Intravitreal injection of fluorochrome-conjugated peanut agglutinin results in specific and reversible labeling of mammalian cones in vivo.
OBJECTIVE
To investigate whether intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo and to evaluate its clinical potential.


METHODS
Fluorescein- or rhodamine-conjugated PNA (0.005%-0.5%) was intravitreally injected into anesthetized mouse, guinea pig, or monkey and retinas were removed at various intervals for fluorescence microscopy. Immunofluorescence and TUNEL assay were carried out to investigate whether PNA injection adversely affected other retinal neurons. Gross visual function was studied in a visual cliff test. The retina of an N-methyl, N-nitrosourea (MNU)-induced mouse model of retinal degeneration was stained 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 the 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 restained 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 the center to the periphery.


CONCLUSIONS
Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo without causing any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina.
Krishnamoorthy
Vidhyasankar
V
National Brain Research Centre, Manesar Haryana, India.
Jain
Varsha
V
Cherukuri
Pitchaiah
P
Baloni
Sonia
S
Dhingra
Narender K
NK
D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D004195:Disease Models, Animal; D005452:Fluoresceins / Q000378:metabolism* / Q000633:toxicity; D006168:Guinea Pigs; D020287:In Situ Nick-End Labeling; D007267:Injections; D008253:Macaca mulatta; D008770:Methylnitrosourea / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D019887:Peanut Agglutinin / Q000378:metabolism* / Q000633:toxicity; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017949:Retinal Cone Photoreceptor Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000139:chemically induced / Q000175:diagnosis / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*; D013194:Staining and Labeling; D014822:Vitreous Body
D000477:Alkylating Agents; D015415:Biomarkers; D005452:Fluoresceins; D019887:Peanut Agglutinin; D012235:Rhodamines; C468515:fluorescein isothiocyanate-peanut agglutinin; D008770:Methylnitrosourea