Intravitreal Injection of Fluorochrome-Conjugated Peanut Agglutinin Results in Specific and Reversible Labeling of Mammalian Cones In Vivo
Vidhyasankar Krishnamoorthy 1,
Varsha Jain 1,
Pitchaiah Cherukuri 2,
Sonia Baloni 1,
and
Narender K. Dhingra 1*
1 National Brain Research Centre, Manesar, India
2 National Brain Research Centre, Manesar, Haryana, India
* To whom correspondence should be addressed. E-mail: naren{at}nbrc.ac.in.
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Abstract |
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PURPOSE. To investigate if intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo, and to evaluate its clinical potential.
METHODS. We injected fluorescein- or rhodamine-conjugated PNA (0.005-0.5%) intravitreally into anesthetized mouse, guinea pig or monkey, and removed the retinas at various intervals for fluorescence microscopy. Immunofluorescence, and TUNEL assay were carried out to investigate if PNA injection adversely affected other retinal neurons. Gross visual function was studied in a Visual Cliff test. We stained retina of a N-methyl, N-Nitrosourea (MNU)-induced mouse model of retinal degeneration with PNA to evaluate how spatiotemporal pattern of the staining would reflect the progression of degeneration.
RESULTS. Intravitreally-injected PNA resulted in specific labeling of cone outer and inner segments, and cone pedicles within 30 minutes, over entire retina and in all tested species. The labeling was reversible: cones did not show any labeling 3 weeks after the injection, but could be re-stained with PNA. TUNEL signal and expression pattern of several retinal proteins in PNA-injected mouse retina were indistinguishable from normal. Similarly, visual behavior of mouse 10 hours after the injection was normal. The pattern of PNA labeling in mice with MNU-induced retinal degeneration showed progressive disappearance of cones from center to periphery.
CONCLUSIONS. Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo, without causing at least any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina.
Key Words:
cones, retinal degeneration, interphotoreceptor matrix, in vivo, intravitreal, peanut agglutinin
