title: Neuroprotective effect of water-dispersible hesperetin in retinal ischemia reperfusion injury.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
authors.forename: Akito
authors.initials: A
authors.lastname: Shimouchi
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan. atokoy18@asahikawa-med.ac.jp.
authors.forename: Harumasa
authors.initials: H
authors.lastname: Yokota
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Ono
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Matsumoto
authors.affiliation: Institute of Health Sciences, Ezaki Glico Co., Ltd, Osaka, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Tamai
authors.affiliation: Institute of Health Sciences, Ezaki Glico Co., Ltd, Osaka, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Takumi
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Subbadra P
authors.initials: SP
authors.lastname: Narayanan
authors.affiliation: Division of Immune Pathology, Department of Pathology, Asahikawa Medical University, Asahikawa, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Kimura
authors.affiliation: Division of Immune Pathology, Department of Pathology, Asahikawa Medical University, Asahikawa, Japan.
authors.forename: Hiroya
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Ruth B
authors.initials: RB
authors.lastname: Caldwell
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Nagaoka
authors.affiliation: Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
authors.forename: Akitoshi
authors.initials: A
authors.lastname: Yoshida
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D004195:Disease Models, Animal; D006569:Hesperidin / Q000627:therapeutic use*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007274:Injections, Intraperitoneal; D053583:Interleukin-1beta / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018696:Neuroprotective Agents / Q000627:therapeutic use*; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015427:Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control*; D012164:Retinal Diseases / Q000378:metabolism / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000473:pathology; D012171:Retinal Vessels / Q000187:drug effects*; D012995:Solubility; D014867:Water
chemical_list: D015415:Biomarkers; D053583:Interleukin-1beta; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D014867:Water; D006569:Hesperidin; D020928:Mitogen-Activated Protein Kinases; C013015:hesperetin
keywords: Hesperetin; Inflammation; Ischemia–reperfusion; Neuroprotection; Retina