Experimental Eye Research 127 (2014) 1e8

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Role of nitric oxide synthase isoforms for ophthalmic artery reactivity in mice Panagiotis Laspas a, Evgeny Goloborodko a, Jan J. Sniatecki a, Marcin L. Kordasz a, Caroline Manicam a, Leszek Wojnowski b, Huige Li b, Andreas Patzak c, Norbert Pfeiffer a, Adrian Gericke a, * a b c

Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany Department of Pharmacology, University Medical Center, Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 67, 55131, Mainz, Germany €tsmedizin Berlin, Charit
Institute of Vegetative Physiology, Charit
e-Universita eplatz 1, 10117 Berlin, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 May 2014 Accepted in revised form 19 June 2014 Available online 10 July 2014

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. © 2014 Elsevier Ltd. All rights reserved.

Keywords: acetylcholine knockout mouse nitric oxide ophthalmic artery retinal ganglion cell layer vasodilation

1. Introduction Nitric oxide (NO) is a key messenger molecule regulating various functions in the eye, including neurotransmission, immune

Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; IOP, intraocular pressure; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; L-NAME, Nu-nitro-L-arginine methyl ester; RGC, retinal ganglion cell layer. * Corresponding author. Tel.: þ49 6131 17 5741; fax: þ49 6131 17 7680. E-mail addresses: [email protected], adrian.gericke@unimedizin-mainz. de (A. Gericke). http://dx.doi.org/10.1016/j.exer.2014.06.018 0014-4835/© 2014 Elsevier Ltd. All rights reserved.

activity, and vasodilation (Linden et al., 2005; Toda and NakanishiToda, 2007). Three isoforms of nitric oxide synthase (NOS) have been characterized; neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) (Forstermann and Sessa, 2012). All three NOS isoforms were reported to be involved in regulation of vascular tone. For example, nNOS was shown to be expressed in blood vessels and to modulate vascular reactivity by both endothelium-dependent and independent mechanisms (Capettini et al., 2008; Forstermann et al., 1993; Seddon et al., 2009). While expression of iNOS can be induced in a wide range of cells and tissues by cytokines and other agents (Schoonover et al., 2000), its existence has also been demonstrated in the retina and

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P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8

choroid under physiological conditions (Berra et al., 2005; Bhutto et al., 2010; Mishra and Newman, 2010; Steinle et al., 2009). Some studies suggested that iNOS is physiologically involved in regulation of vascular tone (Berra et al., 2005; Fagan et al., 1999). However, the majority of reports indicates that induction of iNOS expression is associated with impairment of vascular responses (Gunnett et al., 1998, 2001; Ichihara et al., 2000; Mishra and Newman, 2010). The third isoform, eNOS, is critically involved in endothelium-dependent regulation of vascular tone and blood flow (Faraci and Sigmund, 1999; Pollock et al., 1993). Its activity can be triggered by mechanical forces (Busse and Fleming, 1998) or by vasoactive substances (Forstermann et al., 1994), e.g., acetylcholine. Moreover, endothelium-derived NO is involved in numerous vasoprotective mechanisms, such as regulation of endothelial growth and permeability, inhibition of thrombocyte aggregation and of vascular smooth muscle cell proliferation, and limitation of LDL cholesterol oxidation (Forstermann and Munzel, 2006). Disturbances of vascular NO production in the retina and optic nerve have been implicated in the pathophysiology of various ocular diseases, including glaucoma and diabetic retinopathy (Polak et al., 2007; Schmetterer et al., 1997; Toda and NakanishiToda, 2007). However, it is still unknown whether the chronic blockade or lack of a specific NOS isoform leads to impaired reactivity of ocular blood vessels and to a decrease of neuron number in the retina, as typically seen in ocular hypoxia-ischemia (Hein et al., 2012; Kaur et al., 2008). Thus, the major goal of the present study was to test the hypothesis that the chronic lack of an individual NOS isoform affects vascular responses in murine ophthalmic arteries. To examine the role of NOS in endothelium-dependent vasodilation, we examined responses to acetylcholine, an endothelium-dependent vasodilator in mouse ocular blood vessels (Gericke et al., 2011, 2014). To this end, we used mice with targeted disruption of individual NOS genes and various NOS inhibitors. Another purpose of this study was to examine whether mice lacking an individual NOS gene displayed alterations in neuron density in the retinal ganglion cell (RGC) layer. 2. Materials and methods 2.1. Animals All mice were treated in accordance with the EU Directive 2010/ 63/EU for animal experiments, and all experiments were approved by the Animal Care Committee of Rhineland-Palatinate, Germany. The following mouse strains were used in this study: B6.129P2NOS3tm1Unc/J (eNOS
/
) (Shesely et al., 1996), B6.129P2Nos2tm1Lau/J (iNOS
/
) (Laubach et al., 1995), C57BL/6J (B6) wild type mice, 129S-Nos1tm1P1h (nNOS
/
) (Huang et al., 1993), and B6129F2/J wild type mice, all from The Jackson Laboratory, Bar Harbour, ME, USA. The eNOS
/
and iNOS
/
mice were backcrossed with C57BL/6J B6 wild type mice and kept as inbreeding of homozygous animals (eNOS
/
and iNOS
/
, respectively). C57BL/6J B6 mice served as wild type controls for eNOS
/
and iNOS
/
mice. The nNOS
/
mice were obtained by breeding of heterozygous (±) mice, obtained from crossing 129S-Nos1tm1P1h and B6129F2/J wild type mice. Homozygous (þ/þ) mice of this breeding served as controls. The breeding was continuously monitored by assessing the genetic status of each animal by PCR using DNA isolated from tail biopsies according to the protocols of The Jackson Laboratory. The age of mice was 314 ± 8 days and 313 ± 5 days in the nNOS
/
(n ¼ 8) and the respective wild type group (n ¼ 8), 232 ± 8 days and 236 ± 6 days in the iNOS
/
(n ¼ 8) and the respective wild type group (n ¼ 8), and 262 ± 7 days and 260 ± 8 days in the eNOS
/
(n ¼ 8) and the respective wild type

