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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

Agarose gel electrophoresis of proteoglycans extracted from human corneal explants. The left (L) cornea of each pair was submitted to LASIK, and the right (R) cornea was its matched control. The corneal explants were maintained under tissue culture conditions for 24 h in the presence of 35S-sulfate for the metabolic labeling of proteoglycans. The proteoglycans were extracted as described in Methods, and aliquots (5 μl) were submitted to agarose gel electrophoresis, as also described in Methods. The proteoglycans were fixed in the gel and stained by toluidine blue (A), and the 35S-labeled proteoglycans were localized by radioautography (B). In the images, S indicates a mixture of standard glycosaminoglycans containing chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each. KS indicates keratan sulfate. The numbers 1, 3, 9, and 12 refer to the corneal pairs described in Table 1.
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f1: Agarose gel electrophoresis of proteoglycans extracted from human corneal explants. The left (L) cornea of each pair was submitted to LASIK, and the right (R) cornea was its matched control. The corneal explants were maintained under tissue culture conditions for 24 h in the presence of 35S-sulfate for the metabolic labeling of proteoglycans. The proteoglycans were extracted as described in Methods, and aliquots (5 μl) were submitted to agarose gel electrophoresis, as also described in Methods. The proteoglycans were fixed in the gel and stained by toluidine blue (A), and the 35S-labeled proteoglycans were localized by radioautography (B). In the images, S indicates a mixture of standard glycosaminoglycans containing chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each. KS indicates keratan sulfate. The numbers 1, 3, 9, and 12 refer to the corneal pairs described in Table 1.

Mentions: Figure 1 shows a representative electrophoresis of proteoglycans extracted from control (right, R) and LASIK-treated (left, L) corneal explants, stained with toluidine blue (total proteoglycans) and detected by radioautography of the agarose gel slabs (metabolically labeled 35S-proteoglycans, synthesized during the last 24 h). The main labeled band, observed in all samples, migrated slightly less than the standard heparan sulfate. A minor band of faster migration was observed in some samples (2 and 4 in Figure 1). The total amounts of proteoglycans extracted for each cornea pair were roughly the same, based on toluidine blue staining, indicating that no variations occurred in the extraction yields. The 35S-labeling was found to be greatly decreased in LASIK-treated corneas (see radioautogram, "R" versus "L" for each pair).


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)

Agarose gel electrophoresis of proteoglycans extracted from human corneal explants. The left (L) cornea of each pair was submitted to LASIK, and the right (R) cornea was its matched control. The corneal explants were maintained under tissue culture conditions for 24 h in the presence of 35S-sulfate for the metabolic labeling of proteoglycans. The proteoglycans were extracted as described in Methods, and aliquots (5 μl) were submitted to agarose gel electrophoresis, as also described in Methods. The proteoglycans were fixed in the gel and stained by toluidine blue (A), and the 35S-labeled proteoglycans were localized by radioautography (B). In the images, S indicates a mixture of standard glycosaminoglycans containing chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each. KS indicates keratan sulfate. The numbers 1, 3, 9, and 12 refer to the corneal pairs described in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Agarose gel electrophoresis of proteoglycans extracted from human corneal explants. The left (L) cornea of each pair was submitted to LASIK, and the right (R) cornea was its matched control. The corneal explants were maintained under tissue culture conditions for 24 h in the presence of 35S-sulfate for the metabolic labeling of proteoglycans. The proteoglycans were extracted as described in Methods, and aliquots (5 μl) were submitted to agarose gel electrophoresis, as also described in Methods. The proteoglycans were fixed in the gel and stained by toluidine blue (A), and the 35S-labeled proteoglycans were localized by radioautography (B). In the images, S indicates a mixture of standard glycosaminoglycans containing chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each. KS indicates keratan sulfate. The numbers 1, 3, 9, and 12 refer to the corneal pairs described in Table 1.
Mentions: Figure 1 shows a representative electrophoresis of proteoglycans extracted from control (right, R) and LASIK-treated (left, L) corneal explants, stained with toluidine blue (total proteoglycans) and detected by radioautography of the agarose gel slabs (metabolically labeled 35S-proteoglycans, synthesized during the last 24 h). The main labeled band, observed in all samples, migrated slightly less than the standard heparan sulfate. A minor band of faster migration was observed in some samples (2 and 4 in Figure 1). The total amounts of proteoglycans extracted for each cornea pair were roughly the same, based on toluidine blue staining, indicating that no variations occurred in the extraction yields. The 35S-labeling was found to be greatly decreased in LASIK-treated corneas (see radioautogram, "R" versus "L" for each pair).

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