{"id":{"repo_id":"cadiz","oai_identifier":"oai:rodin.uca.es:10498/39657"},"canonical_url":"https://search.dev.ndltd.org/etd/cadiz/oai:rodin.uca.es:10498/39657","repository":{"repo_id":"cadiz","name":"Universidad de Cadiz","base_url":"https://rodin.uca.es/oai/request"},"display":{"title":"Central and peripheral mechanisms of pain modulationthe role of the Locus coeruleus and pupillary dynamics in neuropathic and inflammatory pain","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["López Martín, Carolina"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Berrocoso Domínguez, Esther María","González Saiz, Francisco Manuel"],"committee_chairs":[],"committee_members":[],"year":2026,"date_issued":"2026","date_published":"2026","updated_at":"2026-07-24T01:29:24Z","subjects":[],"languages":["eng"],"rights":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"],"rights_urls":["http://creativecommons.org/licenses/by-nc-nd/4.0/"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10498/39657","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Berrocoso Domínguez, Esther María","González Saiz, Francisco Manuel"]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Neurociencias"]},{"key":"dc:creator","label":"Author","values":["López Martín, Carolina"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-05-26T09:50:49Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-05-26T09:50:49Z"]},{"key":"dc:date.issued","label":"Date","values":["2026"]},{"key":"dc:type","label":"Dc Type","values":["doctoral thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://creativecommons.org/licenses/by-nc-nd/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10498/39657"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.","El dolor es una experiencia sensorial y emocional compleja que resulta de la interacción entre la entrada nociceptiva periférica y el procesamiento central en el sistema nervioso. A pesar de su relevancia clínica y su impacto social, el abordaje del dolor, especialmente del dolor crónico, sigue siendo un desafío significativo. Los tratamientos actuales suelen proporcionar un alivio insuficiente, y solo un número limitado de hallazgos procedentes de la investigación básica se han traducido de forma exitosa en terapias efectivas. Esta brecha traslacional pone de manifiesto la necesidad de mejorar los modelos preclínicos y de desarrollar biomarcadores fisiológicos objetivos que reflejen de manera fiable el procesamiento nociceptivo y la eficacia de los tratamientos. Un factor adicional y a menudo subestimado en la investigación del dolor es el sexo biológico, que influye de manera notable en la percepción del dolor, su prevalencia y las respuestas al tratamiento. Los estudios epidemiológicos muestran de forma consistente que las mujeres se ven afectadas con mayor frecuencia que los hombres por enfermedades crónicas e inflamatorias que cursan con dolor. Los mecanismos subyacentes son multifactoriales e incluyen la modulación hormonal, las respuestas inmunitarias y diferencias en el sistema nervioso central. Entre estos factores, el locus coeruleus (LC), el principal núcleo noradrenérgico del tronco encefálico, presenta un marcado dimorfismo sexual tanto en su estructura como en su función. El LC es un nodo clave en la modulación del dolor, la activación y el estrés, y proyecta hacia regiones ampliamente distribuidas del encéfalo y de la médula espinal. Las diferencias en la organización del LC, la densidad neuronal y la sensibilidad hormonal entre machos y hembras pueden contribuir a variaciones dependientes del sexo en la modulación del dolor y en la regulación autonómica. El objetivo principal de esta tesis doctoral fue investigar los mecanismos neurofisiológicos centrales y periféricos que subyacen a la modulación del dolor en modelos de lesión nerviosa e inflamación. Para ello, se evaluó la contribución del LC como nodo central y la dinámica pupilar como indicador periférico, con el fin último de establecer las respuestas pupilares como biomarcador traslacional para evaluar el dolor y la eficacia de intervenciones analgésicas. Para ello se diseñaron tres estudios complementarios. El primero se centró en el papel de los circuitos del LC en el dolor neuropático utilizando un modelo de lesión por constricción crónica (CCI) del nervio actividad del LC y de sus proyecciones