Sex-specific acceleration of Alzheimer's pathogenesis by chronic sleep-deprivation methods
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Home-cage assessment
Noldus PhenoTypers served as home cages with overhead cameras to continuously record and track mouse movement. Mice were placed individually in the PhenoTypers at 3 p.m. on the concluding day of the 2-week SD. Following a 2.5-h habituation, recordings began at 6:30 p.m. and continued for 25 h. Mice were removed from the PhenoTyper at 9 a.m. the following day after a total of 42 h. Home-cage assessments were recorded and analyzed in EthoVision XT15 software. For analysis, the mean velocity of the mouse center point was recorded, and time was binned into 10-s intervals. A mouse was considered to be "attempting sleep" if their average velocity was under 0.1 cm/s for four straight 10-s intervals. Additionally, nests were scored at 18, 24, and 42 h from 1 to 5 based on previously published protocol, and the unshredded portion of...
Novel Object Recognition test
Four days after the conclusion of the SD, mice were subjected to a Novel Object Recognition (NOR) test to examine recognition memory. Mice were habituated to the testing room for 30 min prior to experimentation. Subsequently, mice were individually placed in empty PhenoTypers to habituate to the arena for 10 min. After a 1-h break, the familiarization phase was conducted where mice were placed in the same PhenoTyper containing either two LEGO towers or two T25 cell culture flasks filled with bedding and allowed to freely explore the objects for 10 min. After another 1-h break, the NOR test was conducted with one object being swapped out for a novel one (a LEGO tower or T25 flask) and mice being allowed to explore the two objects for 10 min. All trials were recorded using EthoVision XT software and scored manually.
Elevated plus maze
A week after the conclusion of the SD, anxiety-like behavior was assessed using an elevated plus maze (EPM). The EPM with arm lengths of 30 cm, widths of 7 cm, and 17.75-cm-high walls in the closed arms was used, and recordings were taken with overhead cameras. Mice were habituated to the testing room for a minimum of 30 min prior to experimentation. The mouse was then placed in the center of the maze and allowed to freely explore for 10 min. Recorded videos were analyzed using EthoVision XT software to determine the time spent on the open arms with no walls (entire mouse body) and in the closed arms (walls) or center of the maze.
Y-Maze
Nine days after SD, mice were subjected to a Y-maze test to assess their working memory. Mice were habituated to the testing room for a minimum of 30 min prior to experimentation. A Y-maze with individual arm lengths of 30 cm was used with an overhead camera and dim lighting. Mice were placed in one arm facing the arm's end and allowed to explore the maze for 5 min. Trials were recorded with EthoVision XT, and each arm entry was recorded manually from the obtained videos. An arm entry was indicated when the entire mouse had entered the arm. A correct alternation was the mouse entering each of the three arms in sequence without returning to the previously investigated arm. For example, ABC or BAC would be a correct alternation, whereas BCC or ABA would be an incorrect alternation. The spontaneous alternation of the mouse was calculated as: No. of Correct Alternations/(...
Tissue preparation for immunoblotting and immunofluorescent staining
Following the behavioral period, the mice were euthanized. They were perfused with ice-cold heparinized phosphate-buffered saline (PBS). Following perfusion, brains were cut in half along the sagittal plane, with the right hemisphere being post-fixed in 4% paraformaldehyde in PBS overnight, transferred to 30% sucrose solution, and stored at 4°C. Brains were sectioned coronally at 40 µm on a sliding microtome (Leica SM2000R), with the prefrontal cortex (∼Bregma 0 to -0.20 mm), hypothalamus (POA: ∼Bregma 0 to -0.20 mm; LH: ∼Bregma -1.30 to -1.50), HPC (∼Bregma -1.30 to -3.20), lateral EC (∼Bregma -3.10 to -3.40), and LC (∼Bregma -5.30 to -5.50) being collected, and stored at -20°C in tissue cryoprotectant in a sealed plate. The left hemi&#...
Immunoblotting
Total protein concentration of the hippocampal homogenate was determined using a bicinchoninic acid assay (Pierce BCA Protein Assay Kits, Thermo Fisher Scientific, Catalog No.: 23227). Samples were prepared with dithiothreitol, loading dye, and ddH 2 O, heated at 70°C for 6 min, and placed on wet ice for 5 to 10 s. Samples were loaded on 4% to 20% Tris-glycine polyacrylamide gels with tris-glycine running buffer under non-reducing conditions or 4% to 12% Bis-Tris gels with 2-(N-morpholino)ethanesulfonic acid buffer. Gel electrophoresis was run at a constant 125 V for approximately 75 min and transferred to a nitrocellulose membrane at a constant 200 mA for 2 h. Membranes were submerged in Ponceau S stain for 5 min and subsequently washed with ddH 2 O to ensure proper protein transfer. Subsequently, membranes were blocked with 5%...
