Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat

Abstract

We examined the neuroprotective efficacy of the poly-arginine peptide R18 and its D-enantiomer R18D in a perinatal hypoxic-ischaemic (HI) model in P7 Sprague-Dawley rats. R18 and R18D peptides were administered intraperitoneally at doses of 30, 100, 300 or 1000 nmol/kg immediately after HI (8% O₂/92%N₂ for 2.5 h). The previously characterised neuroprotective JNKI-1-TATD peptide at a dose of 1000 nmol/kg was used as a control. Infarct volume and behavioural outcomes were measured 48 h after HI. For the R18 and R18D doses examined, total infarct volume was reduced by 25.93% to 43.80% (P = 0.038 to < 0.001). By comparison, the JNKI-1-TATD reduced lesion volume by 25.27% (P = 0.073). Moreover, R18 and R18D treatment resulted in significant improvements in behavioural outcomes, while with JNKI-1-TATD there was a trend towards improvement. As an insight into the likely mechanism underlying the effects of R18, R18D and JNKI-1-TATD, the peptides were added to cortical neuronal cultures exposed to glutamic acid excitotoxicity, resulting in up to 89, 100 and 71% neuroprotection, respectively, and a dose dependent inhibition of neuronal calcium influx. The study further confirms the neuroprotective properties of poly-arginine peptides, and suggests a potential therapeutic role for R18 and R18D in the treatment of HIE.

Keywords: Cationic arginine-rich peptides (CARPs); Hypoxia-ischaemia; Hypoxic-ischaemic encephalopathy; Neuroprotection; Poly-arginine peptides; R18.

Fig. 1

Percentage infarct volume; percentage infarct volume, representative images of coronal brain slices and percentage infarct volume in brain slices for the different treatment groups as determined 48 h after HI. Treatments were administered intraperitoneally (saline or R18, R18D and JNKI-1-TATD; doses in nmol/kg) immediately after hypoxia. a Percentage of infarct volume when compared to total brain volume. b Representative TTC coronal brain slice 2, from saline and peptide treated animals. Minor adjustments to brightness and contrast has been made to improve digital images. c Infarct volume analysis in 2 mm coronal brain slices (slices numbered 1–6 from rostral to caudal) from saline and peptide treated animals. Statistical significance is expressed in the table. Values are mean ± SE; *P < 0.05, **P < 0.01 when compared to saline. JNKD = JNKI-1-TATD

Fig. 2

Behavioural measurements using righting reflex, negative geotactic response, wire-hang test and weight gain 48 h after HI. Treatments were administered intraperitoneally (saline, R18, R18D or JNKI-1-TATD; doses in nmol/kg). The sham procedure group was assessed 48 h following sham-surgery. a R18 righting reflex percentage improvement from 0 to 48 h. b R18D righting reflex percentage improvement from 0 to 48 h. c R18 negative geotactic response percentage improvement from 0 to 48 h. d R18D negative geotactic response percentage improvement from 0 to 48 h. e R18 wire-hang percentage improvement from 0 to 48 h. f R18D wire-hang percentage improvement from 0 to 48 h. g R18 weight percentage improvement from 0 to 48 h. h R18D weight percentage improvement from 0 to 48 h. Values are mean ± SE. *P < 0.05 when compared to saline. JNKD = JNKI-1-TATD

Fig. 3

Glutamic acid excitotoxicity model; R18, R18D and JNKI-1-TATD dose response study. Peptides present in neuronal cultures 10 min before and during 5 min glutamic acid exposure. Neuronal viability measured 24 h after glutamic acid exposure. Concentration of peptide in μmol/L. MTS data were expressed as percentage neuronal viability with no insult (100% viability) and glutamic acid control (5% viability). Values are mean ± SE; n = 4; *P < 0.05 when compared to no glutamic acid control. Cont = no treatment control. Glut = glutamic acid control. JNKD = JNKI-1-TATD

Fig. 4

Intracellular calcium assessment using Fura-2 AM after glutamic acid exposure in primary neuronal cultures. a Fluorescent Fura-2 AM tracers; fluorescent intensity (FI) of neuronal cultures 30 s before and after the addition (arrow) of glutamic acid (100 μM final concentration). Peptides (1, 2 and 5 μM) or glutamate receptor blockers (MK801/CNQX; 5 μM/5 μM) were added to neuronal cultures for 10 min and removed (time = 0) before glutamic acid addition (time = 30 s). Values are mean ± SE; n = 3. b Trapezoidal area under the curve (AUC) approximation of calcium kinetic tracers. AUC is determined at 35 s (point after glutamic acid addition. Values are mean ± SE; n = 3; *P < 0.05 when compared to glutamic acid control. Cont = no treatment control. Glut = glutamic acid control. Blkrs = glutamate receptor blockers (MK801/CNQX; 5 μM/5 μM). JNKD = JNKI-1-TATD