Mouse Offspring Exposed to Preeclampsia/Eclampsia-like Symptoms Exhibit Cerebral Hypoperfusion & Mild Cognitive Impairment at 2 months of age

Abstract

Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of organ damage, after the 20^th week of pregnancy. Children born to mothers with preeclampsia or eclampsia (new-onset seizures during pregnancy) are more likely to develop learning and memory deficits and are more susceptible to neurovascular diseases compared to those born from normal pregnancies. The contributing mechanisms are unknown. In this study, we assessed whether exposure to reduced uteroplacental perfusion (RUPP), modeling placental hypoperfusion and preeclampsia, with or without pentylenetetrazol (PTZ) injection (to induce seizures and model eclampsia), results in cognitive impairment, Alzheimer’s disease markers, and regional cerebral perfusion changes in adult offspring. On gestational day (GD)13.5, pregnant C57BL/6 mice (n=22) underwent Sham or RUPP surgery followed by injection or no treatment with PTZ (40 mg/kg) on GD18.5. At 2 months of age, spatial learning and cerebral perfusion were measured in randomly selected offspring or averaged to obtain mean data per sex, per litter (n=4-6 data points per group/treatment). RUPP-exposed offspring took a longer distance and made more errors navigating the Barnes maze. Cerebral perfusion was reduced in offspring exposed to RUPP, specifically in the prefrontal cortex, superior sagittal sinus, and whole brain. There was a significant reduction in perfusion in seizure-exposed offspring in the superior sagittal and transverse sinuses, whole brain, and cerebellum. Our results support the hypothesis that exposure to preeclampsia/eclampsia-like symptoms leads to mild learning impairment through reduced cerebral perfusion to cortical regions and decreased drainage of waste from the brain via the cerebral sinuses.

New & Noteworthy:

• This is the first study to assess vascular-related cognitive function and Alzheimer’s disease markers in young adult mice exposed to preclinical preeclampsia and eclampsia-like conditions.

• We show that mild learning impairments occur in the presence of cerebral hypoperfusion and reduced venous drainage into the brain sinuses.

• We also report that as early as 2 months of age, Alzheimer’s disease markers are increased in the brains of offspring exposed to seizures.

Graphical Abstract

Introduction

Preeclampsia (PE) is a complex hypertensive syndrome of pregnancy that affects approximately 5–8% of pregnancies globally, with rates reaching up to 15% among Black American women (1). PE is characterized by elevated blood pressure, proteinuria, and systemic endothelial dysfunction and can lead to severe complications, including stroke, seizure, and multi-organ damage (2). If left unmanaged, PE may progress to eclampsia, a life-threatening condition defined by new-onset seizures. Although delivery remains the only effective intervention, the adverse effects of PE/E extend beyond the perinatal period, contributing to long-term health complications for both mother and offspring. While the immediate maternal and neonatal outcomes of PE/E have been extensively studied, growing evidence suggests that these conditions may have lasting effects on the neurodevelopment of the offspring. In particular, children born to mothers with hypertensive disorders of pregnancy exhibit higher risks for cognitive impairments, including deficits in memory, learning, and executive functioning (3-7). These outcomes are likely mediated by impaired placental function, hypoxic injury, and disruptions in cerebral perfusion during critical windows of fetal brain development.

Reduced placental perfusion can lead to global changes in the offspring, as supported by a recent study showing a clear relationship between placental vascular resistance and stunted growth in the resulting child (8). Further evidence shows that reduced blood flow to the developing fetal-placental unit, using the preclinical reduced uterine perfusion (RUPP) model, results in reduced baseline cerebral perfusion in 5-day-old RUPP-exposed neonates (9). Regulation of cerebral blood flow (CBF) ensures relatively constant blood flow to the brain despite changes in systemic blood pressure, protecting the fragile microvasculature from damage (10). Additionally, CBF is regulated by neuronal activity, with increased CBF during periods of high neuronal activity (10). The arteries supplying blood to cortical areas of the brain are located in the Circle of Willis. The vertebra-basilar system supplies blood to the cerebellum, occipital lobe, and brain stem, while the internal carotid arteries supply blood primarily to the cerebrum. Using a preclinical model of preeclampsia, one study found that neonates exposed to preeclampsia-like conditions had reduced perfusion of the cerebral microvasculature (9). The mechanisms underlying reduced cerebral perfusion after exposure to preeclampsia-like conditions remain poorly understood.

