Developmental and interventional plasticity of motor maps after perinatal stroke.

Within the perinatal stroke field, there is a need to establish preclinical models where putative biomarkers for motor function can be examined. In a mouse model of perinatal stroke, we evaluated motor map size and movement latency following optogenetic cortical stimulation against three factors of post-stroke biomarker utility: 1) Correlation to chronic impairment on a behavioral test battery; 2) Amenability to change using a skilled motor training paradigm; 3) Ability to distinguish individuals with potential to respond well to training. Thy1-ChR2-YFP mice received a photothrombotic stroke at postnatal day 7 and were evaluated on a battery of motor tests between days 59-70. Following a cranial window implant, mice underwent longitudinal optogenetic motor mapping both before and after 3 weeks of skilled forelimb training. Map size and movement latency of both hemispheres was positively correlated with impaired spontaneous forelimb use, whereas only ipsilesional hemisphere map size was correlated with performance in skilled reaching. Map size and movement latency did not show groupwise changes with training; however, mice with the smallest pre-training map sizes and worst impairments demonstrated the greatest expansion of map size in response to skilled forelimb training. Overall, motor map size showed utility as a potential biomarker for impairment and training-induced modulation in specific individuals. Future assessment of the predictive capacity of post-stroke motor representations for behavioral outcome in animal models opens the possibility of dissecting how plasticity mechanisms contribute to recovery following perinatal stroke.SIGNIFICANCE STATEMENTWe investigated the utility of two cortical motor representation measures (motor map size and movement onset latency) as potential biomarkers for post-stroke motor recovery in a mouse model of perinatal stroke. Both motor map size and movement latency were associated with functional recovery after perinatal stroke, with map size showing an additional association between training responsiveness and severity of impairment. Overall, both motor map size and movement onset latency show potential as neurophysiological correlates of recovery. As such, future studies of perinatal stroke rehabilitation and neuromodulation should include these measures in order to help explain neurophysiological changes that might be occurring in response to treatment.

Mesoscale Mapping of Mouse Cortex Reveals Frequency-Dependent Cycling between Distinct Macroscale Functional Modules.

Connectivity mapping based on resting-state activity in mice has revealed functional motifs of correlated activity. However, the rules by which motifs organize into larger functional modules that lead to hemisphere wide spatial-temporal activity sequences is not clear. We explore cortical activity parcellation in head-fixed, quiet awake GCaMP6 mice from both sexes by using mesoscopic calcium imaging. Spectral decomposition of spontaneous cortical activity revealed the presence of two dominant frequency modes (<1 and ∼3 Hz), each of them associated with a unique spatial signature of cortical macro-parcellation not predicted by classical cytoarchitectonic definitions of cortical areas. Based on assessment of 0.1-1 Hz activity, we define two macro-organizing principles: the first being a rotating polymodal-association pinwheel structure around which activity flows sequentially from visual to barrel then to hindlimb somatosensory; the second principle is correlated activity symmetry planes that exist on many levels within a single domain such as intrahemispheric reflections of sensory and motor cortices. In contrast, higher frequency activity >1 Hz yielded two larger clusters of coactivated areas with an enlarged default mode network-like posterior region. We suggest that the apparent constrained structure for intra-areal cortical activity flow could be exploited in future efforts to normalize activity in diseases of the nervous system.SIGNIFICANCE STATEMENT Increasingly, functional connectivity mapping of spontaneous activity is being used to reveal the organization of the brain. However, because the brain operates across multiple space and time domains a more detailed understanding of this organization is necessary. We used in vivo wide-field calcium imaging of the indicator GCaMP6 in head-fixed, awake mice to characterize the organization of spontaneous cortical activity at different spatiotemporal scales. Correlation analysis defines the presence of two to three superclusters of activity that span traditionally defined functional territories and were frequency dependent. This work helps define the rules for how different cortical areas interact in time and space. We provide a framework necessary for future studies that explore functional reorganization of brain circuits in disease models.

Individualized tracking of self-directed motor learning in group-housed mice performing a skilled lever positioning task in the home cage.