group (n ¼ 8). The mean age of each NOS knockout genotype and its respective wild type control did not differ significantly. Mice were housed under standardized conditions with a 12 h light/dark cycle, temperature of 22 ± 2  C, humidity of 55 ± 10%, and with free access to food and tap water. 2.2. Intraocular pressure measurement Non-invasive measurements of intraocular pressure (IOP) were performed in conscious mice using the Icare® Tonolab rebound tonometer (Bon Optic, Lübeck, Germany) especially designed for rats and mice. Proparacaine 0.5% eye drops (URSAPHARM Arzneimittel GmbH, Saarbrücken, Germany) were administered to each eye before examination. Twelve IOP measurements have been taken per eye, and the overall mean of all 24 measurements was calculated for each mouse. 2.3. Measurements of vascular reactivity After mice had been killed by CO2 inhalation, the eyes were immediately removed together with the retrobulbar tissue and placed in ice-cold Krebs buffer with the following ionic composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11 glucose (all chemicals were from Carl Roth GmbH, Karlsruhe, Germany). Ophthalmic arteries were isolated under a dissecting microscope, placed in an organ chamber filled with cold Krebs buffer and cannulated and sutured onto micropipettes, as reported previously (Gericke et al., 2009). Vessels were pressurized via the micropipettes to 50 mm Hg under no-flow conditions using two reservoirs filled with Krebs buffer and imaged using a video camera mounted on an inverted microscope. Video sequences were captured to a personal computer for off-line analysis. The organ chamber was continuously perfused with oxygenated and carbonated Krebs buffer at 37  C and pH 7.4. Arteries were allowed to equilibrate for 30 min before further intervention. Viability of vessels was assessed as satisfactory when at least 50% constriction from resting diameter in response to high KCl solution (100 mM) was achieved. Next, cumulative concentrationeresponse curves were obtained for the a1-adrenoceptor agonist phenylephrine (10-8e10
4 M, SigmaeAldrich, Munich, Germany). In arteries preconstricted with phenylephrine to 50%e70% of the initial vessel diameter, concentrationeresponse curves for the endothelium-dependent vasodilator acetylcholine (10-9e10
4 M, SigmaeAldrich) and for the endothelium-independent vasodilator nitroprusside (10-9e10
4 M, SigmaeAldrich) have been obtained. Because the lack of a single NOS isoform has been reported to be functionally compensated in other vascular beds (Lamping et al., 2000; Meng et al., 1998), we used various inhibitors to examine whether such a compensation occured in endothelium-dependent vasodilation. After a concentrationeresponse curve for acetylcholine (10
9e10
4 M) had been obtained in arteries preconstricted with phenylephrine, vessels were washed for 10 min and then incubated with either the arginine analog Nu-nitro-L-arginine methyl ester (L-NAME, 10
4 M, SigmaeAldrich), a non-isoformselective NOS inhibitor, 7-nitroindazole (10
5 M, SigmaeAldrich), a selective blocker of neuronal NOS (nNOS), or aminoguanidine (3  10
4 M, SigmaeAldrich), a selective inhibitor of inducible NOS (iNOS). Experiments with the three inhibitors were conducted in all mouse genotypes. Since no highly selective inhibitors for eNOS are yet known, the contribution of eNOS to vasodilation responses had to be derived from experiments using nNOS, iNOS and nonspecific NOS inhibition. The used concentrations of all three NOS inhibitors were proven effective in other vascular preparations (Boulanger et al., 1998; Gunnett et al., 1998, 2001; Patzak et al., 2008; Ren et al., 2001; Stapleton et al., 2007). The incubation time for each

P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8

inhibitor was 30 min. After incubation, the respective vessel was again preconstricted with phenylephrine to a similar level as before and again a concentrationeresponse curve for acetylcholine (10
9e10
4 M) was obtained. In the absence of an inhibitor, there were no differences in vascular responsiveness to sequential administrations of increasing acetylcholine concentrations.

3

diameter, whereas responses to acetylcholine and nitroprusside are presented as percent change in luminal diameter from the preconstricted diameter. Statistical significances of concentrationresponses were calculated using the Brunner test for nonparametric analysis of longitudinal data. For comparisons of IOP and neuron density, the unpaired t-test was used. The level of significance was set at 0.05.

