Restricted accessResearch articleFirst published online 2025-6
Isoform-selective and non-selective rho-kinase inhibitors do not affect collagenase-induced intracerebral hemorrhage outcomes in mice: Influence of sex and circadian cycle
Rho-associated protein kinase (ROCK) inhibitors are therapeutic candidates in ischemic stroke and subarachnoid hemorrhage. However, their efficacy in intracerebral hemorrhage (ICH) is unknown. Here, we tested the efficacy of fasudil (10 mg/kg), an isoform-nonselective ROCK inhibitor, and NRL-1049 (10 mg/kg), a novel inhibitor with 43-fold higher selectivity for ROCK2 isoform compared with ROCK1, in a collagenase-induced ICH model in mice. Both short (1–3 days) and prolonged (14 days) therapeutic paradigms were tested using robust sample sizes in both males and females and in active and inactive circadian stages. Outcome readouts included weight loss, mortality, hematoma volume, hemispheric swelling, brain water content, BBB permeability to large molecules, and sensorimotor and cognitive function. We found the treatments safe but not efficacious in improving the hematoma volume, BBB disruption, or neurological deficits in this collagenase-induced ICH model. Intriguingly, however, induction of ICH during the active circadian stage was associated with worse tissue and behavioral outcomes compared with the inactive stage.
CordonnierCDemchukAZiaiW, et al.
Intracerebral haemorrhage: current approaches to acute management. Lancet2018;
392: 1257–1268.
2.
GodoyDAPiñeroGDi NapoliM.Predicting mortality in spontaneous intracerebral hemorrhage: can modification to original score improve the prediction?Stroke2006;
37: 1038–1044.
3.
KeepRFHuaYXiG.Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol2012;
11: 720–731.
4.
ShaoZTuSShaoA.Pathophysiological mechanisms and potential therapeutic targets in intracerebral hemorrhage. Front Pharmacol2019;
10: 1079.
5.
Magid-BernsteinJGirardRPolsterS, et al.
Cerebral hemorrhage: pathophysiology, treatment, and future directions. Circ Res2022;
130: 1204–1229.
6.
ChenYChenSChangJ, et al.
Perihematomal edema after intracerebral hemorrhage: an update on pathogenesis, risk factors, and therapeutic advances. Front Immunol2021;
12: 740632.
7.
HartmannSRidleyAJLutzS.The function of rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease. Front Pharmacol2015;
6: 276.
ChrissobolisSSobeyCG.Recent evidence for an involvement of rho-kinase in cerebral vascular disease. Stroke2006;
37: 2174–2180.
10.
VesterinenHMCurrieGLCarterS, et al.
Systematic review and stratified meta-analysis of the efficacy of RhoA and rho kinase inhibitors in animal models of ischaemic stroke. Syst Rev2013;
2: 33.
11.
SladojevicNYuBLiaoJK.ROCK as a therapeutic target for ischemic stroke. Expert Rev Neurother2017;
17: 1167–1177.
12.
FujiiMDurisKAltayO, et al.
Inhibition of rho kinase by hydroxyfasudil attenuates brain edema after subarachnoid hemorrhage in rats. Neurochem Int2012;
60: 327–333.
13.
SharmaPRoyK.ROCK-2-selective targeting and its therapeutic outcomes. Drug Discov Today2020;
25: 446–455.
14.
ShibuyaMSuzukiYSugitaK, et al.
Dose escalation trial of a novel calcium antagonist, AT877, in patients with aneurysmal subarachnoid haemorrhage. Acta Neurochir (Wien)1990;
107: 11–15.
15.
ShibuyaMSuzukiYSugitaK, et al.
Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Results of a prospective placebo-controlled double-blind trial. J Neurosurg1992;
76: 571–577.
16.
ZhaoJZhouDGuoJ, et al.
Effect of fasudil hydrochloride, a protein kinase inhibitor, on cerebral vasospasm and delayed cerebral ischemic symptoms after aneurysmal subarachnoid hemorrhage. Neurol Med Chir (Tokyo)2006;
46: 421–428.
17.
LiuGJWangZJWangYF, et al.
Systematic assessment and meta-analysis of the efficacy and safety of fasudil in the treatment of cerebral vasospasm in patients with subarachnoid hemorrhage. Eur J Clin Pharmacol2012;
68: 131–139.
18.
ShibuyaMHiraiSSetoM, et al.
Effects of fasudil in acute ischemic stroke: results of a prospective placebo-controlled double-blind trial. J Neurol Sci2005;
238: 31–39.
19.
FuZChenYQinF, et al.
Increased activity of rho kinase contributes to hemoglobin-induced early disruption of the blood-brain barrier in vivo after the occurrence of intracerebral hemorrhage. Int J Clin Exp Pathol2014;
7: 7844–7853.
20.
AlhadidiQNashKMAlaqelS, et al.
Cofilin knockdown attenuates hemorrhagic brain injury-induced oxidative stress and microglial activation in mice. Neuroscience2018;
383: 33–45.
21.
LiLLouXZhangK, et al.
Hydrochloride fasudil attenuates brain injury in ICH rats. Transl Neurosci2020;
11: 75–86.
22.
ManaenkoAYangPNowrangiD, et al.
Inhibition of stress fiber formation preserves blood-brain barrier after intracerebral hemorrhage in mice. J Cereb Blood Flow Metab2018;
38: 87–102.
23.
HuangBKrafftPRMaQ, et al.
Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice. Neurobiol Dis2012;
46: 204–214.
24.
AkhterMQinTFischerP, et al.
Rho-kinase inhibitors do not expand hematoma volume in acute experimental intracerebral hemorrhage. Ann Clin Transl Neurol2018;
5: 769–776.
25.
