Pain is defined by the International Association for the Study of Pain (IASP) as an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage [1]. From a biological perspective, pain is an essential protective mechanism against noxious stimuli, as it helps prevent further injury and promotes tissue repair processes [2]. Pathological pain results from cellular, molecular, and structural modifications of the networks that modulate pain perception and response, altering their function under different conditions. Consequently, pain loses its adaptive-protective character, becoming chronic, debilitating, and sometimes disabling [3][4]. Chronic pain affects approximately 20% of the world’s population, according to the U.S. Centers for Disease Control and Prevention [5]-[7]. In México, the number of people suffering from chronic pain is unknown. However, in 2010, it was estimated that approximately 28 million Mexicans suffered from this type of pain, out of a population of 112 million at that time [8]. Pain perception begins with the detection of noxious stimuli by nociceptors [9][10]. These neurons possess a high activation threshold for distinguishing between innocuous and noxious stimuli [9][11] and express specialized receptors and ion channels for various stimuli [12][13]. Nociceptors are classified into three fiber types according to their characteristics: C fibers, which are slow and unmyelinated; Aδ fibers, which are faster and lightly myelinated; and a subgroup of Aβ fibers, which conduct noxious stimuli at high speed [14]-[16]. Nociceptors transduce stimuli into action potentials that propagate to the dorsal horn of the spinal cord [12][17]. The arrival of the action potential at the central terminal then induces the release of neuromodulators such as glutamate and substance P [12]. In this way, nociceptive information ascends to the thalamus and brainstem via specific tracts [18][19]. These projections integrate nociceptive activity with homeostatic processes [19]. Finally, third-order neurons project to various brain areas to generate the sensory, emotional, and cognitive components of pain [20].

Pain can be classified according to its duration, function, and neurobiological mechanism. Based on its duration, acute pain has a limited course, generally less than three months, and tends to resolve once the underlying cause is treated. In contrast, chronic pain persists for extended periods, exceeding three months, and can continue even after the initial damage has been resolved, often requiring continuous and multidisciplinary therapeutic management [2]. Based on its function, pain is classified as adaptive and maladaptive. Adaptive pain is defined as a physiological and protective response of the nervous system to a real or potential noxious stimulus, whose purpose is to preserve bodily integrity and promote tissue recovery. It warns of damage, promotes avoidance behaviors, and facilitates reparative processes. It originates from the transient activation of peripheral nociceptors and the transmission of the pain impulse to the central nervous system via Aδ and C fibers, without altering pain modulation mechanisms. Its duration is limited and proportional to the magnitude of the injury or inflammation [21]. Maladaptive pain is defined as pain that loses its original protective function and persists in the absence of an active noxious or inflammatory stimulus, becoming a pathological condition. It is associated with phenomena of peripheral and central sensitization, abnormal synaptic plasticity, persistent microglial activation, and dysfunction of descending inhibitory systems. As a result, non-noxious or low-intensity stimuli can produce allodynia or hyperalgesia, generating a disproportionate pain perception independent of the initial tissue injury [22].

According to itr neurobiological mechanism, it is classified as: nociceptive pain, inflammatory pain and neuropatic pain.

Chronic pain involves mechanisms that include central sensitization, peripheral sensitization, and GABAergic disinhibition. The IASP defines central sensitization as an increased responsiveness of nociceptive neurons in the central nervous system to their normal or subthreshold afferent input [33]-[35]. The main causes of central neuropathic pain include spinal cord injuries, Multiple Sclerosis (MS), chemotherapy [36], traumatic brain injuries, and strokes [37]. Central sensitization involves an alteration in the synaptic plasticity of neurons in the dorsal horn of the spinal cord and supraspinal structures such as the thalamus and somatosensory cortex. This process is characterized by sustained neuronal hyperexcitability caused by the excessive release of glutamate and the activation of NMDA, AMPA, and kainate receptors, which promote calcium influx into neurons and trigger intracellular signaling cascades dependent on Protein Kinase C (PKC) and MAPK/ERK [38]. These pathways activate the phosphorylation of ion channels and modify gene expression, promoting synaptic potentiation and the persistence of the nociceptive signal. Furthermore, the activation of microglia and astrocytes releases proinflammatory mediators such as IL-1β, TNF-α, and BDNF, which reduce GABAergic inhibition and exacerbate hypersensitivity [39]. As a result, clinical phenomena such as allodynia, secondary hyperalgesia, and persistent pain in the absence of peripheral stimuli occur. Peripheral sensitization refers to the increased responsiveness and reduced threshold of nociceptive neurons in the periphery to stimulation of their receptive fields [35][40][41]. Some causes of peripheral neuropathic pain include diabetic neuropathy [42], postherpetic neuralgia [43], trigeminal neuralgia [44], phantom limb pain [45], and spinal nerve compression neuropathy [46]. Mediators such as prostaglandins, bradykinin, histamine, serotonin, ATP, Interleukins (IL-1β, IL-6), and Tumor Necrosis Factor alpha (TNF-α) induce a decrease in the activation threshold of nociceptors and an increase in discharge frequency, promoting peripheral sensitization [47].