principales, con el fin de examinar su contribución a conductas espontáneas de tipo doloroso. El segundo estudio realizó un seguimiento longitudinal de los cambios pupilares en ratones machos y hembras tras el CCI, para caracterizar la evolución temporal de la dinámica pupilar e identificar posibles diferencias dependientes del sexo. Los registros pupilares se obtuvieron en animales anestesiados, bajo condiciones controladas y en múltiples puntos temporales, y se correlacionaron con evaluaciones conductuales sensoriales, rendimiento cognitivo y comportamiento de tipo depresivo. El tercer estudio utilizó un modelo de dolor inflamatorio inducido por formalina para analizar la asociación entre el comportamiento nociceptivo y las respuestas pupilares, con el fin de evaluar el potencial de la pupilometría como biomarcador traslacional para monitorizar la eficacia de compuestos analgésicos con distintos mecanismos de acción. Los resultados mostraron que el LC contribuye a la modulación del dolor mediante mecanismos que dependen de los circuitos neuronales específicos reclutados, así como del contexto fisiopatológico, incluyendo el tipo y la fase del dolor y el sexo biológico del individuo. Durante el dolor neuropático, los patrones de activación del LC cambiaron a lo largo del curso de la lesión, mostrando contribuciones diferenciadas de sus proyecciones ascendentes y descendentes. La manipulación quimiogenética demostró que la activación o inhibición selectiva de las vías del LC producía efectos distintos sobre los comportamientos nociceptivos, lo que respalda la idea de que el LC no actúa como una estructura homogénea, sino como una red distribuida cuyos componentes pueden facilitar o inhibir el dolor. Además, la estimulación optogenética de las neuronas del LC provocó de manera consistente dilatación pupilar y comportamientos de tipo ansioso, confirmando una relación causal entre la actividad del LC, la salida autonómica y el estado emocional. El análisis longitudinal de la dinámica pupilar tras el CCI, reveló diferencias claras dependientes del sexo y del tiempo. Las hembras mostraron una mayor dilatación pupilar que los machos durante la estimulación nociceptiva repetida, lo que indica una sensibilización aumentada. En fases tardías tras la CCI, esta mayor reactividad pupilar persistió, mientras que no se observaron diferencias relacionadas con el sexo en los parámetros afectivos o cognitivos. Estos hallazgos coinciden con las observaciones clínicas sobre la mayor prevalencia del dolor en mujeres y podrían reflejar diferencias intrínsecas relacionadas con el sexo en la estructura o función del LC. La inclusión de ambos sexos y la caracterización detallada de sus respuestas aportan información novedosa sobre cómo el sexo biológico modula los correlatos neurales y autonómicos del dolor. En el modelo de dolor inflamatorio, la dinámica pupilar reflejó de forma precisa la progresión del comportamiento nociceptivo y presentó cambios tras la administración de fármacos analgésicos. Los fármacos de acción central atenuaron las respuestas pupilares inducidas por el dolor., lo que respalda la utilidad de la pupilometría como indicador cuantitativo y objetivo de la eficacia analgésica central. La reproducibilidad y el carácter no invasivo de este método subrayan su potencial traslacional para conectar la investigación preclínica y clínica del dolor. En conjunto, los estudios presentados en esta tesis apoyan un modelo en el que el LC actúa como un regulador dinámico del procesamiento del dolor, cuya influencia depende de los circuitos neuronales específicos reclutados, del tipo de dolor y del sexo biológico del sujeto. El uso combinado de herramientas optogenéticas con medidas conductuales y fisiológicas ha proporcionado nueva información sobre las interacciones complejas entre la actividad neuronal, la función del sistema autónomo y el estado afectivo. Además, este trabajo amplía el conocimiento en el campo y sugiere que la pupilometría puede servir como un biomarcador sensible y fiable para evaluar el procesamiento nociceptivo y los efectos de analgésicos de acción central. En resumen, esta tesis contribuye a una comprensión más profunda de cómo la actividad noradrenérgica del LC modula el dolor y las respuestas autonómicas, enfatizando la importancia de considerar el sexo como variable biológica tanto en la investigación básica como en la traslacional. La integración del análisis a nivel de circuitos, la evaluación conductual y el seguimiento fisiopatológico ofrece un enfoque más completo para el estudio del dolor y respalda el desarrollo de estrategias más objetivas e individualizadas para su evaluación y manejo."