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Proteoglycan synthesis by human corneal explants submitted to laser in situ keratomileusis (LASIK).

Martins SA, Campos MQ, Vidal BC, Berto AG, Aguiar JA, Michelacci YM - Mol. Vis. (2007)

Bottom Line: After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases.Histopathological and birefringence analysis were performed in fixed tissue slices.Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Oftalmologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil. suyreboucas@uol.com.br <suyreboucas@uol.com.br>

ABSTRACT

Purpose: To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of proteoglycans (PGs) and collagen fibril organization in human corneal explants.

Methods: Human corneas that had been rejected for transplants were obtained at Banco de Olhos of Hospital São Paulo. For each eye pair, one cornea was submitted to refractive surgery, and the other was used as its matched control. After surgery, the corneas were excised from the eyes and immediately placed in a Ham F-12 nutrient mixture containing (35)S-sulfate for the metabolic labeling of PGs. After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases. Histopathological and birefringence analysis were performed in fixed tissue slices.

Results: A marked decrease in (35)S-sulfate incorporation in PGs was observed in corneal explants that received LASIK, especially concerning dermatan sulfate-PGs, with keratan sulfate- and heparan sulfate-PG synthesis reduced to a lower degree. Only low molecular weight PGs were present in the corneas, both before and 24 h after LASIK. No sign of wound healing processes were observed, but a marked change in corneal birefringence was seen following LASIK treatment.

Conclusions: Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

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Related in: MedlinePlus

Polyacrylamide gel electrophoresis of proteoglycans extracted from human corneal explants. Aliquots (5 μl) of proteoglycans extracted from pair number 1 (see Table 1) of human corneal explants were submitted to polyacrylamide gel electrophoresis, as described in Methods. The gel was stained with coomassie blue (A), and the 35S-labeled compounds were localized by radioautography (B). St: molecular weight standard proteins; C: control cornea; L: LASIK submitted cornea.
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f5: Polyacrylamide gel electrophoresis of proteoglycans extracted from human corneal explants. Aliquots (5 μl) of proteoglycans extracted from pair number 1 (see Table 1) of human corneal explants were submitted to polyacrylamide gel electrophoresis, as described in Methods. The gel was stained with coomassie blue (A), and the 35S-labeled compounds were localized by radioautography (B). St: molecular weight standard proteins; C: control cornea; L: LASIK submitted cornea.

Mentions: Figure 5 shows a representative polyacrylamide gel electrophoresis of the proteoglycans [34] extracted from human corneal explants after LASIK. This analysis was performed for all samples. It showed that only low molecular weight proteoglycans (probably of the decorin/biglycan and lumican/mimecan families) are present in human cornea, both before and after LASIK.


Proteoglycan synthesis by human corneal explants submitted to laser in situ keratomileusis (LASIK).

Martins SA, Campos MQ, Vidal BC, Berto AG, Aguiar JA, Michelacci YM - Mol. Vis. (2007)

Polyacrylamide gel electrophoresis of proteoglycans extracted from human corneal explants. Aliquots (5 μl) of proteoglycans extracted from pair number 1 (see Table 1) of human corneal explants were submitted to polyacrylamide gel electrophoresis, as described in Methods. The gel was stained with coomassie blue (A), and the 35S-labeled compounds were localized by radioautography (B). St: molecular weight standard proteins; C: control cornea; L: LASIK submitted cornea.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533033&req=5

f5: Polyacrylamide gel electrophoresis of proteoglycans extracted from human corneal explants. Aliquots (5 μl) of proteoglycans extracted from pair number 1 (see Table 1) of human corneal explants were submitted to polyacrylamide gel electrophoresis, as described in Methods. The gel was stained with coomassie blue (A), and the 35S-labeled compounds were localized by radioautography (B). St: molecular weight standard proteins; C: control cornea; L: LASIK submitted cornea.
Mentions: Figure 5 shows a representative polyacrylamide gel electrophoresis of the proteoglycans [34] extracted from human corneal explants after LASIK. This analysis was performed for all samples. It showed that only low molecular weight proteoglycans (probably of the decorin/biglycan and lumican/mimecan families) are present in human cornea, both before and after LASIK.

Bottom Line: After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases.Histopathological and birefringence analysis were performed in fixed tissue slices.Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Oftalmologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil. suyreboucas@uol.com.br <suyreboucas@uol.com.br>

ABSTRACT

Purpose: To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of proteoglycans (PGs) and collagen fibril organization in human corneal explants.

Methods: Human corneas that had been rejected for transplants were obtained at Banco de Olhos of Hospital São Paulo. For each eye pair, one cornea was submitted to refractive surgery, and the other was used as its matched control. After surgery, the corneas were excised from the eyes and immediately placed in a Ham F-12 nutrient mixture containing (35)S-sulfate for the metabolic labeling of PGs. After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases. Histopathological and birefringence analysis were performed in fixed tissue slices.

Results: A marked decrease in (35)S-sulfate incorporation in PGs was observed in corneal explants that received LASIK, especially concerning dermatan sulfate-PGs, with keratan sulfate- and heparan sulfate-PG synthesis reduced to a lower degree. Only low molecular weight PGs were present in the corneas, both before and 24 h after LASIK. No sign of wound healing processes were observed, but a marked change in corneal birefringence was seen following LASIK treatment.

Conclusions: Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

Show MeSH
Related in: MedlinePlus