Poly-arginine-18 peptides do not exacerbate bleeding, or improve functional outcomes following collagenase-induced intracerebral hemorrhage in the rat

Autoři: Lane Liddle aff001;  Ryan Reinders aff001;  Samantha South aff002;  David Blacker aff003;  Neville Knuckey aff003;  Frederick Colbourne aff001;  Bruno Meloni aff003
Působiště autorů: Department of Psychology, University of Alberta, Edmonton, Alberta, Canada aff001;  Office of Research Enterprise, The University of Western Australia, Western Australia, Australia aff002;  Perron Institute for Neurological and Translational Science, Nedlands, Western Australia, Australia aff003;  Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Nedlands, Western Australia, Australia aff004;  Department of Neurology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia aff005;  Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia aff006;  Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Alberta, Canada aff007
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224870



Cationic arginine-rich peptides (CARPs) have demonstrated neuroprotective and/or behavioural efficacy in ischemic and hemorrhagic stroke and traumatic brain injury models. Therefore, in this study we investigated the safety and neuroprotective efficacy of the CARPs poly-arginine-18 (R18; 18-mer of arginine) and its D-enantiomer R18D given in the acute bleeding phase in an intracerebral hemorrhage (ICH) model.


One hundred and fifty-eight male Sprague-Dawley rats received collagenase-induced ICH. Study 1 examined various doses of R18D (30, 100, 300, or 1000 nmol/kg) or R18 (100, 300, 1000 nmol/kg) administered intravenously 30 minutes post-collagenase injection on hemorrhage volume 24 hours after ICH. Study 2 examined R18D (single intravenous dose) or R18 (single intravenous dose, plus 6 daily intraperitoneal doses) at 300 or 1000 nmol/kg commencing 30 minutes post-collagenase injection on behavioural outcomes (Montoya staircase test, and horizontal ladder test) in the chronic post-ICH period. A histological assessment of tissue loss was assessed using a Nissl stain at 28 days after ICH.


When administered during ongoing bleeding, neither R18 or R18D exacerbated hematoma volume or worsened functional deficits. Lesion volume assessment at 28 days post-ICH was not reduced by the peptides; however, animals treated with the lower R18D 300 nmol/kg dose, but not with the higher 1000 nmol/kg dose, demonstrated a statistically increased lesion size compared to saline treated animals.


Overall, both R18 and R18D appeared to be safe when administered during a period of ongoing bleeding following ICH. Neither peptide appears to have any statistically significant effect in reducing lesion volume or improving functional recovery after ICH. Additional studies are required to further assess dose efficacy and safety in pre-clinical ICH studies.

Klíčová slova:

Brain damage – Collagenases – Hemorrhagic stroke – Histology – Ischemic stroke – Rats – Carps


1. Sacco S, Marini C, Toni D, Olivieri L, Carolei A. Incidence and 10-year survival of intracerebral hemorrhage in a population-based registry. Stroke. 2009; 40:394–9. doi: 10.1161/STROKEAHA.108.523209 19038914

2. van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ. Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol. 2010; 9:167–76. doi: 10.1016/S1474-4422(09)70340-0 20056489

3. Qureshi AI, Mendelow AD, Hanley DF. Intracerebral haemorrhage. Lancet (London, England). 2009; 373:1632–44. doi: 10.1016/S0140-6736(09)60371-8 19427958

4. Anderson CS, Heeley E, Huang Y, Wang J, Stapf C, Delcourt C, et al. Rapid blood-pressure lowering in patients with acute intracerebral hemorrhage. N Engl J Med. 2013; 368:2355–65. doi: 10.1056/NEJMoa1214609 23713578

5. Mayer SA, Brun NC, Begtrup K, Broderick J, Davis S, Diringer MN, et al. Recombinant activated factor VII for acute intracerebral hemorrhage. N Engl J Med. 2005; 352:777–85. doi: 10.1056/NEJMoa042991 15728810

