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Potential circadian effects on translational failure for neuroprotection

An Author Correction to this article was published on 13 June 2020

This article has been updated

Abstract

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches—normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-d-aspartic acid (NMDA) antagonist MK801—in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in ‘active-phase’ than in ‘inactive-phase’ rodent neurons. αPBN and MK801 reduced neuronal death only in ‘inactive-phase’ neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.

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Fig. 1: Neuroprotection in rodent models of stroke.
Fig. 2: Comparisons of penumbra after focal cerebral ischaemia.
Fig. 3: Effects of circadian cycles on response to OGD and neuroprotection.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Source Data associated with Figures and Extended Data are available online.

Change history

  • 13 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This study was supported in part by grants from the National Institutes of Health (K99MH120053 (I.Ş.), R01NS091230 and R01MH111359 (S.S.)), the Rappaport Foundation (E.H.L.), the Chinese Ministry of Education (X.J.), and the National Research Foundation of Korea (J.-H.P.). The authors thank J. Lipton, M. Ning and W. Deng for discussions, Y. Sun and all team members of the MGH 149-8 animal facility for help with light schedule switching, and M. Ali and K. R. Lees for generous assistance and expert analysis of the VISTA database.

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Contributions

Performed experiments and/or analysed data: (E.E., W.L., E.T.M., J.-H.P., I. Ş., S.G., J.S., J. Lan, J. Lee, K.H.); designed experiments (E.E., W.L., E.T.M., J.-H.P., I. Ş., S.S., E.H.L.); wrote and/or revised manuscript (E.E., W.L., E.T.M., K.H., X.J., E.H.L.); funding and support (J.-H.P., S.S., X.J., E.H.L.).

Corresponding author

Correspondence to Eng H. Lo.

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The authors declare no competing interests.

Additional information

Peer review information Nature thanks John Hogenesch, Costantino Iadecola and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Clot lysis.

Rates of tissue-plasminogen activator-induced clot lysis were not significantly different in blood drawn from day-time (ZT3–9, n = 5) or night-time (ZT15–21, n = 8) male C57BL/6 mice. Mean ± s.e.m., repeated-measures ANOVA; P = 0.80. Source Data

Extended Data Fig. 2 Effects of αPBN on reperfusion.

Laser Doppler flowmetry showed that αPBN (100 mg kg−1) did not affect reperfusion profiles after 60-min transient focal ischaemia in ZT3–9 or ZT15–21 male C57BL/6 mice. Mean ± s.e.m., n = 4 per group, repeated-measures ANOVA; P = 0.87. Source Data

Extended Data Fig. 3 MK801 did not significantly affect body temperature after permanent focal cerebral ischaemia.

White bars, ZT3–9; grey bars, ZT15–21; male C57BL/6 mice. Mean ± s.e.m., n = 4 per group, repeated-measures ANOVA; P = 0.97. Source Data

Extended Data Fig. 4 Physiological parameters for laser speckle imaging experiments in Fig. 2.

n = 4 mice per group; mean ± s.e.m., two-tailed t-test. Source Data

Extended Data Fig. 5 Quantification of blood flow.

Speckle imaging data were used to quantify blood flow in terms of absolute blood flow (ml per 100 g per min). Blood flow histograms show the presence of cerebral ischaemia in the ipsilateral hemispheres (top). Thresholded areas between 25 and 55 ml per 100 g per min (see Methods) were significantly smaller in ZT17–19 than in ZT5–7 C57BL/6 male mice (bottom; n = 4 per group, two-tailed t-test). Source Data

Extended Data Fig. 6 Circadian gene expression and patterns in different species.

Top, comparison of circadian gene patterns between mice, rats, nonhuman primates and humans. Bottom, expression of selected circadian genes in C57BL/6 male mouse and Sprague–Dawley male rat somatosensory cortex (n = 4 per group). Source Data

Extended Data Fig. 7 Blood cortisol levels.

No significant differences were detected in blood cortisol levels of ZT3–9 versus ZT15–21 C57BL/6 male mice subjected to similar handling procedures as cerebral ischaemia mice. Mean ± s.e.m., n = 5 mice per group, two-tailed t-test. Source Data

Extended Data Table 1 Physiological parameters, laser doppler flow, mortality, inclusion and exclusions for neuroprotection experiments in Fig. 1

Supplementary information

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This file contains the uncropped gel images.

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Esposito, E., Li, W., T. Mandeville, E. et al. Potential circadian effects on translational failure for neuroprotection. Nature 582, 395–398 (2020). https://doi.org/10.1038/s41586-020-2348-z

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