Neuroimagen molecular preclínica en estimulación cerebral profunda

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Farmacología, leída el 28/01/2021. Tesis formato europeo (compendio de artículos)Deep brain stimulation (DBS) is a very powerful neurostimulation therapy for the palliative treatment of, mainly, resistant cases of motor disorders. The fruitful results obtained in the neurological scenario led to explore the possibility of extending its application to different psychiatric pathologies. Thus, DBS emerges as a potential alternative to the traditional neurosurgical interventions, which consist of local or major ablative procedures, performed in specific brain regions involved in the symptomatology with the aim to palliate it. In this sense, although it is indeed an invasive technique, it is actually a focal therapy, with potential reversibility of the effects induced by the electric stimulation, and offering the possibility of adjusting the stimulation parameters according to the patient’s needs. Nevertheless, despite the huge efforts and extensive research history in the DBS field, the specific mechanism of action of this therapy remains unknown. Several theories have tried to explain the effects obtained under specific pathologies and stimulation circumstances. However, regardless of DBS undoubted efficacy, a wide number of unanswered questions remain open regarding the specific physiological effects and long-term consequences. Therefore, the application of DBS in the majority of the proposed neuropsychiatric pathologies to potentially benefit from this treatment remains at a research level. Objectives: The main aim of this thesis is to evaluate the effects of DBS, by means of in vivo functional neuroimaging techniques, in healthy rats and in two models of murine obesity. In particular, the main contribution of this thesis is to describe the neuromodulation consequences of DBS on brain metabolism using [18F]FDG-PET at different stages: 1st) after the electrodes insertion, 2nd) during the electrical stimulation, 3rd) after a chronic and intermittent DBS protocol, and 4th) after a chronic and continuous DBS protocol...La estimulación cerebral profunda (DBS) es una potente terapia de neuroestimualción dedicada al tratamiento paliativo de, en su mayoría, casos avanzados de patologías motoras. Los fructíferos resultados obtenidos dentro del ámbito de la neurología llevaron a explorar la posibilidad de extender la aplicación de la DBS a diferentes patologías psiquiátricas. Así, la DBS emerge como una alternativa a las tradicionales intervenciones neuroquirúrgicas, las cuales consisten en procedimientos de ablación, local o extensa, realizados en regiones cerebrales específicas involucradas en la sintomatología a paliar. En este sentido, aunque la DBS es de hecho una técnica invasiva, se trata de una terapia local, con potencial reversibilidad de los efectos inducidos por la estimulación eléctrica, y que proporciona la posibilidad de ajustar in vivo los parámetros de estimulación de acuerdo a las necesidades del paciente. Sin embargo, a pesar de los enormes esfuerzos y la extensa historia de investigación en el campo de la DBS, los mecanismos de acción específicos de esta terapia todavía se desconocen. Varias teorías han intentado explicar los efectos obtenidos bajo circunstancias patológicas y de estimulación concretas. No obstante, a pesar de la indudable eficacia de la DBS, un amplio número de preguntas sin responder permanecen abiertas en relación a los efectos fisiológicos específicos de la DBS, así como a las consecuencias a largo plazo de la misma. Por este motivo, la aplicación de la DBS en la mayoría de las patologías neuropsiquiátricas que han sido propuestas de poder potencialmente beneficiarse de su tratamiento, permanecen en una etapa de investigación. Objetivos: La principal motivación de esta tesis consiste en evaluar los efectos de la DBS, por medio de técnicas de imagen funcional in vivo, en ratas sanas y en dos modelos murinos de obesidad. En concreto, la principal contribución de este trabajo radica en describir las consecuencias de la neuromodulación inducida por la DBS sobre el metabolismo cerebral, utilizando [18F]FDG-PET en diferentes niveles: 1º) después de la inserción de los electrodos; 2º) durante la estimulación eléctrica; 3º) después de un protocolo crónico e intermitente de DBS; y 4º) tras un protocolo crónico y continuo de DBS...Fac. de MedicinaTRUEunpu

