An initial experiment was performed using 18 females C57BL/6J mice, aged 6–7 months, that had not previously had litters (Jackson Laboratory, Bar Harbor, ME). Mice were randomized into three groups: oophorectomized (OVX, n = 6), UTI inoculated (Sham, n = 6), and oophorectomized and UTI inoculated (n = 6). For the associated timeline see Fig. 1A. In the primary experiment (see Fig. 2A for experimental design schematic), a total of 48 female C57BL/6J mice, aged 6–7 months (no prior litters) (Jackson Laboratory, Bar Harbor, ME), were utilized. Mice were randomized to one of the following groups: oophorectomized (OVX), UTI inoculated, and treated with 7β-estradiol (n = 24); or OVX, UTI inoculated and treated with a vehicle (sesame oil, n = 24). We utilized a total of 4 cohorts (n = 6/group/cohort, 24 total mice/group) to restrict the time of day at which behavioral assessments were performed—thus, minimizing any variability due to natural circadian variations. In each of 4 cohorts, treatment groups were equally represented, and mice were randomized across the two groups. In each cohort methods were identical, including close approximations of time of day of each procedure. All dependent variables were measured in cohorts 1–3 with the following exception: novel object recognition test was measured in cohorts 3 and 4 only. CC3 was evaluated in all 4 cohorts. Data were analyzed and displayed combining all 4 cohorts where applicable. All mice were housed in Cedars-Sinai’s AAALAC accredited animal facility under standard conditions (kept in ventilated cages at approximately 21 °C, 40–70% humidity, a 12-h light/dark cycle, with food and water available to the animals ad libitum). All procedures herein follow the recommendations in the ARRIVE guidelines for research involving the use of animals. For testing procedures, order was randomized within a day to control for potential confounders.
UTI model and 17β-estradiol treatment
We have previously published the following methods (including all following subsections) and the original source of the method descriptions can be found elsewhere13. Here, we replicate the methods and provided details of any modifications and/or additions. Static cultures of E. coli (Migula) Castellani and Chalmers (ATCC® 700928™) strain (CFT073) were grown for 24 h at 37 °C. Bacterial cultures were harvested by centrifugation (4000 rpm for 10 min at 4 °C) and resuspended in sterile endotoxin-free phosphate-buffered saline (PBS; PromoCell, Heidelberg, Germany) at a concentration of 1 × 109 colony forming unit/mL (CFU/mL). Mice were inoculated transurethrally as previously described13,24. Briefly, 6–7-month-old female C57BL/6J mice were quarantined for a minimum of 1 week prior to inoculation and allowed food and water ad libitum. On Day 0 (Fig. 2A), mice were anesthetized with isoflurane USP (Clipper Distribution Company LLC, St. Joseph, MO) delivered by a vaporizer (Vet Equip Inc., Pleasanton, CA) set at 2% isoflurane and 0.8 L of oxygen per minute. The inoculum was delivered via a 1-ml syringe connected to a 1/2-in., 30-gauge needle. The catheters were prepared with sterile 0.61-mm (internal diameter) polyethylene tubing (PE 10, Becton, Dickinson, Sparks, MD). The needle was inserted into the catheter, and 100 µl of inoculum containing 1 × 108 CFU of E. coli was delivered. Urine samples were collected and processed bacterial enumeration and determination of the intensity of pyuria. Following inoculation with E. coli mice were treated with either vehicle (200 µl of sesame oil) or 17β-estradiol (7 µg in 200 µl of sesame oil; mean = 0.28 mg/kg, range 0.24–0.32 mg/kg) via intraperitoneal injection once a day for 3 days. The 17β-estradiol dose was selected based on previously observed protective effects on behavior and neuronal function in mouse studies25,26. After 3 days, mice were euthanized by perfusion, while deeply anesthetized with a combination of ketamine and dexmedetomidine, followed by a physical method for tissue and blood collection, and the brain was aseptically removed. Plasma collected at the time of tissue harvest was utilized for the quantification of IL-6 and IL-6R.
