AIMS:To determine how lesion size and location shape longitudinal functional connectivity (FC) changes after stroke. METHODS:Adult male mice underwent atlas-based resting-state fMRI at baseline and 1, 2, and 4 weeks after either a small photothrombotic cortical stroke (N = 25) or a larger transient cortico-striatal MCAO stroke (N = 6). FC was quantified across 98 atlas regions, focusing on sensorimotor cortex, striatum, and thalamus, including intra- and inter-hemispheric connectivity matrices and regional seed strength. RESULTS:Cortical stroke caused widespread hyperconnectivity at Weeks 1-2, with about 90% of connections increased, followed by partial normalization by Week 4. This effect declined most strongly in the ischemic hemisphere and remained more sustained contralesionally. In contrast, cortico-striatal stroke induced global hypoconnectivity at Week 1, with more than 90% of connections decreased, a modest and heterogeneous shift toward baseline at Week 2, and widespread decreases persisting at Week 4. A subset of sensorimotor connections showing opposite changes in the two models robustly separated groups at all post-stroke time points. Regional lesion involvement scaled with the magnitude of baseline-referenced FC alterations. CONCLUSION:Lesion topography drives distinct longitudinal FC trajectories after stroke and may help define network biomarkers and optimal windows for targeted interventions.

Beyond immediate neuronal damage,functional and structural connectivity is altered brain-wide with implications for functional deficits and recovery in stroke.It remains unclear,however,if the level of axonal damage,as well as compensatory plasticity,i.e.,axonal sprouting and remyelination,depend on the lesion size and topology.This study compared two different stroke models in adult male mice,with the aim of uncovering the dynamics in white matter changes.Repetitive diffusion magnetic resonance imaging was acquired over 4 weeks post photothrombotic cortical(1.41％±0.92％of brain volume)and middle cerebral artery occlusion cortico-striatal(11.53％±2.8％of brain volume)strokes.Structural connectivity changes were mapped over time at the whole-brain level.We quantified inter-and intra-hemispheric seed strength changes over time,with seed strength reflecting how strongly each region was connected to the rest of the brain.Differences between groups and time points were assessed using a mixed model corrected for multiple comparisons.The results showed that large cortico-striatal lesions led to increased structural connectivity in sensorimotor regions,whereas small cortical lesions induced asymmetric connectivity changes:an increase extending globally from the ischemic hemisphere and a decrease expanding globally from the healthy hemisphere.These findings highlight that stroke severity and lesion size significantly affect the temporal dynamics and spatial distribution of connectivity disruptions,emphasizing the need for targeted monitoring of neural changes post-stroke.

Transcranial direct current stimulation (tDCS) is a clinically promising neuromodulatory therapy, capable of promoting function and motor recovery after stroke. Beyond the primary stroke lesion, remote networks disturbances, e.g., stroke-induced secondary neurodegeneration (SND), are related to long-term disabilities. Under the hypothesis that tDCS promotes recovery by supporting neuroprotection, we investigated the effects of tDCS on thalamic SND after stroke. Three days after cortical stroke, induced by photothrombosis, cathodal tDCS over the lesioned cortex was performed daily for ten days (39.6 kC/m2). SND, i.e., neuronal loss, and inflammation in the ipsilesional thalamus were evaluated ex vivo 28 days after stroke. Parameters of functional thalamic network integration measured by resting-state functional magnetic resonance imaging (rs-fMRI) were conducted longitudinally. To assess the effects of tDCS on glucose metabolism, positron emission tomography (PET) was performed after a similar tDCS regimen in healthy mice. Repetitive tDCS decreased the ipsilateral thalamic glucose metabolism in unlesioned animals. Four weeks after cortical stroke, secondary glial scaring was found in the ipsilesional thalamus, its extent correlating to the cortical lesion size (R2 = 0.54, p < 0.001). Notably, while it did not affect glial scaring, tDCS reduced thalamic neurodegeneration by over 60 % (p < 0.05), being reflected by parameters of functional thalamic integration as assessed by rs-fMRI. Additionally, tDCS downregulated the pro-inflammatory polarization of microglia. Overall, tDCS ameliorated the stroke-induced remote SND, in parallel to mitigating sustained neuroinflammation. Thus, the data show that tDCS exerts previously unknown effects on remote brain regions after stroke.

