The hippocampus is a brain structure of the medial temporal lobe known primarily for its role in supporting learning and memory functions (Jin and Maren, 2015; Joshi et al., 2020; Knierim, 2015; Maren et al., 2013; Wixted and Squire, 2011). Work with rodents and human case studies with selective hippocampal damage (e.g., patient H.M.; Squire, 2009) has thoroughly documented the structure and function of the hippocampus (Bartsch and Wulff, 2015; Coburn, 2018; Knierim, 2015; Lupien and Lepage, 2001; Phillips and LeDoux, 2021; Preston-Ferrer and Burgalossi, 2018; Tatu and Vuillier, 2014; Witter et al., 2017). Comprised of several subfields with specialized cytoarchitecture, connectivity, and function including Cornu Ammonis (CA) 1–4, dentate gyrus (DG), presubiculum, subiculum, and parasubiculum, the hippocampus is integral for a myriad of mnemonic functions, such as the formation of fear memory traces (e.g., Coburn, 2018; El-Falougy and Benuska, 2006; Haukvik et al., 2018; Radonjic et al., 2014; Witter et al., 2017).

A substantial body of literature indicates the hippocampus is particularly vulnerable to stress from exposure to stress hormones produced by activity of the hypothalamic-pituitary-adrenal (HPA) axis (Bartsch and Wulff, 2015; Kim et al., 2015; Lupien and Lepage, 2001; McEwen et al., 2016; Miller and O’Callaghan, 2005; Ortiz and Conrad, 2018). Morphological, structural, and functional changes of the hippocampus have been reported in an array of psychological disorders, including posttraumatic stress disorder (PTSD; Besnard and Sahay, 2016; Coburn, 2018; Hayes et al., 2017; Lazarov et al., 2017; Logue et al., 2018; Malivoire et al., 2018; Rangaprakash et al., 2017; Tural et al., 2018; van Rooij et al., 2018). PTSD, which may develop as a consequence of experiencing a trauma, is a debilitating psychiatric disorder (Fenster et al., 2018; Mahan and Ressler, 2012). Symptoms include re-experiencing the traumatic event (e.g., flashbacks), avoidance of stimuli associated with the event, negative affect and cognition, and heightened arousal (American Psychiatric Association, 2013). Differences in hippocampus volume, as well as function, are theorized to underly memory issues frequently present in individuals with PTSD (Joshi et al., 2020; Liberzon and Sripada, 2007; McEwen et al., 2016; Shin, 2006).

A number of scholars have suggested hippocampal volume is a biomarker of risk for PTSD development (i.e., vulnerability factor; Gilbertson et al., 2002; Gurvits et al., 2006; Kremen et al., 2012; Wang et al., 2010; Xie et al., 2018) and/or asserted that changes in volume track with PTSD symptoms (i.e., are caused by the resulting psychological sequela; Apfel et al., 2011; Gurvits et al., 1996; Woon and Hedges, 2008). Although more sparse, additional work has demonstrated null findings, suggesting smaller hippocampus volume is neither a risk factor nor a consequence of PTSD (e.g., Bonne et al., 2001). Notably, the majority of this work has referenced the whole volume (Bonne et al., 2001; Chen et al., 2018).

Closer examination of hippocampal subfields may afford greater precision to the utility of the region as a biomarker of PTSD. In addition, each subfield may play a differential role in symptom development. Impaired extinction of fear memories and over-consolidation of fear, are two hallmarks of PTSD development which may result from specific subfield functional and/or structural abnormalities (Mahan and Ressler, 2012). Select studies, with both adolescents and adults, have segmented the hippocampus into its subfields and demonstrated that PTSD symptom severity is associated with smaller dentate gyrus (Hayes et al., 2017; Postel et al., 2019; Wang et al., 2010), CA1 (Chen et al., 2018), hippocampus-amygdala transition area (Ahmed-Leitao et al., 2016; Averill et al., 2017); , and parasubiculum (Ahmed-Leitao et al., 2016).