6. LoPresti MA, Bruce SS, Camacho E, Kunchala S, Dubois BG, Bruce E, et al. Hematoma volume as the major determinant of outcomes after intracerebral hemorrhage. J Neurol Sci. 2014; 345:3–7. doi: 10.1016/j.jns.2014.06.057 25034055

7. Pandey AS, Xi G. Intracerebral hemorrhage: a multimodality approach to improving outcome. Vol. 5, Translational stroke research. United States; 2014. p. 313–5. doi: 10.1007/s12975-014-0344-z 24764218

8. Meloni BP, Brookes LM, Clark VW, Cross JL, Edwards AB, Anderton RS, et al. Poly-arginine and arginine-rich peptides are neuroprotective in stroke models. J Cereb Blood Flow Metab. 2015; 35:993–1004. doi: 10.1038/jcbfm.2015.11 25669902

9. Meloni BP, Milani D, Edwards AB, Anderton RS, O’Hare Doig RL, Fitzgerald M, et al. Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties. Pharmacol Ther. 2015; 153:36–54. doi: 10.1016/j.pharmthera.2015.06.002 26048328

10. Instrum R, Sun H. Restoring neuroprotection through a new preclinical paradigm: translational success for NA-1 in stroke therapy. Acta Pharmacol Sin. 2013; 34:3–5. doi: 10.1038/aps.2012.175 23262667

11. Ballarin B, Tymianski M. Discovery and development of NA-1 for the treatment of acute ischemic stroke. Acta Pharmacol Sin. 2018; 39:661–8. doi: 10.1038/aps.2018.5 29565039

12. Chiu LS, Anderton RS, Cross JL, Clark VW, Edwards AB, Knuckey NW, et al. Assessment of R18, COG1410, and APP96-110 in excitotoxicity and traumatic brain injury. Transl Neurosci. 2017; 8:147–57. doi: 10.1515/tnsci-2017-0021 29177102

13. Moutal A, Francois-Moutal L, Brittain JM, Khanna M, Khanna R. Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides. Front Cell Neurosci. 2014; 8:471. doi: 10.3389/fncel.2014.00471 25674050

14. Brittain JM, Chen L, Wilson SM, Brustovetsky T, Gao X, Ashpole NM, et al. Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2). J Biol Chem. 2011; 286:37778–92. doi: 10.1074/jbc.M111.255455 21832084

15. Szeto HH, Liu S, Soong Y, Wu D, Darrah SF, Cheng F-Y, et al. Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury. J Am Soc Nephrol. 2011; 22:1041–52. doi: 10.1681/ASN.2010080808 21546574

16. Birk A V, Chao WM, Liu S, Soong Y, Szeto HH. Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia. Biochim Biophys Acta. 2015; 1847:1075–84. doi: 10.1016/j.bbabio.2015.06.006 26071084

17. Kacprzak MM, Peinado JR, Than ME, Appel J, Henrich S, Lipkind G, et al. Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine. J Biol Chem. 2004; 279:36788–94. doi: 10.1074/jbc.M400484200 15197180

18. Cameron A, Appel J, Houghten RA, Lindberg I. Polyarginines are potent furin inhibitors. J Biol Chem. 2000; 275:36741–9. doi: 10.1074/jbc.M003848200 10958789

19. Rosenberg GA. Neurological diseases in relation to the blood-brain barrier. J Cereb Blood Flow Metab. 2012; 32:1139–51. doi: 10.1038/jcbfm.2011.197 22252235

20. Righy C, Bozza MT, Oliveira MF, Bozza FA. Molecular, Cellular and Clinical Aspects of Intracerebral Hemorrhage: Are the Enemies Within? Curr Neuropharmacol. 2016; 14:392–402. doi: 10.2174/1570159X14666151230110058 26714583

21. Hazell AS. Excitotoxic mechanisms in stroke: an update of concepts and treatment strategies. Neurochem Int. 2007; 50:941–53. doi: 10.1016/j.neuint.2007.04.026 17576023

22. Qureshi AI, Ali Z, Suri MFK, Shuaib A, Baker G, Todd K, et al. Extracellular glutamate and other amino acids in experimental intracerebral hemorrhage: an in vivo microdialysis study. Crit Care Med. 2003; 31:1482–9. doi: 10.1097/01.CCM.0000063047.63862.99 12771622