Previous studies have shown that vascular risk factors and reduced CBF are linked to increased risk of cognitive impairment and Alzheimer’s disease (AD); however, the possible moderating effects of age and vascular risk burden on CBF in late life remain understudied (11). Additionally, reduced CBF was associated with worse performance on cognitive tasks, increasing the time to complete the task (11). Other studies have shown that neonatal and fetal offspring exposed to preeclampsia-like conditions have alterations in CBF and exhibit abnormal levels of cerebral proangiogenic and antiangiogenic activity (9). Furthermore, the most common treatment for preeclampsia is delivery, which often results in preterm birth and may adversely affect neurodevelopment.

Studies suggest that vascular dysfunction and hypoperfusion precede or exacerbate the development of Alzheimer’s disease (AD) pathology, particularly in individuals with elevated vascular risk factors (12-18). AD, the most common form of dementia, is characterized by progressive cognitive decline, amyloid beta (Aβ) plaque accumulation, and tau protein hyperphosphorylation (19). The early pathophysiology of AD is thought to involve impaired clearance of neurotoxic proteins, a function mediated in part by the glymphatic system, a glial-dependent waste clearance mechanism that relies on adequate cerebral perfusion (20). While the relationship between maternal hypertension and late-life neurodegenerative disease in offspring remains understudied, emerging data point to a potential link. Children born to mothers with PE/E may be more vulnerable to neurovascular dysregulation and cognitive dysfunction, possibly increasing their long-term susceptibility to diseases like AD (7, 9, 21).

The goal of the current study was to determine whether exposure to PE/E-like conditions leads to a reduction in offspring cerebrovascular perfusion, cognitive impairment, and increases in Alzheimer’s disease markers in the cortex. The overall hypothesis is that exposure to preclinical preeclampsia/eclampsia-like conditions leads to cerebral hypoperfusion, learning impairment, and increased Alzheimer’s disease markers.

METHODS

Animal Subjects

Twenty-two (22) Timed-pregnant C57BL/6 mice (RRID: IMSR_JAX:000664; n=5-6 per group/treatment) were ordered from Jackson Laboratories and housed in the Center for Comparative Research Animal Facilities at the University of Mississippi Medical Center. Mice were housed 2 per cage until surgery day when they were individually housed, and had ad libitum access to food and water. Animal rooms were maintained on a 12h light, 12h dark cycle, and temperature (22-23°C) and humidity (50-54%) were controlled. Behavioral experiments were performed in the Animal Behavior Core facility, with room conditions maintained similar to the housing rooms. All animal procedures were approved by the Institutional Animal Care and Use Committee (Protocols 1509A and 2023-1263) at UMMC before initiating studies.

RUPP and Sham surgeries

On gestational day (GD) 13.5, mice were weighed and matched by weight to the Sham or RUPP groups (Figure 1). Mice were anesthetized with isoflurane (3% induction, 1.5% maintenance), and the hair on the abdomen was removed with depilatory cream. The surgical site was prepared using 70% isopropyl alcohol and betadine. An incision was made along the midline, and the uterine horn with the placenta and fetuses was exteriorized and counted. The uterine horn was returned to the abdominal cavity for the Sham group of mice. For the mice in the RUPP group, the uterine vessels between the ovaries and the first pup were ligated using 5.0 silk sutures. The uterus was returned to the abdominal cavity, and the muscular and skin layers were sutured closed with 6.0 sutures. Mice were injected using 5mg/kg Carprofen (s.c.) and had access to Rimadyl tablets for post-surgery analgesia. Dams were individually housed after surgery.

Figure 1. Study Design and Timeline.

Timed-pregnant C57BL/6 mice underwent Sham or RUPP surgery on GD 13.5. On GD 18.5, mice were administered 40mg/kg PTZ. Pregnant dams gave birth, and at 2 months of age, learning and memory were assessed using the Barnes maze. Cerebral perfusion was also measured before euthanasia and tissue harvest under isoflurane anesthesia. GD – gestational day, PND – postnatal day. This figure was created with BioRender.com.

Seizure Induction

On GD 18.5, mice were injected with 40mg/kg pentylenetetrazol (i.p, dissolved using sterile saline) and video-monitored for 30 minutes (Figure 1). A vehicle control was not used in this study. Dams experienced mild-to-moderate seizures during the 30-minute monitoring period. After 30 minutes of observation, mice were returned to their home cages. Mice were allowed to deliver, and pups were weaned on postnatal day 21 (2-4 per cage). No litters were culled. Based on observations in our mouse colonies, manipulating pups before weaning increases cannibalism by the dams.

