Post-Traumatic Stress Disorder (PTSD) develops in a subset of people who experience a traumatic event with or without physical trauma. As a result of the trauma, patients with PTSD develop symptoms such as nightmares, intrusive memories, flashbacks, and disruptions in activities of daily living which persist for more than 30 days after trauma exposure (American Psychiatric Association, 2013). The presence of intrusive memories and flashbacks suggests that PTSD is, in part, a memory disorder and substantiates the view that it is the most severe of fear disorders (Sher and Vilens, 2010; Izquierdo et al., 2016). They also have a higher likelihood of developing cardiovascular disease (Edmondson and von Känel, 2017) and are at a significant risk of disability and suicide (Mitchell et al., 2021).

Since trauma is unpredictable and PTSD is difficult to treat (Sessa, 2017), it is medically important to identify susceptibility factors in order to improve therapeutic treatment regimens that can confer resilience against developing PTSD. We define susceptibility factors as conditions that exist before trauma and that increase the probability of developing and maintaining PTSD after trauma and which can be altered to change PTSD outcomes (Alexander et al., 2020). We have previously shown that animals susceptible to developing a PTSD-like phenotype have impaired hippocampal function prior to experiencing trauma (Nalloor et al., 2014) and have difficulty in learning safety (impaired extinction) after trauma (Nalloor et al., 2011) similar to what is seen in humans (Jovanovic et al., 2010, 2012). In addition, PTSD patients have pre-frontal cortical (PFC) dysfunction marked by dysregulation of emotion, inappropriate recall of fear memories, impaired learning of safety, and flashbacks of the traumatic experience (Gamwell et al., 2015; Hourani et al., 2015; Izquierdo et al., 2016; Gobin et al., 2017).

It is well documented that patients with established PTSD also have increased levels of circulating pro-inflammatory cytokines, such as TNFα, IL-1β, IFN-γ, and IL-6 (Passos et al., 2017; Speer et al., 2018; Wang et al., 2019). Inflammation may be involved in the maintenance of PTSD pathology as inflammatory markers between people with PTSD experiencing symptom remission and no-PTSD controls are similar (O’Donovan et al., 2017). Additionally, some studies report a chronic inflammation in people with PTSD (Hori and Kim, 2019), as evidenced by high levels of the anti-inflammatory cytokine IL-10 (a negative feedback mechanism toward an ongoing inflammation) and of the pro-inflammatory cytokine IL-6 (de Oliveira et al., 2018). However, in contrast to these studies, others have reported decreased circulating levels of pro-inflammatory cytokines and chemokines in people with PTSD, including IL-6 (Agorastos et al., 2019) and CXCL1 (Bam et al., 2016), respectively. Moreover, a peri-trauma study found that lower levels of peripheral TNF and IFN-γ were associated with a higher risk of persistent PTSD (Lalonde et al., 2021), suggesting that higher levels of these pro-inflammatory cytokines during trauma can be protective. It would be ideal, if the inflammatory state of the central nervous system (neuroinflammation) in PTSD patients could be inferred from the measurement of peripheral blood markers. However, despite of the fact that the majority of findings from recent studies using peripheral markers point toward a higher proinflammatory state in people with PTSD, a study using post-mortem brains of people with PTSD intriguingly found lower levels of IL-1α gene expression in the prefrontal cortex (PFC) compared to controls (Morrison et al., 2019). As such, in the absence of peripheral and central markers measured in the same subjects it is possible that peripheral markers may not be adequate to infer neuroinflammation in PTSD and rather local markers should be assessed. Taken together, these opposing findings from the different studies reported above document that inflammation in people with PTSD is highly complex. This stresses the need for more research investigating the role of systemic (peripheral) and local (neuro) inflammation in the pathogenesis and maintenance of PTSD.

Furthermore, it is not clear whether peripheral inflammation or neuroinflammation represent susceptibility factors. Few studies have examined inflammation prior to trauma exposure. A study in humans found that high levels of circulating C-reactive protein (CRP) prior to military deployment were associated with developing PTSD after combat exposure (Eraly et al., 2014) suggesting that inflammation is a potential susceptibility factor. Preclinical studies of inflammation and stress also support the idea that inflammation plays a role in susceptibility. Notably, it has been shown that early life inflammation induced by lipopolysaccharide (LPS) in rats is associated with impaired fear extinction at 2 months of age (Doenni et al., 2017). In mice, social defeat stress was associated with increased serum levels of TNFα and IL-6 (Azzinnari et al., 2014). However, the hypothesis that inflammation is a susceptibility factor has not been experimentally tested in the context of PTSD.

