Gynecological surgery in adulthood imparts cognitive and brain changes in rats: A focus on hysterectomy at short-, moderate-, and long-term intervals after surgery

All procedures were identical for each age cohort. Three cohorts of 40 five-month-old female, non-sexually experienced, reproductive tract-intact Fischer-344-CDF rats from the National Institute on Aging colony at Charles Rivers Laboratories (Raleigh, NC) were utilized in this experiment ( N = 120). Upon arrival at the Arizona State University animal facility, rats were pair-housed, provided free access to food and water, and were maintained in a temperature- and humidity- controlled environment on a 12-hour light/dark cycle (lights on at 7:00 am, lights off at 7:00 pm). Experimental surgeries commenced after one week of acclimation to the facility. All experimental procedures were approved by the Arizona State University Institutional Animal Care and Use Committee and adhered to National Institutes of Health standards. Fig. 1 depicts the experimental timeline for each Age cohort (Adult, Early Middle-Age, and Late Middle-Age) of rats and the variations in surgical manipulations performed. All rats in this study were five months old at the beginning of the experiment. For each Age cohort of rats ( N = 40 per Age cohort), 10 subjects were randomly assigned to each of the following surgical conditions: Sham, Ovx, Hysterectomy, or Ovx-Hysterectomy. Surgeries were performed one week (±1 day) after arriving at the animal facility. The Adult cohort was tested six weeks after surgery when rats were approximately 6 months of age, and hormones, ovaries, and brains were assessed after euthanasia which was 10 weeks after surgery at 8 months of age. The Early Middle-Age cohort was tested 7 months after surgery when rats were approximately 12 months of age, and hormones, ovaries, and brains were assessed after euthanasia which was 8 months after surgery at 13 months of age. The Late Middle-Age cohort was tested 12 months after surgery, when rats were approximately 17 months of age, and hormones, ovaries, and brains were assessed after euthanasia which was 13 months after surgery at 18 months of age. The anesthetic was vaporized inhaled 3–5 % isoflurane in otherwise pure oxygen for induction of anesthesia, and 1–3 % isoflurane for anesthesia maintenance. Each rat received subcutaneous injections of 1.0 mg/kg meloxicam (a non-steroidal anti-inflammatory drug), 1.2 mg/kg buprenorphine SR-LAB (an extended-release opioid analgesic), and 5.0 mL of Lactated Ringer’s Solution to maintain hydration. Following sterilization of the surgical field, all rats received a ventral midline incision through the skin and muscle. The Sham group underwent only these incisions; abdominal viscera were not further manipulated. For the Ovx group, the ovary, oviduct, and tip of the uterine horn were ligated bilaterally with dissolvable Vicryl sutures and removed. For the Hysterectomy group, each uterine horn was ligated and cut below the ovary and oviduct. The uterine body was then separated from the abdominal fat and ligated at the utero-cervical junction prior to excision. For the Ovx-Hysterectomy group, the ovaries, oviducts, and uterus were separated from the abdominal fat; ovarian vessels were crushed and the uterine body was ligated at the utero-cervical junction and removed. Next, the muscle incision was closed with dissolvable Vicryl suture, and 0.25 % bupivacaine (Marcaine, Pfizer Pharmaceutical, Hospira Inc., Lake Forest, IL) was applied topically prior to closing the skin with surgical staples. Rats recovered under an escapable heat immediately following surgery and were then single-housed for one week prior to being re-pair-housed with a cage mate for the remainder of the study. Following surgery, two rats died from the Early Middle-Age Cohort (two Ovx-Hysterectomy rats), and three rats died from the Late Middle-Age Cohort (two Ovx rats and one Hysterectomy rat). Over the course of the year-long experiment, some natural attrition occurred in the Early Middle-Age and Late Middle-Age cohorts as well. The final n/group utilized in the statistical analyses is noted below. Baseline weight was recorded for each rat at the time of surgery. Body weights were then recorded weekly for the entirety of the experiment, as well as on the day of euthanasia. All rats in each Age cohort underwent vaginal cytology monitoring throughout the experiment. The Adult cohort was assessed two weeks after surgery for eight consecutive days. The Early Middle-Age cohort was assessed for eight consecutive days two weeks after surgery, and again for eight consecutive days six months after surgery, prior to the Early Middle-Age cohort’s behavior testing. The Late Middle-Age cohort was assessed for eight consecutive days two weeks after surgery, for eight consecutive days six months after surgery, and for eight consecutive days 12 months after surgery, prior to starting behavior testing. All animals were also smeared 24 h prior to euthanasia, and on the day of euthanasia. Vaginal cytology classification was completed to assess normal estrous cyclicity in rats with intact ovaries (i.e., the Sham and Hysterectomy groups) as well as confirm Ovx status in rats without intact ovaries (i.e., the Ovx and Ovx-Hysterectomy groups). Smear cytology was classified as proestrus, estrus, metestrus, or diestrus, according to Goldman et al. (2007) and Koebele and Bimonte-Nelson (2016) . Proestrus smears were characterized by round nucleated epithelial cells with or without some cornified cells. Estrus was classified as cornified cells. Metestrus was considered a combination of cornified, needle-like, leukocytes, and round epithelial cells. Diestrus consisted of the presence of leukocytes, with or without cornified cells ( Goldman et al., 2007 ; Koebele and Bimonte-Nelson, 2016 ). The water radial-arm maze (WRAM) was a water-escape, win-shift task that evaluated spatial working and reference memory, as previously described ( Bimonte-Nelson et al., 2015 ; Bimonte and Denenberg, 1999 ; Koebele et al., 2019 ). The maze apparatus had a circular center with eight evenly sized arms (38.1 cm long x 12.7 cm wide) radiating from the center arena in a circular fashion. The maze was filled with 18–20 °C water made opaque with nontoxic black paint. Four of the eight arms had platforms submerged ~2 cm below the water’s surface. The pattern of arm locations for the hidden platforms stayed the same within a rat across all testing days, but varied among rats, and platform location patterns were counterbalanced across treatment groups. The testing rooms had spatial cues around the room to facilitate spatial navigation. Rats were given four trials per day for 12 baseline days. During each daily testing session, at the beginning of the first trial, the rat was released into the non-platformed start arm and allotted three minutes to locate a platform. If a platform was not discovered after the maximum allotted trial time, the experimenter gently guided the rat to the nearest platform using a lead stick. Once the rat was on the platform, it remained on it for 15 s before the experimenter removed the rat from the maze and placed it into its testing cage for a 30-s inter-trial-interval (ITI). During those 30 s, the experimenter removed the just-found platform from the maze and used a net to stir the water to distribute potential olfactory cues and remove debris from the water. The rat was then placed back in the water for the second trial with the three remaining platforms. This procedure was repeated until all four platforms were located within the daily testing session. The experimenter manually recorded arm entries during all trials. An arm entry was quantified when the rat’s snout passed an 11 cm mark on the outside of a given arm, which was visible to the experimenter and not the rat. On day 13 of testing, a six-hour delay was incorporated between trials two and three in order to assess delayed working memory retention. Errors were classified into three error sub-types. Working memory correct (WMC) errors were arm entries made on trials 2–4 into arms that previously contained a platform during the daily testing session. Reference memory (RM) errors were the first entries into a never-platformed arm within a day. Working memory incorrect (WMI) errors were quantified when a rat subsequently re-entered a never-platformed arm within the daily testing session. Following one day of rest after completing the WRAM, all rats were tested on the spatial reference memory Morris water maze (MM) task as previously described ( Bimonte-Nelson et al., 2015 ; Morris, 2015 ; Morris et al., 1982 ). The apparatus was a circular tub (188 cm in diameter). The tub was filled with 18–20 °C water made opaque with nontoxic powdered black paint. One weighted platform (11 cm in diameter) was submerged ~2 cm below the water’s surface in the northeast quadrant of the maze, where it remained constant across all days and baseline trials. Obvious spatial cues were placed around the room to aid in spatial navigation. The rats received four trials per day for five days. For each trial within a daily testing session, the rats were each dropped off from one of four possible starting points (north, south, east, or west). The trial drop-off location order varied across days but was the same for all rats within a day. The rat was allotted a maximum of 60 s to locate the submerged platform. If the rat did not find the platform after 60 s, the experimenter gently guided the rat to the platform with a lead stick, where the rat remained for 15 s before being returned to its heated testing cage for an ITI of approximately 10 min. Between each trial the water was stirred and cleared of any debris. On day five of the task, after the four baseline trials were complete, an additional probe trial was administered. During the probe trial, the platform was removed from the maze, and each rat was allowed to swim for 60 s to test whether the rats had successfully spatially localized to the fixed platform’s previous location within this maze. A digital video camera and Ethovision tracking system (Ethovision; Noldus Instruments, Wageningen, Netherlands) tracked the rat’s swim distance and path (cm) to the platform. The day following the completion of the MM task, all rats were tested on the control Visible Platform task which evaluated the motoric and visual competency to successfully solve a water maze task. The apparatus was a rectangular tub (100 cm × 60 cm) surrounded by opaque curtains to