Learning and memory assessment

At 2 months of age, mice were habituated to the Barnes maze (20 holes) in our animal behavior core facility for 2 minutes, the day before the training trials. Mice were placed in the center of the maze and guided to the escape hole, where they remained for 2 minutes. For the next 4 days, mice navigated the maze in 4 daily trials, with an intertrial interval of 20 minutes. Mice were motivated to find the escape box hidden beneath one of the holes (the target hole) by bright light and white noise. Mice used cues posted on the walls around the maze to navigate. On day 5, short-term memory was tested using a probe trial. The escape box was removed, and the mice were allowed to navigate the maze for 90 seconds. For learning acquisition, escape latency (the time taken to enter the escape box), path length, and the number of errors (visits to non-target holes) were assessed. The duration in the target quadrant and the frequency of each hole were evaluated for the probe trial.

Cerebral perfusion measurement and analysis

Twelve days after the last training day on the Barnes maze, mice were anesthetized using isoflurane using the SomnoSuite system (Kent Scientific) combined with room air and prepared for cerebral perfusion imaging. A midline incision was made on the head, and the skin was pulled away, exposing the skull. Isoflurane was reduced to 1.5%, and paw sensors were connected to the left hind paw. The paw sensors measured heart rate and oxygen saturation (sPO2), which were recorded using a PowerLab system and LabChart software (version 8, ADInstruments). The PeriCam PSI HR Laser Speckle Imager (Perimed) was moved over the head of the mouse and positioned 10cm above the skull so that the entire brain surface was visible. Baseline perfusion was measured via PIMSoft software (Perimed) for 3 minutes. Regions of interest (Figure 2) were drawn to identify the superior sagittal sinus, left and right parietal cortices, transverse sinus, prefrontal cortex, cerebellum, and whole brain. Perfusion (perfusion units) was divided by the area of each region of interest to obtain normalized regional perfusion.

Figure 2. Representative perfusion and intensity images.

The regions of interest used to measure perfusion are shown on the intensity image. Olf – olfactory area, SSS – superior sagittal sinus, PFCx – prefrontal cortex, LPCx – left parietal cortex, RPCx – right parietal cortex, and Tr. Sinus – transverse sinus. The whole brain comprised the area around all the regions of interest.

Euthanasia and tissue collection

Following cerebral perfusion measurements, isoflurane was increased to 3%, and blood was collected via cardiac puncture. Hearts and kidneys were harvested and weighed. Brains were then collected and hemisected after the cerebellum was removed. The right hemisphere was transferred to 4% paraformaldehyde, and the left hemisphere was dissected into the anterior cerebrum (prefrontal cortex), posterior cortex, hippocampus, and striatum, weighed, and flash-frozen in liquid nitrogen. Brain regions were stored at −80°C until further processing.

Measurement of AD markers

The left hippocampus and posterior cortex were homogenized using a bead homogenizer in RIPA buffer containing proteinase inhibitors. Protein concentration was measured using a BCA kit. Total tau (Catalog number: KMB7011, ThermoFisher), AB40 (Catalog number: KMB3481, ThermoFisher), and AB42 (Catalog number: KMB3441, ThermoFisher) were measured using a commercially available ELISA kit following the manufacturer’s instructions.

Statistical Analysis

Data were expressed as factors of RUPP (RUPP vs. Sham) and PTZ exposure (−PTZ vs. +PTZ). Datasets were tested for normality using the Shapiro-Wilks test, and cerebral perfusion changes were averaged within the different groups. The study followed a 2x2 design with the factors of RUPP and PTZ. A Two-Way ANOVA was used to test statistical differences (main effects of the factors and interactions between the two factors). For learning acquisition in the Barnes maze, a 3-way ANOVA was used (RUPP, seizure, and day). Pair-wise comparisons were made using Tukey’s post hoc analysis. A p-value of <0.05 was considered statistically significant. Data were reported as mean ± standard deviation. Graphs and statistical analyses were conducted using GraphPad Prism software (version 10.6.1).

RESULTS

General Characteristics:

There was a main effect of RUPP to reduce litter size at birth (F(1, 18) = 5.742; p=0.028), and a trend for an effect of seizures to reduce litter size (F(1, 18) = 3.535; p=0.076 – Table 1). Because not all pups survived to weaning, we also counted the number of offspring that did and found no significant effect of RUPP and seizure exposure on pup survival to weaning. There was no effect of RUPP or seizure exposure on the offspring’s body weight (p>0.05) at 2 months of age (Table 1). RUPP exposure significantly affected offspring brain weight (F(1, 15) = 5.710; p = 0.030), whereas seizure exposure had no effect. There was no effect of RUPP or seizure exposure on heart weight. We also assessed hematocrit and found a significant effect of seizure exposure to decrease hematocrit in the offspring (F(1, 15)=15.34; p = 0.001). Specifically, a significant decrease in hematocrit was observed in Sham-exposed offspring exposed to seizures (p=0.004). Seizure exposure significantly increased blood oxygen saturation (F(1, 15) = 22.26; p<0.001) with significant increases in Sham (p=0.026) and RUPP (p=0.002) -exposed offspring. Heart rate, measured using a paw sensor, was not affected by RUPP or seizure exposure (p>0.05). Lastly, seizure-exposed offspring had a significant decrease in cerebellum water content (F(1, 15) = 9.594; p=0.007), with a significant reduction in Sham-exposed offspring (p=0.034) compared to Sham-PTZ group.