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Central and peripheral mechanisms of pain modulationthe role of the Locus coeruleus and pupillary dynamics in neuropathic and inflammatory pain"]}]}],"canonical_facts":{"dc:contributor.advisor":["Berrocoso Domínguez, Esther María","González Saiz, Francisco Manuel"],"dc:contributor.other":["Neurociencias"],"dc:creator":["López Martín, Carolina"],"dc:date.accessioned":["2026-05-26T09:50:49Z"],"dc:date.available":["2026-05-26T09:50:49Z"],"dc:date.issued":["2026"],"dc:description.abstract":["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.","El dolor es una experiencia sensorial y emocional compleja que resulta de la interacción entre la entrada nociceptiva periférica y el procesamiento central en el sistema nervioso. A pesar de su relevancia clínica y su impacto social, el abordaje del dolor, especialmente del dolor crónico, sigue siendo un desafío significativo. Los tratamientos actuales suelen proporcionar un alivio insuficiente, y solo un número limitado de hallazgos procedentes de la investigación básica se han traducido de forma exitosa en terapias efectivas. Esta brecha traslacional pone de manifiesto la necesidad de mejorar los modelos preclínicos y de desarrollar biomarcadores fisiológicos objetivos que reflejen de manera fiable el procesamiento nociceptivo y la eficacia de los tratamientos. Un factor adicional y a menudo subestimado en la investigación del dolor es el sexo biológico, que influye de manera notable en la percepción del dolor, su prevalencia y las respuestas al tratamiento. Los estudios epidemiológicos muestran de forma consistente que las mujeres se ven afectadas con mayor frecuencia que los hombres por enfermedades crónicas e inflamatorias que cursan con dolor. Los mecanismos subyacentes son multifactoriales e incluyen la modulación hormonal, las respuestas inmunitarias y diferencias en el sistema nervioso central. Entre estos factores, el locus coeruleus (LC), el principal núcleo noradrenérgico del tronco encefálico, presenta un marcado dimorfismo sexual tanto en su estructura como en su función. El LC es un nodo clave en la modulación del dolor, la activación y el estrés, y proyecta hacia regiones ampliamente distribuidas del encéfalo y de la médula espinal. Las diferencias en la organización del LC, la densidad neuronal y la sensibilidad hormonal entre machos y hembras pueden contribuir a variaciones dependientes del sexo en la modulación del dolor y en la regulación autonómica. El objetivo principal de esta tesis doctoral fue investigar los mecanismos neurofisiológicos centrales y periféricos que subyacen a la modulación del dolor en modelos de lesión nerviosa e inflamación. Para ello, se evaluó la contribución del LC como nodo central y la dinámica pupilar como indicador periférico, con el fin último de establecer las respuestas pupilares como biomarcador traslacional para evaluar el dolor y la eficacia de intervenciones analgésicas. Para ello se diseñaron tres estudios complementarios. El primero se centró en el papel de los circuitos del LC en el dolor neuropático utilizando un modelo de lesión por constricción crónica (CCI) del nervio actividad del LC y de sus proyecciones principales, con el fin de examinar su contribución a conductas espontáneas de tipo doloroso. El segundo estudio realizó un seguimiento longitudinal de los cambios pupilares en ratones machos y hembras tras el CCI, para caracterizar la evolución temporal de la dinámica pupilar e identificar posibles diferencias dependientes del sexo. Los registros pupilares se obtuvieron en animales anestesiados, bajo condiciones controladas y en múltiples puntos temporales, y se correlacionaron con evaluaciones conductuales sensoriales, rendimiento cognitivo y comportamiento de tipo depresivo. El tercer estudio utilizó un modelo de dolor inflamatorio inducido por formalina para analizar la asociación entre el comportamiento nociceptivo y las respuestas pupilares, con el fin de evaluar el potencial de la pupilometría