Brain isolation and treatment
Mice were deeply anesthetized and perfused with room temperature PBS with 0.5 mM ethylenediaminetetraacetic acid (10 mL). Right hemispheres were collected and fixed by submerging in ice-cold PBS buffered 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min, and then cryo-protected in 2% paraformaldehyde + 30% sucrose at 4 °C for 24–48 h. Free-floating, 30-μm-thick coronal brain cryosections were prepared and stored at 4 °C in PBS + 0.02% sodium azide until staining. Cortices were separated from the left hemispheres and were immediately frozen on dry ice. They were then stored at − 80 °C until protein extraction.
Immunohistochemistry and microscopy
Sections were affixed to slides by air drying and subjected to heat-induced epitope retrieval for 10 min in antigen-retrieval solution (pH 6.0; Invitrogen) before permeabilization/blocking in 5% BSA + 0.25% Triton X-100 in PBS for 1 h at room temperature. Sections were then incubated at 4 °C overnight with primary antibodies diluted in 1% BSA + 0.01% Triton X-100 in PBS (Ab Diluent). After washing, sections were incubated with a combination of the appropriate secondary antibody (Alexa Fluor Plus conjugated; Invitrogen) diluted to 4 µg/mL in Ab Diluent for 1 h at room temperature. After washing, sections were incubated in 0.05% Sudan black B in 70% ethanol for 10 min to reduce tissue autofluorescence. Sections were mounted using ProLong Glass with DAPI (Invitrogen, Carlsbad, CA, USA). Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labeling. A Carl Zeiss AxioImager Z.2 epi-fluorescence microscope equipped with standard filter sets/mercury arch lamp, an Apotome 2.0, and an Axiocam HRm camera controlled by Zen Blue Pro (version 2.3) software was used to acquire and process images. Images of damage marker (i.e., CC3) staining were acquired with a 10× objective (NA 0.3, Zeiss) as a 5 × 5 tiled image that encompassed the frontal cortex and hippocampus of each section. All acquisition and display settings are the same between groups and settings were optimized using the UTI group. All images within a figure are set to the same scale.
Fiji (ImageJ v. 1.53c) software was used for image analysis and semi-quantitation. Three coronal sections containing the cortex and hippocampus were analyzed (one ventral, one mid, and one dorsal) per animal. For damage marker analysis, two different regions of interest (ROIs) were drawn on tiled images of sections: a ROI around the cortex (with an average area of 315 µm2), or a ROI encompassing the entire hippocampus. A threshold was set to exclude background pixels per ROI based on the pixel intensity histogram, and the number of positive pixels was measured and then expressed as percent area of the ROI (e.g., see Fig. 1B). For cytokine analysis, a single field z-stack projection was analyzed per section. Background pixels were excluded per field based on the pixel intensity histogram and the intensity of the remaining pixels was used to calculate percent area. Values for each protein from the triplicate sections were averaged to yield one number per animal. Analysis was performed by assessors blinded to group allocation.
Locomotor activity, anxiety, and memory were evaluated by subjecting individual mice to a series of behavioral tests prior to and following UTI induction and dose 2 of 17β-estradiol treatment (or vehicle) (Fig. 2A). Note that, to prevent any habituation to the apparatus and lack of motivation that may occur during subsequent exposures, novel object recognition and Y-maze behavioral tests were only performed post UTI induction and 17β-estradiol (or vehicle, sesame oil) treatment. Collectively, these behavioral tests (Fig. 2) are thought to model delirium-like phenotypes observed in humans: psychomotor agitation, inattentiveness, and short-term memory impairment3. We have previously utilized a subset of these behavioral tests to model delirium-like phenotypes in a UTI mouse model13.
Broadly, the open field test measures locomotor activity and exploratory behavior. Delirium-relevant behavioral features that can be assessed with the open field test include altered level of consciousness, psychomotor agitation, or retardation27. In normal mice, locomotor activity decreases over time due to habituation to the testing environment. Failure to habituate to the testing environment indicates the presence of altered consciousness while increased time spent in the periphery as opposed to the center of the open field maze indicates increased anxiety-like behavior. Locomotor activity and anxiety-related emotional behaviors were evaluated by placing individual mice in the center of an opaque Plexiglas (40 × 40 × 40 cm) arena and allowed to explore for 45 min27,28. The open field was divided into a 20 × 20 cm central zone (center) and a surrounding border zone (periphery) during the analysis (Fig. 2B). Activity in the open field was recorded by a computer-operated camera system (Stoelting Digital USB 2.0 CMOS Camera). Total distance or locomotor activity (m), movement time (sec), time in the center zone (sec), and time in the periphery (sec) were collected using ANY-maze Video Tracking Software version 7.1 (Stoelting Co., Wood Dale, IL, USA). Average speed (m/s) and time immobile (sec) were calculated. Total distance (m) and total time immobile (sec) were analyzed as a percent of baseline activity. Baseline activity was defined as the distance traveled or time spent immobile during the first five minutes of the 45-min test.