Response to deep brain stimulation in three brain targets with implications in mental disorders: a PET study in rats

Objective: To investigate metabolic changes in brain networks by deep brain stimulation (DBS) of the medial prefrontal cortex (mPFC), nucleus accumbens (NAcc) and dorsomedial thalamus (DM) using positron emission tomography (PET) in naïve rats. Methods: 43 male Wistar rats underwent stereotactic surgery and concentric bipolar platinum-iridium electrodes were bilaterally implanted into one of the three brain sites. [18F]-fluoro-2-deoxy-glucose-PET (18FDG-PET) and computed tomography (CT) scans were performed at the 7th (without DBS) and 9th day (with DBS) after surgery. Stimulation period matched tracer uptake period. Images were acquired with a small-animal PET-CT scanner. Differences in glucose uptake between groups were assessed with Statistical Parametric Mapping. Results: DBS induced site-specific metabolic changes, although a common increased metabolic activity in the piriform cortex was found for the three brain targets. mPFC-DBS increased metabolic activity in the striatum, temporal and amygdala, and reduced it in the cerebellum, brainstem (BS) and periaqueductal gray matter (PAG). NAcc-DBS increased metabolic activity in the subiculum and olfactory bulb, and decreased it in the BS, PAG, septum and hypothalamus. DM-DBS increased metabolic activity in the striatum, NAcc and thalamus and decreased it in the temporal and cingulate cortex. Conclusions: DBS induced significant changes in 18FDG uptake in brain regions associated with the basal ganglia-thalamo-cortical circuitry. Stimulation of mPFC, NAcc and DM induced different patterns of 18FDG uptake despite interacting with the same circuitries. This may have important implications to DBS research suggesting individualized target selection according to specific neural modulatory requirements.This research was conducted under the EraNet Neuron framework (DBS_F20rat) and supported by the Federal Ministry of Education and Research, Germany (BMBF 01EW1103), the Ministry of Economy and Competitiveness ISCIII-FIS grants (PI14/00860, CPII/00005) co-financed by ERDF (FEDER) Funds from the European Commission, "A way of making Europe", Fundación Mapfre and Comunidad de Madrid (BRADE S2013/ICE-2958)

Exploratory study of the long-term footprint of deep brain stimulation on brain metabolism and neuroplasticity in an animal model of obesity.

Deep brain stimulation (DBS) is a powerful neurostimulation therapy proposed for the treatment of several neuropsychiatric disorders. However, DBS mechanism of action remains unclear, being its effects on brain dynamics of particular interest. Specifically, DBS reversibility is a major point of debate. Preclinical studies in obesity showed that the stimulation of the lateral hypothalamus (LH) and nucleus accumbens (NAcc), brain centers involved in satiety and reward circuits, are able to modulate the activity of brain structures impaired in this pathology. Nevertheless, the long-term persistence of this modulation after DBS withdrawal was unexplored. Here we examine the in vivo presence of such changes 1 month after LH- and NAcc-DBS, along with differences in synaptic plasticity, following an exploratory approach. Thus, both stimulated and non-stimulated animals with electrodes in the NAcc showed a common pattern of brain metabolism modulation, presumably derived from the electrodes' presence. In contrast, animals stimulated in the LH showed a relative metabolic invariance, and a reduction of neuroplasticity molecules, evidencing long-lasting neural changes. Our findings suggest that the reversibility or persistence of DBS modulation in the long-term depends on the selected DBS target. Therefore, the DBS footprint would be influenced by the stability achieved in the neural network involved during the stimulation.This research was supported by the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III (projects PI14/00860 and PI17/01766, and grant CPII14/00005), co-financed by European Regional Development Fund (ERDF), “A way of making Europe”, CIBERSAM, Delegación del Gobierno para el Plan Nacional sobre Drogas (2017/085), Fundación Mapfre and Fundación Alicia Koplowitz. MCV was supported by Fundación Tatiana Pérez de Guzmán el Bueno as scholarship holder of this institution. DRM was supported by Consejería de Educación e Investigación, Comunidad de Madrid, co-funded by European Social Fund “Investing in your future” (grant, PEJD-2018-PRE/BMD-7899). NLR was supported by Instituto de Investigación Sanitaria Gregorio Marañón, "Programa Intramural de Impulso a la I+D+I 2019”. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV‐2015‐0505). Support for Nacher’s lab came from Ministry of Economy and Competitiveness (SAF2015-68436-R), Generalitat Valenciana (PROMETEO2013/069) and Fundación Alicia Koplowitz (FAK2012/01).S

