Loren J Martin, Department of Psychology, University of Toronto, Canada
Robert E Sorge, Department of Psychology, University of Alabama at Birmingham, Birmingham, USA
It is widely recognised that there are sex differences in the prevalence of chronic pain conditions with females greatly outnumbering males in diagnosis and treatment. This justifies the mandatory inclusion of females in clinical trials from 1993. However, preclinical researchers have been neglectful and reluctant to include both sexes in their experimental design and analyses – despite glaring disparities in the respective clinical conditions. To that end, the bulk of preclinical investigations, across multiple disciplines, including pain, have primarily utilised male rodents. The reliance on male rodents may have spawned from inertia or incorrect assumptions regarding variability with respect to ovarian hormones. In any case, key differences between males and females have been observed forming a basis for equal inclusion among the sexes.
Biologically based sex differences
According to the biopsychosocial model, biological, psychological and social variables interact to alter an individual’s health and well-being. With a phenomenon such as pain, the belief is often that injury to the body is reflected in a clear biological response and differences in the pain experience are underpinned by a sex-specific biological mechanism. Thus, animal models are employed to explore biologically based mechanisms, potentially simplifying the investigations. In rodents, female animals show more hypersensitivity after injury (Nicotra et al., 2014) and more nociceptive behaviours across a host of inflammatory tests (Nasir et al., 2016), suggesting that they experience more pain in a manner similar to human females. Additionally, primary afferent neurons from female rats were twice as likely to respond to mechanical stimulation than those from males (Ross et al., 2018), suggesting fundamental differences in the pain-sensing system between males and females.
The immune system plays a key role in the mediation of chronic pain through direct interactions with neurons via inflammatory signals. While this integral role has been recognised for some time, we were the first to demonstrate that microglia, a type of neuro-immune cell, is critical for chronic pain hypersensitivity in male mice, but not females. In short, we demonstrated that inhibitors of microglial activation (or their receptors) could alleviate hypersensitivity in male mice when delivered into the spinal cord, but these treatments did not work in females. In contrast, females appear to utilize T cells. Consequently, removal of T cells or administration of testosterone shifts the balance to the “male” system (Sorge et al., 2015). This work has been replicated with rats and other injury models to date (Mapplebeck et al., 2018; Taves et al., 2015). To support this work, there are clear sex differences in immune cell responses (Wegner et al., 2015) and proportion of immune cells (Abdullah et al., 2012) in humans that resemble those seen in animal models. Interestingly, the sex differences in T cell populations seen in rodents and humans appear to be conserved across a wide range of species from fruit flies and sea urchins, to kestrels and macaques (Klein and Flanagan, 2016). There are clear biologically-based differences in the mechanism of pain sensation that is evident in animal models. However, the experience of pain is rarely simply biological.
Sex differences in learning mechanisms
Typically, the ability to predict the likelihood of pain or other unpleasant events by learning from prior experience is an important adaptive behaviour in healthy organisms (McNally and Westbrook, 2006) and can cause disabling fear and avoidance in patients with persistent pain states (Fritz et al., 2001). Pain caused either by an injury or illness is suspected to leave a memory trace within brain and spinal structures (Ji et al., 2003). In most cases this pain memory is implicit and not subject to conscious awareness, but may lead to behavioural and perceptual changes such as hypersensitivity. We have recently highlighted the importance of sex differences for learned pain responses by demonstrating that male mice and men exhibit more pain when returned to a location previously associated with pain, but females do not (Martin et al., 2019). We serendipitously uncovered these responses by routinely testing males and females for pain sensitivity and stratifying our statistical analyses by sex. Testosterone was required for male-specific “pain memory” in mice, and testosterone administration to female mice evoked pain memory. Interestingly, an injection of zeta inhibitory peptide (ZIP), an inhibitor of atypical protein kinase C (aPKC), which has been shown to prevent long-lasting plasticity also prevented conditioned pain hypersensitivity in male mice but had no effect on female responses (Martin et al., 2019). The ZIP findings are in line with sexual dimorphisms in aPKC action in mouse models of chronic pain (Nasir et al., 2016) and open a wider debate on sex-specific plasticity mechanisms. In addition, since we developed parallel mouse and human procedures for testing the same phenomena, we assessed physiological similarities between the two species and corroborated that males – of both species – were stressed by the expectation of impending pain.