block potential extra-maze cues and was filled with 18–20 °C clear water. A visible black platform (10 cm in diameter) protruded about 4 cm above the water’s surface. Each rat underwent six trials within one day. The visible platform location varied across trials (left, center, or right side of the north wall of the tub), but the location within a given trial was the same for all rats. Each rat was dropped off from a fixed start location in the middle of the south wall of the tub and had a maximum of 90 s to locate the visible platform. Once the platform was located, the rat remained on it for 15 s before being returned to its heated testing cage for an ITI of approximately 10 min while other rats were tested. The experimenter recorded latency (s) to reach the visible platform for each subject and trial. After two days of rest following the Visible Platform task, all rats underwent testing in the open field task (OFT), a measure of locomotor activity and anxiety-like behavior. Task performance has been reported to be sensitive to the presence and absence of ovarian hormones ( Hiroi and Neumaier, 2006 ). The open field apparatus was a 100 cm × 100 cm square arena made of black plexiglass. Twenty-four hours prior to the task, the arena was thoroughly cleaned with Odormute, an enzyme cleaner used to eliminate odors. A red light visible to the experimenter was used in the room, such that the task was run in darkness for the rats, as has been previously done ( Hiroi and Neumaier, 2006 ). Rats were allowed to acclimate in the anteroom of the testing area for at least 30 min in their individual testing cages prior to OFT testing. Each rat was brought into the testing room individually. The experimenter gently placed the rat in the arena from the center of the north wall and quietly exited the room. The rat was allowed to explore the box freely for a 10-min trial. Ethovision was used to record and track the rat’s movements in the OFT. Twenty-five equally spaced squares were digitally overlaid onto the arena in order to track distance moved and time spent in the small center (inner most square), center (inner nine squares), corners, and along the perimeter of the arena. Following the 10-min trial, the rat was removed from the arena. Any fecal boli were removed from the arena; the experimenter wiped the arena with water and then dried it prior to starting the next rat’s trial. This was done in order to evenly distribute any odors left by a previously tested rat throughout the arena. One week following the OFT, rats were euthanized. The Adult cohort was approximately 8 months old, the Early Middle-Age cohort was approximately 13 months old, and the Late Middle-Age cohort was approximately 18 months old, at euthanasia. All rats were deeply anesthetized using inhaled isoflurane anesthesia. Blood was obtained via cardiocentesis and allowed to clot at 4 °C for a minimum of 30 min (Vacutainer 367,986, Becton Dickinson and Company, Franklin Lakes, NJ) prior to centrifugation at 2000 rpm at 4 °C for 20 min. Serum was extracted and stored at −20 °C until analysis. Rats were decapitated and brains rapidly removed. The right hemisphere of the brain was rapidly raw dissected for frontal cortex, dorsal hippocampus, and entorhinal cortex. The raw dissected tissues were weighed, snap frozen on dry ice, and stored at −70 °C until analysis. Ovaries were collected from ovary-intact groups (i.e., the Sham and Hysterectomy groups), trimmed of excess fat, weighed, and transferred to 10 % buffered formalin for 48 h prior to being stored in 70 % ethanol at 4 °C until analysis. Uteri were collected from uterus-intact groups (i.e., the Sham and Ovx groups), trimmed of excess fat, and wet weight was obtained. All serum hormone assays were completed by the Ligand Assay and Analysis Core of the Center for Research in Reproduction at the University of Virginia. Samples were run in duplicate unless otherwise noted. 17β-Estradiol (E2) levels were determined with a commercially available ELISA kit (CAT# ES180S-100, CALBIOTECH, Spring Valley, CA). E2 inter-assay coefficients of variation averaged 9.7 % (intra-assay 6.7 %) with a functional sensitivity of 3 pg/mL and a reportable range of 3–300 pg/mL. Progesterone levels were determined with a commercially available ELISA kit (CAT# IB79105, Immuno-Biological Laboratories Inc., Minneapolis, MN). Inter-assay coefficients of variation averaged 10.5 % (intra-assay 6.6 %) with a functional sensitivity of 0.045 ng/mL and a reportable range of 0.15–40.00 ng/mL. Androstenedione levels were determined with a commercially available ELISA kit (CAT# 11-ANRHU-E01, ALPCO, Salem, NH). Inter-assay coefficients of variation averaged 9.1 % (intra-assay 5.9 %) with a functional sensitivity of 0.04 ng/mL and a reportable range of 0.10–10.0 ng/mL. E2 and androstenedione levels were measured for ovary-intact rats only. Progesterone levels were obtained for all subjects. Serum LH levels were assessed using a sensitive two-site sandwich immunoassay ( Fallest et al., 1995 ; Haavisto et al., 1993 ) with monoclonal antibodies against bovine LH (no. 581B7; RRID:AB_2665514) and against the human LH-beta subunit (no. 5303: Medix, Kauniainen, Finland; RRID:AB_2665513) as previously described ( Haavisto et al., 1993 ). Dr. Janet Roser (Department of Animal Science, University of California, Davis) ( Matteri et al., 1987 ) kindly provided the tracer antibody (no. 518B7), which was iodinated by the chloramine T method, and purified on Sephadex G-50 columns. The capture antibody (no. 5303) was biotinylated and immobilized on avidin-coated polystyrene beads (7 mm; Epitope Diagnostics, Inc., San Diego, CA). The standard was a mouse LH reference prep (AFP5306A; provided by Dr. A.F. Parlow and the National Hormone and Peptide program). The assay had a sensitivity of 0.04 ng/mL and a reportable range of 0.04–75.0 ng/mL. Serum FSH levels were obtained via RIA utilizing reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program, as described previously ( Gay et al., 1970 ). Mouse FSH reference prep AFP5308D was used for assay standards. The primary antibody was a Mouse FSH antiserum (guinea pig; AFP-1760191; RRID:AB_2665512) diluted to a final concentration of 1:400,000. The secondary antibody was purchased from Equitech-Bio, Inc. and diluted to a final concentration of 1:25. The assay had a sensitivity of 2.0 ng/mL and <0.5 % cross-reactivity with other pituitary hormones. The reportable range for the initial assay was 2.5–37.7 ng/mL. Because rats without ovaries were above the detection range of the original undiluted assay, samples for all Ovx and Ovx-Hysterectomy groups were rerun in singlet at a dilution of 1:2 or 1:5. This assay had a reportable range of 1.6–46.0 ng/mL. Thus, all Ovx and Ovx-Hysterectomy values used singlet values for the reported statistical analysis. For all serum hormone assays, if values fell outside of the reportable range, a placeholder value was inserted that was ±0.1 beyond the reportable range as a conservative estimate (progesterone placeholder = 40.1 ng/mL; androstenedione placeholder = 0.09 ng/mL; E2 placeholder = 2.9 pg/mL; FSH placeholder = 2.4 ng/mL); however, the actual circulating level values are unknown (D. Haisenleder, Director of the Ligand Assay and Analysis Core of the Center for Research in Reproduction at the University of Virginia, personal communication, April 5, 2021). All substitutions are noted in the results section . Thus, serum results should be interpreted with caution, as we cannot state the true magnitude of difference between groups due to technical assay limitations, only that there is a statistical difference when placeholder values are included. Only subjects included in behavioral analyses were included in serum assessments. All ovarian histology and quantification was completed using methods previously described ( Koebele et al., 2017 , 2019 , 2020a ). Briefly, for each Age cohort, one ovary from each Sham and Hysterectomy subject (i.e., all rats that had ovaries at the end of the experiment) was randomly selected for histological evaluation and quantification of healthy primordial, primary, secondary, and antral follicles, as well as corpora lutea. Following fixation, ovarian tissues were paraffin embedded and sectioned at 5 μm. Every 10th section was mounted on a slide and stained with hematoxylin and eosin Y-phloxine B. Primordial, primary, secondary, and antral follicles were counted at 20× magnification for every 20th section. Corpora lutea were counted at a lower magnification (Pannoramic DESK; 3D Histech, Budapoest, Hungary). The following formula was used to calculate the total number of follicles in the ovary: N t = (N 0 x S t x t s ) / (S 0 x d 0 ), where N t = total calculated number of follicles, N 0 = number of follicles observed in the ovary, S t = total number of sections in the ovary, t s = thickness of the section (μm), S 0 = total number of sections observed, and d 0 = mean diameter of the nucleus ( Gougeon and Chainy, 1987 ). Ovarian follicles were classified using criteria from Haas et al., 2007 . Primordial cells were counted when there was a single layer of squamous granulosa cells surrounding the oocyte. Primary follicles contained a single layer of cuboidal granulosa cells. Secondary follicles required several granulosa cell layers surrounding the oocyte. Antral follicles were counted when the follicle contained two or more layers of granulosa cells and had a fluid-filled antral space in the follicle ( Haas et al., 2007 ). Atretic/unhealthy follicles were not counted. Ovary weights were compared between groups that retained their ovaries throughout the experiment (i.e., Sham and Hysterectomy groups) after euthanasia. Left and right ovaries were weighed separately. There were no differences in weight between left and right ovaries for any cohort, so ovary weight was summed for analysis. Uterine weights were compared between groups that retained their uterus throughout the experiment (i.e., Sham and Ovx rats) after euthanasia. Frontal cortex, dorsal hippocampus, and entorhinal cortex from the right hemisphere were processed for FOSB expression and ΔFOSB expression via western blot protein analysis for all subjects that survived until the end of the experiment. Frozen raw tissue samples were suspended in a 1:25 weight-to-volume RIPA buffer solution (150 mM NaCl, 1% Triton X-100, 0.1 % SDS, 0.5 % sodium deoxycholate, 50-mM Tris HCl, protease inhibitor (Millipore-Sigma, CAT#5892791001), and phosphatase inhibitor (Millipore-Sigma, CAT#524625) and