Limits...
Retinol dehydrogenase 13 protects the mouse retina from acute light damage.

Wang H, Cui X, Gu Q, Chen Y, Zhou J, Kuang Y, Wang Z, Xu X - Mol. Vis. (2012)

Bottom Line: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice.Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice.The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: To investigate whether retinol dehydrogenase 13 (RDH13) can protect the retina from acute light-induced damage.

Methods: We generated Rdh13 knockout mice using molecular biologic methods and assessed the associated morphological and functional changes under room-light conditions by hematoxylin-eosin (H&E), transmission electron microscopy (TEM), and scotopic electroretinography. Then, the light-damage model was established by exposure to diffuse white light (3,000 lx) for 48 h. Twenty-four h after light exposure, H&E was used for the histological evaluation. The thickness of the outer-plus-inner-segment and the outer nuclear layer was measured on sections parallel to the vertical meridian of the eye. An electroretinography test was performed to assess the functional change. Furthermore, the impairment of mitochondria was detected by TEM. Finally, the expression of cytochrome c (CytC) and other apoptosis-related proteins was detected by western blot.

Results: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice. In Rdh13(-/-) mice subjected to intense light exposure, the photoreceptor outer-plus-inner-segment and outer nuclear layer were dramatically shorter, and the amplitudes of a- and b-waves under scotopic conditions were significantly attenuated. Distinctly swollen mitochondria with disrupted cristae were observed in the photoreceptor inner segments of Rdh13(-/-) mice. Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice.

Conclusions: Rdh13 can protect the retina against acute light-induced retinopathy. The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

Show MeSH

Related in: MedlinePlus

Generation of Rdh13 knockout mice. A: This is the graphic representation of the Rdh13 gene knockout strategy for the deletion of Rdh13 exons 2 and 3 in embryonic stem cells. Exons are shown in boxes. The targeting vector was designed to delete exon 2 and exon 3. The targeting vector contained a 2.9 kb 5′ arm and 3.2 kb 3′ arm. PGK-Neo and HSV-TK cassettes were used for positive and negative selections, respectively. The genomic positive of the PCR primers for genotyping are indicated by arrows. B: Genomic DNA from ES cell clones was isolated and analyzed by PCR. The successfully targeted embryonic stem cell DNA was amplified into 6.2 kb and 3.5 kb products for the 5′ arm and 3′ arm, respectively. C: The genotype of Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was detected by PCR. D: Rdh13 transcripts in mouse liver from Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was analyzed by reverse-transcription PCR. E: The expression pattern of RDH13 protein in Rdh13+/+, Rdh13+/−, and Rdh13−/− mouse liver was revealed by western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351414&req=5

f2: Generation of Rdh13 knockout mice. A: This is the graphic representation of the Rdh13 gene knockout strategy for the deletion of Rdh13 exons 2 and 3 in embryonic stem cells. Exons are shown in boxes. The targeting vector was designed to delete exon 2 and exon 3. The targeting vector contained a 2.9 kb 5′ arm and 3.2 kb 3′ arm. PGK-Neo and HSV-TK cassettes were used for positive and negative selections, respectively. The genomic positive of the PCR primers for genotyping are indicated by arrows. B: Genomic DNA from ES cell clones was isolated and analyzed by PCR. The successfully targeted embryonic stem cell DNA was amplified into 6.2 kb and 3.5 kb products for the 5′ arm and 3′ arm, respectively. C: The genotype of Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was detected by PCR. D: Rdh13 transcripts in mouse liver from Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was analyzed by reverse-transcription PCR. E: The expression pattern of RDH13 protein in Rdh13+/+, Rdh13+/−, and Rdh13−/− mouse liver was revealed by western blot.

Mentions: A targeting vector was constructed by replacing the mouse Rdh13 genomic 1,661 bp fragment, covering exons 2 and 3, with the 1,904 bp phosphoglycerate kinase-neomycin resistance cassette (PGK-Neo cassette) for positive selection. An external herpes simplex virus-1-thymidine kinase cassette (HSV-TK cassette) was used for negative selection (Figure 2A). The targeting vector contained 2.9 kb of homologous DNA upstream to the PGK-Neo cassette and 3.2 kb downstream as homologous recombination arms. The embryonic stem (ES) cells that had undergone homologous recombination were identified by PCR (Figure 2B) using two pairs of primers, whose direction and position is depicted in Figure 2A. The primers used for the 5′-arm recombination were (P1) 5′-CTT CTG CTT CTT GCC TAG TTC TTC TCA-3′ and (P2) 5′-AAT TGC ATC GCA TTG TCT GAG TAG G-3′. The 3′-arm primers were (P3) 5′-CCA GAG GCC ACT TGT GTA GCG-3′ and (P4) 5′-GAA GCA AAG AAC CAA CCC CTC TGA-3′. The correctly recombined ES cells were subsequently microinjected into blastocysts, which were in turn implanted into pseudopregnant female recipients to generate chimeric mice. The F1 mice with germ-line transmission of the Rdh13 knockout (KO) allele were heterozygous. These heterozygous mice were identified by PCR using mouse tails and primers depicted in Figure 2A, (P5) 5′-CAG GAG GCA ACG TCA TTC TG-3′, (P6) 5′-GCT CAA TGA CAC TCC AGC AA-3′, (P7) 5′-TGG CTG GAC GTA AAC TCC TC-3′.


