Hajime Kawamura

ATP: a vasoactive signal in the pericyte-containing microvasculature of the rat retina.

In this study we tested the hypothesis that extracellular ATP regulates the function of the pericyte‐containing retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial‐to‐vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated‐patch pipettes, measured intracellular calcium levels with the use of fura‐2, and visualized microvascular contractions with the aid of time‐lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X7 receptors and UTP‐activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte‐containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X7 receptors inhibited cell‐to‐cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.

Pharmacokinetics of bevacizumab and its effect on vascular endothelial growth factor after intravitreal injection of bevacizumab in macaque eyes.

PURPOSE To evaluate the pharmacokinetics of intravitreally injected bevacizumab in the systemic circulation and the aqueous humor and its effect on vascular endothelial growth factor (VEGF) in the aqueous humor. METHODS Bevacizumab (1.25 mg/50 microL) was injected into the vitreous cavity of the right eyes of three cynomolgus macaques. Aqueous humor and serum were obtained from the macaques just before injection and on days 1, 3, and 7 and weeks 2, 4, 6, and 8 after injection. The bevacizumab and VEGF concentrations were measured using enzyme-linked immunosorbent assay. RESULTS Aqueous VEGF concentrations ranged from 63.2 to 106 pg/mL (mean, 80.0 +/- 22.6 pg/mL) before injection; decreased to <31.2 pg/mL, the lower limit of detection, in all eyes between 1 and 28 days after injection; and returned to the preinjection concentration at 42 days. Aqueous VEGF concentrations in the fellow eyes did not change throughout the experiment. Aqueous bevacizumab concentrations in the treated eyes reached a mean peak concentration of 49,500 +/- 10,900 ng/mL the day after injection and gradually declined, whereas those in the untreated eyes peaked at 3 days, with a mean concentration of 18.5 +/- 25.5 ng/mL, and declined to below 0.156 ng/mL, the limit of detection at 2 weeks. A maximum mean bevacizumab concentration of 1430 +/- 186 ng/mL was achieved in the serum 1 week after injection. CONCLUSIONS Intravitreal injection of bevacizumab decreased the VEGF concentration in the treated eyes for at least 4 weeks and had no or a minimal effect on the untreated fellow eyes.

Effects of angiotensin II on the pericyte‐containing microvasculature of the rat retina

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte‐containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated‐patch pipettes, measured pericyte calcium levels with fura‐2 and visualized pericyte contractions and lumen constrictions by time‐lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium‐activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage‐dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT1 angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium‐activated chloride and voltage‐dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction‐mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

Comparison of macular thickness between Cirrus HD-OCT and Stratus OCT.

BACKGROUND AND OBJECTIVE To compare macular thicknesses in healthy subjects measured with spectral domain optical coherence tomography (SD-OCT) (Cirrus; Carl Zeiss Meditec, Inc., Dublin, CA) with measurements using time domain (TD-OCT) (Stratus; Carl Zeiss Meditec, Inc.). PATIENTS AND METHODS Macular thickness was measured five times in the same eye of 10 healthy subjects with both Cirrus and Stratus to assess reliability and then once in the same eye of 50 healthy subjects with both Cirrus and Stratus to compare the average obtained by each device. RESULTS Using TD-OCT, the coefficient of variations (CV) of the macular thicknesses within a 1-mm central area ranged from 0.7% to 3.3% (mean, 1.33%); with SD-OCT, the range was 0.2% to 1.3% (mean, 0.66%). The mean CV with SD-OCT was significantly smaller than with TD-OCT (P < .05). The average macular thicknesses with TD-OCT and SD-OCT were 197.2 +/- 17.8 microm and 257.6 +/- 19.6 microm, respectively. However, the correlation was significant (correlation coefficient, 0.916, P<.001). CONCLUSION Cirrus showed better reliability than Stratus. Using SD-OCT, the macula was 60-microm thicker than when measured with TD-OCT. Attention should be given to comparing data obtained using different OCT machines.

Effect of vitrectomy on aqueous VEGF concentration and pharmacokinetics of bevacizumab in macaque monkeys.

PURPOSE To evaluate the effect of vitrectomy on the concentration of vascular endothelial growth factor (VEGF) and the pharmacokinetics of intravitreally injected bevacizumab in the aqueous humor in cynomolgus macaques. METHODS Pars plana lensectomy and a standard three-port vitrectomy were performed in one eye each of six macaques. After a minimal 12-week healing period, the vitrectomized eyes received an intravitreal injection of bevacizumab (1.25 mg/50 μL). Aqueous humor and venous blood samples were obtained from the macaques just before vitrectomy, just before injection of bevacizumab, on days 1, 3, and 7, and during weeks 2, 4, 6, and 8 after the injection. The bevacizumab and VEGF concentrations were measured by using enzyme-linked immunosorbent assay. RESULTS The VEGF concentrations in the aqueous humor ranged from 52.6 to 113.9 pg/mL (mean ± standard deviation [SD], 81.7 ± 27.0 pg/mL) before vitrectomy and 20.7 to 72.7 pg/mL (mean ± SD, 51.4 ± 20.5 pg/mL) 3 months after vitrectomy, a difference that reached significance (P = 0.03). The aqueous VEGF concentrations decreased to less than 9.0 pg/mL, the lower limit of detection, in all eyes between 1 and 7 days after injection of bevacizumab. The mean half-life of 1.25 mg intravitreally injected bevacizumab was 1.5 ± 0.6 days (range, 1.0-2.4 days) in the aqueous humor. CONCLUSIONS The VEGF concentration in the aqueous humor decreased and the half-life of the intravitreally injected bevacizumab was shorter in vitrectomized eyes.

