DOI: 10.5061/dryad.95x69p900
title: Lemborexant reduces infarct volume and improves long-term functional recovery in a murine Model of Ischemic stroke

description: Recovery following ischemic stroke is highly variable and often
 incomplete, underscoring the urgent need to develop novel targeted
 poststroke treatments. While the mechanisms underlying poststroke recovery
 remain incompletely understood, sleep fragmentation, a common consequence
 of stroke, has been linked to worse patient outcomes. Lemborexant is a
 dual orexin receptor antagonist that promotes sleep by suppressing
 wakefulness and enhancing sleep continuity. We hypothesized that
 lemborexant would reduce poststroke sleep disturbances and promote
 recovery in a rodent model of stroke. We examined the effects of
 lemborexant (10 mg/kg and 30 mg/kg) and zolpidem (30 mg/kg) on sleep
 macrostructure, fragmentation, and EEG spectra in both healthy mice and in
 stroke model mice, which underwent photothrombotic ischemia of the
 forelimb somatosensory cortex. We also evaluated whether 12 days of drug
 administration altered infarct volume and functional recovery following
 the experimental induction of stroke in model mice. Lemborexant treatment
 (30mg/kg) increased the percentage of NREM sleep, while preserving sleep
 continuity, in both healthy and stroke model mice. In contrast, zolpidem
 increased NREM sleep after stroke, but also increased sleep fragmentation
 in both groups. Lemborexant treatment at 10 mg/kg and 30 mg/kg
 significantly reduced infarct volume eight weeks after the induction of
 stroke. In addition, lemborexant-treated mice showed greater use of the
 impaired limb four weeks after stroke. These preclinical findings suggest
 that lemborexant stabilizes sleep and promotes structural and functional
 recovery following the experimental induction of stroke in model mice,
 supporting its potential as a novel therapeutic intervention following
 ischemic stroke.
description: # Lemborexant reduces infarct volume and improves long-term functional
 recovery in a murine Model of Ischemic stroke Dataset DOI:
 [10.5061/dryad.95x69p900](https://doi.org/10.5061/dryad.95x69p900) ##
 Description of the data and file structure **Aim 1:** Healthy, male,
 wild-type mice were administered Lemborexant (10 mg/kg, N = 7),
 Lemborexant (30 mg/kg, N = 7), Zolpidem (30 mg/kg, N = 7), or vehicle (N =
 7) via Jell-O in a randomized order, with a three-day washout period
 between each dose. EEG/EMG signals were recorded continuously, and the
 24-hour period following each dose was scored for NREM, REM, and Wake
 states. Analyses included vigilance-state quantification, EEG power, sleep
 fragmentation, sleep latency, and circadian index. **Aim 2:**
 Photothrombotic stroke was induced in male, wild-type mice, which were
 then given a single dose of Lemborexant (10 mg/kg, N = 13), Lemborexant
 (30 mg/kg, N = 14), Zolpidem (30 mg/kg, N = 12), or vehicle (N = 16) via
 Jell-O. EEG/EMG signals were recorded continuously, and the subsequent
 24-hour period was scored for NREM, REM, and Wake states. Analyses
 included vigilance-state quantification, EEG power, sleep fragmentation,
 sleep latency, and circadian index. **Aim 3:** Photothrombotic stroke was
 induced in male, wild-type mice, which then underwent a 12-day dosing
 regimen of Lemborexant (10 mg/kg, N = 7), Lemborexant (30 mg/kg, N = 7),
 Zolpidem (30 mg/kg, N = 7), or vehicle (N = 7). Behavioral tests assessing
 asymmetry during exploratory behavior were conducted before stroke
 induction and again at 48 hours, 2 weeks, 4 weeks, and 8 weeks
 post-stroke. At the 8-week time point, mice were sacrificed and infarct
 volume was quantified. ### **Files and variables** **File:
 Figure_1_KMI.prism** **Description:** For all data tables in Figure 1, the
 independent variables are the four drug types the mice received in a
 randomized order in aim 1 of the study: Vehicle (VEH), Zolpidem 30 mg/kg
 (ZOL30), Lemborexant 10 mg/kg (LEM10), and Lemborexant 30 mg/kg (LEM30).
