Two waves of neutrophil emigration in response to corneal epithelial abrasion: distinct adhesion molecule requirements

PURPOSE: Corneal abrasion results in an inflammatory response characterized by leukocyte emigration into the corneal stroma. Adhesion molecules play a critical role in leukocyte emigration to wound sites, but differences are evident in different vascular beds. In this study, the contributions of two families of adhesion molecules to neutrophil emigration into the cornea were investigated. METHODS: Re-epithelialization, patterns of neutrophil influx and CXC chemokine production were assessed in C57Bl/6 mice after removal of a 2-mm diameter area of central corneal epithelium. Comparisons were made between wild-type (WT) mice and mice with targeted deletions of genes for CD18 (CD18(-/-)) or P- and E-selectin (P/E-sel(-/-)) or in mice with antibody-induced neutropenia. RESULTS: Wild-type mice exhibited neutrophil emigration in two waves, the first peaking at 18 hours and the second at 30 hours after wounding, 6 hours after epithelial wound closure and peak levels of corneal CXCL1. In CD18(-/-) animals, only a single wave of neutrophil influx was seen, and it was temporally and quantitatively equivalent to the second wave in WT. In P/E-sel(-/-) mice, neutrophil influx was markedly depressed throughout the 48-hour observation period. Re-epithelialization was significantly delayed in mice with adhesion molecule deletions and in neutropenic animals. Transfer of wild-type leukocytes into CD18(-/-) mice resulted in neutrophil emigration into the injured cornea within 18 hours of wounding and improved closure of the epithelium. CONCLUSIONS: Neutrophil emigration into corneal stroma after epithelial abrasion occurs in two waves. The first is dependent on CD18 integrins and selectins, whereas the second is CD18-independent but requires selectins. Early leukocyte emigration appears to promote re-epithelialization.     

P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响

目的观察P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响。方法选用P-选择素基因敲除小鼠与野生型小鼠进行对比,观察角膜上皮创伤修复过程,同时用免疫组织化学方法观察中性粒细胞在角膜的迁移,并用酶联免疫吸附测定方法检测角膜中细胞因子和趋化因子的变化。结果与野生型小鼠相比,P-选择素基因敲除小鼠的角膜创伤修复延迟,向角膜迁移的中性粒细胞数量减少;中性粒细胞的迁移与趋化因子和细胞因子有密切的关系。结论P-选择素表达缺陷可导致角膜上皮创伤修复的延迟。这种延迟可能与创伤后中性粒细胞向创伤区迁移数量减少有关。     

Effects of salicylate and steroid on neutrophil migration and corneal blood vessel growth

Cauterization of the cornea results in emigration of neutrophils from the limbal blood vessels into the corneal tissue. Blood vessel proliferation follows, the stimulus for which is unknown. In this study, 0.1 M sodium salicylate drops administered topically to cauterized rat corneas over a 48-h period had an inhibitory effect on the migration of neutrophils from the limbal vessels 6 h after injury, but this was not maintained at 48 h. After 6 days of treatment, the salicylate had no effect on vessel growth into the cauterized rat cornea. Application of prednisolone disodium phosphate ointment to cauterized corneas also inhibited neutrophil migration at 6 h, but increased the extravascular neutrophils at 48 h. After 6 days of treatment, corneal blood vessel growth was significantly reduced. It was concluded that there is no consistent relation between the number of extravascular neutrophils at the corneal limbus and the extent of corneal blood vessel growth.     

Impaired eosinophil recruitment to the cornea in P-selectin-deficient mice in Onchocerca volvulus keratitis (River blindness)

PURPOSE: A murine model of helminth-induced keratitis (river blindness) that is characterized by a biphasic recruitment of neutrophils (days 1-3) and eosinophils (days 3+) to the cornea has been developed. The purpose of this study was to determine the relative contribution of P- and E-selectin in recruitment of these inflammatory cells from limbal vessels to the corneal stroma. METHODS: P- and E-selectin gene knockout (-/-) mice were immunized with antigens extracted from the parasitic helminth Onchocerca volvulus. One week after the last immunization, parasite antigens were injected directly into the corneal stroma. Mice were killed on days 1 and 3 postchallenge, and eyes were immunostained with either anti-eosinophil major basic protein (MBP) or with anti-neutrophil Ab. The number of cells in the cornea was determined by direct counting. RESULTS: Recruitment of eosinophils to the cornea was significantly impaired in P-selectin(-/-) mice (63.9% fewer eosinophils on day 1 [P: = 0.0015], and 61% fewer on day 3 [P: < 0.0001]) compared with control C57BL/6 mice. In contrast, P-selectin deficiency had no effect on neutrophil recruitment to the cornea. There was no inhibition of eosinophil and neutrophil migration to the corneas of E-selectin(-/-) mice, indicating that there is no direct role for this adhesion molecule in helminth-induced keratitis. CONCLUSIONS: The present study demonstrates that P-selectin is an important mediator of eosinophil recruitment to the cornea. P-selectin interactions may therefore be potential targets for immunotherapy in eosinophil-mediated ocular inflammation.     