Skilled forelimb function in mice is traditionally studied through behavioral paradigms that require extensive training by investigators and are limited by the number of trials individual animals are able to perform within a supervised session. We developed a skilled lever positioning task that mice can perform within their home cage. The task requires mice to use their forelimb to precisely hold a lever mounted on a rotary encoder within a rewarded position to dispense a water reward. A Raspberry Pi microcomputer is used to record lever position during trials and to control task parameters, thus making this low-footprint apparatus ideal for use within animal housing facilities. Custom Python software automatically increments task difficulty by requiring a longer hold duration, or a more accurate hold position, to dispense a reward. The performance of individual animals within group-housed mice is tracked through radio-frequency identification implants, and data stored on the microcomputer may be accessed remotely through an active internet connection. Mice continuously engage in the task for over 2.5 mo and perform ~500 trials/24 h. Mice required ~15,000 trials to learn to hold the lever within a 10° range for 1.5 s and were able to further refine movement accuracy by limiting their error to a 5° range within each trial. These results demonstrate the feasibility of autonomously training group-housed mice on a forelimb motor task. This paradigm may be used in the future to assess functional recovery after injury or cortical reorganization induced by self-directed motor learning. NEW & NOTEWORTHY We developed a low-cost system for fully autonomous training of group-housed mice on a forelimb motor task. We demonstrate the feasibility of tracking both end-point, as well as kinematic performance of individual mice, with each performing thousands of trials over 2.5 mo. The task is run and controlled by a Raspberry Pi microcomputer, which allows for cages to be monitored remotely through an active internet connection.

Displacement of sensory maps and disorganization of motor cortex after targeted stroke in mice.

BACKGROUND AND PURPOSE: Recovery from stroke is hypothesized to involve the reorganization of surviving cortical areas. To study the functional organization of sensorimotor cortex at multiple time points before and after stroke, we performed longitudinal light-based motor mapping of transgenic mice expressing light-sensitive channelrhodopsin-2 in layer 5 cortical neurons. METHODS: Pulses of light stimulation were targeted to an array of cortical points, whereas evoked forelimb motor activity was recorded using noninvasive motion sensors. Intrinsic optical signal imaging produced maps of the forelimb somatosensory cortex. The resulting motor and sensory maps were repeatedly generated for weeks before and after small (0.2 mm3) photothrombotic infarcts were targeted to forelimb motor or sensory cortex. RESULTS: Infarcts targeted to forelimb sensory or motor areas caused decreased motor output in the infarct area and spatial displacement of sensory and motor maps. Strokes in sensory cortex caused the sensory map to move into motor cortex, which adopted a more diffuse structure. Stroke in motor cortex caused a compensatory increase in peri-infarct motor output, but did not affect the position or excitability of sensory maps. CONCLUSIONS: After stroke in motor cortex, decreased motor output from the infarcted area was offset by peri-infarct excitability. Sensory stroke caused a new sensory map to form in motor cortex, which maintained its center position, despite becoming more diffuse. These data suggest that surviving regions of cortex are able to assume functions from stroke-damaged areas, although this may come at the cost of alterations in map structure.

Localizing Microemboli within the Rodent Brain through Block-Face Imaging and Atlas Registration.

Brain microinfarcts are prevalent in humans, however because of the inherent difficulty of identifying and localizing individual microinfarcts, brain-wide quantification is impractical. In mice, microinfarcts have been created by surgically introducing microemboli into the brain, but a major limitation of this model is the absence of automated methods to identify and localize individual occlusions. We present a novel and semi-automated workflow to identify the anatomic location of fluorescent emboli (microspheres) within the mouse brain through histologic processing and atlas registration. By incorporating vibratome block-face imaging with the QuickNII brain registration tool, we show that the anatomic location of microspheres can be accurately registered to brain structures within the Allen mouse brain (AMB) atlas (e.g, somatomotor areas, hippocampal region, visual areas, etc.). Compared with registering images of slide mounted sections to the AMB atlas, microsphere location was more accurately determined when block-face images were used. As a proof of principle, using this workflow we compared the distribution of microspheres within the brains of mice that were either perfused or immersion fixed. No significant effect of perfusion on total microsphere number or location was detected. In general, microspheres were distributed brain-wide, with the largest density found in the thalamus. In sum, our block-face imaging workflow enables efficient characterization of the widespread distribution of fluorescent microemboli, facilitating future investigation into the impact of microinfarct load and location on brain health.