 For "A1 NREM_3hr" and "A1 REM_3hr", the total duration
 of NREM and REM, respectively, is reported for the first three hours after
 drug administration as a percentage. "A1 NREM Latency" and
 "A1 REM Latency" represent the time until the first NREM or REM
 bout, respectively, post-drug administration. "A1 #Arousal_3hr"
 represents the number of arousals (events) within the first three hours
 post-drug administration; arousals were defined as transitions from NREM
 or REM to Wake lasting less than 300 seconds. "A1 meanREM_3hr"
 and "A1 meanNREM_3hr" represent the mean bout duration of REM
 and NREM, in minutes, within the first three hours post-drug
 administration. "Aim 1 Circadian Index" represents the circadian
 index of wakefulness for the 24 hours post-drug administration. Circadian
 index of wakefulness was calculated using the following formula: CI =
 (Percent Wake Dark Period – Percent Wake Light Period) / Percent Wake
 24-Hour. "A1 PSD NREM," "A1 PSD REM," and "A1 PSD
 Wake" are power spectral density arrays depicting the effect of the
 four drug types on the EEG power of each vigilance state within the first
 three hours post-drug administration. Power values were plotted on a
 logarithmic scale. **File: Figure_2_KMI.prism** **Description:** For all
 data tables in Figure 2, the independent variables are the four drugs each
 group of mice received after undergoing photothrombotic stroke in aim 2:
 Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). "A2 NREM_3hr" and "A2
 REM_3hr" quantify the change in total duration, in percentage, of
 NREM and REM sleep, respectively, from 48–51 hours post-stroke to 72–75
 hours post-stroke (first three hours post-drug administration). "NREM
 Latency (Normalized)" and "REM Latency (Normalized)"
 represent the change in time to the first NREM or REM bout, respectively,
 from 48–51 hours post-stroke to 72–75 hours post-stroke.
 "#Arousal_3hr" represents the change in number of arousals
 (events)—defined as transitions from NREM or REM to Wake lasting less than
 300 seconds—from 48–51 hours post-stroke to 72–75 hours post-stroke.
 "meanNREM_3hr" and "meanREM_3hr" represent the change
 in bout duration, in minutes, of NREM and REM, respectively, across the
 same time periods. "Aim 2 Change in Circadian Index" represents
 the change in circadian index of wakefulness (calculated using the formula
 described in Figure 1) from 48–72 hours post-stroke to 72–96 hours
 post-stroke. **File: Figure_3_KMI.prism** **Description:** For all data
 tables in Figure 3, the independent variables are the four drugs each
 group of mice received daily during the 12-day dosing period in aim 3:
 Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). Each group started with N = 16 mice, but
 several were excluded due to not having a stroke or not reaching the end
 of the study. The blank cells in some drug groups reflect these
 exclusions. "Infarct Volume" represents the stroke volume (µm³)
 in each mouse 8 weeks post-stroke following cresyl violet staining.
 "MD-2wk Score all groups," "MD-4wk Score all groups,"
 and "MD-8wk Score all groups" represent the change in forelimb
 asymmetry score from a stroke-sensitive behavior test called cylinder
 rearing, from Max Deficit (48 hours post-stroke) to 2 weeks, 4 weeks, and
 8 weeks post-stroke, respectively. Asymmetry score was calculated as
 percent difference in left vs. right forelimb use during rearing: ((left
 rears – right rears) / (left rears + right rears + both forepaw rears)) ×
 100. **File: Supplementary_Figure_1_KMI.prism** **Description:** For all
 data tables in Supplementary Figure 1, the independent variables are the
 four drug types the mice received in a randomized order in aim 1: Vehicle
 (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10), and
 Lemborexant 30 mg/kg (LEM30). "A1 REM TimeCourse" and "A1
 NREM TimeCourse" indicate the total duration of REM and NREM,
 respectively, as a percentage of hours elapsed over the 24 hours post-drug
 administration and depict drug effects across light and dark cycles.
