Introduction

Major eye disorders, like glaucoma, diabetic retinopathy, age-dependent macular degeneration, are associated with abnormal retinal blood flow. Glaucoma is the first leading cause of irreversible blindness (Koustenis et al., 2017, Linsenmeier et al., 2017). Based on estimation, there would be 60.5 million people with open angle glaucoma (OAG) and angle closure glaucoma (ACG) in 2010, increasing to 79.6 million by 2020 (Quigley et al., 2006). Patients with normal-tension glaucoma (NTG) had narrower central retinal vessel diameters than did the eyes of normal subjects (Shin et al., 2017). Optical coherence tomography angiography (OCTA) is powerful to diagnose abnormal blood flow in glaucoma eyes, which indicated that glaucoma preferentially affects perfusion in the superficial vascular complex (SVC) in the macula more than the deeper plexuses (Takusagawa et al., 2017). It was reported that blood flow in ophthalmic artery (OA), central retinal artery, and short posterior ciliary artery is all abnormal in primary open-angle glaucoma patients and correlated with the abnormal 24-hour blood pressure (Marjanovic et al., 2016). Diabetes was the seventh leading cause of death in the US in 2010. The prevalence of diabetes among US adults is projected to increase from 14% in 2010 to 21% by 2050, representing a significant burden on the population (Boyle et al., 2010). Diabetic retinopathy (DR), a major microvascular complication of diabetes, is one of the leading causes of vision loss and visual impairment in the working age population worldwide (Tarr et al., 2013). Total retinal blood flow (RBF) is impaired in patients with both non-proliferative DR (NPDR) and proliferative DR (PDR), which could be detected by multiplane en face Doppler optical coherence tomography (OCT) (Lee et al., 2017). In addition, wall shear stress (WSS) could also be altered as the marker of abnormal hemodynamics of diabetic microvasculopathy (Pechauer et al., 2018; Khansari et al., 2017). Hence it is worth to explore more eye diseases and more animal eye disease models where retinal blood flow might be impaired.

The release of endothelium-derived relaxing factors (EDRFs) such as nitric oxide (NO) and prostacyclin (PGI2) in response to stimulations has been well described in various vascular beds in retina (Haefliger et al., 2001; Opatrilova et al., 2018). The rats with experimental early diabetic retinopathy have higher retinal nitric oxide concentration and this concentration is inversely correlated with blood glucose, while nitric oxide is lower than control level in severe diabetic rats (Guthrie et al., 2014). Not only in eye, the increased plasma NO levels are associated with increased severity of diabetic retinopathy indicated with in vivo structural changes in inner segment ellipsoid and pigment epithelium (RPE) (Sharma et al., 2015). The critical role of NO in retinal circulation control is also supported by the observation that neuronal nitric oxide synthase (nNOS) contributes to regulation of the retinal circulation during rest and flicker stimulation in cats (Yoshioka et al., 2015). The soluble guanylyl cyclase/cGMP system plays an important role in the vasodilator response to nitric oxide in most retinal vasculature, but the cyclooxygenase-1/cAMP-mediated pathway could still contribute to the vasodilator effects of NO, for example, prostaglandin I2/prostanoid IP receptor signaling functions in rat retinal arterioles (Mori et al., 2015). In addition to NO and prostacyclin, more EDRFs are explored in various eye diseases, which would also contribute to the pathogenesis of these diseases.

The anti-oxidation function of ascorbate (vitamin C) in eyes has been widely studied. It profoundly impacts on the vasomotor response in retinal microcirculation. As vasodilatators are released from endothelium, l-ascorbic acid 2-phosphate (Asc-2P) extended the proliferation of cultured human corneal endothelial cells (HCECs), partly due to protection against oxidative DNA damage (Shima et al., 2011). It was also demonstrated that ascorbate restored nitric oxide-dependent vasodilatation following its impairment by oxidant stress in isolated arterial rings (Dudgeon et al., 1998; Fontana et al., 1999). This protective effect might be mediated by the clearance of superoxide anion which destroys nitric oxide (Gryglewski et al., 1986; Rubanyi et al., 1986), or mediated by the elevation of tetrahydrobiopterin, the cofactor of nitric oxide synthase (Heller et al., 2001; Huang et al., 2000). It was further observed that ascorbate in aqueous humor promoted NO production in macrophages by stabilizing tetrahydrobiopterin and increasing intracellular arginine (McKenna et al., 2013). Accordingly, studies about ascorbate in multiple models of retinal microcirculation are required to further understand its effects and regulation.