Elevated plus maze
Anxiety related behavior and altered level of consciousness was assessed utilizing an elevated plus maze (EPM)28,29,30. The EPM crossed arms are approximately 30 cm above the floor during testing, with open (no walls) and closed arms (walled) crossed perpendicularly to each other (Fig. 2D). Mice with delirium spend more time in the closed arms of the maze—avoidance of the open arms reflects higher anxiety-like behavior. A video camera was placed above the apparatus and ANY-Maze Video Tracking Software 7 (Stoelting Co., Wood Dale, IL, USA) was used to record movements. Individual mice were placed in the center of the crossed arms facing an open arm and allowed to freely explore the entire maze for 5 min. The EPM was cleaned with 70% EtOH solution between animals to eliminate odor traces. Time spent in open and closed arms of the elevated plus maze was quantified and time spent in open arms (sec) was analyzed.
Novel object recognition
To test non-spatial memory performance we utilized a novel object recognition memory test (NOR)31. Prior to testing the arena was cleaned with 70% EtOH before use. The training phase for the known object exposure occurred 24 h prior to and following UTI induction. During the training phase, a single object (known) was presented. During the training phase if mice exhibited biases for regions of the arena or did not explore the objects presented, they were excluded from the testing portion utilizing the novel object. The novel object, or test phase exposure, occurred after the treatment of 17β-estradiol (or vehicle), 48 h after UTI induction. During the test phase, in addition to the known object, a novel object was also presented (Fig. 2E). In normal mice, there is typically a preference for the novel object as the known object has been previously investigated. In mice with delirium, or altered acute memory, less preference for the novel object is demonstrated. All data was collected and scored using the AnyMaze (Stoelting Co., Wood Dale, IL, USA) data acquisition system. During all phases mice were habituated to the open field arena for at least 30 min prior to object exposure to minimize fear and/or anxiety. All objects were fixed to the floor before all exposure phases. The known and novel objects were both placed in the arena with enough distance between the wall and each other so that mice could freely explore the objects from all angles. Mice were allowed to explore objects for 10 min total during all phases and were not disturbed during this period. Object explorations were counted once the mouse was oriented towards the object, the snout was within 2 cm of the object, and the midpoint of the animal’s body was beyond 2 cm from the object. During the test phase the objects were placed in the same position as the training phase. We assessed novel object recognition by examining the time oriented toward each object.
The Y-Maze test was used to assess attentional, cognitive, and short-term memory28,32,33. The Y-maze consists of three arms with an angle of 120 degrees between each arm (Fig. 2F). Mice typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited, as shown by alternated exploration of the 3 arms. In mice with delirium, we expect fewer alterations into a new arm as a percent of the total visits. Tests were performed in the manner previously described in13. Briefly, after treatment of 17β-estradiol or vehicle, mice are placed in the maze and allowed to freely explore the three arms for 10 min. AnyMaze (Stoelting Co., Wood Dale, IL, USA) tracking software was used to analyze the number and the sequence of arm entries, and the percentage of spontaneous alternations was calculated. Alternations are defined as the consecutive visits of the three different arms without returning to an arm previously visited. The Y-maze was cleaned with 70% EtOH solution between each mouse to eliminate odor traces.