This process results from the release of inflammatory and neurogenic mediators such as prostaglandins, bradykinin, histamine, serotonin, ATP, Interleukins (IL-1β, IL-6), and TUMOR necrosis Factor alpha (TNF-α), which interact with G protein-coupled receptors (EP, B2, 5-HT, P2Y) and ion channels such as TRPV1, TRPA1, ASICs, and P2X3. Its activation causes phosphorylation and sensitization of sodium (Nav1.7, Nav1.8) and calcium (Cav2.2) channels, generating spontaneous ectopic discharges and an increased firing rate in C and Aδ fibers [9][47]. These increased afferent signals reinforce central excitability, creating a feedback loop that perpetuates pain.

GABAergic disinhibition is another crucial mechanism in the pathophysiology of chronic pain. Under physiological conditions, inhibitory interneurons that release GABA and glycine regulate nociceptive transmission in the dorsal horn of the spinal cord, preventing excessive propagation of the pain signal. However, in chronic pain, there is a functional loss of these interneurons or a decrease in GABAergic synaptic efficacy, caused by reduced GABA synthesis, alteration of the chloride ion gradient, or internalization of GABA_A receptors [48][49]. Increased Brain-Derived Neurotrophic Factor (BDNF) released by microglia alters the expression of the KCC2 chloride cotransporter, reversing the anion gradient and transforming the inhibitory action of GABA into an excitatory response [50]. This phenomenon amplifies central sensitization and contributes to the maintenance of persistent pain.

Research in animal models is fundamental to understanding the neurobiological basis of pain, from its sensory processing to the sexual dimorphism that influences its regulation [51][52]. Pain models are classified according to their underlying pathophysiological mechanism, the affected tissue, or the temporal course of the pain [53].

Nociceptive Pain: Animal models of nociceptive pain are primarily based on spinal reflexes, and the behavioral responses used for their assessment include tail flicking, paw withdrawal or elevation, trembling, licking, nipping, cowering, or decreased weight-bearing [54]. On the other hand, nociceptive pain can also be assessed by measuring the frequency of withdrawal of the injected limb, vertical activity (rearing), or horizontal locomotion, indicators of the degree of discomfort or hypersensitivity [53].

Inflammatory pain: Most models of inflammatory pain are based on the induction of an immune response or the administration of inflammatory mediators. Among the irritant chemical agents used to reproduce this type of pain in rodents are carrageenan, capsaicin, and Freund’s complete adjuvant (CFA), which induce local or systemic inflammation [55].

Neuropathic pain: Nociplastic pain arises from altered nociception despite no clear evidence of actual or potential tissue damage causing peripheral nociceptor activation or evidence of disease or injury to the somatosensory system causing pain [56]. One of the most widely used models for its study is Spinal Nerve Ligation (SNL), which consists of ligating the L5 or L6 roots of the spinal nerve [57]. The contralateral, uninjured leg serves as a control, and the animals are evaluated using mechanical hyperalgesia and allodynia tests, analyzing behavioral changes in response to tactile, thermal, or cold stimuli applied to the affected limb [53]. To determine the mechanical threshold of response, von Frey filaments are used, applied with progressively increasing force until the withdrawal reflex is elicited. These models reproduce with high accuracy the neuropathic abnormalities detected in human neuropathic pain, making them widely used in the exploration of new therapeutic strategies [58].

