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Sanjoy K Bhattacharya, Teresia Carreon, Ayman Aljohani, Giovanni Gregori, Jianhua Wang; Quantification of cochlin in DBA/2J mice in vivo using optical coherence tomography.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3272.
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© ARVO (1962-2015); The Authors (2016-present)
To repeatedly detect and quantify cochlin in real time in DBA/2J mice anterior chamber in vivo to correlate its level with non-invasively measured intraocular pressure (IOP).
All experiments were performed adhering to ARVO statement for the use of animals.The spectroscopic and magnetomotive optical coherence tomography (OCT) approaches was used for in vivo cochlin quantification. An OCT instrument was custom built with a two light sources (780, 840nm) and a magnet arm (10T) . Cochlin detection was optimized using polymeric beads, purified recombinant cochlin (1-5 µg) and anti-cochlin chicken polyclonal antibodies (1-5 µg) coupled with fluorophore with specific absorbance at 780nm or magnetic nanoparticles (150-250 nm sizes were found optimal). The optimized procedure was used in vivo in DBA/2J (n= 240; 40 mice per group at 3, 6, 7, 8, 9 and 10 months of age) by periodic injection (once a week) of the antibody in the anterior chamber angles. IOP was measured daily using a Tonolab. A dye injection in the cornea and limiting edges of the angle were used as landmarks. Images were processed using Matlab software programs. At endpoints, the animals were euthanized, eyes enucleated, fixed, embedded in cutting medium and 10µm sections were stained with hematoxylin-eosin. The stained images were compared with OCT images using duel landmarks (for stained images and for OCT images) for confirmation of measurements along with Western blot estimation of quantities of cochlin from alternate sections.
The spectroscopic (SOCT) and magnetomotive (MMOCT) successfully detected cochlin and enabled quantification of cochlin in polymeric beads and in mouse eyes. In living mouse eyes, cochlin-antibody OCT signal remains stable for up to 24 hours as seen at 3.5 hours after initial injection of cochlin antibody allowing repeated quantification. The endpoint Western blot and other quantitation approaches correlate with OCT measurements.
It is possible to detect and quantify cochlin in the living mouse eyes by injecting infrared dye coupled or magnetic bead coupled antibodies. The OCT quantification correlates with endpoint Western blot analyses. Our measurements suggest that expression of cochlin in the DBA/2J mice trabecular meshwork precedes the elevation in intraocular pressure in these mice.
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