Improved post-stroke spontaneous recovery by astrocytic extracellular vesicles

Spontaneous recovery after a stroke accounts for a major part of the neurological recovery in patients. However limited, the spontaneous recovery is mechanistically driven by axonal restorative processes for which several molecular cues have been previously described. We report the acceleration of spontaneous recovery in a preclinical model of ischemia/reperfusion in rats via a single intracerebroventricular administration of extracellular vesicles released from primary cortical astrocytes. We used MRI, confocal and multiphoton microscopy to correlate the structural remodeling of the corpus callosum and striatocortical circuits with neurological performance over 21 days. We also evaluated the functionality of the corpus callosum by repetitive recordings of compound action potentials to show that the recovery facilitated by astrocytic extracellular vesicles was both anatomical and functional. Our data provide compelling evidence that astrocytes can hasten the basal recovery that naturally occurs post-stroke through the release of cellular mediators contained in extracellular vesicles.


Introduction
The epidemiology of stroke has dynamically changed over recent decades, during which a constant improvement in clinical care and increased preventive measures, acute treatment, and timely neurorehabilitation have reduced the fatality rate and transformed into a prevailing cause of chronically disabling disease across the developed world 1 and several developing nations, particularly in Latin America 2,3 . Acute ischemic stroke impacts cognition, sensation, vision, language, and motor performance, with prominent signs of hemianopsia (loss of sight in half of the visual field), diplopia (double vision of the same object), speech deficits, paresis (muscular weakness), paresthesia (abnormal sensations of the skin) and other motor and sensory deficits 4 .
Despite impactful neurological impairment, most patients regain some of their lost neurological functions without intervention via a phenomenon of spontaneous recovery, which is determined exclusively by the passage of time 5 . Such spontaneous recovery occurs rapidly, at the level of impairment, and is driven by plasticity mechanisms initiated by the stroke 6 . Functional motor gains related to spontaneous recovery are considered to be due to increased connectivity of motor network areas that were initially disjointed by the ischemic attack 7 . However, the extent of recovery varies among patients, and the level of neurological regain is considered to be proportional to the initial deficit 8 . Rehabilitation during a narrow time window can enhance functional recovery, primarily through compensation. However, most clinical trials have failed to show differences in outcome endpoints between experimental and control interventions 9 .
Studies in animal models attempted to elucidate a time-limited period of heightened plasticity after focal brain injury and the mechanisms behind spontaneous recovery 6,10 . Cumulative data indicate that axonal repair, neurogenesis, and inflammation resolution mechanistically intervene in this recovery 11,12 . Particularly, axonal remodeling is a principal mechanism in which surviving neurons in the peri-infarct cortex establish new connections within the motor, somatosensory, and premotor areas within the ipsilesional hemisphere, with some reaching contralesional sites 13,14 , while others even innervate the frontal motor regions to the brainstem or spinal cord 15 . However, these new projections alter the topography of cortical projections in the somatosensory system and change the aggregate map of projections 16,17 .
Astrocytes and astrocytic gliosis are known to impede the recovery of injured tissue by blocking axonal sprouting through the production of structural astrocytic proteins such as glial fibrillary acidic protein (GFAP) and vimentin 18 , as well as neurite-inhibiting signaling molecules such as Nogo-A 19 . Nonetheless, astrocytes have multiple essential support functions, whose loss can precipitate or contribute to neurodegeneration 20 . Astrocytes are more resilient against ischemia than neurons; 21,22 therefore, astrocyte survival holds the potential to restore neuronal integrity and promote a functional improvement, especially in the ischemic penumbra 23 .
Here we evaluated in a preclinical stroke model caused by the transient occlusion of the middle cerebral artery (MCAO) in rat whether astrocytes might influence spontaneous recovery through signaling mechanisms mediated by the release of extracellular vesicles (EVs), which are known to contribute to the modulation of CNS physiology and pathology 24 .

EV release from astrocytes changes after hypoxia.
We isolated EVs released from primary astrocytes cultured under normoxic conditions to characterize their effects on stroke evolution. We also subjected astrocytes to a hypoxic challenge to assess whether EVs would retain their molecular activities in response to ischemia. We first ran a physical and molecular characterization of the EVs released from cultured primary cortical astrocytes obtained from P2 newborn rats. The isolation method employed in this study, which involves differential centrifugation, produces a homogeneous EV population with a size consistent with exosomes, as analyzed by transmission electron microscopy ( Figure 1a) and nanoparticle tracking analysis (Figure 1c, and d). The isolated EVs also expressed the canonical exosomal marker CD63 (Figure 1b). When we subjected the astrocytic cultures to hypoxia for six h, the number of exosomes collected in 48 h-conditioned media was significantly lower than the normoxic conditions (Figure 1 c, d, and e), so that a physiological outcome to ischemic stress would be reducing the release of EVs.
Administration of astrocyte-derived EVs in the rat brain.
In a pilot experiment, using a pulled glass microcapillary pipette, we administered stereotaxic injections of EVs collected from astrocyte-conditioned media resuspended in 0.1 mM PB into the lateral ventricle of the rat brain. We evaluated EV boluses with total protein concentrations of 200 and 400 ng determined by bicinchoninic acid protein assay in a volume of 4 µl. The higher concentration (400 ng) yielded a larger difference in infarct volume respective to vehicle-injected controls and was therefore used for the rest of the study. On average, we injected approximately 8.5×10 7 vesicles in each administration. Subsequently, we explored the distribution of EVs in the rat brain. We noted that exogenous EVs reached the striatum ( Figure 1f  1) and were preferentially localized in the perinuclear region. Using these experimental parameters, we studied the effects of EVs produced by astrocytes cultured under normoxic conditions for 48 h (NxEV) and those released by astrocytes over the same period after culturing under hypoxic conditions for six h (HxEV) to determine whether hypoxia modifies the neuroprotective potential of astrocyte EVs.

Spontaneous recovery in the preclinical model of stroke.
We began by determining the temporal evolution of infarct assessed with MRI. Figure 2a shows coronal slices from a T2-weighted MRI sequence of stroke-challenged animals at 24 h with NxEV or HxEV administration compared to control animals injected with vehicle alone. Evident at 24 h, there is a significant reduction in the average infarct volume in animals that received either type of EVs compared to the control group (p = 0.0157 for NxEV and p = 0.0163 for HxEV two-way ANOVA with Tukey, Figure 2a and 2c). At this time, there was no difference between NxEV and HxEV (p=0.9997), but a difference became apparent over time. Specifically, by day 21 after stroke, p values were 0.0144 for NxEV and <0.0001 for HxEV compared to the control and 0.002 between NxEV and HxEV (Figure 2b and 2c), demonstrating that a single injection of EVs at the beginning of the reperfusion phase contributed to the gradual reduction of the infarct volume. These observations correlated with the natural occurrence of spontaneous recovery in the rat after MCAO. We assessed the neurological performance of subjects on a series of tests that assess the integrity of sensory, motor, and sensorimotor circuits (Table 1)