subsequently kept on ice at all times. Tissues were homogenized using a probe sonicator (Ultrasonic Processor, Cole Parmer, IL, USA) and then centrifuged at 10,000 rpm for 10 min at 4 °C. Cleared supernatants were collected, aliquoted, and frozen at −70 °C until analysis. Protein concentration for each sample was determined using a bicinchoninic acid protein assay (Thermo-Fisher Scientific, Pittsburg, PA, USA). Treatment groups within each Age cohort were counterbalanced and equally represented on each gel run. Samples for each region were loaded at an equal protein concentration and run on a 4–12 % NuPAGE Bis-Tris gel in an XCell SureLock Mini-Cell (Invitrogen, Carlsbad, CA, USA) with MOPS running buffer. After transferring to an Immobilon polyvinylidene difluoride membrane, the membrane was incubated in Ponceau stain to confirm protein transfer. The membrane was washed in 1xTBST and then blocked in 5 % nonfat milk for 1 h at room temperature. Following blocking, the membrane was washed in TBST and incubated with the primary antibodies for either anti-FOSB (1:1000; Abcam, ab184938), anti-phosphorylated (p)- ERK1/2 (1:2000; Cell Signaling, #4370), anti-total ERK1/2 (1:20,000; Cell Signaling, #9102), or anti-beta-tubulin (1:20,000; Cell Signaling, #2128S) in 5 % nonfat milk/TBST on a shaker at 4 °C overnight, washed in TBST, and then incubated with anti-rabbit HRP secondary antibodies (1:500; Cell Signaling #7074) for 1 h at room temperature in 5 % nonfat milk. The membrane was washed with TBST and secondary antibodies were detected using chemiluminescence (Lumiglo and peroxide, Cell Signaling, #7003S) and a film developer (Konica SRX-101A Film Processor, Tokyo, Japan). FOSB bands were observed at approximately 50 kDa and ΔFOSB bands were observed at approximately 38 kDa. Beta-tubulin was observed at approximately 52 kDa. Bands for ERK1 and ERK2 were present at approximately 44 kDa and 42 kDa, respectively. Densitometry analyses of high-resolution scans were completed using ImageJ ( Gallo-Oller et al., 2018 ). Phosphorylated- ERK1 and ERK2 were normalized to Total ERK1 and ERK2, respectively. Total ERK1/2 were also normalized to beta-tubulin to assess changes in Total ERK1/2. FOSB and ΔFOSB were normalized to beta-tubulin. Statview software was utilized to complete all statistical analyses. The independent variable for all analyses was Surgery type (Sham, Ovx, Hysterectomy, or Ovx-Hysterectomy). All analyses were two-tailed tests with an alpha level set to 0.05. Because our primary question was to understand how hysterectomy with ovarian conservation impacted cognition compared to intact sham-control rats, as well as how hysterectomy with ovarian conservation impacted cognition compared to other surgical variations, two-group a priori planned comparisons were completed for the Hysterectomy vs. Sham control group, as well as Hysterectomy compared to each other experimental surgery group. The Sham group was compared to the Ovx group to assess the role of surgical removal of the ovaries only, and the Ovx group was compared to the Ovx-Hysterectomy group to understand the impact of the presence or absence of the uterus when ovaries are not intact. As previously reported, to provide optimal comparability between analysis types ( Koebele et al., 2019 , 2020a , 2020b , 2022 ), effect size for repeated measures ANOVA was calculated as generalized eta squared ( η G 2 ) ( Bakeman, 2005 ; Olejnik and Algina, 2003 ). For non-repeated measures ANOVAs with one between-subjects factor, eta squared (η 2 ) was calculated. Standard effect size guidelines are used to interpret η G 2 and η 2 , with 0.02 as a small effect, 0.13 as a medium effect, and 0.26 as a large effect) ( Bakeman, 2005 ; Olejnik and Algina, 2003 ). WRAM data were analyzed with Trials within Days as the repeated measures and Errors as the dependent variable. WRAM data were divided into two blocks, similar to our previously reported procedures; Day 1 was considered training and not included in analyses; Days 2–7 were the Learning Phase, and Days 8–12 were the Asymptotic Phase ( Bimonte et al., 2000 ; Bimonte and Denenberg, 1999 ; Braden et al., 2017 ; Koebele et al., 2017 , 2020a ; Mennenga et al., 2015b ; Prakapenka et al., 2018 ). Within the Learning Phase and Asymptotic Phase, a priori planned two-group analyses compared all trials, as well as moderate (Trial 3) and maximum (Trial 4) working memory load trials alone. These analyses were based on previous publications revealing exacerbated working memory deficits when working memory load becomes taxed in rats, including recent findings of hysterectomy-induced working memory deficits six weeks post-surgery ( Koebele et al., 2019 ). Separate analyses were run for each WRAM error type (i.e., WMC, WMI, RM). MM data were evaluated using two-group planned comparison repeated measures ANOVA, with Trials within Days as repeated measures, and the dependent variable as Swim Distance (cm) to Platform for all baseline trials. For the probe trial, Proportion Total Swim Distance in the target versus the opposite quadrant was compared. Each Surgery group was evaluated independently for probe trial performance. Descriptive statistics for each group’s Visible Platform performance showed that each surgery group within all Age cohorts showed competence to perform the procedural components of water maze testing, as reflected in the low trial times to locate the visible platform by the end of the session ( Mennenga et al., 2015a ) ( Table 1 ). OFT distance and time measures were analyzed using ANOVA for all planned comparisons. Serum ovarian hormone levels, ovarian follicle counts, body weights, ovary weights, uterine weights, and western blot analyses were also analyzed using ANOVA with each of those respective measurements as the dependent variable for all planned comparisons. All subjects from the Adult cohort were included in the analyses (final n/group in the Adult cohort: Sham = 10, Ovx = 10, Hysterectomy = 10, Ovx-Hysterectomy = 10). In addition to natural attrition, one Ovx and two Ovx-Hysterectomy rats were excluded from all analyses within the Early Middle-Age cohort due to consistent floating behavior, and thus failure to perform in the WRAM task (final n/group in the Early Middle-Age cohort: Sham = 10, Ovx = 9, Hysterectomy = 9, Ovx-Hysterectomy = 6). Within the Late Middle-Age cohort, in addition to natural attrition, one rat from the Sham group and one rat from the Hysterectomy group were excluded from all analyses within the Late Middle-Age cohort due to deteriorated health; one rat in the Ovx group of the Late Middle-Age cohort was also excluded from all analyses for abnormal vaginal smear and hormone levels indicative of estrogen stimulation at the end of the experiment (final n/group in the Late Middle-Age cohort: Sham = 9, Ovx = 7, Hysterectomy = 6, Ovx-Hysterectomy = 9). Only subjects included in behavioral analyses were evaluated on brain measures.

Hysterectomy rats made more WMC errors than Ovx-Hysterectomy rats on the moderate working memory load trial [main effect: F (1,18) = 6.82, p < 0.05, η G 2 = 0.04 ] ( Fig. 2A ). A Trial x Surgery interaction was present between Sham and Hysterectomy groups for WMI errors across all trials in the Learning Phase [F (3,54) = 3.52, p < 0.05, η G 2 = 0.01 ] ( Fig. 2B ). Hysterectomy rats made more WMI errors on Trial 3 compared to Sham intact control rats [F (1,18) = 8.82, p < 0.01, η G 2 = 0.07 ] ( Fig. 2C ), replicating prior work ( Koebele et al., 2019 ). There were no Surgery effects for RM errors (data not shown), pointing to a working-memory specific impairment in the Hysterectomy group in the Adult cohort. Sham rats made more WMC errors than Ovx rats [main effect of Surgery: F (1,17) = 5.17, p < 0.05, η G 2 = 0.02 ] collapsed across trials 2–4 ( Fig. 2D ). On the moderate working memory load trial, Hysterectomy rats made more WMC errors compared to Ovx rats [F (1,16) = 4.83, p < 0.05, η G 2 = 0.04 ] and compared to Ovx-Hysterectomy rats [F (1,13) = 9.49, p < 0.01, η G 2 = 0.09 ] ( Fig. 2E ). When collapsed across all trials, there were main effects of Surgery with Hysterectomy rats making more WMI errors than Ovx [F (1,16) = 7.23, p < 0.05, η G 2 = 0.02 ], and Ovx-Hysterectomy rats [F (1,13) = 5.26, p < 0.05, η G 2 = 0.02 ]; there was also a Trial x Surgery interaction present between Hysterectomy and Ovx-Hysterectomy rats [F (3,39) = 3.68, p < 0.05, η G 2 = 0.03 ] and between Ovx and Ovx-Hysterectomy rats [F (3,39) = 3.44, p < 0.05, η G 2 = 0.02 ] ( Fig. 2F ). Hysterectomy rats made more WMI errors on the moderate working memory load trial compared to Sham intact rats [F (1,17) = 4.62, p < 0.05, η G 2 = 0.04 ], extending prior findings in adult rats ( Koebele et al., 2019 ); Hysterectomy rats also made more WMI errors on the moderate working memory load trial compared to Ovx rats [F (1,16) = 11.80, p < 0.01, η G 2 = 0.09 ] ( Fig. 2G ). On the maximum working memory load trial, Trial 4, the Hysterectomy group made more WMI errors than the Ovx-Hysterectomy group [F (1,13) = 5.27, p < 0.05] ( Fig. 2H , η G 2 = 0.07 ). During the Learning Phase, there were main effects of Surgery for RM as well, with the Hysterectomy group making more RM errors than the Ovx group [F (1,16) = 26.88, p < 0.0001, η G 2 = 0.01 ] and the Ovx-Hysterectomy group [F (1,13) = 5.29, p < 0.05, η G 2 = 0.01 ]. The Ovx group made fewer RM errors compared to the Sham group during this phase [F(1,17) = 11.04, p < 0.01, η G 2 = 0.01 ] (data not shown). There was a main effect of Surgery for WMI errors for the Ovx vs Hysterectomy comparison [F (1,11) = 9.67, p < 0.01, η G 2 = 0.02 ], as well as Trial x Surgery interactions between Hysterectomy and Sham intact control groups [F (3,39) = 6.58, p < 0.001, η G 2 = 0.04 ], Hysterectomy and Ovx groups [F (3,33) = 7.50, p < 0.001, η G 2 = 0.06 ], and Hysterectomy and Ovx-Hysterectomy groups [F (3,39) = 4.14, p < 0.05, η G 2 = 0.02 ] ( Fig. 2I ). On the maximum working memory load trial, Hysterectomy rats made more WMI errors compared to Sham intact control rats [F (1,13) = 7.11, p < 0.05, η G 2 = 0.06 ], again extending prior findings ( Koebele et al., 2019 ); Hysterectomy rats also made more WMI errors at the highest load compared to Ovx rats [F (1,11) = 12.47, p < 0.01, η G 2 = 0.11 ] during the Learning Phase ( Fig. 2J ). There were no main effects or interactions with Surgery for any memory measure during the Asymptotic Phase, suggesting that all surgery groups in the Adult cohort successfully learned the WRAM task by the latter portion of testing (data