As various vascular beds within the eyes are under tight vasomotor regulation but utilize different mediators, therefore the present study aimed to determine how vasodilatation is mediated in retinal vascular bed.

Materials and Methods

Drugs and chemicals

Acetylcholine chloride (Ach), L-NAME (N^G-nitro-L-arginine methylester, MW 270), Bradykinin acetate, Indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2- methyllindole-3-acetic acid, MW 358), papaverine hydrochloride (MW 376), ascorbate (MW 176) and U46619 (9, 11-dideoxy-11a, 9a-epoxy-methanoprostaglandin F_2a) were all obtained from Sigma chemical corporation (Poole, UK). All drugs were dissolved and diluted in distilled water except indomethacin (3×10^-3 M stock solution), which was dissolved in 10ml of Na₂CO₃ (3×10^-3M) solution.

Preparation of the bovine isolated arterially perfused eye

The retinal vascular bed of the bovine eye was perfused using constant flow perfusion method of Wilson et al. (1993). In brief, Bovine eyes obtained from a local abattoir within 1 h of killing were cannulated so as to perfuse the retinal artery. The ophthalmic artery was dissected free from connective tissue. In some eyes, the ophthalmic artery bifurcates into two long posterior ciliary arteries approximate 2-3 cm proximal to the sclera. In other eyes, the bifurcation occurs close to the sclera. The retinal is a branch of one long posterior ciliary artery and enters the retina via the optic nerve. The perfusion of the very small retinal artery was achieved by cannulating either the long posterior ciliary artery or the ophthalmic artery as appropriate. In the first case, a cannula was inserted into ciliary artery which appeared to be closer to the optic nerve. The other ciliary artery was tied off with thread. In the second case, the bifurcation occurs too close to the optic nerve for safe dissection, so the cannula was inserted into the ophthalmic artery. In this latter situation, thread was tied around whichever ciliary artery appeared to be distal from the optic nerve. In both these situations, the ciliary which was judged to be supplying the retinal artery was also tied off at a point distal to the optic nerve (usually about 1 cm beyond the nerve).

The retinal vasculature was perfused at 37ºC with modified Krebs solution (NaCl 118 mM, KCl 4.7 mM, KH2PO4, 1.2 mM, MgSO4 1.2 mM, NaHCO3 25.0 mM, CaCl2 2.5 mM) with 5 % CO₂. Flow was commenced at _~0.2- 0.5 ml min ^-1 and was raised in 2-5 increments to a final constant rate of 0.8- 1.1 ml min ^-1 over a 10 min period. After the final flow rate was achieved, eyes were perfused for the equilibration period of 10-20 min. Perfusion pressure was measured using Could Statham P32 ID transducers contact to Grass Polygraph via a side arm located immediately proximal to the inflow cannula. Only eyes that had a basal perfusion pressure of 20-30 mmHg after the equilibration period were used for further study.

Experimental protocols

After the equilibration period, drugs were added either to the Krebs reservoir for continuous infusion, or as bolus doses immediately proximal to the cannula. The first part of the experiments involved constructing cumulative concentration-response curves to the thromboxane A_2-mimetic, U46619. Vasoconstrictor responses to each concentration of U46619 were allowed to stabilize before a higher concentration was added and continuous infusion of U46619 at a concentration of 2x10^-7 M was chosen to achieve a sub-maximal perfusion pressure (_~60-180 mmHg). At first, 2 ml L-NAME (N^G-nitro-L-arginine methyl ester) at a concentration of 10^{-2 M} was added into 200ml Krebs solution. After 10-15 minutes, the pressure of the perfused retinal vasculature started to increase little by little. 30-40 minutes later, the perfusion pressure achieved a sub-maximal value (_~160mmHg) around which is suitable for conducting experiments with vasodilators. Once this perfusion pressure was established, vasodilator responses to acetylcholine and bradykinin were assessed by varying bolus doses (3, 10, 30, 100, 300, 1000 pmol of bradykinin and 0.1, 1, 10 nmol of acetylcholine) injected proximal to the cannula with a Hamilton micro-syringe.

