Sex Differences in Memory Performance after Traumatic Brain Injury in Rats

Introduction

While males are approximately 40% more likely to suffer a traumatic brain injury (TBI) than females, ¹ the relationship between biological sex and outcome is complex and severity-dependent. A systematic review showed that across all injury severities, 47% of clinical studies found worse outcomes in women. ² However, this pattern shifted when stratified by severity, with 60% of mild-moderate and 34% of moderate-severe TBI studies reporting worse outcomes in women. ² At the severe end of the spectrum, women have been found to be approximately 1.75 times as likely to die following TBI and 1.6 times more likely to experience severe disability or persistent vegetative states. ³ These findings suggest that injury severity interacts with biological sex to determine outcome. Despite these discrepancies and the fact that sex hormones are known to be involved in neuroprotective mechanisms, most preclinical research has only utilized males.

Clinically, memory deficits are among the most disabling cognitive consequences of TBI, with significant gender differences reported in outcome. ⁴ In preclinical models, TBI-exposed male mice show more prominent learning impairment, while deficits in females occur predominantly in spatial memory performance. ⁵

This study compares long-term memory performance of male and female rats following moderate-severe fluid percussion injury (FPI), a model shown to produce lasting and measurable cognitive deficits.

Methods

Behavioral assessments

A timeline summarizing behavioral experiments may be found in Figure 1A.

OPEN IN VIEWER

FIG. 1.

Sex- and injury-dependent effects on open field test (OFT) and recognition memory. (A) Experimental timeline, with vaginal lavage sample collection indicated by stars. (B) Percentage locomotion in the OFT, (C) novel object location (NOL) index scores, and (D) novel object recognition (NOR) index scores shown by sex (female, male) and injury group (naive and TBI). Bars represent mean ± SEM. Statistical significance at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SEM, standard error; TBI, traumatic brain injury.

Open Field, Novel Object Location, and Novel Object Recognition Tests

The open field test (OFT) was used to assess locomotion and served as habituation for subsequent novel object location (NOL) and novel object recognition (NOR) tests (Day 14). The NOL and NOR tests included a familiarization session (Day 15), during which the animals were placed in the OFT arena with two identical objects for 5 min. On Day 16, animals were exposed to the same arena with one of the objects relocated to the opposite side (NOL) for 5 min. Then, 2 h later, one of the familiar objects was replaced by a novel one, and the animals were once again allowed 5 min to explore the arena (NOR). The NOL and NOR indices were calculated according to the formula: (duration of novel location or object exploration—duration of familiar location or object exploration)/total exploration time. ⁶

Barnes Maze

Barnes maze (BM) habituation (Day 18) and training trials (Days 19 and 20; 2 trials/day) recorded latency to locate the escape box(s), number of errors, and search strategy. The latter was scored based on the BM unbiased strategy (BUNS) classification algorithm. ⁷ Higher scores reflect greater reliance on spatial memory-guided, goal-directed navigation. In the probe trial (Day 21), the escape box was removed and replaced with a false bottom. Parameters collected included percentage of time spent in target quadrant (%) and search strategy. For analysis, strategies were grouped into nonspatial (random and serial; scores ≤ 0.25) and spatial (long correction, focused search, correction, and direct; scores ≥ 0.5). This dichotomization was used to distinguish reliance on spatial memory-guided navigation from nonspatial or systematic search behaviors.

Results

Barnes maze

Overall, no significant sex or TBI effects were noticed during the training sessions for the BM test (Fig. 2A). During the probe, no significant sex (F(1,57) = 0.358, p = 0.552) or TBI (F(1,57) = 1.511, p = 0.224) effects were found for the percentage time spent in target quadrant, despite a trend toward significance in the sex × TBI interaction (F(1,57) = 3.452, p = 0.068, Fig. 2B).

OPEN IN VIEWER

FIG. 2.

Barnes maze probe performance. (A) Learning curves based on the number of errors in each trial, (B) percentage of time spent in the target quadrant, (C) percentage of animals employing a spatial search strategy. Results shown by sex (male and female) and injury group (naïve and TBI). Bars represent mean ± SEM. For spatial strategy use, the naïve versus TBI group, but not sex differences were found to be significant. SEM, standard error; TBI, traumatic brain injury.

As for the strategy, we found that a greater proportion of naive animals employed a spatial strategy (17/28, 60.7%) compared to TBI animals (5/32, 15.6%), and this difference was statistically significant (p = 0.0008). There was no significant difference in strategy use between sexes (p = 1).

Discussion

This study examined spatial and recognition memory in female and male rats following TBI using NOL/NOR and BM paradigms. As expected, TBI impaired performance across tasks, with naive animals consistently outperforming injured animals. Importantly, the pattern and magnitude of some deficits were sex- and task-dependent, highlighting distinct vulnerabilities in males and females.

