DOI: 10.5061/dryad.6djh9w1cw
title: Data from: Surgery impairs glymphatic activity and cognitive function in aged mice

description: Delirium is a common complication in elderly surgical patients and is
 associated with an increased risk of dementia. Although advanced age is a
 major risk factor, the mechanisms underlying postoperative delirium remain
 poorly understood. The glymphatic system, a brain-wide network of
 perivascular pathways, facilitates cerebrospinal fluid (CSF) flow and
 supports the clearance of metabolic waste. Impairments in glymphatic
 function have been observed in aging brains and various neurodegenerative
 conditions. Using in vivo two-photon imaging, we examined the effects of
 surgery (laparotomy) on glymphatic function in adult (6 months) and aged
 (18 months) mice 24 h post-surgery. In adult mice, CSF tracer entry into
 the brain parenchyma along periarteriolar spaces occurred rapidly
 following intracisternal tracer injection, with no significant differences
 between sham and surgery groups. In contrast, aged mice exhibited delayed
 tracer influx, with further impairments observed in the surgery group
 compared to sham controls. This glymphatic dysfunction correlated with
 poorer T-maze performance in aged mice. These findings suggest that
 surgery exacerbates glymphatic impairment in aging brains, potentially
 hindering brain waste clearance and contributing to postoperative
 delirium.
description: <strong>Animals</strong> Adult (6
 months old) and aged (18 months old) C57BL/6J mice were purchased from the
 Jackson Laboratory (Bar Harbor, ME, USA). Mice were group-housed in
 temperature- and humidity-controlled rooms on a 12-h light/dark cycle at
 the Columbia University animal facility (New York, NY, USA) and were
 randomly assigned to different treatment groups. Both male and female mice
 were included in this study. Since no significant sex differences were
 observed, data from both sexes were combined for analysis. All experiments
 were conducted in accordance with protocols approved by the Columbia
 University Institutional Animal Care and Use Committee (IACUC) and in
 compliance with the National Institutes of Health (NIH) Guidelines for the
 Care and Use of Laboratory Animals.
 <strong>Anesthesia and surgery</strong>
 Mice in the surgery group underwent a laparotomy under isoflurane
 anesthesia. Anesthesia was induced with 1.5% isoflurane in air, delivered
 via a transparent acrylic chamber. Once the mice were anesthetized,
 anesthesia was maintained using a cone device. A longitudinal midline
 incision was made, extending from the xiphoid process to 0.5 cm proximal
 to the pubic symphysis, penetrating the skin, abdominal muscles, and
 peritoneum. The incision was closed in layers using 5-0 vicryl sutures,
 and EMLA cream (lidocaine 2.5% and prilocaine 2.5%) was applied to the
 incision site to provide postoperative analgesia. The procedure lasted
 approximately 15 min. Throughout the procedure, the animal’s rectal
 temperature was maintained at 37 ± 0.5°C using a feedback-controlled
 temperature controller (WPI, Sarasota, FL, USA). After recovering from
 anesthesia, the mice were returned to their home cages with <em>ad
 libitum</em> access to food and water. Sham-operated mice underwent
 the same anesthesia protocol but did not receive a surgical
 incision. <strong>T maze
 test</strong> The T maze test was performed using
 a T maze chamber (Stoelting, Wood Dale, IL, USA) with three arms of equal
 length. On day 0, prior to the laparotomy surgery, mice were trained by
 being allowed to explore two of the three arms for 2 min, while access to
 the third arm was blocked using a removable door. 24 h post-surgery, mice
 were tested by granting them free access to all three arms for 2 min. The
 time spent in each arm was automatically recorded using ANY-Maze video
 tracking software (Stoelting). Novel arm preference was calculated as the
