Neutrophil chemotactic factors derived from conjunctival epithelial cells: preliminary biochemical characterization

We have previously reported on the release of neutrophil chemotactic factors (NCF) from injured conjunctival tissue. The present study was designed to biochemically characterize these conjunctiva-derived chemotactic factors and determine their biological activities. Bulbar conjunctiva was surgically removed from a rabbit eye and incubated with 250 microL of minimal essential medium (MEM) for 6 hours at 37 degrees C in a 5% CO2 atmosphere. Chemotactic activity was assayed using modified Boyden chambers with rabbit peritoneal neutrophils as indicator cells. Following treatment with subtilisin protease for 90 minutes, chemotactic activity of the conjunctival factors was reduced by 74%. Similarly, activity was lost after heating at 56 degrees C for 60 minutes (41% inhibition). Using ultrafiltration techniques, we showed that the majority of the chemotactic activity remained above a 100 kilodalton filter, suggesting the existence of high molecular weight factors. We also showed that the conjunctival factors are not glycoproteins and bind to both anion and cation exchange resins. When 100 microL of conjunctival supernatant was injected in the superior tarsal conjunctiva of rabbits, significant recruitment of neutrophils was evident by 4 hours. Control rabbits injected with MEM did not show neutrophil recruitment. Results of these studies indicate that NCF from traumatized conjunctival tissue are proteins (and not glycoproteins) of high molecular weight, heat labile, exhibit anionic and cationic charges, and are active in vivo.     

M-K medium and postmortem cytologic damage.

Freshly enucleated rabbit eyes were refrigerated at +4 degrees C under standard eye bank conditions for 2, 6, 9, and 21 days. One group of corneas with a scleral rim were excised and placed in M-K medium, stored for 18, 24, or 48 hr at +4 degrees C; they were then removed, and endothelial cell viability was evaluated with nitroblue tetrazolium. The cells were examined by light microscopy and scanning and transmission electron microscopy. A second group of corneas were similarly obtained and then used as donor corneas from 6 mm transplants. Each recipient rabbit was evaluated daily by slit-lamp biomicroscopy and corneal pachometry. Fourteen days postoperatively, the rabbits were sacrificed, the eyes enucleated, and the excised corneas were evaluated in a fashion similar to those of group 1. M-K medium storage protected the morphology and functional integrity of the rabbit corneal endothelium up to 48 hr beyond moist chamber storage for 2, 6, and 9 days. However, M-K medium appeared to have no such effect on corneas that had been moist chamber--stored for 21 days. These results suggest that some human corneas with a prolonged time from death to moist chamber storage may be utilized for corneal transplantation after further storage in M-K medium.     

Inflammatory mediators in alkali-burned corneas: preliminary characterization

Leukocyte chemotactic factors (LCF) are important inflammatory mediators which activate and recruit leukocytes from the circulation into sites of tissue damage. These factors were recently detected in the tear fluid of inflamed eyes induced by alkali burn. It remains unclear, however, whether the detected LCF are released from injured corneal tissues or leaked from the circulation. Using a corneal cup model developed in our laboratory, we began examining the capability of corneal tissues to produce LCF in response to alkali injury. We also evaluated the influence of citric acid on the production of LCF from corneas preinjured by the alkali sodium hydroxide (NaOH). For these studies, the epithelial surfaces of corneas isolated from bovine and human eyes were exposed to 1N NaOH for 35 seconds at room temperature. The NaOH was then removed and the epithelial surfaces washed once with buffer and incubated with culture medium for 1, 2, 4, and 6 hours at 37 degrees C/5% CO2 atmosphere. Our results showed that (1) NaOH2 induced corneal epithelial cell injury ranging from cell discoloration and moderate damage of the upper half of the epithelium (1-4 hrs) to total destruction of the epithelium (6 hrs); (2) NaOH-injured corneas (2 hr incubation post injury) produced significant levels of chemotactic activity (via checkerboard analysis) specific for neutrophils (115% maximum chemotactic response [MCR]) and mononuclear cells (94% MCR); (3) preliminary characterization of these factors revealed that they are protease and heat sensitive, extractable by organic solvents, and possess molecular weight values greater than 100,000 daltons; and (4) incubation of NaOH-pretreated corneas with 0.01% citric acid for 2 hours markedly inhibited the production of LCF for both neutrophils (98% inhibition) and mononuclear cells (91% inhibition). Results of these studies indicate that alkali-burned bovine and human corneas generate leukocyte chemoattractants which differ in their biochemical characteristics from previously known low molecular weight chemotactic factors such as C5a, interleukin-1, or leukotriene B4.     

