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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

Effect of LASIK upon the synthesis of 35S-dermatan sulfate, 35S-keratan sulfate, and 35S-heparan sulfate by human corneal explants. Shown are the 35S-sulfate incorporation (A; cpm, mean±standard error, three determinations for each sample) and the ratio between LASIK and control (B). The identification of each glycosaminoglycan was based on a combination of agarose gel electrophoresis and enzymatic degradation with specific glycosaminoglycan lyases (chondroitinases, keratanase and heparitinase).
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f4: Effect of LASIK upon the synthesis of 35S-dermatan sulfate, 35S-keratan sulfate, and 35S-heparan sulfate by human corneal explants. Shown are the 35S-sulfate incorporation (A; cpm, mean±standard error, three determinations for each sample) and the ratio between LASIK and control (B). The identification of each glycosaminoglycan was based on a combination of agarose gel electrophoresis and enzymatic degradation with specific glycosaminoglycan lyases (chondroitinases, keratanase and heparitinase).

Mentions: To confirm the glycosaminoglycan identification, these compounds were incubated with specific glycosaminoglycan lyases. Upon the action of F. heparinum chondroitinase B, dermatan sulfate was degraded to oligosaccharides and disaccharides that were not fixed in the gel [33]. The band migrating as heparan sulfate disappeared upon the action of heparitinase II, and upon the action of keratanase, keratan sulfate was digested. F. heparinum chondroitinase AC had no effect upon corneal glycosaminoglycan, confirming that chondroitin sulfate was not present and corneal dermatan sulfate is predominantly a L-iduronic acid containing polymer. Figure 4 shows the mean±standard error of all the samples (three determinations for each sample), obtained after release of the glycosaminoglycan chains by proteolysis of the 35S-labeled proteoglycans. The synthesis of all glycosaminoglycans decreased after LASIK, but the synthesis of dermatan sulfate decreased more, leading to a decrease in the relative proportion of this glycosaminoglycan. The degradation products formed after the action of the glycosaminoglycan lyases were the same for LASIK-treated and control corneas, indicating that this decrease in 35S-sulfate incorporation is not due to the synthesis of undersulfated proteoglycans.


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)

Effect of LASIK upon the synthesis of 35S-dermatan sulfate, 35S-keratan sulfate, and 35S-heparan sulfate by human corneal explants. Shown are the 35S-sulfate incorporation (A; cpm, mean±standard error, three determinations for each sample) and the ratio between LASIK and control (B). The identification of each glycosaminoglycan was based on a combination of agarose gel electrophoresis and enzymatic degradation with specific glycosaminoglycan lyases (chondroitinases, keratanase and heparitinase).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effect of LASIK upon the synthesis of 35S-dermatan sulfate, 35S-keratan sulfate, and 35S-heparan sulfate by human corneal explants. Shown are the 35S-sulfate incorporation (A; cpm, mean±standard error, three determinations for each sample) and the ratio between LASIK and control (B). The identification of each glycosaminoglycan was based on a combination of agarose gel electrophoresis and enzymatic degradation with specific glycosaminoglycan lyases (chondroitinases, keratanase and heparitinase).
Mentions: To confirm the glycosaminoglycan identification, these compounds were incubated with specific glycosaminoglycan lyases. Upon the action of F. heparinum chondroitinase B, dermatan sulfate was degraded to oligosaccharides and disaccharides that were not fixed in the gel [33]. The band migrating as heparan sulfate disappeared upon the action of heparitinase II, and upon the action of keratanase, keratan sulfate was digested. F. heparinum chondroitinase AC had no effect upon corneal glycosaminoglycan, confirming that chondroitin sulfate was not present and corneal dermatan sulfate is predominantly a L-iduronic acid containing polymer. Figure 4 shows the mean±standard error of all the samples (three determinations for each sample), obtained after release of the glycosaminoglycan chains by proteolysis of the 35S-labeled proteoglycans. The synthesis of all glycosaminoglycans decreased after LASIK, but the synthesis of dermatan sulfate decreased more, leading to a decrease in the relative proportion of this glycosaminoglycan. The degradation products formed after the action of the glycosaminoglycan lyases were the same for LASIK-treated and control corneas, indicating that this decrease in 35S-sulfate incorporation is not due to the synthesis of undersulfated proteoglycans.

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