Nociplastic pain: Due to its functional nature, nociplastic pain presents an experimental challenge, as its pathophysiology involves central mechanisms of sensitization, synaptic dysfunction, and neuroimmune alterations. Among the preclinical models used for its study, several approaches stand out that reproduce processes of central sensitization and emotional dysregulation associated with this type of pain. The Chronic Mild Stress model seeks to simulate the effects of persistent emotional and physiological stress, capable of inducing generalized hyperalgesia and central sensitization [59]. The Reserpine-Induced Pain Model is based on the repeated administration of reserpine, an irreversible inhibitor of the Vesicular Monoamine Transporter 2 (VMAT2), which causes sustained monoamine depletion and dysfunction of descending pain inhibitory pathways. As a result, animals develop diffuse hypersensitivity, mechanical and thermal hyperalgesia, anhedonia, and decreased locomotor activity, along with glial activation and increased proinflammatory cytokines [60]. Another relevant model is sleeping deprivation-induced central hypersensitivity. Partial sleep restriction for several days increases spontaneous activity of nociceptive neurons in the dorsal horn, reduces spinal serotonin concentration, and generates diffuse allodynia, reproducing the sensory alterations typical of fibromyalgia [61]. Finally, the Repeated Acid Injection Model involves the repeated administration of intramuscular injections of an acidic solution (pH 4.0 - 4.5) into the gastrocnemius or masseter muscle. After two to three weeks, the animals develop diffuse mechanical and thermal hypersensitivity [62]. These models reproduce the phenomena of central sensitization, monoaminergic dysfunction, and neuroinflammation that characterize nociplastic pain, constituting fundamental tools for exploring sex-dependent differences in pain modulation and expression.

Taken together, animal models of pain allow us to understand the pathophysiological mechanisms underlying different pain modalities and offer a solid experimental basis for studying sex-dependent variations in nociceptive perception and modulation. Accumulated evidence suggests that such differences are not solely due to factors ot only genetic or neuroanatomical factors, but also the modulating influence of sex hormones on the neuronal circuits and glial cells involved in pain transmission and regulation. In this context, the interaction between estrogen, testosterone, and progesterone with their respective receptors plays a crucial role, modulating both nociceptive sensitivity and the associated inflammatory and emotional response. Therefore, it is essential to delve deeper into the analysis of the sex hormones involved in the pain process to fully understand sexual dimorphism in its expression and treatment.

Various studies have demonstrated that biological sex is a determining factor in the perception, modulation, and response to pain, influenced by hormonal, neuroendocrine, immunological, and genetic differences. In this context, experimental models in rodents represent a fundamental tool for studying sexual dimorphism in nociception. This is because they allow the control of biological and environmental variables, the reproduction of different types of pain (nociceptive, neuropathic, inflammatory or nociplastic) and the evaluation of changes in the expression of receptors associated with the transmission and modulation of pain.

Sexual dimorphism refers to the differences between individuals of the same species [63]. Several factors are involved in this phenomenon; some of the differences are attributed to the influence of sex hormones and their receptors, which modulate sensitivity and response to pain [63][64].

In addition to hormonal factors, the neuroimmunological mechanisms involved in pain modulation have been identified as differing between males and females, reflecting a qualitative divergence in how the nervous system processes nociceptive stimuli [65][66]. In male rodents, spinal microglial activation plays a fundamental role in the generation and maintenance of central sensitization. Activation of Toll-Like Receptor 4 (TLR4) in microglia induces the release of proinflammatory cytokines (IL-1β, TNF-α, IL-6) and Brain-Derived Neurotrophic Factor (BDNF), as well as activation of the p38 MAPK pathway, resulting in GABAergic disinhibition and increased neuronal excitability [67][68]. In contrast, in female rodents, pain sensitization does not depend on microglia, but rather on the activation of CD4⁺ T lymphocytes that infiltrate the spinal cord and release proinflammatory cytokines and growth factors, indirectly modulating nociceptive transmission [69][70]. In experimental models, T-cell depletion induces the use of microglial pathways in females, pathways typically observed in males, confirming the existence of alternative, sex-dependent cellular mechanisms in pain modulation [69].

