Central and peripheral mechanisms of pain modulationthe role of the Locus coeruleus and pupillary dynamics in neuropathic and inflammatory pain
Pain is a complex sensory and emotional experience that results from the interaction between peripheral nociceptive input and central processing in the nervous system. Despite its clinical relevance and social impact, the management of pain, particularly chronic pain remains a significant challenge. Current treatments often provide insufficient relief, and only a limited number of findings from basic research have successfully translated into effective therapies. This translational gap underscores the need for improved preclinical models and for objective physiological biomarkers that can reliably reflect nociceptive processing and treatment efficacy. An additional and often underestimated factor in pain research is biological sex, which strongly influences pain perception, prevalence, and treatment outcomes. Epidemiological studies consistently show that women are more frequently affected by chronic and inflammatory pain disorders than men. The underlying mechanisms are multifactorial, involving hormonal modulation, immune responses, and central nervous system differences. Among these, the locus coeruleus (LC), the main noradrenergic nucleus in the brainstem, exhibits pronounced sexual dimorphism in both its structure and function. The LC is a key node in the modulation of pain, arousal, and stress, and its projections reach widespread regions of the brain and spinal cord (SC). Differences in LC organization, neuronal density, and hormonal sensitivity between males and females may contribute to sex-dependent variations in pain modulation and autonomic regulation. The main objective of this doctoral thesis was to investigate the central and peripheral neurophysiological mechanisms underlying pain modulation in models of nerve injury and inflammation. This was achieved by assessing the contribution of the LC as a central node and pupillary dynamics as a peripheral readout, with the ultimate goal of establishing pupillary responses as a translational biomarker for the assessment of pain and the efficacy of analgesic interventions. Three complementary studies were designed to address these aims. The first study focused on the role of LC circuits in neuropathic pain using a model of chronic constriction injury (CCI) of the sciatic nerve. Chemogenetic tools were used to selectively modulate LC activity and its major projections in order to examine their contribution to spontaneous pain-like behavior. The second study implemented longitudinal pupillometry in male and female mice following CCI to characterize the temporal evolution of pupil dynamics and to identify possible sex-dependent differences. Pupil recordings were obtained, in anaesthetized animals, under controlled conditions at multiple time points and were correlated with sensory behavioral assessments, cognitive performance, and depression-like behavior. The third study used a formalin-induced inflammatory pain model to investigate the association between nociceptive behavior and pupillary responses, in order to evaluate the potential of pupillometry as a translational biomarker for monitoring the efficacy of analgesic compounds with different mechanisms of action. The results revealed that the LC contributed to pain modulation through mechanisms that depend on the specific neural circuits engaged, as well as on the pathophysiological context, including the type and stage of pain and the biological sex of the subject. During neuropathic pain, LC activation patterns changed with the stage of injury, showing distinct contributions of ascending and descending projections. Chemogenetic manipulation demonstrated that selective activation or inhibition of LC pathways produced different effects on nociceptive behaviors, supporting the idea that the LC does not function as a homogeneous structure but rather as a distributed network whose components can either facilitate or inhibit pain. Moreover, optogenetic stimulation of LC neurons elicited consistent pupil dilation and anxiety-like behaviors, confirming a causal relationship between LC activity, autonomic output, and emotional state. Longitudinal analysis of pupil dynamics after CCI revealed clear sex- and time-dependent differences. Female mice exhibited greater pupil dilation than males during repeated nociceptive stimulation, indicating enhanced sensitization. At long-term stages after CCI, this enhanced pupillary reactivity persisted, whereas no sex-related differences were observed in affective or cognitive outcomes. These findings are consistent with clinical observations of higher pain prevalence in women and may reflect intrinsic sex-related differences in LC structure or function. The inclusion of both sexes and the detailed characterization of their responses provide novel insight into how biological sex shapes the neural and autonomic correlates of pain. In the inflammatory pain model, the dynamics of the pupil accurately reflected the progression of nociceptive behavior and exhibited changes following the administration of analgesic drugs. Centrally acting compounds attenuated the pain-induced pupillary responses., supporting the utility of pupillometry as a quantitative and objective indicator of central analgesic efficacy. The reproducibility and non-invasive nature of this method highlight its translational potential for bridging preclinical and clinical pain research. Taken together, the studies presented in this thesis support a model in which the LC acts as a dynamic regulator of pain processing, whose influence depends on the specific neural circuits engaged, the type of pain, and the biological sex of the subject. The combined use of optogenetic tool with behavioral and physiological measures has provided new insight into the complex interactions between neural activity, autonomic function, and affective state. Furthermore, this work advances our understanding of the field and suggests that pupillometry may serve as a sensitive and reliable biomarker for assessing nociceptive processing and the effects of centrally acting analgesics. In summary, this thesis contributes to a deeper understanding of how the LC noradrenergic system modulates pain and autonomic responses, emphasizing the importance of considering sex as a biological variable in both basic and translational research. The integration of circuit-level analysis, behavioral assessment, and patophysiological monitoring offers a more comprehensive approach to studying pain and supports the development of more objective and individualized strategies for its evaluation and management.
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