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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 glycosaminoglycans released by proteolysis from proteoglycans extracted from human corneal explants after LASIK. The proteoglycans extracted from pair number 12 (see Table 1) of human corneal explants underwent proteolysis with papain for release of the glycosaminoglycan chains. Aliquots of the incubation mixtures were submitted to agarose gel electrophoresis. Total proteoglycans (PG) and glycosaminoglycans (GAG) were stained by toluidine blue (A), and the 35S-labeled glycosaminoglycans (35S-GAG) were localized by radioautography (B). S: A mixture of standard glycosaminoglycans containing chondroitin sulfate (CS) dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each; KS, keratan sulfate; PG, intact proteoglycans.
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f3: Agarose gel electrophoresis of glycosaminoglycans released by proteolysis from proteoglycans extracted from human corneal explants after LASIK. The proteoglycans extracted from pair number 12 (see Table 1) of human corneal explants underwent proteolysis with papain for release of the glycosaminoglycan chains. Aliquots of the incubation mixtures were submitted to agarose gel electrophoresis. Total proteoglycans (PG) and glycosaminoglycans (GAG) were stained by toluidine blue (A), and the 35S-labeled glycosaminoglycans (35S-GAG) were localized by radioautography (B). S: A mixture of standard glycosaminoglycans containing chondroitin sulfate (CS) dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each; KS, keratan sulfate; PG, intact proteoglycans.

Mentions: The electrophoretic migration of the glycosaminoglycan chains released from the core proteins by proteolysis is shown in Figure 3. Upon incubation of the proteoglycans with papain, the band(s) corresponding to proteoglycans completely disappeared, and one band, migrating as dermatan sulfate and keratan sulfate, appeared upon toluidine blue staining. Nevertheless, on radioautogram, a second band, migrating as heparan sulfate, could also be detected. Chondroitin sulfate was not found.


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 glycosaminoglycans released by proteolysis from proteoglycans extracted from human corneal explants after LASIK. The proteoglycans extracted from pair number 12 (see Table 1) of human corneal explants underwent proteolysis with papain for release of the glycosaminoglycan chains. Aliquots of the incubation mixtures were submitted to agarose gel electrophoresis. Total proteoglycans (PG) and glycosaminoglycans (GAG) were stained by toluidine blue (A), and the 35S-labeled glycosaminoglycans (35S-GAG) were localized by radioautography (B). S: A mixture of standard glycosaminoglycans containing chondroitin sulfate (CS) dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each; KS, keratan sulfate; PG, intact proteoglycans.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Agarose gel electrophoresis of glycosaminoglycans released by proteolysis from proteoglycans extracted from human corneal explants after LASIK. The proteoglycans extracted from pair number 12 (see Table 1) of human corneal explants underwent proteolysis with papain for release of the glycosaminoglycan chains. Aliquots of the incubation mixtures were submitted to agarose gel electrophoresis. Total proteoglycans (PG) and glycosaminoglycans (GAG) were stained by toluidine blue (A), and the 35S-labeled glycosaminoglycans (35S-GAG) were localized by radioautography (B). S: A mixture of standard glycosaminoglycans containing chondroitin sulfate (CS) dermatan sulfate (DS), and heparan sulfate (HS), 5 μg each; KS, keratan sulfate; PG, intact proteoglycans.
Mentions: The electrophoretic migration of the glycosaminoglycan chains released from the core proteins by proteolysis is shown in Figure 3. Upon incubation of the proteoglycans with papain, the band(s) corresponding to proteoglycans completely disappeared, and one band, migrating as dermatan sulfate and keratan sulfate, appeared upon toluidine blue staining. Nevertheless, on radioautogram, a second band, migrating as heparan sulfate, could also be detected. Chondroitin sulfate was not found.

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