Potential circadian effects on translational failure for neuroprotection

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 humans^1,2 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.

Extended Data 2:

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 C57BL6 mice (mean ± SEM, repeated-measures ANOVA p=0.80).

Extended Data 3:

Laser Doppler flowmetry showed that αPBN (100 mg/kg) did not affect reperfusion profiles after 60 min transient focal ischemia in ZT3-9 versus ZT15-21 male C57BL6 mice (mean ± SEM, n=4 per group, repeated-measures ANOVA p=0.87).

Extended Data 4:

MK801 did not significantly affect body temperature after permanent focal cerebral ischemia in ZT3-9 (white bars) versus ZT15-21 (gray bars) male C57BL6 mice (mean ± SEM, n=4 per group, repeated-measures ANOVA p=0.97).

Extended Data 5:

Physiologic parameters for laser speckle imaging experiments in Figure 2 (n=4 mice per group). All values are mean ± SEM, P values in data from 2-tailed t test comparison.

Extended Data 6:

Quantitation of blood flow was checked by calculating speckle imaging data in terms of absolute blood flow (mL/100g/min). Blood flow histograms show the presence of cerebral ischemia in the ipsilateral hemispheres (top panel). Thresholded areas between 25-55 mL/100g/min (see Methods) were significantly smaller in ZT17-19 versus ZT5-7 C57BL6 male mice (n=4 per group, comparisons via 2-tailed t test).

Extended Data 7:

Expression of selected circadian genes in C57BL6 male mouse and Sprague-Dawley male rat somatosensory cortex (n=4 per group), and comparison of circadian gene patterns between mice, rats, nonhuman primates and humans.

Extended Data 8:

No significant differences were detected in blood cortisol levels of ZT 3-9 versus ZT15-21 C57BL6 male mice subjected to similar handling procedures as cerebral ischemia mice. All values are mean ± SEM, n= 5 mice per group, comparisons via 2-tailed t test.

Figure 1.. Neuroprotection in rodent models of stroke.

a. Opposite circadian cycles in nocturnal rodents versus diurnal humans. b. 100 min middle cerebral artery occlusion (MCAO) in male Sprague-Dawley rats. Controls received 30% O₂. NBO group received 100% O₂ (for 85% of ischemic period). NBO reduced infarction at ZT3-9 but not ZT15-21. c. NBO (for 85% of ischemic period) reduced infarction after 60 min MCAO in Sprague-Dawley rats during ZT3-9. d. αPBN (100 mg/kg IP immediately and 2 hrs post-reperfusion) reduced infarction after 60 min MCAO in ZT3-9 but not ZT15-21 mice. e. MK801 (2.5 mg/kg IP, 1 hr pre-occlusion) reduced infarction after permanent MCAO in ZT3-9 but not ZT15-21 mice. f. Infarct volumes were smaller in 60 min transient versus permanent MCAO in both ZT3-9 and ZT15-21 mice. All values in Figure 1 are mean ± SEM; comparisons via 2-tailed t-test. Physiologic parameters, laser-doppler flow, inclusion/exclusion/mortality in Extended Data 1. White bars: ZT3-9; gray bars: ZT15-21. Infarct volumes quantified with triphenyltetrazolium (TTC) staining. (n) indicate animals per group. tMCAO: transient MCAO; pMCAO: permanent MCAO; dMCAO permanent distal MCAO.

Figure 2.. Comparisons of penumbra after focal cerebral ischemia.

a. Laser speckle imaging at 25 mins post-MCAO in ZT5-7 versus ZT17-19 C57BL6 mice. Rectangular area for quantifying data from 4 independent experiments/mice per group in (b) and (c). Scale: 1 mm. b. Ipsilateral blood flow gradients were steeper in ZT17-19 versus ZT5-7 images (line is mean, shaded areas are SEM, left panel; area-under-curve, right panel). c. The blood flow penumbra was operationally defined as average cortical width or area-under-curve between 30-50% of normal levels, based on a lower threshold of infarction and an upper threshold of gene expression and protein synthesis inhibition (25-55 mL/100g/min, see Methods and References^,). The penumbra was narrower in ZT17-19 versus ZT5-7 mice. Physiologic parameters were similar across all animals (Extended Data 5). Penumbral perfusion was not correlated with blood pressure or pCO₂ (r² = 0.006 and 0.171 respectively). d. Infarct growth (12 to 72 hrs) after 60 min MCAO was smaller in ZT15-21 (n=8) versus ZT3-9 (n=9) mice. e. Representative TUNEL, fluoro-jade and DAPI immunostaining in penumbral cortex at 24 hrs after 60 min MCAO in mice. Scale: 50 μm. f. Percentages of TUNEL/DAPI and TUNEL plus fluoro-jade/fluoro-jade were lower in ZT15-21 versus ZT3-9 penumbra (n=4 mice per group). All values in Figure 2 are mean ± SEM; comparisons via 2-tailed t-test.

Figure 3.. Effects of circadian cycles on response to oxygen-glucose deprivation and neuroprotection.

a. Primary mouse cortical neurons were treated for 2 hrs with dexamethasone (Dex). Per1/2 levels at 6 and 12 hrs matched in vivo circadian cycles for rats and mice at ZT15-21 (upregulated Per1/2) and ZT3-9 (downregulated Per1/2). *P=0.0003 (Per1), *P=0.0026 (Per2), n=3 independent experiments in triplicate. b. Oxygen-glucose deprivation (OGD) with αPBN or MK801 in neurons after induction of circadian-like cycles in vitro. c. αPBN was neuroprotective when Per1/2 were downregulated (n=4 independent experiments in triplicate). d. MK801 was neuroprotective when Per1/2 were downregulated (n=4 independent experiments in triplicate). e. After 3 hrs OGD, glutamate release and ROS production were lower during times with upregulated Per1/2 (n=3 independent experiments in triplicate). f. Representative western blots of cleaved caspase-3, caspase-3, phosphorylated RIP3 kinase and RIP3 kinase at 24 hrs after OGD (gel source data in Supplementary Information; n=5 independent experiments for densitometry). g. Quantitation of cleaved caspase-3/caspase-3. h. Quantitation of phosphorylated RIP3 kinase/RIP3 kinase. i. “Resistance to neuroprotection” after combination treatment with MK801 (10 μM), NBQX (50 μM), αPBN (1μM), zVAD-fmk (50 μM) and Necrostatin-1 (100 μM) (n=3 independent experiments in triplicate). All data in Figure 3 are mean ± SEM; two-way ANOVA with Bonferroni adjustment (a, c, d, e) or one-way ANOVA with post-hoc Tukey adjustment (g, h) or unpaired 2-tailed t-test (i).