evaluated for degrees of redness, swelling(chemosis), and discharge. The cornea was evaluated for de-gree and area of opacity. The iris was assessed for increasedprominence of iridal folds, congestion, swelling, etc. Themaximum total score possible was 110 (conjunctiva=20, cor-nea=80, iris=10).

Neovascularization scoring: Corneal neovascularizationwas observed by slit lamp microscopy after corneal surgeriesfrom D1 to D9 (five eyes in each group) then until D30 (threeeyes). To give an overall assessment of its extent,neovascularization was scored from zero to four according toa previous study [21]. The scores were recorded by the lengthof newly formed corneal vessels: zero, no vessels; one, ves-sels only in the peripheral cornea and extension less than 1mm; two, vessel extension less than 2 mm from the limbusbut higher than 1 mm; three, vessel extension between 2 and 3mm; four, vessel extension greater than 3 mm.

In vivo confocal microscopy evaluation: A recently de-veloped laser scanning IVCM, the Heidelberg Retina Tomo-graph (HRT) II/Rostock Cornea Module (RCM, HeidelbergEngineering GmbH, Heidelberg, Germany), was used, as de-scribed previously, on humans [22,23] and on animals[16,20,24]. Briefly, the HRT II camera was disconnected fromthe head rest and maintained in a vertical position. The objec-tive of the microscope, magnification 60X, numerical aper-ture 0.90 (Olympus, Hamburg, Germany), covered by a poly-methyl methacrylate cap, was used to evaluate the corneal in-juries. Images comprise 384x384 pixels covering an area of400x400 µm with a transversal optical resolution of approxi-mate 1 µm/pixel and an acquisition time of 0.024 s (Heidel-berg Engineering). The x-y position and the depth of the opti-

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cal section were controlled manually and the focus position(µm) was automatically calculated by the HRT II/RCM.

Rabbits were positioned on their side and held in place toalign the central cornea parallel to the objective tip. A drop ofgel tear substitute (Lacrigel®, carbomer 0.2%; Europhta, Mo-naco) was placed on the tip of the objective lens. For all eyes,at least ten confocal microscopic images of each layer wererecorded by focusing the microscope on the cornea.

Using the Cell Count® software associated with the HRTII/RCM, we counted the number of infiltrating inflammatorycells (lymphocytes, polymorphonuclear cells, or dendritic-likecells) in corneal stroma and basal epithelium as in our previ-ous study [16]. The number of marks of each image wascounted by the computer and cellular densities were expressedas cells per square millimeter (cells/mm2). Cellular densitieswere calculated on the five to ten confocal microscopy im-ages for each corneal layer of each eye and the final scoreswere the average of three (D9 to D30) to five (M30 to D9)rabbits.

Immunohistology on cryosections: Two rabbits in eachgroup were euthanized with a lethal dose of pentobarbital atD9, the day following the last saline or LPS instillation. Enucle-ated eyes were fixed in 4% paraformaldehyde, embedded inan optimal cutting-temperature (OCT) compound (Tissue-Tek®; Miles Inc., Bayer Diagnostic, Puteaux, France). The 8µm cryosections were incubated with antibodies to TNF-α(1:50; clone 6402; R&D Systems, Wiesbaden-Nordenstadt,Germany), TNFR1 (1:50; clone 16803; R&D Systems), TLR4/MD2 (1:50; Santa Cruz Biotech, Santa Cruz, CA), class IIantigen RLA-DR (1:50; clone TAL.1B5; DAKO, Copenhagen,Denmark), ICAM-1 (1:200; clone 6.5B5; DAKO), CD11b,CD11c (Immunotech, Marseilles, France), KI 67 (1:50;Nuclear Antigen Ki-67, Immunotech), and with mouse IgG1as negative control (Immunotech). Sections were stained withAlexa Fluor®488 goat anti-mouse as secondary antibody(1:250; Molecular Probes, Montluçon, France) for one h andlater with propidium iodide (PI, Sigma Chemical Co., SaintLouis, MO). Images were digitized using an Olympus BX-UCB fluorescent microscope (Olympus, Melville, NY),equipped with DP70 Olympus digital camera and image analy-sis software, to determine the total number of cells positive tothe different markers. Cells were counted in a masked mannerin at least five 100x100 µm areas for corneal stroma and 100µm long for epithelial layers.

