Comparison of neuroprotective efficacy of poly-arginine R18 and R18D (D-enantiomer) peptides following permanent middle cerebral artery occlusion in the Wistar rat and in vitro toxicity studies

Abstract

We have previously demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties, with poly-arginine peptide R18 identified as being highly effective at reducing infarct volume following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. Since peptides synthesised using D-isoform amino acids have greater stability than L-isoform peptides due to increased resistance to proteolytic degradation, they represent potentially more effective peptide therapeutics. Therefore we compared the neuroprotective efficacy of R18 and its D-enantiomer R18D following permanent MCAO in the Wistar rat. Furthermore, as increased peptide stability may also increase peptide toxicity, we examined the effects of R18 and R18D on cultured cortical neurons, astrocytes, brain endothelial cells (bEND.3), and embryonic kidney cells (HEK293) following a 10-minute or 24-hour peptide exposure duration. The in vivo studies demonstrated that R18D resulted in a greater reduction in mean infarct volume compared to R18 (33%, p = 0.004 vs 12%, p = 0.27) after intravenous administration at 300 nmol/kg 30 minutes after MCAO. Both R18D and R18 reduced cerebral hemisphere swelling to a comparable degree (27%, p = 0.03 and 30%, p = 0.02), and improved neurological assessment scores (1.5, p = 0.02 and 2, p = 0.058 vs 3 for vehicle). No abnormal histological findings specific to peptide treatments were observed in hematoxylin and eosin stained sections of kidney, liver, spleen, lung and heart. In vitro studies demonstrated that R18 and R18D were most toxic to neurons, followed by astrocytes, HEK293 and bEND.3 cells, but only at high concentrations and/or following 24-hour exposure. These findings further highlight the neuroprotective properties of poly-arginine peptides, and indicate that R18D at the dose examined is more potent than R18 in Wistar rats, and justify continued investigation of the R18 peptide as a novel neuroprotective agent for stroke.

Fig 1. Infarct volume analysis, cerebral hemisphere swelling and representative images of coronal brain slices as assessed 24-hours after permanent MCAO.

Treatments were administered intravenously (saline vehicle, or R18 and R18D at 300 nmol/kg) 30 min after MCAO. A: Infarct volume measurements. ^†Denotes animal that died several hours before the 24-hour post-MCAO study end-point, but was still included in the final infarct volume analysis. Unfilled circles, triangles and squares represent infarct volumes obtained from outlier animals excluded from the study. B: Percentage hemisphere swelling for vehicle and the R18 and R18D treatment groups. C: Representative images of TTC stained coronal brain slices from vehicle, R18 and R18D treatment groups. Values are mean ± SD; n = 9–10. *p < 0.05 when compared to vehicle.

Fig 2. Neurological score and rota-rod measurements, 24-hours after permanent MCAO.

A. Neurological grading scores (0 = no deficit, 5 = major deficit) for vehicle (saline), R18 and R18D (300 nmol/kg) treatment groups. Vertical lines on graph indicate the range and median for neurological score; n = 7–10. *p < 0.05, when compared to the vehicle. Note: one R18 treated animal inadvertently did not undergo neurological assessment. B. Rota-rod performance for vehicle, R18 and R18D treatment groups. Sec = seconds. Values are mean ± SD; n = 8–10.

Fig 3. Adhesive tape test measurements 24-hours after permanent MCAO.

Treatment groups are vehicle (saline), R18 and R18D (300 nmol/kg). Time (sec = seconds) to detect tape, time to remove tape and number of attempts to remove tape. Values are mean ± SD; n = 8–10. Maximum time allowed for adhesive tape removal was 120 seconds.

Fig 4. Cell viability and cell death in cortical neuronal cell cultures following exposure to different concentrations of R18 or R18D peptide for either 10 minutes or 24 hours as measured by MTS and LDH assays.

Peptide concentration in μM. Cell viability was measured 24-hours after the addition of peptide. A, B, C, D: R18 and E, F, G, H: R18D. *p < 0.05 when compared to control [Cont = 100% cell viability (MTS assay) or 0% cell death (LDH assay)]. SDS = wells treated with SDS [SDS = 0% cell viability (MTS assay) or 100% cell death (LDH assay)]. Values are means ± SE; n = 4.

Fig 5. Cell viability and cell death in astrocyte cell cultures following exposure to different concentrations of R18 or R18D peptide for either 10 minutes or 24 hours as measured by MTS and LDH assays.

Peptide concentration in μM. Cell viability was measured 24-hours after the addition of peptide. A, B, C, D: R18 and E, F, G, H: R18D. *p < 0.05 when compared to control [Cont = 100% cell viability (MTS assay) or 0% cell death (LDH assay)]. SDS = wells treated with SDS [SDS = 0% cell viability (MTS assay) or 100% cell death (LDH assay)]. Values are means ± SE; n = 4.

Fig 6. Cell viability and cell death in b.End3 brain endothelial cell cultures following exposure to different concentrations of R18 or R18D peptide for either 10 minutes or 24 hours as measured by MTS and LDH assays.

Peptide concentration in μM. Cell viability was measured 24-hours after the addition of peptide. A, B, C, D: R18 and E, F, G, H: R18D. *p < 0.05 when compared to control [Cont = 100% cell viability (MTS assay) or 0% cell death (LDH assay)]. SDS = wells treated with SDS [SDS = 0% cell viability (MTS assay) or 100% cell death (LDH assay)]. Values are means ± SE; n = 4.

Fig 7. Cell viability and cell death in HEK293 embryonic kidney cell cultures following exposure to different concentrations of R18 or R18D peptide for either 10 minutes or 24 hours as measured by MTS and LDH assays.

Peptide concentration in μM. Cell viability was measured 24-hours after the addition of peptide. A, B, C D: R18 and E, F, G, H: R18D. *p < 0.05 when compared to control [Cont = 100% cell viability (MTS assay) or 0% cell death (LDH assay)]. SDS = wells treated with SDS [SDS = 0% cell viability (MTS assay) or 100% cell death (LDH assay)]. Values are means ± SE; n = 4.