Mice are of a size at which passive joint and muscle forces should be important in leg movements. To investigate this issue, we measured, in anesthetized mice, hind leg passive movements in response to changes in animal orientation relative to gravity and to manual deflections of the leg. Changing gravity orientation did not rotate leg joints to their physiological extremes, indicating that passive responses limit joint rotation range. The manual leg deflections were sufficient to achieve joint angles overlapping those present in published descriptions of mouse locomotion. Upon release from these deflections, the legs returned to intermediate postures. These results show that passive responses are (1) present at locomotory joint angles and (2) sufficiently large, at these angles, to move the leg. Return amplitude depended linearly on deflection amplitude. The slope of this dependence was the same across leg joints, suggesting it is evolutionarily or developmentally selected for. Combining the extremes of our passive response data and published descriptions of joint angles during mouse locomotion (e.g., most-flexed passive response mouse with most-extended published locomotion pattern) allowed determining when in a locomotory cycle passive responses could be definitely extending or flexing. In three of these four combinations, only extending passive responses could be definitely present in the locomotory patterns. In the fourth, alternatively, both extending and flexing passive responses could be definitely present. Passive responses thus likely act during mouse hind leg locomotion, but their amplitude and even sign may vary across individual mice.

The proportional Recovery Rule (PRR) has been frequently used to predict recovery of lost motor function in acute stroke patients. However, it still needs to be explored whether the same concept applies to preclinical, i.e. animal models of stroke recovery. To address this question, we investigated behavioral data from 125 adult male C57Bl/6 J mice with photothrombotic strokes in the sensorimotor cortex. Lesion size and location were determined in the first week using in vivo T2-weighted MRI. Motor recovery was evaluated repeatedly over four weeks using the cylinder, grid walk, and rotating beam test. Recovery trajectories were analyzed using a newly formulated Mouse Recovery Rule (MRR), comparing it against the traditional PRR. Initial findings indicated variable recovery patterns, which were separated using a stepwise linear regression approach resulting in two clusters: 47 % PRR and 53 % MRR. No significant correlation was found between recovery patterns and lesion size or location, suggesting that other biological factors drive individual differences in recovery. Of note, in the MRR cluster, animals recovered to 90 % of their initial behavioral state within the first four weeks post-stroke, which is higher than the 70 % recovery usually reported in human PRR studies. This study demonstrates the complexity of translating the PRR to stroke recovery models in mice and underscores the need for species-specific recovery models. Our findings have implications for designing and interpreting therapeutic strategies for stroke recovery in preclinical settings, with the potential to improve the predictive accuracy of stroke recovery assessments.

Degeneration of dopaminergic neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinson's disease. In idiopathic cases, high levels of mitochondrial DNA alterations, leading to mitochondrial dysfunction, are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E variant of the mitochondrial helicase Twinkle in dopaminergic neurons, leading to accelerated mitochondrial DNA mutations. These K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although ∼70% of nigral dopaminergic neurons had perished. Remaining neurons still preserved ∼75% of axon terminals in the dorsal striatum and enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of the surviving neurons. We conclude that a small population of substantia nigra dopaminergic neurons is able to adapt to the accumulation of mitochondrial DNA mutations and maintain motor control.

Magnetic resonance imaging (MRI) is a valuable tool for studying brain structure and function in animal and clinical studies. With the growth of public MRI repositories, access to data has finally become easier. However, filtering large datasets for potential poor-quality outliers can be a challenge. We present AIDAqc, a machine-learning-assisted automated Python-based command-line tool for small animal MRI quality assessment. Quality control features include signal-to-noise ratio (SNR), temporal SNR, and motion. All features are automatically calculated and no regions of interest are needed. Automated outlier detection for a given dataset combines the interquartile range and the machine-learning methods one-class support vector machine, isolation forest, local outlier factor, and elliptic envelope. To evaluate the reliability of individual quality control metrics, a simulation of noise (Gaussian, salt and pepper, speckle) and motion was performed. In outlier detection, single scans with induced artifacts were successfully identified by AIDAqc. AIDAqc was challenged in a large heterogeneous dataset collected from 19 international laboratories, including data from mice, rats, rabbits, hamsters, and gerbils, obtained with different hardware and at different field strengths. The results show that the manual inter-rater agreement (mean Fleiss Kappa score 0.17) is low when identifying poor-quality data. A direct comparison of AIDAqc results, therefore, showed only low-to-moderate concordance. In a manual post hoc validation of AIDAqc output, precision was high (>70%). The outlier data can have a significant impact on further postprocessing, as shown in representative functional and structural connectivity analysis. In summary, this pipeline optimized for small animal MRI provides researchers with a valuable tool to efficiently and effectively assess the quality of their MRI data, which is essential for improved reliability and reproducibility.

Neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography provide unique in vivo data to analyze structural and functional connectivity of the whole brain. Recent advances in small animal neuroimaging have opened new opportunities for the study of structure-function interactions in healthy and diseased brain networks, which are essential to develop therapies targeting network reorganization associated with functional improvement. Based on clinical studies, a common network response to acute neurological insult (e.g., stroke) and neurodegeneration (e.g., Alzheimer’s disease) is hyperconnectivity, i.e., an increase of functional connectivity strength above (healthy) control levels, a process which is, however, not well understood. For example, in resting-state functional MRI (rs-fMRI), which measures the hemodynamic response to neuronal activity, hyperconnectivity would refer to a drastic increase in the correlation between two regional time series. In absence of an absolute threshold defining hyperconnectivity, it relates to a control group or baseline measurement before intervention. It was hypothesized that hyperconnectivity follows a nonlinear distribution representing an interaction between actual demands, injury severity, and resource availability (Hillary and Grafman, 2017). Hyperconnectivity is significantly larger than transient variability in functional networks. The increase in connectivity follows a nonlinear distribution that reaches a maximum and transitions to a state of hypoconnectivity when a critical loss of structural resources is reached (Figure 1). As an extension of this hypothesis, we consider hyperconnectivity as part of an understudied and not well understood compensatory mechanism, which is embedded in the framework of the brain’s resilience capacity to respond to (network) disturbances.

Recent developments in rodent brain imaging have enabled translational characterization of functional and structural connectivity at the whole brain level in vivo. Nevertheless, fundamental questions about the link between structural and functional networks remain unsolved. In this review, we systematically searched for experimental studies in rodents investigating both structural and functional network measures, including studies correlating functional connectivity using resting-state functional MRI with diffusion tensor imaging or viral tracing data. We aimed to answer whether functional networks reflect the architecture of the structural connectome, how this reciprocal relationship changes throughout a disease, how structural and functional changes relate to each other, and whether changes follow the same timeline. We present the knowledge derived exclusively from studies that included in vivo imaging of functional and structural networks. The limited number of available reports makes it difficult to draw general conclusions besides finding a spatial and temporal decoupling between structural and functional networks during brain disease. Data suggest that when overcoming the currently limited evidence through future studies with combined imaging in various disease models, it will be possible to explore the interaction between both network systems as a disease or recovery biomarker.

Degeneration of dopamine neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinson’s disease (PD). In idiopathic cases, high levels of mitochondrial DNA (mtDNA) mutations associated with mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopamine neurons, leading to accelerated mtDNA ageing. K320E-Twinkle DaN mice showed normal motor function at 20 months of age, although already ∼70% of nigral dopamine neurons had perished. The remaining neuron population still preserved ∼75% of axon terminals in the dorsal striatum, which enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of surviving nigral dopamine neurons. We conclude that a small population of substantia nigra neurons can adapt to mtDNA mutations and maintain motor control in mice, holding chances for new treatment strategies in PD patients.

Sharing of data, processing tools, and workflows require open data hosting services and management tools. Despite FAIR guidelines and the increasing demand from funding agencies and publishers, only a few animal studies share all experimental data and processing tools. We present a step-by-step protocol to perform version control and remote collaboration for large multimodal datasets. A data management plan was introduced to ensure data security in addition to a homogeneous file and folder structure. Changes to the data were automatically tracked using DataLad and all data was shared on the research data platform GIN. This simple and cost-effective workflow facilitates the adoption of FAIR data logistics and processing workflows by making the raw and processed data available and providing the technical infrastructure to independently reproduce the data processing steps. It enables the community to collect heterogeneously acquired and stored datasets not limited to a specific category of data and serves as a technical infrastructure blueprint with rich potential to improve data handling at other sites and extend to other research areas.

Stroke patients show some degree of spontaneous functional recovery, but this is not sufficient to prevent long-term disability. One promising approach is to characterize the dynamics of stroke recovery genes in the lesion and distant areas. We induced sensorimotor cortex lesions in adult C57BL/6J mice using photothrombosis and performed qPCR on selected brain areas at 14, 28, and 56 days post-stroke (P14-56). Based on the grid walk and rotating beam test, the mice were classified into two groups. The expression of cAMP pathway genes Adora2a, Pde10a, and Drd2, was higher in poor- compared to well-recovered mice in contralesional primary motor cortex (cl-MOp) at P14&56 and cl-thalamus (cl-TH), but lower in cl-striatum (cl-Str) at P14 and cl-primary somatosensory cortex (cl-SSp) at P28. Plasticity and axonal sprouting genes, Lingo1 and BDNF, were decreased in cl-MOp at P14 and cl-Str at P28 and increased in cl-SSp at P28 and cl-Str at P14, respectively. In the cl-TH, Lingo1 was increased, and BDNF decreased at P14. Atrx, also involved in axonal sprouting, was only increased in poor-recovered mice in cl-MOp at P28. The results underline the gene expression dynamics and spatial variability and challenge existing theories of restricted neural plasticity.

