Philanthotoxin-343 attenuates retinal and optic nerve injury, and protects visual function in rats with N-methyl-D-aspartate-induced excitotoxicity

Abstract

Retinal ganglion cell (RGC) loss and optic neuropathy, both hallmarks of glaucoma, have been shown to involve N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity. This study investigated the neuroprotective effects of Philanthotoxin (PhTX)-343 in NMDAinduced retinal injury to alleviate ensuing visual impairments. Sprague-Dawley rats were divided into three; Group I was intravitreally injected with phosphate buffer saline as the control, Group II was injected with NMDA (160 nM) to induce retinal excitotoxic injury, while Group III was injected with PhTX-343 (160 nM) 24 h prior to excitotoxicity induction with NMDA. Rats were subjected to visual behaviour tests seven days post-treatment and subsequently euthanized. Rat retinas and optic nerves were subjected to H&E and toluidine blue staining, respectively. Histological assessments showed that NMDA exposure resulted in significant loss of retinal cell nuclei and thinning of ganglion cell layer (GCL). PhTX-343 pre-treatment prevented NMDA-induced changes where the RGC layer morphology is similar to the control. The numbers of nuclei in the NMDA group were markedly lower compared to the control (p<0.05). PhTX-343 group had significantly higher numbers of nuclei within 100 μm length and 100 μm 2 area of GCL (2.9-and 1.7-fold, respectively) compared to NMDA group (p<0.05). PhTX-343 group also displayed lesser optic nerve fibres degeneration compared to NMDA group which showed vacuolation in all sections. In the visual behaviour test, the NMDA group recorded higher total distance travelled, and lower total immobile time and episodes compared to the control and PhTX-343 groups (p<0.05). Object recognition tests showed that the rats in PhTX-343 group could recognize objects better, whereas the same objects were identified as novel by NMDA rats despite multiple exposures (p<0.05). Visual performances in the PhTX-343 group were all comparable with the control

Neuroprotection Against NMDA-Induced Retinal Damage by Philanthotoxin-343 Involves Reduced Nitrosative Stress

“…Prior to administering the treatments, animals were anaesthetized through intraperitoneal administration of ketamine (80 mg/kg) and xylazine (12 mg/kg) mixture (Ilium Troy Laboratories, Glendenning, New South Wales, Australia). All treatments were administrated via intravitreal injection in both eyes ( Lambuk et al, 2019b ; Fazel et al, 2020 ). Rats in group 1 were intravitreally injected with phosphate buffer saline (PBS) (Sigma-Aldrich, St. Louis, MO, United States ) and served as the negative control group.…”

“…Rats in group 1 were intravitreally injected with phosphate buffer saline (PBS) (Sigma-Aldrich, St. Louis, MO, United States ) and served as the negative control group. Group 2 rats were injected with NMDA (Sigma-Aldrich) at a concentration of 160 nM to induce retinal excitotoxic injury ( Lambuk et al, 2019a ; Fazel et al, 2020 ). Rats in group 3 were treated with PhTX-343, 24 h prior to NMDA administration as the experimental group.…”

“…The procedure was conducted slowly over 2 min to prevent pressure-induced retinal damage. Neomycin and Polymyxin B Sulfates ophthalmic suspension (Bausch + Lomb, Quebec, Canada) were applied post-injection ( Fazel et al, 2020 ). After 7 days of treatment, the rats were subjected to visual behaviour tests which were conducted between 8.00 and 10.00 am, during which the animals demonstrate the highest behavioural activities, and then were sacrificed.…”

“…In recent years, natural and synthetic bioactive compounds have been intensively explored and studied for their therapeutic potential against RGC loss in glaucomatous animal models ( Lambuk et al, 2017 ; Chou et al, 2018 ; Jafri et al, 2018 ; Lambuk et al, 2019a ; Jafri et al, 2019 ; Chou et al, 2020 ; Fazel et al, 2020 ; Jafri et al, 2020 ; Romano et al, 2020 ; Conti et al, 2021 ). Philanthotoxin (PhTX) is a potent NMDAR modulator ( Piek, 1982 ) representing a relevant therapeutic option to counteract excitotoxicity and prolong RGC survival in glaucoma.…”

“…A variety of synthetic PhTX analogues have been developed where structural modifications have been shown to improve affinity and selectivity of the compounds for NMDAR ( Mellor et al, 2003 ; Strømgaard et al, 2005 ; Frølund et al, 2010 ; Kachel et al, 2019 ; Banke and Barria, 2020 ). PhTX-343 is one of the synthetic analogues that is structurally similar to PhTX-433 ( Kachel et al, 2019 ), and it retains the active pharmacological properties of the parent compound ( Eldefrawi et al, 1988 ; Fazel et al, 2020 ). In the PhTX-343 molecule, the thermospermine tail (polyamine component) has been exchanged for the symmetrical spermine.…”