The second part of experiments involved adding 1 ml 50 µM ascorbate into 200ml Krebs solution, and then injects 100pmol bradykinin with a Hamilton micro syringe in every 20 minutes during the following 100 minutes. After 100 minutes, acetylcholine and bradykinin were again tested by varying doses (100, 300, 1000 pmol of bradykinin and 0.1, 1, 10 nmol of acetylcholine) with a Hamilton micro-syringe.

The last part of the experiments involved adding 5 ml papaverine hydrochloride at the concentration of 10^-2 M into 100Krebs solution in order to confirm the maximum relaxation which could be achieved in the artery. Responses were measured using baseline which was established by using papaverine (10^-4M) to produce full relaxation of the vasculatine at the end of the experiment.

Vasoconstrictor responses are given in mmHg and vasodilator responses are expressed as percentage (%) reduction of U46619-induced perfusion pressure.

Statistical analysis

Results are expressed as the mean ± s.e.mean of n separate observations, each from a separate eye, Graphs were drawn and statistical comparisons made (Student’s t-test, or one-way analysis of variance with Benferroni’s post-test, as appropriate) using the computer package Prism (GraphPad, San Diego, USA). A probability (P) less than or equal to 0.05 was considered significant. (SF-1)

Results

Acetylcholine and bradykinin induce vasodilatation in retinal arteries of the bovine isolated perfused eye

Acetylcholine and bradykinin were used for retinal microcirculation models for vasodilatation (McNeish et al., 2001), hence the effects of these to stimulators were detected in our samples. The basal perfusion pressure of the retinal vascular bed of the bovine isolated perfused eye preparation was detected to be stable at the constant flow of 0.8-1.1 ml min-1 was 22±2.8 mmHg (n=8). The Thromboxane A2-mimetic, U46619 (2x10-7M), produced a concentration-dependent rise in perfusion pressure, as similar as previous reports using it for vasoconstriction (Hou et al., 2004). Under this situation, the addition of acetylcholine (Figure 1A) and bradykinin (Figure 1B) significantly reduced perfusion pressure in samples of retinal vascular bed of the bovine isolated perfused eye, and this effects were dose-dependent (ach: 0/0.1/1/10 nmol; BK: 3/10/30/100/300/1000 pmol).

Nitric oxide is required for neither basal perfusion pressure nor acetylcholine- and bradykinin-induced vasodilatation

To determine if nitric oxide was involved in Ach- and BK-induced vasodilatation, addition of L-NAME to the perfusate had no effect on this basal perfusion pressure (change of -0.75±0.53 mmHg, n=8). After NO treatment and U46619 vasoconstriction. Ach (Figure 2A) and BK (Figure 2B) still induced vasodilatation indicated by the reduction of perfusion pressure, and the reduction of perfusion pressure for each dose of Ach or BK was similar to the reduction when L-NAME was absent.

Ascorbate blocks Acetylcholine-induced vasodilatation

To determine if ascorbate effects on Ach- and BK-induced vasodilatation in samples of retinal vascular bed of the bovine isolated perfused eye, the ascorbate (50µM) was added to the samples. Consistent with the reduction of ROS species, ascorbate treatment significantly attenuated Ach (0.1/1/10 nmol)-induced vasodilatation, and even high concentration of Ach did not change the perfusion pressure in our samples (Figure 3). In the samples without ascorbate treatment, repeat Ach addition still induced vasodilatation.

Ascorbate blocks bradykinin-induced vasodilatation

In addition to the observation for Ach-induced vasodilatation, we also determine the effects of ascorbate on BK-induced vasodilatation. Consistently, ascorbate treatment significantly attenuated BK (100/300/1000 pmol)-induced vasodilatation, and even high concentration of BK did not change the perfusion pressure in our samples (Figure 4), indicating that Ach and BK regulates retinal vasodilatation with same mediators in our model, and ascorbate could block both of the vasodilators. In the samples without ascorbate treatment, repeat Ach addition still induced vasodilatation.

