Takafumi Yoshioka

Autoregulation of retinal blood flow in response to decreased ocular perfusion pressure in cats: comparison of the effects of increased intraocular pressure and systemic hypotension.

PURPOSE To investigate the regulatory mechanisms responsible for autoregulation of retinal blood flow (RBF) during periods of decreased ocular perfusion pressure (OPP). METHODS The effects of acute reductions in OPP on RBF were assessed using laser Doppler velocimetry in cats. The OPP decreased from 90 to 40 mm Hg by increasing the IOP (elevated IOP) or by decreasing the systemic blood pressure via exsanguination (systemic hypotension). The contributions of nitric oxide (NO), adenosine, and/or N-methyl-D-aspartic acid (NMDA) in regulation of the retinal arteriolar hemodynamics during decreased OPP was determined at 120 minutes after intravitreal injection of various inhibitors or PBS. RESULTS Following PBS injection, the flow velocity decreased in proportion to the decrease in OPP; however, the retinal arteriolar diameter gradually increased. Consequently, the RBF was maintained near baseline levels when the OPP exceeded 70 mm Hg but decreased significantly (P < 0.01) when the OPP fell to less than or equal to 60 mm Hg due to elevated IOP or systemic hypotension. Adenosine receptor blocker 8-(p-sulfophenyl)theophylline, significantly (P < 0.01) enhanced decreases in RBF induced by elevated IOP and systemic hypotension at OPP from 80 to 40 mm Hg, whereas NO synthase inhibitor N(G)-nitro-L-arginine-methyl ester and NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid only significantly (P < 0.01) enhanced reductions in RBF induced by elevated IOP. CONCLUSIONS These results indicate that adenosine contributes to autoregulation of RBF during systemic hypotension, whereas adenosine, NO, and NMDA receptors autoregulate the RBF after elevated IOP. Different vasoregulatory factors might contribute to autoregulation of RBF after decreases in OPP induced by elevated IOP and systemic hypotension.

Role of Ca2+-dependent and Ca2+-sensitive mechanisms in sphingosine 1-phosphate-induced constriction of isolated porcine retinal arterioles in vitro

Although sphingosine 1-phosphate (S1P), a bioactive lipid derived from activated platelets, has a variety of physiologic effects on vessels, no reports have described the effect of S1P on the retinal circulation. We examined the effect and underlying mechanism of the vasomotor action of S1P on porcine retinal arterioles. The porcine retinal arterioles were isolated, cannulated, and pressurized without flow for in vitro study. S1P-induced diameter changes were recorded using videomicroscopic techniques. S1P elicited concentration-dependent (1 nM-10 μM) vasoconstriction of the retinal arterioles that was abolished by the S1P receptor 2 (S1PR2) antagonist JTE-013. S1P-induced vasoconstriction was abolished by the Rho kinase (ROCK) inhibitor H-1152 and was inhibited partly by the protein kinase C (PKC) inhibitor Gö-6983. The inhibition of phospholipase C by U73122 and L-type voltage-operated calcium channels (L-VOCCs) by nifedipine inhibited S1P-induced vasoconstriction; a combination of both inhibitors abolished S1P-induced vasoconstriction. Furthermore, inhibition of myosin light chain kinase (MLCK) by ML-9 significantly blocked S1P-induced vasoconstriction; further coadministration of ML-9 with H-1152 or Gö-6983 abolished S1P-induced vasoconstriction. The current data suggest that S1P elicits vasoconstriction of the retinal arterioles via S1PR2 in vascular smooth muscle cells and this vasoconstriction may be mediated by the Ca2+ -sensitive pathway via activation of PKC leading to activation of ROCK and the Ca2+ -dependent pathway via activation of L-VOCCs resulting in activation of MLCK.

Role of Glial Cells in Regulating Retinal Blood Flow During Flicker-Induced Hyperemia in Cats.