The Poly I:C maternal immune stimulation model shows unique patterns of brain metabolism, morphometry, and plasticity in female rats

Introduction: Prenatal infections are associated with an increased risk of the onset of schizophrenia. Rodent models of maternal immune stimulation (MIS) have been extensively used in preclinical studies. However, many of these studies only include males, omitting pathophysiological features unique to females. The aim of this study is to characterize the MIS model in female rats using positron emission tomography (PET), structural magnetic resonance imaging (MR), and neuroplasticiy studies. Methods: In gestational day 15, Poly I:C (or Saline) was injected into pregnant Wistar rats to induce the MIS model. Imaging studies: [18F]-fluoro-2-deoxy-D-glucose-PET scans of female-offspring were acquired at post-natal day (PND) 35 and PND100. Furthermore, T2-MR brain images were acquired in adulthood. Differences in FDG uptake and morphometry between groups were assessed with SPM12 and Regions of Interest (ROI) analyses. Ex vivo study: The density of parvalbumin expressing interneurons (PV), perineuronal nets (PNN), and parvalbumin expressing interneurons surrounded by perineuronal nets (PV-PNN) were evaluated in the prelimbic cortex and basolateral amygdala using confocal microscopy. ROIs and neuroplasticity data were analyzed by 2-sample T-test and 2-way-ANOVA analyses, respectively. Results: A significant increase in brain metabolism was found in all animals at adulthood compared to adolescence. MIS hardly modified brain glucose metabolism in females, highlighting a significant hypometabolism in the thalamus at adulthood. In addition, MIS induced gray matter (GM) enlargements in the pituitary, hippocampus, substantia nigra, and cingulate cortex, and GM shrinkages in some thalamic nuclei, cerebelar areas, and brainstem. Moreover, MIS induced white matter shrinkages in the cerebellum, brainstem and corpus callosum, along with cerebrospinal fluid enlargements in the lateral and 4th ventricles. Finally, MIS reduced the density of PV, PNN, and PV-PNN in the basolateral amygdala. Conclusion: Our work showed in vivo the differential pattern of functional and morphometric affectation in the MIS model in females, as well as the deficits caused at the synaptic level according to sex. The differences obtained highlight the relevance of including both sexes in psychiatric research in order to consider their pathophysiological particularities and successfully extend the benefits obtained to the entire patient population.MS-M was supported by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (PI17/01766, BA21/0030); co-financed by European Regional Development Fund (ERDF), “A way to make Europe”; project PID2021_128862OB-I00 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE; Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM; project number CB07/09/0031); Delegación del Gobierno para el Plan Nacional sobre Drogas (project number 2017/085); and Fundación Alicia Koplowitz. MC-V was supported by Fundación Tatiana Pérez de Guzmán el Bueno as scholarship holder of this institution, and EU Joint Programme—Neurodegenerative Disease Research (JPND). DR-M was supported by Consejería de Educación e Investigación, Comunidad de Madrid, co-funded by European Social Fund “Investing in your future” (grant number PEJD-2018-PRE/BMD-7899). NL-R was supported by Instituto de Investigación Sanitaria Gregorio Marañón, “Programa Intramural de Impulso a la I+D+I 2019”. MD’s work was supported by Ministerio de Ciencia e Innovación (MCIN) and Instituto de Salud Carlos III (PT20/00044). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). JN was supported by the project RTI2018-098269-B-I00 and PID2021-127595OB-I00 financed by the Spanish Ministry of Science and Innovation/AEI/10.13039/501100011033/(“FEDER Una manera de hacer Europa”) and the Generalitat Valenciana (PROMETEU/2020/024)