Apoptosis evaluation with terminal deoxynucleotidyltransferase-mediated dUTP-nick end labeling staining: A ter-minal deoxynucleotidyl transferase-mediated dUTP-nick endlabeling (TUNEL) assay (Roche Diagnostics, Meylan, France)was used according to the manufacturer’s instructions.Cryosections were permeabilized with a 0.1% Triton X-100-0.1% sodium citrate (2V:1V) solution for two min and thenincubated with an apoptosis detection kit including the 10 µlTUNEL enzymes and 90 µl TUNEL label at 37 °C for one h.After three washes in PBS, the slides were stained with DAPI,observed, and counted under the fluorescence microscope.

Statistical analysis: The groups were compared usingthe Mann-Whitney test for Draize test and neovascularization

scores and factorial analysis of variance (ANOVA) followedby the Bonferroni/Dunnet method for cell counts (Statview V;SAS Institute Inc., Cary, NC).

RESULTSSaline- or lipopolysaccharide-instilled corneal incision mod-els: Clinically, 4 h after corneal incision, compared to salineinstillation (Figure 1A), LPS-instilled corneas presented moreredness and purulent secretions (Figure 1B). At D9, the salineplus incision eyes returned to a normal aspect (Figure 1C)whereas LPS plus incision eyes still presented slight cornealopacity (Figure 1D) with delayed corneal wound healing. Be-tween D1 and D15, according to the Draize test, LPS instilla-tion provoked more ocular inflammation than did saline (Fig-ure 2A, p<0.05). The saline plus incision eyes presented analmost normal ocular surface aspect at D4, which remaineduntil the end of the experiment with no obvious ocular irrita-tion. However, in the LPS plus incision model, animals pre-sented a higher Draize score during instillation time, whichstarted to return to a normal aspect at D15 and continued re-turning normal until the end of the experiment. In the twoincision models, no corneal neovascularization was observedat D9 (Figure 3) or during the entire experimental process.

In the corneal incision model, IVCM (Figure 4) showedthat the earliest cellular changes (H1-H4) were keratocytesadjacent to the cut edge becoming invisible (H4; Figure 4A).Infiltration of inflammatory cells started already at H1 in theLPS-instilled incision. Within three to four h, abundant in-flammatory cells appeared near the incision (H4; Figure 4B).However, the saline-instilled model showed mild and delayedinflammatory infiltrates. We counted inflammatory cell infil-trates at H4 in the cornea stroma (Figure 5A) and found ahighly significant difference between the two models:745.8±55.5 cells/mm2 for LPS plus incision versus 55.3±11.5cells/mm2 for saline plus incision (p<0.0001). By D1-D2, cor-neal reepithelization had already occurred in the lesional zoneboth in saline- (D1; Figure 4A) and LPS-instilled eyes (D1;Figure 4B). At this time, more numerous inflammatory cellscould still be found inside at the edge and outside the incisionallesion in eyes that had received LPS (953.3±127.0 cells/mm2

for LPS versus 144.3±20.2 cells/mm2 for saline, p<0.0001).At D9, compared to the early time points, the inflammatoryinfiltrates decreased but still remained significantly different:320.5±26.0 cells/mm2 for LPS plus incision (D9; Figure 4B)versus 70.3±11.1 cells/mm2 for the saline plus incision model(D9; Figure 4A; p<0.001). Later, from D15 to the end of theexperiment, there was no obvious inflammatory infiltrationobserved near the two incisions (Figure 5A). In basal epithe-lial layers, the inflammatory cell counts were consistent withthose found in the stroma (Figure 6): from D2 to D6, LPS plusincision induced significantly more inflammatory infiltrationthan did saline plus incision (p<0.01 from D2 to D6). FromD9 to D30, no obvious inflammatory infiltration in the sub-epithelial layer was observed in the two incision models.