Retinol dehydrogenase 13 protects the mouse retina from acute light damage.

Wang H, Cui X, Gu Q, Chen Y, Zhou J, Kuang Y, Wang Z, Xu X - Mol. Vis. (2012)

Generation of Rdh13 knockout mice. A: This is the graphic representation of the Rdh13 gene knockout strategy for the deletion of Rdh13 exons 2 and 3 in embryonic stem cells. Exons are shown in boxes. The targeting vector was designed to delete exon 2 and exon 3. The targeting vector contained a 2.9 kb 5′ arm and 3.2 kb 3′ arm. PGK-Neo and HSV-TK cassettes were used for positive and negative selections, respectively. The genomic positive of the PCR primers for genotyping are indicated by arrows. B: Genomic DNA from ES cell clones was isolated and analyzed by PCR. The successfully targeted embryonic stem cell DNA was amplified into 6.2 kb and 3.5 kb products for the 5′ arm and 3′ arm, respectively. C: The genotype of Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was detected by PCR. D: Rdh13 transcripts in mouse liver from Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was analyzed by reverse-transcription PCR. E: The expression pattern of RDH13 protein in Rdh13+/+, Rdh13+/−, and Rdh13−/− mouse liver was revealed by western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351414&req=5

f2: Generation of Rdh13 knockout mice. A: This is the graphic representation of the Rdh13 gene knockout strategy for the deletion of Rdh13 exons 2 and 3 in embryonic stem cells. Exons are shown in boxes. The targeting vector was designed to delete exon 2 and exon 3. The targeting vector contained a 2.9 kb 5′ arm and 3.2 kb 3′ arm. PGK-Neo and HSV-TK cassettes were used for positive and negative selections, respectively. The genomic positive of the PCR primers for genotyping are indicated by arrows. B: Genomic DNA from ES cell clones was isolated and analyzed by PCR. The successfully targeted embryonic stem cell DNA was amplified into 6.2 kb and 3.5 kb products for the 5′ arm and 3′ arm, respectively. C: The genotype of Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was detected by PCR. D: Rdh13 transcripts in mouse liver from Rdh13+/+, Rdh13+/−, and Rdh13−/− mice was analyzed by reverse-transcription PCR. E: The expression pattern of RDH13 protein in Rdh13+/+, Rdh13+/−, and Rdh13−/− mouse liver was revealed by western blot.
Mentions: A targeting vector was constructed by replacing the mouse Rdh13 genomic 1,661 bp fragment, covering exons 2 and 3, with the 1,904 bp phosphoglycerate kinase-neomycin resistance cassette (PGK-Neo cassette) for positive selection. An external herpes simplex virus-1-thymidine kinase cassette (HSV-TK cassette) was used for negative selection (Figure 2A). The targeting vector contained 2.9 kb of homologous DNA upstream to the PGK-Neo cassette and 3.2 kb downstream as homologous recombination arms. The embryonic stem (ES) cells that had undergone homologous recombination were identified by PCR (Figure 2B) using two pairs of primers, whose direction and position is depicted in Figure 2A. The primers used for the 5′-arm recombination were (P1) 5′-CTT CTG CTT CTT GCC TAG TTC TTC TCA-3′ and (P2) 5′-AAT TGC ATC GCA TTG TCT GAG TAG G-3′. The 3′-arm primers were (P3) 5′-CCA GAG GCC ACT TGT GTA GCG-3′ and (P4) 5′-GAA GCA AAG AAC CAA CCC CTC TGA-3′. The correctly recombined ES cells were subsequently microinjected into blastocysts, which were in turn implanted into pseudopregnant female recipients to generate chimeric mice. The F1 mice with germ-line transmission of the Rdh13 knockout (KO) allele were heterozygous. These heterozygous mice were identified by PCR using mouse tails and primers depicted in Figure 2A, (P5) 5′-CAG GAG GCA ACG TCA TTC TG-3′, (P6) 5′-GCT CAA TGA CAC TCC AGC AA-3′, (P7) 5′-TGG CTG GAC GTA AAC TCC TC-3′.

Bottom Line: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice.Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice.The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: To investigate whether retinol dehydrogenase 13 (RDH13) can protect the retina from acute light-induced damage.

Methods: We generated Rdh13 knockout mice using molecular biologic methods and assessed the associated morphological and functional changes under room-light conditions by hematoxylin-eosin (H&E), transmission electron microscopy (TEM), and scotopic electroretinography. Then, the light-damage model was established by exposure to diffuse white light (3,000 lx) for 48 h. Twenty-four h after light exposure, H&E was used for the histological evaluation. The thickness of the outer-plus-inner-segment and the outer nuclear layer was measured on sections parallel to the vertical meridian of the eye. An electroretinography test was performed to assess the functional change. Furthermore, the impairment of mitochondria was detected by TEM. Finally, the expression of cytochrome c (CytC) and other apoptosis-related proteins was detected by western blot.

Results: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice. In Rdh13(-/-) mice subjected to intense light exposure, the photoreceptor outer-plus-inner-segment and outer nuclear layer were dramatically shorter, and the amplitudes of a- and b-waves under scotopic conditions were significantly attenuated. Distinctly swollen mitochondria with disrupted cristae were observed in the photoreceptor inner segments of Rdh13(-/-) mice. Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice.

Conclusions: Rdh13 can protect the retina against acute light-induced retinopathy. The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

Show MeSH
Related in: MedlinePlus