Discussions

It was well known that endothelium-derived relaxing factors (EDRFs) such as nitric oxide (NO) and prostacyclin (PGI2) plays essential roles in vasomotor control in various vascular beds in retina (Hardy et al., 2000; Mori et al., 2007). Inhibition of inducible nitric oxide synthase has already been shown to be effective in various animal models of diabetic eye disease (Carr et al., 2017). For example, administration of shikonin, a major red naphthoquinone pigment, attenuated diabetes/hypoxia-induced production of inflammatory proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (Liao et al., 2017). On the other hand, beraprost sodium, a stable PGI2 analogue, BPS elicits endothelium-dependent and -independent dilation of the retinal arterioles mediated by NO induced by activation of PKA in the endothelium and the KATP channel activation in the vascular smooth muscle, respectively (Ono et al., 2014). According to previous studies (Nelli et al, 2003), we developed the model system in retinal vascular bed of the bovine isolated perfused eye where acetylcholine- and bradykinin-induced vasodilatation could be readily recorded. Interestingly, neither NOS inhibitor (L-NAME) nor cyclooxygenase inhibitor (indomethacin) suppressed the vasodilatation. This is consistent with the ciliary vascular bed of the bovine isolated perfused eye, where vasodilatation induced by acetylcholine or bradykinin was not sensitive to L-NAME (McNeish et al., 2001).

Though the nitric oxide and prostacyclin are traditionally considered as endothelium-derived relaxing factors, some of the endothelium-dependent vasodilations are resistant to the blockade of NO synthase and cyclooxygenase. The nature of these endothelium-derived hyperpolarizing factor (EDHF) is still unknown, but possible candidates include a cytochrome P450 metabolite, an epoxyeicosatrienoic acid an endogenous cannabinoid or potassium ions (Edwards et al., 2000). The endothelium plays an essential role in the dilation of porcine retinal arterioles to histamine via H1- and H2-receptor activation. The EDHF derived from cytochrome P450 contributed in part to this vasodilation via Ca2+ -activated K+ (KCa) channel activation, in addition to the endothelial release of NO and prostanoids (Otani et al., 2016). It was reported that EDHF is able to relax ocular ciliary artery vascular tone in rats via large-conductance calcium-activated K(+) channel and this ability is injured in spontaneous hypertensive rats (SHR) (Dong et al., 2010). Acetylcholine- and bradykinin-induced vasodilatation in the ciliary vascular bed of the bovine isolated eye was also reported to be independent of nitric oxide and hance might be mediated by an unknown EDHF, (McNeish et al., 2001) which is similarly observed in the present study on bovine retinal arteries.

Therefore, the present study provided opportunity to explore how a EDHF-mediated vasodilatation could be pharmacologically targeted. The anti-oxidation function of ascorbate (vitamin C) in eyes has been widely studied. As ascorbate scavenges superoxide anion, it might be responsible for clearance of the toxic reactive oxygen species (ROS) as the levels of superoxide dismutases are found to be extremely low in eyes. This was observed in patients treated with vitrectomy that the level of ascorbate in the vitreous fluid was significantly reduced and increased oxygen from the retina dramatically induced lens oxidation and protein aggregation in these patients. (Yan et al., 2017). Benefited by the anti-oxidation capacity, ascorbate could block agonist-induced vasodilator responses mediated by EDHP. In the ciliary vascular bed of the bovine isolated perfused eye, EDHF-mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate, reminiscent to the effects of two other reducing agents, N-acetyl-L-cysteine and dithiothreitol (McNeish et al., 2002).

In summary, our present study developed a model in retinal vascular bed of the bovine isolated perfused eye to study agonist-induced vaso-relaxation. In the present study, acetylcholine and bradykinin efficiently stimulated a non-traditional vaso-relaxation that neither NO nor prostacyclin is involved, providing insights to EDHF-mediated vasomotor response. As a concentrated anti-oxidant in eyes, ascorbate (vitamin C) showed capability to block acetylcholine and bradykinin-induced vasodilatation in bovine retinal arteries. However, further studies are warranted to unveil the detail mechanism of this process.

Author Contributions

All authors equally contributed for experimental and data analysis.