In the OFT, females exhibited greater locomotor activity than males. Of note, this effect was seen in both naive and TBI groups, suggesting that injury did not differentially alter locomotor activity. These results corroborate previous work suggesting that baseline locomotion and exploratory behavior in rodents vary significantly between sexes.^10,11 In both NOL and NOR, TBI rats showed significantly worse performance, but the magnitude of the deficits appeared to differ by sex. Females appeared to be particularly susceptible to deficits in rapid, novelty-driven memory tasks, as they presented a trend toward a lower NOL index and a significantly reduced NOR index compared to TBI-exposed males. Our NOR results for naive animals are also consistent with the broader literature, which reports a female advantage in object-based recognition memory across extended retention intervals.^12,13 In the BM, TBI effects were less clear, but trends suggested that male TBI rats exhibited reduced probe performance relative to their naive counterparts. TBI shifted both sexes towards the use of a nonspatial search strategy, indicating impaired cognition and a reduced use of hippocampal-dependent memory. In addition, male TBI animals also appeared to acquire the BM task more slowly, with elevated error rates persisting across training, suggesting an impairment in spatial learning rather than an isolated deficit in memory retention. These findings indicate domain-specific sex differences after TBI, with males showing greater impairments in learning-dependent spatial navigation and females in novelty-driven recognition memory.

Rapid novelty-driven tasks, such as the NOL and NOR, engage hippocampal circuits that support one-trial learning and rely heavily on exploratory and novelty-seeking behaviour. ¹⁴ These tasks appear more disrupted in females. In contrast, strategy-dependent and aversive stimulus-driven tasks like the BM rely on multiday learning and executive processes that appear more susceptible in males.

In addition to the sex differences described above, we found that the estrous cycle phase during testing did not significantly affect performance across tasks. Our initial hypothesis was that females in high-hormone phases (proestrus/estrus) might have shown relatively preserved memory, given the proposed neuroprotective and neuromodulatory roles of estrogen and progesterone. Understanding whether endogenous hormonal fluctuations modulate cognitive recovery after TBI may have a translational relevance, as recovery and rehabilitation could be different in pre- and postmenopausal women, in women taking different hormone formulations, as well as during different phases of the cycle. In naive females, there was a nonsignificant trend toward lower NOL performance during high-hormone phases, consistent with previous reports of reduced spatial recognition during elevated ovarian hormone states. ¹³ This pattern was not observed after TBI or in any other tasks, suggesting that injury-related effects may overshadow more subtle hormonal modulation of cognition in this dataset. Lavages were not collected during BM training to avoid compounding stress during task acquisition. As a result, the estrous state was only characterized at the probe trial (Day 21), and hormonal influences on spatial learning across training could not be fully assessed.

The present findings align with and extend prior preclinical studies demonstrating sex-dependent cognitive outcomes following TBI. Notably, our results parallel those reported by Santos et al., ⁵ who showed that male mice exhibited greater impairments in learning after controlled cortical impact-TBI, whereas females displayed more pronounced deficits during memory retrieval. Together, these convergent patterns suggest that TBI may differentially disrupt learning and memory processes in males and females, rather than producing uniform cognitive deficits across sexes. Our findings also reflect clinical observations that, despite a higher incidence of TBI in men, women often experience worse long-term cognitive outcomes. ² In particular, memory impairments are among the most persistent and disabling sequelae reported after TBI. The greater deficits observed in female rats on recognition- and location-memory-based tasks in the present study are consistent with these reports, supporting the translational relevance of sex-specific vulnerability in memory systems. Importantly, the task-dependent nature of deficits observed here may help explain variability in clinical findings, where outcomes differ depending on the cognitive domain assessed.

Our study was not without limitations. The injury pressure used to generate TBI in females was lower than in males, as pilot testing indicated that the use of male-equivalent pressure ranges in females resulted in an unacceptably high mortality rate. Pressure therefore was adjusted by sex to ensure animal welfare and permit meaningful post-injury behavioral assessment. Importantly, despite these differences in mechanical injury parameters, acute physiological indicators of injury severity, including postinjury apnea duration and postoperative weight loss, were comparable between sexes. Although no sham group was included in the present study, we have previously validated this FPI model against sham-operated animals, demonstrating that the surgical procedure for implanting the hub did not produce substantial lasting behavioral deficits at comparable time points. ⁶ Additionally, females and naïve animals exhibited greater total exploration time during NOL and NOR testing. While discrimination indices are calculated as a ratio of novel-to-total exploration and are therefore partially normalized for overall activity, reduced exploration in TBI animals and males may have limited opportunities to encode object information, potentially contributing to lower indices independent of memory per se. In our study, memory was tested 2 weeks after TBI. While it is possible that animals presented a certain degree of recovery, we were interested in studying more chronic behavioral changes. In the BM, animals were also relatively undertrained (i.e., two sessions/day over 2 days). This was chosen based on our previous report in which males tested 4 days after TBI had clear memory deficits compared to naïve controls. ⁶ It is possible that behavioral deficits may be more striking in fully trained animals. However, as this study was designed to address sex differences, the number of training sessions was unlikely to alter our conclusions.

Conclusions

Our study showed that male rats exposed to TBI were more vulnerable to deficits in learning-dependent spatial navigation, whereas females showed greater impairment in novelty-driven recognition memory. In addition, no influence of the estrous cycle was found on behavioral results. Future studies should aim to disentangle learning-related versus memory-specific impairments, use expanded cognitive batteries, and investigate potential mechanisms and substrates underlying sex-specific behavioral patterns.

Authors’ Contributions

I.K.R., T.K.R., T.H.A.S., and E.M. conducted behavioral and neurochemical experiments. T.K.R., I.K.R., and C.H. analyzed the results. I.K.R. and C.H. wrote the article. S.M.N., M.G., and N.L. critically revised the article. All authors approved the final version.

Data Sharing

Researchers with a sound proposal can request access to this data by contacting the authors, which reserve the right to share it at their discretion.

Supplemental Material