Immunofluorescent staining
Generally, sections were washed three times for 10 min with PBS, blocked with 2% goat serum, 0.1% triton X-100, and 1% bovine serum albumin in PBS for 1 h, and incubated at 4°C overnight with primary antibody diluted in blocking solution: GFAP (Dako, Catalog No.: Z0334), LAMP1 (Biolegend, Catalog No.: 121602), NeuN (Millipore Sigma, Catalog No.: MAB377), Orexin-A (Santa Cruz Biotechnology, Catalog No.: SC-80263), p62 (Abcam, Catalog No.: AB109012 ), PHF1 (Albert Einstein College of Medicine, Catalog No.: AB_2315150), and TH (Abcam, Catalog No.: AB76442). Primary antibodies are described in detail in Table. Following primary incubation, sections were washed three times with PBS and incubated at room temperature for 2 h in the secondary antibody listed in Table: goat anti-rabbit 488 (1:200, Invitrogen, MA, USA, Catalog No.: A11008), goat antiR...
Plasma collection and corticosterone measurements
Blood samples were obtained via saphenous vein collection immediately prior to and following the 2-week SD. Mice were restrained inside 50-mL conical tubes to gain access to the saphenous vein and pierced with a 23-gauge needle, and blood was collected using Sarstedt Microvette CB300 K2 EDTA tubes. Collections were done in a rapid fashion to reduce stress associated with restraint. Blood was subsequently centrifuged at 6000 × g and 4°C for 8 min. Plasma was isolated and stored at -80°C until use. Plasma samples were run in duplicate on the Arbor Assays corticosterone multiformat enzyme-linked immunosorbent assay kit from Cedarlane (K014-H5) according to the provided protocol and analyzed using the MyAssays Corticosterone Enzyme Immunoassay software.
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Noldus PhenoTypers served as home cages with overhead cameras to continuously record and track mouse movement. Mice were placed individually in the PhenoTypers at 3 p.m. on the concluding day of the 2-week SD. Following a 2.5-h habituation, recordings began at 6:30 p.m. and continued for 25 h. Mice were removed from the PhenoTyper at 9 a.m. the following day after a total of 42 h. Home-cage assessments were recorded and analyzed in EthoVision XT15 software. For analysis, the mean velocity of the mouse center point was recorded, and time was binned into 10-s intervals. A mouse was considered to be "attempting sleep" if their average velocity was under 0.1 cm/s for four straight 10-s intervals. Additionally, nests were scored at 18, 24, and 42 h from 1 to 5 based on previously published protocol, and the unshredded portion of the nestlet was weighed after 42 h.
Four days after the conclusion of the SD, mice were subjected to a Novel Object Recognition (NOR) test to examine recognition memory. Mice were habituated to the testing room for 30 min prior to experimentation. Subsequently, mice were individually placed in empty PhenoTypers to habituate to the arena for 10 min. After a 1-h break, the familiarization phase was conducted where mice were placed in the same PhenoTyper containing either two LEGO towers or two T25 cell culture flasks filled with bedding and allowed to freely explore the objects for 10 min. After another 1-h break, the NOR test was conducted with one object being swapped out for a novel one (a LEGO tower or T25 flask) and mice being allowed to explore the two objects for 10 min. All trials were recorded using EthoVision XT software and scored manually.
A week after the conclusion of the SD, anxiety-like behavior was assessed using an elevated plus maze (EPM). The EPM with arm lengths of 30 cm, widths of 7 cm, and 17.75-cm-high walls in the closed arms was used, and recordings were taken with overhead cameras. Mice were habituated to the testing room for a minimum of 30 min prior to experimentation. The mouse was then placed in the center of the maze and allowed to freely explore for 10 min. Recorded videos were analyzed using EthoVision XT software to determine the time spent on the open arms with no walls (entire mouse body) and in the closed arms (walls) or center of the maze.
Nine days after SD, mice were subjected to a Y-maze test to assess their working memory. Mice were habituated to the testing room for a minimum of 30 min prior to experimentation. A Y-maze with individual arm lengths of 30 cm was used with an overhead camera and dim lighting. Mice were placed in one arm facing the arm's end and allowed to explore the maze for 5 min. Trials were recorded with EthoVision XT, and each arm entry was recorded manually from the obtained videos. An arm entry was indicated when the entire mouse had entered the arm. A correct alternation was the mouse entering each of the three arms in sequence without returning to the previously investigated arm. For example, ABC or BAC would be a correct alternation, whereas BCC or ABA would be an incorrect alternation. The spontaneous alternation of the mouse was calculated as: No. of Correct Alternations/(No. of Arm Entries - 2) × 100%.
