Real-Time Measurement of Antiglaucoma Drugs in Porcine Eyes Using Boron-Doped Diamond Microelectrodes

The primary treatment for glaucoma, the most common cause of intermediate vision impairment, involves administering ocular hypotensive drugs in the form of topical eye drops. Observing real-time changes in the drugs that pass through the cornea and reach the anterior chamber of the eye is crucial for improving and developing safe, reliable, and effective medical treatments. Traditional methods for measuring temporal changes in drug concentrations in the aqueous humor employ separation analyzers such as LC–MS/MS. However, this technique requires multiple measurements on the eyes of various test subjects to track changes over time with a high temporal resolution. To address this issue, we have developed a measurement method that employs boron-doped diamond (BDD) microelectrodes to monitor real-time drug concentrations in the anterior chamber of the eye. First, we confirmed the electrochemical reactivity of 13 antiglaucoma drugs in a phosphate buffer solution with a pH of 7.4. Next, we optimized the method for continuous measurement of timolol maleate (TIM), a sympathetic beta-receptor antagonist, and generated calibration curves for each BDD microelectrode using aqueous humor collected from enucleated porcine eyes. We successfully demonstrated the continuous ex vivo monitoring of TIM concentrations in the anterior chambers of these enucleated porcine eyes. The results indicate that changes in intracameral TIM concentrations can be monitored through electrochemical measurements using BDD microelectrodes. This technique holds promise for future advancements in optimizing glaucoma treatment and drug administration strategies

Comparison of lesion size between wild-type (WT) and transgenic mice lacking lymphocytes at day 7 after laser injury.

<p>(A) There were no differences in mean choroidal neovascularization (CNV) area between WT and <i>CD4</i>^{<i>−/−</i>} mice or (B) between WT and <i>Rag2</i>^{<i>−/−</i>} mice. (C) Representative micrographs of CNV (white arrows) in RPE-choroid flatmounts. Scale bars, 100 μm. n = 8 for all groups.</p

Choroidal Neovascularization Is Inhibited in Splenic-Denervated or Splenectomized Mice with a Concomitant Decrease in Intraocular Macrophage

<div><p>Purpose</p><p>To determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model.</p><p>Methods</p><p>We investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6C^hi, Ly6C^int, and Ly6C^lo) after laser injury in mice using flow cytometry, and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the β3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6C^hi cells were transferred into Ly5.2 mice, the proportions of intraocular Ly5.1⁺Ly6C^hi cells were compared.</p><p>Results</p><p>In WT mice, the proportion of CD4⁺ T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8⁺ T cells did not change significantly. Proportions of B220⁺ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6C^hi and Ly6C^lo) peaked at day 3 following laser injury. In contrast, Ly6C^int/loCD64⁺ subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between <i>CD4</i>^{<i>–/–</i>}or <i>Rag2</i>^{<i>–/–</i>}mice and controls, whereas lesion size was significantly reduced in <i>CCR2</i>^{<i>−/−</i>} mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with β3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6C^hi macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6C^hi cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6C^hi cells recruited into the eye were reduced in splenic-denervated and splenectomized mice.</p><p>Conclusions</p><p>Although lymphocytes had little effect on CNV formation, Ly6C^hi macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.</p></div

Changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenectomized mice after laser injury.

<p>(A) Less Ly6C^hi cells were recruited into the eye in splenectomized mice than those in controls both before laser treatment and at day 3 after laser injury, and intraocular Ly6C^int/loCD64⁺ cells were also reduced by splenectomy from before laser photocoagulation up to 7 days after laser, whereas, there were no significant changes in Ly6C^lo cells between two groups with or without laser injury. (B) In the peripheral blood, a lower proportion of Ly6C^int cells was observed before laser treatment, at day 3 and day 7 after laser injury, and Ly6C^lo cells were also reduced at day 7 after laser injury. In contrast, peripheral blood Ly6C^hi monocytes showed no differences between controls and splenectomized mice. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Comparison of lesion size between wild-type mice and mice lacking macrophages at the inflammatory sites or in the systemic system at day 7 after laser injury.

<p>(A) Lesion size was significantly smaller in <i>CCR2</i>^{<i>−/−</i>} mice compared with WT mice and (B) in mice injected with clodronate liposomes compared with controls. (C) Representative micrographs of CNV (white arrows) in RPE-choroid flatmounts. Scale bars, 100 μm. *P < 0.05 with Wilcoxon rank-sum test. n = 8 for all groups.</p

Lesion size was significantly reduced at day 7 after laser injury in mice with sympathetic nerves blockade.

<p>(A) CNV lesion size was significantly reduced in splenic-denervated mice compared with controls and (B) in mice injected with a β3 receptor antagonist injection compared with controls. (C) Splenectomized mice also showed a decreased lesion size after laser injury compared with controls. (D) Representative micrographs of CNV (white arrows) in RPE-choroid flatmounts. Scale bars, 100 μm. *P < 0.05 with Wilcoxon rank-sum test. n = 6 for all groups.</p

Changes in the proportions of circulating monocyte/macrophage subtypes in peripheral blood after laser injury.

<p>(A) Representative flow cytometry plots from WT mice. Monocyte subpopulations were gated based on Ly6C and CD43 expression to determine the proportions of classical (Ly6C^hiCD43^lo), intermediate (Ly6C^intCD43^hi) and nonclassical (Ly6C^loCD43^hi) monocytes per total leukocytes. Hi, int, and lo correspond to Ly6C^hi, Ly6C^int, and Ly6C^lo cells, respectively. (B) The proportion of circulating Ly6C^hi cells were significantly higher at day 3 after laser injury, whereas there were no changes in the proportions of Ly6C^lo and Ly6C^int cells. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Change in the proportions of inflammatory cells recruited into the posterior segment of the eye in wild-type mice following laser photocoagulation.

<p>The proportion of CD4⁺ T cells increased progressively from day 3 after laser injury and greatly increased until day 7. Proportions of B220⁺ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6C^hi and Ly6C^lo) peaked at day 3 after laser injury, whereas Ly6C^int/loCD64⁺ subtype showed a significantly higher percentage at 7 days after laser injury. In contrast, there were no changes in the proportion of CD8⁺ T cells after injury. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Comparison of Ly5.1⁺Ly6C^hi cell infiltration into the eye of Ly5.2 mice at day 3 after laser photocoagulation.

<p>We injected CD11b⁺CD115⁺Ly6C^hi cells harvested from the spleens of Ly5.1 mice into Ly5.2 mice before laser photocoagulation, and compared the proportion of donor-derived intraocular Ly6C^hi macrophages between the controls, splenic-denervated mice or splenectomized mice three days later. Compared with controls, splenic-denervated mice and splenectomized mice showed a significantly lower percentage of donor- derived Ly6C^hi cells in the eye after laser.</p

Changes in the proportions of intraocular macrophages and peripheral blood monocytes in <i>CCR2</i>^<i>-/-</i> mice after laser injury.

<p>(A) Ly6C^hi macrophage recruitment into the eye was inhibited in <i>CCR2</i>^<i>-/-</i> mice compared with controls without laser treatment, at 3 days and 7 days after laser injury. In contrast, there were no differences in intraocular Ly6C^lo and Ly6C^int/loCD64⁺ cells between two groups with or without laser injury. (B) The recruitment of both Ly6C^hi and Ly6C^int monocyte from bone marrow into the peripheral blood were also significantly inhibited by the absence of CCR2 expression with or without laser injury, whereas there were no significant changes in Ly6C^lo monocytes between two groups. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p