Remote Ischemic Conditioning and Stroke Recovery.

Remote ischemic conditioning (RIC) is a noninvasive procedure whereby several periods of ischemia are induced in a limb. Although there is growing interest in using RIC to improve stroke recovery, preclinical RIC research has focused exclusively on neuroprotection, using male animals and the intraluminal suture stroke model, and delivered RIC at times not relevant to either brain repair or behavioral recovery. In alignment with the Stroke Recovery and Rehabilitation Roundtable, we address these shortcomings. First, a standardized session (5-minute inflation/deflation, 4 repetitions) of RIC was delivered using a cuff on the contralesional hindlimb in both male and female Sprague-Dawley rats. Using the endothelin-1 stroke model, RIC was delivered once either prestroke (18 hours before, pre-RIC) or poststroke (4 hours after, post-RIC), and infarct volume was assessed at 24 hours poststroke using magnetic resonance imaging. RIC was delivered at these times to mimic the day before a surgery where clots are possible or as a treatment similar to tissue plasminogen activator, respectively. Pre-RIC reduced infarct volume by 41% compared with 29% with post-RIC. RIC was neuroprotective in both sexes, but males had a 46% reduction of infarct volume compared with 23% in females. After confirming the acute efficacy of RIC, we applied it chronically for 4 weeks, beginning 5 days poststroke. This delayed RIC failed to enhance poststroke behavioral recovery. Based on these findings, the most promising application of RIC is during the hyperacute and early acute phases of stroke, a time when other interventions such as exercise may be contraindicated.

Neuroprotection by Remote Ischemic Conditioning in Rodent Models of Focal Ischemia: a Systematic Review and Meta-Analysis.

Remote ischemic conditioning (RIC) is a promising neuroprotective therapy for ischemic stroke. Preclinical studies investigating RIC have shown RIC reduced infarct volume, but clinical trials have been equivocal. Therefore, the efficacy of RIC in reducing infarct volume and quality of current literature needs to be evaluated to identify knowledge gaps to support future clinical trials. We performed a systematic review and meta-analysis of preclinical literature involving RIC in rodent models of focal ischemia. This review was registered with PROSPERO (CRD42019145441). Eligibility criteria included rat or mice models of focal ischemia that received RIC to a limb either before, during, or after stroke. MEDLINE and Embase databases were searched from 1946 to August 2019. Risk of bias was assessed using the SYRCLE risk of bias tool along with construct validity. Seventy-two studies were included in the systematic review. RIC was shown to reduce infarct volume (SMD - 2.19; CI - 2.48 to - 1.91) when compared to stroke-only controls and no adverse events were reported with regard to RIC. Remote ischemic conditioning was shown to be most efficacious in males (SMD - 2.26; CI - 2.58 to - 1.94) and when delivered poststroke (SMD - 1.34; CI - 1.95 to - 0.73). A high risk of bias was present; thus, measures of efficacy may be exaggerated. A limitation is the poor methodological reporting of many studies, resulting in unclear construct validity. We identified several important, but under investigated topics including the efficacy of RIC in different stroke models, varied infarct sizes and location, and potential sex differences.

Rodent models of intracerebral hemorrhage.

The collagenase and whole blood intracerebral hemorrhage (ICH) models are widely used to identify mechanisms of injury and to evaluate treatments. Despite preclinical successes, to date, no treatment tested in phase III clinical trials has benefited ICH patients. These failures call into question the predictive value of current ICH models. By highlighting differences between these common rodent models of ICH, we sought to help investigators choose the more appropriate model for their study and to encourage the use of both whenever possible. For instance, we previously reported substantial differences in the bleeding profile, progression of cell death, and functional outcome between these models. These and other differences influence the efficacy and mechanisms of action of various treatment modalities. Thus, in this review, we also summarize neuroprotective and rehabilitation findings in each model. We conclude that differences between ICH models along with our current inability to identify the more clinically predictive model necessitate that preclinical assessments should normally be done in both. Such an approach, coupled with better assessment practices, will likely improve chances of future clinical success.