Following the behavioral period, the mice were euthanized. They were perfused with ice-cold heparinized phosphate-buffered saline (PBS). Following perfusion, brains were cut in half along the sagittal plane, with the right hemisphere being post-fixed in 4% paraformaldehyde in PBS overnight, transferred to 30% sucrose solution, and stored at 4°C. Brains were sectioned coronally at 40 µm on a sliding microtome (Leica SM2000R), with the prefrontal cortex (∼Bregma 0 to -0.20 mm), hypothalamus (POA: ∼Bregma 0 to -0.20 mm; LH: ∼Bregma -1.30 to -1.50), HPC (∼Bregma -1.30 to -3.20), lateral EC (∼Bregma -3.10 to -3.40), and LC (∼Bregma -5.30 to -5.50) being collected, and stored at -20°C in tissue cryoprotectant in a sealed plate. The left hemi-brain was subdissected immediately following perfusion and stored at -80°C until use. To prepare for immunoblotting and proteomics, the HPC was sonicated at a 30% duty cycle for 5 s three times in radioimmunoprecipitation assay buffer (10 µL/mg) containing 1 mM phenylm...
Total protein concentration of the hippocampal homogenate was determined using a bicinchoninic acid assay (Pierce BCA Protein Assay Kits, Thermo Fisher Scientific, Catalog No.: 23227). Samples were prepared with dithiothreitol, loading dye, and ddH 2 O, heated at 70°C for 6 min, and placed on wet ice for 5 to 10 s. Samples were loaded on 4% to 20% Tris-glycine polyacrylamide gels with tris-glycine running buffer under non-reducing conditions or 4% to 12% Bis-Tris gels with 2-(N-morpholino)ethanesulfonic acid buffer. Gel electrophoresis was run at a constant 125 V for approximately 75 min and transferred to a nitrocellulose membrane at a constant 200 mA for 2 h. Membranes were submerged in Ponceau S stain for 5 min and subsequently washed with ddH 2 O to ensure proper protein transfer. Subsequently, membranes were blocked with 5% milk in Tris-buffered saline 0.05% Tween-20 (TBS-T) for 1 h at room temperature. Following three 5-min TBS-T washes, membranes were incubated overnight at 4°C with primary antibodies described in Table in SuperBlock/TBS solution: ATG5 (Invitrogen, Catalog No.:...
Generally, sections were washed three times for 10 min with PBS, blocked with 2% goat serum, 0.1% triton X-100, and 1% bovine serum albumin in PBS for 1 h, and incubated at 4°C overnight with primary antibody diluted in blocking solution: GFAP (Dako, Catalog No.: Z0334), LAMP1 (Biolegend, Catalog No.: 121602), NeuN (Millipore Sigma, Catalog No.: MAB377), Orexin-A (Santa Cruz Biotechnology, Catalog No.: SC-80263), p62 (Abcam, Catalog No.: AB109012 ), PHF1 (Albert Einstein College of Medicine, Catalog No.: AB_2315150), and TH (Abcam, Catalog No.: AB76442). Primary antibodies are described in detail in Table. Following primary incubation, sections were washed three times with PBS and incubated at room temperature for 2 h in the secondary antibody listed in Table: goat anti-rabbit 488 (1:200, Invitrogen, MA, USA, Catalog No.: A11008), goat anti-rabbit 647 (1:200, Invitrogen, MA, USA, Catalog No.: A21245), goat anti-mouse 568 (1:200, Invitrogen, MA, USA, Catalog No.: A11004), goat anti-mouse 647 (1:200, Invitrogen, MA, USA, Catalog No.: A21236), goat anti-rat 647 (1:200, Invitrogen, MA, USA, Catalog No.: A21247), or goa...
Blood samples were obtained via saphenous vein collection immediately prior to and following the 2-week SD. Mice were restrained inside 50-mL conical tubes to gain access to the saphenous vein and pierced with a 23-gauge needle, and blood was collected using Sarstedt Microvette CB300 K2 EDTA tubes. Collections were done in a rapid fashion to reduce stress associated with restraint. Blood was subsequently centrifuged at 6000 × g and 4°C for 8 min. Plasma was isolated and stored at -80°C until use. Plasma samples were run in duplicate on the Arbor Assays corticosterone multiformat enzyme-linked immunosorbent assay kit from Cedarlane (K014-H5) according to the provided protocol and analyzed using the MyAssays Corticosterone Enzyme Immunoassay software.
Machine-readable layer
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"headline": "Sex-specific acceleration of Alzheimer's pathogenesis by chronic sleep-deprivation",
"datePublished": "2026",
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"name": "Wai Haung Yu"
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"identifier": "10.1002/alz.71099"
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