Hastened recovery of structural alterations by the administration of astrocyte-derived EVs.
Using MRI, we studied the impact of EVs from cultured astrocytes on the evolution of the brain's physical recovery from stroke in a longitudinal study. We measured mean diffusivity, which is a quantitative measure that reflects cellular and membrane density-an increase of mean diffusivity results from edema and necrosis. The basal values of mean diffusivity ranged around 6×10 -4 mm 2 /ms in the corpus callosum, 3 ×10 -4 mm 2 /ms in the striatum, and 4 ×10 -4 mm 2 /ms in the cortex.
The values almost doubled in stroke-challenged rats at 24 h after the infarction, irrespective of treatment (Figure 3a), across all brain structures analyzed. Notably, these values declined gradually and were decisively lower at 7 d post-stroke in the animals that received EVs. This was particularly evident in the group that received HxEV, which showed baseline mean diffusivity values by day 21. The corpus callosum and striatum exhibited the most robust recovery rates in this parameter, coincidentally with structures with a lower cellular density than the cortex.
As noted above, the underlying processes of spontaneous recovery are firmly bound to axonal damage recovery. Axial diffusivity results from the structural integrity of axons and a decrease of this value mirrors axonal damage. We found that at 24 h post-stroke, all animals displayed a significant reduction in axial diffusivity that was more severe in the striatum>cortex>corpus callosum, which corresponds to the hierarchical structural damage produced by MCAO ( Figure   3b). Values in this parameter indicate a time-dependently recovery across all of the groups.
However, such recovery was more robust in HxEV, followed by the NxEV group. By day 21 poststroke, the axial diffusivities of HxEV in the striatum and cortex reached the baseline values; the overall recovery trend of the HxEV group was significantly different from that of the control group (p = 0.0001 for striatum and p < 0.0001 for cortex), while the group with NxEV showed only significant differences in the cortex (p = 0.4259 for striatum and p < 0.0001 for cortex).
Radial diffusivity is another DTI parameter that evaluates axonal integrity, which is increased with demyelination and reduced axonal density. We found this value to be doubled in the corpus callosum, almost quadrupled in the striatum and tripled in the cortex 24 h post-stroke ( Figure 3c).
The last parameter that we assessed with DTI was fractional anisotropy (FA), which is related to how freely water molecules move along axons and reflects their structural integrity. In contrast, HxEV did promote a significant improvement that evolved rapidly at 7 d post-stroke and exhibited almost normal values by day 14. Finally, in the cortex, with a more heterogeneous architecture than the corpus callosum and the striatum, the stroke-induced changes were of lesser magnitude by day 7. HxEV almost fully mitigated them at this early time point (Figure 3d), whereas NxEV did not affect the spontaneous recovery.
Stroke notably impacts the brain as a whole, and structural damage appears not to be restricted to the only sites where blood supply was impeded. Our MRI study shows that the brain hemisphere contralateral to the lesion had structural changes that resolved over time, and the administration of intact brains shows a complete reconstruction of the tract segments. In contrast, in the control group, the number of fibers that could be reconstructed was significantly limited, and the shape of reconstructed tracts is highly disorganized and atrophied. In the reconstructions of NxEV and HxEV groups, the length of assembled tracts was longer than that of their respective controls, and the number of fibers was also significantly increased.

Structural correlations of anatomical fibers with MRI.
We evaluated the integrity of brain neuronal structural fibers by immunolabeling microtubuleassociated protein 2 (MAP2) and βIII-tubulin (Tuj1). In control animals, there was a near total loss of dendritic staining with MAP2 and all neuronal processes with TUJ1 at 7 d post-stroke ( Figure   4). A gradual time-dependent recovery from this loss occurred across all groups, but only to a decidedly limited extent under control conditions. This limit was surpassed with the aid of astrocyte EVs, especially in the cortex (Figure 4). Also illustrated in Figure 4, the recovery of dendritic arborizations was greatly disorganized in the striatum of the control group animals. EVs did not help shape a better organization of the fibers by day 21, as indicated by the directionality of the tracts (Supplementary Figure 5). Overall, the appearance of the neuronal processes labeled with MAP2 and Tuj1 positively correlates with the MRI parameters described above, providing a histological grounding to the determinations made by indirect measurements of DTI.
Although the administration of EVs facilitated the recovery of the gross neuronal architectural conformation of the brain, we intended to discover whether this treatment also enabled the outgrowth of axons from the lesion core in the striatum towards the innervated cortical areas. We Next, we determined how the innervation of the ipsilateral cortex from the dorsal striatum was modified by stroke, using two-photon laser scanning confocal microscopy of the entire ipsilateral hemisphere. To achieve this, 20×20 binned images were converted to single pixels and mapped to the cortex in a polar plot with the center located at an anatomical point identifiable by the first major split of the M4 segment of the superior MCA trunk. Overlapping the image of a slice of the corresponding preparation of the contralateral side (corrected for anatomical symmetry) stained with cytochrome C oxidase to distinguish the barrel cortex allowed us to map the general territories of the motor and somatosensory cortices (Supplementary Figure 7). As expected, we found that the stroke caused a drastic loss of striatal projections to the somatosensory and motor cortices.
Whereas the administration of EVs did not necessarily rescue the lost innervations, it promoted the reorganization of innervated cortical areas that instead preferentially targeted the parietal somatosensory cortex ( Figure 5). The patterns reorganized by EVs were notably similar between normoxia and hypoxia. The motor cortex was predominantly spared in our experiments, possibly owing to the small infarct size on days 14 and 21 ( Figure 2).

Functional axonal regeneration of the corpus callosum.
The corpus callosum is the most prominent white matter tract in the mammalian brain, making this The I/O curves for N1 and N2 amplitudes in the control group were significantly shifted downwards relative to all other groups, indicating an injury-induced reduction of evoked action potentials in the myelinated and unmyelinated axon populations, respectively. In rats from the NxEV and HxEV groups, the N1 and N2 CAP amplitudes were significantly elevated above those of the control group, indicating a favorable degree of neurorestoration of myelinated and unmyelinated fibers. This was more prominent in non-myelinated fibers, revealing a greater susceptibility of myelinated axons.
The results obtained through the I/O curve measurements reveal a similar recovery pattern to that observed in FA analysis of samples from the corpus callosum (21 d, i). Likewise, there was a remarkable correspondence between electrophysiological results (CAPs for N1 at 10X, ii) and the functional recovery (combined neurological score at 21 d, iii) reported in the present study.

Identification of molecular mediators of axonal remapping within astrocyte-derived EVs cargo.
For the last part of this study, we set out to identify the possible molecular mediators of axonal regeneration contained in EVs released from astrocytes. To determine this, we performed a metaanalysis of three protein datasets from previously published astrocyte exosome-derived proteomes with n = 19 26 , n = 107 27 , and n = 219 28 identified proteins ( Figure 7a). We also performed a gene ontology (GO) over-representation test with R (3.6.3) package clusterProfiler 29 . Subsequently, we selected eight biological processes (BP) GO terms related to axonal growth and synaptogenesis, including synapse organization, response to axon injury, regulation of synapse structure or activity, postsynaptic cytoskeleton organization, postsynapse organization, neuron projection extension, modification of synaptic structure, and axonogenesis ( Figure 7b). Unique proteins belonging to these eight BP GO terms (n= 39) were analyzed using STRING with a minimum interaction score of 0.900 and k-means clustering of 3 ( Figure 7c).

Discussion
How astrocytes react to ischemic damage after stroke is a complex biological process that involves a coordinated response from a heterogeneous cell population across multiple phases. Here we report that astrocytes produce and release EVs loaded with chemical cues that instigate the axonal remapping of cortical areas damaged by stroke. Importantly, we determined that astrocytes subjected to hypoxic stress release EVs with an increased capacity for repair, although in significantly fewer numbers. Lastly, we found that this mechanism of intercellular communication accelerated the spontaneous recovery of experimental subjects in the subacute phase following stroke. The molecular link between astrocyte-produced biomolecules transmitted in EVs and functional recovery from stroke has not been established before.
The MCAO procedure used in the present study primarily affects motor coordination and stimuli perceptual integration, coded in the circuits of the somatosensory cortex and thalamus 30 . The subcortical damage affects functional connectivity in the somatosensory cortex, which correlates with cortical activations after electrical stimulation of the affected forelimbs 31 . Previous work shows that distinct sensorimotor pathways have a significant loss of connectivity two weeks after stroke in the rat 32 .
During spontaneous recovery, the reorganization of axonal connections and the overall histological patterns of the brain involve either compensation or repair, and the latter is considered to reflect functional recovery 33 . With work performed on stroke preclinical models, we know that the mechanisms for spontaneous recovery include promoting new brain cortical maps through axon sprouting, remyelination, and blockade of extracellular inhibitory signals 34,35 . After a stroke, axonal growth and repair mechanisms establish new projections in the contra-lesional hemisphere.
The axonal sprouting triggered by stroke generates new local intra-cortical projections as well as long inter-hemispheric projections 36 , which have been associated with functional recovery 37 .
Through the engagement of both the ipsilateral and contralateral hemispheres, the rostral and caudal portions of the motor cortex are involved in coordinating the skilled reaching performance in the rat 38 . In humans, the contra-lesional motor cortex plays a central role in the recovery of motor function 39 , but acute cortical reorganization following focal ischemia appears to occur less rapidly than in rodents 40 .
Regarding the activators of these repair processes, endogenous cellular and molecular processes occurring during a limited time window promote a regenerative microenvironment in the postacute ischemic phase. Physiologically, the axon growth cone that covers the terminal zone of neuritic processes is assembled, and along with the reorganization of the cytoskeleton in the proximal axon stump, initiates the regeneration process 41 . Growth cone formation and axon growth progression are regulated by extracellular factors and intracellular signaling molecules 42,43 .
In addition, it is known that neurons endure profound changes in their transcriptional profile in the subacute phase after stroke, which enables them to undergo plasticity changes 44 , and astrocytes activate a transcriptional profile that favors the expression of repairing genes 45 . We hypothesize that astrocytes signal neurons to activate the regenerative mechanisms through intercellular signaling driven by EVs. This type of communication mechanism has recently been shown to be capable of directing these processes. Astrocyte-derived exosomes with prostaglandin D2 synthase expression contribute to axonal outgrowth and functional recovery in stroke by inhibiting the axon growth blocker Semaphorin 3A 46 , providing evidence that the release of EVs from astrocyte does contribute to axonal growth signaling.
In our meta-analysis of astrocytic EV proteomes, we identified several proteins that regulate axon outgrowth and guidance. In this regard, TUBB is a beta-tubulin protein that is expressed in the developing CNS and is involved in neuronal proliferation, migration, differentiation, and axon guidance 47 . ACTG1 is a critical protein for axogenesis, axon guidance, and synaptogenesis 48 .
RhoA has been shown to restrict the initiation of neuronal polarization and axon outgrowth during development 49 , and its knockdown promotes axon regeneration 50 . RTN4 is a myelin-associated axon growth inhibitor that impairs axon regeneration in the adult mammalian CNS 51 . Cdc42 targets the cytoskeleton, cell adhesion, and polarity regulators 52 , and it is involved in axon guidance 53 . TUBA4A and PFN1 regulate cytoskeletal dynamics 54 . SPARC is a secreted protein involved in synapse pruning during development 55 . Rab11A is involved in axon outgrowth 56 .
Ephrin signaling is known to modulate axon guidance and synaptic plasticity and promote longterm potentiation 57,58 , and its inhibition has been targeted to augment recovery after stroke 59 .
EVs also regulate several physiological processes by delivering miRNAs to their target cells.
Numerous species of these molecules, which were previously found in astrocyte-shed EVs 28 are known to regulate axon growth and guidance. miRNAs in distal axons of cortical neurons, like miR15b, miR195, and miR26b, are known to integrate network regulatory systems that promote axonal growth 60 . Other species that block these processes, like miR203a and miR29a 60,61 , are decreased in EVs released from astrocytes subjected to proinflammatory signals. Yet other miRNA species, such as miR130a, are known to indirectly regulate VEGFR2 expression 62 , which is also involved in neuroadaptation that follows stroke 63 . The axon growth and guidance prompted by EVs most likely result from combining all these different regulatory molecules within the same vesicle. The exact mechanism of action of each miRNA contained within astrocytic EVs requires elucidation.
We enhanced input signals for axonal growth from astrocytes under stress (HxEV) and basal conditions (NxEV), which allowed us, under a permissive environment generated by the stroke, to potentiate the endogenous process of neurite growth and cortical remapping that underlies functional recovery. The endogenous production of EV content within the brain-resident astrocytes changes dynamically throughout recovery after the ischemic insult. By delivering these vesicles 30 min after initiation of the reperfusion phase, we are likely shortening the time taken for the endogenous reparative mechanisms to commence, which is a plausible explanation for the recovery accelerating effect induced by administering astrocytic EVs. The astrocytes in culture were free from the influence of classically activated microglia, which is known to induce A1 reactive astrocytes that promote the death of neurons and oligodendrocytes 20 .
One of the most important findings of the present study is that astrocytes convey signals that directly reshape the innervation of affected cortical brain areas and potentiate axonal communication in impaired neuronal tracts through EVs. These effects are accompanied by neurological improvement after stroke. Previous studies have shown that astrocytes are highly heterogeneous cells, with typical types accounting for ̴ 70% of astrocytes in culture 64 , promoting neuron adhesion and neurite growth, while atypical astrocytes inhibit such processes 65 . Reactive astrocytes, like those that form glial scars after brain damage, potentially limit axonal growth after stroke, mainly by producing an extrinsic inhibitory environment. Our results might also reflect the enhancement of soluble signals released by repair-promoting astrocytes, and by the external administration of these EVs, we could surpass the physical inhibitory barriers promoted by reactive gliosis.
Given the dire need for suitable biomarkers for post-stroke plasticity mechanisms, we propose that astrocyte-derived particles, which can be isolated from human plasma 66,67 , may serve as functional markers for brain plasticity, especially in the chronic phase after stroke when neurological restoration is minimal and can only possibly be obtained by proper neurorehabilitative interventions.
In conclusion, understanding the biological basis of neurological function restoration after stroke is critical for designing intervention therapies by the exploitation of endogenously coded mechanisms for the repair of the damaged brain. The use of isolated EVs from cultured astrocytes, even if unmodified, may shorten the time requires for neurological recuperation, or even more so, extend the very limited time window of spontaneous recovery and increase the proportion of functional gains in patients. Additional studies are warranted to explore the optimal time point for astrocyte-derived EVs administrations and the optimally effective and safe way to deliver them to the damaged brain.

Animals
This study used young 6-week-old (270-290 g) wild-type Wistar rats subjected to MCAO as

Study design
For MRI studies, animals were randomly divided into four groups (n = 4 / group). The sample size was calculated based on a previous study from our group used as pilot 63  This study was limited to assess effects on male rats to limit potential confounds of estrogenmediated neuroprotective actions present in female rodents.

MCAO
Ischemic stroke was performed as previously reported with slight modifications 63 . Briefly, rats were subjected to isoflurane anesthesia (5 % for induction followed by ≤1.5 % during surgery) with oxygen as the carrier. Normal ventilation was autonomously maintained. A nylon monofilament with a silicone-covered tip (403734, Doccol, Sharon, MA) was inserted through the ligated left external carotid artery, and intra-luminally advanced through the internal carotid artery until it occluded the MCA. The occlusion was maintained for 60 min after which the monofilament was removed. Body temperature was maintained at 37 °C with a heating pad for the duration of surgery. At the end of the procedure, the neck's skin was sutured, and rats were returned to their cages. During the entire experimental procedure, the cerebral blood flow (CBF) was monitored in the territory irrigated by the MCA with laser-Doppler flowmetry at the following stereotaxic coordinates; AP -1.5 L +3.5 from Bregma, with a laser-Doppler probe (model 407, Perimed, Järfälla, Sweden) connected to a Periflux System 5010 (Perimed). CBF was continuously monitored with an acquisition interval of 0.3 s using the Perisoft software (Perimed).

Primary astrocyte cell culture
Primary cortical astrocyte cell cultures were prepared and maintained using methods similar to those described previously 28 . Briefly, astrocytes were isolated from the cerebral cortices of postnatal day 1-2 Wistar rats. The tissue was digested with trypsin and subsequently mechanically dissociated in Hanks' balanced salt solution. The cell suspension was plated in poly-D-lysine culture flasks containing Dulbecco's modified Eagle's medium/F-12 medium (Gibco BRL) and 10% fetal bovine serum (FBS) (Gibco BRL). Cultures were shaken to remove less-adherent cells, with remaining cells determined to be ̴ 98% GFAP + astrocytes. Experiments were performed between 3 to 5 passages.

EVs isolation and characterization
EVs were purified from astrocyte cell culture supernatants under two experimental conditions: incubated for 48 h under normoxic conditions (NxEV) or for 6 h under hypoxia, followed by 42 h for recovery in normoxia (HxEV). For hypoxia, cultures were incubated inside a hypoxic chamber (Stemcell, Stemcell Technologies Inc., Canada) with a 100% N2 atmosphere for 6 h at 37°C. For the collection of exosomes secreted exclusively by astrocytes, a conditioned medium was prepared with exosome-free FBS. The conditioned medium was recovered and filtered with a 220 nm pore membrane to remove large cell debris, microvesicles derived from plasma membrane with a diameter greater than 220 nm, and large apoptotic bodies. The supernatant was collected and sequentially ultracentrifuged at 50,000 x g for 30 min, followed by 100,000 x g for 70 min. The purified vesicles were resuspended in 100 µl PBS.  The preparations were examined by confocal microscopy with an LSM 800 microscope (Zeiss, JENA, Germany) using a 63 X oil immersion objective.

Behavioral testing
Animals were evaluated with a battery of neurological tests to assess sensorimotor deficits at 24 h, 7, 14, and 21 d after stroke. The severity of functional deficits was scored by assessing eight items described in Table 1. All evaluations were cross-validated by a trained observer blinded to the experimental treatment that analyzed the tests' recorded videos.

Diffusion tensor imaging (DTI)
The diffusion tensor was fitted at each voxel for DTI analysis using DSI Studio (http://dsistudio.labsolver.org/Manual/diffusion-mri-indices; 12/6/2015). Next, tractography was performed using a deterministic fiber-tracking algorithm 68 . Seed regions were placed at the striatum and corpus callosum. The anisotropy threshold was 0.21, and the angular threshold was 30 degrees.
The step size was 0.7 mm, averaging the propagation direction with 30 % of the previous direction smoothed the fiber trajectories. A total of reconstructed streamlines (tracts), AD, MD, and RD, were calculated by area.

Immunofluorescence and confocal microscopy
For immunohistological analyses, three rats per group were anesthetized with pentobarbital (100 mg/kg) and transcardially perfused with 200 mL ice-cold 0.  Figure 6). This point was considered as the origin of the polar transformation. All the processing and plotting were undertaken with ad hoc Python scripts available at https://github.com/TYR-LAB-MX/PolarPlot_HerasRomero_2021.

In vivo electrophysiological recording
Input-output (I/O) curves were assessed 21 d after stroke. For this, animals (n=4 per group) were anesthetized with isoflurane 5% for induction, followed by ≤2% during surgery. Body temperature was maintained at 35° C with heating pads. Rats were placed on a stereotaxic frame, and the skull was exposed. A constant current was delivered by direct and unilateral stimulation of the corpus callosum using a bipolar stainless-steel electrode placed at the stereotaxic coordinates AP +0.2, ML -1.0, DV -3.7 from Bregma, with a Grass S48 stimulator and a photoelectric stimulus isolation unit Grass PSIU6 (Grass Instrument Co. Quincy, MS). Corpus callosal responses were recorded unilaterally with a monopolar stainless-steel electrode (127 μm diameter) placed at the stereotaxic coordinates AP +0.2, ML +1.0, DV -3.7 from Bregma. The evoked responses were measured with the compound action potentials (CAP) amplitude, measured in negative peak 1 (N1) and peak 2 (N2). I/O curves were built with threshold folding of intensity (1-10 X) to determine the axonal conduction for a range of stimulation intensities. The threshold was defined at the stimulation intensity required to produce a 0.10 mV amplitude response in N1. The electric signal was digitalized, stored, and analyzed using the software DataWave SciWorks (Broomfield, CO, USA).

Statistics
GraphPad Prism 8 was used to analyze all data. The normal distribution in each data set was corroborated using the Shapiro-Wilk normality test. Neurological scores and DTI parameter changes among experimental groups and over time within each group were tested on a 2-way analysis of variance ANOVA with repeated measures based on a general linear model, followed by Tukey's post hoc test. Data were considered significant at α ≤ 0.05 level.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Statement
All the animal experimentation was carried out at the Universidad Nacional Autónoma de México in Mexico City and Juriquilla Qro., Mexico, in accordance with the Mexican law for the use and

Conflict of interest
The research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest.

Neurological assessment
Reflex Score

Spontaneous activity
Exploring an open arena for more than 20 s 3 Exploring between 10 to 20 s 2 Exploring less than 10 s