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.     

Corneal endothelial regeneration and tissue engineering

Human corneal endothelial cells (HCECs) have a limited proliferative capacity. Descemet stripping with automated endothelial keratoplasty (DSAEK) has become the preferred method for the treatment of corneal endothelial deficiency, but it requires a donor cornea. To overcome the shortage of donor corneas, transplantation of cultured HCEC sheets has been attempted in experimental studies. This review summarizes current knowledge about the mechanisms of corneal endothelial wound healing and about tissue engineering for the corneal endothelium. We also discuss recent work on tissue engineering for DSAEK grafts using cultured HCECs and HCEC precursor cell isolation method (the sphere-forming assay). DSAEK grafts (HCEC sheets) were constructed by seeding cultured HCECs on human amniotic membrane, thin human corneal stroma, and collagen sheets. The pump function of the HCEC sheets thus obtained was approximately 75%–95% of that for human donor corneas. HCEC sheets were transplanted onto rabbit corneas after DSAEK. While the untransplanted control group displayed severe stromal edema, the transplanted group had clear corneas throughout the observation period. The sphere-forming assay using donor human corneal endothelium or cultured HCECs can achieved mass production of human corneal endothelial precursors. These findings indicate that cultured HCECs transplanted after DSAEK can perform effective corneal dehydration in vivo and suggest the feasibility of employing the transplantation of cultured HCECs to treat endothelial dysfunction. Additionally, corneal endothelial precursors may be an effective strategy for corneal endothelial regeneration.     

Effect of indocyanine green intraocular stain on human and rabbit corneal endothelial structure and viability. An in vitro study

PURPOSE: To evaluate the direct effect of intraocular indocyanine green (ICG) on endothelial cell function, ultrastructure, and viability in human and rabbit corneas. SETTING: A laboratory evaluation study. METHODS: Paired human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion. One corneal endothelium was perfused with 25 mg ICG dissolved in 0.5 mL aqueous solvent in 4.5 mL balanced salt solution (BSS(R)) for 3 minutes followed by washout with a control solution. The percentage of ICG exposed to the corneal endothelium was 0.5%. The paired cornea was perfused with the same solution without ICG, followed by the washout. The corneas were fixed for scanning and transmission electron microscopy (TEM). In another group, the endothelial viability was determined using a live cell/dead cell assay. RESULTS: In rabbit corneas, the mean corneal swelling rate was 12.9 microm/h +/- 1.2 (SEM) in the ICG corneas and 2.8 +/- 1.9 microm/h in the controls. Scanning electron microscopy and TEM revealed a normal endothelial cell mosaic. The control electron micrographs were similar. In human corneas, the mean swelling rate was 19.1 +/- 2.8 microm/h in the ICG corneas and 19.2 +/- 2.6 microm/h in the controls. Scanning electron microscopy and TEM revealed intact junctions with slight cellular vacuolization, similar to that in the controls. In the live cell/dead cell subgroup, the mean damage was 17.3% +/- 1.7% in the ICG-exposed corneas and 22.0% +/- 8.9% in the controls. CONCLUSIONS: Three-minute exposure to ICG in BSS had no adverse effect on corneal endothelial function, ultrastructure, or viability in human and rabbit corneas. This study provides a safety profile for the corneal endothelium when ICG is used as an intraocular tissue stain in ophthalmic surgery.     

In vivo effects of thimerosal on the rabbit corneal endothelium: an ultrastructural study

To assess ultrastructural alterations to the corneal endothelium, eye drops containing vehicle alone or vehicle plus 0.004% thimerosal with or without 0.1% disodium ethylenediaminetetraacetate (Na2EDTA) were administered hourly over a 2-day period to rabbit corneas in vivo. Twenty of the corneas received a 5-mm keratectomy that removed epithelium and superficial stroma and 10 corneas were left intact. The vehicle alone had no effect on the endothelium of normal corneas. Thimerosal drops with or without Na2EDTA caused some clarification of the cytoplasm and dilatation of the endoplasmic reticulum of endothelial cells. The mitochondria had dilated cristae and membrane condensations. After keratectomy and vehicle only drops, the central endothelium showed a marked increase in microvilli and mitochondrial changes. Addition of either thimerosal or thimerosal plus Na2EDTA resulted in a slight decrease in membrane activity but an increase in intracellular edema and dilatation and damage to organelles. The peripheral areas were less affected than the center. Although the endothelium was stressed, most changes were considered reversible.     