Table 1:

General Characteristics of Offspring at 2 months of age

General Characteristics	Sham − PTZ	RUPP − PTZ	Sham + PTZ	RUPP + PTZ
Litter size (birth) †	7 ± 1	5 ± 3	6 ± 2	3 ± 2
Litter size (weaned)	1 ± 2	2 ± 3	4 ± 3	2 ± 2
Total Pups (male)	6 (4)	12 (5)	21 (13)	9 (4)
Body Weight (g)	25.0 ± 3.2	23.8 ± 3.2	23.9 ± 3.2	24.0 ± 2.4
Brain weight (mg) †	435 ± 10	449 ± 14	436 ± 11	448 ± 10
Heart Weight (mg)	120 ± 21	123 ± 14	117 ± 15	117 ± 10
Hematocrit (%) ‡	45 ± 2	42.8 ± 2.6	39.8 ± 0.9	41.5 ± 1.6
Oxygen saturation (%) ‡	63 ± 2.6	59.8 ± 8.3	77.7 ± 10.4	77.8 ± 4.8
Heart rate (bpm)	573 ± 15	558 ± 22	547 ± 45	553 ± 59
Cerebellum water content (%) ‡	76.6 ± 0.2	76.5 ± 0.5	76.0 ± 0.3	76.0 ± 0.3

Data represent Mean ± standard deviation.

^†main effect of RUPP,

^‡main effect of seizure

Offspring exposed to RUPP during pregnancy have modest learning impairments on the Barnes Maze at 2 months of age.

At 2 months of age, male and female offspring were tested on the Barnes Maze to assess spatial learning acquisition over 4 days (Figure 3). Learning was assessed by quantifying the distance traveled to enter the escape box (Figure 3A and 3B), the time taken to enter the escape box (Figure 3C and 3D), the number of errors made before escaping (Figure 3E and 3F), and the time taken to find the target hole the first time (Figure 3G and 3H). There was no effect of RUPP (F(1, 16) = 0.171; p=0.685) or seizure-exposure (F(1, 16) = 0.116; p=0.738) on path length. Pairwise comparisons indicated that offspring exposed to both RUPP and seizures took a more efficient path to the escape box on Day 1 compared to those exposed to RUPP only (Figure 3B). For latency to enter the escape box, there was a significant Day × Seizure interaction (F(2.09, 34.4) = 3.829; p = 0.031), with offspring exposed to both RUPP and seizures demonstrating significantly shorter times to escape compared to the Sham and RUPP-exposed offspring (Figure 3D). There were no significant effects of RUPP or seizure exposure on total errors (Figure 3F) or primary latency (Figure 3H). For all the above learning endpoints, learning improved with additional training days.

Figure 3. Changes in spatial learning acquisition on the Barnes maze.

Over the 4 days of training, (A) path length to escape box per trial and (B) averaged per day, (C) escape latency per trial, (D) averaged escape latency, (E) total errors per trial, (F) average errors per day, (G) primary latency per trial, and (H) primary latency per day were assessed. Data for the line graphs represent mean ± SEM. Box-and-whisker plots showing individual data, along with the minimum and maximum datapoint, upper and lower quartiles, and median for each group. Data were analyzed using Mixed Effects analysis for 3-Way ANOVA. *p<0.05 Sham-PTZ vs. RUPP-PTZ, ^fp<0.05 Sham-PTZ vs. Sham+PTZ, $p<0.05 RUPP-PTZ vs RUPP+PTZ, and #p<0.05 Sham+PTZ vs. RUPP+PTZ.

Exposure to RUPP and Seizures led to negligible memory deficits in the offspring

On Day 5, a probe trial was conducted in which the escape box was removed, and the animals were tested for whether they remembered the general location of the target hole. Figure 4A shows representative heatmaps for each group, indicating that mice displayed overall good recollection of the escape box’s prior location, spending the most time in that area. We counted the number of times the mice visited each hole and plotted the frequency of head pokes in each hole (Figure 4B). While the mice visited the target hole most frequently, those exposed to RUPP during pregnancy visited the adjacent holes more often than the other groups. The Barnes maze was separated into quadrants, with the target and 2 adjacent holes on either side designated as the target quadrant. There was a significant interaction between RUPP and Seizure effects (F(1, 16) = 6.038; p=0.026), with RUPP-exposure leading to a directional decrease in time spent in the target quadrant, and seizure exposure increasing the time spent in the target quadrant (Figure 4C). Lastly, we assessed the latency to the target during the memory probe and found no significant differences between groups (Figure 4D).