Dopamine activates ATP-sensitive K+ currents in rat retinal pericytes.

The relatively sparse vasculature of the retina minimizes obstruction to incoming light, but also poses a challenge to fulfilling the metabolic demands of retinal neurons. An efficient process for distributing energy supplies to areas of need is likely to involve neuron-derived vasoactive signals. However, knowledge of the mechanisms by which capillary perfusion is regulated by neuron-to-vascular signaling is limited. Potential targets of vasoactive molecules released from nerve cells are the pericytes, which are positioned on the endothelial walls of microvessels and are thought to play a role in controlling the microcirculation. In this study, we assessed the effect of dopamine on pericyte physiology. Because dopaminergic neurites are closely associated with microvessels that express dopamine receptors, this molecule is a putative neuron-to-capillary signal, as well as neurotransmitter. We used the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina. In 43% (58/134) of the sampled pericytes, we found that dopamine reversibly activated a hyperpolarizing current, which increased the membrane potential by 19 +/- 1 mV. This dopamine-induced current was inhibited by the ATP-sensitive potassium (KATP) channel blocker, glibenclamide. Consistent with a signaling pathway involving D1 dopamine receptors, adenylate cyclase and protein kinase A (PKA), the selective D1 antagonist, SCH23390, inhibited the hyperpolarizing effect of dopamine; the activator of adenylate cyclase, forskolin, mimicked the dopaminergic effect, and H89, which inhibits PKA, significantly reduced the hyperpolarization induced by dopamine. Taken together, our experiments indicate that a mechanism involving D1 dopamine receptors, adenylate cyclase, and PKA activates KATP currents in retinal pericytes. Our observations support the hypothesis that, in addition to being a neuromodulator, dopamine also serves as a signal linking neuronal activity with the function of the pericyte-containing microvasculature.

Electrotonic transmission within pericyte-containing retinal microvessels

Objective: Little is known about the electrotonic architecture of the pericyte‐containing retinal microvasculature. Here, the authors focus on the cell‐to‐cell transmission of hyperpolarization, which can induce abluminal pericytes to relax and lumens to dilate.

Prospective comparisons of intravitreal injections of triamcinolone acetonide and bevacizumab for macular oedema due to branch retinal vein occlusion

Purpose:  To compare the efficacy of intravitreal injections of triamcinolone acetonide (TA) and that of bevacizumab for macular oedema because of branch retinal vein occlusion (BRVO).

Scleral imbrication combined with vitrectomy and gas tamponade for refractory macular hole retinal detachment associated with high myopia.

Purpose: To evaluate scleral imbrication with vitrectomy and gas tamponade for refractory macular hole retinal detachment associated with high myopia. Methods: We retrospectively reviewed the medical records of eight eyes with macular hole retinal detachment and high myopia treated with temporal scleral imbrication, pars plana vitrectomy, and gas tamponade for refractory macular hole retinal detachment with history of pars plana vitrectomy. Retinal reattachment and macular hole closure were assessed. Postoperative changes in axial length and surgically induced astigmatism were evaluated. Results: The retinas were reattached in all eyes and the macular holes closed in 6 (75%) eyes. The mean baseline logarithm of the minimum angle of resolution best-corrected visual acuity of 1.43 ± 0.48 significantly (P < 0.01) improved to 0.87 ± 0.34 at the final visit (889 ± 173 postoperative days). The mean baseline axial length of 29.5 ± 1.3 mm decreased significantly (P < 0.01) to 27.1 ± 1.9 mm 1 month after scleral imbrication and 28.1 ± 1.7 mm at the final visit (P < 0.05 vs. baseline, P = 0.13 vs. 1 month). The mean 1-month surgically induced astigmatism of 3.6 ± 1.4 diopters (D) after scleral imbrication significantly (P < 0.05) decreased to 2.4 ± 1.5 D at the final visit. Conclusion: Scleral imbrication with vitrectomy and gas tamponade resulted in high reattachment and macular hole closure rates for treating refractory macular hole retinal detachment.

Vascular endothelial growth factor in the aqueous humour in eyes with myopic choroidal neovascularization

Purpose:  To determine the concentration of vascular endothelial growth factor (VEGF) in the aqueous humour of eyes with myopic choroidal neovascularization (mCNV).