23. Kim-Han JS, Kopp SJ, Dugan LL, Diringer MN. Perihematomal mitochondrial dysfunction after intracerebral hemorrhage. Stroke. 2006; 37:2457–62. doi: 10.1161/01.STR.0000240674.99945.4e 16960094

24. Power C, Henry S, Del Bigio MR, Larsen PH, Corbett D, Imai Y, et al. Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases. Ann Neurol. 2003; 53:731–42. doi: 10.1002/ana.10553 12783419

25. Wang J, Tsirka SE. Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage. Brain. 2005; 128:1622–33. doi: 10.1093/brain/awh489 15800021

26. Li L, Ke K, Tan X, Xu W, Shen J, Zhai T, et al. Up-regulation of NFATc4 involves in neuronal apoptosis following intracerebral hemorrhage. Cell Mol Neurobiol. 2013; 33:893–905. doi: 10.1007/s10571-013-9955-2 23852416

27. MacLellan CL, Silasi G, Poon CC, Edmundson CL, Buist R, Peeling J, et al. Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion. J Cereb Blood Flow Metab. 2008; 28:516–25. doi: 10.1038/sj.jcbfm.9600548 17726491

28. Beray-Berthat V, Delifer C, Besson VC, Girgis H, Coqueran B, Plotkine M, et al. Long-term histological and behavioural characterisation of a collagenase-induced model of intracerebral haemorrhage in rats. J Neurosci Methods. 2010; 191:180–90. doi: 10.1016/j.jneumeth.2010.06.025 20600312

29. Milani D, Cross JL, Anderton RS, Blacker DJ, Knuckey NW, Meloni BP. Neuroprotective efficacy of poly-arginine R18 and NA-1 (TAT-NR2B9c) peptides following transient middle cerebral artery occlusion in the rat. Neurosci Res. 2017; 114:9–15. doi: 10.1016/j.neures.2016.09.002 27639457

30. Edwards AB, Cross JL, Anderton RS, Knuckey NW, Meloni BP. Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat. Mol Brain. 2018; 11:8. doi: 10.1186/s13041-018-0352-0 29426351

31. Rosenberg GA, Mun-Bryce S, Wesley M, Kornfeld M. Collagenase-induced intracerebral hemorrhage in rats. Stroke. 1990; 21:801–7. doi: 10.1161/01.str.21.5.801 2160142

32. Choudhri TF, Hoh BL, Solomon RA, Connolly ESJ, Pinsky DJ. Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice. Stroke. 1997; 28:2296–302. doi: 10.1161/01.str.28.11.2296 9368579

33. Williamson MR, Dietrich K, Hackett MJ, Caine S, Nadeau CA, Aziz JR, et al. Rehabilitation Augments Hematoma Clearance and Attenuates Oxidative Injury and Ion Dyshomeostasis After Brain Hemorrhage. Stroke. 2017; 48:195–203. doi: 10.1161/STROKEAHA.116.015404 27899761

34. Montoya CP, Campbell-Hope LJ, Pemberton KD, Dunnett SB. The “staircase test”: a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods. 1991; 36:219–28. doi: 10.1016/0165-0270(91)90048-5 2062117

35. MacLellan CL, Auriat AM, McGie SC, Yan RHY, Huynh HD, De Butte MF, et al. Gauging recovery after hemorrhagic stroke in rats: implications for cytoprotection studies. J Cereb Blood Flow Metab. 2006; 26:1031–42. doi: 10.1038/sj.jcbfm.9600255 16395282

36. Metz GA, Whishaw IQ. Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and co-ordination. J Neurosci Methods. 2002; 115:169–79. doi: 10.1016/s0165-0270(02)00012-2 11992668

37. Li Q, Weiland A, Chen X, Lan X, Han X, Durham F, et al. Ultrastructural characteristics of neuronal death and white matter injury in mouse brain tissues after intracerebral hemorrhage: coexistence of ferroptosis, autophagy, and necrosis. Front Neurol. 2018; 9.