One available tool for examining susceptibility factors is the Revealing Individual Susceptibility to PTSD-like phenotype (RISP) model. The RISP model classifies male rats as susceptible (Sus), resilient (Res), or intermediate using behavioral tests of anxiety and startle 4 days after exposure to a mild stressor but before exposure to a traumatic experience (Nalloor et al., 2011; Alexander et al., 2020). The mild stressor in the RISP model is a necessary step for revealing susceptibility. Sus animals (∼20%) have high anxiety and startle responses and Res animals (∼30%) have low anxiety and startle responses. Res and Sus classifications correlate with a post-trauma PTSD-like phenotype such that Sus animals have impaired fear extinction and maintain or increase their startle response for at least 3 weeks after trauma (Nalloor et al., 2011; Alexander et al., 2020). In addition to behavioral differences between Sus and Res before trauma, Sus animals have impaired hippocampal function as measured by plasticity related immediate early genes Arc and Homer1a compared to Res (Nalloor et al., 2014). Thus, classification with the RISP model is predictive of post-trauma phenotype yet functional and phenotypical differences exist and can be identified before trauma.

To determine whether systemic or neuroinflammation represent susceptibility factors to developing PTSD, we measured pre-trauma levels of the pro-inflammatory cytokines IL-6, IL-1β, TNFα, and IFN-γ, the anti-inflammatory cytokine IL-10, and the pro-inflammatory neutrophil-attractant chemokine KC/GRO/CINC-1 in the serum and pre-frontal cortex of rats classified as Sus or Res according to the RISP model. We tested the hypothesis that Sus rats would have higher pro-inflammatory cytokines compared to Res. We predicted that there would be an elevation in at least one of the pro-inflammatory cytokines in either serum and/or PFC, no change or decrease in IL-10, and no change in KC/GRO/CINC-1 as we do not expect there to be involvement of the adaptive immune response.

It is well-documented that PTSD is associated with inflammation, however, there is limited information on the role of inflammation in susceptibility to developing PTSD. Because PTSD is difficult to treat, it is important to understand what factors contribute to susceptibility to tailor interventions that can confer resilience. Here we examined cytokine and chemokine levels in animals classified as Susceptible (Sus) and Resilient (Res) using ASR, EPM, and the RISP model before trauma in serum and prefrontal cortex (PFC).

We used three different classification methods in male Sprague-Dawley rats because it is an outbred strain with a broad genetic background suitable for revealing individual differences in a PTSD-like phenotype. Although we present the data from all three classifications, we want to stress the data from the RISP model which was designed to incorporate multiple aspects of PTSD that can be modeled in rodents (anxiety, startle, impaired learning of safety) and classifies rats before trauma for susceptibility to a post-trauma PTSD-like phenotype. Thus, it models best the complex characteristics of PTSD. ASR or EPM classifications alone show singular aspects of this phenotype- Sus-ASR rats show only elevated long-lasting startle, while Sus-EPM rats show impaired extinction but not long-lasting elevation in startle compared to the Res-ASR and Res-EPM rats, respectively after trauma exposure (Nalloor et al., 2011; Alexander et al., 2020). Additionally, we have previously shown that animals identified as Sus with the RISP model have altered hippocampal function before trauma (Nalloor et al., 2014).

Our data demonstrate that Sus rats classified with the RISP model have significantly higher levels of IL-6 (Figure 2B) and nearly significantly higher levels of IL-1β in PFC (Figure 2A), but not serum, before trauma compared to Res rats which supports the hypothesis that neuroinflammation, rather than peripheral inflammation, is a susceptibility factor. We also found a tendency, albeit not significant, of higher serum levels of IL-1β, IL-6, and TNFα in Res rats compared to Sus before trauma (Figures 2G–I). This may be residual inflammation from the mild stressor exposure (cat hair) and/or ASR and EPM tests that are required in the RISP model for classification (Nalloor et al., 2011). Because the cat hair exposure is required to reveal susceptibility, in its absence virtually all animals behave as resilient, it is likely that the mild stressor (cat hair) is affecting Res rats with peripheral inflammation (Figure 2H), whereas Sus rats develop neuroinflammation (Figure 2B). Combined, the current findings suggest that the behavioral differences that emerge after cat hair exposure in the RISP classification are influenced by inflammatory status (Nalloor et al., 2011; Alexander et al., 2020). The results demonstrate that the role and location of inflammation in susceptibility to PTSD-like conditions can be different between susceptible and resilient animals.