Cryopreservation studies with porcine corneas.

PURPOSE. A new technique for the cryopreservation of rabbit corneas in 20% w/w dimethylsulfoxide, which has been shown to preserve significant structural and functional integrity of the endothelium, was tested in porcine corneas. METHODS. The characteristics of uptake of dimethylsulfoxide into porcine corneas were measured using proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy. The effect on structural integrity of exposure to 20% w/w dimethylsulfoxide without freezing was first assessed using vital staining (acridine orange and propidium iodide), and optimum temperature conditions for addition and removal of the cryoprotectant were derived. The effects on structural integrity of cryopreservation in 15% and 20% w/w dimethylsulfoxide, and of reducing the degree of cell swelling during cryoprotectant removal following cryopreservation, were then evaluated. RESULTS. The characteristics of uptake of dimethylsulfoxide from a 10% w/w solution fitted a single exponential, resulting in a maximum tissue concentration of 14.6% when the addition occurred on ice, and 18.5% when the addition took place at room temperature. The toxic effects of dimethylsulfoxide in porcine corneas were highly temperature dependent and only evident after removal of the cryoprotectant. Unlike rabbit corneas, cryopreservation of porcine corneas in 15% and 20% w/w dimethylsulfoxide induced substantial endothelial injury which was not improved by reducing the degree of cell swelling that occurred during removal of the cryoprotectant. CONCLUSIONS. Porcine corneas were substantially more susceptible to the toxic effects of dimethyl sulfoxide, and to cryopreservation injury, than rabbit corneas. These results underline the importance of species variation in animal studies aimed at the cryopreservation of human tissue for transplantation.     

Effect of irradiation on vascularization of corneas grafted onto chorioallantoic membranes

Several studies have shown that total body irradiation decreases the angiogenic response to corneal cauterization. This inhibition could be due to alterations in angiogenic stimuli within injured corneas and/or to a decreased ability of irradiated animals to respond to such stimuli. To determine whether total body irradiation specifically affects angiogenic stimuli within injured corneal tissue, cauterized corneas from mice exposed to 900 rads of total body irradiation and from non-irradiated controls were grafted onto the chorioallantoic membranes (CAM) of chick embryos and their abilities to stimulate the ingrowth of healthy embryonic blood vessels were compared. Cauterized corneas incorporated into CAM mesenchymal tissue were invaded by blood vessels in 34.6% of the irradiated group and in 75% of the non-irradiated controls. This difference in the two groups was statistically significant (P less than 0.03). Total body irradiation significantly decreased the frequency of vascular invasion of cauterized corneal tissues by healthy CAM blood vessels. This finding suggests that total body irradiation can specifically affect the stimulus for angiogenesis within cauterized corneas.     

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.     

Evidence for a role of the plasminogen activator--plasmin system in corneal ulceration

Plasminogen is present in the cornea andcan be activated to plasmin by plasminogen activator. Plasmin is able, in turn, to activate latent collagenase. This system could initiate and perpetuate the collagen degradation of corneal ulceration. This report details evidence for such a system in the cornea. Plasmin has been found to activate latent collagenase from organ cultures of ulcerating rabbit corneas and from fibroblast cultures derived from such corneas. As in the case of activation by trypsin, activation by plasmin results in the conversion of the 40,000 MW latent form to an active species of 23,000 MW. Explants of normal or alkali-burned, ulcerating corneas demonstrated plasminogen-dependent lysis of fibrin clots; frozen sections of such corneas demonstrated that lysis begins in the superficial stroma near the periphery of the cornea. Multiply freeze-thawed ulcerating corneas, but not normal corneas, showed initial lysis, not peripherally but at the ulcer region containing polymorphonuclear leukocytes. The fact that the peripheral lytic pattern existed in corneas that were obtained from eyes prefrozen in liquid nitrogen before excision of the corneas would suggest that plasminogen activator is normally contained in cells in vivo and is not made only in response to tissue injury. There was no correlation between the location of blood vessels or the presence of the corneal endothelium and the plasminogen-dependent lysis. Plasminogen activator from the ulcerating cornea and from fibroblasts was characterized by sodium dodecyl sulfate--gel electrophoresis of its cleavage products of plasminogen. The activator cleaves plasminogen into heavy- and light-chain fragments similar to those produced from plasminogen by urokinase. Plasminogen activator activity was quantitated by a new assay that restricts diffusion of the enzyme to one dimension into a narrow bore tube. The addition of plasminogen daily to cultures of ulcerating corneas resulted in earlier rises of plasminogen activator, collagenase, and collagen degradation fragments in the culture media. Although total plasminogen activator levels were not increased by the addition of plasminogen to culture, levels of both collagenase and solubilized collagen were approximately doubled. It is concluded that the plasminogen activator--plasmin system might play an important role in the destruction of stromal matrix in corneal ulceration.     

Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium

Previous light microscopic autoradiographic results from our laboratory have shown a quantitative decrease in ATPase pump site density in dysfunctional corneal endothelium. In order to develop antibody techniques to correlate these findings with electron microscopic localization of pump sites, three types of corneas with dysfunctional endothelium (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas with functional endothelium (age matched eye bank and keratoconus) were obtained at the time of transplant surgery. Corneas were fixed, frozen and cryostat sections were incubated in a rabbit kidney ATPase primary antibody followed by incubation in an HRP-labeled secondary antibody. Functional endothelia showed dense antibody labeling along the lateral cell membranes and there was a substantial reduction in labeling along the lateral membranes of dysfunctional endothelia. Positive tissue controls of rabbit kidney showed HRP reaction product in the convoluted tubules. Control tissue, incubated in either non-immune serum or primary antibody preabsorbed with ATPase, confirmed the specificity by having diminished or no reaction product. These results provide immunohistochemical confirmation of our autoradiographic data showing a quantitative reduction in pump site density on dysfunctional corneal endothelium.     

Low-dose ultraviolet-B irradiation of donor corneal endothelium and graft survival

Donor rabbit corneal endothelium was pretreated with different doses of ultraviolet (UV-B) irradiation (302 nm) before grafting to test whether allograft survival could be favorably affected in comparison with untreated corneas grafted into the same recipients. Endothelial rejection was observed in 19 of 32 (59%) eyes that received no treatment compared with five of 32 (16%) eyes that received UV-B (P less than 0.001), and increasing doses of UV-B were associated with lower rejection rates (P less than 0.05). Although exposure of donor endothelium significantly reduced endothelial rejection at all doses tested, it resulted in primary graft failure in a substantial proportion of corneas treated at high doses. Class II (Ia) antigen staining of corneal tissue was present in conjunction with clinical evidence of rejection, and the magnitude of staining correlated with the histologic extent of inflammation. Scanning electron microscopy revealed various endothelial cell surface irregularities and membrane defects in high-dose UV-treated corneas. Endothelial cell cultures exposed in vitro to UV-B light showed a dose-dependent loss in cell viability. These data suggest that UV-B pretreatment of donor corneal endothelium prolongs graft survival but that toxic side effects must be carefully controlled.     

Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP

PURPOSE: Human corneal endothelium, a neural crest-derived tissue, has a very limited regenerative capacity and may depend on trophic factors for its survival throughout life, as well as after injury and during storage before transplantation. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a 28-amino acid neurotrophic factor present in human aqueous humor, promotes the survival of corneal endothelium in corneal organ cultures, and whether VIP is produced by the corneal endothelium. METHODS: Thirteen viable human donor corneas that had been received from the Central Florida Lions Eye Bank and stored in preservation medium (Optisol-GS; Chiron Vision, Irvine, CA) at 4 degrees C for 8 to 17 days were bisected. Each half was treated with either 0 or 10 nM VIP (15 minutes) and subjected to H(2)O(2) (1.4 mM, 30 minutes) treatment at 37 degrees C. The numbers of live and dead corneal endothelial (CE) cells isolated from the corneas were then determined under fluorescence microscopy using a live-dead viability-cytotoxicity assay conducted by an observer uninformed of the treatment. The effect of VIP (10(-16) to 10(-6) M) on CE cell survival was also studied in fresh bovine corneas in situ, by using the same assay. The presence of VIP in the corneal endothelium in fresh human donor and bovine eyes was examined by immunocytochemistry, in situ hybridization, and Western blot analysis, whereas VIP in the bovine aqueous humor was assessed by radioimmunoassay. RESULTS: VIP (10 nM) significantly increased CE survival in 10 of 13 human corneas. The mean survival of CE cells (+/-SEM) was 42% +/- 3% in control corneas versus 59% +/- 3% in VIP-treated corneas (P < 0.001). In bovine corneas, VIP at concentrations as low as 10(-10) M demonstrated a significant protective effect. The mean number of dead CE cells on bovine corneas was maximally decreased by 10(-6) M VIP from 46 +/- 5 to 18 +/- 3 per field. In CE cells from fresh human and bovine corneas, VIP immunoreactivity and mRNA were detected. VIP was also present in bovine aqueous humor at 40 +/- 8 pM. CONCLUSION: VIP may be an autocrine trophic factor that protects CE cells from H(2)O(2) in normal aqueous humor and possibly from other oxidative insults.     