Isoliertes Carcinoma in situ der Kornea

We present the case of an 83-year-old patient with an isolated epithelial dysplasia of the cornea. After corneal abrasion the lesion reoccurred 14 days later. The abrasion was then increased to cover the whole corneal epithelium and adjacent limbal and conjunctival areas were also biopsied. Histology revealed a corneal epithelial dysplasia stage 3, whereas the limbal and conjunctival biopsies showed normal epithelium. After resection two cycles of local mitomycin C application (2 cycles of 14 days each) were administered and 9 months after the second intervention the cornea remained clear with good vision. The investigation for human papillomavirus showed a type 6, which is not associated with an increased risk of malignancy.     

中性粒细胞在不同品系正常小鼠角膜的分布及其与角膜创伤修复的关系

背景 以往人们通常认为角膜无血管、无髓系来源的免疫细胞存在.C57BL/6J小鼠、BALB/c小鼠和裸鼠是眼科免疫学基础研究的常用模型,这些小鼠的角膜是否存在天然免疫的关键细胞——中性粒细胞是值得关注的问题. 目的 研究实验室常用的C57BL/6J鼠、BALB/c鼠和裸鼠正常角膜的中性粒细胞分布特征及其与角膜创伤修复的关系,为相关研究提供依据. 方法 选取角膜正常、10 ～ 12周龄的SPF级雄性C57BL/6J鼠、BALB/c鼠和BALB/c背景的裸鼠各16只,取3种小鼠各8只制备成中央区相连的4瓣角膜铺片,并以角膜周边血管缘为界向内以3个同心圆分区.分别用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体对角膜中性粒细胞和血管进行免疫荧光染色,用免疫荧光显微镜和AR软件测量角膜血管面积并计数中性粒细胞.选取3种小鼠各8只制备角膜创伤模型,用无菌手术刀片以角膜中央为中心做十字划痕,深度至角膜前弹力层.创伤后即刻（0 h）、12h、24 h用2 g/L荧光素钠点眼,荧光显微镜下以AR软件计算不同时间点的创伤面积.于划痕后24 h取小鼠角膜制备铺片,用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体行荧光染色,比较3种创伤小鼠角膜的血管分布、中性粒细胞的数量和分布情况.实验动物的饲养与使用均遵循美国视觉与眼科研究协会制定的科研动物使用规范. 结果 正常C57BL/6J小鼠、BALB/c小鼠和裸鼠角膜中性粒细胞总数分别为（1 733±237）、（353±96）和（1 601 ±223）个/角膜,BALB/c小鼠角膜中性粒细胞数明显少于C57BL/6J小鼠和裸鼠,差异均有统计学意义（P＜0.01）.3种正常小鼠角膜缘均可见血管分布,BALB/c小鼠角膜缘血管面积明显小于C57BL/6J小鼠和裸鼠,C57BL/6J小鼠角膜缘血管面积明显大于裸鼠,差异均有统计学意义（P＜0.01）.3种正常小鼠中性粒细胞的数量与血管面积均呈明显正相关（C57BL/6J：r=0.936,P=0.001;BALB/c：r =0.939,P=0.001;     

培养角膜缘上皮细胞深低温保存后异体移植实验研究

目的深低温保存培养的角膜缘上皮细胞,复苏后进行异体移植实验,为临床应用提供实验依据。方法将浅层角膜基质载体上培养的角膜缘上皮细胞深低温保存,同时制作兔角膜缘干细胞损伤模型,分别用新鲜培养的角膜缘上皮细胞和冷冻复苏后培养的细胞及角膜基质进行异体移植实验。结果角膜缘上皮细胞能成功地培养于载体上,将培养细胞保存于-196℃的液氮罐中二月,可见深低温保存前后细胞的形态结构及生物学特性无显著差异。异体移植实验显示：新鲜培养的细胞及深低温保存后的细胞异体移植后,模型兔角膜表面逐渐正常上皮化,而角膜基质组异体移植后,角膜表面仍结膜化并可见大量新生血管生长,各项检测结果与培养细胞组相比差异显著（P〈0．05）。结论以浅层角膜基质为载体培养的角膜缘上皮细胞深低温保存后仍具有上皮细胞特性并具有高增殖能力,可用以进行异体移植治疗角膜缘干细胞缺陷的角膜病。     