not shown). There were no main effects or interactions with Surgery for any memory measure during the Asymptotic Phase, suggesting that all surgery groups in the Early Middle-Age cohort successfully learned the WRAM task by the latter portion of testing (data not shown). Within the Late Middle-Age cohort, there was a Trial x Surgery interaction between Hysterectomy and Ovx groups for WMC errors [F (2,22) = 4.37, p < 0.05, η G 2 = 0.02 ] ( Fig. 2K ). However, there were no differences on the moderate or maximum working memory load trials when evaluated separately. Sham rats did not show a delay-induced memory impairment ( Fig. 3A ). There were delay-induced impairments for all other groups ( Fig. 3B – D ), with a main effect of Delay present for Ovx rats [F (9,1) = 15.00, p < 0.01, η G 2 = 0.28 ], Hysterectomy rats [F (9,1) = 6.43, p < 0.05, η G 2 = 0.26 ], and Ovx-Hysterectomy rats [F(9,1) = 14.23, p < 0.01, η G 2 = 0.52 ]. Sham, Ovx, and Hysterectomy groups did not exhibit a delay-induced memory impairment ( Fig. 3E – G ). The Ovx-Hysterectomy group made more WMC errors on Trial 3 following the six-hour delay compared to the previous day [F (5,1) = 7.35, p < 0.05, η G 2 = 0.13 ; Fig. 3H ]. Sham rats made more WMC errors on Trial 3 after the delay compared to Trial 3 on the last day of baseline testing [F (8,1) = 16.00, p < 0.01, η G 2 = 0.33 , Fig. 3I ]. Ovx, Hysterectomy, and Ovx-Hysterectomy groups performed similarly on the post-delay trial compared to the previous day ( Fig. 3J – L ). The MM learning curve for the Adult cohort is visualized in Fig. 4A . There was a main effect of Day within each comparison (Hysterectomy versus Sham: [F (4,72) = 37.22, p < 0.0001, η G 2 = 0.27 ]; Hysterectomy versus Ovx: [F (4,72) = 30.23, p < 0.0001, η G 2 = 0.24 ]; Hysterectomy versus Ovx-Hysterectomy: [F (4,72) = 18.05, p < 0.0001, η G 2 = 0.18 ]; Sham versus Ovx [F (4,72) = 50.76, p < 0.0001, η G 2 = 0.32 ]; Ovx versus Ovx-Hysterectomy [F (4,72) = 23.88, p < 0.0001, η G 2 = 0.22 ]), indicating learning across the five days of baseline testing. Each Surgery group was evaluated independently for probe trial performance. There was a main effect of Quadrant for each group (Sham: [F (9,1) = 80.79, p < 0.0001, η G 2 = 0.89 ]; Ovx: [F (9,1) = 103.59, p < 0.0001, η G 2 = 0.90 ]; Hysterectomy: [F(9,1) = 118.30, p < 0.0001, η G 2 = 0.91 ]; Ovx-Hysterectomy: [F(9,1) = 125.63, p < 0.0001, η G 2 = 0.91 ]), whereby all groups swam a greater proportion of total swim distance in the previously platformed quadrant compared to the opposite quadrant, indicating spatial localization to the platform location ( Fig. 4B ). The MM learning curve for the Early Middle-Age cohort is visualized in Fig. 4C . There was a main effect of Day within each comparison (Hysterectomy versus Sham: [F (4,68) = 12.64, p < 0.0001, η G 2 = 0.15 ]; Hysterectomy versus Ovx: [F (4,64) = 8.73, p < 0.0001, η G 2 = 0.12 ]; Hysterectomy versus Ovx-Hysterectomy: [F (4,52) = 17.20, p < 0.0001, η G 2 = 0.19 ]; Sham vs Ovx [F(4,68) = 9.71, p < 0.0001, η G 2 = 0.12 ]; Ovx vs Ovx-Hysterectomy [F (4,52) = 12.83, p < 0.0001, η G 2 = 0.16 ]), indicating learning across the five days of baseline testing. There was a main effect of Quadrant for each group during the probe trial (Sham: [F (9,1) = 29.12, p < 0.001, η G 2 = 0.68 ]; Ovx: [F(8,1) = 67.11, p < 0.0001, η G 2 = 0.84 ]; Hysterectomy: [F(8,1) = 38.59, p < 0.001, η G 2 = 0.76 ]; Ovx-Hysterectomy: [F (5,1) = 14.10, p < 0.05, η G 2 = 0.69 ]), whereby all rats swam a greater proportion of total swim distance in the previously platformed quadrant compared to the opposite quadrant, indicating spatial localization to the platform location ( Fig. 4D ). The MM learning curve for the Late Middle-Age cohort is visualized in Fig. 4E . There was a main effect of Day for each comparison (Hysterectomy versus Sham: [F (4,52) = 15.77, p < 0.0001, η G 2 = 0.23 ]; Hysterectomy versus Ovx: [F (4,44) = 21.14, p < 0.0001, η G 2 = 0.28 ]; Hysterectomy versus Ovx-Hysterectomy: [F (4,52) = 18.85, p < 0.0001, η G 2 = 0.22 ]; Sham versus Ovx: [F(4,56) = 17.06, p < 0.0001, η G 2 = 0.22 ]; Ovx versus Ovx-Hysterectomy [F (4,56) = 19.96, p < 0.0001, η G 2 = 0.20 ]), indicating learning across the five days of baseline testing. For the probe trial, there was a main effect of Quadrant for Sham: [F (8,1) = 11.75, p < 0.01, η G 2 = 0.54 ], Hysterectomy [F(5,1) = 11.25, p < 0.05, η G 2 = 0.66 ], and Ovx-Hysterectomy [F (8,1) = 30.49, p < 0.001, η G 2 = 0.76 ]) groups. The Ovx group Quadrant effect was p = 0.05 [F (6,1) = 5.81, η G 2 = 0.40 ]. Overall, all groups swam a greater proportion of total swim distance in the previously platformed quadrant compared to the opposite quadrant, indicating spatial localization to the platform location ( Fig. 4F ). Ovx rats traveled less Total Distance compared to Ovx-Hysterectomy rats [F(1,18) = 4.31, p < 0.05, η 2 = 0.20] and Sham rats [F(1,18) = 4.65, p < 0.05, η 2 = 0.21]. Ovx rats also covered less distance along the perimeter of the arena compared to Hysterectomy rats [F (1,18) = 5.42, p < 0.05, η 2 = 0.25] (data not shown). There were no significant effects for any OFT measure in the Early Middle-Age cohort (data not shown). There were no significant effects for any OFT measure in the Late Middle-Age cohort (data not shown). Two weeks after surgery, all Ovx and Ovx-Hysterectomy rat displayed diestrus-like or blank smears across all evaluation days, indicating successful ovary removal and a lack of uterine stimulation. Sham and Hysterectomy groups exhibited normal cyclic estrous activity consisting of 4–5-day estrous cycles. Vaginal smears collected one day prior to euthanasia and on the day of euthanasia showed that Ovx and Ovx-Hysterectomy groups continued to display diestrus-like or blank smears, and Sham and Hysterectomy rats were in various phases of the estrous cycle, such that all phases (proestrus, estrus, metestrus, and diestrus) were represented in both ovary-intact groups on the day of euthanasia. Two weeks after surgery, Ovx and Ovx-Hysterectomy rats displayed diestrus-like or blank smears, and Sham and Hysterectomy groups showed normal 4–5-day cyclic estrous activity. Eight consecutive days prior to behavior testing, vaginal smears were collected again for this cohort, when rats were approximately 12 months of age. All Ovx and Ovx-Hysterectomy rats continued to display diestrus-like or blank vaginal smears. All Sham and Hysterectomy rats displayed regular estrous cycle activity at this time point as well. Thus, rats with ovaries had normal cycling at all time points examined, while those without ovaries were acyclic. Lastly, after behavior testing was complete, vaginal smears were also collected one day prior to euthanasia and on the day of euthanasia. Ovx and Ovx-Hysterectomy groups continued to display diestrus-like or blank smears, and Sham and Hysterectomy rats were in various phases of the estrous cycle, such that all phases (proestrus, estrus, metestrus, and diestrus) were each represented in both ovary-intact groups within 24 h of euthanasia. Two weeks after surgery, Ovx and Ovx-Hysterectomy rats displayed diestrus-like or blank smears. Sham and Hysterectomy groups demonstrated normal cyclic estrous activity consisting of 4–5-day estrous cycles. Six months later, vaginal smears were collected for this cohort for eight consecutive days, when rats were approximately 12 months of age. All Ovx and Ovx-Hysterectomy rats continued to display diestrus-like or blank vaginal smears. All Sham rats displayed regular estrous cycle activity at this time point. Seven of nine Hysterectomy rats displayed estrous cyclicity; the remaining two Hysterectomy rats were observed to be in persistent diestrus across all eight days of vaginal smear collection at this early middle-aged time point for the Late Middle-Age cohort. Eight consecutive days prior to behavior testing, vaginal smears were again collected for this cohort, when rats were approximately 17 months of age. Seven of eight Ovx rats displayed diestrus or blank smears; one Ovx rat displayed a persistent estrus phenotype across all eight days of evaluation. All Ovx-Hysterectomy rats displayed blank or diestrus-like smears. Two of ten Sham rats displayed persistent diestrus smears across all eight days of evaluation; the remaining eight Sham subjects displayed elongated estrous cycles with most days in diestrus and variable/intermittent cycling. Three of seven Hysterectomy rats displayed persistent diestrus smears; the remaining four exhibited elongated estrous cycling with most days in diestrus and variable/intermittent cycling. After behavior testing was complete, vaginal smears were collected one day prior to euthanasia and on the day of euthanasia. Ovx-Hysterectomy rats displayed diestrus-like or blank smears. Ovx rats included in the analyses displayed blank or diestrus-like smears, and one Ovx rat (the same rat described above) remained in persistent estrus and thus was excluded from the study and all statistical analyses. All Sham rats were in diestrus prior to euthanasia. Five of seven Hysterectomy rats were in diestrus, and two Hysterectomy rats displayed estrus or metestrus-like cytology within 24 h of euthanasia. Body weights for the Adult cohort across the experimental period are depicted in Fig. 5A . At the end of the experiment, Hysterectomy rats weighed less than Ovx rats [F (1,18) = 96.35, p < 0.0001, η 2 = 0.84] and Ovx-Hysterectomy rats [F (1,18) = 72.35, p < 0.0001, η 2 = 0.80]. Ovx rats weighed more than Sham rats [F (1,18) = 47.82, p < 0.0001, η 2 = 0.73], and the ovary-intact groups (Sham vs Hysterectomy) did not differ from each other ( Fig. 5B ). Body weights for the Early Middle-Age cohort across the experimental period are shown in Fig. 5C . At the end of the experiment, Hysterectomy rats weighed less than Ovx rats [F (1,16) = 23.13, p < 0.001, η 2 = 0.59] and Ovx-Hysterectomy rats [F (1,13) = 31.50, p < 0.0001, η 2 = 0.71]. Sham rats also weighed less than Ovx rats [F (1,17) = 28.39, p < 0.0001, η 2 = 0.63] ( Fig. 5D ). Body weights for the Late Middle-Age cohort across the experimental period are denoted in Fig. 5E . At the end of the experiment, Ovx rats weighed more than Sham rats [F (1,14) = 6.02, p < 0.05, η 2 = 0.30; Fig. 5F ]. There were no other group differences in body weight at the Late Middle-Age time point when rats were approximately 18 months old. Uterine weights were lower in Ovx rats compared to Sham rats [F (1,18) = 58.56, p < 0.0001, η 2 = 0.77] ( Fig. 6A ), in accordance with prior findings ( Engler-Chiurazzi