The Home-Cage Automated Skilled Reaching Apparatus (HASRA): Individualized Training of Group-Housed Mice in a Single Pellet Reaching Task.

The single pellet reaching task is commonly used in rodents to assess the acquisition of fine motor skill and recovery of function following nervous system injury. Although this task is useful for gauging skilled forelimb use in rodents, the process of training animals is labor intensive and variable across studies and labs. To address these limitations, we developed a single pellet reaching paradigm for training and testing group housed mice within their home cage. Mice enter a training compartment attached to the outside of the cage and retrieve millet seeds presented on a motorized pedestal that can be individually positioned to present seeds to either forelimb. To identify optimal training parameters, we compared task participation and success rates between groups of animals that were presented seeds at two different heights (floor vs mouth height) and at different intervals (fixed-time vs trial-based). The mouth height/fixed interval presentation style was most effective at promoting reaching behavior as all mice reached for seeds within 5 d. Using this paradigm, we assessed stroke-induced deficits in home-cage reaching. Following three weeks of baseline training, reaching success rate was ∼40%, with most trials performed during the dark cycle. A forelimb motor cortex stroke significantly decreased interaction with presented seeds within the first 2 d and impaired reaching success rates for the first 7 d. Our data demonstrate that group-housed mice can be efficiently trained on a single pellet reaching task in the home cage and that this assay is sensitive to stroke induced motor impairments.

Social and olfactory experiences modify neuronal morphology of orbital frontal cortex.

Structural modifications in the dendritic morphology of neurons occur following many forms of experience, including exposure to drugs, complex housing, and training in specific behavioral tasks. The present study examined morphological changes in orbitofrontal (OFC) and medial prefrontal cortex (mPFC) neurons of female rats following experience with a variety of social partners or nonsocial olfactory stimuli. We reasoned that experience with various social partners or olfactory stimuli, and the associated behavioral adaptations, would drive structural modifications in prefrontal cortex neurons engaged by these stimuli. Social experience was manipulated by providing rats with a novel cage-mate or housing the animal with the same cage-mate throughout the study. Similarly, olfactory experience was manipulated by introducing novel, nonsocial odors in the home cage or exposing the animals to the same home-cage odor throughout the study. Both forms of experience resulted in altered dendritic morphology in OFC neurons, whereas morphological changes in mPFC were comparatively small and limited to changes in spine density. These observations indicate that OFC and mPFC neurons respond differently to social and nonsocial olfactory stimulation in adulthood and join the growing body of data illustrating differential effects of experience on structural plasticity in OFC and mPFC. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Longitudinal monitoring of mesoscopic cortical activity in a mouse model of microinfarcts reveals dissociations with behavioral and motor function.

Small vessel disease is characterized by sporadic obstruction of small vessels leading to neuronal cell death. These microinfarcts often escape detection by conventional magnetic resonance imaging and are identified only upon postmortem examination. Our work explores a brain-wide microinfarct model in awake head-fixed mice, where occlusions of small penetrating arterioles are reproduced by endovascular injection of fluorescent microspheres. Mesoscopic functional connectivity was mapped longitudinally in awake GCaMP6 mice using genetically encoded calcium indicators for transcranial wide-field calcium imaging. Microsphere occlusions were quantified and changes in cerebral blood flow were measured with laser speckle imaging. The neurodeficit score in microinfarct mice was significantly higher than in sham, indicating impairment in motor function. The novel object recognition test showed a reduction in the discrimination index in microinfarct mice compared to sham. Graph-theoretic analysis of functional connectivity did not reveal significant differences in functional connectivity between sham and microinfarct mice. While behavioral tasks revealed impairments following microinfarct induction, the absence of measurable functional alterations in cortical activity has a less straightforward interpretation. The behavioral alterations produced by this model are consistent with alterations observed in human patients suffering from microinfarcts and support the validity of microsphere injection as a microinfarct model.

Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.