Acknowledgment

None declared

References

Boyle JP, Thompson TJ, Gregg EW, Barker LE, Williamson DF. Projection of the year 2050 burden of diabetes in the US adult population: dynamic modeling of incidence, mortality, and prediabetes prevalence. Population health metrics. 2010;8:29. Epub 2010/10/26. doi: 10.1186/1478-7954-8-29. PubMed PMID: 20969750; PubMed Central PMCID: PMCPMC2984379.
https://doi.org/10.1186/1478-7954-8-29
PMid:20969750 PMCid:PMC2984379
 
Carr BC, Emigh CE, Bennett LD, Pansick AD, Birch DG, Nguyen C. TOWARDS A TREATMENT FOR DIABETIC RETINOPATHY: Intravitreal Toxicity and Preclinical Safety Evaluation of Inducible Nitric Oxide Synthase Inhibitors. Retina (Philadelphia, Pa). 2017;37(1):22-31. Epub 2016/07/06. doi: 10.1097/iae.0000000000001133. PubMed PMID: 27380429; PubMed Central PMCID: PMCPMC5177480.
https://doi.org/10.1097/IAE.0000000000001133
PMid:27380429 PMCid:PMC5177480
 
Dong Y, Watabe H, Cui J, Abe S, Sato N, Ishikawa H, et al. Reduced effects of endothelium-derived hyperpolarizing factor in ocular ciliary arteries from spontaneous hypertensive rats. Experimental eye research. 2010;90(2):324-9. Epub 2009/11/28. doi: 10.1016/j.exer.2009.11.009. PubMed PMID: 19941853.
https://doi.org/10.1016/j.exer.2009.11.009
PMid:19941853
 
Dudgeon S, Benson DP, MacKenzie A, Paisley-Zyszkiewicz K, Martin W. Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta. British journal of pharmacology. 1998;125(4):782-6. Epub 1998/12/01. doi: 10.1038/sj.bjp.0702120. PubMed PMID: 9831915; PubMed Central PMCID: PMCPMC1571043.
https://doi.org/10.1038/sj.bjp.0702120
PMid:9831915 PMCid:PMC1571043
 
Edwards G, Thollon C, Gardener MJ, Feletou M, Vilaine J, Vanhoutte PM, et al. Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery. British journal of pharmacology. 2000;129(6):1145-54. Epub 2000/03/22. doi: 10.1038/sj.bjp.0703188. PubMed PMID: 10725263; PubMed Central PMCID: PMCPMC1571957.
https://doi.org/10.1038/sj.bjp.0703188
PMid:10725263 PMCid:PMC1571957
 
Fontana L, McNeill KL, Ritter JM, Chowienczyk PJ. Effects of vitamin C and of a cell permeable superoxide dismutase mimetic on acute lipoprotein induced endothelial dysfunction in rabbit aortic rings. British journal of pharmacology. 1999;126(3):730-4. Epub 1999/04/03. doi: 10.1038/sj.bjp.0702331. PubMed PMID: 10188985; PubMed Central PMCID: PMCPMC1565839.
https://doi.org/10.1038/sj.bjp.0702331
PMid:10188985 PMCid:PMC1565839
 
Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320(6061):454-6. Epub 1986/04/03. doi: 10.1038/320454a0. PubMed PMID: 3007998.
https://doi.org/10.1038/320454a0
PMid:3007998
 
Guthrie MJ, Osswald CR, Kang-Mieler JJ. Inverse relationship between the intraretinal concentration of bioavailable nitric oxide and blood glucose in early experimental diabetic retinopathy. Investigative ophthalmology & visual science. 2014;56(1):37-44. Epub 2014/12/17. doi: 10.1167/iovs.14-15777. PubMed PMID: 25503458.
https://doi.org/10.1167/iovs.14-15777
PMid:25503458
 
Haefliger IO, Flammer J, Beny JL, Luscher TF. Endothelium-dependent vasoactive modulation in the ophthalmic circulation. Progress in retinal and eye research. 2001;20(2):209-25. Epub 2001/02/15. PubMed PMID: 11173252.
https://doi.org/10.1016/S1350-9462(00)00020-3
 
Hardy P, Dumont I, Bhattacharya M, Hou X, Lachapelle P, Varma DR, et al. Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy. Cardiovascular research. 2000;47(3):489-509. Epub 2000/08/30. PubMed PMID: 10963722.
https://doi.org/10.1016/S0008-6363(00)00084-5
 
Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner ER. L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. The Journal of biological chemistry. 2001;276(1):40-7. Epub 2000/10/07. doi: 10.1074/jbc.M004392200. PubMed PMID: 11022034.
https://doi.org/10.1074/jbc.M004392200
PMid:11022034
 
Hou X, Roberts LJ, 2nd, Gobeil F, Jr., Taber D, Kanai K, Abran D, et al. Isomer-specific contractile effects of a series of synthetic f2-isoprostanes on retinal and cerebral microvasculature. Free radical biology & medicine. 2004;36(2):163-72. Epub 2004/01/28. PubMed PMID: 14744628.
https://doi.org/10.1016/j.freeradbiomed.2003.10.024
PMid:14744628
 