38. Wilkinson CM, Fedor BA, Aziz JR, Nadeau CA, Brar PS, Clark JJA, et al. Failure of bumetanide to improve outcome after intracerebral hemorrhage in rat. PLoS One. 2019; 14:e0210660. doi: 10.1371/journal.pone.0210660 30629699

39. Meloni BP, Brookes LM, Clark VW, Cross JL, Edwards AB, Anderton RS, et al. Poly-arginine and arginine-rich peptides are neuroprotective in stroke models. J Cereb Blood Flow Metab. 2015; 35:993–1004. doi: 10.1038/jcbfm.2015.11 25669902

40. Marshall J, Wong KY, Rupasinghe CN, Tiwari R, Zhao X, Berberoglu ED, et al. Inhibition of N-methyl-D-aspartate-induced retinal neuronal death by polyarginine peptides is linked to the attenuation of stress-induced hyperpolarization of the inner mitochondrial membrane potential. J Biol Chem. 2015; 290:22030–48. doi: 10.1074/jbc.M115.662791 26100636

41. da Silva-Candal A, Vieites-Prado A, Gutierrez-Fernandez M, Rey RI, Argibay B, Mirelman D, et al. Blood glutamate grabbing does not reduce the hematoma in an intracerebral hemorrhage model but it is a safe excitotoxic treatment modality. J Cereb Blood Flow Metab. 2015; 35:1206–12. doi: 10.1038/jcbfm.2015.28 25735920

42. Bye N, Habgood MD, Callaway JK, Malakooti N, Potter A, Kossmann T, et al. Transient neuroprotection by minocycline following traumatic brain injury is associated with attenuated microglial activation but no changes in cell apoptosis or neutrophil infiltration. Exp Neurol. 2007; 204:220–33. doi: 10.1016/j.expneurol.2006.10.013 17188268

43. MacLellan CL, Girgis J, Colbourne F. Delayed onset of prolonged hypothermia improves outcome after intracerebral hemorrhage in rats. J Cereb Blood Flow Metab. 2004; 24:432–40. doi: 10.1097/00004647-200404000-00008 15087712

44. Davis SM, Broderick J, Hennerici M, Brun NC, Diringer MN, Mayer SA, et al. Hematoma growth is a determinant of mortality and poor outcome after intracerebral hemorrhage. Neurology. 2006; 66:1175–81. doi: 10.1212/01.wnl.0000208408.98482.99 16636233

45. Adeoye O, Clark JF, Khatri P, Wagner KR, Zuccarello M, Pyne-Geithman GJ. Do current animal models of intracerebral hemorrhage mirror the human pathology? Transl Stroke Res. 2011; 2:17–25. doi: 10.1007/s12975-010-0037-1 24323583

46. Larrue V, von Kummer R, del Zoppo G, Bluhmki E. Hemorrhagic transformation in acute ischemic stroke. Potential contributing factors in the European Cooperative Acute Stroke Study. Stroke. 1997; 28:957–60. doi: 10.1161/01.str.28.5.957 9158632

47. Paciaroni M, Agnelli G, Corea F, Ageno W, Alberti A, Lanari A, et al. Early hemorrhagic transformation of brain infarction: rate, predictive factors, and influence on clinical outcome: results of a prospective multicenter study. Stroke. 2008; 39:2249–56. doi: 10.1161/STROKEAHA.107.510321 18535273

48. Balami JS, Chen RL, Grunwald IQ, Buchan AM. Neurological complications of acute ischaemic stroke. Lancet Neurol. 2011; 10:357–71. doi: 10.1016/S1474-4422(10)70313-6 21247806

49. Zhang J, Yang Y, Sun H, Xing Y. Hemorrhagic transformation after cerebral infarction: current concepts and challenges. Ann Transl Med. 2014; 2:81. doi: 10.3978/j.issn.2305-5839.2014.08.08 25333056