2.4. Retinal wholemounts and neuron counting 3. Results After isolation of the ophthalmic artery, the eye globes were fixed in 4% paraformaldehyde (SigmaeAldrich, Munich, Germany) solution for 1 h. Next, retinas were isolated in phosphate buffered solution (PBS, Invitrogen, Karlsruhe, Germany) using fineepoint tweezers and microscissors, and wholemounts were prepared and stained with cresyl blue using a standard protocol (Laspas et al., 2011). Briefly, after de- and rehydration using increasing and decreasing concentrations of ethanol (70%e100%), the wholemounts were placed in distilled water and stained with 2% cresyl blue (Merck, Darmstadt, Germany). Subsequently, the wholemounts were dehydrated in ethanol, incubated in xylene, and embedded in quick-hardening mounting medium (Eukitt, SigmaeAldrich). Next, wholemounts were examined under a light microscope (Vanox-T, Olympus, Hamburg, Germany) connected to a Hitachi CCD camera (Hitachi, Düsseldorf, Germany) and equipped with Diskus €nigswinter, Germany). Per wholemount, software (Carl H. Hilgers, Ko 16 pre-defined areas, 8 central and 8 peripheral, of 150 mm  200 mm were photographed (Fig. 1A). The proximal border of a central area was localized 0.75 mm from the center of the papilla. This distance corresponded to 5 heights of a photographed area. Each proximal border of a peripheral area was localized 0.75 mm from the distal border of a central photographed area. Thus, the distance from the center of the papilla and the proximal border of a peripheral area was 1.65 mm. In each photograph (Fig. 1B), cells were counted manually using the cell counter plug-in for ImageJ software (NIH, http://rsb. info.nih.gov/ij/). Cells were subdivided into three types based on morphological criteria: Neurons (cells rich in Nissl substance with irregular outlines, a prominent nucleolus, and diameter larger than 8 mm), glial cells (rounded cells with regular outlines, darker stain and diameter smaller than 8 mm), and endothelial cells (long cells positioned along a blood vessel and with a pale uniform stain). The mean density of neurons in the RGC layer per wholemount was then calculated and presented as cells/mm2.

3.1. Intraocular pressure IOP was similar in all NOS knockout mouse genotypes and respective wild type controls. In none of the groups, differences between the right and the left eye were observed. Mean IOP was 12.8 ± 0.5 mm Hg and 12.7 ± 0.5 mm Hg in nNOS
/
(n ¼ 8) and wild-type mice (n ¼ 8), respectively (P ¼ 0.88; Fig. 2A), 11.8 ± 0.5 mm Hg and 11.3 ± 0.6 mm Hg in iNOS
/
(n ¼ 8) and wild-type mice (n ¼ 8), respectively (P ¼ 0.54; Fig. 2B), and 12.4 ± 0.9 mm Hg and 11.4 ± 0.6 mm Hg in eNOS
/
(n ¼ 8) and wild-type mice (n ¼ 8), respectively (P ¼ 0.41; Fig. 2C). 3.2. Responses of ophthalmic arteries Phenylephrine evoked concentration-dependent vasoconstriction of ophthalmic arteries that was similar in all NOS knockout mouse genotypes and their respective wild type controls (n ¼ 8 per concentration and genotype; Fig. 3A, D and G). Both the endothelium-independent vasodilator nitroprusside (n ¼ 7e8 per concentration and genotype; Fig. 3B, E and H) and the endotheliumdependent vasodilator acetylcholine (n ¼ 8 per concentration and genotype; Fig. 3C, F and I) produced concentration-dependent dilation, which did also not differ between arteries from individual NOS knockout mouse genotypes and their respective wild type controls. After incubation with the non-isoform-selective NOS blocker LNAME, responses to acetylcholine were not affected in eNOS
/
mice, but were markedly reduced in all other mouse genotypes tested (n ¼ 6e7 per concentration and genotype; Fig. 4A, D and G). Neither the selective nNOS blocker 7-nitroindazole (n ¼ 5e6 per concentration and genotype; Fig. 4B, E and H) nor the selective iNOS inhibitor aminoguanidine (n ¼ 5e6 per concentration and genotype; Fig. 4C, F and I) had any effect on acetylcholine-induced ophthalmic artery responses in the tested mouse genotypes.

2.5. Statistical analysis 3.3. Neuron density in the retinal ganglion cell layer Data are presented as mean ± SE, and n represents the number of mice per group. Vasoconstriction responses to phenylephrine are presented as percent change in luminal artery diameter from resting

Neuron density in the RGC layer did not differ between individual NOS knockout mouse genotypes and respective wild type

Fig. 1. Mouse retinal wholemount stained with cresyl blue. Microphotographs were taken from 16 pre-defined areas of each wholemount (A). In each photograph (B), the neuron number was counted (E: endothelial cell; G: glial cell; N: neuron).