Huang A, Vita JA, Venema RC, Keaney JF, Jr. Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. The Journal of biological chemistry. 2000;275(23):17399-406. Epub 2000/04/06. doi: 10.1074/jbc.M002248200. PubMed PMID: 10749876.
https://doi.org/10.1074/jbc.M002248200
PMid:10749876
 
Khansari MM, Wanek J, Tan M, Joslin CE, Kresovich JK, Camardo N, et al. Assessment of Conjunctival Microvascular Hemodynamics in Stages of Diabetic Microvasculopathy. Scientific reports. 2017;7:45916. Epub 2017/04/08. doi: 10.1038/srep45916. PubMed PMID: 28387229; PubMed Central PMCID: PMCPMC5384077.
https://doi.org/10.1038/srep45916
PMid:28387229 PMCid:PMC5384077
 
Koustenis A, Jr., Harris A, Gross J, Januleviciene I, Shah A, Siesky B. Optical coherence tomography angiography: an overview of the technology and an assessment of applications for clinical research. The British journal of ophthalmology. 2017;101(1):16-20. Epub 2016/10/07. doi: 10.1136/bjophthalmol-2016-309389. PubMed PMID: 27707691.
https://doi.org/10.1136/bjophthalmol-2016-309389
PMid:27707691
 
Lee B, Novais EA, Waheed NK, Adhi M, de Carlo TE, Cole ED, et al. En Face Doppler Optical Coherence Tomography Measurement of Total Retinal Blood Flow in Diabetic Retinopathy and Diabetic Macular Edema. JAMA ophthalmology. 2017;135(3):244-51. Epub 2017/02/15. doi: 10.1001/jamaophthalmol.2016.5774. PubMed PMID: 28196198; PubMed Central PMCID: PMCPMC5784830.
https://doi.org/10.1001/jamaophthalmol.2016.5774
PMid:28196198 PMCid:PMC5784830
 
Liao PL, Lin CH, Li CH, Tsai CH, Ho JD, Chiou GC, et al. Anti-inflammatory properties of shikonin contribute to improved early-stage diabetic retinopathy. Scientific reports. 2017;7:44985. Epub 2017/03/23. doi: 10.1038/srep44985. PubMed PMID: 28322323; PubMed Central PMCID: PMCPMC5359562.
https://doi.org/10.1038/srep44985
PMid:28322323 PMCid:PMC5359562
 
Linsenmeier RA, Zhang HF. Retinal oxygen: from animals to humans. Progress in retinal and eye research. 2017;58:115-51. Epub 2017/01/23. doi: 10.1016/j.preteyeres.2017.01.003. PubMed PMID: 28109737; PubMed Central PMCID: PMCPMC5441959.
https://doi.org/10.1016/j.preteyeres.2017.01.003
PMid:28109737 PMCid:PMC5441959
 
Marjanovic I, Marjanovic M, Martinez A, Markovic V, Bozic M, Stojanov V. Relationship between blood pressure and retrobulbar blood flow in dipper and nondipper primary open-angle glaucoma patients. European journal of ophthalmology. 2016;26(6):588-93. Epub 2016/10/30. doi: 10.5301/ejo.5000789. PubMed PMID: 27338118.
https://doi.org/10.5301/ejo.5000789
PMid:27338118
 
McKenna KC, Beatty KM, Scherder RC, Li F, Liu H, Chen AF, et al. Ascorbate in aqueous humor augments nitric oxide production by macrophages. Journal of immunology (Baltimore, Md : 1950). 2013;190(2):556-64. Epub 2012/12/18. doi: 10.4049/jimmunol.1201754. PubMed PMID: 23241881; PubMed Central PMCID: PMCPMC3538947.
https://doi.org/10.4049/jimmunol.1201754
PMid:23241881 PMCid:PMC3538947
 
McNeish AJ, Wilson WS, Martin W, editors. Ascorbic acid attenuates EDHF-mediated vasodilatation in the bovine isolated perfused eye. BPS meeting 2002a; Imperial College London.
 
