HISTAMINE H3 RECEPTOR LIGANDS IN PROGRESSIVE OPTIC NEUROPATHY

Glaucoma is the second leading cause of blindness worldwide. It is characterized by progressive optic nerve atrophy and loss of retinal ganglion cells (RGCs) caused by ocular hypertension (OHT). Intra-ocular pressure (IOP) reduction is the only therapeutic approach to preserve visual function in glaucomatous patients. The first line of treatment consists of topical IOP-lowering medications. The target IOP reduction of 25% is not easily reached/maintained, especially at night. Long-term IOP circadian fluctuation is associated with poor prognosis. The histaminergic tone is reduced at night and nocturnal IOP is higher than diurnal one. The aim of this work was to develop a reliable in vivo model of glaucoma and to investigate the role of the histaminergic system in IOP modulation; to study H3 receptor (H3R) antagonists’ hypotensive action; the long-term baroprotective effects on ocular hemodynamics, oxidative stress and inflammatory response in retinal glia and microglia. Transient and stable OHT models were induced by injection of 50 µl of hypertonic saline (5% NaCl in sterile water) into the vitreous or 100 µl carbomer (0.1%) in the anterior chamber of New Zealand albino rabbits’ eyes. IOP was measured with a Tono-Pen prior to saline or carbomer injection (baseline), 60, 120 and 240 minutes after transient OHT induction and, every day for 12 days, in stable OHT model before drug dosing. Color Doppler ultrasound examination was performed to evaluate changes in central retinal artery (CRA) Resistivity Index (RI) before and after repeated dosing of ciproxifan, DL-76, GSK189254 and timolol (1%). Oxidative stress was assessed by 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) determination using an ELISA kit and fluorescent dye dihydroethidium (DHE) staining. Morphological changes in retinal layers were assessed on histological sections by chemical (hematoxylin/eosin) and fluorescent staining (CD11B/IBA1 and GFAP/VEGFA double labeling). RT-PCR and Western blot analysis were performed to identify respectively mRNA and protein expression of histaminergic receptors, immunofluorescence staining from retinal and ciliary body biopsies to localize them. Selective histamine H3R antagonists Ciproxifan and DL76 and timolol lowered IOP both in transient and stable OHT in a statistically significant way, GSK189254 was less effective. Chronic treatment with H3R antagonists improved the vascular performance of central retinal artery. All histamine receptors were localized in retinal slides in the trabecular meshwork. H3R signal was present in trabecular meshwork cells as well. A clear correlation between IOP increase, vascular impairment of CRA and hypoxia-induced inflammation (VEGFA) was found, with the consequent increase of activated microglial cells and oxidative stress. These processes could be modulated by a baroprotective strategy involving H3R antagonism. In conclusion, these results demonstrated that the histaminergic system participates in IOP regulation and that H3R antagonists represent a future promising therapy for glaucoma.