Many therapies have shown promise in preclinical stroke studies, but few benefit patients. A greater understanding of stroke pathophysiology is needed to successfully develop therapies, and this depends on appropriate animal models. The collagenase and blood infusion models of intracerebral hemorrhage (ICH) are widely used; yet, investigators often prefer using one model for a variety of reasons. Thus, we directly compared these to highlight advantages and limitations of each as well as the assessment approach. An ICH was created by infusing blood or bacterial collagenase into the rats' striatum. We matched initial hematoma volume in each model (Experiment 1) and assessed the time course of bleeding (Experiment 2). Functional deficits and the progression of injury were tracked over 6 weeks using behavior, magnetic resonance imaging, and histology (Experiment 3). Despite similar initial hematoma volumes, collagenase-induced ICH resulted in a greater blood-brain barrier breakdown and more damage to the striatum, substantia nigra, white matter, and cortex. Magnetic resonance imaging revealed faster hematoma resolution in the blood model, and little increase in the volume of tissue lost from 1 to 6 weeks. In contrast, tissue loss continued over 4 weeks in the collagenase model. Finally, functional deficits recovered more quickly and completely in the blood model. This study highlights key differences between these models and that neither closely replicates the human condition. Thus, both should be used whenever possible taking into account the significant differences between these models and their limitations. Furthermore, this work illustrates significant weaknesses with several outcome measures.

Social instability blocks functional restitution following motor cortex stroke in rats.

Social interactions have previously been shown to influence stroke outcome. In the current experiment we investigated the effects of a changing social environment on anatomical and behavioral recovery following motor cortex stroke in rats. Adult rats were trained on the Whishaw single pellet reaching task prior to receiving a devascularizing stroke lesion of the motor cortex. During the post-stroke testing period half of the rats were exposed to a form of social experience that has previously been shown to stimulate synaptic plasticity in frontal cortex circuitry, whereas the remaining rats were housed in pairs, in standard cages. At the end of the experiment the brains were processed for Golgi-Cox staining and dendritic length was measured in layer V of the intact forelimb motor area, layer III of Zilles' area Cg3 and layer II/III of Zilles' area AID. Social experience was found to completely block the normal spontaneous behavioural restitution in the lesion animals. Anatomically, whereas social experience selectively increased dendritic length in AID in rats that had not undergone behavioral training or the stroke procedure, this was not seen in the lesion animals, as the lesion alone produced an increase in dendritic length in both AID and Cg3. The findings are discussed in terms of the role of social experiences, including stress, on spontaneous plasticity that occurs following unilateral motor cortex stroke, and the effectiveness of inducing synaptic plasticity to promote behavioural recovery.

An Automated Home-Cage System to Assess Learning and Performance of a Skilled Motor Task in a Mouse Model of Huntington's Disease.

Behavioral testing is a critical step in assessing the validity of rodent models of neurodegenerative disease, as well as evaluating the efficacy of pharmacological interventions. In models of Huntington's disease (HD), a gradual progression of impairments is observed across ages, increasing the need for sensitive, high-throughput and longitudinal assessments. Recently, a number of automated systems have been developed to perform behavioral profiling of animals within their own home-cage, allowing for 24-h monitoring and minimizing experimenter interaction. However, as of yet, few of these have had functionality for the assessment of skilled motor learning, a relevant behavior for movement disorders such as HD. To address this, we assess a lever positioning task within the mouse home-cage. Animals first acquire a simple operant response, before moving to a second phase where they must learn to hold the lever for progressively longer in a rewarded position range. Testing with this paradigm has revealed the presence of distinct phenotypes in the YAC128 mouse model of HD at three early symptomatic time points. YAC128 mice at two months old, but not older, had a motor learning deficit when required to adapt their response to changes in task requirements. In contrast, six-month-old YAC128 mice had disruptions of normal circadian activity and displayed kinematic abnormalities during performance of the task, suggesting an impairment in motor control. This system holds promise for facilitating high throughput behavioral assessment of HD mouse models for preclinical therapeutic screening.

Targeted ischemic stroke induction and mesoscopic imaging assessment of blood flow and ischemic depolarization in awake mice.

