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. 2022 Dec 16:13:1082997.
doi: 10.3389/fphar.2022.1082997. eCollection 2022.

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

Shisi Hu  1   2   3   4 , Yaoyao Li  1   2   3 , Yuanjie Zhang  1   2   3 , Ruyi Shi  1   2   3 , Ping Tang  1   2   3 , Di Zhang  1   2   3 , Xiuli Kuang  1   2   3 , Jiangfan Chen  1   2   3 , Jia Qu  2   3 , Ying Gao  1   2   3
Affiliations

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

Shisi Hu et al. Front Pharmacol. .

Abstract

Adenosine A2A receptors (A2ARs) appear early in the retina during postnatal development, but the roles of the A2ARs in the morphogenesis of distinct types of retinal ganglion cells (RGCs) during postnatal development and neonatal inflammatory response remain undetermined. As the RGCs are rather heterogeneous in morphology and functions in the retina, here we resorted to the Thy1-YFPH transgenic mice and three-dimensional (3D) neuron reconstruction to investigate how A2ARs regulate the morphogenesis of three morphologically distinct types of RGCs (namely Type I, II, III) during postnatal development and neonatal inflammation. We found that the A2AR antagonist KW6002 did not change the proportion of the three RGC types during retinal development, but exerted a bidirectional effect on dendritic complexity of Type I and III RGCs and cell type-specifically altered their morphologies with decreased dendrite density of Type I, decreased the dendritic field area of Type II and III, increased dendrite density of Type III RGCs. Moreover, under neonatal inflammation condition, KW6002 specifically increased the proportion of Type I RGCs with enhanced the dendrite surface area and volume and the proportion of Type II RGCs with enlarged the soma area and perimeter. Thus, A2ARs exert distinct control of RGC morphologies to cell type-specifically fine-tune the RGC dendrites during normal development but to mainly suppress RGC soma and dendrite volume under neonatal inflammation.

Keywords: 3D reconstruction; adenosine A2A receptor; development; morphology; neonatal inflammation; retinal ganglion cell.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The effect of A2AR antagonist KW6002 on the proportion of different morphological types of RGCs during the development. (A) Timeline of KW6002 (or vehicle) administration in transgenic Thy-1 YFPH mice. (B) Representative flat-mounted whole retina showing three different morphological types of RGCs in different colors from transgenic Thy-1 YFPH mice (red, Type I; blue, Type II; green, Type III). The morphology of an individual RGC was revealed by immunohistochemistry and reconstructed by the Neurolucida system. Scale bar, 500 μm. (C) Representative 3D reconstructions of the three morphological types of RGCs as shown in B. Scale bar, 100 μm.(D,E) Quantitative evaluation of dendritic field area (D) and dendrite density (E) among different morphological types of RGCs. (F) The proportion of different morphological types of RGCs from the control and KW6002-treated mice during the development. Values are mean ± SEM; **p < 0.01, ***p < 0.001. 54 RGCs from six retinas (3 vehicle-treated mice) and 66 RGCs from eight retinas (4 KW6002-treated mice) were analyzed in the control and KW6002-treated group, respectively. On average, nine RGCs were analyzed in each eye in the control group, while 8.25 RGCs per eye were analyzed in KW6002-treated group.
FIGURE 2
FIGURE 2
The effect of KW6002 on the somatic morphology of the three RGC types during the development. (A) Representative pictures of somata in each RGC type examined from vehicle -treated and KW6002-treated group. Scale bar, 20 μm. (B,C) Comparative analysis of soma area (B) and perimeter (C) of different RGC types between vehicle-treated and KW6002-treated mice. Data represent mean ± SEM. *p < 0.05.
FIGURE 3
FIGURE 3
KW6002 differentially affected the dendritic morphology of RGCs during the development. (A) Representative 3D reconstructions of the three RGC types from the vehicle-treated and KW6002-treated mice. (B–G) Comparative analysis of dendritic field area (B), dendrite density (C), dendrite segment number (D), dendrite length (E), dendrite surface area (F) and dendrite volume (G) in each RGC type between the vehicle-treated and KW6002-treated group. Data represent mean ± SEM. *p < 0.05; **p < 0.01. Scale bar, 100 μm.
FIGURE 4
FIGURE 4
The dendritic complexity of RGCs was differentially altered by KW6002 during the development.(A–C) Quantitative Sholl analysis of dendrite intersection numbers in each RGC type from the vehicle-treated and KW6002-treated group during the development. Inset, a series of concentric circles with increasing radius at a 10 μm step were superimposed on the RGC. Data represent mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 5
FIGURE 5
KW6002 changed the proportion of RGC morphological types after neonatal inflammation. (A) Timeline of KW6002 (or vehicle) administration with an intraperitoneal injection of LPS in transgenic Thy-1 YFPH mice. (B) The proportion of different morphological types of RGCs from the control and KW6002-treated mice after neonatal LPS exposure. *p < 0.05. 63 RGCs from 14 retinas (7 vehicle-treated mice) and 68 RGCs from 12 retinas (6 KW6002-treated mice) in the control and KW6002-treated group after neonatal LPS exposure, respectively. On average, 4.5 and 5.67 RGCs per eye were analyzed in each group.
FIGURE 6
FIGURE 6
KW6002 enlarged the soma of Type II RGCs after neonatal LPS exposure. (A) Representative pictures of somata in each RGC type examined from vehicle -treated and KW6002-treated group after neonatal LPS exposure. Scale bar, 20 μm. (B,C) Comparative analysis of soma area (B) and perimeter (C) of different RGC types between vehicle-treated and KW6002-treated mice after neonatal LPS exposure. Data represent mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 7
FIGURE 7
The effect of KW6002 on the dendritic morphology of RGCs after neonatal LPS exposure. (A) Representative 3D reconstructions of the three RGC types from the vehicle-treated and KW6002-treated mice after neonatal LPS exposure. (B–G) Comparative analysis of dendritic field area (B), dendrite density (C), dendrite segment number (D), dendrite length (E), dendrite surface area (F) and dendrite volume (G) in each RGC type between the vehicle-treated and KW6002-treated group after neonatal LPS exposure. Data represent mean ± SEM. *p < 0.05; **p < 0.01. Scale bar, 100 μm.
FIGURE 8
FIGURE 8
KW6002 decreased the dendritic complexity of Type III RGCs after neonatal LPS exposure. (A–C) Quantitative Sholl analysis of dendrite intersection numbers in each RGC type from the vehicle-treated and KW6002-treated group after neonatal LPS exposure. Data represent mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 9
FIGURE 9
Summary of RGC morphological changes by KW6002 under physiological and pathological conditions The A2AR antagonist KW6002 differentially altered these morphological parameters of different RGC types during normal and neonatal inflammation. The arrow means significant up or down-regulation by KW6002, while horizontal line means no significant difference.
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