como biomarcador traslacional para monitorizar la eficacia de compuestos analgésicos con distintos mecanismos de acción. Los resultados mostraron que el LC contribuye a la modulación del dolor mediante mecanismos que dependen de los circuitos neuronales específicos reclutados, así como del contexto fisiopatológico, incluyendo el tipo y la fase del dolor y el sexo biológico del individuo. Durante el dolor neuropático, los patrones de activación del LC cambiaron a lo largo del curso de la lesión, mostrando contribuciones diferenciadas de sus proyecciones ascendentes y descendentes. La manipulación quimiogenética demostró que la activación o inhibición selectiva de las vías del LC producía efectos distintos sobre los comportamientos nociceptivos, lo que respalda la idea de que el LC no actúa como una estructura homogénea, sino como una red distribuida cuyos componentes pueden facilitar o inhibir el dolor. Además, la estimulación optogenética de las neuronas del LC provocó de manera consistente dilatación pupilar y comportamientos de tipo ansioso, confirmando una relación causal entre la actividad del LC, la salida autonómica y el estado emocional. El análisis longitudinal de la dinámica pupilar tras el CCI, reveló diferencias claras dependientes del sexo y del tiempo. Las hembras mostraron una mayor dilatación pupilar que los machos durante la estimulación nociceptiva repetida, lo que indica una sensibilización aumentada. En fases tardías tras la CCI, esta mayor reactividad pupilar persistió, mientras que no se observaron diferencias relacionadas con el sexo en los parámetros afectivos o cognitivos. Estos hallazgos coinciden con las observaciones clínicas sobre la mayor prevalencia del dolor en mujeres y podrían reflejar diferencias intrínsecas relacionadas con el sexo en la estructura o función del LC. La inclusión de ambos sexos y la caracterización detallada de sus respuestas aportan información novedosa sobre cómo el sexo biológico modula los correlatos neurales y autonómicos del dolor. En el modelo de dolor inflamatorio, la dinámica pupilar reflejó de forma precisa la progresión del comportamiento nociceptivo y presentó cambios tras la administración de fármacos analgésicos. Los fármacos de acción central atenuaron las respuestas pupilares inducidas por el dolor., lo que respalda la utilidad de la pupilometría como indicador cuantitativo y objetivo de la eficacia analgésica central. La reproducibilidad y el carácter no invasivo de este método subrayan su potencial traslacional para conectar la investigación preclínica y clínica del dolor. En conjunto, los estudios presentados en esta tesis apoyan un modelo en el que el LC actúa como un regulador dinámico del procesamiento del dolor, cuya influencia depende de los circuitos neuronales específicos reclutados, del tipo de dolor y del sexo biológico del sujeto. El uso combinado de herramientas optogenéticas con medidas conductuales y fisiológicas ha proporcionado nueva información sobre las interacciones complejas entre la actividad neuronal, la función del sistema autónomo y el estado afectivo. Además, este trabajo amplía el conocimiento en el campo y sugiere que la pupilometría puede servir como un biomarcador sensible y fiable para evaluar el procesamiento nociceptivo y los efectos de analgésicos de acción central. En resumen, esta tesis contribuye a una comprensión más profunda de cómo la actividad noradrenérgica del LC modula el dolor y las respuestas autonómicas, enfatizando la importancia de considerar el sexo como variable biológica tanto en la investigación básica como en la traslacional. La integración del análisis a nivel de circuitos, la evaluación conductual y el seguimiento fisiopatológico ofrece un enfoque más completo para el estudio del dolor y respalda el desarrollo de estrategias más objetivas e individualizadas para su evaluación y manejo."],"dc:format":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10498/39657"],"dc:language.iso":["eng"],"dc:rights":["Attribution-NonCommercial-NoDerivatives 4.0 Internacional"],"dc:rights.uri":["http://creativecommons.org/licenses/by-nc-nd/4.0/"],"dc:title":["Central and peripheral mechanisms of pain modulationthe role of the Locus coeruleus and pupillary dynamics in neuropathic and inflammatory pain"],"dc:type":["doctoral thesis"]},"updated_at":"2026-07-24T01:29:24Z"}