Immunohistology was used at the end of instillation toinvestigate markers of inflammation, proliferation, andapoptosis in rabbit corneas. Figure 7A shows the results in


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cryosections of saline plus incision and LPS plus incision eyesat D9. There were no significant differences between the twogroups in inflammatory (TNF-α, TNFR1, TLR4/MD2, RLA-DR, ICAM-1, CD11b, and CD11c), proliferating (KI 67), orapoptotic (TUNEL) cells. We observed that even in LPS plusincision eyes, there were few cells positive for TLR4/MD2(Figure 8B) or TUNEL (Figure 8D) immunostainings.

Saline- or lipopolysaccharide-instilled corneal scrapingmodels: In scraping models at H4, compared to saline-treatedcorneas (Figure 1E), the LPS-instilled eyes (Figure 1F) werecharacterized by severe inflammatory reactions with chemo-sis, redness, and especially abundant purulent secretions inthe ocular surface and adjacent to the eyelid. During the entireinstillation period (H1 to D8), LPS induced more ocular in-flammation and redness than did saline (Figure 2B; p<0.05).From D4 to D9 (Figure 1G for saline plus scraping at D9, andFigure 1H for LPS plus scraping at D9), LPS also induced asignificantly higher neovascularization score (Figure 3A) thandid saline (p<0.05). After stopping saline or LPS instillations,there was no difference in neovascularization scores in thetwo models until the end of the experiment.

In the scraping model eyes observed by IVCM, we foundthat at H4 the first reactions in response to mechanical scrap-ing were keratocytes becoming larger and more reflective andthe presence of numerous hyper-reflective stellate structures(H4; Figure 4C). Compared to saline-treated eyes at H4, theLPS-instilled eyes presented more inflammatory infiltrationwith long strands of inflammatory cells stretching from thescraping periphery toward the lesion center (H4; Figure 4D).From H4 to D4, at all time points, LPS induced significantlymore inflammatory cell infiltrates in corneal stroma than didsaline (Figure 5B, p<0.0001). From D6 (1,096.5±147.2 cells/mm2 in LPS plus scraping versus 894±114.1 cells/mm2 in sa-line plus scraping) to the end of the experiments (D30), differ-ences were no longer significant between saline- and LPS-treated scraping models (Figure 5B). At D9, the two modelsdeveloped neovascularization (D9; Figure 4C,D) but with nodifference in inflammatory infiltration cells until the end ofobservation times (Figure 5B). In basal epithelial layers, weobserved numerous dendritic-like inflammatory cells duringthe entire LPS instillation procedure. Their number reachedmaximal values at D9 (Figure 6): 265±9 cells/mm2 in LPSplus scraping versus 114±17 cells/mm2 in saline plus scraping(p<0.0001). A significant difference between saline- or LPS-instilled inflammatory infiltrates in basal epithelial layers wasobserved throughout the experimental period even at D30, 22days after stopping LPS instillation (Figure 6, p<0.0001).

Histologically, all inflammatory, proliferating, andapoptotic markers showed significant differences betweensaline- and LPS-instilled scraping eyes (Figure 7B, p<0.01).


Figure 1. Clinical photos in the saline- or LPS-instilled corneal in-jury models. Clinical features of the saline-instilled (A,C) or LPS-instilled (B,D) incision model, the saline-instilled (E,G) or LPS-in-stilled (F,H) scraping model, and the saline-instilled (I ,K ) or LPS-instilled (J,L ) suture model at H4 (A ,B,E,F,I ,J) and D9(C,D,G,H,K ,L ).

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Figure 2. Draize test evaluation in thesaline- or LPS-instilled corneal injurymodels. The Draize test evaluation in thesaline- or LPS-instilled incision (A),scraping (B) and suture (C) models. Theasterisk indicates that p<0.05 comparedto the corresponding saline-instilled mod-els at the same time point.

Figure 3. Mean neovascularization scoresin the saline- or LPS-instilled scrapingand suture models. This figure shows themean neovascularization scores in the sa-line- or LPS-instilled scraping (A) andsuture (B) models. The two incision mod-els did not induce neovascularization.The asterisk indicates that p<0.05 com-pared to the corresponding saline-in-stilled models.