Specific neurocircuitry within the hippocampus, as described in animal models, would suggest particular behavioral effects (i.e., aberrant memory formation, consolidation, retrieval) emerge when different hippocampal subfields are perturbed (Phillips and LeDoux, 2021; Preston-Ferrer and Burgalossi, 2018). For example, decreased volume in the dentate gyrus, a region proposed to underlie pattern separation processes, may contribute to overgeneralization of fear, a common theoretical model of PTSD (Hayes et al., 2017). Together the dentate gyrus and CA3 also work together to encode and retrieve spatial information, while the CA1 is essential for a myriad of mnemonic tasks, including autobiographical memory (Bartsch et al., 2011). The parasubiculium is also linked to processing spatial information (Dalton and Maguire, 2017). Although future work is required, these findings collectively suggest PTSD is linked with decreased volume of hippocampal subfields responsible for holistic representations of scenes and offer a potential mechanism by which trauma impacts hippocampal activity and memory (Miller and Wiener, 2014).

The current evidence suggests that differences in hippocampal subfield volumes may reflect a predisposition to PTSD as well as correspond to post-trauma symptom trajectories. However, the current literature is lacking evidence as to whether hippocampal subfield volumes measured acutely post-trauma are prospectively related to PTSD symptoms. If subfield volumes are to be a useful biomarker for post-trauma individual risk and resilience, the measurement of the volumes must be valid – capture the individual differences associated with PTSD – and be reliably measured (Dhama et al., 2019; Lehrner and Yehuda, 2014; Mayeux, 2004). Therefore, reliable measurement of hippocampal structure and subfields is important for accurate monitoring of morphological and volumetric changes that accompany PTSD (Bartsch and Wulff, 2015; Burke and Barnes, 2010; Fröhner et al., 2019).

Although measurement of the whole hippocampus has proven reliable (Ahmed-Leitao et al., 2016; Brown et al., 2020; Hsu et al., 2002; Mulder et al., 2014; Schmidt et al., 2018), until recently, the small size of the subfields made assessing volumes challenging (Brown et al., 2020). Manual segmentation of the hippocampus and its subfields used to be the gold standard for segmentation despite the highly subjective process that depends heavily on the expertise of the evaluator (Dill et al., 2015; Yushkevich et al., 2015a,b). However, enhanced resolution of structural magnetic resonance imaging (MRI) technology and new segmentation programs have allowed for more quantitative approaches using atlases and probabilistic features of structural MRI data, making automated pipelines for hippocampal subfield segmentation a commonly used analytic tool (Dill et al., 2015). Although higher resolution images typically offer the most accurate segmentation (Wisse et al., 2016; Yushkevich et al., 2009), previous work has concluded automatic segmentation of hippocampal subfields in lower resolution images yields accurate measurements compared to manual edits (Yushkevich et al., 2015a,b).

FreeSurfer is perhaps the most widely used tool for automated tissue parcellation and cortical and subcortical segmentation (Fischl et al., 2002). Hippocampal subfield reliability processed through FreeSurfer has been evaluated across scanners (Marizzoni et al., 2015; Quattrini et al., 2020; Whelan et al., 2016) and across time on the scale of several months (Brown et al., 2020) to a year (Mulder et al., 2014). In the few studies that have assessed subfield reliability (Brown et al., 2020; Buser et al., 2020; Mulder et al., 2014), the majority appear to have moderate to good reliability, with the poorest reliability reported for the hippocampal fissure, which separates the dentate gyrus from the subiculum (Brown et al., 2020; Buser et al., 2020; Haładaj, 2020; Whelan et al., 2016). However, to our knowledge, day-to-day reliability, when a difference in hippocampal volume would be least expected, has not been evaluated. Moreover, reliability of hippocampal subfields has been predominately assessed in aging or healthy populations (Flores et al., 2015; Schmidt et al., 2018).