Cortical neuron culture and treatment
Primary rat cortical neurons were obtained from ThermoFisher Scientific (Gibco) and grown according to the manufacture’s protocol. Briefly, a 24-well plate precoated with poly-d-lysine (100 µg/mL for a minimum of 1 h at room temperature) was seeded with 4 × 105 live cells (measured by BioRad TC10 Cell Counter, trypan blue-positive cells excluded) per well in 2 mL of growth medium. Neurons were grown in an incubator set to 37 °C and 5% CO2 in complete Neurobasal Plus medium (Neurobasal™ Plus base medium with 2% B-27™ Plus supplement, 2 mM GlutaMax™, and 1 × Antibiotic–Antimycotic), and half volume medium exchanges occurred every 24 h for 7 days. On the 7th day, neurons were treated with one of following conditions: (1) vehicle (growth medium), (2) 10 ng/mL IL-6 and 40 ng/mL sIL-6R (both from PeproTech, Cat# 200-06 and Cat# 200-06RC respectively), (3) IL-6 & sIL-6R plus 5 ng/mL 17β-estradiol (Millipore Sigma, Cat# 3301). Treatment medium (2 mL) was first allowed to equilibrate to the incubator for 30 min prior to treatment. The growth medium was then completely replaced with the pre-equilibrated treatment medium, and the plate was then incubated for 1 h, after which time protein extraction occurred.
Tissue culture plates were placed on ice, culture media was removed, and wells were washed two times with ice-cold PBS. Wells were then extracted with 95uL of ice-cold RIPA buffer with inhibitors. Protein concentrations were measured with Pierce, BCA protein assay kit (Thermo Scientific, USA). Equal amounts of proteins (8 μg for primary culture extracts) were denatured (95 °C for 5 min in SDS-sample buffer), separated on a SDS 4–12% polyacrylamide gel, and then transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA, USA). Blots were blocked for 1 h at room temperature with 5% (w/v) BSA in PBST (PBS + 0.01% TX-100). Then the membrane was incubated at 4 °C with specific primary antibodies overnight. Primary antibodies used are listed in Table S1. After 3 wash steps with PBST, the blot was then incubated with the corresponding horseradish peroxidase‐conjugated secondary antibody (1:30,000). The blot was developed using Pierce Super Signal West Pico (Thermo Scientific, USA). Western blot images were acquired by iBright Western Blot Imaging Systems (Invitrogen, Carlsbad, CA, USA) and analyzed by iBright Analysis Software Version 3.1.3.
As previously described13, based on preliminary analysis using CC3, a power analysis with one-way ANOVA and Tukey’s post-hoc test yielded greater than 95% power at the 0.05 significance level for an effect size of 1.1317 with a minimum of n = 8/group.
Prism 9.4.1 (GraphPad, https://www.graphpad.com/) was used for statistical analyses and analyses were performed by assessors blinded to group allocation. For the primary experiment (Fig. 2A), differences between experimental groups were evaluated using independent sample t-tests, while within group differences across time points (pre and post UTI) were evaluated using paired sample t-tests. For some open field behaviors (Fig. 2B) a two-way repeated measures ANOVA was utilized with main effects of time, (5 min sampling windows over 45 min) treatment (17β-estradiol vs. vehicle), and time-by-treatment interaction. Post-hoc group comparisons were evaluated using a Fisher’s LSD test. Regression analyses were utilized to examine putative causal relationships between variables. For the in vitro data presented in Fig. 6, a one-way ANOVA (with post-hoc analysis) was utilized to determine differences between treatment groups. Data are presented as means ± SD (unless otherwise noted) and p < 0.05 was always considered statistically significant. Statistically significant outliers were determined (Grubbs’ method with an Alpha = 0.05) and excluded from analyses, with corresponding analyses of complete data sets provided in the Supplementary Information. Data were reported missing when a sample could not reliably be obtained and are appropriately acknowledged in each figure legend. Exclusions for behavioral analyses were based on visual identification of compromised (e.g., acute minor injury) or abnormal (e.g., excessive jumping or attempting to exit the apparatus) locomotor behavior presumed unrelated to infection and/or treatment. Across 4 cohorts, there were 3 such cases in the vehicle group and 3 cases in the 17β-estradiol group. These individuals were excluded from all behavioral analyses.
Ethics approval and consent to participate
All the experiments were performed according to the National Institutes of Health guidelines and regulations. The Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center (protocol #7914) approved all the experimental procedures. All animals were kept under regular barrier conditions at room temperature with exposure to light for 12 h and dark for 12 h. Food and water were provided ad libitum. All efforts were made to ameliorate the suffering of animals. Deeply anesthetized mice were euthanized by perfusion followed by a physical method for tissue collection.