Despite advances in acute care, ischemic stroke remains a major cause of long-term disability. Approaches targeting both neuronal and glial responses are needed to enhance recovery and improve long-term outcome. The complement C3a receptor (C3aR) is a regulator of inflammation with roles in neurodevelopment, neural plasticity, and neurodegeneration. Using mice lacking C3aR (C3aR-/-) and mice overexpressing C3a in the brain, we uncovered 2 opposing effects of C3aR signaling on functional recovery after ischemic stroke: inhibition in the acute phase and facilitation in the later phase. Peri-infarct astrocyte reactivity was increased and density of microglia reduced in C3aR-/- mice; C3a overexpression led to the opposite effects. Pharmacological treatment of wild-type mice with intranasal C3a starting 7 days after stroke accelerated recovery of motor function and attenuated astrocyte reactivity without enhancing microgliosis. C3a treatment stimulated global white matter reorganization, increased peri-infarct structural connectivity, and upregulated Igf1 and Thbs4 in the peri-infarct cortex. Thus, C3a treatment from day 7 after stroke exerts positive effects on astrocytes and neuronal connectivity while avoiding the deleterious consequences of C3aR signaling during the acute phase. Intranasal administration of C3aR agonists within a convenient time window holds translational promise to improve outcome after ischemic stroke.

BACKGROUND: Beyond focal effects, stroke lesions impact the function of distributed networks. We here investigated (1) whether transcranial direct current stimulation (tDCS) alters the network changes induced by cerebral ischemia and (2) whether functional network parameters predict the therapeutic efficacy of tDCS in a mouse model of focal photothrombotic stroke. METHODS: Starting 3 days after stroke, cathodal tDCS (charge density=39.6 kC/m²) was applied over 10 days in male C57Bl/6J mice under light anesthesia over the lesioned sensory-motor cortex. Functional connectivity (resting-state functional magnetic resonance imaging) was evaluated for up to 28-day poststroke, with global graph parameters of network integration computed. RESULTS: Ischemia induced a subacute increase in connectivity accompanied by a significant reduction in characteristic path length, reversed by 10 days of tDCS. Early measures of functional network alterations and the network configuration at prestroke baseline predicted spontaneous and tDCS-augmented motor recovery. DISCUSSION: Stroke induces characteristic network changes throughout the brain that can be detected by resting-state functional magnetic resonance imaging. These network changes were, at least in part, reversed by tDCS. Moreover, early markers of a network impairment and the network configuration before the insult improve the prediction of motor recovery.

Graph theory allows assessing changes of neuronal connectivity and interactions of brain regions in response to local lesions, e.g., after stroke, and global perturbations, e.g., due to psychiatric dysfunctions or neurodegenerative disorders. Consequently, network analysis based on constructing graphs from structural and functional MRI connectivity matrices is increasingly used in clinical studies. In contrast, in mouse neuroimaging, the focus is mainly on basic connectivity parameters, i.e., the correlation coefficient or fiber counts, whereas more advanced network analyses remain rarely used. This review summarizes graph theoretical measures and their interpretation to describe networks derived from recent in vivo mouse brain studies. To facilitate the entry into the topic, we explain the related mathematical definitions, provide a dedicated software toolkit, and discuss practical considerations for the application to rs-fMRI and DTI. This way, we aim to foster cross-species comparisons and the application of standardized measures to classify and interpret network changes in translational brain disease studies.

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS). In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

PurposeTo support acquisition of accurate, reproducible and high-quality preclinical imaging data, various standardisation resources have been developed over the years. However, it is unclear the impact of those efforts in current preclinical imaging practices. To better understand the status quo in the field of preclinical imaging standardisation, the STANDARD group of the European Society of Molecular Imaging (ESMI) put together a community survey and a forum for discussion at the European Molecular Imaging Meeting (EMIM) 2022. This paper reports on the results from the STANDARD survey and the forum discussions that took place at EMIM2022. ProceduresThe survey was delivered to the community by the ESMI office and was promoted through the Society channels, email lists and webpages. The survey contained seven sections organised as generic questions and imaging modality-specific questions. The generic questions focused on issues regarding data acquisition, data processing, data storage, publishing and community awareness of international guidelines for animal research. Specific questions on practices in optical imaging, PET, CT, SPECT, MRI and ultrasound were further included. ResultsData from the STANDARD survey showed that 47% of survey participants do not have or do not know if they have QC/QA guidelines at their institutes. Additionally, a large variability exists in the ways data are acquired, processed and reported regarding general aspects as well as modality-specific aspects. Moreover, there is limited awareness of the existence of international guidelines on preclinical (imaging) research practices. ConclusionsStandardisation of preclinical imaging techniques remains a challenge and hinders the transformative potential of preclinical imaging to augment biomedical research pipelines by serving as an easy vehicle for translation of research findings to the clinic. Data collected in this project show that there is a need to promote and disseminate already available tools to standardise preclinical imaging practices.