Effect of leukocytes on corneal cellular proliferation and wound healing

PURPOSE: To establish whether fucoidin, by blocking the adhesion of leukocytes on the limbal vascular endothelium, prevents extravasation of the cells from the blood stream into the limbal stroma and the wounded area after corneal injury. Successful leukocyte blocking enabled investigation of the influence of leukocytes on corneal cellular proliferation after corneal wounding. METHODS: Thirty-two New Zealand White rabbits were used. Photorefractive keratectomy (PRK) and a standardized alkali corneal wound were used as models in two sets of experiments. In half of the injured rabbits fucoidin was used to prevent leukocytes from leaving the local vessels. The efficiency of the blocking technique was evaluated by counting the number of leukocytes in the limbal and wounded corneal areas. Proliferating cell nuclear antigen (PCNA) was used as a marker for proliferative activity. RESULTS: The infiltration of leukocytes into the limbus and the cornea after PRK and alkali injuries can be blocked by fucoidin. The healing rate of corneal epithelium after alkali burn was retarded in the absence of leukocytes. PCNA expression was enhanced in the presence of leukocytes. Fucoidin per se had no influence on corneal cell proliferation and wound healing. CONCLUSIONS: Polymorphonuclear leukocytes (PMNs) can be prevented from entering the cornea in vivo by fucoidin after PRK and after alkali burn. The corneal epithelial healing rate is delayed in the absence of PMNs in vivo, and PCNA expression increases in the presence of leukocytes.     

Synchronization of the G1/S transition in response to corneal debridement.

PURPOSE: This study's intention was to examine the progression of ocular surface epithelium through the G1/S transition of the cell cycle after corneal epithelial debridement. METHODS: Three-millimeter debridements were made in central rat cornea and allowed to heal 4 to 48 hours in vivo. Unwounded contralateral eyes served as controls. Two hours before the animals were killed, 5-bromo-2-deoxyuridine (BrdU) was injected to detect S-phase cells. Incorporated BrdU was visualized by indirect immunofluorescence microscopy, and expression of G1 cell-cycle markers cyclins D and E was examined by indirect immunofluorescence and immunoblotting. RESULTS: The number of BrdU-labeled cells in conjunctival, limbal, and peripheral epithelium peaked at 28 hours after wounding (3.9-, 4.5-, and 3.2-fold increases, respectively). In unwounded eyes, cyclin D showed diffuse cytoplasmic localization with occasional basal cells exhibiting a nuclear localization, while anti-cyclin E showed intense localization in limbal and conjunctival basal cells but only minimal labeling in corneal epithelium. Within 8 to 12 hours after wounding, the nuclei of most corneal basal cells outside the wound area were bound intensely by anti-cyclins D and E. Immunoblotting revealed that cyclin D and E protein levels increased 4.5- and 12.1-fold after wounding, respectively. Epithelium migrating into the wound area did not incorporate BrdU and did not exhibit nuclear localization of cyclins D and E. CONCLUSIONS: Corneal epithelial debridement stimulates basal cells outside the wound area to synchronously enter the cell cycle. However, cells migrating to cover the wound area do not progress through the cell cycle. These data suggest a compartmentalization of the proliferative and migratory phases of wound repair.     

The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding

PURPOSE: Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding. METHODS: To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding. RESULTS: The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells. CONCLUSIONS: The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.     

角膜缘部干细胞对角膜上皮创伤愈合的影响

利用角膜缘上皮移植，结膜移植和非手术的方法分别对角膜上皮创伤伴随膜缘损伤的兔眼进行实验研究，结果表明角膜缘上皮移植组上皮愈合时间短（平均８．５天），且无杯状细胞，无或极少量新生血管；结膜移植组，上皮愈合时间延长（平均１１．５天），含有杯状细胞和新生血管，非手术组形成角膜血管翳性混浊。而不件随角膜缘损伤仅有角膜中央皮皮创伤的兔眼愈合时间最短（平均３．５天），愈合后无新生血管及怀状细胞。结果证实角膜部     

The effects of hypertonic salicylate on neutrophil migration and vascularization in the rat cornea