 ratio of time spent in the novel arm to the total time spent in the novel
 and familiar arms. <strong>Two-photon imaging of
 CSF tracer</strong> <em>In vivo</em>
 imaging of cerebrospinal fluid (CSF) tracer dynamics was conducted using a
 Scientifica two-photon system (Uckfield, East Sussex, UK) equipped with a
 Ti:Sapphire laser (Vision S, Coherent, Santa Clara, CA, USA). Mice were
 anesthetized with 100 mg/kg ketamine and 15 mg/kg xylazine. After creating
 a thinned-skull cranial window and performing cisternal magna cannulation,
 the mice were head-fixed under the two-photon microscope. Prior to
 imaging, 10 µL of 0.5% fluorescein isothiocyanate-dextran (FITC-dextran;
 D3305, Thermo Fisher Scientific) was infused into the cisternal magna at a
 rate of 2 µL/min using a syringe pump (UMP3T-1, WPI). Additionally, 0.1 mL
 of Texas Red™-dextran 70 kDa (D1830, Thermo Fisher Scientific) was
 injected retro-orbitally. The two-photon laser was tuned to 920 nm to
 simultaneously excite both FITC and Texas Red. Images were acquired using
 a 25× objective lens (1.05 N.A.) immersed in artificial CSF, with a
 digital zoom of 1× and a resolution of 512×512 pixels. Time-lapse Z-stack
 images were captured at 10-min intervals, spanning from the cortical
 surface to a depth of 250 µm with a step size of 5 µm.
 Imaging data were analyzed using NIH ImageJ software. Optical
 sections at a depth of 100 µm below the pial surface were selected to
 quantify CSF tracer kinetics. Penetrating arterioles were differentiated
 from venules based on morphological features, such as less branching and
 their superficial course relative to veins at shallower cortical depths.
 To assess CSF and interstitial fluid (ISF) exchange, a donut-shaped region
 of interest (ROI) was defined, with an outer diameter of 150 pixels and an
 inner diameter of 50 pixels, centered on penetrating arterioles. The mean
 pixel intensity within the ROI was calculated for each time point.
 Perivascular ROI values for each animal and time point were averaged to
 generate a single biological replicate. Tracer dynamics along penetrating
 arterioles were compared using two-way ANOVA followed by Bonferroni’s
 <em>post hoc</em> test for multiple comparisons.
 <strong>Statistical Analysis</strong>
 Statistical analysis was performed using Prism 10.0 software
 (GraphPad; San Diego, CA, USA). Data are presented as mean ± SEM (standard
 error of the mean). Comparisons between two groups were performed using an
 unpaired, two-tailed Student’s <em>t</em>-test. For
 multiple-group comparisons, two-way ANOVA was used, followed by
 Bonferroni’s <em>post hoc</em> test. No data points were
 excluded from the analysis, and variance was similar between compared
 groups. Statistical significance was set at <em>P</em> ≤
 0.05.
description: # Data from: Surgery impairs glymphatic activity and cognitive function in
 aged mice
 [https://doi.org/10.5061/dryad.6djh9w1cw](https://doi.org/10.5061/dryad.6djh9w1cw) ## Description of the data and file structure This dataset comprises the source data for the figures published in the following paper: Chen K, Du X, Chao MA, Xie Z, Yang G. Surgery impairs glymphatic activity and cognitive function in aged mice. Mol Brain. 2025 Jan 24;18(1):7. doi: 10.1186/s13041-025-01177-y ### Files and variables Figure 1. Surgery impairs glymphatic activity and cognitive function in aged mice. Source_data.xlsx contains data for Figure 1 panels c, d, and e. Sheet Fig. 1c, Quantification of CSF tracer intensity in periarteriolar spaces in adult and aged mice, with and without surgery (n = 8 mice per group). Measurements are in arbitrary units (a.u.). Sheet Fig. 1d, Quantification of novel arm preference (%) in the T-maze test in adult and aged mice, with and without surgery (n = 8 mice per group). Sheet Fig. 1e, Fluorescent tracer intensity measured at 60 minutes post-injection in aged mice and their corresponding novel arm preference (%) in the T-maze test.