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We observed that there were more TLR4/MD2-positive cells,especially in the corneal stroma, in the eyes of LPS plus scrap-ing (Figure 8F) than those of saline plus scraping (Figure 8E).These cells could be keratocytes or inflammatory cells. Theother inflammatory markers such as TNFα, TNFR1, RLA-DR, ICAM-1, CD11b, and CD11c were also found more abun-dantly in corneal layers in LPS-associated scraping than insaline-treated eyes. KI-67-positive cells were especially nu-merous in basal epithelial layers in the two scraping models,but the number was much higher in LPS plus scraping (Figure7B, 740±48 cells/mm2 versus 460±74 cells/mm2 in saline plusscraping, p<0.01). At D9, TUNEL-positive apoptotic cells insaline plus scraping (Figure 8G) were notably located in thebasal epithelial layer or anterior stroma whereas, in the eyessubjected to LPS-instilled scraping, the cells were found moreabundantly and were located in all corneal (Figure 8H). Theseapoptotic cells were possibly keratocytes or inflammatory cells,but were not endothelial cells of new blood vessels (doublestaining of TUNEL and CD31 immunostaining, data notshown).

Saline- or lipopolysaccharide-instilled corneal suturemodels: As in the eyes that underwent scraping, four h aftercorneal sutures, compared to eyes receiving saline (Figure 1I),the LPS-treated suture eyes (Figure 1J) were characterized bya more pronounced inflammatory reaction with chemosis, red-

ness, and substantial purulent secretions. The Draize test (Fig-ure 2C) showed that ocular inflammation from H1 to D6 weresignificantly greater in LPS plus suture than in saline plus su-ture eyes (p<0.05). At D9, the LPS plus suture eyes (Figure1L) still showed more active inflammation than did saline-treated eyes (Figure 1K). During the observation time, LPSplus suture induced an earlier onset of neovascularization (start-ing at D3-D4) than did saline (starting at D5-D6; Figure 3B).During the experiments, LPS treatment induced higherneovascularization scores (p<0.05 versus saline treatment fromD9 to D20). Twelve days after stopping instillations (at D20),we still found higher neovascularization scores in LPS-instilledeyes (Figure 3B; p<0.05 at D20 versus saline treatment). AtD30, the two models presented almost the sameneovascularization scores.

In the saline plus suture eyes, no obvious inflammatoryinfiltration was observed at H4 by IVCM (H4; Figure 4E).However, numerous inflammatory cells, dendritiform in shapeor round and hyper-reflective and most likely PMNs, accu-mulated near the LPS-instilled suture at H4 (H4; Figure 4F)and their number increased at D1 (Figure 5C; p<0.0001). Elon-gated, bright, and spindle-shaped structures were also foundnear the suture one day (D1; Figure 4E) after suture place-ment, suggesting migratory fibroblast-like cells in responseto a suture-induced mechanical attraction. In LPS-instilled


Figure 4. Heidelberg Retina tomography II in vivo confocal microscopy images in the saline- or LPS-instilled corneal injury models. Heidel-berg Retina Tomograph II in vivo confocal microscopy images illustrates the results of the saline- or LPS-instilled incision models (A,B: 80-120 µm from the most superficial epithelial layer), of scraping models (C,D: 50-70 µm from the most superficial epithelial layer), and ofsuture models (E,F: 95-135 µm from the superficial epithelium layer) at H4 (line 1), D1 (line 2), and D9 (line 3). All LPS-receiving injurymodels (B,D,F) showed more inflammatory infiltration than saline-receiving models (A,C,E). In all LPS-receiving models (B,D,F), theinflammatory infiltrating cells were more abundant than in all saline-instilled models (A,C,E). Weakly reflective star-like cells in H4 (C) andH4 (E) are keratocytes whereas inflammatory elements, whatever their shape, appear as highly reflective cells. Corneal new blood vessels areclearly visible in scraping models (D9; C and D) and in suture models (D9; E andF). The black line in H4 E to D9 E and H4 F to D9 Fcorrespond to nylon sutures.