McNeish AJ, Wilson WS, Martin W. Dominant role of an endothelium-derived hyperpolarizing factor (EDHF)-like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye. British journal of pharmacology. 2001;134(4):912-20. Epub 2001/10/19. doi: 10.1038/sj.bjp.0704332. PubMed PMID: 11606333; PubMed Central PMCID: PMCPMC1573020.
https://doi.org/10.1038/sj.bjp.0704332
PMid:11606333 PMCid:PMC1573020
 
Mori A, Namekawa R, Hasebe M, Saito M, Sakamoto K, Nakahara T, et al. Involvement of prostaglandin I(2) in nitric oxide-induced vasodilation of retinal arterioles in rats. European journal of pharmacology. 2015;764:249-55. Epub 2015/07/08. doi: 10.1016/j.ejphar.2015.07.009. PubMed PMID: 26151307.
https://doi.org/10.1016/j.ejphar.2015.07.009
PMid:26151307
 
Mori A, Saito M, Sakamoto K, Narita M, Nakahara T, Ishii K. Stimulation of prostanoid IP and EP(2) receptors dilates retinal arterioles and increases retinal and choroidal blood flow in rats. European journal of pharmacology. 2007;570(1-3):135-41. Epub 2007/07/14. doi: 10.1016/j.ejphar.2007.05.052. PubMed PMID: 17628525.
https://doi.org/10.1016/j.ejphar.2007.05.052
PMid:17628525
 
Nelli S, Wilson WS, Laidlaw H, Llano A, Middleton S, Price AG, et al. Evaluation of potassium ion as the endothelium-derived hyperpolarizing factor (EDHF) in the bovine coronary artery. British journal of pharmacology. 2003;139(5):982-8. Epub 2003/07/04. doi: 10.1038/sj.bjp.0705329. PubMed PMID: 12839872; PubMed Central PMCID: PMCPMC1573923.
https://doi.org/10.1038/sj.bjp.0705329
PMid:12839872 PMCid:PMC1573923
 
Ono S, Nagaoka T, Omae T, Tanano I, Kamiya T, Otani S, et al. Beraprost sodium, a stable prostacyclin analogue, elicits dilation of isolated porcine retinal arterioles: roles of eNOS and potassium channels. Investigative ophthalmology & visual science. 2014;55(9):5752-9. Epub 2014/08/02. doi: 10.1167/iovs.14-14902. PubMed PMID: 25082887.
https://doi.org/10.1167/iovs.14-14902
PMid:25082887
 
Opatrilova R, Kubatka P, Caprnda M, Busselberg D, Krasnik V, Vesely P, et al. Nitric oxide in the pathophysiology of retinopathy: evidences from preclinical and clinical researches. Acta ophthalmologica. 2018;96(3):222-31. Epub 2017/04/10. doi: 10.1111/aos.13384. PubMed PMID: 28391624.
https://doi.org/10.1111/aos.13384
PMid:28391624
 
Otani S, Nagaoka T, Omae T, Tanano I, Kamiya T, Ono S, et al. Histamine-Induced Dilation of Isolated Porcine Retinal Arterioles: Role of Endothelium-Derived Hyperpolarizing Factor. Investigative ophthalmology & visual science. 2016;57(11):4791-8. Epub 2016/09/13. doi: 10.1167/iovs.15-19038. PubMed PMID: 27618417.
https://doi.org/10.1167/iovs.15-19038
PMid:27618417
 
Pechauer AD, Hwang TS, Hagag AM, Liu L, Tan O, Zhang X, et al. Assessing total retinal blood flow in diabetic retinopathy using multiplane en face Doppler optical coherence tomography. The British journal of ophthalmology. 2018;102(1):126-30. Epub 2017/05/13. doi: 10.1136/bjophthalmol-2016-310042. PubMed PMID: 28495904; PubMed Central PMCID: PMCPMC5800769.
https://doi.org/10.1136/bjophthalmol-2016-310042
PMid:28495904 PMCid:PMC5800769
 
Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. The British journal of ophthalmology. 2006;90(3):262-7. Epub 2006/02/21. doi: 10.1136/bjo.2005.081224. PubMed PMID: 16488940; PubMed Central PMCID: PMCPMC1856963.
https://doi.org/10.1136/bjo.2005.081224
PMid:16488940 PMCid:PMC1856963
 
Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. The American journal of physiology. 1986;250(5 Pt 2):H822-7. Epub 1986/05/01. doi: 10.1152/ajpheart.1986.250.5.H822. PubMed PMID: 3010744.
https://doi.org/10.1152/ajpheart.1986.250.5.H822
PMid:3010744
 