Figure 4. Changes in memory on the Barnes maze.

(A) Heatmap showing the relative time spent in each area of the maze on day 5 (probe trial). Hotter colors indicate more time spent in that area. (B) Frequency of visits to each hole of the 20-hole maze. T represents the target hole. (C) Time spent in the target quadrant. (D) Latency to first visit the target hole. Box-and-whisker plots are shown in C. Dots represent the behavior of averaged datapoints per sex per litter. Data were analyzed using Mixed Effects analysis for 3-Way ANOVA

Exposure to RUPP or Seizures reduced brain drainage in the Superior Sagittal Sinus and Transverse Sinus of offspring.

We used a laser Speckle imager to assess baseline cerebral perfusion under isoflurane anesthesia. In the superior sagittal sinus, subjects exposed to RUPP had a significant decrease in cerebral perfusion (Figure 5A; F(1,16) = 14.95, p = 0.001), with a significant reduction in the RUPP compared to sham-exposed offspring (p=0.049), and RUPP+PTZ-exposed groups compared to Sham+PTZ (p=0.016). In the transverse sinus, subjects exposed to seizures showed decreased perfusion (Figure 5B; F(1,16) = 6.34, p = 0.023), with no pairwise differences between groups. In the cerebellum, seizure (F(1,16) = 19.96, p < 0.001) led to reduced perfusion (Figure 5C), with a significant reduction in offspring exposed to RUPP and seizures. Together, these findings indicate that exposure to RUPP or seizures reduces venous drainage into the cerebral sinuses.

Figure 5. Changes in perfusion of the sinuses and cerebellum.

Baseline perfusion of (A) superior sagittal, (B) transverse sinus, and (C) cerebellum of offspring exposed to RUPP and/or PTZ during pregnancy. Individual points represent averaged data per sex per litter. Sex symbols are used to differentiate male and female data points. Data were analyzed using Two-Way ANOVA and Sidak’s multiple comparison tests.

Offspring born from RUPP dams had reduced perfusion of the right parietal cortex and whole brain, with no effect on the left parietal cortex.

We next assessed perfusion in the cortical regions. There was a significant interaction between RUPP and seizure exposure on perfusion of the left parietal cortex (Figure 6A, F(1, 16) = 4.598; p = 0.048), with no significant effect of RUPP or seizure exposure. In the right parietal cortex, there was no significant effect of RUPP or seizure exposure (Figure 6B). In the prefrontal cortex, there was a significant interaction between RUPP and seizure exposure (Figure 6C, F(1, 16) = 5.157; p = 0.037), with a trend toward an effect of RUPP exposure (F(1, 16) = 4.439, p = 0.051). In the whole brain, there was a significant interaction between RUPP and seizure exposure (F(1, 16) = 7.299; p = 0.016), a main effect of RUPP exposure to reduce perfusion (Figure 6D; F(1,16) = 6.921, p = 0.018), and a main effect of seizure exposure to reduce perfusion (F(1, 16) = 14.02; p = 0.002).

Figure 6: Changes in cerebral perfusion in cortical regions and the whole brain.

Changes in (A) left parietal cortex, (B) right parietal cortex, (C) prefrontal cortex, and (D) whole brain in response to prenatal RUPP and seizure exposure. Individual points represent averaged data per sex per litter. Sex symbols are used to differentiate male and female data points. Data were analyzed using Two-Way ANOVA and Sidak’s multiple comparison tests.

Offspring exposed to RUPP and/or seizures exhibit changes in AD markers in the hippocampus and posterior cortex.

There was no effect of RUPP or seizure exposure on cortical Aβ40 expression (Figure 7A). However, offspring exposed to seizures showed a significant increase (F(1, 16) = 12.80; p=0.003) in cortical Aβ42, with a significant increase in Sham and RUPP offspring exposed to seizures (Figure 7B). Additionally, offspring exposed to seizures showed a significant increase (F(1, 16) = 12.53; p=0.003) in cortical Aβ42/40 ratio, with a significant increase in Sham and RUPP offspring exposed to seizures (Figure 7C). Next, we assessed changes in total and phosphorylated Tau. Seizure exposure significantly increased cortical tau (F(1,15)=5.39; p=0.035), with a trend for an increase in offspring exposed to both RUPP and seizures (Figure 7D). Lastly, we measured phosphorylated Tau and found no difference in Tau[pS199] concentrations in cortical samples of offspring exposed to both RUPP and seizures during pregnancy (Figure 7E).