et al., 2012 ; Koebele et al., 2019 ; Mennenga et al., 2015b ; Prakapenka et al., 2018 ; Westerlind et al., 1998 ). When uterine weights were corrected for body weight, the main effect of Surgery persisted [F (1,18) = 53.12, p < 0.0001, η 2 = 0.75]. There were no differences in total ovary weight between Sham and Hysterectomy groups in the Adult cohort ( Fig. 6B ). Uterine weights were lower in Ovx rats compared to Sham rats [F (1,17) = 51.77, p < 0.0001, η 2 = 0.75] ( Fig. 6C ). When uterine weights were corrected for body weight, the main effect of Surgery persisted [F (1,17) = 51.53, p < 0.0001, η 2 = 0.75]. There were no differences in total ovary weight between Sham and Hysterectomy groups in the Early Middle-Age cohort ( Fig. 6D ). Uterine weights were lower in Ovx rats compared to Sham rats, again, as expected [F (1,14) = 148.49, p < 0.0001, η 2 = 0.91] ( Fig. 6E ). When uterine weights were corrected for body weight, the main effect of Surgery persisted [F (1,14) = 91.52, p < 0.0001, η 2 = 0.87]. For this Late Middle-Age cohort, there was a main effect of Surgery for ovary weight [F (1,13) = 7.35, p < 0.05, η 2 = 0.33], such that Hysterectomy rats’ ovaries weighed more than Sham rats’ ovaries. However, when ovary weight was corrected for body weight, this effect was no longer significant ( Fig. 6F ). There were no effects of Surgery for any follicle type or corpora lutea count in the Adult cohort ( Fig. 7A – E ), Early Middle-Age cohort ( Fig. 7F – J ) or Late Middle-Age cohort ( Fig. 7K – O ). One Sham subject and one Hysterectomy subject had progesterone serum values above the detectable range of the assay (0.15 ng/mL – 40.00 ng/mL), and were assigned a placeholder value of 40.1 ng/mL. Hysterectomy rats had higher progesterone levels compared to Ovx rats [F(1,18) = 10.15, p < 0.01, η 2 = 0.36] and Ovx-Hysterectomy rats [F(1,18) = 9.33, p < 0.01, η 2 = 0.34]. Sham rats also had higher progesterone levels compared to Ovx rats [F (1,18) = 11.61, p < 0.01, η 2 = 0.39] ( Fig. 8A ), indicating that Ovx decreased progesterone levels compared to rats that retained their ovaries. The Ovx and Ovx-Hysterectomy groups did not differ from each other. Three Sham subjects and two Hysterectomy subjects had serum androstenedione values below the detectable range of the assay (0.10–10.0 ng/mL), and were assigned a placeholder value of 0.09 ng/mL. One value in the Sham group was based on a singlet value, as the other singlet was outside the detectable range. There were no differences in androstenedione levels between Sham and Hysterectomy rats ( Fig. 8B ). One Sham and one Hysterectomy subject had serum E2 values below of the detectable range of the assay (3.0 pg/mL – 300 pg/mL), and were assigned a placeholder value of 2.9 pg/mL. One value in the Hysterectomy group was based on a singlet value, as the other singlet was outside the detectable range. There were no differences in E2 levels between Sham and Hysterectomy rats ( Fig. 8C ). One Sham rat was excluded from the LH analyses due to insufficient serum volume. Hysterectomy rats had lower LH compared to Ovx rats [F (1,18) = 117.87, p < 0.0001, η 2 = 0.88] and compared to Ovx-Hysterectomy rats [F (1,18) = 56.08, p < 0.0001, η 2 = 0.76]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other, nor did Ovx versus Ovx-Hysterectomy groups. Sham rats had lower LH compared to Ovx rats [F (1,17) = 109.73, p < 0.0001, η 2 = 0.87] ( Fig. 8D ). Two Sham rats and two Hysterectomy rats had FSH values below the detectable range of the assay (2.5–37.7 ng/mL for intact groups), and were assigned a placeholder value of 2.4 ng/mL. Three values from the Sham group and two values from the Hysterectomy group were based on a singlet value, as the other singlet was outside the detectable range. Hysterectomy rats had lower FSH compared to Ovx rats [F (1,18) = 507.03, p < 0.0001, η 2 = 0.97] and Ovx-Hysterectomy rats [F (1,18) = 343.77, p < 0.0001, η 2 = 0.95]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other for FSH, nor did the Ovx versus Ovx-Hysterectomy groups. As expected, Ovx rats had higher FSH values compared to Sham rats [F (1,18) = 468.69, p < 0.0001, η 2 = 0.96] ( Fig. 8E ). Two Sham rats and two Hysterectomy rats had progesterone serum values above the detectable range of the assay (0.15 ng/mL – 40.00 ng/mL), and were assigned a placeholder value of 40.1 ng/mL. One Ovx rat was excluded from the progesterone analyses due to a technical issue. Hysterectomy rats had higher progesterone levels compared to Ovx rats [F(1,15) = 9.73, p < 0.01, η 2 = 0.39] and Ovx-Hysterectomy rats [F(1,13) = 8.23, p < 0.05, η 2 = 0.39]. Sham rats also had more progesterone compared to Ovx rats [F (1,16) = 11.50, p < 0.01, η 2 = 0.42] ( Fig. 8F ). Ovx groups did not differ from each other. One Hysterectomy subject had serum androstenedione values below the detectable range of the assay (0.10–10.0 ng/mL), and was assigned a placeholder value of 0.09 ng/mL. Furthermore, two values in the Sham group and one value in the Hysterectomy group were based on a singlet value, as the other singlet was outside the detectable range. There were no differences in androstenedione levels between Sham and Hysterectomy rats ( Fig. 8G ). Six Sham subjects and four Hysterectomy subjects had serum E2 values below the detectable range of the assay (3.0 pg/mL – 300 pg/mL), and were assigned a placeholder value of 2.9 pg/mL. Additionally, one value in the Hysterectomy group was based on a singlet value, as the other singlet was outside the detectable range. There were no differences in E2 levels between Sham and Hysterectomy rats ( Fig. 8H ). All rats included in behavioral analyses had detectable serum LH levels. Hysterectomy rats had lower LH compared to Ovx rats [F (1,16) = 64.58, p < 0.0001, η 2 = 0.80] and Ovx-Hysterectomy rats [F (1,13) = 507.85, p < 0.0001, η 2 = 0.98]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other, nor did Ovx versus Ovx-Hysterectomy groups. Sham rats had lower LH compared to Ovx rats [F(1,17) = 73.66, p < 0.0001, η 2 = 0.81] ( Fig. 8I ). Three Hysterectomy rats had FSH values below the detectable range of the assay (2.5–37.7 ng/mL for intact groups), and were assigned a placeholder value of 2.4 ng/mL. One value from the Sham group was based on a singlet value, as the other singlet was outside the detectable range. Hysterectomy rats had lower FSH compared to Ovx rats [F (1,16) = 128.65, p < 0.0001, η 2 = 0.89] and Ovx-Hysterectomy rats [F (1,13) = 534.51, p < 0.0001, η 2 = 0.98]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other, nor did Ovx versus Ovx-Hysterectomy groups. Sham rats had lower FSH compared to Ovx rats [F(1,17) = 140.36, p < 0.0001, η 2 = 0.89] ( Fig. 8J ). Seven out of nine Sham rats, and four out of six Hysterectomy rats, had progesterone serum values above of the detectable range of the assay (0.15 ng/mL – 40.00 ng/mL), and were assigned a placeholder value of 40.1 ng/mL. Hysterectomy rats had higher progesterone levels compared to Ovx rats [F (1,11) = 122.04, p < 0.0001, η 2 = 0.92] and Ovx-Hysterectomy rats [F (1,13) = 28.42, p < 0.0001, η 2 = 0.69]. Sham rats also had higher progesterone levels than Ovx rats [F (1,14) = 102.803, p < 0.0001, η 2 = 0.88]. Progesterone levels were not significantly different between Ovx and Ovx-Hysterectomy groups ( Fig. 8K ). There were no significant differences in serum androstenedione levels for Sham versus Hysterectomy rats at the Late Middle-Age time point ( Fig. 8L ). Three Sham subjects and three Hysterectomy subjects had serum E2 values below the detectable range of the assay (3.0 pg/mL – 300 pg/mL), and were assigned a placeholder value of 2.9 pg/mL. Additionally, three values from the Sham group and three values from the Hysterectomy group were based on a singlet value, as the other singlet value registered outside the detectable range of the assay. There were no significant differences in serum E2 levels at this Late Middle-Age time point ( Fig. 8M ). All rats included had detectable serum LH levels. Hysterectomy rats had lower LH levels compared to Ovx rats [F (1,11) = 35.18, p < 0.0001, η 2 = 0.76] and compared to Ovx-Hysterectomy rats [F (1,13) = 85.72, p < 0.0001, η 2 = 0.87]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other, nor did Ovx versus Ovx-Hysterectomy groups, for LH levels. Sham rats had lower LH compared to Ovx rats [F(1,14) = 38.37, p < 0.0001, η 2 = 0.73] ( Fig. 8N ). All rats included had detectable serum FSH levels. Hysterectomy rats had lower FSH levels compared to Ovx rats [F (1,11) = 457.19, p < 0.0001, η 2 = 0.98] and Ovx-Hysterectomy rats [F (1,13) = 144.81, p < 0.0001, η 2 = 0.92]. Ovary intact groups (Sham and Hysterectomy) did not differ from each other for FSH levels, nor did Ovx versus Ovx-Hysterectomy groups. Sham rats had lower FSH compared to Ovx rats [F(1,14) = 726.74, p < 0.0001, η 2 = 0.98] ( Fig. 8O ). All subjects evaluated in behavioral analyses were included in the brain analyses; two subjects in the Adult cohort had insufficient tissue for analysis and were therefore not included in the brain analyses (Adult cohort: Sham n = 10, Ovx n = 10, Hysterectomy n = 9, Ovx-Hysterectomy n = 9; Early Middle-Age cohort: Sham n = 10, Ovx n = 9, Hysterectomy n = 9, Ovx-Hysterectomy n = 6; Late Middle-Age cohort: Sham n = 9, Ovx n = 7, Hysterectomy n = 6, Ovx-Hysterectomy n = 9). There were no main effects of Surgery for FOSB expression or ΔFOSB expression detected at any time point in the Dorsal Hippocampus or Frontal Cortex for the Adult, Early Middle-Age, or Late Middle-Age cohorts (data not shown). There were no effects of Surgery for FOSB expression or ΔFOSB expression in the Entorhinal Cortex of rats 10 weeks after surgery (i.e., the Adult cohort) when rats were 8 months old ( Fig. 9A – B ). However, in the Early Middle-Age cohort, eight remained ovary-intact did not differ from each other. **** p < 0.0001, **p < 0.01, * p < 0.05. months after surgery when rats were 13 months old, Hysterectomy rats had greater FOSB expression compared to Ovx rats [F (1,16) = 7.18, p < 0.05, η 2 = 0.31] and Ovx-Hysterectomy rats [F (1,13) = 4.81, p < 0.05, η 2 = 0.27] ( Fig. 9C ). Hysterectomy rats also had greater ΔFOSB expression compared to Ovx rats [F (1,16) = 6.16, p < 0.05, η 2 = 0.28] ( Fig. 9D ). In the Late Middle-Age cohort, 13 months after surgery, when rats were 18 months old, there were no significant Surgery differences in FOSB expression or ΔFOSB expression in the Entorhinal Cortex for any two-group comparison ( Fig. 9E – F ). Representative blots for FOSB, ΔFOSB, and beta-tubulin for each Age cohort are shown in Fig. 9 . Since the Entorhinal Cortex showed unique changes in FOSB and ΔFOSB expression at the early middle-aged time point, we additionally evaluated phosphorylated- and total- ERK1/2 expression in the Entorhinal Cortex in each cohort. Representative blots for phosphorylated-ERK1/2, Total ERK1/2, and beta-tubulin for each Age cohort are shown in Fig. 9 . There were no differences in total ERK1/2 normalized to beta-tubulin (data not shown). In the Adult cohort, Ovx rats had relatively less phosphorylated ERK1 (p44) [F (1,17) = 5.16, p < 0.05, η 2 = 0.23] ( Fig. 9G ) and less phosphorylated ERK2 (p42) [F (1,17) = 4.97, p < 0.05, η 2 = 0.23] ( Fig. 9H ) when compared to Ovx-Hysterectomy rats.