The Poly I:C maternal immune stimulation model shows unique patterns of brain metabolism, morphometry, and plasticity in female rats

Introduction: Prenatal infections are associated with an increased risk of the onset of schizophrenia. Rodent models of maternal immune stimulation (MIS) have been extensively used in preclinical studies. However, many of these studies only include males, omitting pathophysiological features unique to females. The aim of this study is to characterize the MIS model in female rats using positron emission tomography (PET), structural magnetic resonance imaging (MR), and neuroplasticiy studies. Methods: In gestational day 15, Poly I:C (or Saline) was injected into pregnant Wistar rats to induce the MIS model. Imaging studies: [18F]-fluoro-2-deoxy-D-glucose-PET scans of female-offspring were acquired at post-natal day (PND) 35 and PND100. Furthermore, T2-MR brain images were acquired in adulthood. Differences in FDG uptake and morphometry between groups were assessed with SPM12 and Regions of Interest (ROI) analyses. Ex vivo study: The density of parvalbumin expressing interneurons (PV), perineuronal nets (PNN), and parvalbumin expressing interneurons surrounded by perineuronal nets (PV-PNN) were evaluated in the prelimbic cortex and basolateral amygdala using confocal microscopy. ROIs and neuroplasticity data were analyzed by 2-sample T-test and 2-way-ANOVA analyses, respectively. Results: A significant increase in brain metabolism was found in all animals at adulthood compared to adolescence. MIS hardly modified brain glucose metabolism in females, highlighting a significant hypometabolism in the thalamus at adulthood. In addition, MIS induced gray matter (GM) enlargements in the pituitary, hippocampus, substantia nigra, and cingulate cortex, and GM shrinkages in some thalamic nuclei, cerebelar areas, and brainstem. Moreover, MIS induced white matter shrinkages in the cerebellum, brainstem and corpus callosum, along with cerebrospinal fluid enlargements in the lateral and 4th ventricles. Finally, MIS reduced the density of PV, PNN, and PV-PNN in the basolateral amygdala. Conclusion: Our work showed in vivo the differential pattern of functional and morphometric affectation in the MIS model in females, as well as the deficits caused at the synaptic level according to sex. The differences obtained highlight the relevance of including both sexes in psychiatric research in order to consider their pathophysiological particularities and successfully extend the benefits obtained to the entire patient population.MS-M was supported by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (PI17/01766, BA21/0030); co-financed by European Regional Development Fund (ERDF), “A way to make Europe”; project PID2021_128862OB-I00 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE; Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM; project number CB07/09/0031); Delegación del Gobierno para el Plan Nacional sobre Drogas (project number 2017/085); and Fundación Alicia Koplowitz. MC-V was supported by Fundación Tatiana Pérez de Guzmán el Bueno as scholarship holder of this institution, and EU Joint Programme—Neurodegenerative Disease Research (JPND). DR-M was supported by Consejería de Educación e Investigación, Comunidad de Madrid, co-funded by European Social Fund “Investing in your future” (grant number PEJD-2018-PRE/BMD-7899). NL-R was supported by Instituto de Investigación Sanitaria Gregorio Marañón, “Programa Intramural de Impulso a la I+D+I 2019”. MD’s work was supported by Ministerio de Ciencia e Innovación (MCIN) and Instituto de Salud Carlos III (PT20/00044). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). JN was supported by the project RTI2018-098269-B-I00 and PID2021-127595OB-I00 financed by the Spanish Ministry of Science and Innovation/AEI/10.13039/501100011033/(“FEDER Una manera de hacer Europa”) and the Generalitat Valenciana (PROMETEU/2020/024)

Interaction between maternal immune activation and peripubertal stress in rats: impact on cocaine addiction-like behaviour, morphofunctional brain parameters and striatal transcriptome.