These findings were not expected and are even more intriguing when you consider that learned pain sensitivity was found only in males – of both species – but that learning mechanisms may contribute to pain chronicity (Apkarian, 2008) and chronic pain conditions are more prevalent in females (Berkley, 1997). There is also precedent to support the hypothesis that male and female subjects may use different learning strategies or incorporate the most salient features of stimuli to alter the expression of behaviour. For instance, in the fear conditioning literature, female rats express fear responses differently than male rats by engaging in darting and escape responses, whereas male rats display much higher levels of freezing behavior (Gruene et al., 2015). Thus, an important consideration in sex-based comparisons is to include the observation of relevant behaviours during experimentation, such as facial expressions, licking and escape behaviour.
Sex differences in psychological variables affecting pain
There appear to be consistent and translatable sex differences in pain between males and females when examining biological underpinnings or innate learning mechanisms, but it is likely that there are human-specific psychological variables that play a significant role. Pain is a subjective experience and many non-drug strategies are employed to reduce pain. In humans, females are more likely to endorse rumination (Meints et al., 2017) and rely on social support (Rovner et al., 2017) than males. Perhaps these coping strategies are the result of female patients reporting greater levels of pain dismissal (Igler et al., 2017) and perceived psychologically based pain (Miller et al., 2018) than males. Unfortunately, despite repeated demonstrations that females are more sensitive to painful stimuli (Bartley and Fillingim, 2013), female pain is often discounted at the clinical level.
It is unfortunate that many researchers use “sex” and “gender” interchangeably. Sex is based on chromosomal biology and the result of hormones during development, while gender identity is individually determined and based on socially constructed roles. In preclinical models, sex is the variable of interest; it is easily determined by outward appearance and there is no evidence that rodents have a gender that is different from their natal sex. In humans, this assumption is woefully incorrect and invalid. In one of the only studies of its kind, gender role expectation of pain accounts for more variability in human pain sensation than genetic sex (Robinson et al., 2001). Therefore, the strength of the belief in gender roles contributes more to an individual’s pain expression than their genetic sex, suggesting that psychological variables play a much larger role than are given credit. Along a similar line, it has been reported that brain connectivity for transgender individuals matches more closely to that of their identified gender (Kruijver et al., 2000), providing further support for an emphasis on psychological forces driving biological responses. However, it should be noted that a study of transgender adults with pain reported that transgender women reported pain following the start of hormone treatment, whereas transgender men were more likely to report pain reductions following hormone therapy (Aloisi et al., 2007). Together, these data demonstrate the complicated interaction of psychology and biology.
“Sex” differences are simple and complicated
According to the model outlined above, experiences are the result of a variety of influences. When comparing sexes at the biological level, there are identified differences in neuronal function, immune cell reactivity and brain responses that appear to be greatly conserved across species and related to hormonal levels. For preclinical scientists, these similarities provide hope for translation of treatments from the bench to the bedside. However, this path becomes tenuous when one considers that the human experience of pain is rarely a pure biological phenomenon. Humans are highly social and cognitive, adding a complex layer to the perception of pain that is difficult to model at the preclinical level. Sex differences should continue to be studied in animals to further our understanding of the complex interaction of hormones and various physiological responses. In contrast, gender differences should be the focus of clinical investigations to emphasise the critical role that psychological and social processes play in perception. The use of more inclusive language as part of demographic information will permit researchers to explore many more aspects of the human experience and allow for more appropriate treatment recommendations.
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