Neuron type-specific optogenetic stimulation for differential stroke recovery in chronic capsular infarct.

Cortical neuromodulation (CNM) is widely used to promote recovery after stroke. Despite the beneficial results of CNM, the roles played by different neuron types in the effects of current CNM techniques are unable to be differentiated. Our aim was to use selective optogenetic cortical stimulation to explore how different subpopulations of neuronal cells contribute to poststroke recovery. We transduced the sensory-parietal cortex (SPC) of rats with CamKII-ChR2 (pyramidal neurons), PV-ChR2 (parvalbumin-expressing inhibitory neurons), or hSyn-ChR2 (pan-neuronal population) before inducing photothrombotic capsular infarct lesions. We found that selective stimulation of inhibitory neurons resulted in significantly greater motor recovery than stimulation of excitatory neurons or the pan-neuronal population. Furthermore, 2-deoxy-2-[18F] fluoro-D-glucose microPET (FDG-microPET) imaging revealed a significant reduction in cortical diaschisis and activation of the corticostriatal neural circuit, which were correlated with behavioral recovery in the PV-ChR2 group. The spatial pattern of brain-derived neurotrophic factor (BDNF) expression was evident in the stimulated cortex and underlying cortico-subcortical circuit. Our results indicate that the plasticity of inhibitory neurons is crucial for functional recovery after capsular infarct. Modifying CNM parameters to potentiate the stimulation of inhibitory neurons could improve poststroke outcomes.

Excessive Astrocytic GABA Causes Cortical Hypometabolism and Impedes Functional Recovery after Subcortical Stroke.

Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke.

Ultrastructural Dendritic Changes Underlying Diaschisis After Capsular Infarct.

Diaschisis has been described as functional depression distant to the lesion. A variety of neuroscientific approaches have been used to investigate the mechanisms underlying diaschisis. However, few studies have examined the pathological changes in diaschisis at ultrastructural level. Here, we used a rat model of capsular infarct that consistently produces diaschisis in ipsilesional and contralesional motor and sensory cortices. To verify the occurrence of diaschisis and monitor time-dependent changes in diaschisis, we performed longitudinal 2-deoxy-2-[18F]-fluoro-d-glucose microPET (FDG-microPET) study. We also used light and electron microscopy to identify the microscopic and ultrastructural changes at the diaschisis site at 7, 14, and 21 days after capsular infarct modeling (CIM). FDG-microPET showed the occurrence of diaschisis after CIM. Light microscopic examinations revealed no significant histopathological changes at the diaschisis site except a mild degree of reactive astrogliosis. However, electron microscopy revealed swollen, hydropic degeneration of axial dendrites and axodendritic synapses, although the neuronal soma (including nuclear chromatin and cytoplasmic organelles) and myelinated axons were relatively well preserved up to 21 days after injury. Furthermore, number of axodendritic synapses was significantly decreased after CIM. These data indicate that a circumscribed subcortical white-matter lesion produces ultrastructural pathological changes related to the pathogenesis of diaschisis.

Three-axis Modification of Coordinates Enables Accurate Stereotactic Targeting in Non-human Primate Brains of Different Sizes.

The brain grows with age in non-human primates (NHPs). Therefore, atlas-based stereotactic coordinates cannot be used directly to target subcortical structures if the size of the animal's brain differs from that used in the stereotactic atlas. Furthermore, growth is non-uniform across different cortical regions, making it difficult to simply apply a single brain-expansion ratio. We determined the skull reference lines that best reflect changes in brain size along the X, Y, and Z axes and plotted the changes in reference-line length against the changes in body weight. The skull reference lines had a linear relationship with body weight. However, comparison of skull reference lines with body weight confirmed the non-uniform skull growth during postnatal development, with skull growth more prominent in the X and Y axes than the Z axis. Comparing the differences between the atlas-based lengths and those calculated empirically from plot-based linear fits, we created craniometric indices that can be used to modify stereotactic coordinates along all axes. We verified the accuracy of the corrected stereotactic targeting by infusing dye into internal capsule in euthanized and preserved NHP brains. Our axis-specific, craniometric-index-adjusted stereotactic targeting enabled us to correct for targeting errors arising from differences in brain size. Histological verification showed that the method was accurate to within 1 mm. Craniometric index-adjusted targeting is a simple and relatively accurate method that can be used for NHP stereotactic surgery in the general laboratory, without the need for high-resolution imaging.

