J Cereb Blood Flow Metab. 2015 Mar;35(3):454-60.

U0126 attenuates cerebral vasoconstriction and improves long-term neurologic outcome after stroke in female rats.

Ahnstedt H1, Mostajeran M1, Blixt FW1, Warfvinge K1, Ansar S1, Krause DN2, Edvinsson L1.
  • 1Division of Experimental Vascular Research, Department of Clinical Sciences, Lund University, Lund, Sweden.
  • 2Department of Pharmacology, School of Medicine, University of California, Irvine, California, USA.

 

Abstract

Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.

PMID: 25492115

 

Supplements:

Stroke is the leading cause of disability worldwide [1]. Blood flow to an area of the brain is cut off either by a blood clot (ischemic stroke) or by rupture and bleeding from a blood vessel (hemorrhagic stroke). Within the first minutes, cells in affected brain regions are permanently damaged [2]. The longer time with restricted blood flow, the greater the brain injury. Thus it is essential to restore and control brain blood flow to limit further damage.

Our research focuses on the large arteries of the brain that play a key role in the regulation of brain blood flow. In our previous studies, the middle cerebral artery, one of the three major arteries of the brain and the most common site of human stroke, constricted more after ischemic stroke in rats. This is hypothesized to contribute to brain blood flow deficits and augment stroke damage. This increased constriction was seen after activation of receptors that bind endothelin-1, a powerful constricting peptide. In parallel, the number of endothelin-1 type B receptors in the smooth muscle cells of the vascular wall was increased. After ischemic stroke, increased constriction and changes in the number of endothelin-1 type B receptors, has been associated with activation of the RAF/MEK/ERK signaling pathway (Figure 1). In male rats, treatment with an inhibitor of this pathway, U0126, resulted in decreased brain damage and improved functional outcome, suggesting that preventing constriction of the middle cerebral artery after stroke is beneficial. However, this has until now not been investigated in female rats.

 

20150714_Figure World Front BiomedFigure 1. Ischemic stroke results in activation of the signaling pathway RAF/MEK/ERK and increased number of endothelin-1 type B receptors in the smooth muscle cells of the vascular wall leading to enhanced constriction. This is hypothesized to further decrease brain blood flow and augment tissue damage after stroke. The present study investigates the potential benefit of treatment with U0126, an inhibitor of the RAF/MEK/ERK signaling pathway, after ischemic stroke in female rats.

 

Women with stroke suffer from worse outcomes and higher mortality. Sex differences in response to stroke treatments have been observed both in animals and in humans, highlighting the importance of including both sexes in research studies. Therefore, the present study aimed to investigate the potential benefit of treatment with U0126, previously not investigated in female rats, after ischemic stroke. Long-term effects on behavioral outcome were assessed up to two weeks after the stroke. An experimental model of stroke was used in female rats that involves transient (two hours) blocking of the middle cerebral artery by insertion of a thin filament that decreases blood flow to a specific region of the brain. The rats were allowed to recover for either two or fourteen days before they were used experimentally. In a laboratory setting, isolated middle cerebral arteries were studied with drugs that activate specific receptors in the vessel wall and cause vasoconstriction. Similar to previous findings in male rats, we found that constriction after activation of the endothelin-1 type B receptor was increased in the middle cerebral arteries two days after stroke in female rats. Treatment with the drug U0126 prevented this increased constriction and was associated with improved functional outcome as seen with behavioral testing up to two weeks after the stroke.

This study shows for the first time that increased constriction of the middle cerebral arteries after ischemic stroke, previously only demonstrated in male rats, also occurs in females. Treatment with U0126, a specific inhibitor of the RAF/MEK/ERK signaling pathway, prevented the constriction and improved long-term functional outcome. Taken together with previous studies using male rats, treatment with U0126 is suggested to improve stroke outcome in both sexes. MEK1/2 inhibition might be a potential target for stroke treatment in humans.

 

References

  1. Mozaffarian, D., et al., Heart disease and stroke statistics–2015 update: a report from the American Heart Association. Circulation, 2015. 131(4): p. e29-322.
  2. Dirnagl, U., C. Iadecola, and M.A. Moskowitz, Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci, 1999. 22(9): p. 391-7.

Acknowledgments: This work was supported by the Swedish Research council, the Swedish Heart-Lung Foundation (20130271), the ‘Agreement for Medical Education and Research’ (ALF) foundation (M2011/1816), the Flight Attendant Medical Research Institute (ID#123071-CIA), and the Lundbeck Foundation in Denmark (center of excellence grant 2011–2015).

 

Hilda Ahnstedt2Contact:

Hilda Ahnstedt, Ph.D., M.Sc.

Division of Experimental Vascular Research, Department of Clinical Sciences, Lund University

Sölvegatan 17, BMC A13, Lund 22184, Sweden

hilda.ahnstedt@gmail.com

http://civi.dk/

 

 

 

 

 

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