Substance use disorders are more prevalent in schizophrenia, but the causal links between both conditions remain unclear. Maternal immune activation (MIA) is associated with schizophrenia which may be triggered by stressful experiences during adolescence. Therefore, we used a double-hit rat model, combining MIA and peripubertal stress (PUS), to study cocaine addiction and the underlying neurobehavioural alterations. We injected lipopolysaccharide or saline on gestational days 15 and 16 to Sprague-Dawley dams. Their male offspring underwent five episodes of unpredictable stress every other day from postnatal day 28 to 38. When animals reached adulthood, we studied cocaine addiction-like behaviour, impulsivity, Pavlovian and instrumental conditioning, and several aspects of brain structure and function by MRI, PET and RNAseq. MIA facilitated the acquisition of cocaine self-administration and increased the motivation for the drug; however, PUS reduced cocaine intake, an effect that was reversed in MIA + PUS rats. We found concomitant brain alterations: MIA + PUS altered the structure and function of the dorsal striatum, increasing its volume and interfering with glutamatergic dynamics (PUS decreased the levels of NAA + NAAG but only in LPS animals) and modulated specific genes that could account for the restoration of cocaine intake such as the pentraxin family. On its own, PUS reduced hippocampal volume and hyperactivated the dorsal subiculum, also having a profound effect on the dorsal striatal transcriptome. However, these effects were obliterated when PUS occurred in animals with MIA experience. Our results describe an unprecedented interplay between MIA and stress on neurodevelopment and the susceptibility to cocaine addiction.This work has been funded by the Spanish Ministry of Economy and Competitiveness (Project no.: PSI2016-80541-P to EA and AH-M); Ministry of Science (PID2019- 104523RB-I00 to A-HM and PID2019-111594RB-100 to EA), Spanish Ministry of Health, Social Services and Equality (Network of Addictive Disorders - Project no.: RTA-RD16/ 020/0022 of the Institute of Health Carlos III and National Plan on Drugs, Project no.: 2016I073 to EA and 2017I042 to A H-M); The BBVA Foundation (Leonardo Grants) to AH-M; The European Union (Project no.: JUST- 2017- AG- DRUG-806996-JUSTSO) to EA; and the UNED (Plan for the Promotion of Research) to EA and AH-M. MLS-M was supported by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (project PI17/01766), co-financed by the European Regional Development Fund (ERDF), ‘A way to make Europe’; project PID2021-128862OB-I00 funded by MCIN/AEI/ 10.13039/501100011033/FEDER, UE, CIBER de Salud Mental - Instituto de Salud Carlos III (project number CB07/09/0031); Delegación del Gobierno para el Plan Nacional sobre Drogas (project number 2017/085, 2022/008917); and Fundación Alicia Koplowitz. Fundación Tatiana Pérez de Guzmán el Bueno supported MC-V. MD’s work was supported by Ministerio de Ciencia e Innovación (MCIN) and Instituto de Salud Carlos III (PT20/00044). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

The Poly I:C maternal immune stimulation model shows unique patterns of brain metabolism, morphometry, and plasticity in female rats