Gynecological surgery can occur at any point during a woman’s lifespan, and a younger age at surgery can increase the risk of developing a multitude of diseases later in life, including dementia, coronary heart disease, diabetes, depression, Parkinson’s disease, and some cancers ( Adelman and Sharp, 2018 ; Benedetti et al., 2001 ; Bove et al., 2014 ; Crawford, 2022 ; Gierach et al., 2014 ; Rocca et al., 2011 , 2012 , 2021 ; Uldbjerg et al., 2022 ; Wilson et al., 2018). If oophorectomy occurs before age 35, or hysterectomy occurs before age 40, these surgeries increase the risk of all-cause mortality (for review, see: Adelman and Sharp, 2018 ; Gierach et al., 2014 ). Women can live a substantial proportion of their lives post-reproductive surgery; thus, it is clinically relevant to elucidate how gynecological surgeries impact the brain longitudinally. Furthermore, given the frequency at which gynecological surgeries occur, it is important to identify neurobiological changes associated with gynecological surgery that may underlie an increased risk for pathological brain aging. Our previous report showed that hysterectomy with ovarian conservation in adulthood led to cognitive impairments within two months after surgery in a rat model ( Koebele et al., 2019 ). In the current series of experiments, we replicated and extended these evaluations, showing that hysterectomy in adulthood can yield detrimental impacts on working memory, and that these impairing effects last not just 6 weeks, but 7 months, and even 12 months, after surgery. The data reported here are the first to demonstrate a hysterectomy-induced cognitive impairment that extends into late middle-age, indicating a potentially life-long change after hysterectomy. At all three post-surgical time points assessed, rats that received hysterectomy alone exhibited impaired spatial working memory during WRAM learning when memory load was taxed compared with ovary-intact rats that received sham control surgery where no reproductive organs were removed. The 6-week post-surgery timepoint is a replication of hysterectomy-induced impairments versus intact sham controls ( Koebele et al., 2019 ). The current data demonstrate the novel finding that hysterectomy-induced working memory impairment versus ovary-intact sham controls extends to Early Middle-Age and Late Middle-Age timepoints after surgery in adulthood. Beyond hysterectomy’s impairing working memory relative to sham control subjects, rats with hysterectomy also performed worse than rats with Ovx-hysterectomy and with Ovx alone on several working memory measures during WRAM learning. For groups that received variations in gynecological surgery, group differences emerged depending on the amount of time after surgery. Six weeks post-surgery, during learning, hysterectomy rats performed worse than Ovx-hysterectomy (WMC), 7 months post-surgery hysterectomy rats performed worse than Ovx-hysterectomy (WMC, WMI, RM) or Ovx alone (WMC, WMI, RM), and 12 months post-surgery hysterectomy rats performed worse than Ovx alone (WMI). Notably, the most widespread impairments were seen 7 months after surgery, in early middle-age; this was the only post-surgical timepoint evaluated that showed hysterectomy-induced impairments on all three memory measures tested in the WRAM, including two orthogonal working memory measures as well as a reference memory measurement. Since the WRAM tests working and reference memory simultaneously, it permits insight into learning on a complex task requiring multiple memory demands ( Bernaud et al., 2021 ). These findings suggest that while hysterectomy negatively affects working memory at short-, moderate-, and long- term timepoints after surgery, the reference memory domain can also be negatively impacted by hysterectomy when tested concurrently with working memory when evaluated in early middle-age, 7 months after surgery. This may reflect an especially sensitive window in early middle-age wherein cognition becomes particularly vulnerable to alterations in physiology, and behavioral changes become evident. It is noteworthy to discuss that rats that experienced a combined Ovx and hysterectomy had divergent cognitive effects compared to hysterectomy alone at Adult and Early Middle-Age time points for several working memory measures. We previously reported a similar effect during the Asymptotic Phase of WRAM testing when tested 6 weeks after surgery, where Ovx-Hysterectomy rats did not exhibit the same impairments as rats that received hysterectomy with ovarian conservation ( Koebele et al., 2019 ). Although the biological mechanism underlying this divergent outcome currently remains elusive, the replication and expansion of this finding underscores that the presence or absence of uterine tissue plays a unique role in cognition during aging. We hypothesize that uterine removal with ovarian conservation may result in unique dysregulated signaling between the brain-reproductive tract axis, while complete removal of the reproductive tract may lead to a unique or protective reorganization of brain-endocrine signaling. Long-term brain and behavioral changes have been reported after hormone-mediated events, including but not limited to puberty ( Juraska et al., 2013 ), pregnancy ( Barha et al., 2015 ; Barha and Galea, 2011 ; Duarte-Guterman et al., 2019 ; Eid et al., 2019 ; Gatewood et al., 2005 ), and exogenous hormone exposures ( Braden et al., 2011 ). Systematic investigation into the effect of Ovx versus Ovx-hysterectomy is an important future direction to better understand the role of the uterus in cognitive aging. Delayed memory retention effects were most evident in the adult cohort for rats that received gynecological surgery interventions. Though the Ovx-Hysterectomy group showed memory impairment for an extended delay in early middle-age and the Sham group showed memory impairment for an extended delay in late middle-age, errors were low overall, which may suggest less overall exploration (i.e., arm entries) after an extended delay as aging ensues, as well as a changing relationship between endogenous ovarian hormone milieu and working memory performance with age. Spatial reference memory, without the concomitant requirements of working memory, as evaluated in the MM, is not measurably influenced by Ovx, hysterectomy, or Ovx-hysterectomy surgical manipulations or sham surgery after short-, moderate-, or long- term lengths of time following surgery. Collectively, our behavioral results demonstrate that hysterectomy with ovarian conservation is associated with cognitive detriments for spatial working memory at short-, moderate-, and long- term time points. Effects are observed when compared to reproductive tract intact sham-control surgery, as well as compared to other gynecological surgery variations including Ovx and Ovx-hysterectomy. These findings provide translational insight into the role of hysterectomy and its effect on cognition during aging, particularly when the surgery occurs during adulthood, in the reproductive years. At Adult and Early Middle-Age time points, ovary-intact rats (i.e., Sham and Hysterectomy groups) had lower body weights compared to rats without ovaries (i.e., Ovx and Ovx-Hysterectomy groups) at euthanasia. At the Late Middle-Age time point, all rats had progressively gained weight with age, such that only Sham rats weighed less than Ovx rats by the end of the experiment. Hysterectomy rats weighed similar to Ovx and Ovx-Hysterectomy rats. This is particularly interesting, as a prospective research study reported greater weight gain in women with hysterectomy one year after surgery ( Moorman et al., 2009 , but see also: Fitzgerald et al., 2009 ), indicating uterus removal with ovarian conservation has a unique effect on metabolism and/or the gut microbiome. This association is of interest to systematically investigate in future research, given our recent report that Ovx and exogenous estrogen alter gut microbial composition ( Zeibich et al., 2021 ). Uterine weights were greater in Sham rats compared to Ovx rats at all time points in the current study, indicative of endogenous estrogen stimulation of the uterus in ovary-intact rats, an effect that has been consistently reported in the literature ( Engler-Chiurazzi et al., 2012 ; Koebele et al., 2019 ; Mennenga et al., 2015b ; Prakapenka et al., 2018 ; Westerlind et al., 1998 ). Ovarian weights did not differ for any Age cohort after correcting for body weight. Ovarian follicle counts estimated the number of healthy follicles present in the ovaries, including non-growing ovarian follicle reserve as well as growing follicles that are steroidogenic. Corpora lutea were also quantified as a measure of ovulatory activity, as they reflect recent ovulation. There were no differences between rats with intact reproductive tracts and rats that underwent hysterectomy with ovarian conservation for any follicle type or corpora lutea counts for any cohort. Overall, this suggests that the non-atretic ovarian reserve or healthy growing follicles did not undergo divergent morphological changes following hysterectomy compared to age-matched sham controls following short-, moderate-, or long-term time intervals after surgery. This finding challenges the notion that ovarian dysfunction or accelerated ovarian aging occurs after uterus removal, and may point to a primary role for the presence or absence of the uterus in regulating cognition that is not secondary to ovarian changes. However, further analysis of ovarian cellular signaling and atretic follicles may provide additional insight into the mechanisms underlying cognitive impairment following hysterectomy with ovarian conservation. Serum progesterone, androstenedione, E2, LH, and FSH levels did not statistically differ between ovary-intact groups