**1. Introduction**

Acute pain is inevitable and important following injury as it protects the individual against further tissue damage. However, pain that persists after tissue injury has healed, so-called "chronic pain," is not protective and has a significant, negative effect on the quality of life [1]. Chronic pain is defined as pain persisting after surgery or trauma for greater than three months [2]. Previous studies from our group and others have found that the incidence of chronic pain in traumatically injured patients can be as high as 70% and there is a strong correlation between pain severity and life interference [3–5]. Unlike acute pain, chronic pain is not primarily related to tissue injury [6] and traditional therapies for pain, including opioids, have poor efficacy in their treatment [7,8]. Not all injured individuals develop chronic pain, and few reliable or clinically significant biomarkers have been identified that predict the progression of acute to chronic pain in the traumatically injured population. Thus, this research could contribute to an improvement in our ability to predict who will develop chronic pain through the validation of biomarkers. A secondary long-term goal of this research project is to better understand the biological factors that contribute to the development of chronic pain, which could improve our ability to treat or prevent this transition.

**Citation:** Trevino, C.M.; Hillard, C.J.; Szabo, A.; deRoon-Cassini, T.A. Serum Concentrations of the Endocannabinoid, 2-Arachidonoylglycerol, in the Peri-Trauma Period Are Positively Associated with Chronic Pain Months Later. *Biomedicines* **2022**, *10*, 1599. https://doi.org/10.3390/ biomedicines10071599

Academic Editor: Wesley M. Raup-Konsavage

Received: 20 May 2022 Accepted: 30 June 2022 Published: 5 July 2022

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Preclinical research suggests a role for the endocannabinoid signaling system (ECSS) in pain [9,10]. The endogenous ligands for cannabinoid (CB) receptors, called endocannabinoids (eCBs), are *N*-arachidonoylethanolamine (AEA or anandamide) and 2-arachidonoylglycerol (2-AG). Noxious stimuli and tissue injury increase eCB mobilization [11,12] and chronic pain can up-regulate the expression of CB1 [13] and CB2 [14] receptors. Although preclinical studies largely support the hypothesis that endogenous activation of CB receptors reduces acute pain [9,10], eCBs produced by intense nociceptive stimuli render nociceptive neurons in the spinal cord excitable by non-painful stimuli, suggesting that eCB/CB signaling can also promote pain sensitization and thus contribute to the development of chronic pain [15].

Multiple studies have examined the relationships between the concentrations of circulating eCBs and related lipids with the presence of chronic pain in humans. Circulating concentrations of 2-AG are higher compared to pain-free control groups in several types of chronic pain, including fibromyalgia, irritable bowel syndrome, and neuropathic pain [16–18]. Similarly, circulating AEA concentrations in individuals with fibromyalgia are three times higher than in matched controls [19]. Patients with complex regional pain syndrome (CRPS), a neuropathic pain syndrome precipitated by extremity injury, have significantly higher plasma concentrations of AEA than controls [20]. Women with endometriosis-associated pain, compared to those without endometriosis, exhibited elevated concentrations of both AEA and 2-AG [21]. Thus, while preclinical data indicate that eCB-mediated signaling reduces pain sensation at multiple sites within the neuronal pain circuit, the peripheral pool of eCBs is positively associated with chronic pain in humans. However, the presence of an association is not indicative of a causal relationship; it is possible that the high circulating eCBs are a physiological response to the stress of the pain experience.

Human studies also support the role of cortisol in developing chronic pain [22–24]. The imposition of acute pain (such as the cold pressor test) in healthy individuals elicits cortisol secretion, which is in accord with pain as a stressor [25]. On the other hand, inappropriate activation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with the presence or development of chronic pain [26,27]. In particular, inappropriately low concentrations of cortisol have been associated with a diagnosis of chronic pain from fibromyalgia and low back pain [28,29].

Both the HPA axis [30] and the ECSS [31] are activated by exposure to physical and psychological stress, and these systems have diverging and converging effects on the stress response. HPA axis activation via actions of cortisol and the ECSS reduce pain and inflammation. In the brain, there are considerable data that cortisol increases the 2-AG synthesis and that CB1 receptor activation links brain cortisol to changes in synaptic activity. On the other hand, ECSS activation in the brain reduces HPA axis activation by stress and enhances recovery to baseline following stress. Importantly, both the ECSS and HPA axis are downregulated in situations of chronic stimulation, so excessive activation of either system can lead to loss of critical homeostatic processes.

This study aimed to develop a model of the relationships between circulating cortisol and eCBs, and the development of chronic pain after a traumatic injury. Our working hypothesis is that both the HPA axis and the ECSS are mobilized by the severe stress that accompanies traumatic injury and both contribute to reduced pain at that time point. However, we hypothesize that excessive concentrations of both at the time of injury will increase the risk for chronic pain development. The specific hypotheses for this study were: (1) circulating concentrations of the eCBs positively correlate with circulating cortisol concentrations at the time of injury, both reflecting the physical and psychological stress of the injury; (2) at the time of injury, pain measures negatively correlate with circulating eCB and cortisol concentrations due to the ability of both mediators to reduce pain; and (3) circulating eCBs concentrations at the time of hospitalization positively correlate with measures of pain months after the injury, possibly due to down-regulation of the ECSS.

#### **2. Materials and Methods**
