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Functional connectivity analysis in health and brain disease using in vivo widefield calcium imaging
Functional connectivity analysis in health and brain disease using in vivo widefield calcium imaging
Stroke is one of the leading causes of death and its prevalence is still raising with aging society in future. Despite its major impact, only two specific therapies are approved in clinical practice, today.Thus, hundreds of possible therapies were identified in experimental research, none of them has proven efficiency on human patients. To address this loss in translation from experimental research to clinical practise, several fronts can be scrutinized. Among several options, the establishment of translational methods to assess functional clinical outcome in preclinical research is inevitable. To approach this one option is to develop modalities of functional imaging of the brain activity. Functional brain imaging not only allows to assess translational parameters for functional regeneration after stroke but also to investigate pathophysiological mechanisms in the brain. Hence, the analysis of functional brain activity in experimental stroke research could both identify new therapeutic targets and validate their effectiveness by creating a translational read-out. Functional brain imaging is a frequently used method which strongly advanced our knowledge in neuroscience and as well in human stroke research. Its aim in general is a better understanding of brain functions, identification of functionally connected brain regions and their dynamic changes under certain conditions. In stroke research, the dynamic changes of functional network and its association with regeneration is of major interest. To investigate functional brain activity, functional magnetic resonance imaging (fMRI) is predominantly used in human research. fMRI faces great technical challenges and essential limitations for use in small rodents such as laboratory mice which are the most frequently used animals to study brain disease. This is why there is interest and need for alternative imaging modalities in experimental research. To benefit of the insights from human research in experimental research, we adapted and evolved the imaging modality of in vivo widefield calcium imaging. This imaging modality is based on transgenic animals who permit to investigate brain activity directly via GCaMP fluorescence. GCaMP is a genetically encoded calcium sensor which is well-known to mirror calcium fluctuations during action potential and with this neuronal activity. Via a customized imaging system, it is possible to acquire cortical neuronal activity and analyse it with comparable methods as used in human brain research. Hence, this method allows the repetitive investigation of brain activity in vivo in a translational manner. In three studies we adapted and enhanced existing protocols to establish a reliable transgenic approach to assess functional brain connectivity. In a first study, we investigated the effect of anaesthesia on brain function and characterized the relationship of different frequency-based imaging parameters, functional connectivity and depth of anaesthesia. Subsequently, we established a stringent protocol for light sedation which is easy to use and results in reproducible imaging parameters. In a second study, we identified functional brain areas by using independent vector analysis (IVA) on resting state imaging data. Therefore, we validated the identified areas with help of an anatomical atlas and stimulus-evoked brain activity. This validation justifies the usage of our unbiasedly selected cortical areas as functional seeds. Finally, we implemented the assessment of functional connectivity values after stroke. In this third study, we investigated repetitively the changes in functional connectivity up to 56 days after an ischemic lesion in the motor cortex induced by a photothrombotic model. We demonstrate both acute and chronic effects of ischemia to cortical functional connectivity. In the acute phase on the first day after stroke we demonstrate transient increase in contralateral functional connectivity. A second transient effect is the increase in contralateral motor cortex size. Third, chronic reduction in interhemispheric functional connectivity is present only in functionally but not anatomically close regions of the brain. And last, changes in both functional connectivity values and the size of contralateral motor cortex size are associated with the deficits assessed by behavioural testing. Hence, the identified parameters are of major relevance for the clinical outcome. The results establish two major facts: preclinical investigation of brain function is possible on a routinely basis and adds additional insight on pathophysiological mechanisms in brain disease which are associated with behavioural outcome. Consequently, the application of this translational imaging modality will not only be of great interest to stroke research but also to several brain diseases where pathophysiological mechanisms still need to be elucidated.
experimental stroke, calcium imaging, functional connectivity
Cramer, Julia Verena
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Cramer, Julia Verena (2020): Functional connectivity analysis in health and brain disease using in vivo widefield calcium imaging. Dissertation, LMU München: Faculty of Medicine
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Abstract

Stroke is one of the leading causes of death and its prevalence is still raising with aging society in future. Despite its major impact, only two specific therapies are approved in clinical practice, today.Thus, hundreds of possible therapies were identified in experimental research, none of them has proven efficiency on human patients. To address this loss in translation from experimental research to clinical practise, several fronts can be scrutinized. Among several options, the establishment of translational methods to assess functional clinical outcome in preclinical research is inevitable. To approach this one option is to develop modalities of functional imaging of the brain activity. Functional brain imaging not only allows to assess translational parameters for functional regeneration after stroke but also to investigate pathophysiological mechanisms in the brain. Hence, the analysis of functional brain activity in experimental stroke research could both identify new therapeutic targets and validate their effectiveness by creating a translational read-out. Functional brain imaging is a frequently used method which strongly advanced our knowledge in neuroscience and as well in human stroke research. Its aim in general is a better understanding of brain functions, identification of functionally connected brain regions and their dynamic changes under certain conditions. In stroke research, the dynamic changes of functional network and its association with regeneration is of major interest. To investigate functional brain activity, functional magnetic resonance imaging (fMRI) is predominantly used in human research. fMRI faces great technical challenges and essential limitations for use in small rodents such as laboratory mice which are the most frequently used animals to study brain disease. This is why there is interest and need for alternative imaging modalities in experimental research. To benefit of the insights from human research in experimental research, we adapted and evolved the imaging modality of in vivo widefield calcium imaging. This imaging modality is based on transgenic animals who permit to investigate brain activity directly via GCaMP fluorescence. GCaMP is a genetically encoded calcium sensor which is well-known to mirror calcium fluctuations during action potential and with this neuronal activity. Via a customized imaging system, it is possible to acquire cortical neuronal activity and analyse it with comparable methods as used in human brain research. Hence, this method allows the repetitive investigation of brain activity in vivo in a translational manner. In three studies we adapted and enhanced existing protocols to establish a reliable transgenic approach to assess functional brain connectivity. In a first study, we investigated the effect of anaesthesia on brain function and characterized the relationship of different frequency-based imaging parameters, functional connectivity and depth of anaesthesia. Subsequently, we established a stringent protocol for light sedation which is easy to use and results in reproducible imaging parameters. In a second study, we identified functional brain areas by using independent vector analysis (IVA) on resting state imaging data. Therefore, we validated the identified areas with help of an anatomical atlas and stimulus-evoked brain activity. This validation justifies the usage of our unbiasedly selected cortical areas as functional seeds. Finally, we implemented the assessment of functional connectivity values after stroke. In this third study, we investigated repetitively the changes in functional connectivity up to 56 days after an ischemic lesion in the motor cortex induced by a photothrombotic model. We demonstrate both acute and chronic effects of ischemia to cortical functional connectivity. In the acute phase on the first day after stroke we demonstrate transient increase in contralateral functional connectivity. A second transient effect is the increase in contralateral motor cortex size. Third, chronic reduction in interhemispheric functional connectivity is present only in functionally but not anatomically close regions of the brain. And last, changes in both functional connectivity values and the size of contralateral motor cortex size are associated with the deficits assessed by behavioural testing. Hence, the identified parameters are of major relevance for the clinical outcome. The results establish two major facts: preclinical investigation of brain function is possible on a routinely basis and adds additional insight on pathophysiological mechanisms in brain disease which are associated with behavioural outcome. Consequently, the application of this translational imaging modality will not only be of great interest to stroke research but also to several brain diseases where pathophysiological mechanisms still need to be elucidated.