Role of nitric oxide synthase isoforms for ophthalmic artery reactivity in mice.

Nitric oxide synthases (NOS) are involved in regulation of ocular vascular tone and blood flow. While endothelial NOS (eNOS) has recently been shown to mediate endothelium-dependent vasodilation in mouse retinal arterioles, the contribution of individual NOS isoforms to vascular responses is unknown in the retrobulbar vasculature. Moreover, it is unknown whether the lack of a single NOS isoform affects neuron survival in the retina. Thus, the goal of the present study was to examine the hypothesis that the lack of individual nitric oxide synthase (NOS) isoforms affects the reactivity of mouse ophthalmic arteries and neuron density in the retinal ganglion cell (RGC) layer. Mice deficient in one of the three NOS isoforms (nNOS-/-, iNOS-/- and eNOS-/-) were compared to respective wild type controls. Intraocular pressure (IOP) was measured in conscious mice using rebound tonometry. To examine the role of each NOS isoform for mediating vascular responses, ophthalmic arteries were studied in vitro using video microscopy. Neuron density in the RGC layer was calculated from retinal wholemounts stained with cresyl blue. IOP was similar in all NOS-deficient genotypes and respective wild type controls. In ophthalmic arteries, phenylephrine, nitroprusside and acetylcholine evoked concentration-dependent responses that did not differ between individual NOS-deficient genotypes and their respective controls. In all genotypes except eNOS-/- mice, vasodilation to acetylcholine was markedly reduced after incubation with L-NAME, a non-isoform-selective inhibitor of NOS. In contrast, pharmacological inhibition of nNOS and iNOS had no effect on acetylcholine-induced vasodilation in any of the mouse genotypes. Neuron density in the RGC layer was similar in all NOS-deficient genotypes and respective controls. Our findings suggest that eNOS contributes to endothelium-dependent dilation of murine ophthalmic arteries. However, the chronic lack of eNOS is functionally compensated by NOS-independent vasodilator mechanisms. The lack of a single NOS isoform does not appear to affect IOP or neuron density in the RGC layer.

Effect of the M1 Muscarinic Acetylcholine Receptor on Retinal Neuron Number Studied with Gene-Targeted Mice.

Pharmacological activation of the M1 muscarinic receptor subtype was suggested to promote the survival of retinal neurons. We examined the hypothesis that the M1 receptor is crucial for retinal neuron survival in vivo by using mice devoid of the M1 receptor gene. Muscarinic receptor gene expression was determined in the retina using real-time PCR. The amount of neurons in the retinal ganglion cell layer and of axons in the optic nerve was determined in retinal wholemounts stained with cresyl blue and in optic nerve cross-sections stained with toluidine blue, respectively. mRNA of all five muscarinic receptor subtypes (M1-M5) was detected in the retina from wild-type mice. Remarkably, M2 and M3 receptor mRNA were most abundant. In retinas from M1 receptor-deficient mice, M4 receptor mRNA expression was increased compared to that of wild-type mice, while no marked changes in the mRNA expression levels of the other muscarinic receptor subtypes were observed. The amount of cells in the retinal ganglion cell layer and the amount of axons in the optic nerve did not differ between M1 receptor-deficient and wild-type mice. The present findings suggest that the M1 receptor is not essential for the survival of retinal neurons in vivo.

Role of α1-adrenoceptor subtypes in pupil dilation studied with gene-targeted mice.

PURPOSE: The α1A-adrenoceptor (α1A-AR) subtype was suggested to mediate contraction and trophic effects in the iris dilator muscle, and thus its pharmacological blockade may be involved in intraoperative floppy iris syndrome. We tested the hypothesis that the α1A-AR mediates pupil dilation and trophic effects in the mouse iris. METHODS: The α1-AR subtype mRNA expression was quantified in iris tissue by real-time PCR. To assess the role of individual α1-ARs for mediating pupil dilation, the α1-AR agonist phenylephrine was topically applied to the ocular surface of mice deficient in one of the three α1-AR subtypes (α1A-AR(-/-), α1B-AR(-/-), α1D-AR(-/-), respectively) and wild-type controls. Changes in pupil diameter were measured under a microscope in restrained mice. Moreover, iris and iris muscle thickness were determined in cryosections. RESULTS: Messenger RNA for all three α1-AR subtypes was detected the iris of wild-type mice with a rank order of abundance of α1A ≥ α1B > > α1D. The lack of a single α1-AR gene did not affect mRNA expression of the remaining two receptor subtypes. Phenylephrine induced pupil dilation in wild-type mice that was reduced in extent and duration in α1A-AR(-/-) and, less so, in α1B-AR(-/-) but not in α1D-AR(-/-) mice. The lack of a single α1-AR subtype had no effect on iris or iris muscle thickness. CONCLUSIONS: The α1-AR-induced mydriasis in mice is mediated mainly by the α1A-AR, with a smaller contribution of the α1B-AR, matching the relative abundance of these subtypes at the mRNA level. The lack of a single α1-AR subtype does not appear to cause atrophy in the mouse iris.

Functional role of α1-adrenoceptor subtypes in murine ophthalmic arteries.

PURPOSE: To identify the α(1)-adrenoceptor (α(1)-AR) subtypes mediating vascular adrenergic responses in murine ophthalmic arteries. METHODS: Expression of mRNA was quantified for individual α(1)-AR subtypes in murine ophthalmic arteries using real-time PCR. To assess the functional relevance of α(1)-ARs for mediating vascular responses, ophthalmic arteries from mice deficient in one of the three α(1)-AR subtypes (α(1A)-AR(-/-), α(1B)-AR(-/-), and α(1D)-AR(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal artery diameter in response to the α(1)-AR agonist phenylephrine, the sympathetic transmitter noradrenaline, and to the nonadrenergic vasoconstrictor arginine vasopressin (AVP) were measured by video microscopy. RESULTS: Using real-time PCR, mRNA for all three α(1)-AR subtypes was detected in ophthalmic arteries from wild-type mice. In functional studies, phenylephrine and noradrenaline produced dose-dependent constriction of ophthalmic arteries that was similar in wild-type, α(1B)-AR(-/-), and α(1D)-AR(-/-) mice. Strikingly, responses to phenylephrine and noradrenaline were almost completely abolished in α(1A)-AR(-/-) mice. In contrast, the nonadrenergic agonist AVP produced dose-dependent vasoconstrictor responses that did not differ between any of the mouse genotypes tested. CONCLUSIONS: These findings provide evidence that the α(1A)-AR subtype mediates adrenergic vasoconstriction in murine ophthalmic arteries.