Introduction: Prenatal infections are associated with an increased risk of the onset of schizophrenia. Rodent models of maternal immune stimulation (MIS) have been extensively used in preclinical studies. However, many of these studies only include males, omitting pathophysiological features unique to females. The aim of this study is to characterize the MIS model in female rats using positron emission tomography (PET), structural magnetic resonance imaging (MR), and neuroplasticiy studies.Methods: In gestational day 15, Poly I:C (or Saline) was injected into pregnant Wistar rats to induce the MIS model. Imaging studies: [18F]-fluoro-2-deoxy-D-glucose-PET scans of female-offspring were acquired at post-natal day (PND) 35 and PND100. Furthermore, T2-MR brain images were acquired in adulthood. Differences in FDG uptake and morphometry between groups were assessed with SPM12 and Regions of Interest (ROI) analyses. Ex vivo study: The density of parvalbumin expressing interneurons (PV), perineuronal nets (PNN), and parvalbumin expressing interneurons surrounded by perineuronal nets (PV-PNN) were evaluated in the prelimbic cortex and basolateral amygdala using confocal microscopy. ROIs and neuroplasticity data were analyzed by 2-sample T-test and 2-way-ANOVA analyses, respectively.Results: A significant increase in brain metabolism was found in all animals at adulthood compared to adolescence. MIS hardly modified brain glucose metabolism in females, highlighting a significant hypometabolism in the thalamus at adulthood. In addition, MIS induced gray matter (GM) enlargements in the pituitary, hippocampus, substantia nigra, and cingulate cortex, and GM shrinkages in some thalamic nuclei, cerebelar areas, and brainstem. Moreover, MIS induced white matter shrinkages in the cerebellum, brainstem and corpus callosum, along with cerebrospinal fluid enlargements in the lateral and 4th ventricles. Finally, MIS reduced the density of PV, PNN, and PV-PNN in the basolateral amygdala.Conclusion: Our work showed in vivo the differential pattern of functional and morphometric affectation in the MIS model in females, as well as the deficits caused at the synaptic level according to sex. The differences obtained highlight the relevance of including both sexes in psychiatric research in order to consider their pathophysiological particularities and successfully extend the benefits obtained to the entire patient population

Understanding Deep Brain Stimulation: In Vivo Metabolic Consequences of the Electrode Insertional Effect

Deep brain stimulation (DBS) is a neurosurgery technique widely used in movement disorders, although its mechanism of action remains unclear. In fact, apart from the stimulation itself, the mechanical insertion of the electrode may play a crucial role. Here we aimed to distinguish between the insertional and the DBS effects on brain glucose metabolism. To this end, electrodes were implanted targeting the medial prefrontal cortex in five adult male Wistar rats. Positron Emission Tomography (PET) studies were performed before surgery (D0) and seven (D7) and nine days (D9) after that. DBS was applied during the (18)FDG uptake of the D9 study. PET data were analysed with statistical parametric mapping. We found an electrode insertional effect in cortical areas, while DBS resulted in a more widespread metabolic pattern. The consequences of simultaneous electrode and DBS factors revealed a combination of both effects. Therefore, the insertion metabolic effects differed from the stimulation ones, which should be considered when assessing DBS protocols.The authors thank Kenia Martinez for her help in performing the statistical analyses and the interpretation of the results; Alexandra de Francisco and Yolanda Sierra for their support in stereotaxic surgery, animal handling, and acquisition of imaging studies. This research was supported by Fundacion Mapfre, Alicia Koplowitz [FAK16/01], CIBER de Salud Mental (CIBERSAM), the Ministry of Economy and Competitiveness ISCIII-FIS Grants [PI14/00860, CPII14/00005, and PI17/01766], and Delegacion del Gobierno para el Plan Nacional sobre Drogas [PNSD 2017/085] and cofinanced by ERDF (FEDER) Funds from the European Commission, ``A way of Making Europe,´´ and Comunidad de Madrid [BRADE-CMS2013/ICE-2958].S

Understanding Deep Brain Stimulation: In Vivo Metabolic Consequences of the Electrode Insertional Effect

Deep brain stimulation (DBS) is a neurosurgery technique widely used in movement disorders, although its mechanism of action remains unclear. In fact, apart from the stimulation itself, the mechanical insertion of the electrode may play a crucial role. Here we aimed to distinguish between the insertional and the DBS effects on brain glucose metabolism. To this end, electrodes were implanted targeting the medial prefrontal cortex in five adult male Wistar rats. Positron Emission Tomography (PET) studies were performed before surgery (D0) and seven (D7) and nine days (D9) after that. DBS was applied during the 18FDG uptake of the D9 study. PET data were analysed with statistical parametric mapping. We found an electrode insertional effect in cortical areas, while DBS resulted in a more widespread metabolic pattern. The consequences of simultaneous electrode and DBS factors revealed a combination of both effects. Therefore, the insertion metabolic effects differed from the stimulation ones, which should be considered when assessing DBS protocols