Herein, we examined the relationship between hippocampal subfield volumes and PTSD in a longitudinal study of psychological outcomes following a traumatic injury, using the probabilistic atlas-based procedure within FreeSurfer (version 6.0). As a secondary aim, we assessed the reliability of hippocampal subfield measurement. The participants in the study were scanned at three times, on two consecutive days approximately 2-weeks after their traumatic injuries (time 1: T1, and time 2: T2), and 6 months (time 3: T3) after their injury. This design allowed us to address four critical aims: 1) assess the reliability of hippocampal subfields on two consecutive days of scanning (T1 – T2), 2) determine whether hippocampal subfield measurements acutely post-trauma (T1) prospectively relate to future PTSD (T3), 3) examine the more routinely investigated cross-sectional association between subfield measurements at follow-up (T3) and current PTSD symptoms (T3), and 4) evaluate whether change in hippocampal volume (T1 – T3) relates to future PTSD symptoms (T3).

Based on the aforementioned research, we hypothesized that smaller global hippocampal volume (T1) would prospectively relate to T3 PTSD symptoms (Gilbertson et al., 2002; Gurvits et al., 2006; Kremen et al., 2012; Wang et al., 2010; Xie et al., 2018). We also hypothesized smaller global hippocampal volume (Apfel et al., 2011; Gurvits et al., 1996; Woon and Hedges, 2008), as well as smaller dentate gyrus/CA4 and CA1 (measured at T3) would be significantly related to T3 PTSD symptoms (Hayes et al., 2017). Finally, we anticipated there would be a significant change between T1 and T3 volumes, such that decreases in dentate gyrus and CA1 volume would track with PTSD symptoms (Chen et al., 2018; Hayes et al., 2017).

We assessed the relationship between subfields and the development of PTSD symptoms and the stability of hippocampal subfield volumes after trauma in a traumatically injured sample. Our longitudinal design, which consisted of two consecutive scans acutely post-trauma (T1 and T2) and one scan 6-months post-injury (T3), provided a unique opportunity to evaluate measurement reliability and utilize both the cross-sectional and longitudinal processing streams within FreeSurfer. We found the associations (although nonsignificant after correcting for multiple comparisons) between subfields and PTSD symptoms varied depending on whether the measurement was acquired acutely post-trauma (T1) or at follow-up (T3) and whether the analysis used the cross-sectional or longitudinal pipeline.

Reliability between Tl and T2 scans of hippocampal subfields ranged from good to excellent, with all ICC values over 0.83 (Koo and Li, 2016). Change in volume did not significantly relate to future PTSD symptoms, therefore, we were also interested in measurement differences between T1 – T3. Reliability between T1 and T3 (Supplemental Material) also ranged from good to excellent with ICC values over 0.86 for both FreeSurfer processing streams (Koo and Li, 2016). In both sets of reliability analyses (T1 – T2 and T1 – T3), we replicated previous work showing excellent reliability in the whole hippocampus and the molecular layer (Brown et al., 2020) with poorer reliability in the hippocampal fissure (Quattrini et al., 2020). Percent volume difference metrics revealed similar outcomes; the lowest percent difference between T1 and T2 was in the whole hippocampus and molecular layer whereas the hippocampal fissure, HATA, and parasubiculum had the largest differences. Using the longitudinal preprocessing pipeline (T1 – T3) revealed the smallest percent differences; subfields demonstrating highest consistency (PVD < 3%) included the bilateral hippocampal tail, subiculum, CA1, molecular layer, and whole hippocampal volume. For both processing streams, the bilateral fissure, parasubiculum, and HATA showed the least consistency (PVD > 5%).