50. Nogueira RG, Gupta R, Jovin TG, Levy EI, Liebeskind DS, Zaidat OO, et al. Predictors and clinical relevance of hemorrhagic transformation after endovascular therapy for anterior circulation large vessel occlusion strokes: a multicenter retrospective analysis of 1122 patients. J Neurointerv Surg. 2015; 7:16–21. doi: 10.1136/neurintsurg-2013-010743 24401478

51. Sussman ES, Connolly E Jr. Hemorrhagic transformation: A review of the rate of hemorrhage in the major clinical trials of acute ischemic stroke. Front Neurol. 2013; 4 JUN:69. doi: 10.3389/fneur.2013.00069 23772220

52. Wang W, Li M, Chen Q, Wang J. Hemorrhagic Transformation after Tissue Plasminogen Activator Reperfusion Therapy for Ischemic Stroke: Mechanisms, Models, and Biomarkers. Mol Neurobiol. 2015; 52:1572–9. doi: 10.1007/s12035-014-8952-x 25367883

53. Borsello T, Clarke PGH, Hirt L, Vercelli A, Repici M, Schorderet DF, et al. A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med. 2003; 9:1180–6. doi: 10.1038/nm911 12937412

54. Corbett D, Carmichael ST, Murphy TH, Jones TA, Schwab ME, Jolkkonen J, et al. Enhancing the alignment of the preclinical and clinical stroke recovery research pipeline: Consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable translational working group. Neurorehabil Neural Repair. 2017; 31:699–707. doi: 10.1177/1545968317724285 28803530

55. DeBow SB, Davies MLA, Clarke HL, Colbourne F. Constraint-induced movement therapy and rehabilitation exercises lessen motor deficits and volume of brain injury after striatal hemorrhagic stroke in rats. Stroke. 2003; 34:1021–6. doi: 10.1161/01.STR.0000063374.89732.9F 12649509

56. Guptill JT, Raja SM, Boakye‐Agyeman F, Noveck R, Ramey S, Tu TM, et al. Phase 1 randomized, double‐blind, placebo‐controlled study to determine the safety, tolerability, and pharmacokinetics of a single escalating dose and repeated doses of CN‐105 in healthy adult subjects. J Clin Pharmacol. 2017; 57:770–6. doi: 10.1002/jcph.853 27990643

57. Michel-Monigadon D, Bonny C, Hirt L. C-Jun N-terminal kinase pathway inhibition in intracerebral hemorrhage. Cerebrovasc Dis. 2010; 29:564–70. doi: 10.1159/000306643 20375499

58. Laskowitz DT, Lei B, Dawson HN, Wang H, Bellows ST, Christensen DJ, et al. The apoE-mimetic peptide, COG1410, improves functional recovery in a murine model of intracerebral hemorrhage. Neurocrit Care. 2012; 16:316–26. doi: 10.1007/s12028-011-9641-5 21989844

59. Lei B, Mace B, Bellows ST, Sullivan PM, Vitek MP, Laskowitz DT, et al. Interaction between sex and apolipoprotein e genetic background in a murine model of intracerebral hemorrhage. Transl Stroke Res. 2012; 3:94–101. doi: 10.1007/s12975-012-0176-7 23935764

60. Lei B, James ML, Liu J, Zhou G, Venkatraman TN, Lascola CD, et al. Neuroprotective pentapeptide CN-105 improves functional and histological outcomes in a murine model of intracerebral hemorrhage. Sci Rep. 2016; 6:34834. doi: 10.1038/srep34834 27713572

61. Bao H, Yang X, Huang Y, Qiu H, Huang G, Xiao H, et al. The neuroprotective effect of apelin-13 in a mouse model of intracerebral hemorrhage. Neurosci Lett. 2016; 628:219–24. doi: 10.1016/j.neulet.2016.06.046 27343409

62. Wang Z, Chen Z, Yang J, Yang Z, Yin J, Duan X, et al. Treatment of secondary brain injury by perturbing postsynaptic density protein-95-NMDA receptor interaction after intracerebral hemorrhage in rats. J Cereb Blood Flow Metab. 2018; doi: 10.1177/0271678X18762637 29513122

Článek vyšel v časopise


2019 Číslo 11