for any Age cohort. Due to the technical detection limitations of the assays used, hormone data, particularly those using singlet values and placeholder values, should be interpreted with caution and in terms of relative estimated levels. That reproductive hormone serum levels did not differ between rats that had intact reproductive tracts and rats that underwent hysterectomy with ovarian conservation further suggests that the ovaries continue to synthesize sex hormones and the pituitary releases gonadotropins in a similar fashion to reproductive-tract-intact ovaries following uterus removal, at least at the post-surgical time points assessed. In all cohorts, progesterone was decreased, and gonadotropins were increased, in Ovx groups compared to ovary-intact groups; these effects are expected following surgical ovary removal. Our prior publication reported a marginal decrease in E2, a decrease in androstenedione, and increases in progesterone and FSH levels between rats that have intact reproductive tracts and those that underwent hysterectomy with ovarian conservation. In the current report, endogenous E2 levels were overall substantially lower compared with previously published data. Here, samples were analyzed with a commercially available ELISA kit (CAT# ES180S-100, CALBIOTECH, Spring Valley, CA) via a Core Endocrine Laboratory. In our prior experiment ( Koebele et al., 2019 ), RIA was used to determine E2 levels. However, RIA measurements of E2 may unreliably produce E2 results several fold higher than values obtained via ELISA, or gas chromatography/tandem mass spectrometry (GC/MSMS)-based measurements of E2, which produce similar results to each other ( Haisenleder et al., 2011 ; Nilsson et al., 2015 ). Androstenedione and progesterone were measured by ELISA kits in both cases; however, kit-to-kit variability (in the case of androstenedione) and different kit use (in the case of progesterone) at two different facilities may explain the variation in serum hormone levels. It is worth noting that sex hormone levels vary across the normal estrous cycle, and fluctuate irregularly as aging ensues, and are therefore not the optimal indicator of a given endogenous hormone milieu. This is also true of the erratic fluctuations of serum steroid hormone levels in women under-going the transition to menopause. In both rats and humans, LH and FSH levels rise at a steadier rate, and may be a better measure overall of ovarian status during the transition to reproductive senescence as FSH levels in serum are typically used as a marker for human menopause status in clinical settings ( Chan et al., 2005 ; Kaiser et al., 1989 ). Expression and activation of activity-dependent signaling components, ERK1/2 and FOSB, were measured following gynecological surgery and subsequent behavioral testing. Observed effects were localized to the entorhinal cortex, which acts as an information gateway between the hippocampus and other cortical areas, and is one of the first regions affected by Alzheimer’s disease pathology ( Braak and Braak, 1991 ). FOSB and ΔFOSB expression changes in the entorhinal cortex were specific to Ovx and Ovx-Hysterectomy groups compared with Hysterectomy in the Early Middle-Age timepoint, where FOSB was decreased after ovarian removal compared to uterus removal alone, and lower ΔFOSB expression was evident between Ovx with uterine conservation versus hysterectomy with ovarian conversation. Though most of what is known about FOSB and ΔFOSB expression changes comes from literature associated with addiction ( Nestler et al., 2001 ; Ruffle, 2014 ), this research provides a framework for measurable long-term alterations in neural activity markers associated with changes in physiology and behavior. Indeed, FOSB/ΔFOSB-immunoreactive cells have been shown to increase in the rat hippocampus after a spatial MM compared to maze-naïve rats and rats that experienced a non-spatial MM ( Eagle et al., 2015 ). Viral-mediated silencing and overexpression of ΔFOSB impaired learning and memory in that experiment ( Eagle et al., 2015 ). Furthermore, a dentate gyrus ΔFOSB knock-out mouse model exhibited neurogenesis and learning impairments compared to wildtype littermates ( Manning et al., 2019 ), further signifying a role for ΔFOSB in learning and memory. Given that ΔFOSB also has a potential role in hypothalamic-pituitary-adrenal function ( Ruffle, 2014 ), it is plausible that disrupting neural circuitry with gynecological surgery may lead to unique changes in FOSB/ΔFOSB expression in the early middle-aged time point when females are particularly susceptible to endogenous hormone fluctuations, which in turn may impact learning and memory performance. Within a shorter timeframe between surgery and behavior and brain assessment, at 10 weeks after surgery, Adult Ovx rats had less phosphorylated-ERK1 and phosphorylated-ERK2 in the entorhinal cortex compared to rats that had both their ovaries and uterus removed. Given that ERK1/2 is a ubiquitous kinase involved in many signal transduction pathways, including those that are associated with estrogen receptor binding and impacted by hormones, aging, and learning ( Atkins et al., 1998 ; Frick, 2015 ; Pinceti et al., 2016 ; Witty et al., 2012 ), it is notable that differences were observed in phosphorylated-ERK1 and -ERK2 in Ovx rats compared with Ovx-hysterectomy rats. Findings should be interpreted with caution given that levels were not different versus ovary-intact sham controls; however, that phosphorylated- ERK1 and -ERK2 were reduced in rats that received Ovx with uterine conservation may provide insight into an effect of the uterus on activity-dependent neurobiological markers when the ovaries are removed, and should be systematically evaluated in the future. Our observation that markers of long-term change in activity were altered in the entorhinal cortex by gynecological surgery supports a role for uterine and ovary-derived cues in regulating this critical region involved in spatial learning and memory. These findings suggest it may be interesting to target the entorhinal cortex in investigations manipulating the adult and middle-aged brain following gynecological surgery. It will also be important to examine how uterine and ovary derived cues modulate neural circuit activity and signaling in specific regions to improve our understanding of the complex regulatory processes and downstream changes that alter cognition. This is the first preclinical report comparing gynecological surgery variants including hysterectomy across multiple post-surgical time frames evaluating cognition and other physiological markers. Here, we show that at multiple time points after surgery, hysterectomy induced cognitive impairments compared with other surgical variants and intact rats. Hysterectomy with ovarian conservation had a unique, negative impact on spatial working memory six weeks after surgery, in adulthood (a replication: Koebele et al., 2019 ); seven months after surgery, in early middle-age; and 12 months after surgery, in late middle-age. Variations in gynecological surgery likely result in reorganizational processes of important neural circuits, including those associated with learning and memory. Considering life history factors (e.g., number of pregnancies and births, lifetime access to healthcare) and identifying prospective brain indicators of long-term, chronic changes with age and gynecological surgery will aid in developing targeted therapies to delay, attenuate, or prevent deleterious cognitive effects associated with hysterectomy and other variations in surgical menopause. Of note, the life history factor of exogenous hormone exposure, including hormone therapy, will likely play out to be critical in this regard. Indeed, hormone therapy has been shown to mitigate some health-associated risks as well as all-cause mortality of gynecological surgeries including oophorectomy and hysterectomy, especially if occurring before the average age of natural menopause ( Adelman and Sharp, 2018 ; Gierach et al., 2014 ; Wilson et al., 2018b ). Mounting scientific evidence demonstrates that taking into account an individual’s age and life histories optimizes health outcomes, further underscoring the importance of a personalized approach to manage menopause-related decisions. A broader and deeper understanding of the short- and long- term corollaries of gynecological surgery could not only drive future preclinical and clinical research directives, and medical care options, but will also further empower and support informed decision making by patients and their health care providers. It will be vital to investigate whether the age at which hysterectomy occurs differentially impacts cognitive outcomes. For example, one study reported that hysterectomy after the natural menopause transition did not affect the relative risk of developing dementia and may in fact be neuroprotective ( Imtiaz et al., 2014 ). We have previously reported that the age at the onset of follicular depletion (i.e., a model of transitional menopause) is a paramount factor in cognitive outcomes ( Koebele et al., 2017 ); thus, it is of particular interest to evaluate age as a central variable in hysterectomy model outcomes. Systematic evaluation into the direct role of the uterus on cognition is also a critical next step, including investigation into the direct sensory and autonomic nervous system innervation of the nonpregnant rat uterus ( Brauer and Smith, 2015 ; Gnanamanickam and Llewellyn-Smith, 2011 ). Although this may not be surprising given the intricate neural control associated with parturition, the collective evidence described herein alludes to a significant role of the uterus-brain connection beyond its role in pregnancy and birth. Nervous system plasticity occurs naturally throughout the reproductive cycle and is mediated, in large part, by reproductive hormones ( Pritschet et al., 2021 ; Woolley and McEwen, 1993 ); this plasticity is interrupted following gynecological surgery. It is likely that neural signaling is impacted by hysterectomy, even when ovaries are preserved, resulting in longitudinal effects on the trajectory of brain aging. This novel model of hysterectomy affords significant advancements in the field of behavioral neuroendocrinology by highlighting the importance of the non-pregnant uterus, opening new doors to many more questions revolving around the ovary-uterus-brain connection, through which we will better our comprehensive understanding of reproductive hormones, aging, and cognition.