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P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8

Fig. 2. IOP determined in conscious mice using rebound tonometry. There were no differences in IOP between nNOS
/
and wild type mice (A), between iNOS
/
and wild type mice (B), and between eNOS
/
and wild type mice (C). Data are presented as mean ± SE (n ¼ 8 per group).

controls. The density was 7393 ± 235 cells/mm2 and 7356 ± 200 cells/mm2 in nNOS
/
(n ¼ 8) and wild type mice (n ¼ 8), respectively (P ¼ 0.91; Fig. 5A), 7604 ± 202 cells/mm2 and 8027 ± 198 cells/mm2 mmHg in iNOS
/
(n ¼ 8) and wild type mice (n ¼ 8), respectively (P ¼ 0.16; Fig. 5B), 7495 ± 255 cells/mm2 and 7904 ± 231 cells/mm2 in eNOS
/
(n ¼ 8) and wild type mice (n ¼ 8), respectively (P ¼ 0.25; Fig. 5C).

4. Discussion There are several major new findings in this study. First, the lack of a single NOS isoform did not affect vasoconstriction or vasodilation in mouse ophthalmic arteries. Second, dilation of mouse ophthalmic arteries to the endothelium-dependent vasodilator acetylcholine involved in part eNOS and in part NOS-independent

Fig. 3. Ophthalmic artery responses to phenylephrine, nitroprusside, and acetylcholine did not differ between individual NOS knockout mouse genotypes and respective wild-type controls. Data are presented as mean ± SE (n ¼ 7e8 per concentration and group).

P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8

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Fig. 4. Ophthalmic artery responses to acetylcholine before and after incubation with different NOS inhibitors. After incubation with the non-isoform-selective NOS blocker LNAME, responses to acetylcholine were markedly reduced in all mouse genotypes, but in eNOS
/
mice (A, D, G). *P < 0.05, L-NAME-treated versus non-treated. Neither the selective nNOS blocker 7-nitroindazole (B, E, H) nor the selective iNOS inhibitor aminoguanidine (C, F, I) had any effect on acetylcholine-induced ophthalmic artery responses. Data are presented as mean ± SE (n ¼ 5e7 per concentration and group).

mechanisms, and neither nNOS nor iNOS appeared to contribute significantly to these responses. Remarkably, the chronic lack of eNOS was functionally compensated by NOS-independent vasodilator pathways, because the non-isoform-selective NOS inhibitor LNAME had no effect on cholinergic vasodilation in ophthalmic arteries from eNOS
/
mice as opposed to all other mouse genotypes tested. Third, the lack of a single NOS isoform did not affect IOP or neuron density in the retinal ganglion cell layer. Previous studies reported that NO derived from NOS is involved in regulation of ocular perfusion. For example, intravenous administration of L-arginine, the substrate for NOS, was shown to increase retinal and choroidal blood flow in humans (Garhofer et al., 2005). Moreover, non-isoform-selective NOS inhibitors were shown to reduce blood flow or vessel diameter in the retina, uvea, and in retrobulbar blood vessels of various species, including humans (Dorner et al., 2003; Granstam and Granstam, 1999; Harino et al., 1999; Kiel et al., 2001; Nagaoka et al., 2002). NOS were also shown to be involved in mediating responses of retinal blood vessels during hypoxia, hyperoxia, and hypercapnia (Izumi et al., 2008; Kringelholt et al., 2013; Sato et al., 2003). We previously reported that cholinergic vasodilation responses in retinal arterioles and the ophthalmic artery of mice are endothelium-dependent (Gericke et al., 2011, 2014). Using specific

NOS inhibitors and gene-targeted mice, we recently demonstrated that eNOS mediates acetylcholine-induced vasodilation in mouse retinal arterioles (Gericke et al., 2013). The findings of the present study extend our previously reported observations by showing that eNOS is the only NOS isoform significantly contributing to endothelium-dependent vasodilation also in the mouse ophthalmic artery. In addition, we show in the present study that a NOS-independent vasodilator pathway may be also involved in acetylcholine-induced vasodilator responses. Several studies reported that apart from eNOS, also nNOS and iNOS can modulate endothelium-dependent and independent vascular responses. For example, in the mouse aorta nNOS localized in endothelial cells was suggested to mediate acetylcholine-induced vasorelaxation via hydrogen peroxide production (Capettini et al., 2008). In mouse renal afferent arterioles the nNOS inhibitor 7-nitroindazole enhanced angiotensin II-induced vasoconstriction, suggesting that nNOS counteracts vasoconstriction (Patzak et al., 2008). Induction of iNOS expression was reported to blunt vasoconstriction and vasodilation responses to various agents, including acetylcholine (Gunnett et al., 1998, 2001). In contrast, some other studies reported that iNOS contributed to vascular responses in ocular and nonocular blood vessels of healthy rodents (Berra et al., 2005; Fagan et al., 1999).

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P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8

Fig. 5. Neuron density in the RGC layer did not differ between nNOS
/
and wild type mice (A), between iNOS
/
and wild type mice (B), and between eNOS
/
and wild type mice (C). Data are presented as mean ± SE (n ¼ 8 per group).

