Neuroprotective effect of water-dispersible hesperetin in retinal ischemia reperfusion injury

Akito Shimouchi, Harumasa Yokota, Shinji Ono, Chiemi Matsumoto, Toshihiro Tamai, Hiroko Takumi, Subbadra P. Narayanan, Shoji Kimura, Hiroya Kobayashi, Ruth B. Caldwell, Taiji Nagaoka, Akitoshi Yoshida

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


Purpose: To determine whether water-dispersible hesperetin (WD-Hpt) can prevent degeneration of ganglion cell neurons in the ischemic retina. Methods: Ischemia reperfusion (I/R) injury was induced by increasing the intraocular pressure of mice to 110 mmHg for 40 min. Mice received daily intraperitoneal injections with either normal saline (NS, 0.3 ml/day) or WD-Hpt (0.3 ml, 200 mg/kg/day). Reactive oxygen species (ROS) was assessed by dihydroethidium and nitrotyrosine formation. Inflammation was estimated by microglial morphology in the retina. Lipopolysaccharide (LPS)-stimulated BV-2 cells were used to explore the anti-inflammatory effect of WD-Hpt on activated microglia by quantifying the expression of IL-1β using real-time quantitative reverse transcription-polymerase chain reaction. Ganglion cell loss was assessed by immunohistochemistry of NeuN. Glial activation was quantified with glial fibrillary acidic protein (GFAP) immunoreactivity. Apoptosis was evaluated with a terminal deoxynucleotidyl transferase (TUNEL) assay and immunohistochemistry of cleaved caspase-3. Phosphorylation of extracellular signal-regulated kinase (p-ERK) was surveyed by western blotting. Results: WD-Hpt decreased I/R-induced ROS formation. WD-Hpt alleviated microglial activation induced by I/R and reduced mRNA levels of IL-1β in LPS-stimulated BV-2. I/R resulted in a 37 % reduction in the number of ganglion cells in the NS-treated mice, whereas the reduction was only 5 % in the WD-Hpt-treated mice. In addition, WD-Hpt mitigated the immunoreactivity of GFAP, increased expression of cleaved caspase-3, increased number of TUNEL positive cells and p-ERK after I/R. Conclusions: WD-Hpt protected ganglion cells from I/R injury by inhibiting oxidative stress and modulating cell death signaling. Moreover, WD-Hpt had an anti-inflammatory effect through the suppression of activated microglia.

Original languageEnglish (US)
Pages (from-to)51-61
Number of pages11
JournalJapanese Journal of Ophthalmology
Issue number1
StatePublished - Jan 1 2016


  • Hesperetin
  • Inflammation
  • Ischemia–reperfusion
  • Neuroprotection
  • Retina

ASJC Scopus subject areas

  • Ophthalmology


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