Figure 7. Changes in Alzheimer’s disease markers in the posterior cortex.

Changes in (A) Amyloid beta (1-40), (B) Amyloid beta (1-42), (C) Aβ42/40 ratio, (D) total Tau, and (E) phosphorylated Tau concentration in cortical samples. Individual points represent the concentration for a randomly selected mouse from each group. The sex of the mouse is indicated by its symbol. Data were analyzed using Two-Way ANOVA and Sidak’s multiple comparison tests.

DISCUSSION

Preeclampsia and eclampsia are serious pregnancy complications with limited treatment options beyond delivery of the offspring (7). Unfortunately, delivery neither alleviates maternal physiological and emotional stress during pregnancy nor addresses the potential long-term developmental effects on offspring. Growing evidence suggests that children exposed prenatally to these disorders are at increased risk for cognitive and behavioral problems such as attention deficits, anxiety, emotional dysregulation, and impairments in learning and memory (7). There is also speculation that these early life insults may predispose individuals to neurodegenerative conditions, including Alzheimer’s disease (22). However, the mechanisms underlying these effects remain poorly understood, underscoring the need for preclinical models to explore both immediate and long-term neurological outcomes. In this study, we found that at 2 months of age, corresponding roughly to late teens to early adulthood, mice exposed to preeclampsia and eclampsia-like symptoms exhibited mild learning impairment, cerebral hypoperfusion, and, in the eclampsia-like conditions, increased Alzheimer’s disease markers.

Rationale for Animal Models and Experimental Design

The current study compares C57BL/6 offspring at 2 months of age, in which exposure to reduced uterine perfusion (RUPP) and/or pentylenetetrazol (PTZ) resulted in reductions in cerebral perfusion and evidence of cognitive impairment. Prior human studies have demonstrated that regional cerebral blood flow positively correlates with cognitive performance; for example, Kilroy et al. (2011) found that typically developing children and adolescents with higher cerebral perfusion in key cortical regions exhibited higher IQ scores, supporting the biological plausibility that reductions in cerebral perfusion can impair cognition (23). Similarly, lower cerebral perfusion has been implicated in cognitive decline in adults with conditions such as vascular cognitive impairment and Alzheimer’s disease (AD) (14) (24), suggesting that even modest disruptions in blood flow during development could have lasting neurocognitive consequences.

To investigate these effects in a controlled setting, we employed a modified mouse RUPP model adapted from Fushima et al. (2017), which replicates key clinical features of preeclampsia (25). Our laboratory has shown that this model results in fetal hypoxia, fetal demise, and elevated maternal anti-angiogenic markers (sFlt-1), without evidence of placental ischemia/hypoxia (26). While the majority of current RUPP models are performed in rats (27-30), the use of a mouse RUPP model facilitates genetic manipulations that may elucidate molecular pathways involved in offspring brain development and that may affect features of clinical preeclampsia such as increased blood pressure, increased fetal demise, evidence of hypoxia in pups, and an imbalance in the presence of maternal angiogenic factors. To mimic eclampsia-related seizures, pregnant dams received the proconvulsant pentylenetetrazol (PTZ), the most common convulsant used in pregnancy studies (26, 31-38). Combining these insults allowed us to model the overlapping but distinct impacts of placental hypoperfusion and seizure-induced injury on offspring neurodevelopment. The RUPP model was used in this study to simulate the reduced utero-placental perfusion observed in some cases of preeclampsia, especially in women with early-onset preeclampsia. Ongoing studies use the preclinical mouse model of superimposed preeclampsia (blood pressure high 5, BPH/5), in which symptoms are more spontaneous (39). Our hypothesis is that reduced utero-placental perfusion may underlie learning and memory impairment, a concept that seems very likely but has not been tested directly before.

The study by Rajabzadeh et al. (2012) supports the use of pentylenetetrazol (PTZ) as an effective tool to model seizure activity in pregnant animals, demonstrating that maternal seizures can significantly disrupt neurodevelopment in offspring (37). Specifically, they found that PTZ-induced seizures during pregnancy reduced hippocampal expression of PSA-NCAM, a molecule critical for neuronal plasticity, synaptic connectivity, and cognitive development. Given the hippocampus’s central role in memory and learning, this disruption suggests a mechanistic link between seizure exposure and long-term cognitive deficits in offspring. Applying this model to the context of preeclampsia/eclampsia, where seizures (eclampsia) are a hallmark of disease severity, PTZ serves as a valuable proxy to replicate seizure-associated neurological stress in utero. Therefore, PTZ can help determine whether seizure exposure in the setting of preeclampsia-like conditions exacerbates alterations in cerebral perfusion and contributes to impaired neurodevelopment in offspring, especially in regions critical for cognition, such as the hippocampus and prefrontal cortex. This approach will enable this study to distinguish and evaluate the combined or interacting effects of placental hypoperfusion and seizure-induced injury on brain function.