These results replicate and further support the reliability of FreeSurfer hippocampal subfield segmentation as demonstrated in other studies comparing varying sample sizes, scanners, and time intervals between scans (Brown et al., 2020; Whelan et al., 2016). Moreover, our traumatically injured sample yields a unique measurement of hippocampal volumes post-trauma that would not otherwise be reported in a healthy sample. Thus, reliable measurement across both sets of time-points is important in disentangling volumetric differences in subfields attributed to trauma-related outcomes rather than measurement biases over time.

Decreased bilateral dentate gyrus/CA4 volume (T1) did not relate to greater PTSD symptom severity (T3). Though the dentate gyrus has been demonstrated to be associated with current PTSD symptoms (Hayes et al., 2017), our results suggest that, in this sample, the dentate gyrus is not prospectively related to, or associated with PTSD symptoms. The size of the dentate gyrus may not be predisposing of PTSD, rather it may be sensitive to the stress associated with PTSD in specific samples, particularly those that are comprised of highly symptomatic participants or individuals who experienced sustained trauma exposure (e.g., combat veterans; Zimmerman et al., 2016).

Chronic stress in the environment that individuals return to after trauma may impact hippocampal volumes (Haddad et al., 2015). The majority of neuroimaging work has been conducted with predominately White participants. Our sample is distinctly comprised of participants from diverse racial, ethnic, and socioeconomic backgrounds. As more data emerges on the neural impact of socioeconomic position (e.g., Johnson et al., 2016; Noble et al., 2012), racism and race-based stressors (Carter, 2007), and chronic exposure to environmental/societal stress (e.g., community violence, environmental toxins, etc.), we encourage future neuroscience research to consider how other forms of traumatic and stressful exposures (e.g., racism, sexism, poverty) may be impacting brain regions highly vulnerable to stress such as the hippocampus.

Previous work has demonstrated smaller whole hippocampus volume is associated with PTSD (e.g., Logue et al., 2018; Salminen et al., 2019; Xie et al., 2018). Surprisingly, we did not find a bivariate association between hippocampal volume and PTSD symptoms, nor was global hippocampal volume a significant term in the regression analysis. It is important to note that a number of studies have not demonstrated a relationship between whole hippocampal volume and PTSD (e.g., Bonne et al., 2001; Chen et al., 2018; see meta-analysis by Logue et al., 2018); perhaps indicating the association is not as robust as widely assumed and that trauma type and timing of measurement are important factors.

Our results, regardless of processing pipeline, do not clearly align with the framework describing differences in hippocampal subfields as either a vulnerability factor of PTSD development or as part of the subsequent post-trauma neurobiological changes. Rather, they suggest the two hypotheses may not be mutually exclusive. Our unique experimental design also stressed the importance of considering timing of structural measurements. The lack of consensus between our results and the majority of previously published findings (c.f. Bonne et al., 2001) is less surprising given a large recent study found that major depressive disorder, a common co-morbid diagnoses with PTSD, was a better predictor of hippocampal subfields than PTSD (Salminen et al., 2019). Future research should attempt to disentangle the effects of PTSD and depression on hippocampal structure and should extend research efforts across various post-trauma timepoints.

The current study demonstrated excellent reliability of FreeSurfer 6.0 hippocampal subfield segmentation, on scans acquired on two consecutive days and six months apart, within a large trauma-exposed sample. Findings replicate and extend previous work examining FreeSurfer reliability by using a larger sample and time points not previously examined. Reliability of automated hippocampal subfield segmentations is crucial to research examining diseases and disorders affecting the hippocampus. Though ongoing validation is necessary, the current results contribute to the promise of robust methodology within FreeSurfer in examining brain-related changes associated with trauma exposure.

Although in our sample the hippocampal subfields volumes did not prospectively relate to or track with PTSD symptoms, future work should still consider how the function and structure of the distinct subfields may underlie pathogenesis of PTSD symptoms. Elucidating the role of hippocampal subfields in PTSD may lead to more effective treatments of specific symptoms (e.g., impaired extinction and over-consolidation of fear).