PURPOSE To investigate how glial cells participate in retinal circulation during flicker stimulation in cats. METHODS Using laser Doppler velocimetry, we measured the vessel diameter and blood velocity simultaneously and calculated the retinal blood flow (RBF) in feline first-order retinal arterioles. Twenty-four hours after intravitreal injections of L-2-aminoadipic acid (LAA), a gliotoxic compound, and the solvent of 0.01 N hydrochloric acid as a control, we examined the changes in RBF in response to 16-Hz flicker stimulation for 3 minutes. We also measured the changes in RBF 2 hours after intravitreal injection of Nω-propyl-L-arginine (L-NPA), a selective neuronal nitric oxide synthase inhibitor, in LAA-treated eyes. To evaluate the effects of LAA on retinal neuronal function, ERGs were monitored. Immunohistochemical examinations were performed. RESULTS In LAA-treated eyes, histologic changes selectively occurred in retinal glial cells. There were no significant reductions in amplitude or elongation of implicit time in ERG after LAA injections compared with controls. In control eyes, the RBF gradually increased and reached the maximal level (53.5% ± 2.5% increase from baseline) after 2 to 3 minutes of flicker stimulation. In LAA-treated eyes, the increases in RBF during flicker stimulation were attenuated significantly compared with controls. In LAA-treated eyes 2 hours after injection of L-NPA, flicker-evoked increases in RBF decreased significantly compared with LAA-treated eyes. CONCLUSIONS The current results suggested that increases in RBF in response to flicker stimulation were regulated partly by retinal glial cells.

Role of neuronal nitric oxide synthase in regulating retinal blood flow during flicker-induced hyperemia in cats.

PURPOSE To investigate how neuronal nitric oxide synthase (nNOS) contributes to regulation of the retinal circulation during rest and flicker stimulation in cats. METHODS Using laser Doppler velocimetry, we measured the vessel diameter and blood velocity simultaneously and calculated the retinal blood flow (RBF) in feline first-order retinal arterioles. After intravitreal injections of Nω-Nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, and Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, we continuously monitored the retinal circulation without any perturbations for 2 hours. We then examined the changes in the RBF in response to 16-Hz flicker stimuli for 3 minutes at 2 hours after intravitreal injection of phosphate-buffered saline (PBS) as a control, L-NAME, L-NPA, and thromboxane A2 (TXA2) analogue U46619 as a basal tone-adjusted control. RESULTS After intravitreal injection of L-NAME and L-NPA, the baseline RBF decreased gradually in a dose-dependent manner. In the PBS group, the RBF increased gradually and reached a maximal level after 2 to 3 minutes of flicker stimuli. After 3 minutes of 16-Hz flicker stimuli, the RBF increased by 53.5% ± 3.4% compared with baseline. In the L-NAME and L-NPA groups, the increases in RBF during flicker stimulation were attenuated significantly compared with the PBS group. In the TXA2 group, the reduction in the flicker-induced increase in RBF was comparable to that in the PBS group. CONCLUSIONS The current results suggested that increased RBF in response to flicker stimulation may be mediated by nitric oxide (NO) production via nNOS activation.

Evaluation of Retinal Circulation Using Segmental-Scanning Doppler Optical Coherence Tomography in Anesthetized Cats

PURPOSE To study retinal blood flow (RBF) measurement reproducibility using segmental-scanning Doppler optical coherence tomography (DOCT) in vitro in glass capillaries and in vivo in anesthetized cats. METHODS As a preliminary study, the flow rates of human blood through glass capillaries were changed by using an infusion pump and measured at 13 preset velocities by DOCT. For in vivo measurement, the cats were anesthetized using sevoflurane. The flow in the parent vessel was compared with the sum of the flow values in the two daughter vessels. The RBF was measured using two different instruments: bidirectional laser Doppler velocimetry (LDV) and DOCT. The reproducibility of the measurements was assessed by calculating the coefficients of variation (CVs) for repeated measurements of RBF at the superior retinal arterioles and venules. RESULTS In vitro, the flow velocities measured by DOCT agreed well with the preset velocities. In vivo, the flow in the parent vessel agreed with the sum of the flow values in the two daughter vessels. In addition, there were no significant differences in the mean averaged CVs of the RBF in both the arterioles and venules between LDV and DOCT. CONCLUSIONS The newly developed segmental-scanning DOCT revealed the accuracy of the measurement in in vitro glass capillaries and reproducibility of the measurements of blood velocity in both the retinal arterioles and venules in anesthetized cats.