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eyes, because of the strong global hyper-reflectivity of the tis-sue resulting from the brightness of abundant inflammatorycells, we could not clearly observe these elongated spindle-shaped fibroblast-like structures. Inflammatory cells also ar-ranged in line beside the suture, possibly influenced by mi-gratory fibroblast arrangement (D1; Figure 4F). On D9, in thesaline plus suture eyes near the suture point, aligned inflam-matory cells were observed with no obvious neovascularizationin the suture area (D9; Figure 4E). However, at that time,neovascularization in LPS plus suture eyes had already reachedand even passed the suture (D9; Figure 4F) with more numer-ous inflammatory cells (Figure 5C; 1,161.3±145.1 cells/mm2

versus 862.3±52.5 cells/mm2 in saline plus suture eyes,p<0.01). From H4 to D20, LPS-instilled suture treatment al-ways induced more inflammatory cells in the cornea stromathan did saline (p<0.01). After that time point, we counted nodifference in stromal inflammatory infiltrates in the two mod-els until the end of the experiment (D30). In basal epitheliallayers, consistent with the eyes subjected to scraping, LPSinduced more inflammatory cell infiltration (Figure 6, 386±11cells/mm2 at D9) than did saline (157±17 cells/mm2, p<0.0001

at D9) and this difference in inflammatory infiltration lasteduntil the end of the experiment (p<0.0001 at all time points).

Immunohistology also showed important inflammatory,proliferative, and apoptotic markers in LPS-instilled suturedeyes (Figure 7C, p<0.0001 compared to saline-instilled su-tured eyes). In corneal cryosections of saline plus suture eyes(Figure 8I), TLR4/MD2-positive cells were observed espe-cially in the neovascularization area. However, in LPS plussuture eyes (Figure 8J), they were found not only in theneovascularization area but also in the other corneal stromalayers. In LPS plus suture eyes, the other inflammatory mark-ers (TNFα, TNFR1, RLA-DR, ICAM-1, CD11b, and CD11C)were also found in the corneal epithelium and stroma espe-cially near the neovascularization area and their numbers werehigher than those found in saline-instilled sutured eyes (Fig-ure 7C, p<0.0001). The proliferative cell marker, KI-67, wasfound chiefly located near the nylon suture andneovascularization areas and the number of positive cells washigher in LPS plus suture (987±154 cells/mm2) than in thesaline plus suture eyes (566±99 cells/mm2, p<0.0001).TUNEL-positive apoptotic cells were observed mainly near


Figure 5. Inflammatory cell countsin corneal stroma using the IVCMCell Count® software. Using theIVCM Cell Count® software, cor-neal stroma inflammatory cellcounts in the saline- or LPS-instilledincision (A), scraping (B), or suture(C) models at H1, H4, and from D1to D30 is shown. The asterisk indi-cates That p<0.0001 and the sharp(hash mark) indicates that p<0.01compared to the corresponding sa-line-instilled models.

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Figure 6. Inflammatory cell counts in basal corneal epithelium using the IVCM Cell Count® software. Inflammatory cell counts in basalcorneal epithelium is illustrated using the IVCM Cell Count® software. The asterisk indicates that p<0.0001 and the sharp (hash mark)indicates that p<0.01 compared to the corresponding saline-instilled models.

Figure 7. Illustrations of positivecell counts in the saline- or LPS-in-stilled corneal injury models. Theseare illustrations of positive cellcounts for inflammatory, prolifera-tive, and apoptotic markers in thesaline- or LPS-instilled incision (A),scraping (B), and suture (C) mod-els at D9 using anti-TNF-α, TNFR1,TLR4/MD2, RLA-DR, ICAM-1,CD11b, CD11c, Ki67 antibodiesand TUNEL assay. The asterisk in-dicates that p<0.0001 and the sharp(hash mark) indicates that p<0.01compared to the corresponding sa-line-instilled models.

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the nylon stitch in the saline plus suture eyes (Figure 8K)whereas they were observed more abundantly not only nearthe nylon suture but also at a distance in the corneal stroma inthe LPS-instilled eyes (Figure 8L).