Thermal cauterization of the center of the rat cornea results in emigration of neutrophils into the extravascular limbal tissue and blood vessel growth into the cornea. In this study, 1.0 M sodium salicylate, 1.0 M sodium chloride, and ointment vehicle were administered to normal and cauterized rat corneas for periods of 6, 48, and 144 h. When applied to the normal cornea, salicylate resulted in a marked increase in neutrophils in the limbal tissue at 6 h, but an inhibition at 48 h. Similarly, for the cauterized corneas, administration of salicylate increased the extent of neutrophil emigration at 6 h, but this effect was not sustained at 48 h. Neither vehicle nor sodium chloride had any effect on the extravascular neutrophil population. After 6 days, administration of the vehicle resulted in a slight increase in vascular growth into the cornea, whereas sodium salicylate caused a decrease. These findings indicate that hypertonic (1 M) sodium salicylate does not inhibit the emigration of neutrophils from limbal vessels of cauterized rat corneas, but does appear to have a cytotoxic effect on the tissues and on blood vessel endothelial cells.     

Limbus transplantation for reconstruction of the ocular surface

Proliferation of the corneal epithelium originates in undifferentiated, long-lived stem cells that are located in the basal limbal epithelium. Stem cells are important for corneal epithelial regeneration and wound healing. Depletion of stem cells due to accidents as well as malfunctions of stem cells due to inborn or inflammatory diseases result in limbal stem cell deficiency. Limbal deficiency is characterized by conjunctivalization of the cornea with vascularization and opacification. Partial limbal deficiency can be treated by removing ingrown conjunctival epithelium thus allowing normal limbal epithelium to repopulate the cornea. Unilateral limbus-derived stem cell disease requires either limbal autograft transplantation from the healthy partner eye or kerato-limbal allograft transplantation. Several modifications of the latter technique have been performed including large kerato-limbal lamellar grafts and central penetrating kerato-limbal allografts. All homologous procedures render a very high risk of immunological reactions that require long term systemic immunosuppression. The use of amniotic membrane, better pharmacological drugs for immunosuppression and improvements in the HLA-matching of limbal allografts as well as ex vivo expansion of corneal stem cells should allow for better reconstruction of the ocular surface in limbal deficiency.     

Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

Effect of vitronectin on the healing of rabbit corneal epithelial damage]

Vitronectin is one of the major cell-adhesive glycoproteins in mammalian plasma. We investigated the effect of topically applied vitronectin on the healing of rabbit corneal epithelial damage. Vitronectin was purified from rabbit serum by heparin-Sepharose affinity chromatography and autoclaved at 121 degrees C for 20 min after adjusting the concentration to 0.2 mg/ml with saline. Rabbit corneal epithelium was abraded with ethanol and a laser blade in the central portion of the cornea which had been demarcated with a 6.5 mm trephine. Vitronectin eye drops were instilled into the right eye, and sterile saline drops into the left eye as control. The dimensions of the circular epithelial defect were measured at various times after wounding. The area of epithelial damage of vitronectin-treated eyes was smaller than that of control eyes at 2hr-12hr after abrasion (p less than 0.01). These results suggested that topically applied vitronectin might accelerate the corneal epithelial wound healing at an early stage. Vitronectin can be considered as a candidate for treatment of corneal epithelial damage.     

Regeneration of rabbit cornea following excimer laser photorefractive keratectomy: a study on gap junctions, epithelial junctions and epidermal growth factor receptor expression in correlation with cell proliferation

Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.     

Localizations of epidermal growth factor receptor and proliferating cell nuclear antigen during corneal wound healing

The localization of epidermal growth factor receptor and proliferating cell nuclear antigen was demonstrated immunohistochemical to be similar in the corneal, limbal, and bulbar conjunctival epithelium, i.e., located adjacent to the artificially made corneal epithelial defect. In the course of regeneration of the corneal epithelium soon after the wounding, both epidermal growth factor receptor and proliferating cell nuclear antigen were expressed in the epithelial cells in the limbal area adjacent to the epithelial defect. After the defect was covered with several layers of regenerated epithelium, the main site of the expression of epidermal growth factor receptor and proliferating cell nuclear antigen moved to the basal layer of regenerated epithelium. The present study indicated that epidermal growth factor receptor, as expressed on the epithelial cell surface, can be considered to play an important role in epithelial cell proliferation, which is an indispensable process in corneal wound healing.     

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1^−/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1^−/− corneas was reduced to  ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1^−/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1^−/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.