Behavioral Outcomes: Learning and Memory Deficits

Offspring were tested at two months using the Barnes Maze, a dry-land spatial memory task minimizing stress relative to water-based mazes (40). During the acquisition phase, RUPP-exposed offspring traveled a longer distance and made more errors in locating the escape box, indicating impaired acquisition. However, over time, these mice eventually found the target, suggesting that gross memory consolidation remained intact and that the primary deficit was in learning rather than memory retention. Importantly, exposure to RUPP and/or PTZ altered regional cerebral perfusion, particularly in the cerebellum and right parietal cortex – brain regions important for spatial navigation, sensorimotor integration, and executive function (41, 42).

Offspring exposed to RUPP alone exhibited significant delays in early learning, traveling longer paths, and making more errors on day 1 of training compared to controls. However, performance across groups converged by the end of the training period, suggesting that initial learning was impaired but memory consolidation was preserved. Conversely, offspring exposed to RUPP and PTZ showed impaired memory retention during the probe phase, spending less time in the target quadrant and exhibiting more erratic maze navigation. The PTZ-only group performed similarly to controls, suggesting a more substantial impact of preeclampsia-like ischemia on early learning, with eclampsia-like seizures exerting subtler or delayed cognitive effects.

Our findings are supported by previous human studies, such as Ratsep et al. (2016), who demonstrated that adolescents born to preeclamptic pregnancies exhibit significant cognitive impairments, particularly in working memory and oculomotor control, compared with those to mothers with uncomplicated pregnancies (3). Specifically, psychometric and eye-tracking evaluations revealed that PE-exposed offspring performed worse on measures of executive function, attention, and visuospatial processing, and exhibited abnormalities in saccadic eye movements. These cognitive and sensorimotor deficits may be linked to altered brain perfusion and disrupted neurovascular development during critical periods of fetal brain maturation. Together, these findings support the hypothesis that prenatal exposure to preeclampsia/eclampsia-like conditions contributes to persistent cerebrovascular and cognitive dysfunction in the offspring, aligning with broader evidence indicating a heightened risk of neurodevelopmental disorders such as autism spectrum disorder, intellectual disability, attention-deficit/hyperactivity disorder, and learning disabilities (7).

Cerebral Perfusion and Neurovascular Impairment

Because learning impairments often occur in the context of hypoperfusion (43), we assessed changes in regional cerebral perfusion under baseline conditions. We found significant reductions in RUPP- and PTZ-exposed offspring in the superior sagittal and transverse sinuses, major venous drainage pathways that clear metabolic waste. Reduced clearance capacity in these sinuses has been implicated in Alzheimer’s pathology (44) and increases the susceptibility for intracranial hypertension and impaired venous outflow (45). We observed that in offspring exposed to RUPP or PTZ, there was no difference in perfusion of the parietal cortex, a brain region critical for spatial perception, visuomotor control, and attention (46). Moreover, seizure-exposed offspring demonstrated decreased cerebellar perfusion, a region that, when damaged, is associated with motor coordination deficits, manifesting as clumsiness and an ataxic gait (47). Seizure exposure led to reduced cerebral perfusion in the prefrontal cortex, which may reflect maladaptive responses amid global hypoperfusion, potentially predisposing offspring to emotional dysregulation or developmental vulnerabilities (48). Our results of global hypoperfusion support the finding of reduced perfusion in RUPP-exposed neonates at postnatal day 5 (9). It is worth noting that the most consistently affected group was the offspring exposed to both RUPP and seizures, highlighting an exacerbated response following seizures. These findings suggest that prenatal insults disrupt cerebrovascular integrity and drainage, potentially contributing to impaired neurodevelopment and vulnerability to neurodegeneration.