It is estimated that nearly half of women will experience some form of gynecological surgery in their lifetime ( Merrill, 2008 ). Despite advances in alternative therapies for benign uterine conditions, hysterectomy remains the most common non-obstetrical gynecological surgical procedure ( Carlson et al., 1993 ; Centers for Disease Control and Prevention, 2010 ; McPherson et al., 2013 ). By age 60, nearly one-third of US women will experience uterus removal ( Centers for Disease Control and Prevention, 2019 ; Corona et al., 2015 ; Wright et al., 2013 ), often performed prior to natural menopause onset ( Hoffman et al., 2012 ; NAMS, 2014 ). To avoid premature surgical menopause in adulthood, the ovaries are retained in about half of hysterectomy cases, especially when women are at low-risk for certain cancers ( Asante et al., 2010 ; Lowder et al., 2010 ; Pocobelli et al., 2012 ; The American College of Obstetrics and Gynecology, 2008 ; Whiteman et al., 2008 ). There is evidence that the ovaries function normally following hysterectomy if the surgery occurs when a woman is still of reproductive age ( Beavis et al., 1969 ; Chalmers et al., 2002 ; Corson et al., 1981 ; Findley et al., 2013 ). However, individuals who undergo hysterectomy with ovarian conservation have been reported to experience the menopause transition three to four years earlier than uterus-intact individuals ( Chan et al., 2005 ; Gallicchio et al., 2006 ; Kaiser et al., 1989 ; Kritz-Silverstein et al., 2000 ; Moorman et al., 2011 ; Read et al., 2010 ). Given that average menopause onset occurs around age 52—and potentially at a younger age in women with hysterectomy—women who undergo variations in gynecological surgery in adulthood typically live many decades in a post-surgical, and eventually post-menopausal, state. How hysterectomy with and without ovarian conservation impacts health outcomes, including long-term effects on cognitive aging, remains an open, yet imperative, question. A premature loss of ovarian hormones, such as with surgical ovary removal, can have manifold repercussions on health and quality of life ( Erekson et al., 2013 ; Faubion et al., 2015 ; Matthews, 2013 ; Parker, 2014 ; Parker et al., 2009 ; Rodriguez and Shoupe, 2015 ), including negatively impacting cognition as demonstrated in both humans and animal models ( Bimonte and Denenberg, 1999 ; Bove et al., 2014 ; Farrag et al., 2002 ; Gibbs and Johnson, 2008 ; Nappi et al., 1999 ; Rocca et al., 2007 , 2009 , 2010 , 2011 , 2012 , 2021 ; Talboom et al., 2008 ; Uldbjerg et al., 2022 ; Wallace et al., 2006 ). Any emergent cognitive deficits following gynecological surgery are typically attributed to ovarian-associated changes. This is because the non-pregnant uterus is traditionally thought to be a “quiescent” and “useless” organ as an independent structure (p. 294) ( Navot and Williams, 1991 ) that only serves to “accommodate and support a fetus” ( Rosen and Cedars, 2007 ). Yet, the non-pregnant uterus has a unique, dynamic microbiota ( Agostinis et al., 2019 ); a well-characterized capacity to regulate and respond to immune challenges ( Agostinis et al., 2019 ; Lea and Clark, 1989 ; Turner et al., 2012 ); and direct innervation to the central nervous system ( Brauer and Smith, 2015 ; Collins et al., 1999 ; Gnanamanickam and Llewellyn-Smith, 2011 ; Ortega-Villalobos et al., 1990 ). Premenopausal hysterectomy –with and without ovarian conservation– has been shown to increase the relative risk of developing neurodegenerative diseases such as dementia ( Phung et al., 2010 ; Rocca et al., 2007 , 2012 ) and Parkinson’s disease ( Benedetti et al., 2001 ) compared to reproductive-tract–intact women of a similar age. The magnitude of this increased relative risk of dementia rises with a younger age at the time of surgery ( Phung et al., 2010 ). Though the uterus has traditionally been considered an endocrine target, but otherwise dormant when independent of its direct communication with the ovaries, this reported association between hysterectomy and neurodegenerative disease has piqued interest into how the non-pregnant uterus influences non-reproductive functions, including cognitive processes. Furthermore, given that two-thirds of Alzheimer’s disease cases are in females ( Alzheimer Association, 2019 ; Riedel et al., 2016 ), this is a critical area to investigate. Many of the neural circuits impacted by reproductive hormone alterations are the same circuits affected by aging (for review, see: Koebele and Bimonte-Nelson, 2017 ). Significant reproductive system events, such as pregnancy, menopause, and gynecological surgery intervention may also alter the trajectory of neural reorganizational processes to influence health outcomes ( Bimonte-Nelson et al., 2021 ; Koebele and Bimonte-Nelson, 2015 ). The brain utilizes adaptive, plasticity-associated mechanisms to respond to changing conditions across the lifespan. Due to the chronic nature of aging and hormone loss following variations in surgical menopause, it is difficult to pinpoint the specific changes and related time course within a given neural circuit or system. It is nonetheless imperative to uncover indicators of long-term changes in the brain that underlie the increased risk of cognitive decline and dementia in females. One candidate marker for long-term neuronal changes in response to menopause and a changing hormone milieu is the altered expression and processing of activity-dependent immediate early genes (IEGs). In general, IEGs are rapidly and transiently activated genes following extracellular activity or stimulation ( Morgan and Curran, 1989 ; Sheng and Greenberg, 1990 ). IEGs are necessary for many neurobiological processes, including synaptic plasticity and memory ( Minatohara et al., 2016 ; Sheng and Greenberg, 1990 ). One such gene, FOSB, can undergo alternative splicing to produce full-length FOSB and a truncated form, ΔFOSB. Because of the stability and extended half-life of ΔFOSB when compared to other IEG transcription factors, it is thought to act as a sustained molecular switch in neurons for weeks to months following initial activation ( Carle et al., 2007 ; Chen et al., 1997 ; Ulery-Reynolds et al., 2009 ). The sustained changes in FOSB expression lend themselves well to the longitudinal nature of our surgical manipulations and extended behavioral testing protocols. As such, we can leverage this distinguishing characteristic to assess FOSB and ΔFOSB expression as a candidate biomarker for long-term changes in the activation of brain areas important to learning and memory following variations in surgical menopause and with age. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) serve as upstream effectors of FOSB expression ( Fasano et al., 2009 ), and are also potential targets for altered activation with surgery manipulations and during aging given their broad influence on cellular signaling cascades and interactions with reproductive hormones ( Fortress et al., 2015 ; Frick, 2015 ; Tuscher et al., 2015 , 2018 ), including on learning and memory processes ( Atkins et al., 1998 ; Bozon et al., 2003 ; Fasano and Brambilla, 2011 ). Together, FOSB and ERK1/2 may serve as putative activity-dependent markers for reorganizational effects associated with surgical menopause and aging processes. A significant gap in the basic science literature exists regarding the effects of hysterectomy in the context of gynecological surgery, menopause, and cognition ( Bimonte-Nelson et al., 2021 ; Brinton, 2012 ). To address this knowledge gap, our laboratory developed a novel rat model of hysterectomy to systematically evaluate effects of variations in gynecological surgery on cognition. We reported that hysterectomy with ovarian conservation in adulthood resulted in a unique, detrimental effect on spatial working memory when assessments were approximately two months after surgery ( Koebele et al., 2019 ). These findings should be interpreted in the context of a short-term time point after surgery. The goal of the current experiment was to expand on these findings by investigating whether the cognitive effects following hysterectomy in adulthood were transient and reverse with time, or the observed cognitive changes were the beginning of a long-term cognitive impairment. Three cohorts of rats received sham, ovariectomy (Ovx), hysterectomy, or Ovx-hysterectomy surgery at five months of age. The first cohort was tested six weeks after surgery, in adulthood; the second cohort was tested seven months after surgery, in early middle-age; the third cohort was tested 12 months after surgery, in late middle-age. Rats underwent cognitive testing on a series of behavior tasks evaluating spatial working memory, spatial reference memory, and anxiety-like behavior to elucidate the long-term impact of variations in gynecological surgery on several cognitive domains. Serum steroid hormone levels, gonadotropin levels, and ovarian follicle counts were quantified to understand how gynecological surgery could impact ovarian steroidogenesis and follicular development. Full-length FOSB and ΔFOSB protein expression were assessed in cognitive brain regions including the frontal cortex, dorsal hippocampus, and entorhinal cortex, and phosphorylated- and total- ERK1/2 protein expression were evaluated in the entorhinal cortex to explore putative markers of long-term neurobiological changes after gynecological surgery and cognitive testing.