RosenKMAm RuschelJMcKerracherL, et al. Rho kinase inhibitor BA-1049 (R) and active metabolites thereof. United States Patent 10,106,525. USPTO Patent Full-Text and Image Database, 2018.
26.
GreenJCaoJBandarageUK, et al.
Design, synthesis, and structure-activity relationships of pyridine-based rho kinase (ROCK) inhibitors. J Med Chem2015;
58: 5028–5037.
27.
MulderIAAbbinantiMWollerSA, et al.
The novel ROCK2 selective inhibitor NRL-1049 preserves the blood-brain barrier after acute injury. J Cereb Blood Flow Metab2024;
44: 1238–1252.
28.
McKerracherLShenkarRAbbinantiM, et al.
A brain-targeted orally available ROCK2 inhibitor benefits mild and aggressive cavernous angioma disease. Transl Stroke Res2020;
11: 365–376.
29.
Percie Du SertNHurstVAhluwaliaA, et al.
The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. J Cereb Blood Flow Metab2020;
40: 1769–1777.
30.
RikitakeYKimHHHuangZ, et al.
Inhibition of rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke2005;
36: 2251–2257.
31.
KoumuraAHamanakaJKawasakiK, et al.
Fasudil and ozagrel in combination show neuroprotective effects on cerebral infarction after murine middle cerebral artery occlusion. J Pharmacol Exp Ther2011;
338: 337–344.
32.
TakaseSLiaoJLiuY, et al.
Antipsychotic-like effects of fasudil, a rho-kinase inhibitor, in a pharmacologic animal model of schizophrenia. Eur J Pharmacol2022;
931: 175207.
33.
AykanSAXieHZhengY, et al.
Rho-kinase inhibition improves the outcome of focal subcortical white matter lesions. Stroke2022;
53: 2369–2376.
34.
DingJLiQYYuJZ, et al.
Fasudil, a rho kinase inhibitor, drives mobilization of adult neural stem cells after hypoxia/reoxygenation injury in mice. Mol Cell Neurosci2010;
43: 201–208.
35.
SheikhAMYanoSMitakiS, et al.
Rho-kinase inhibition decreases focal cerebral ischemia-induced glial activation in rats. J Cent Nerv Syst Dis2022;
14: 11795735221123910.
36.
MulherkarSFiroziKHuangW, et al.
RhoA-ROCK inhibition reverses synaptic remodeling and motor and cognitive deficits caused by traumatic brain injury. Sci Rep2017;
7: 10689.
37.
FujitaYYamashitaT.Axon growth inhibition by RhoA/ROCK in the central nervous system. Front Neurosci2014;
8: 338.
38.
FournierAETakizawaBTStrittmatterSM.Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci2003;
23: 1416–1423.
39.
RuanJYaoY.Behavioral tests in rodent models of stroke. Brain Hemorrhages2020;
1: 171–184.
40.
SadeghianHLacosteBQinT, et al.
Spreading depolarizations trigger caveolin-1-dependent endothelial transcytosis. Ann Neurol2018;
84: 409–423.
41.
ShiYZhangLPuH, et al.
Rapid endothelial cytoskeletal reorganization enables early blood-brain barrier disruption and long-term ischaemic reperfusion brain injury. Nat Commun2016;
7: 10523.
42.
AksoyDBammerRMlynashM, et al.
Magnetic resonance imaging profile of blood-brain barrier injury in patients with acute intracerebral hemorrhage. J Am Heart Assoc2013;
2: e000161.
43.
JiaPHeJLiZ, et al.
Profiling of blood-brain barrier disruption in mouse intracerebral hemorrhage models: collagenase injection vs. autologous arterial whole blood infusion. Front Cell Neurosci2021;
15: 699736.
44.
LieszAMiddelhoffMZhouW, et al.
Comparison of humoral neuroinflammation and adhesion molecule expression in two models of experimental intracerebral hemorrhage. Exp Transl Stroke Med2011;
3: 11.
45.
MacLellanCLSilasiGPoonCC, et al.
Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion. J Cereb Blood Flow Metab2008;
28: 516–525.
46.
GibsonCLSrivastavaKSpriggN, et al.
Inhibition of rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions. J Neurochem2014;
129: 816–826.
47.
LoEHAlbersGWDichgansM, et al.
Circadian biology and stroke. Stroke2021;
52: 2180–2190.
48.
MergenthalerPBalamiJSNeuhausAA, et al.
Stroke in the time of circadian medicine. Circ Res2024;
134: 770–790.
49.
EspositoELiWT MandevilleE, et al.
Potential circadian effects on translational failure for neuroprotection. Nature2020;
582: 395–398.
50.
FodorDMMartaMMPerju-DumbravaL.Implications of circadian rhythm in stroke occurrence: certainties and possibilities. Brain Sci2021;
11: 865.
51.
YaoXWuBXuY, et al.
Day-night variability of hematoma expansion in patients with spontaneous intracerebral hemorrhage. J Biol Rhythms2015;
30: 242–250.
52.
GongYZhangGLiB, et al.
BMAL1 attenuates intracerebral hemorrhage-induced secondary brain injury in rats by regulating the Nrf2 signaling pathway. Ann Transl Med2021;
9: 1617.
ShewardWJNaylorEKnowles-BarleyS, et al.
Circadian control of mouse heart rate and blood pressure by the suprachiasmatic nuclei: behavioral effects are more significant than direct outputs. PLoS One2010;
5: e9783.
55.
CurtisAMChengYKapoorS, et al.
Circadian variation of blood pressure and the vascular response to asynchronous stress. Proc Natl Acad Sci U S A2007;
104: 3450–3455.
56.
ZhangSLLahensNFYueZ, et al.
A circadian clock regulates efflux by the blood-brain barrier in mice and human cells. Nat Commun2021;
12: 617.
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