Fig 1.
Flowcharts of retinal explant and retinal ganglion cell cultures.
(A) Retinas were isolated and divided into four pieces. The retinal explants were then cultured in a 12-well plate with the medium being replaced daily and the herbal extract added each day. (B) Retinas were isolated and collected in a tube. RGCs were purified via immunopanning and subsequently cultured in a 24-well plate. The herbal extract was added at Day 1 (DIV 1) in vitro. At DIV 2, half of the fresh culture medium was replaced and the herbal extract was added.
Fig 2.
Schematic diagrams of the quantification of the neurite outgrowth of retinal explants and retinal ganglion cells.
(A) The retinal explant images were split into two channels: blue (DAPI) and green (TUJ1). [1] The area of the retinal explant was defined using the blue channel, and the tissue perimeter was measured. [2] The total area of neurites expanded from the retinal explant was quantified. [3] The area outside the explant was divided into three regions: 100 μm (yellow), 100-200 μm (pink), > 200 μm (blue). Neurite areas grown out from the explants were measured in each of these regions. (B) The immunostained image of retinal ganglion cells consisted of blue (DAPI) and green (TUJ1) signals. [1] Neurite length was traced using Neuron J. [2] The length of grown neurite from the retinal ganglion cells was measured.
Fig 3.
C. cicadae mycelium extracts, mainly the aqueous extract, promote neurite outgrowth of retinal explants in P8 mice.
(A)-(D) Confocal images of retinal explants from P8 mice cultured without and with N6-(2-Hydroxyethyl) Adenosine (HEA) at concentrations of 1, 10, and 100 μg/mL. TUJ1 (green) shows the neurite outgrowth of retinal explants, and DAPI (blue) is a nuclear marker. (E) HEA did not affect the neurite outgrowth of retinal explants (n = 15 for the control; n = 8 for 1 μg/mL; n = 7 for 10 μg/mL; n = 7 for 100 μg/mL) (F)-(I) Confocal images of retinal explants cultured without and with the ethanol extract of C. cicadae (Cc-EtOH) at concentrations of 1, 10, and 100 μg/mL. (J) Cc-EtOH did not affect the neurite outgrowth of retinal explants (n = 15 for the control; n = 6 for 1 μg/mL; n = 6 for 10 μg/mL; n = 6 for 100 μg/mL). (K)-(N) Confocal images of retinal explants cultured without and with the aqueous extract of C. cicadae (Cc-H2O) at concentrations of 1, 10, and 100 μg/mL. (O) Cc-H2O at the concentration of 100 μg/mL enhanced neurite outgrowth of retinal explants (n = 15 for the control; n = 7 for 1 μg/mL; n = 7 for 10 μg/mL; n = 7 for 100 μg/mL). Note: Variability in TUJ1 signal intensity in certain regions likely reflects technical limitations in antibody penetration in areas directly contacting the coverslip, rather than biological variability. Scale bar 100 μm. *p < 0.05; Error bars, mean ± SEM.
Fig 4.
H. erinaceus extracts, mainly Erinacine S and the ethanol extract, promote neurite outgrowth of retinal explants in P8 mice.
(A)-(E) Confocal images of retinal explants cultured with Erinacine A (HeA) at concentrations of 0.05, 0.5, 5, and 10 μg/mL. TUJ1 (green) shows the neurite outgrowth of retinal explants, and DAPI (blue) is a nuclear marker. (F) Erinacine A did not affect the neurite outgrowth of retinal explants (n = 15 for the control; n = 10 for 0.05 μg/mL; n = 11 for 0.5 μg/mL; n = 8 for 5 μg/mL; n = 6 for 10 μg/mL). (G)-(K) Confocal images of retinal explants cultured with Erinacine S (HeS) at concentrations of 0.05, 0.5, 5, and 10 μg/mL. (L) Erinacine S at the concentration of 5 μg/mL promoted the highest neurite outgrowth in retinal explants, but there was no neurite outgrowth at a concentration of 10 μg/mL. (n = 15 for the control; n = 8 for 0.05 μg/mL; n = 7 for 0.5 μg/mL; n = 7 for 5 μg/mL; n = 7 for 10 μg/mL). (M)-(P) Confocal images of retinal explants cultured without and with the ethanol extract of H. erinaceus (He-EtOH) at concentrations of 1, 10, and 100 μg/mL. (Q) He-EtOH at the concentration of 100 μg/mL enhance the neurite outgrowth of retinal explants (n = 15 for the control; n = 10 for 1 μg/mL; n = 8 for 10 μg/mL; n = 8 for 100 μg/mL). Note: Variability in TUJ1 immunostaining intensity in certain areas is attributed to limited antibody penetration at the coverslip interface and does not reflect inconsistency in culture or staining procedures. Scale bar 100 μm. *p < 0.05; **p < 0.01; Error bars, mean ± SEM.
