Alleviating sleep disturbances and modulating neuronal activity after ischemia: Evidence for the benefits of zolpidem in stroke recovery

Abstract

Aims: Sleep disorders are prevalent among stroke survivors and impede stroke recovery, yet they are still insufficiently considered in the management of stroke patients, and the mechanisms by which they occur remain unclear. There is evidence that boosting phasic GABA signaling with zolpidem during the repair phase improves stroke recovery by enhancing neural plasticity; however, as a non-benzodiazepine hypnotic, the effects of zolpidem on post-stroke sleep disorders remain unclear.

Method: Transient ischemic stroke in male rats was induced with a 30-minute middle cerebral artery occlusion. Zolpidem or vehicle was intraperitoneally delivered once daily from 2 to 7 days after the stroke, and the electroencephalogram and electromyogram were recorded simultaneously. At 24 h after ischemia, c-Fos immunostaining was used to assess the effect of transient ischemic stroke and acute zolpidem treatment on neuronal activity.

Results: In addition to the effects on reducing brain damage and mitigating behavioral deficits, repeated zolpidem treatment during the subacute phase of stroke quickly ameliorated circadian rhythm disruption, alleviated sleep fragmentation, and increased sleep depth in ischemic rats. Immunohistochemical staining showed that in contrast to robust activation in para-infarct and some remote areas by 24 h after the onset of focal ischemia, the activity of the ipsilateral suprachiasmatic nucleus, the biological rhythm center, was strongly suppressed. A single dose of zolpidem significantly upregulated c-Fos expression in the ipsilateral suprachiasmatic nucleus to levels comparable to the contralateral side.

Conclusion: Stroke leads to suprachiasmatic nucleus dysfunction. Zolpidem restores suprachiasmatic nucleus activity and effectively alleviates post-stroke sleep disturbances, indicating its potential to promote stroke recovery.

Keywords: MCAO; c-Fos; circadian rhythm; sleep disorder; suprachiasmatic nucleus; zolpidem.

FIGURE 1

Zolpidem reduces infarct volume and improves motor functions after ischemic injury. (A) Timeline of procedures for treatments in rats. (B) Representative Fluoro‐Jade B staining in coronal sections of rats at day 10 after middle cerebral artery occlusion (MCAO). The bright green areas circled by white dotted lines indicate infarct areas. (C) Zolpidem significantly reduced the infarct volume in ischemic injured rats on day 10 after MCAO (n = 6). (D) and (E) The wire test (n = 12) and rotarod test (n = 8) revealed that the latencies to fall in zolpidem‐treated rats were significantly increased compared to those of the vehicle‐treated group at day 10 after MCAO. Data are presented as the mean ± SEM, n = 6–12/group. *p < 0.05, assessed by two‐tailed unpaired Student's t‐test. Vehi, vehicle; Zol, zolpidem.

FIGURE 2

Effects of Zolpidem on the vigilance states of rats with 30‐min middle cerebral artery occlusion (MCAO) following different ischemic/reperfusion (I/R) days. (A) Examples of the relative EEG power, EEG/EMG traces, and corresponding hypnograms following vehicle or zolpidem injection at 9 a.m. in MCAO rats on I/R d3. (B) Hourly time course of wakefulness of rats in basal states, and on I/R d1–d7 after a 30 min‐MCAO. During I/R d2–d7, vehicle and zolpidem were administrated daily at 9 a.m. Each circle represents the mean hourly amount of wakefulness. The horizontal open and filled bars on the x‐axis indicate 12‐h light and 12‐h dark period, respectively. The gray‐shaded areas represent dark periods. (C) The changes in the CI of wakefulness (Δ CI_wakefulness = treatment CI_wakefulness − baseline CI_wakefulness) in ischemic rats after vehicle or zolpidem treatment on I/R d2–d7. (D) non‐rapid eye movement sleep latency of ischemic rats after vehicle or zolpidem administration at 9 a.m. on I/R d2–d7. Data are presented as the mean ± SEM, n = 6/group. *p < 0.05, **p < 0.01, assessed by two‐tailed unpaired Student's t‐test between vehicle‐ and zolpidem‐treated stroke animals at each time point.

FIGURE 3

Effects of Zolpidem on changes in the amounts of each sleep/wake stage in rats with 30‐min middle cerebral artery occlusion following different I/R days. Total Δ amounts of wakefulness, REM sleep, and non‐rapid eye movement sleep during the light period, dark period, and over the 24‐h period of each I/R day, with vehicle or zolpidem daily administration at 9 a.m. Data are presented as the mean ± SEM, n = 6/group. *p < 0.05, **p < 0.01, assessed by two‐tailed unpaired Student's t‐test between vehicle‐ and zolpidem‐treated stroke animals at each time point.