Alzheimer’s Disease-Related Biomarkers

To explore long-term disease susceptibility, cortical samples were analyzed for Alzheimer’s disease markers, including total tau and amyloid beta isoforms (Aβ40, Aβ42). PTZ exposure was associated with elevated cortical Aβ42 and tau levels, consistent with prior evidence linking seizures to amyloidogenic and tauopathy processes (49). While some protein changes were subtle - potentially due to the relatively young age of the mice, which approximates adolescence in humans - these preliminary findings indicate that eclampsia-like seizures may more strongly influence neurodegenerative pathways. In contrast, preeclampsia-like ischemia primarily affects early cognitive development. The current study also showed that offspring exposed to PTZ-induced seizures exhibit a significant increase in cortical Aβ42/40 ratio, cortical Aβ42, and cortical tau, suggesting elevations are consistent with studies demonstrating that PTZ-induced seizures can increase oxidative stress, neuronal apoptosis, and amyloidogenic processes, leading to AD-like changes in rodent models (50, 51). These results suggest that prenatal exposure to ischemic and seizure-related insults may differentially affect amyloid accumulation and tau expression, providing a potential mechanistic link between early-life cerebrovascular compromise and future neurocognitive risk (52).

Limitations and Future Directions

Our study has several strengths. First, we utilized a well-characterized preclinical preeclampsia mouse model, including groups that differentiated preeclampsia from eclampsia-like symptoms. Furthermore, using the laser speckle imager, we could measure multiple relevant brain regions, rather than a single area, as a Doppler would have allowed. Our study had some limitations. The young age of the offspring may have precluded detection of robust AD biomarker accumulation, which typically manifests at an older age. Moreover, the sample size was limited, particularly for analyses by sex, due to the limited availability of offspring. As a result, the study was underpowered to detect subtle or sex-specific differences in cerebral perfusion and cognitive function; thus, inherent sex differences may influence the observed outcomes, either contributing to group differences or masking other potential effects. Future studies with larger cohorts will be necessary to validate and expand upon these findings. Second, although laser speckle contrast imaging provides a valuable method for assessing cerebral blood flow at the cortical surface, it cannot capture perfusion changes in the deeper brain structures, such as the hippocampus or amygdala, which are critical for cognition and emotional processing. This technical limitation may underestimate the full extent of cerebrovascular alterations following exposure to preeclampsia/eclampsia-like conditions. In this study, we did not utilize a vehicle control and could not isolate the effect of the injection from that of the PTZ. Lastly, systemic blood pressure was not directly measured in dams or their offspring. Because hypertension is a hallmark of preeclampsia and can independently affect cerebrovascular function, future studies incorporating longitudinal blood pressure measurements will be essential to better understand the contribution of systemic hemodynamic changes to the observed cerebral perfusion abnormalities.

The findings from the current study suggest that offspring exposed to preeclampsia- and eclampsia-like conditions are at increased risk for disruptions in cerebral perfusion and associated cognitive impairments. These observations align with previous clinical evidence indicating that children born to preeclamptic pregnancies exhibit deficits in working memory, attention, and visuospatial processing [16]. While the current study focused on regional cerebral blood flow, future investigations will incorporate measures of capillary density and cerebrovascular architecture to elucidate further the microvascular mechanisms underlying these impairments. Assessing vascular parameters, such as capillary rarefaction, vessel diameter, and arterial wall thickness, may reveal structural vulnerabilities that predispose offspring to long-term cognitive dysfunction.

Conclusion

In conclusion, prenatal exposure to preeclampsia- and eclampsia-like conditions disrupts cerebral perfusion and impairs early learning and memory in offspring, with possible long-term implications for neurodegenerative disease risk. This study supports the hypothesis that such prenatal insults result in persistent alterations in cerebrovascular function, particularly in brain regions essential for spatial navigation, executive function, and motor coordination, including the prefrontal cortex and cerebellum. These region-specific reductions in perfusion likely contribute to the cognitive deficits observed in affected offspring, as cerebral blood flow is closely tied to neural function and cognitive performance across the lifespan. Importantly, these findings raise critical questions about the underlying mechanisms driving the observed vascular changes. Whether these deficits stem from disruptions in microvascular architecture, such as decreased capillary density, vessel rarefaction, or structural remodeling, remains to be determined. Furthermore, it is unknown whether these impairments persist or worsen with age, potentially increasing susceptibility to neurodegenerative diseases such as Alzheimer’s. Preliminary biomarker analyses suggest seizure exposure may influence amyloid and tau pathology, while ischemia may more robustly impair early learning and memory, indicating distinct but intersecting neuropathological trajectories. Together, these results underscore the long-term neurodevelopmental consequences of fetal exposure to maternal hypertensive and seizure conditions. They also highlight the urgent need for early detection and targeted interventions to mitigate the adverse outcomes associated with preeclampsia and eclampsia. Continued research into cerebrovascular development, neuroinflammation, and protein pathology in exposed offspring will be critical for advancing our understanding and developing future therapies.