DISCUSSION Our design of this work was aimed to mimic the clinical dif-ferences between an infected and non-infected corneal wound.In a previous study, an intrastromal injection of LPS in rabbitsinduced severe keratitis characterized by edema and PMN in-filtration [14]. Pseudomonas aeruginosa endotoxin-inducedkeratitis in a mouse-ablated epithelium model was regulatedby TLR4-dependent expression of PECAM-1 and MIP-2,which are essential for recruitment of neutrophils [8]. Com-pared with these previous studies, we observed in vivo in-flammatory infiltration using IVCM, a very promising tech-nique meeting the criteria of the guidelines for the design ofanimal experiments that recommend minimizing the numberof animals and refining the tests used on animals (Statementfor the Use of Animals in Ophthalmic and Visual Research).The HRT II/RCM used for IVCM offers a high definition pro-viding histology-like images and could help us choose themaximum reaction time point and then optimize the time cho-sen for the histological studies requiring animal sacrifice. Thehigh-resolution images given by HRT II/RCW provided non-invasive evaluations at a cellular level in these animal mod-els. This technique can be performed repeatedly in vivo tofollow the course of a disease or a healing process. We ob-served a strong correlation between standard immunohistol-ogy with non-invasive HRT and its major advantage for cellu-lar analyses of healthy or pathological corneas by providingin vivo histologic-like images. HRT II/RCM clearly showedthat in corneal tissue, LPS derived from E. coli was a power-ful inflammatory cell inducer in the corneal stroma and alsoin basal epithelial layers. IVCM images showed that, com-pared to saline instillation, this special layer was more sensi-tive to LPS instillation as we found an abundant number ofinflammatory cells especially dendritic-like cells, even 20 daysafter stopping LPS instillation. We thus observed that the sig-nificant increase in inflammatory infiltrates induced by LPSinstillation remained for a longer duration in basal epitheliallayers (until D30) than in the stroma (until D20). We observed,however, that the total inflammatory cell counts were always


Figure 8. Immunohistological images in the saline- or LPS-instilledcorneal injury models. Immunohistological images of the saline-instilled incision (A,C) or LPS-instilled incision (B,D), saline-instilledscraping (E,G) or LPS-instilled scraping (F,H), and saline-instilledsuture (I ,K ) or LPS-instilled incision (J,L ) immunostained by anti-bodies to TLR4/MD2 (A ,B,E,F,I ,J), and TUNEL assay(C,D,G,H,K ,L ). Nuclei are counterstained in red by propidium io-dide or in blue by DAPI. Note that in LPS plus incision model (B,D),even though there were more cells in the stroma than in the saline-instilled eyes (A,C), they were not positive for inflammatory orapoptotic markers. However, in the scraping (E to H) or suture (I toL ) models, the immunopositive cells for TLR/MD2 or TUNEL wereconsistently more abundant in the LPS-receiving eyes (F,H,J,L ) thanin saline-instilled eyes (E,G,I ,K ). Note the neovascularization in Iand J.

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higher in the corneal stroma than in the basal corneal epithe-lium.

In this study, we observed the enhancement of inflamma-tion, apoptosis, and neovascularization under the influence ofLPS from E. coli instillation in three types of corneal injury.The three types of injury corresponding to clinical ophthal-mological conditions, i.e. incision, scraping, and intrastromalsuture, were all sensitive to LPS instillation, showing moreinflammatory cell infiltrates than to saline instillation withIVCM and immunohistology. Among the three types of cor-neal injury, the suture model showed higher inflammatory in-filtration than did the scraping and incision models. This couldbe due to the persistence of the non-absorbable suture, whichcould prolong the duration of LPS contact at the corneal sur-face and within the stroma. Even though the deep transversalincision model also injured the stroma, cornealreepithelialization at D1-D2 seemed to prevent the effect ofLPS instillation.

