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. 2024 Apr 10;44(1):173.
doi: 10.1007/s10792-024-03095-9.

Axonal protection by combination of ripasudil and brimonidine with upregulation of p-AMPK in TNF-induced optic nerve degeneration

Mizuki Otsubo  1   2 , Kana Sase  3 , Chihiro Tsukahara  3 , Naoki Fujita  3 , Ibuki Arizono  4   3 , Naoto Tokuda  3 , Yasushi Kitaoka  4   3
Affiliations

Axonal protection by combination of ripasudil and brimonidine with upregulation of p-AMPK in TNF-induced optic nerve degeneration

Mizuki Otsubo et al. Int Ophthalmol. .

Abstract

Purpose: The ROCK inhibitor ripasudil hydrochloride hydrate was shown to have axonal protective effects in TNF-induced optic nerve degeneration. The α2-adrenoreceptor agonist brimonidine was also shown to exert axonal protection. The current study aimed to elucidate whether additive axonal protection was achieved by the simultaneous injection of ripasudil and brimonidine and examine the association with AMPK activation.

Methods: Intravitreal administration was performed in the following groups: PBS, TNF, or TNF with ripasudil, with brimonidine, or with a combination of ripasudil and brimonidine. Axon numbers were counted to evaluate the effects against axon loss. Immunoblot analysis was performed to examine phosphorylated AMPK expression in optic nerves, and immunohistochemical analysis was performed to evaluate the expression levels of p-AMPK and neurofilament in the optic nerve.

Results: Both ripasudil alone or brimonidine alone resulted in significant neuroprotection against TNF-induced axon loss. The combination of ripasudil and brimonidine showed additive protective effects. Combined ripasudil and brimonidine plus TNF significantly upregulated p-AMPK levels in the optic nerve compared with the TNF groups. Immunohistochemical analysis revealed that p-AMPK is present in axons and enhanced by combination therapy.

Conclusion: The combination of ripasudil and brimonidine may have additive protective effects compared with single-agent treatment alone. These protective effects may be at least partially associated with AMPK activation.

Keywords: AMPK; Brimonidine; Glaucoma; Ripasudi.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
The combination of ripasudil and brimonidine exerted additive axonal effects in TNF-induced axon loss. Histologic findings 2 weeks following injection of A PBS, B TNF, C TNF + 20 pmol ripasudil, D TNF + 2 pmol brimonidine, or E TNF + 20 pmol ripasudil and 2 pmol brimonidine. Scale bar = 10 μm F Quantification of axon numbers. Each column represents mean ± SD; seven rats were used in each group except PBS group. PBS group was from contralateral left eye in the TNF group. Total 28 rats were used. *p < 0.0001 compared with PBS injection; #p < 0.005 compared with TNF injection; †p < 0.001 compared with TNF injection; ##p < 0.0001 compared with TNF injection; ‡p < 0.001 compared with TNF + ripasudil injection; §p < 0.01 compared with TNF + brimonidine injection
Fig. 2
Fig. 2
Protein expression in optic nerves 1 week following intravitreal injection. A p-AMPK and AMPK expressions after injection of PBS, TNF, TNF + ripasudil, TNF + brimonidine, or TNF + ripasudil + brimonidine. B Immunoblotting values of p-AMPK are normalized to AMPK. C Immunoblotting values of p-AMPK are normalized to β-actin. D Immunoblotting values of AMPK are normalized to β-actin. Values are expressed as percentages of control and represent mean ± SEM n = 5–7 *p < 0.05 compared with TNF injection alone
Fig. 3
Fig. 3
Immunohistochemical analysis of the optic nerve 1 week after injection. The p-AMPK immunoreactivity was colocalized with neurofilament-positive fibers in TNF-treated optic nerves (arrows). Abundant p-AMPK immunoreactivity was found with neurofilament-positive fibers in the TNF + ripasudil + brimonidine treatment group (arrows). Scale bar = 50 µm; n = 4 per experimental group
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References

    1. Inoue T, Tanihara H (2013) Rho-associated kinase inhibitors: a novel glaucoma therapy. Prog Retin Eye Res 37:1–12. 10.1016/j.preteyeres.2013.05.002 - PubMed
    1. Sit AJ, Gupta D, Kazemi A, McKee H, Challa P, Liu KC, Lopez J, Kopczynski C, Heah T (2021) Netarsudil improves trabecular outflow facility in patients with primary open angle glaucoma or ocular hypertension: a phase 2 study. Am J Ophthalmol 226:262–269. 10.1016/j.ajo.2021.01.019 - PubMed
    1. Kaneko Y, Ohta M, Inoue T, Mizuno K, Isobe T, Tanabe S, Tanihara H (2016) Effects of K-115 (ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm’s canal endothelial cells. Sci Rep 19(6):19640. 10.1038/srep19640 - PMC - PubMed
    1. Vidal-Sanz M, Lafuente MP, Mayor S, Imperial JMD, Villegas-Pérez MP (2001) Retinal ganglion cell death induced by retinal ischemia. Neuroprotective effects of two alpha-2 agonists. Surv Ophthalmol 45:S261–S267 - PubMed
    1. Kim HS, Chang YI, Kim JH, Park CK (2007) Alteration of retinal intrinsic survival signal and effect of alpha2-adrenergic receptor agonist in the retina of the chronic ocular hypertension rat. Vis Neurosci 24:127–139. 10.1017/S0952523807070150 - PubMed

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