 "A1 NREM_24hr" and "A1 REM_24hr" represent the total
 duration of NREM and REM, as a percentage of the 24-hour period post-drug
 administration. **File: Supplementary_Figure_2_KMI.prism**
 **Description:** For all data tables in Supplementary Figure 2, the
 independent variables are the four drug types the mice received in a
 randomized order in aim 1: Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30),
 Lemborexant 10 mg/kg (LEM10), and Lemborexant 30 mg/kg (LEM30). "A1
 #Arousal_24hr" represents the number of arousals (events) across the
 24 hours post-drug administration; arousals were defined as transitions
 from NREM or REM to Wake lasting less than 300 seconds. "A1
 MeanNREM_24hr" and "A1 meanREM_24hr" represent mean bout
 duration of NREM and REM, in minutes, across the same 24-hour period.
 **File: Supplementary_Figure_3_KMI.prism** **Description:** For all data
 tables in Supplementary Figure 3, the independent variables are the four
 drug types the mice received in a randomized order in aim 1: Vehicle
 (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10), and
 Lemborexant 30 mg/kg (LEM30). The data tables provide further analysis of
 the power spectral density arrays in Figure 1. To summarize spectral
 shape, we calculated spectral slope/exponent as the regression coefficient
 of the log–log PSD fit and spectral offset as the y-intercept. "A1
 Exponent NREM," "A1 REM Exponent," and "A1 Exponent
 Wake" represent slopes of the EEG power spectral density arrays from
 Figure 1, and "A1 Offset NREM," "A1 Offset REM," and
 "A1 Offset Wake" represent the corresponding y-intercepts.
 **File: Supplementary_Figure_4_KMI.prism** **Description:** For all data
 tables in Supplementary Figure 4, the independent variables are the four
 drugs each group of mice received after photothrombotic stroke in aim 2:
 Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). "A2 NREM_3hr (absolute)" and
 "A2 REM_3hr (absolute)" represent the total duration of NREM and
 REM, as a percentage, during 72–75 hours post-stroke. "NREM Latency
 (absolute)" and "REM Latency (absolute)" represent the time
 in minutes to the first NREM or REM bout, respectively, within 72–75 hours
 post-stroke. **File: Supplementary_Figure_5_KMI.prism** **Description:**
 For all data tables in Supplementary Figure 5, the independent variables
 are the four drugs each group of mice received after photothrombotic
 stroke in aim 2: Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10
 mg/kg (LEM10), and Lemborexant 30 mg/kg (LEM30). "A2 NREM_24hr"
 and "A2 REM_24hr" quantify the change in total duration, in
 percentage, of NREM and REM sleep, respectively, from 48–72 hours
 post-stroke to 72–96 hours post-stroke. "A2 NREM Time Course"
 and "A2 REM Time Course" indicate the total duration of NREM and
 REM, as a percentage of hours elapsed over 72–96 hours post-stroke, and
 depict drug effects across light and dark cycles. **File:
 Supplementary_Figure_6_KMI.prism** **Description:** For all data tables in
 Supplementary Figure 6, the independent variables are the four drugs each
 group of mice received after photothrombotic stroke in aim 2: Vehicle
 (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10), and
 Lemborexant 30 mg/kg (LEM30). "#Arousal_3hr (absolute)"
 represents the number of arousals (events) during 72–75 hours post-stroke;
 arousals were defined as transitions from NREM or REM to Wake lasting less
 than 300 seconds. "meanNREM_3hr (absolute)" and
 "meanREM_3hr (absolute)" represent mean bout duration, in
 minutes, of NREM and REM, respectively, during 72–75 hours post-stroke.