Fig 5.
C. cicadae and H.erinaceus extracts at all concentrations do not affect cell apoptosis of retinal explants.
(A)-(G) Confocal images of P8 retinal explants cultured without and with C. cicadae and H. erinaceus extracts, respectively. Retinal explants were immunostained with cleaved caspase-3 (red) and nuclear marker DAPI (blue). (H) N6-(2-Hydroxyethyl) Adenosine (HEA; 100 μg/mL), C. cicadae-EtOH (Cc-EtOH; 100 μg/mL), C. cicadae-H2O (Cc-H2O; 100 μg/mL), Erinacine A (HeA; 10 μg/mL), Erinacine S (HeS; 5 μg/mL), and H. erinaceus-EtOH (He-EtOH; 100 μg/mL) did not significantly affect the apoptosis of retinal ganglion cells (n = 9 for the control; n = 7 for HEA; n = 8 for CC-EtOH; n = 9 for CC-H2O; n = 6 for HeA; n = 7 for HeS; n = 7 for HE-EtOH). Scale bar 100 μm. Error bars, mean ± SEM.
Fig 6.
The effects of C. cicadae mycelium extracts at different concentrations on neurite outgrowth of retinal ganglion cells in P2-4 mice.
(A)-(D) Confocal images of retinal ganglion cells from P2-4 mice cultured without and with N6-(2-Hydroxyethyl) Adenosine (HEA) at concentrations of 1, 10, and 100 μg/mL. TUJ1 (green) shows the neurite outgrowth of retinal ganglion cells, and DAPI (blue) is a nuclear marker. (E) HEA did not significantly promote neurite outgrowth of retinal ganglion cells, rather it inhibited neurite growth at a higher concentration of 100 μg/mL (n = 13 for the control; n = 7 for 1 μg/mL; n = 7 for 10 μg/mL; n = 9 for 100 μg/mL). (F)-(I) Confocal images of RGCs cultured without and with C. cicadae-EtOH (Cc-EtOH) at concentrations of 1, 10 and 100 μg/mL. (J) C. cicadae-EtOH did not significantly affect neurite outgrowth of RGCs (n = 13 for the control; n = 6 for 1 μg/mL; n = 7 for 10 μg/mL; n = 12 for 100 μg/mL). (K)-(N) Confocal images of RGCs cultured without and with C. cicadae- H2O (Cc-H2O) at concentrations of 1, 10 and 100 μg/mL. (O) C. cicadae-H2O also had no significant effect on neurite outgrowth of retinal ganglion cells, rather it inhibited neurite growth at a concentration of 100 μg/mL (n = 13 for the control; n = 7 for 1 μg/mL; n = 6 for 10 μg/mL; n = 10 for 100 μg/mL). Scale bar 200 μm. *p < 0.05; **p < 0.01; Error bars, mean ± SEM.
Fig 7.
H. erinaceus extracts, mainly Erinacine S, promote neurite outgrowth of retinal ganglion cells in P2-4 mice.
(A)-(D) Confocal images of retinal explants cultured without and with Erinacine A (HeA) at concentrations of 0.05, 0.5, and 5 μg/mL. TUJ1 (green) shows the neurite outgrowth of retinal ganglion cells, and DAPI (blue) is a nuclear marker. (E) Erinacine A did not significantly affect the neurite outgrowth of retinal ganglion cells (n = 13 for the control; n = 7 for 0.05 μg/mL; n = 8 for 0.5 μg/mL; n = 9 for 5 μg/mL). (F)-(I) Confocal images of retinal ganglion cells cultured without and with Erinacine S (HeS) at concentrations of 0.05, 0.5, and 5 μg/mL. (J) Erinacine S at the concentration of 0.05 μg/mL promoted neurite outgrowth of retinal ganglion cells, but inhibited neurite growth at the concentration of 5 μg/mL. (n = 13 for the control; n = 6 for 0.05 μg/mL; n = 8 for 0.5 μg/mL; n = 11 for 5 μg/mL). (K)-(N) Confocal images of cultured retinal ganglion cells without and with the ethanol extract of H. erinaceus (He-EtOH) at concentrations of 1, 10, and 100 μg/mL. (O) H. erinaceus-EtOH did not significantly affect the neurite outgrowth of retinal ganglion cells, rather it inhibited neurite growth at the concentrations of 10 μg/mL and 100 μg/mL (n = 13 for the control; n = 6 for 1 μg/mL; n = 7 for 10 μg/mL; n = 11 for 100 μg/mL). Scale bar 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001; Error bars, mean ± SEM.