Simplified adaptor for stereotactic surgery in non-human primates.

BACKGROUND: It is challenging for researchers performing stereotactic procedures to transition from small animals to non-human primate (NHP) experiments. The NHP stereotactic atlas is based on ear-bar zero (EBZ), which is an anatomical reference frame that is not visible during surgery. Most current NHP stereotactic systems require high-cost MRI or CT imaging and complex computer processing to determine the stereotactic coordinates, limiting the procedure to those with significant expertise. NEW METHOD: We have designed a simplified adaptor consisting of a circular arc for coronal tilt, a carrier for electrodes or cannulas, and an anchor to attach the adaptor to a conventional stereotactic frame. Our adaptor allows easy identification of the EBZ with the help of an anchor notch, and provides digital distance sensors without the need for imaging data or computer processing. Our system enables the use of trajectories that avoid injury to important structures and vessels. RESULTS: We tested the accuracy of our system using simulated targeting with phantoms, and demonstrated sub-millimeter accuracy. Infusion of methylene blue also showed satisfactory staining in target structures deep in the brain. COMPARISON WITH EXISTING METHODS: This system does not require high-cost imaging and extra training to determine EBZ. Once EBZ is set automatically by the system itself, targeting is similar to that in small animal stereotactic procedure. CONCLUSION: Our simple adaptor will aid researchers who plan to conduct experiments involving stereotactic surgery in NHPs.

An Experimental Infarct Targeting the Internal Capsule: Histopathological and Ultrastructural Changes.

BACKGROUND: Stroke involving the cerebral white matter (WM) has increased in prevalence, but most experimental studies have focused on ischemic injury of the gray matter. This study was performed to investigate the WM in a unique rat model of photothrombotic infarct targeting the posterior limb of internal capsule (PLIC), focusing on the identification of the most vulnerable structure in WM by ischemic injury, subsequent glial reaction to the injury, and the fundamental histopathologic feature causing different neurologic outcomes. METHODS: Light microscopy with immunohistochemical stains and electron microscopic examinations of the lesion were performed between 3 hours and 21 days post-ischemic injury. RESULTS: Initial pathological change develops in myelinated axon, concomitantly with reactive change of astrocytes. The first pathology to present is nodular loosening to separate the myelin sheath with axonal wrinkling. Subsequent pathologies include rupture of the myelin sheath with extrusion of axonal organelles, progressive necrosis, oligodendrocyte degeneration and death, and reactive gliosis. Increase of glial fibrillary acidic protein (GFAP) immunoreactivity is an early event in the ischemic lesion. WM pathologies result in motor dysfunction. Motor function recovery after the infarct was correlated to the extent of PLIC injury proper rather than the infarct volume. CONCLUSIONS: Pathologic changes indicate that the cerebral WM, independent of cortical neurons, is highly vulnerable to the effects of focal ischemia, among which myelin sheath is first damaged. Early increase of GFAP immunoreactivity indicates that astrocyte response initially begins with myelinated axonal injury, and supports the biologic role related to WM injury or plasticity. The reaction of astrocytes in the experimental model might be important for the study of pathogenesis and treatment of the WM stroke.

Remodeling of Neuronal Circuits After Reach Training in Chronic Capsular Stroke.

BACKGROUND: Subcortical capsular stroke has a poor prognosis, and it is not yet fully understood how and under what circumstances reach training contributes to motor recovery. Objective This study was performed to investigate changes in neuronal circuits and motor recovery in a chronic capsular stroke model in the presence or absence of reach training. METHOD: We generated photothrombotic capsular lesions in 42 Sprague-Dawley rats and evaluated motor recovery with or without daily training in a single-pellet reaching task (SPRT). We used 2-deoxy-2-[18F]-fluoro-D-glucose-microPET (positron emission tomography) to assess remodeling of neuronal circuits. RESULTS: SPRT training was selectively beneficial only for the group with incomplete capsular destruction (P < .05), suggesting the relevance of plasticity in the remaining capsular fibers for motor recovery. Groups that did not receive SPRT training showed no motor recovery at all. The microPET analysis demonstrated that motor recovery was correlated with a reduction in cortical diaschisis in ipsilesional motor and sensory cortices and in the contralesional sensory cortex (Pearson's correlation, P < .05). We also observed training-dependent subcortical activation in the contralesional red nucleus, the internal capsule, and the ventral hippocampus (P < .0025; false discovery rate q < 0.05). The groups without reach training did not show the same degree of reduction in diaschisis or activation of the red nucleus. CONCLUSIONS: Our results suggest that motor recovery and remodeling of neuronal circuits after capsular stroke depend on the magnitude of the capsular lesion and on the presence or absence of reach training. Task-specific training is strongly indicated only when there is incomplete destruction of the capsular fibers.