By using knockout mice for each NOS isoform, we demonstrated here that the lack of a single isoform does not affect vasoconstriction or vasodilation in the ophthalmic artery, and by using isoformselective and non-selective NOS inhibitors, we found that eNOS is involved in endothelium-dependent vasodilation in the ophthalmic artery of wild type, nNOS
/
, and iNOS
/
mice. Previous studies in mice suggested that the lack of a single NOS isoform could be functionally compensated by another isoform or other enzyme systems. For example, in mouse coronary arteries, the lack of eNOS was shown to be compensated by nNOS and cyclooxygenase products (Godecke et al., 1998; Lamping et al., 2000). Moreover, in pial arterioles of eNOS-deficient mice, vasodilation in response to acetylcholine was reported to be mediated by nNOS (Meng et al., 1998, 1996). By using isoform-selective NOS inhibitors, we excluded the possibility that the lack of a single NOS isoform was functionally compensated by another isoform in the ophthalmic artery. However, our studies in eNOS
/
mice suggest that acetylcholine-induced vasodilation is retained by a NOSindependent mechanism, because the non-isoform-selective NOS inhibitor L-NAME did not block acetylcholine-induced vasodilation responses. In rats, in vivo, the acetylcholine- and bradykinininduced fall in blood pressure was blocked by acute but not by chronic L-NAME treatment (Desai et al., 2006). Results from this study suggested that an endothelium-dependent hyperpolarizing factor (EDHF) compensates for the chronic lack of functional NOS. The compensatory effect occured within only 24 h after NOS blockade (Desai et al., 2006). Studies in ocular vascular preparations indicate that NOS- and cyclooxygenase-independent EDHFs as well as cyclooxygenase products may contribute to endotheliumdependent vasodilation (Delaey et al., 2007; McNeish et al., 2001; Meyer et al., 1996; Mori et al., 2011; Quinn et al., 2003). However, whether these pathways or another one compensate for the chronic lack of eNOS in ocular blood vessels remains to be determined. Alterations of the NO system were suggested to contribute to changes of ocular perfusion and thus to promote the development of various ocular diseases, including glaucoma and diabetic retinopathy (Polak et al., 2007; Schmetterer et al., 1997; Toda and Nakanishi-Toda, 2007). Remarkably, each of the three NOS isoforms has been implicated in pathophysiological processes in the retina eventually affecting neuron survival. For example, nNOS was suggested to contribute to the decline of retinal ganglion cells in mice with high IOP (Chen et al., 2013). In the diabetic retina, the loss of functional hyperemia was restored by iNOS inhibition, suggesting that iNOS may affect retinal perfusion and thus contribute to the progression of diabetic retinopathy (Mishra and Newman, 2010). Moreover, iNOS was suggested to contribute to retinal and optic nerve head damage following ischemia-reperfusion (Cho et al., 2011). In retinal ischemia, iNOS-positive leukocytes were observed to cause retinal ganglion cell degeneration, which could

be prevented with iNOS inhibitors (Neufeld et al., 2002). Other mechanisms by which excessive amounts of NO produced by iNOS may contribute to neuronal toxicity is peroxynitrite-mediated damage (Li et al., 2005). As for eNOS, some gene variants of the eNOS gene have been associated with the development of glaucoma (Awadalla et al., 2013; Ayub et al., 2010; Kang et al., 2010). Moreover, diabetic eNOS
/
mice were shown to develop more vascular complications than diabetic wild type mice of the same age, indicating that eNOS plays a protective role in diabetes (Li et al., 2010). In the present study, we found no differences in neuron density in the RGC layer between mice lacking a single NOS isoform and respective wild type mice. It seems that the individual NOS isoforms are either not essential for neuron survival in our models, or that their lack can be compensated by other signaling pathways, at least under non-pathologic conditions. One limitation of our in vitro study is that we cannot rule out the possibility that nNOS is involved in regulation of vascular tone in the ophthalmic artery under in vivo conditions. In various vascular beds, nNOS has been reported to contribute to regulation of vascular tone and blood flow, possibly by the release of NO from nitrergic neurons (Ferrer et al., 2004; Nangle et al., 2004; Seddon et al., 2009). Moreover, the mice used in this study were of medium age and were not subjected to any pathophysiological stimuli. Thus, we cannot exclude the possibility that under pathophysiological conditions or at higher age our mouse models may develop changes in vascular function or neuron density in the RGC layer. Although the data do not support our initial hypothesis that the lack of a single NOS isoform affects the reactivity of mouse ophthalmic arteries and neuron survival in the retina, the findings are important because they are the first to demonstrate that eNOS is involved in mediating endothelium-dependent vasodilation in the mouse ophthalmic artery and that the chronic lack of eNOS is functionally compensated by NOS-independent vasodilator pathways. Further studies should help to determine whether such a compensation occurs also in retinal arterioles and in ocular blood vessels of other species.

References Awadalla, M.S., Thapa, S.S., Hewitt, A.W., Craig, J.E., Burdon, K.P., 2013. Association of eNOS polymorphisms with primary angle-closure glaucoma. Investig. Ophthalmol. Vis. Sci. 54, 2108e2114. Ayub, H., Khan, M.I., Micheal, S., Akhtar, F., Ajmal, M., Shafique, S., Ali, S.H., den Hollander, A.I., Ahmed, A., Qamar, R., 2010. Association of eNOS and HSP70 gene polymorphisms with glaucoma in Pakistani cohorts. Mol. Vis. 16, 18e25. Berra, A., Ganzinelli, S., Saravia, M., Borda, E., Sterin-Borda, L., 2005. Inducible nitric oxide synthase subserves cholinergic vasodilation in retina. Vis. Neurosci. 22, 371e377. Bhutto, I.A., Baba, T., Merges, C., McLeod, D.S., Lutty, G.A., 2010. Low nitric oxide synthases (NOSs) in eyes with age-related macular degeneration (AMD). Exp. Eye Res. 90, 155e167.