 **File: Supplementary_Figure_7_KMI.prism** **Description:** For all data
 tables in Supplementary Figure 7, the independent variables are the four
 drugs each group of mice received after photothrombotic stroke in aim 2:
 Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). "#Arousal_24hr" quantifies the
 change in number of arousals (events) from 48–72 hours post-stroke to
 72–96 hours post-stroke. Arousals were defined as transitions from NREM or
 REM to Wake lasting less than 300 seconds. "meanNREM_24hr" and
 "meanREM_24hr" quantify the change in mean bout duration, in
 minutes, of NREM and REM, respectively, across the same periods. **File:
 Supplementary_Figure_8_KMI.prism** **Description:** For all data tables in
 Supplementary Figure 8, the independent variables are the four drugs each
 group of mice received after photothrombotic stroke in aim 2: Vehicle
 (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10), and
 Lemborexant 30 mg/kg (LEM30). "Aim 2 72–96 Hr Circadian Index"
 represents the circadian index of wakefulness for hours 72–96 post-stroke
 (calculated using the formula from Figure 1). **File:
 Supplementary_Figure_9_KMI.prism** **Description:** For all data tables in
 Supplementary Figure 9, the independent variables are the four drugs each
 group of mice received after photothrombotic stroke in aim 2: Vehicle
 (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10), and
 Lemborexant 30 mg/kg (LEM30). The files within Supplementary Figure 9 are
 separated by drug group and vigilance state. They contain EEG power
 spectral density arrays comparing effects of drug at 72–75 hours
 post-stroke to baseline 48–51 hours post-stroke for each vigilance state.
 **File: Supplementary_Figure_10_KMI.prism** **Description:** For all data
 tables in Supplementary Figure 10, the independent variables are the four
 drugs each group of mice received after photothrombotic stroke in aim 2:
 Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). The data tables provide further analysis
 of the power spectral density arrays in Supplementary Figure 9. To
 summarize spectral shape, we calculated spectral slope/exponent as the
 regression coefficient of the log–log PSD fit and spectral offset as the
 y-intercept. "A2 Exponent NREM," "A2 Exponent REM,"
 and "A2 Exponent Wake" quantify the change in slope of the EEG
 power spectral density arrays from Supplementary Figure 9 from 48–51 hours
 post-stroke to 72–75 hours post-stroke. "A2 Offset NREM,"
 "A2 Offset REM," and "A2 Offset Wake" quantify the
 change in y-intercept of the PSDs across the same periods. **File:
 Supplementary_Figure_11_KMI.prism** **Description:** For all data tables
 in Supplementary Figure 11, the independent variables are the four drugs
 each group of mice received daily during the 12-day dosing period in aim
 3: Vehicle (VEH), Zolpidem 30 mg/kg (ZOL30), Lemborexant 10 mg/kg (LEM10),
 and Lemborexant 30 mg/kg (LEM30). Each group began with N = 16 mice, but
 several were excluded due to not having a stroke or not reaching the end
 of the study. The blank cells reflect these exclusions. "STI"
 represents the prevalence, in percentage, of Secondary Thalamic Injury
 within each drug group. **File: Supplementary_Figure_12_KMI.prism**
 **Description:** For all data tables in Supplementary Figure 12, the
 independent variables are the four drugs each group of mice received daily
 during the 12-day dosing period in aim 3: Vehicle (VEH), Zolpidem 30 mg/kg
 (ZOL30), Lemborexant 10 mg/kg (LEM10), and Lemborexant 30 mg/kg (LEM30).
 Each group began with N = 16 mice, but several were excluded due to not
 having a stroke or not reaching the end of the study. The blank cells
 reflect these exclusions. "MD-2wk Score all groups,"
 "MD-4wk Score all groups," and "MD-8wk Score all
 groups" quantify the change in asymmetry score from the Y-maze
 behavior test. Asymmetry scores were calculated as the change in
 percentage of leftward head turns relative to total head turns from
 baseline (24 hours post-stroke) to two, four, and eight weeks post-stroke.
 ## Code/software GraphPad Prism is required to view our data files.