Introduction: Transcranial direct current stimulation (tDCS) non-invasively promotes recovery after stroke ( Braun et al. Exp. Neurol. 2016; Hummel et al. Brain 2005 ). Stroke affects entire networks beyond the focal lesion, impacting post-stroke impairment ( Blaschke et al. Stroke 2021 ). The thalamus constitutes a central hub due to its numerous functional connections but is also prone to secondary injury ( Cao et al. Front. Neurol. 2020 ). Under the hypothesis that tDCS promotes recovery by supporting neuroprotection, we investigated the effects of tDCS on secondary thalamic neurodegeneration after stroke. Methods: Cortical stroke was induced in the sensory-motor cortex (photothrombosis model in mice). Starting three days after stroke, cathodal tDCS over the ipsilesional somatosensory cortex was performed daily for ten days (39.6 kC/m 2 ), sham-stimulated mice served as control. Behavioral tests over time assessed functional recovery. Secondary degeneration of the ipsilesional thalamus was evaluated ex vivo 28 days after stroke. Using an atlas-based registration pipeline ( Pallast et al. J. Neurosc. Methods 2019 ), the absolute number of GFAP+ astrocytes and relative number of ipsilesional NeuN+ neurons compared to the contralateral unaffected thalamus were evaluated. Lesion maps based on T2-weighted Magnetic Resonance Imaging (MRI) before tDCS were used for voxel-based lesion-symptom mapping (VBLSM) to evaluate the effect of the lesion location on remote neurodegeneration. Functional connectivity (FC) between the lesioned sensorimotor cortex and the ipsilesional thalamus was measured by resting-state functional MRI. Additionally, glucose metabolism after a likewise tDCS regimen in healthy mice was measured by positron emission tomography compared to sham stimulation. Results: Repetitive tDCS decreased the ipsilateral thalamic glucose metabolism in unlesioned animals, while accelerating motor recovery after stroke. Four weeks after cortical stroke, secondary glial scaring was found in the ipsilesional thalamus, most pronounced in the posterior (Po), ventral posterolateral (VPL), and ventral posteromedial (VPM) nucleus, its extent correlating to the cortical lesion size (R 2 =0.3, p<0.01). Neurodegeneration was associated with a lesion cluster (VSBLM, p<0.05) extending laterally and including the callosal white matter tracts as well as deeper primary somatosensory layers. A decrease in FC between the primary somatosensory area of the lower limb (SS-LL) and the thalamus correlated with secondary neurodegeneration (R 2 =0.25, p<0.01). Intriguingly, in contrast to glial scaring, tDCS reduced thalamic neurodegeneration by over 60% (p<0.05). Conclusion: Cortical stroke induced network changes and led to remote secondary structural impairment of the thalamus, depending on the affection of corticothalamic connections. TDCS mitigated this remote secondary neurodegeneration. Data suggest previously unknown effects of tDCS on remote brain regions after stroke.

Restoration of functional connectivity is a major contributor to functional recovery after stroke. We investigated the role of reactive astrocytes in functional connectivity and recovery after photothrombotic stroke in mice with attenuated reactive gliosis (GFAP-/-Vim-/-). Infarct volume and longitudinal functional connectivity changes were determined by in vivo T2-weighted magnetic resonance imaging (MRI) and resting-state functional MRI. Sensorimotor function was assessed with behavioral tests, and glial and neural plasticity responses were quantified in the peri-infarct region. Four weeks after stroke, GFAP-/-Vim-/- mice showed impaired recovery of sensorimotor function and aberrant restoration of global neuronal connectivity. These mice also exhibited maladaptive plasticity responses, shown by higher number of lost and newly formed functional connections between primary and secondary targets of cortical stroke regions and increased peri-infarct expression of the axonal plasticity marker Gap43. We conclude that reactive astrocytes modulate recovery-promoting plasticity responses after ischemic stroke.