P. Laspas et al. / Experimental Eye Research 127 (2014) 1e8 Boulanger, C.M., Heymes, C., Benessiano, J., Geske, R.S., Levy, B.I., Vanhoutte, P.M., 1998. Neuronal nitric oxide synthase is expressed in rat vascular smooth muscle cells: activation by angiotensin II in hypertension. Circ. Res. 83, 1271e1278. Busse, R., Fleming, I., 1998. Pulsatile stretch and shear stress: physical stimuli determining the production of endothelium-derived relaxing factors. J. Vasc. Res. 35, 73e84. Capettini, L.S., Cortes, S.F., Gomes, M.A., Silva, G.A., Pesquero, J.L., Lopes, M.J., Teixeira, M.M., Lemos, V.S., 2008. Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor. Am. J. Physiol. Heart Circ. Physiol. 295, H2503eH2511. Chen, C., Xu, Y., Zhang, J., Zhu, J., Zhang, J., Hu, N., Guan, H., 2013. Altered expression of nNOS/NIDD in the retina of a glaucoma model of DBA/2J mice and the intervention by nNOS inhibition. J. Mol. Neurosci. : MN 51, 47e56. Cho, K.J., Kim, J.H., Park, H.Y., Park, C.K., 2011. Glial cell response and iNOS expression in the optic nerve head and retina of the rat following acute high IOP ischemia-reperfusion. Brain Res. 1403, 67e77. Delaey, C., Boussery, K., Breyne, J., Vanheel, B., Van de Voorde, J., 2007. The endothelium-derived hyperpolarising factor (EDHF) in isolated bovine choroidal arteries. Exp. Eye Res. 84, 1067e1073. Desai, K.M., Gopalakrishnan, V., Hiebert, L.M., McNeill, J.R., Wilson, T.W., 2006. EDHF-mediated rapid restoration of hypotensive response to acetylcholine after chronic, but not acute, nitric oxide synthase inhibition in rats. Eur. J. Pharmacol. 546, 120e126. Dorner, G.T., Garhofer, G., Kiss, B., Polska, E., Polak, K., Riva, C.E., Schmetterer, L., 2003. Nitric oxide regulates retinal vascular tone in humans. Am. J. Physiol. Heart Circ. Physiol. 285, H631eH636. Fagan, K.A., Tyler, R.C., Sato, K., Fouty, B.W., Morris Jr., K.G., Huang, P.L., McMurtry, I.F., Rodman, D.M., 1999. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am. J. Physiol. 277, L472eL478. Faraci, F.M., Sigmund, C.D., 1999. Vascular biology in genetically altered mice : smaller vessels, bigger insight. Circ. Res. 85, 1214e1225. Ferrer, M., Sanchez, M., Martin, M.C., Marquez-Rodas, I., Alonso, M.J., Salaices, M., Balfagon, G., 2004. Protein kinase A increases electrical stimulation-induced neuronal nitric oxide release in rat mesenteric artery. Eur. J. Pharmacol. 487, 167e173. Forstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwarz, P., Gath, I., Kleinert, H., 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121e1131. Forstermann, U., Munzel, T., 2006. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113, 1708e1714. Forstermann, U., Nakane, M., Tracey, W.R., Pollock, J.S., 1993. Isoforms of nitric oxide synthase: functions in the cardiovascular system. Eur. Heart J. 14 (Suppl I), 10e15. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829e837. Garhofer, G., Resch, H., Lung, S., Weigert, G., Schmetterer, L., 2005. Intravenous administration of L-arginine increases retinal and choroidal blood flow. Am. J. Ophthalmol. 140, 69e76. Gericke, A., Goloborodko, E., Sniatecki, J.J., Steege, A., Wojnowski, L., Pfeiffer, N., 2013. Contribution of nitric oxide synthase isoforms to cholinergic vasodilation in murine retinal arterioles. Exp. Eye Res. 109, 60e66. Gericke, A., Mayer, V.G., Steege, A., Patzak, A., Neumann, U., Grus, F.H., Joachim, S.C., Choritz, L., Wess, J., Pfeiffer, N., 2009. Cholinergic responses of ophthalmic arteries in M3 and M5 muscarinic acetylcholine receptor knockout mice. Investig. Ophthalmol. Vis. Sci. 50, 4822e4827. Gericke, A., Sniatecki, J.J., Goloborodko, E., Steege, A., Zavaritskaya, O., Vetter, J.M., Grus, F.H., Patzak, A., Wess, J., Pfeiffer, N., 2011. Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles. Investig. Ophthalmol. Vis. Sci. 52, 7479e7484. Gericke, A., Steege, A., Manicam, C., Bohmer, T., Wess, J., Pfeiffer, N., 2014. Role of the M3 muscarinic acetylcholine receptor subtype in murine ophthalmic arteries after endothelial removal. Investig. Ophthalmol. Vis. Sci. 55, 625e631. Godecke, A., Decking, U.K., Ding, Z., Hirchenhain, J., Bidmon, H.J., Godecke, S., Schrader, J., 1998. Coronary hemodynamics in endothelial NO synthase knockout mice. Circ. Res. 82, 186e194. Granstam, E., Granstam, S.O., 1999. Regulation of uveal and retinal blood flow in STZ-diabetic and non-diabetic rats; involvement of nitric oxide. Curr. Eye Res. 19, 330e337. Gunnett, C.A., Chu, Y., Heistad, D.D., Loihl, A., Faraci, F.M., 1998. Vascular effects of LPS in mice deficient in expression of the gene for inducible nitric oxide synthase. Am. J. Physiol. 275, H416eH421. Gunnett, C.A., Lund, D.D., Chu, Y., Brooks 2nd, R.M., Faraci, F.M., Heistad, D.D., 2001. NO-dependent vasorelaxation is impaired after gene transfer of inducible NOsynthase. Arterioscler. Thromb. Vasc. Biol. 21, 1281e1287. Harino, S., Nishimura, K., Kitanishi, K., Suzuki, M., Reinach, P., 1999. Role of nitric oxide in mediating retinal blood flow regulation in cats. J. Ocul. Pharmacol. Ther. 15, 295e303. Hein, T.W., Ren, Y., Potts, L.B., Yuan, Z., Kuo, E., Rosa Jr., R.H., Kuo, L., 2012. Acute retinal ischemia inhibits endothelium-dependent nitric oxide-mediated dilation of retinal arterioles via enhanced superoxide production. Investig. Ophthalmol. Vis. Sci. 53, 30e36. Huang, P.L., Dawson, T.M., Bredt, D.S., Snyder, S.H., Fishman, M.C., 1993. Targeted disruption of the neuronal nitric oxide synthase gene. Cell 75, 1273e1286.