Sharma S, Saxena S, Srivastav K, Shukla RK, Mishra N, Meyer CH, et al. Nitric oxide and oxidative stress is associated with severity of diabetic retinopathy and retinal structural alterations. Clinical & experimental ophthalmology. 2015;43(5):429-36. Epub 2015/02/14. doi: 10.1111/ceo.12506. PubMed PMID: 25675974.
https://doi.org/10.1111/ceo.12506
PMid:25675974
 
Shima N, Kimoto M, Yamaguchi M, Yamagami S. Increased proliferation and replicative lifespan of isolated human corneal endothelial cells with L-ascorbic acid 2-phosphate. Investigative ophthalmology & visual science. 2011;52(12):8711-7. Epub 2011/10/08. doi: 10.1167/iovs.11-7592. PubMed PMID: 21980003.
https://doi.org/10.1167/iovs.11-7592
PMid:21980003
 
Shin YU, Lee SE, Cho H, Kang MH, Seong M. Analysis of Peripapillary Retinal Vessel Diameter in Unilateral Normal-Tension Glaucoma. Journal of ophthalmology. 2017;2017:8519878. Epub 2017/07/01. doi: 10.1155/2017/8519878. PubMed PMID: 28660081; PubMed Central PMCID: PMCPMC5474240.
https://doi.org/10.1155/2017/8519878
PMid:28660081 PMCid:PMC5474240
 
Srinivas S, Tan O, Nittala MG, Wu JL, Fawzi AA, Huang D, et al. ASSESSMENT OF RETINAL BLOOD FLOW IN DIABETIC RETINOPATHY USING DOPPLER FOURIER-DOMAIN OPTICAL COHERENCE TOMOGRAPHY. Retina (Philadelphia, Pa). 2017;37(11):2001-7. Epub 2017/01/19. doi: 10.1097/iae.0000000000001479. PubMed PMID: 28098726.
https://doi.org/10.1097/IAE.0000000000001479
PMid:28098726
 
Takusagawa HL, Liu L, Ma KN, Jia Y, Gao SS, Zhang M, et al. Projection-Resolved Optical Coherence Tomography Angiography of Macular Retinal Circulation in Glaucoma. Ophthalmology. 2017;124(11):1589-99. Epub 2017/07/06. doi: 10.1016/j.ophtha.2017.06.002. PubMed PMID: 28676279; PubMed Central PMCID: PMCPMC5651191.
https://doi.org/10.1016/j.ophtha.2017.06.002
PMid:28676279 PMCid:PMC5651191
 
Tarr JM, Kaul K, Chopra M, Kohner EM, Chibber R. Pathophysiology of diabetic retinopathy. ISRN ophthalmology. 2013;2013:343560. Epub 2014/02/25. doi: 10.1155/2013/343560. PubMed PMID: 24563789; PubMed Central PMCID: PMCPMC3914226.
https://doi.org/10.1155/2013/343560
PMid:24563789 PMCid:PMC3914226
 
Wilson WS, Shahidullah M, Millar C. The bovine arterially-perfused eye: an in vitro method for the study of drug mechanisms on IOP, aqueous humour formation and uveal vasculature. Current eye research. 1993;12(7):609-20. Epub 1993/07/01. PubMed PMID: 7693396.
https://doi.org/10.3109/02713689309001840
PMid:7693396
 
Yan H, Wang D, Ding TB, Zhou HY, Yan WJ, Wang XC. Comparison of lens oxidative damage induced by vitrectomy and/or hyperoxia in rabbits. International journal of ophthalmology. 2017;10(1):6-14. Epub 2017/02/06. doi: 10.18240/ijo.2017.01.02. PubMed PMID: 28149770; PubMed Central PMCID: PMCPMC5225342.
 
Yoshioka T, Nagaoka T, Song Y, Yokota H, Tani T, Yoshida A. Role of neuronal nitric oxide synthase in regulating retinal blood flow during flicker-induced hyperemia in cats. Investigative ophthalmology & visual science. 2015;56(5):3113-20. Epub 2015/03/19. doi: 10.1167/iovs.14-15854. PubMed PMID: 25783603.
https://doi.org/10.1167/iovs.14-15854
PMid:25783603