Despite advances in experimental stroke models, confounding factors such as anesthetics used during stroke induction remain. Furthermore, imaging of blood flow during stroke is not routinely done. We take advantage of in vivo bihemispheric transcranial windows for longitudinal mesoscopic imaging of cortical function to establish a protocol for focal ischemic stroke induction in target brain regions using photothrombosis in awake head-fixed mice. Our protocol does not require any surgical steps at the time of stroke induction or anesthetics during either head fixation or photoactivation. In addition, we performed laser speckle contrast imaging and wide-field calcium imaging to reveal the effect of cortical spreading ischemic depolarization after stroke in both anesthetized and awake animals over a spatial scale encompassing both hemispheres. With our combined approach, we observed ischemic depolarizing waves (3 to [Formula: see text]) propagating across the cortex 1 to 5 min after stroke induction in genetically encoded calcium indicator mice. Measures of blood flow by laser speckle were correlated with neurological impairment and lesion volume, suggesting a metric for reducing experimental variability. The ability to follow brain dynamics immediately after stroke as well as during recovery may provide a valuable guide to develop activity-dependent therapeutic interventions to be performed shortly after stroke induction.

Intact skull chronic windows for mesoscopic wide-field imaging in awake mice.

BACKGROUND: Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. NEW METHOD: We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. RESULTS: The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30min. A single set-screw provides a stable means of attachment (in relation to the measured lateral and axial resolution) for mesoscale assessment without obscuring the cortical field of view. COMPARISON WITH EXISTING METHODS: We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice in longitudinal studies of up to 2 months in duration. CONCLUSIONS: We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors or in transgenic mice expressing genetically encoded sensors of activity.

High-throughput automated home-cage mesoscopic functional imaging of mouse cortex.

Mouse head-fixed behaviour coupled with functional imaging has become a powerful technique in rodent systems neuroscience. However, training mice can be time consuming and is potentially stressful for animals. Here we report a fully automated, open source, self-initiated head-fixation system for mesoscopic functional imaging in mice. The system supports five mice at a time and requires minimal investigator intervention. Using genetically encoded calcium indicator transgenic mice, we longitudinally monitor cortical functional connectivity up to 24 h per day in >7,000 self-initiated and unsupervised imaging sessions up to 90 days. The procedure provides robust assessment of functional cortical maps on the basis of both spontaneous activity and brief sensory stimuli such as light flashes. The approach is scalable to a number of remotely controlled cages that can be assessed within the controlled conditions of dedicated animal facilities. We anticipate that home-cage brain imaging will permit flexible and chronic assessment of mesoscale cortical function.

A mouse model of small-vessel disease that produces brain-wide-identified microocclusions and regionally selective neuronal injury.

We developed a mouse model of small-vessel disease where occlusions are produced through endovascular injection of fluorescent microspheres that target ~12 µm diameter penetrating arterioles and can be localized in histology. Using Thy1-GFP transgenic mice, we visualized the impact of microocclusions on neuronal structure. Microocclusions in the hippocampus produce cell loss or neuronal atrophy (~7% of lodged microspheres led to microinfarcts), while axons within white matter tracts, as well as the striatum and thalamus became blebbed or disrupted. Although the neocortex contained more occlusions than other structures, labeled layer 5 neurons were relatively resistant to structural damage, with <2% of the lodged microspheres producing obvious neuronal damage.

Stroke and the connectome: how connectivity guides therapeutic intervention.

Connections between neurons are affected within 3 min of stroke onset by massive ischemic depolarization and then delayed cell death. Some connections can recover with prompt reperfusion; others associated with the dying infarct do not. Disruption in functional connectivity is due to direct tissue loss and indirect disconnections of remote areas known as diaschisis. Stroke is devastating, yet given the brain's redundant design, collateral surviving networks and their connections are well-positioned to compensate. Our perspective is that new treatments for stroke may involve a rational functional and structural connections-based approach. Surviving, affected, and at-risk networks can be identified and targeted with scenario-specific treatments. Strategies for recovery may include functional inhibition of the intact hemisphere, rerouting of connections, or setpoint-mediated network plasticity. These approaches may be guided by brain imaging and enabled by patient- and injury-specific brain stimulation, rehabilitation, and potential molecule-based strategies to enable new connections.