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Ichihara, A., Hayashi, M., Navar, L.G., Saruta, T., 2000. Inducible nitric oxide synthase attenuates endothelium-dependent renal microvascular vasodilation. J. Am. Soc. Nephrol. 11, 1807e1812. Izumi, N., Nagaoka, T., Sato, E., Sogawa, K., Kagokawa, H., Takahashi, A., Kawahara, A., Yoshida, A., 2008. Role of nitric oxide in regulation of retinal blood flow in response to hyperoxia in cats. Investig. Ophthalmol. Vis. Sci. 49, 4595e4603. Kang, J.H., Wiggs, J.L., Rosner, B.A., Hankinson, S.E., Abdrabou, W., Fan, B.J., Haines, J., Pasquale, L.R., 2010. Endothelial nitric oxide synthase gene variants and primary open-angle glaucoma: interactions with sex and postmenopausal hormone use. Investig. Ophthalmol. Vis. Sci. 51, 971e979. Kaur, C., Foulds, W.S., Ling, E.A., 2008. Hypoxia-ischemia and retinal ganglion cell damage. Clin. Ophthalmol. 2, 879e889. Kiel, J.W., Reitsamer, H.A., Walker, J.S., Kiel, F.W., 2001. Effects of nitric oxide synthase inhibition on ciliary blood flow, aqueous production and intraocular pressure. Exp. Eye Res. 73, 355e364. Kringelholt, S., Holmgaard, K., Bek, T., 2013. Relaxation of porcine retinal arterioles during acute hypoxia in vitro depends on prostaglandin and NO synthesis in the perivascular retina. Curr. Eye Res. 38, 965e971. Lamping, K.G., Nuno, D.W., Shesely, E.G., Maeda, N., Faraci, F.M., 2000. Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthasedeficient mice. Am. J. Physiol. Heart Circ. Physiol. 279, H1906eH1912. Laspas, P., Gramlich, O.W., Muller, H.D., Cuny, C.S., Gottschling, P.F., Pfeiffer, N., Dick, H.B., Joachim, S.C., Grus, F.H., 2011. Autoreactive antibodies and loss of retinal ganglion cells in rats induced by immunization with ocular antigens. Investig. Ophthalmol. Vis. Sci. 52, 8835e8848. Laubach, V.E., Shesely, E.G., Smithies, O., Sherman, P.A., 1995. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. U. S. A. 92, 10688e10692. Li, J., Baud, O., Vartanian, T., Volpe, J.J., Rosenberg, P.A., 2005. Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc. Natl. Acad. Sci. U. S. A. 102, 9936e9941. Li, Q., Verma, A., Han, P.Y., Nakagawa, T., Johnson, R.J., Grant, M.B., CampbellThompson, M., Jarajapu, Y.P., Lei, B., Hauswirth, W.W., 2010. Diabetic eNOSknockout mice develop accelerated retinopathy. Investig. Ophthalmol. Vis. Sci. 51, 5240e5246. Linden, R., Martins, R.A., Silveira, M.S., 2005. Control of programmed cell death by neurotransmitters and neuropeptides in the developing mammalian retina. Prog. Retin. Eye Res. 24, 457e491. McNeish, A.J., Wilson, W.S., Martin, W., 2001. Dominant role of an endotheliumderived hyperpolarizing factor (EDHF)-like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye. Br. J. Pharmacol. 134, 912e920. Meng, W., Ayata, C., Waeber, C., Huang, P.L., Moskowitz, M.A., 1998. Neuronal NOScGMP-dependent ACh-induced relaxation in pial arterioles of endothelial NOS knockout mice. Am. J. Physiol. 274, H411eH415. Meng, W., Ma, J., Ayata, C., Hara, H., Huang, P.L., Fishman, M.C., Moskowitz, M.A., 1996. ACh dilates pial arterioles in endothelial and neuronal NOS knockout mice by NO-dependent mechanisms. Am. J. Physiol. 271, H1145eH1150. Meyer, P., Flammer, J., Luscher, T.F., 1996. Effect of dipyridamole on vascular responses of porcine ciliary arteries. Curr. Eye Res. 15, 387e393. Mishra, A., Newman, E.A., 2010. Inhibition of inducible nitric oxide synthase reverses the loss of functional hyperemia in diabetic retinopathy. Glia 58, 1996e2004. Mori, A., Suzuki, S., Sakamoto, K., Nakahara, T., Ishii, K., 2011. Role of calciumactivated potassium channels in acetylcholine-induced vasodilation of rat retinal arterioles in vivo. Naunyn Schmiedeb. Arch. Pharmacol. 383, 27e34. Nagaoka, T., Sakamoto, T., Mori, F., Sato, E., Yoshida, A., 2002. The effect of nitric oxide on retinal blood flow during hypoxia in cats. Investig. Ophthalmol. Vis. Sci. 43, 3037e3044. Nangle, M.R., Cotter, M.A., Cameron, N.E., 2004. An in vitro investigation of aorta and corpus cavernosum from eNOS and nNOS gene-deficient mice. Pflugers Arch. 448, 139e145. Neufeld, A.H., Kawai, S., Das, S., Vora, S., Gachie, E., Connor, J.R., Manning, P.T., 2002. Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase. Exp. Eye Res. 75, 521e528. Patzak, A., Steege, A., Lai, E.Y., Brinkmann, J.O., Kupsch, E., Spielmann, N., Gericke, A., Skalweit, A., Stegbauer, J., Persson, P.B., Seeliger, E., 2008. Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform of nitric oxide synthase. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R429eR437. Polak, K., Luksch, A., Berisha, F., Fuchsjaeger-Mayrl, G., Dallinger, S., Schmetterer, L., 2007. Altered nitric oxide system in patients with open-angle glaucoma. Arch. Ophthalmol. 125, 494e498. Pollock, J.S., Nakane, M., Buttery, L.D., Martinez, A., Springall, D., Polak, J.M., Forstermann, U., Murad, F., 1993. Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies. Am. J. physiol. 265, C1379eC1387. Quinn, S., O'Brien, C., McLoughlin, P., 2003. Role of cyclooxygenase and haemoxygenase products in nitric oxide-independent vasodilatation in the porcine ciliary artery. Eye (Lond) 17, 628e636. Ren, Y.L., Garvin, J.L., Ito, S., Carretero, O.A., 2001. Role of neuronal nitric oxide synthase in the macula densa. Kidney Int. 60, 1676e1683. Sato, E., Sakamoto, T., Nagaoka, T., Mori, F., Takakusaki, K., Yoshida, A., 2003. Role of nitric oxide in regulation of retinal blood flow during hypercapnia in cats. Investig. Ophthalmol. Vis. Sci. 44, 4947e4953.