Sensory-parietal cortical stimulation improves motor recovery in severe capsular infarct.

The prevalence of subcortical white matter strokes in elderly patients is on the rise, but these patients show mixed responses to conventional rehabilitative interventions. To examine whether cortical electrical stimulation can promote motor recovery after white matter stroke, we delivered stimulation to a small or wide region of sensory-parietal cortex for two weeks in a rodent model of circumscribed subcortical capsular infarct. The sham-operated group (SOG) showed persistent and severe motor impairments together with decreased activation in bilateral sensorimotor cortices and striatum. In contrast, sensory-parietal cortex stimulation significantly improved motor recovery: final recovery levels were 72.9% of prelesion levels in the wide stimulation group (WSG) and 37% of prelesion levels in the small stimulation group (SSG). The microPET imaging showed reversal of cortical diaschisis in both groups: in both hemispheres for the WSG, and in the hemisphere ipsilateral to stimulation in the SSG. In addition, we observed activation of the corpus callosum and subcortical corticostriatal structures after stimulation. The results from the c-Fos mapping study were grossly consistent with the microPET imaging. Sensory-parietal cortex stimulation may therefore be a useful strategy for overcoming the limits of rehabilitative training in patients with severe forms of subcortical capsular infarct.

Longitudinal changes in resting-state brain activity in a capsular infarct model.

Strokes attributable to subcortical infarcts have been increasing recently in elderly patients. To gain insight how this lesion influences the motor outcome and responds to rehabilitative training, we used circumscribed photothrombotic capsular infarct models on 36 Sprague-Dawley rats (24 experimental and 12 sham-operated). We used 2-deoxy-2-[(18)F]-fluoro-D-glucose-micro positron emission tomography (FDG-microPET) to assess longitudinal changes in resting-state brain activity (rs-BA) and daily single-pellet reaching task (SPRT) trainings to evaluate motor recovery. Longitudinal FDG-microPET results showed that capsular infarct resulted in a persistent decrease in rs-BA in bilateral sensory and auditory cortices, and ipsilesional motor cortex, thalamus, and inferior colliculus (P<0.0025, false discovery rate (FDR) q<0.05). The decreased rs-BA is compatible with diaschisis and contributes to manifest the malfunctions of lesion-specific functional connectivity. In contrast, capsular infarct resulted in increase of rs-BA in the ipsilesional internal capsule, and contralesional red nucleus and ventral hippocampus in recovery group (P<0.0025, FDR q<0.05), implying that remaining subcortical structures have an important role in conducting the recovery process in capsular infarct. The SPRT training facilitated motor recovery only in rats with an incomplete destruction of the posterior limb of the internal capsule (PLIC) (Pearson's correlation, P<0.05). Alternative therapeutic interventions are required to enhance the potential for recovery in capsular infarct with complete destruction of PLIC.

A rat model of photothrombotic capsular infarct with a marked motor deficit: a behavioral, histologic, and microPET study.

We present a new method for inducing a circumscribed subcortical capsular infarct (SCI), which imposes a persistent motor impairment in rats. Photothrombotic destruction of the internal capsule (IC) was conducted in Sprague Dawley rats (male; n=38). The motor performance of all animals was assessed using forelimb placing, forelimb use asymmetry, and the single pellet reaching test. On the basis of the degree of motor recovery, rats were subdivided into either the poor recovery group (PRG) or the moderate recovery group (MRG). Imaging assessment of the impact of SCI on brain metabolism was performed using 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) microPET (positron emission tomography). Photothrombotic lesioning using low light energy selectively disrupted circumscribed capsular fibers. The MRG showed recovery of motor performance after 1 week, but the PRG showed a persistent motor impairment for >3 weeks. Damage to the posterior limb of the IC (PLIC) is more effective for producing a severe motor deficit. Analysis of PET data revealed decreased regional glucose metabolism in the ipsilesional motor and bilateral sensory cortex and increased metabolism in the contralesional motor cortex and bilateral hippocampus during the early recovery period after SCI. Behavioral, histologic, and functional imaging findings support the usefulness of this novel SCI rat model for investigating motor recovery.