Glial Endothelin-1 Regulates Retinal Blood Flow During Hyperoxia in Cats

Purpose To investigate the role of endothelin-1 (ET-1) in retinal glial cells in regulating retinal blood flow (RBF) during hyperoxia in cats. Methods We measured the vessel diameter (D), blood velocity (V), and blood flow (F) simultaneously in first-order retinal arterioles using a laser Doppler velocimetry system. The animals were under general anesthesia during hyperoxia (100% oxygen) for 10 minutes 24 hours after intravitreal injection of L-2-aminoadipic acid (LAA), a gliotoxic compound, or diluted hydrochloric acid (0.01 N) used as the vehicle control. We also measured the changes in the RBF after intravitreal injection of BQ-123, a specific ET type A receptor antagonist, in LAA-treated eyes. To examine if endothelin-converting enzyme-1 (ECE-1), as an ET-1-generating enzyme located in retinal glial cells, immunohistochemical examinations with costaining of antiglial fibrillary acidic protein (GFAP) antibody and anti-ECE-1 antibody were performed in whole-mount retinas. Results During hyperoxia, the decreases in D, V, and F in response to hyperoxia were attenuated significantly (P < 0.01 for all comparisons) in the LAA-treated eyes compared with the vehicle control (LAA, D, -8.5 ± 1.5%; V, -13.8 ± 1.5%; F, -27.8 ± 3.0% versus vehicle control, D, -16.8 ± 1.3%; V, -26.3 ± 2.0%; F, -48.9 ± 2.4%). In LAA-treated eyes, intravitreal injections of BQ-123 did not change the rate of hyperoxia-induced RBF compared to LAA-treated eyes. The anti-ECE-1 antibody was costained with anti-GFAP antibody in the whole-mount retinas. Conclusions The current findings suggest that retinal glial ET-1 may play an important role in regulating RBF during hyperoxia in cats.

Repeatability and Reproducibility of Retinal Blood Flow Measurement Using a Doppler Optical Coherence Tomography Flowmeter in Healthy Subjects

Purpose To evaluate the repeatability and reproducibility of retinal blood flow (RBF) measurements in humans by using new auto-alignment and measurement software in a commercially available Doppler optical coherence tomography (DOCT) system. Methods The DOCT flowmeter assessed the intrasession repeatability and the intersession and interobserver reproducibility of the RBF measurements. For intrasession repeatability, the coefficients of variation (CVs) of five repeated RBF measurements were calculated at the retinal arteries and veins in 20 normal eyes of 20 healthy volunteers. For intersession reproducibility, two sets of three measurements obtained by one observer on 2 different days were compared. For interobserver reproducibility, two sets of three measurements obtained by two observers on the same day were compared. Intraclass correlation coefficients (ICCs) also were used to evaluate the repeatability and reproducibility. The relevance of the DOCT flowmeter and laser Doppler velocimetry (LDV) also was assessed. Results Regarding intrasession repeatability, the ICC of the RBF exceeded 0.90 in arterioles and venules (ICC: 0.994 and 0.970, respectively). The CVs of the RBF in the arterioles and venules were 6.0% ± 3.4% and 8.8% ± 5.1%, respectively. The intersession and interobserver RBF values had high reproducibility in the arterioles (ICC: 0.980 and 0.993, respectively) and venules (ICC: 0.982 and 0.986, respectively). The RBF measured with the DOCT flowmeter was correlated strongly with LDV in the arterioles (r = 0.76; P < 0.001). Conclusions The DOCT flowmeter had good reproducibility in the arterioles and venules and precisely measured the RBF as compared to the LDV in the arterioles.