The Nicotinic Cholinergic Pathway Contributes to Retinal Neovascularization in a Mouse Model of Retinopathy of Prematurity

Abstract

Purpose: To investigate the role of nicotinic acetylcholine receptors (nAChRs) in retinal vascular development and ischemia-induced retinal neovascularization (NV).

Methods: The expression of nAChR subtypes and VEGF signaling pathway components was assessed in mice with and without oxygen-induced ischemic retinopathy by comparing expression levels at postnatal day (P) 14 and P17 in mice exposed to 75% oxygen from P7 to P12 and returned to room air versus mice pups that were exposed to ambient oxygen levels during the same period. The effect of topical or intraocular injection of mecamylamine, a nonspecific nAChR antagonist, or targeted deletion of α7- or α9-nAChRs on ischemia-induced retinal NV was determined by comparing the amount of retinal NV at P17 in these mice versus appropriate controls.

Results: The expression of nAChR subunits and components of the VEGF signaling pathways was increased in ischemic retina. Topical application or intraocular injection of mecamylamine decreased retinal NV in this model. Mecamylamine had no effect on normal retinal vascular development or on revascularization of the central retinal area of nonperfusion in mice with ischemic retinopathy. Targeted deletion of α9, but not α7, nAChR receptor subunits reduced retinal NV in mice with ischemic retinopathy.

Conclusion: These data suggest that nAChR signaling, primarily through the α9 nAChR subunit, contributes to ischemia-induced retinal NV, but not retinal vascular development. Mecamylamine or a specific α9 nAChR antagonist could be considered for treatment of retinopathy of prematurity and other ischemic retinopathies.

Figure 1

Mecamylamine reduces rNV in OIR but does not affect normal vascular development. (a) Mouse pup litters (n = 4–9 pups per litter) were treated by intraocular injection of mecamylamine or PBS at the onset of hypoxia (P12). *P < 0.05, **P < 0.001 for control versus treated rNV. (b) Distance from the optic nerve head (asterisk) to the peripheral edge of vascularization (arrow) was measured at P7 in the superficial vascular bed and at P10 and P18 for the deep capillary vascular bed. The distance was measured in each of four quadrants in the flat-mounted retinas and averaged to generate one value per eye. n = 4 to 10 per group. (c) Images of representative retinal quadrants for superficial vessels at P7 of mecamylamine- and PBS-treated eyes.

Figure 2

Gene expression in PECAM (+) and PECAM (−) fractions of retinal homogenates from RA control versus OIR mice at P14 and P17 was compared. (a) The enrichment factor of PECAM expression in bead-selected fractions versus nonselected fraction was measured. n = 12, **P = 2.65 × 10⁻⁵. (b) Vascular endothelial growth factor A and VEGFR2 expression levels were measured at P14 (n = 6) and P17 (n = 3) in both PECAM (+) and PECAM (−) fractions from OIR retinas and RA control retinas. *P < 0.05, **P < 0.01. (c) Expression levels of the eight nAChRα subtypes were measured at P14 (n = 6) and at P17 (n = 3) in the PECAM (−) fraction of retinal homogenate samples from OIR mice and RA controls. *P < 0.05. (d) Expression of the nAChR α subtypes were measured at P14 (n = 6) and P17 (n = 3) in the PECAM (+) fraction of retinal homogenate samples from OIR mice and RA controls. *P < 0.05, **P < 0.01.

Figure 3

Pathologic rNV is reduced in nAChRα9 KO mice but not in nAChRα7 KO mice. (a) Retinal NV was quantified in retinas of P17 OIR that were from nAChR α7 (+/−) or (−/−), nAChR, α9 (+/−) or (−/−), and wild-type control mice. n = 6 to 15. P < 0.01. (b) The central ANP that results from exposure to hyperoxia was measured in nAChR α9 (+/+) and (−/−) mice at P12 and also at P17 as normal vessels grow to revascularize the central retina. n = 3 to 7. (c) Representative images of one quadrant of flat mounted retinas from (+/+), (+/−), and (−/−) nAChR α9 KO mice display the amount of rNV for each group (arrows).

Figure 4

The expression levels of VEGF, PEDF, VEGFR2, and the nAChRα subtypes were compared in P17 OIR retinas from nAChR α9 (+/+) and (−/−) mice. (+/+) n = 8, (−/−) n = 13. **P < 0.01.