FIGURE 4

Effects of zolpidem on the characteristics of sleep–wake episodes in rats with 30‐min middle cerebral artery occlusion following different I/R days. (A) The total amount of Δ non‐rapid eye movement (NREM) sleep to wake transitions (upper panel) and Δ wake to NREM sleep transitions (lower panel) during the light period, dark period, and over the 24‐h period of each I/R day with vehicle or zolpidem daily administration at 9 a.m. (B) Total Δ number of wake, NREM sleep, and REM sleep bouts during the light, the dark period, and over the 24‐h period of each I/R day with vehicle or zolpidem daily administration at 9 a.m. (C) Δ mean duration of wake, NREM sleep, and REM sleep during the light period, dark period, and over the 24‐h period of each I/R day with vehicle or zolpidem daily administration at 9 a.m. Data are presented as the mean ± SEM, n = 6/group. *p < 0.05, **p < 0.01, assessed by two‐tailed unpaired Student's t‐test between vehicle‐ and zolpidem‐treated stroke animals at each time point.

FIGURE 5

Effects of zolpidem on EEG spectral power of non‐rapid eye movement (NREM) sleep in rats with 30‐min middle cerebral artery occlusion following different I/R days. (A) Δ EEG power density of NREM sleep during light and dark periods of each I/R day, with daily administration of vehicle or zolpidem at 9 a.m. The changes in power in each frequency bin were obtained by subtracting the corresponding baseline value from the treatment value. (B) Averaged EEG power density of NREM sleep in 2 h after vehicle or zolpidem administration (9 a.m., i.p.) on each I/R day. (C) Percentage of NREM sleep power density at 0–4, 6–10, and 12–24.5 Hz within 2 h after vehicle or zolpidem administration (9 a.m., i.p.) on each I/R day. Data represented as mean ± SEM. n = 5/group. *p < 0.05, **p < 0.01, assessed by two‐tailed unpaired t‐test.

FIGURE 6

c‐Fos and NeuN immunostaining in adjacent brain slices at striatum level reveal neuronal loss in the ischemia‐involved region of rats with 30‐min middle cerebral artery occlusion (MCAO) following 24 h reperfusion. (A–C) Representative images of c‐Fos immunostaining (black) from a coronal section at the level of the striatum. The black dotted lines indicate the infarct boundary. The boxed regions in (A) are enlarged in (B) and (C), showing the cortex and striatal ischemic border zone after MCAO, respectively. (D–F) Representative images of NeuN immunostaining (brown) from an adjacent brain slice of (A), NeuN‐expression (brown color) was significantly reduced in the infarct area. The black dotted lines indicate the infarct boundary. The boxed regions in (D) are enlarged in (E) and (F), showing the cortex and striatal ischemic border zone after MCAO, respectively. Note that the infarcts elucidated by c‐Fos and NeuN immunostaining are very similar. n = 4. CPu, caudate putamen; gcc, genu of the corpus callosum; LV, lateral ventricle.

FIGURE 7

Changes of c‐Fos expression in the SCN of rats with 30‐min middle cerebral artery occlusion (MCAO) following 24 h reperfusion. Representative images of c‐Fos (green) and VIP (red) immunostaining and DAPI (blue) from a coronal section at SCN level of a rat with 30‐min MCAO following 24 h reperfusion. The border of the SCN was determined with the aid of VIP and DAPI‐stained images. Scale bars = 100 μm. n = 4. 3V, third ventricle; och, optic chiasm; SCN, suprachiasmatic nucleus; VIP, vasoactive intestinal polypeptide.

FIGURE 8

Zolpidem increases the expression of c‐Fos in the SCN and PVN in rats with 30‐min middle cerebral artery occlusion following 24 h reperfusion. Representative images of cresyl violet staining and c‐Fos immunostaining from a vehicle‐treated (A–C) or a zolpidem‐treated (D–F) rat, respectively. Vehicle or zolpidem was administrated 90 min before brain harvest at 24 h after stroke. (G) The number of c‐Fos‐immunoreactive neurons in ipsilateral/contralateral SCN and PVN after vehicle or zolpidem administration. Data are presented as the mean ± SEM, n = 3/group, three slices of the SCN or PVN per animal, respectively. *p < 0.05, **p < 0.01, assessed by one‐way ANOVA followed by Turkey's test (For SCN: F(3, 32) = 4.25, p = 0.0123, Turkey's Test: *p < 0.05. For PVN: F(3, 32) = 8.85, p = 0.0002, Turkey's Test: *p < 0.05 and **p < 0.01). 3V, third ventricle; aca, anterior commissure, anterior part; gcc, genu of the corpus callosum; contra, contralateral; ipsi, ipsilateral; LV, lateral ventricle; och, optic chiasm; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus.