Stefano Lai

Quantitative Kinematic Characterization of Reaching Impairments in Mice After a Stroke

Background and Objective. Kinematic analysis of reaching movements is increasingly used to evaluate upper extremity function after cerebrovascular insults in humans and has also been applied to rodent models. Such analyses can require time-consuming frame-by-frame inspections and are affected by the experimenter’s bias. In this study, we introduce a semi-automated algorithm for tracking forepaw movements in mice. This methodology allows us to calculate several kinematic measures for the quantitative assessment of performance in a skilled reaching task before and after a focal cortical stroke. Methods. Mice were trained to reach for food pellets with their preferred paw until asymptotic performance was achieved. Photothrombosis was then applied to induce a focal ischemic injury in the motor cortex, contralateral to the trained limb. Mice were tested again once a week for 30 days. A high frame rate camera was used to record the movements of the paw, which was painted with a nontoxic dye. An algorithm was then applied off-line to track the trajectories and to compute kinematic measures for motor performance evaluation. Results. The tracking algorithm proved to be fast, accurate, and robust. A number of kinematic measures were identified as sensitive indicators of poststroke modifications. Based on end-point measures, ischemic mice appeared to improve their motor performance after 2 weeks. However, kinematic analysis revealed the persistence of specific trajectory adjustments up to 30 days poststroke, indicating the use of compensatory strategies. Conclusions. These results support the use of kinematic analysis in mice as a tool for both detection of poststroke functional impairments and tracking of motor improvements following rehabilitation. Similar studies could be performed in parallel with human studies to exploit the translational value of this skilled reaching analysis.

Neuroplastic Changes Following Brain Ischemia and their Contribution to Stroke Recovery: Novel Approaches in Neurorehabilitation

Ischemic damage to the brain triggers substantial reorganization of spared areas and pathways, which is associated with limited, spontaneous restoration of function. A better understanding of this plastic remodeling is crucial to develop more effective strategies for stroke rehabilitation. In this review article, we discuss advances in the comprehension of post-stroke network reorganization in patients and animal models. We first focus on rodent studies that have shed light on the mechanisms underlying neuronal remodeling in the perilesional area and contralesional hemisphere after motor cortex infarcts. Analysis of electrophysiological data has demonstrated brain-wide alterations in functional connectivity in both hemispheres, well beyond the infarcted area. We then illustrate the potential use of non-invasive brain stimulation (NIBS) techniques to boost recovery. We finally discuss rehabilitative protocols based on robotic devices as a tool to promote endogenous plasticity and functional restoration.

A Robotic System for Quantitative Assessment and Poststroke Training of Forelimb Retraction in Mice

Background. Neurorehabilitation protocols based on the use of robotic devices have recently shown to provide promising clinical results. However, their efficacy is still limited because of the poor comprehension of the mechanisms at the basis of functional enhancements. Objective. To increase basic understanding of robot-mediated neurorehabilitation by performing experiments on a rodent model of stroke. Methods. Mice were trained to pull back a handle on a robotic platform and their performances in the task were evaluated before and after a focal cortical ischemic stroke. The platform was designed for the quantitative assessment of forelimb function via a series of parameters (time needed to complete the task, t-target; average force; number of sub-movements). Results. The animals rapidly learned the retraction task and reached asymptotic performance by the fifth session of training. Within 2 to 6 days after a small, endothelin-1-induced lesion in the caudal forelimb area, mice showed an increase in t-target and number of sub-movements and a corresponding decrease in the average force exerted. These parameters returned to baseline, pre-lesion values with continued platform training (10-14 days after stroke). Conclusions. These results highlight the utility of the devised platform for characterizing post-infarct deficits and improvements of forelimb performance. Further research is warranted to widen the understanding of device-dependent rehabilitation effects.

Combining robotic training and inactivation of the healthy hemisphere restores pre-stroke motor patterns in mice

Focal cortical stroke often leads to persistent motor deficits, prompting the need for more effective interventions. The efficacy of rehabilitation can be increased by ‘plasticity-stimulating’ treatments that enhance experience-dependent modifications in spared areas. Transcallosal pathways represent a promising therapeutic target, but their role in post-stroke recovery remains controversial. Here, we demonstrate that the contralesional cortex exerts an enhanced interhemispheric inhibition over the perilesional tissue after focal cortical stroke in mouse forelimb motor cortex. Accordingly, we designed a rehabilitation protocol combining intensive, repeatable exercises on a robotic platform with reversible inactivation of the contralesional cortex. This treatment promoted recovery in general motor tests and in manual dexterity with remarkable restoration of pre-lesion movement patterns, evaluated by kinematic analysis. Recovery was accompanied by a reduction of transcallosal inhibition and ‘plasticity brakes’ over the perilesional tissue. Our data support the use of combinatorial clinical therapies exploiting robotic devices and modulation of interhemispheric connectivity.

Post-Stroke Longitudinal Alterations of Inter-Hemispheric Correlation and Hemispheric Dominance in Mouse Pre-Motor Cortex.

Purpose Limited restoration of function is known to occur spontaneously after an ischemic injury to the primary motor cortex. Evidence suggests that Pre-Motor Areas (PMAs) may “take over” control of the disrupted functions. However, little is known about functional reorganizations in PMAs. Forelimb movements in mice can be driven by two cortical regions, Caudal and Rostral Forelimb Areas (CFA and RFA), generally accepted as primary motor and pre-motor cortex, respectively. Here, we examined longitudinal changes in functional coupling between the two RFAs following unilateral photothrombotic stroke in CFA (mm from Bregma: +0.5 anterior, +1.25 lateral). Methods Local field potentials (LFPs) were recorded from the RFAs of both hemispheres in freely moving injured and naïve mice. Neural signals were acquired at 9, 16 and 23 days after surgery (sub-acute period in stroke animals) through one bipolar electrode per hemisphere placed in the center of RFA, with a ground screw over the occipital bone. LFPs were pre-processed through an efficient method of artifact removal and analysed through: spectral,cross-correlation, mutual information and Granger causality analysis. Results Spectral analysis demonstrated an early decrease (day 9) in the alpha band power in both the RFAs. In the late sub-acute period (days 16 and 23), inter-hemispheric functional coupling was reduced in ischemic animals, as shown by a decrease in the cross-correlation and mutual information measures. Within the gamma and delta bands, correlation measures were already reduced at day 9. Granger analysis, used as a measure of the symmetry of the inter-hemispheric causal connectivity, showed a less balanced activity in the two RFAs after stroke, with more frequent oscillations of hemispheric dominance. Conclusions These results indicate robust electrophysiological changes in PMAs after stroke. Specifically, we found alterations in transcallosal connectivity, with reduced inter-hemispheric functional coupling and a fluctuating dominance pattern. These reorganizations may underlie vicariation of lost functions following stroke.

Evaluation of the difference in caries experience in diabetic and non-diabetic children—A case control study

Aim To evaluate the caries prevalence and related variables in Type 1 diabetic and non-diabetic children and among the diabetic children according to their metabolic status. Methods Sixty-eight diabetic and 136 non-diabetic children, matching by gender and age (4–14 years) were enrolled. The diabetic children were divided: a) 20 children in good metabolic control (Hb1ac≤7.5) and b) 48 children in bad metabolic control (Hb1ac>7.5). Dietary and oral hygiene habits were investigated. Caries status was registered using the International Caries Detection and Assessment System. Oral microflora was analysed using the checkerboard DNA-DNA hybridisation method. Plaque acidogenicity was recorded after a sucrose rinse. Results Sugared beverage and snack intake was higher in diabetic group compared to non-diabetic group (p = 0.03 and p = 0.04, respectively) and in subjects in bad metabolic control (p = 0.03 and p<0.01, respectively). Oral hygiene habits were similar, except for the use of fluoridated adjuvants, higher in non-diabetic children (p = 0.04). No statistically significant differences were observed regarding caries figures, but a higher number of caries free subjects was found in diabetic subjects in good metabolic control (p<0.01). Significant difference for the main cariogenic bacteria was found between diabetic and non-diabetic subjects (p<0.05). The pH values showed statistically significant differences between diabetic and non-diabetic subjects and between diabetic subjects in good and bad metabolic control (p<0.01). Conclusions Diabetic children in good metabolic control might even be considered at low caries risk, while those in bad metabolic control showed an oral environment prone to a high caries risk.

