Expression of Collagen I, Smooth Muscle a-Actin, and Vimentin ...

More documents

Recommendations

Info

Expression of Collagen I in Injured Corneas 3323 E 4.0 3.0 2.0 1.0 0 - • • • • i • ' • • i • • • • i • • • • i • • • • i • • • • : I / N ' 1 $ l/f J r 0 5 10 15 days after Injury FIGURE l. Slot-blot hybridization of «1 (I) mRNA from alkaliburned corneas by 32 P-labeled cDNA insert of ARC35. Total RNA was isolated from alkali-burned corneas healed for 1 to 21 days as indicated. Twenty jug of total RNA from each sample were twofold serially diluted and blotted to BASmembranes (10 Mg in the first well). The niters were then hybridized with 32 P-labelecl cDNA insert of ARC35, 22 and autoradiograms were prepared. The relative intensities of the samples were determined with a Helena Quick Scan densitometer (Helena Laboratories, Beaumont, TX). The figures are an average of four corneas; the bars indicate the standard deviations. situ hybridization with 3 H-labeled cDNA probe of cd(I) mRNA as previously described. 18 Figure 2a shows that 1 day after alkali burn, the keratocytes adjacent to the injury express a low level of al(I) mRNA- As the injured corneas heal, the keratocytes migrate into the injured stroma and express high levels of al (I) mRNA. The observation is consistent with the results shown in Figure 1 in which the levels of a 1(1) mRNA increase as alkali-burned corneas heal for up to 21 days. Results of the histologic examination and the bright field of in situ hybridization indicate that retrocorneal fibrillar membranes often form in the alkaliburned corneas healed for more than 1 week (Fig. 2f). The presence of the retrocorneal membrane in alkaliinjured corneas can also be seen in Figures 4 and 7. The formation of a retrocorneal membrane starts at the edge of injury and extends toward the center as the alkali-burned corneas healed progressively. The fibroblastic cells in the retrocorneal membranes in alkaliburned corneas that healed for 1 to 3 weeks also express high levels of al(l) mRNA (Figs. 2b, 2c, 2d, 3f). Figures 3b and 3c show that no specific hybridization signals can be readily identified in the regenerating epithelium of alkali-burned corneas that have healed for 1 day and 3 weeks. The results indicate that the regenerating epithelium either does not express or expresses a very low level of al(i) mRNA. 20 • In an attempt to examine whether the lacerated cornea may heal differently, a series of similar experiments of in situ hybridization was performed with lacerated corneas healed for 1, 2, and 3 weeks. Fibroblastic cells migrate into the wound of the lacerated cornea and express high levels of al(I) mRNA. Figure 2e shows a typical dark-field microgram from a lacerated cornea healed for 2 weeks. Similar results were obtained with lacerated corneas that had healed for 1 and 3 weeks (data not shown). It is of interest to note that lacerated corneas do not form the retrocorneal membrane (Fig. 2e). The endothelial cells in the uninjured portion of the cornea either do not express or express a very low level of al(l) mRNA (Fig. 3d), whereas the epithelial cells in the regenerated epithelial plug have a low level but specific expression of al(l) mRNA (Figs. 2e, 3a). Immunostaining With Antibody Against Collagen I To verify whether collagen I exists in the retrocorneal membrane, goat antibody against collagen I was used in immunohistochemical studies with alkali-burned and lacerated corneas. Figure 4a shows the reaction of anti-collagen I antibody with the denatured collagenous components in the alkali-injured stroma healed for I day. Figure 4b demonstrates the presence of collagen I in the granulomatous tissue between the regenerated epithelium and stroma of alkali-burned corneas healed for 3 weeks. Figure 4c shows that the antibody reacts with the retrocorneal membrane but does not react with the Descemet's membrane. Similar immunohistochemical studies were performed with lacerated corneas healed for 1, 2, and 3 weeks. The results indicate that the lacerated corneas did not form retrocorneal membrane, and no fibrous structure can be detected underneath the endothelium of the uninjured portion of the lacerated corneas (data not shown). Immunostaining of Alkali-Burned Rabbit Corneas With Antibodies Against Smooth Muscle a-Actin, Vimentin, and Desmin To characterize the fibroblastic cells in the alkaliburned corneas, antibodies against a-SMA, vimentin, and desmin were used in immunohistochemical studies with alkali-injured cornea healed for 3 weeks. Figure 5a shows that anti-a-SMA does not react with either keratocytes or epithelial cells in the normal corneas. In alkali-burned cornea healed for 2 and 3 weeks, the antibody reacts with the fibroblastic cells in stroma and retrocorneal membrane (Figs. 5b, 7a, 7c). The anti-a-SMA antibody does not react with the epithelial cells in alkali-burned and lacerated corneas (Figs. 5b, 7a, 7e). The endothelial cells in both normal
3324 Investigative Ophthalmology 8c Visual Science, November 1993, Vol. 34, No. 12 1 f RCM 0.1 mm FIGURE 2 In situ hybridization of injured corneas with 3 H-labeled cDNA of al(I) mRNA. The alkali-injured corneas were allowed to heal for 1 day and for 1, 2, and 3 weeks. The corneas were then subjected to in situ hybridization with 3 H-labeled ARC35 cDNA probes encoding the 3'-end of the al(I) mRNA. Dark-field micrograms: Panel a, alkali-burned corneas healed for 24 hours; panel b, alkali-burned corneas 1 week after injury; panel c, alkali-burned corneas 2 weeks after injury; panel d, alkali-burned corneas 3 weeks after injury; panel e, lacerated cornea healed for 2 weeks; panel f, bright-field microgram of panel b. Large arrows indicate the sites of injury. Small arrows and stars indicate cells hybridized by the 3 H-labeled probe. D, Descemet's membrane; RCM, retrocorneal membrane. (data not shown) and lacerated corneas (Fig. 7g) are not stained by the anti-a-SMA antibody. In another experiment, antibody against vimentin was used to stain the normal, alkali-injured, and lacerated corneas. The anti-vimentin antibody reacts with keratocytes in normal corneas and fibroblastic cells in alkali-injured stroma (Figs. 6, 7b), fibroblastic cells in retrocorneal membranes (Fig. 7d), endothelial cells of normal corneas (data not shown), and in lacerated corneas healed for 2 weeks (Fig. 7h). The epithelial cells in
Page 1 and 2: Expression of Collagen I, Smooth Mu
Page 3: 3322 Investigative Ophthalmology &
Page 7 and 8: 3326 Investigative Ophthalmology &
Page 9: 3328 Investigative Ophthalmology 8c

Expression of Collagen I, Smooth Muscle a-Actin, and Vimentin ...

Create successful ePaper yourself

Delete template?

Save as template?