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Schmetterer, L., Findl, O., Fasching, P., Ferber, W., Strenn, K., Breiteneder, H., Adam, H., Eichler, H.G., Wolzt, M., 1997. Nitric oxide and ocular blood flow in patients with IDDM. Diabetes 46, 653e658. Schoonover, L.L., Stewart, A.S., Clifton, G.D., 2000. Hemodynamic and cardiovascular effects of nitric oxide modulation in the therapy of septic shock. Pharmacotherapy 20, 1184e1197. Seddon, M., Melikian, N., Dworakowski, R., Shabeeh, H., Jiang, B., Byrne, J., Casadei, B., Chowienczyk, P., Shah, A.M., 2009. Effects of neuronal nitric oxide synthase on human coronary artery diameter and blood flow in vivo. Circulation 119, 2656e2662. Shesely, E.G., Maeda, N., Kim, H.S., Desai, K.M., Krege, J.H., Laubach, V.E., Sherman, P.A., Sessa, W.C., Smithies, O., 1996. Elevated blood pressures in mice

lacking endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. 93, 13176e13181. Stapleton, P.A., Goodwill, A.G., James, M.E., Frisbee, J.C., 2007. Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110eR1119. Steinle, J.J., Sharma, S., Smith, C.P., McFayden-Ketchum, L.S., 2009. Normal aging involves modulation of specific inflammatory markers in the rat retina and choroid. J. Gerontol. A Biol. Sci. Med. Sci. 64, 325e331. Toda, N., Nakanishi-Toda, M., 2007. Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy. Prog. Retin. Eye Res. 26, 205e238.