Rehabilitation Restores Cortical Activation Profiles And Stabilizes Synaptic Contacts After Stroke

Neuro-rehabilitative therapy is the most effective treatment for recovering motor deficits in stroke patients. Nevertheless, the neural basis of recovery associated with rehabilitative intervention is debated. Here, we addressed the multiple facets of cortical remodeling induced by rehabilitative therapy. By longitudinal imaging of cortical activity while training, we demonstrated progressive attenuation of motor map dedifferentiation and rise of a more intense and fast-rising calcium response in the peri-infarct area during movement execution. Coupling of the spared cortex to the injured hemisphere was reinforced by rehabilitation, as demonstrated by our all-optical approach. Alongside, profound angiogenic response accompanied the stabilization of peri-infarct micro-circuitry at the synaptic level. The present work, by combining optical tools of visualization and manipulation of neuronal activity, provides the first in vivo evidence of the mechanism of rehabilitation in shaping cortical plasticity.Rehabilitation is the most effective treatment for promoting the recovery of motor deficits after stroke. Despite its importance, the processes associated with rehabilitative intervention are poorly understood. One of the most challenging experimental goals is to unambiguously link specific circuit changes induced by rehabilitation in recovering animals to improved behavior. Here, we investigate which facets of cortical remodeling are induced by rehabilitative therapy after stroke by combining optical imaging and manipulation tools. We demonstrate the progressive restoration of cortical motor maps and of cortical activity in parallel with the reinforcement of inter-hemispheric connectivity after rehabilitation. Furthermore, we reveal that the increase in vascular density goes along with the stabilization of peri-infarct neural circuitry at synaptic level. The present work provides the first evidences that rehabilitation is sufficient to promote the combined recovery of distinct structural and functional features distinctive of healthy neuronal networks. We believe that understanding the plastic changes induced by rehabilitation will lead to more effective interventions to improve recovery after stroke.

Rehabilitation-triggered cortical plasticity after stroke: in vivo imaging at multiple scales (Conference Presentation)

Neurorehabilitation protocols based on the use of robotic devices provide a highly repeatable therapy and have recently shown promising clinical results. Little is known about how rehabilitation molds the brain to promote motor recovery of the affected limb. We used a custom-made robotic platform that provides quantitative assessment of forelimb function in a retraction test. Complementary imaging techniques allowed us to access to the multiple facets of robotic rehabilitation-induced cortical plasticity after unilateral photothrombotic stroke in mice Primary Motor Cortex (Caudal Forelimb Area - CFA). First, we analyzed structural features of vasculature and dendritic reshaping in the peri-infarct area with two-photon fluorescence microscopy. Longitudinal analysis of dendritic branches and spines of pyramidal neurons suggests that robotic rehabilitation promotes the stabilization of peri-infarct cortical excitatory circuits, which is not accompanied by consistent vascular reorganization towards pre-stroke conditions. To investigate if this structural stabilization was linked to functional remapping, we performed mesoscale wide-field imaging on GCaMP6 mice while performing the motor task on the robotic platform. We revealed temporal and spatial features of the motor-triggered cortical activation, shining new light on rehabilitation-induced functional remapping of the ipsilesional cortex. Finally, by using an all-optical approach that combines optogenetic activation of the contralesional hemisphere and wide-field functional imaging of peri-infarct area, we dissected the effect of robotic rehabilitation on inter-hemispheric cortico-cortical connectivity.