r18-peptide and Brain-Edema

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.

Topics: Animals; Astrocytes; Brain Damage, Chronic; Brain Edema; Cells, Cultured; Cerebral Cortex; Drug Evaluation, Preclinical; HEK293 Cells; Humans; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Male; Neurons; Neuroprotective Agents; Peptides; Rats; Rats, Sprague-Dawley; Rats, Wistar; Stereoisomerism

Following positive results with the poly-arginine peptide R18 when administered intravenously 30 or 60min after permanent and/or transient middle cerebral artery occlusion (MCAO; 90min) in the rat, we examined the effectiveness of the peptide when administered 2h after MCAO. R18 was administered intravenously (1000nmol/kg via jugular vein) after permanent MCAO or a transient 3-h MCAO or when administered intra-arterially (100nmol/kg via internal carotid artery) immediately after reperfusion following a transient 2-h MCAO. In the transient MCAO studies, the neuroprotective NA-1 peptide was used as a positive control. Infarct volume, cerebral edema and functional outcomes were measured 24h after MCAO. Following permanent or transient MCAO, neither R18 nor NA-1 significantly reduced infarct volume. However, following permanent MCAO, R18 appeared to reduce cerebral edema (p=0.006), whereas following a transient 3-h MCAO, R18 improved the time to remove adhesive tape (p=0.04) without significantly affecting cerebral edema. There was also a trend (p=0.07) towards improved rota-rod performance with R18 in both permanent and transient 3-h MCAO. Following a transient 2-h MCAO, R18 had no significant effects on cerebral edema or neurological score but did lessen the extent of weight loss. Overall, while R18 had no effect on infarct volume, the peptide reduced cerebral edema after permanent MCAO, and improved some functional outcomes after transient MCAO.

Topics: Animals; Brain Edema; Brain Ischemia; Carotid Artery Injuries; Carotid Artery, Internal; Cerebral Arteries; Cerebrovascular Disorders; Disease Models, Animal; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Ischemic Attack, Transient; Male; Neuroprotective Agents; Peptides; Rats; Reperfusion; Stroke