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. 2020 Mar 18;13(3):390-398.
doi: 10.18240/ijo.2020.03.04. eCollection 2020.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

Qian Zhang  1 , Mei-Fang Chu  1 , Yan-Hong Li  2 , Chun-Hua Li  1 , Run-Jia Lei  2 , Si-Cen Wang  3 , Bao-Jun Xiao  4 , Jian-Gang Yang  2
Affiliations

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

Qian Zhang et al. Int J Ophthalmol. .

Abstract

Aim: To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography (RP-HPLC), and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure (IOP) model.

Methods: The eyes of albino rabbits were administered topically and ipsilaterally with 0.2% moxonidine. A RP-HPLC method was employed for the identification and quantification of moxonidine between 2 and 480min, which presented in the aqueous humor and iris-ciliary body. Flash electroretinography (F-ERG) amplitude and superoxide dismutase (SOD) level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP. Histological and ultrastructural observation underwent to analyze the changes of retinal morphology, the inner retinal layers (IRL) thickness, and retinal ganglion cell (RGC) counting.

Results: Moxonidine was detectable between 2 and 480min after administration, and the peak concentration developed both in the two tissues at 30min, 0.51 µg/mL in aqueous humor and 1.03 µg/g in iris-ciliary body. In comparison to control, F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15 (P<0.01) in the high IOP model; SOD levels were significantly higher at all time-points (P<0.01) with a maximum level of 20.29 U/mgprot at day 15; and RGCs were significantly higher (P<0.05).

Conclusion: Moxonidine is a viable neuroprotective agent with application to high IOP model. All layers of retina, including RGC layer, retinal nerve fiber layer and INL, are more preserved after moxonidine administration. SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Keywords: moxonidine; neuroprotection; retinal ganglion cell; reversed-phase high-performance liquid chromatography; superoxide dismutase.

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Figures

Figure 1
Figure 1. The typical moxonidine separation pattern in aqueous humor and iris-ciliary body on an RP-HPLC column
A: The retention time of the peak in aqueous humor is measured at 12.023min. The peaks are well isolated and not overlapped. There is no interference with impurity peak. a: Standard moxonidine curve; b: An extract of blank aqueous humor; c: An extract of aqueous humor with moxonidine. B: The similar retention time in ciliary body. a: Standard moxonidine curve; b: An extract of blank iris-ciliary body; c: An extract of iris-ciliary body with moxonidine.
Figure 2
Figure 2. RP-HPLC chromatograms and mean concentrations of moxonidine in aqueous humor and iris-ciliary body from 2 to 480min after topical administration of 0.2% moxonidine eyedrop on rabbits' eyes
A: RP-HPLC chromatograms in aqueous humor; B: The mean concentrations in aqueous humor, the peak concentration was 0.51±0.11 µg/mL at 30min; C: RP-HPLC chromatograms in iris-ciliary body; D: The mean concentrations in iris-ciliary body.
Figure 3
Figure 3. Comparison of amplitude recovery rate of F-ERG b-wave and SOD activities between moxonidine and control eyes
A: The alteration of amplitude recovery rate of F-ERG b-wave; B: The alteration of SOD activities.
Figure 4
Figure 4. Hematoxylin-eosin stained retinal cross sections
A-E: The retinal changes at baseline, day 1, 3, 7 and 15 in moxonidine group, respectively; F-J: The structures at the counterpart time-point in control eyes. The retinal thickness, including outer nuclear layer (ONL), inner nuclear layer (INL) and retinal nerve fiber layer (RNFL), was slightly thinned in the moxonidine-treating eyes, while much thinned in control eyes. Magnification: 400×.
Figure 5
Figure 5. Transmission electron micrographs of RGCs after topical administration with moxonidine
The cell ultrastructure was treated with moxonidine (A) and palcebo (D) at baseline, and with moxonidine (B and C) and placebo (E and F) at day 7 in the eyes of high IOP model. Scale bar: 0.5 µm (A, C and F) and 2 µm (B, D and E).
Figure 6
Figure 6. The RGC counting and IRL thickness between moxonidine-induced and control eyes
A: In comparison to control, there are significant increase of RGCs in moxonidine-induced eyes; B: There was difference of IRL between moxonidine-induced and control eyes. On day 1, IRLs in control eyes, because of slightly more obvious edema, were higher than those in the moxonidine-induced eyes. Whereas, IRLs in moxonidine-induced eyes were significantly higher than those in control eyes on day 3, 7 and 15. aP<0.05; bP<0.01.
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