TNF-α and TLRs appear to be the most important media-tors associated with LPS-induced inflammation and apoptosisin various in vivo and in vitro systems. In the endotoxic shockmouse model, LPS induced disseminated endothelial apoptosispredominantly resulting from autocrine secretion of TNF[25,26]. In a previous study, we showed that in the conjuncti-val tissue, LPS could induce substantial ocular surface inflam-mation. Anti-TNF-α neutralizing antibodies could in large partinhibit LPS-induced inflammation and apoptosis thus, empha-sizing the cascade of interactions between LPS and TNF-α[16]. Here, we showed that LPS instillation clearly worsenedthe corneal injuries in whatever model was used, possibly viaTNF-related interactions. Upregulation of TLR4 on inflam-matory cells, CD4+ lymphocytes, eosinophils, and mast cellswas found in vernal keratoconjunctivitis (VKC), suggesting arole played by TLRs and therefore by LPS in allergic keratitisand conjunctivitis. In the ocular epithelia, TLRs might par-ticipate in the defense against environmental microbial agents[27]. Another in vitro study showed that human conjunctivalepithelial cell lines (HCEC Chang and IOBA-NHC cell lines)lack LPS responsiveness due to this deficient expression ofMD2 (an accessory molecule required for TLR4 signaling),and the response to LPS can be restored by interferon-γ (IFNγ)priming or MD2 supplementation but not by TNF-α [2]. TLR4,but not TLR2, was shown to act as an apoptosis-promotingsignal in cultured microglia [28]. In mice and bone-marrow-derived macrophage, apoptosis induced by different bacterialpathogens was dependent on activation of TLR4 [29]. In ourstudy, TLR4/MD2 was expressed at a higher level in cornealinjuries with LPS instillation than in those instilled with sa-line. We assume that TLR4/MD2 complex expression re-sponded to LPS stimulation in corneal injury models, mostlikely as the injury induces inflammatory cascades stimulat-ing both MD2 and TLRs. This could be one explanation forthe accentuation of inflammation, apoptosis, andneovascularization induced by LPS.

Intrastromal injection of LPS in rats has been shown tostimulate corneal neovascularization more than injection ofsaline [30,31]. The reason for a high frequency of allograft


rejection in these rat models was the consistent and prolongedneovascularization of allografts that were placed intointralamellar pockets formed by intrastromal injection of 10-25 µl of 100 µg/ml LPS [31]. In rabbits, implants containing100 ng of LPS also enhanced angiogenic response in cornea[32]. Harmey et al. reported that in murine lung tumor cells,LPS exposure could increase angiogenesis and vascular per-meability. This LPS-related angiogenesis was principally re-lated to two inducers, TNF and TLRs [33]. LPS can induceproliferation of endothelial cells and initiate angiogenesis di-rectly through TNF receptor-associated factor 6 (TRAF6)-de-pendent signaling pathways [34]. Primary cultures of humanlimbal fibroblasts (PCHLFs) participated in the vascular en-dothelial growth factor (VEGF) production induced by LPSthrough the stimulation of TLR4 [35]. Murine macrophageVEGF expression is synergistically upregulated by LPS, act-ing through TLR4 receptors [36]. Consistent with these previ-ous studies, in corneal injury models, we found that LPS in-stillation could accelerate and amplify the neovascularizationprocess. This LPS-related angiogenesis was accompanied bymore TNF and TLR4/MD2 immunopositive cells observed incorresponding cryosections.

LPS stimulates directly or indirectly both TNF- and TLR-associated cascades. TLRs may respond to bacteria by induc-ing the expression of cytokines such as TNF-α, IL-1, IL-6,and IL-12 [37,38]. This LPS/TLR/TNF system is also impli-cated in apoptosis and may play a major role in ocular surfacediseases.

Altogether, our studies point out a direct enhancing roleof LPS in ocular surface inflammation, apoptosis, andneovascularization. We assume a potentially important rela-tionship between bacterial components and inflammatory re-actions, leading to persistent neovascularization that may se-verely compromise visual function. Our findings emphasizethe importance of an aggressive treatment of corneal bacterialinfections to avoid permanent damage and irreversible visualfunction. Infected corneal wounds should be treated rapidlyand aggressively to get rid of the proinflammatory andproapoptotic effects of LPS and minimize future irreversiblecorneal damage. Further studies will investigate the possibleefficacy of blocking strategies for LPS, TNF, or TLR4 in suchsevere corneal inflammatory models.

ACKNOWLEDGEMENTS Granted by Quinze-Vingts National Ophthalmology Hospi-tal and INSERM, UMR S 872

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The print version of this article was created on 17 Jul 2007. This reflects all typographical corrections and errata to the article through that date.Details of any changes may be found in the online version of the article. α

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