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.
Highlights
Stroke is a major cause of death and disability globally, with no clinically effective pharmacological neuroprotective treatments available to reduce brain injury and improve patient outcomes
The primary objective of the present study was to compare the neuroprotective efficacy of Land D-isoforms of the R18D peptide in a permanent middle cerebral artery occlusion (MCAO) stroke model in the Wistar rat
Using a peptide dose of 300 nmol/kg administered 30 minutes after MCAO, R18D demonstrated a greater capacity to reduce infarct volume compared to R18, while both peptides reduce cerebral hemisphere swelling and improve functional outcomes to a similar degree
Summary
Stroke is a major cause of death and disability globally, with no clinically effective pharmacological neuroprotective treatments available to reduce brain injury and improve patient outcomes. We have previously demonstrated that cationic arginine-rich and poly-arginine peptides (here after referred to as CARPs) possess potent neuroprotective properties in both in vitro and in vivo stroke related injury models [1,2,3,4,5,6,7,8,9,10] with peptide arginine content and peptide positive charge being critical for neuroprotection [2,3]; findings confirmed by others [11,12]. Additional studies [6,7] using both transient and permanent middle cerebral artery stroke models demonstrated that R18 has superior neuroprotective efficacy than the previously characterised NR2B9c peptide fused to the cationic cell penetrating peptide TAT (TAT-NR2B9c; known as NA-1) [18,19]. Given that peptides synthesised with D-isoform amino acids have an increased resistance to proteolytic degradation and consequent higher serum stability [22,23,24] they represent potentially even more effective therapeutic agents
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