Based on the RISP model, our data indicate that serum cytokine levels may not be sufficient for determining susceptibility to PTSD, as we found no correlation between serum and PFC cytokine levels in Res or Sus rats (Table 1). Others have also found inconsistent levels of cytokines and chemokines in brain and blood in rats (Zahr et al., 2010; Abramova et al., 2013). Because the blood-brain barrier (BBB) modulates cytokine transport in and out of the brain (Banks et al., 1995; Chen and Reichlin, 1999; Yarlagadda et al., 2009) it is not surprising to find that brain and blood levels of these markers are different or not correlated in young rats with presumably intact BBB. It is known that cytokines affect BBB integrity over time (Saija et al., 1995; Rochfort and Cummins, 2015) and therefore our findings do not contradict that people with established PTSD have, in most studies, higher levels of peripheral pro-inflammatory cytokines which may correlate with neuroinflammation. Coupled with the finding of significantly higher IL-6 in PFC of susceptible rats prior to trauma, this suggests that the role of inflammation in PTSD susceptibility is neurogenic in nature. This is an important consideration because it points to the potential difficulty in assessing susceptibility using peripheral markers in heathy individuals which is currently one of the least invasive and most widely used methods for acquiring biomarkers in humans.

Startle and anxiety are symptoms of PTSD (clusters D&E) (American Psychiatric Association, 2013) and in the RISP model they are collectively predictive of PTSD-like phenotype after trauma. However, there is merit in examining these symptoms individually as well. Anxiety disorders are prevalent and share a high comorbidity with PTSD (Spinhoven et al., 2014). High startle response is a symptom of anxiety, PTSD, and hypersensitivity disorders (Butler et al., 1990; Morgan et al., 1995; Grillon, 2002; Lucker, 2013). Examining ASR and EPM classification criteria individually also yielded interesting findings about PTSD-associated behaviors and their relationship with inflammation. In animals classified based on ASR alone, which only predicts long-lasting elevation in ASR in Sus-ASR rats, we found a significant increase in serum IL-6 in Res-ASR rats (Figure 3H) suggesting that higher levels of circulating IL-6 may be protective against high startle responses and may be worth investigating as a potential marker to track improvement in startle related disorders. Using EPM criteria alone showed that higher levels of circulating IL-1β are associated with lower anxiety-like behavior (Figure 4G). Combined, peripheral inflammation appears to be associated with resilience to a PTSD-like phenotype. EPM classification reveals that higher anxiety-like behavior is associated with chronic neuroinflammation as Sus-EPM rats have high levels of both IL-6 and IL-10 in PFC (Figures 4B, D). Using mice, others have found increased peripheral cytokine levels that were associated with high anxiety-like behavior after restraint stress (Elkhatib et al., 2020) and blocking IL-6 was associated with resilience to social defeat stress (Hodes et al., 2014) which highlights the importance of considering animal model, stressor type and when cytokines are measured in translational extrapolation.

Although the work presented here uses a model that can be used to study a post-trauma phenotype, it can also be used to study pre-trauma differences. However, both cannot be done in the same animals due to the nature of stress. Previous attempts to examine blood before and after trauma in the same animals revealed that blood draws are stressful and impact subsequent behavior skewing it toward Sus phenotype (0% classified as Res, unpublished observations).

The presented data reveal that the role of inflammation in susceptibility to a PTSD-like phenotype is complex. While peripheral inflammation may be protective, neuroinflammation in a brain region implicated in PTSD (PFC) is associated with susceptibility. These findings suggest that pre-trauma anti-inflammatory interventions that decrease neuroinflammation may reduce the likelihood of developing PTSD. Whole body interventions such as exercise and anti-inflammatory diet might be a better approach to reduce susceptibility than targeting individual cytokines or pathways as more information about the pleiotropic effects of individual cytokines emerges (Mocellin et al., 2003; Kishimoto, 2006; Gough and Myles, 2020). Such interventions have been shown to reduce inflammation (Tolkien et al., 2019; Metsios et al., 2020) and have effects on the brain (increased BDNF levels) (Firth et al., 2018).

Regardless, it is critical to apply interventions such as these prior to experiencing trauma or soon after because once the debilitating effects of PTSD occur it is tremendously more difficult to implement. Ongoing studies are evaluating anti-inflammatory interventions in the RISP model to determine how effective they might be in conferring resilience. Additionally, we are currently testing if increasing inflammation will worsen susceptibility. The RISP model was initially developed in male rats and studies in our lab attempting to apply the model to female rats revealed that is it not predictive for females. Female rats display different anxiety-like behavior and startle is not predictive of their post-trauma phenotype (Shanazz et al., 2022). Therefore, a new model for predicting a PTSD-like phenotype in females needs to be developed before this question can be answered in females with tools that account for sexual dimorphism in anxiety and fear responses (Shanazz et al., 2022).

The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

This animal study was reviewed and approved by the Charlie Norwood VA Medical Center IACUC.

KS wrote the manuscript, collected the data, analyzed the data, and helped with experimental design. RN helped with writing, analysis, and experimental design. RL helped with experimental design and editing the manuscript. AV helped with writing manuscript, analyzed the data, designed the experiment, and provided funding. All authors contributed to the article and approved the submitted version.