Modulation of hydrogen peroxide induced injury to corneal endothelium by virus mediated catalase gene transfer

AIM: To examine the effect of catalase gene transfer on survival of corneal endothelial cells (EC) following challenge with hydrogen peroxide (H(2)O(2)) in an ex vivo model of oxidative stress. METHODS: A recombinant adenovirus vector (AdCL) was used to transfer human catalase cDNA into EC of whole thickness rabbit corneas ex vivo. The resulting catalase protein concentration was measured in corneal lysates by ELISA; catalase functional activity in lysates was determined using a H(2)O(2) activity assay. To examine the morphological effects of catalase gene transfer in modulation of H(2)O(2) induced injury, transduced corneas were maintained in ex vivo culture and challenged with H(2)O(2). Laser scanning confocal microscopy was used to image EC injury. Cell density, cell morphology, and ratios of viable to necrotic cells were determined. RESULTS: Following incubation with AdCL, catalase expression reached maximum at 5-7 days. Corneas transduced with AdCL showed increased EC cell survival following challenge with H(2)O(2) on day 3 when compared to null vector control or mock infected corneas. CONCLUSIONS: Ex vivo catalase gene transfer can protect EC from death mediated by H(2)O(2). This gene based approach to the protection of corneal endothelium from oxidative stress may have application in prevention of EC loss in pathological conditions in which H(2)O(2) is involved and in ex vivo donor corneal storage before transplantation.     

人脐带血清器官培养液保存猪角膜的实验研究

目的 观察胎牛血清器官培养液和人脐带血清器官培养液对猪角膜内皮细胞形态学、组织学、超微结构、酶活性及代谢等的影响。方法 器官培养方法：4周以内31℃密闭培养,之后脱水24h。选择猪眼113只,其中100只角膜分为两组进行配对器官培养保存。第1组(50只角膜)：应用培养液Ⅰ(含10％胎牛血清);第2组(50只角膜,第1组的对侧角膜)：应用培养液Ⅱ(含10％人脐带血清)。13只角膜作为正常对照。器官培养每周每组各取出12只角膜进行内皮细胞形态学分析、HE染色、酶组织化学染色。保存2周、4周时行扫描电镜检查。检测保存前后培养液的pH值和葡萄糖、乳酸浓度。器官培养过程中行微生物学检测。结果 器官培养期间角膜内皮细胞单层保持完整,多形性增加,两组之间角膜内皮细胞密度、细胞面积的变异系数和六边形细胞比例在保存4周内差异无显著意义。保存4周的猪角膜与正常猪角膜相比,平均内皮细胞丢失率第1组为10．98％,第2组为10．85％。两组角膜器官培养后的组织学、超微结构、酶活性无明显区别。扫描电镜显示内皮细胞层完整,细胞形态改变。酶组织化学染色显示上皮、内皮细胞酶活性旺盛,基质细胞的酶活性随保存时间的延长而降低。角膜代谢状态良好。器官培养液污染率为6％。结论 两种器官培养液均可以保持角膜内皮活性达4周,推测人脐带血清能够替代胎牛血清作为角膜器官培养液的成分。(中华眼科杂志,2004,40：533-538)     

Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA

PURPOSE: This study was undertaken to investigate the wound healing process of the first 6 weeks after photodynamic cross-linking treatment in the rabbit cornea, using the photosensitizer riboflavin and UVA. METHODS: After removal of the central epithelium, the right corneas of 8 Chinchilla rabbits were cross-linked with a photosensitizing 0.1% riboflavin solution and UVA light (370 nm; irradiance, 3 mW/cm(2); dose, 5.4 J/cm(2)) for 30 minutes. Two animals were euthanized 3 days, 7 days, 4 weeks, and 6 weeks postoperatively. The corneas of the enucleated eyes were evaluated using 4-microm light microscopic sections with routine stains and avidin-biotin complex immunostaining with anti-alpha-smooth muscle actin. RESULTS: By day 3 after treatment, complete apoptotic damage and loss of the endothelial cells and the stromal keratocytes were found in the irradiated area through the entire thickness of the stroma. There was marked stromal edema (850 +/- 66 vs. 332 +/- 43 microm in the untreated controls; P < 0.01). The epithelium was already closed again. At the margins of the lesion, there was a mild inflammatory reaction with scattered macrophages, lymphocytes, and neutrophils. By day 7, the endothelium was already intact again, and keratocyte repopulation of the posterior stroma was noted. By week 4, the keratocyte repopulation of the anterior stroma was observed with some acellular areas between. By week 6, the cytoarchitecture of the cornea seemed normal again. By weeks 4 and 6, alpha-actin-positive keratocytes were identified, especially in the periphery of the irradiated area. CONCLUSIONS: After riboflavin/UVA cross-linking of rabbit cornea, a complete cell loss occurs in the irradiation area with an irradiance of 3 mW/cm(2). The cytotoxic damage is repaired by repopulation after approximately 4-6 weeks. A combination of cross-linking with other procedures such as the implantation of intracorneal rings should be performed only after a sufficient time interval of approximately 2 months, allowing cellular regeneration.     

Role of leukocytes in ocular inflammation of tyrosinemia II

In the animal model of tyrosinemia II only corneas from tyrosine(tyr)-fed rats produce chemoattractants in organ culture. To study the role of neutrophils (PMNs) in production of these chemoattractants, leukocytes (WBCs) were depleted using i.p. cyclophosphamide (CP). Saline (SAL)-treated rats maintained 18,375 +/- 894 WBC/mm3 (mean +/- SEM) with 4168 +/- 424 PMNs. Rats receiving CP (150 mg/kg day 0, 75 mg/kg day 4) has 1565 +/- 170 WBC (565 +/- 129 PMN) on day 3, and 398 +/- 68 WBC (19 +/- 5 PMN) on day 8. Rats ate a low-protein +/- 5% tyr diet on days 4-8. Only SAL-treated tyr-fed rats developed plaque-like gray epithelial lesions; histopathology showed corneal epithelial necrosis, stromal edema, and epithelial and stromal PMN infiltration. Control and CP-treated tyr-fed rat corneas showed no inflammation. On day 8 corneas were cultured in RPMI 1640 + 5% heat-inactivated fetal bovine serum. After 3 days, supernatants were assayed for chemotactic activity (leading front method); data were expressed as the percentage of peritoneal PMN migration relative to 5% zymosan-activated rat serum. The mean total migration toward 75% supernatant from SAL-treated, tyr-fed rat corneas was 79%, whereas migration toward corneal supernatants from controls and CP-treated tyr-fed rats ranged from 42-48%. Corneal extracts were assayed for proteolytic activity. WBC depletion prevented the increase in cathepsin B- and D-like activities present in tyr-fed corneas, suggesting that PMNs were a major source of these enzymes. The data suggest that WBC depletion reduces both corneal inflammation in vivo and the production of chemotactic activity by tyr-fed corneas in culture.     

Intercellular relationships in the synthesis of macromolecules by organ cultures of corneas

Sepharose CL-4B chromatography of guanidine hydrochloride and aqueous extracts of 3H-glucosamine labeled intact corneal tissue reveals four peaks representing proteoglycans and glycoproteins. To evaluate the universality of the 4th peak, hereafter designated as Sepharose CL-4B (IV), its presence was investigated in rabbit, bovine, cat, rhesus monkey, and human corneal preparations. Following incubation in isotopically labeled medium, corneas were extracted with aqueous and/or 4M guanidine hydrochloride and subjected to Sepharose CL-4B chromatography. Sepharose CL-4B (IV) was detected in all species studied; 3H-glucosamine and 14C-amino acids, but not 35SO4, were incorporated into this peak which eluted in the range consistent with an apparent molecular weight of approximately 30,000 D. To determine which layers were involved in the synthesis of Sepharose CL-4B (IV) the layers of the rabbit cornea were incubated separately (stroma scraped of endothelium and/or epithelium, epithelium only, endothelium only). A distinct Sepharose CL-4B (IV) peak was not identified in the chromatographs obtained from organ cultures of corneal epithelium, endothelium, or from corneal stroma scraped of epithelium and/or endothelium. This decrease in Sepharose CL-4B (IV) synthesis occurred even if the scraped cornea was not allowed to expand in volume by compressing it beneath a membrane porous to the incubation medium. Thus, Sepharose CL-4B (IV) synthesis was enhanced significantly by the stroma being in conjunction with other corneal cells as they exist in vivo.