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Retinal thickness measurement obtained with spectral domain optical coherence tomography assisted optical biopsy accurately correlates with ex vivo histology.

Ferguson LR, Grover S, Dominguez JM, Balaiya S, Chalam KV - PLoS ONE (2014)

Bottom Line: The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17.All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida, United States of America.

ABSTRACT

Background: This study determines 'correlation constants' between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods: Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as 'correlation constants'.

Results: Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (± 1.09) and 219.58 µm (± 2.67), respectively. The mean 'correlation constant' for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion: This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

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Related in: MedlinePlus

Cross-sectional views of the C57BL/6 mouse retina by SD-OCT (left) and histology (right).OS/IS/ELM, outer segment/inner segment/external limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL/GC, inner plexiform layer/ganglion cell; RNFL, retinal nerve fiber layer.
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pone-0111203-g002: Cross-sectional views of the C57BL/6 mouse retina by SD-OCT (left) and histology (right).OS/IS/ELM, outer segment/inner segment/external limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL/GC, inner plexiform layer/ganglion cell; RNFL, retinal nerve fiber layer.

Mentions: Animals were euthanized with CO2 inhalation followed by cervical dislocation. The same eyes that had SD-OCT scans were enucleated and then punctured with a 30-gauge needle and submerged into 4% paraformaldehyde (PFA) for 2–4 hours. This was followed by a series of submersions into phosphate buffered saline (PBS) with increasing concentrations of sucrose in the following manner: PBS with 5% sucrose (6 hrs), PBS with 10% sucrose (10 hrs), and PBS with 20% sucrose (10 hrs). Samples were then removed from the PBS with sucrose solution and solidified into optimal cutting temperature compound in a container with dry ice and 2-methylbutane. A cryostat microtome was used to produce sample slices of 10 µm per section. Every twentieth section was selected for tissue staining. The samples were then stained with H&E using standard laboratory protocols. The Zeiss Axioskop 2 Mot Plus (Carl Zeiss MicroImaging, Inc., Thornwood, NY) microscope, with 10x objective lens, was used to evaluate histological sections. All sections selected for light microscopy evaluation contained optic nerve tissue landmarks. The histological section that first demonstrated the appearance of the ONH was considered the histological ‘inferior point’. As illustrated in Figures 1 and 2, histological measurements of retinal sublayers were similarly performed as mentioned for SD-OCT scan thickness measurements. The manual caliper instrument, from the Axiovision 4.8 (Carl Zeiss MicroImaging, Inc., Thornwood, NY) imaging software, was used to measure histological sublayer retinal thickness. TRT was derived from the sum of all measured histological layers up to but not including the retinal pigment epithelium.


Retinal thickness measurement obtained with spectral domain optical coherence tomography assisted optical biopsy accurately correlates with ex vivo histology.

Ferguson LR, Grover S, Dominguez JM, Balaiya S, Chalam KV - PLoS ONE (2014)

Cross-sectional views of the C57BL/6 mouse retina by SD-OCT (left) and histology (right).OS/IS/ELM, outer segment/inner segment/external limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL/GC, inner plexiform layer/ganglion cell; RNFL, retinal nerve fiber layer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216007&req=5

pone-0111203-g002: Cross-sectional views of the C57BL/6 mouse retina by SD-OCT (left) and histology (right).OS/IS/ELM, outer segment/inner segment/external limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL/GC, inner plexiform layer/ganglion cell; RNFL, retinal nerve fiber layer.
Mentions: Animals were euthanized with CO2 inhalation followed by cervical dislocation. The same eyes that had SD-OCT scans were enucleated and then punctured with a 30-gauge needle and submerged into 4% paraformaldehyde (PFA) for 2–4 hours. This was followed by a series of submersions into phosphate buffered saline (PBS) with increasing concentrations of sucrose in the following manner: PBS with 5% sucrose (6 hrs), PBS with 10% sucrose (10 hrs), and PBS with 20% sucrose (10 hrs). Samples were then removed from the PBS with sucrose solution and solidified into optimal cutting temperature compound in a container with dry ice and 2-methylbutane. A cryostat microtome was used to produce sample slices of 10 µm per section. Every twentieth section was selected for tissue staining. The samples were then stained with H&E using standard laboratory protocols. The Zeiss Axioskop 2 Mot Plus (Carl Zeiss MicroImaging, Inc., Thornwood, NY) microscope, with 10x objective lens, was used to evaluate histological sections. All sections selected for light microscopy evaluation contained optic nerve tissue landmarks. The histological section that first demonstrated the appearance of the ONH was considered the histological ‘inferior point’. As illustrated in Figures 1 and 2, histological measurements of retinal sublayers were similarly performed as mentioned for SD-OCT scan thickness measurements. The manual caliper instrument, from the Axiovision 4.8 (Carl Zeiss MicroImaging, Inc., Thornwood, NY) imaging software, was used to measure histological sublayer retinal thickness. TRT was derived from the sum of all measured histological layers up to but not including the retinal pigment epithelium.

Bottom Line: The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17.All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida, United States of America.

ABSTRACT

Background: This study determines 'correlation constants' between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods: Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as 'correlation constants'.

Results: Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (± 1.09) and 219.58 µm (± 2.67), respectively. The mean 'correlation constant' for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion: This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

Show MeSH
Related in: MedlinePlus