In general, sexual dimorphism in the pathophysiology of pain reflects differences in neuroimmune interactions: Males: TLR4-mediated microglial activation → BDNF release → decreased GABAergic inhibition → central sensitization. Females: CD4⁺ T-cell activation → cytokine release → indirect modulation of neuronal excitability. These differences not only underscore the influence of biological sex on pain mechanisms, but also have therapeutic relevance, given that microglial inhibitors such as minocycline show analgesic efficacy in males, while their effect in females is limited, highlighting the need to develop analgesic strategies with a sex-specific approach [69].

Sex hormones play an important role in pain modulation, acting on peripheral and central mechanisms of the nervous system [71]. Fluctuations in circulating levels of estradiol and progesterone in females, and testosterone and dehydroepiandrosterone (DHEA) in males, influence pain perception, tolerance, and analgesic response, determining not only pain intensity but also the efficacy of pharmacological treatments [63][72]. The dual function of sex hormones depends on factors such as hormone concentration, physiological context, tissue type, and the activated molecular signaling pathway [73]. For example, hormonal fluctuations of the estrous cycle are known to modulate these actions, increasing susceptibility to chronic painful diseases such as migraine, fibromyalgia, or neuropathic pain in women [74][75].

Among the main receptors involved in pain are hormonal (ER, AR and GPER1), neuronal (opioid, serotonergic, glutamatergic and cannabinoid), immunological (TLR4, macrophages, T cells, B cells, neutrophils, natural killers) and those belonging to the tyrosine kinase family (TrkB) and neurotrophin receptor (p75), whose interactions support the sex-dependent differences in the perception and modulation of pain [63][66][69][76].

Historically, opiates have largely been the cornerstone of pain treatment, whether acute or chronic, due to their availability and effectiveness. However, with increased use, associated risks have been revealed: tolerance, dependence, and overdose [108]. Their mechanism involves binding to the same opioid receptors in the central nervous system, producing similar analgesic and euphoric effects [109]. Multiple studies have shown that morphine is more effective in males than in females, demonstrating that this variability is due to differences in the location or binding to opiate receptors and to physiological mechanisms or anatomical differences between the sexes, rather than to its pharmacokinetics [110].

Regarding estrogens, estrogen receptors are present throughout the Central Nervous System (CNS): amygdala, thalamus, nociceptive areas, and Anterior Cingulate Cortex (ACC). Previous studies demonstrate that estrogen can regulate the sensory system and pain. Studies in mice have shown that the lack or absence of estrogen triggered by ovariectomy produces hyperalgesia, while its administration eliminates it [76]. In rats exposed to a continuous increase in estrogen as hormone replacement therapy, the analgesic effect of estrogen was suppressed in the early phase of ovariectomy, resulting in an increase in ERα and a decrease in ERβ. On the other hand, in rats whose estrogen levels gradually decreased, both receptors were slightly increased. The results show that a slight increase in both receptors restores the nociceptive threshold, compared to when only one of the receptors remains elevated. In other words, the analgesic effects of estrogens depend on the exposure time and the balance between the different types of receptors that are activated or increased, and their interaction with each other [111].

On the other hand, testosterone has an inhibitory modulatory effect on pain. Preclinical research has shown that the Transient Receptor Potential Vanilloid 1 (TRPV1) receptor is overexpressed in orchiectomized male rats and female rats. When testosterone levels are low or decrease, TRPV1 receptor expression increases, causing increased pain sensitivity. However, in mice treated with testosterone replacement therapy, this receptor decreases, as does musculoskeletal pain [86]. Regarding the TRPV1 receptor, agonists are generally used as a therapeutic measure for pain. One of them is capsaicin, which is the prototype TRPV1 agonist. When the agonist stimulates the receptor, after an initial excitation period, a refractory period of desensitization follows, meaning the neuron stops responding to new stimuli. If this stimulus is applied in high doses, a reversible degeneration of the peripheral nerve terminals occurs, resulting in a decrease in local sensitivity for a time [112].

Consideration of biological sex and hormonal context is crucial in the perception of pain. Preclinical research confirms sexual dimorphism in pain perception. Hormonal and immunological differences explain this variability. Animal pain models validate this sexual difference and suggest possible molecular mechanisms involved. Therefore, preclinical research provides a paradigm for the development of more effective analgesics for pain specific to men and women.