Enriched housing enhances recovery of limb placement ability and reduces aggrecan-containing perineuronal nets in the rat somatosensory cortex after experimental stroke.

Stroke causes life long disabilities where few therapeutic options are available. Using electrical and magnetic stimulation of the brain and physical rehabilitation, recovery of brain function can be enhanced even late after stroke. Animal models support this notion, and housing rodents in an enriched environment (EE) several days after experimental stroke stimulates lost brain function by multisensory mechanisms. We studied the dynamics of functional recovery of rats with a lesion to the fore and hind limb motor areas induced by photothrombosis (PT), and with subsequent housing in either standard (STD) or EE. In this model, skilled motor function is not significantly enhanced by enriched housing, while the speed of recovery of sensori-motor function substantially improves over the 9-week study period. In particular, this stroke lesion completely obliterates the fore and hind limb placing ability when visual and whisker guidance is prevented, a deficit that persists for up to 9 weeks of recovery, but that is markedly restored within 2 weeks by enriched housing. Enriched housing after stroke also leads to a significant loss of perineuronal net (PNN) immunoreactivity; detection of aggrecan protein backbone with AB1031 antibody was decreased by 13-22%, and labelling of a glycan moiety of aggrecan with Cat-315 antibody was reduced by 25-30% in the peri-infarct area and in the somatosensory cortex, respectively. The majority of these cells are parvalbumin/GABA inhibitory interneurons that are important in sensori-information processing. We conclude that damage to the fore and hind limb motor areas provides a model of loss of limb placing response without visual guidance, a deficit also seen in more than 50% of stroke patients. This loss is amenable to recovery induced by multiple sensory stimulation and correlates with a decrease in aggrecan-containing PNNs around inhibitory interneurons. Modulating the PNN structure after ischemic damage may provide new therapies enhancing tactile/proprioceptive function after stroke

Changes in resting-state functional connectivity after stroke in a mouse brain lacking extracellular matrix components

In the brain, focal ischemia results in a local region of cell death and disruption of both local and remote functional neuronal networks. Tissue reorganization following stroke can be limited by factors such as extracellular matrix (ECM) molecules that prevent neuronal growth and synaptic plasticity. The brain's ECM plays a crucial role in network formation, development, and regeneration of the central nervous system. Further, the ECM is essential for proper white matter tract development and for the formation of structures called perineuronal nets (PNNs). PNNs mainly surround parvalbumin/GABA inhibitory interneurons, of importance for processing sensory information. Previous studies have shown that downregulating PNNs after stroke reduces the neurite-inhibitory environment, reactivates plasticity, and promotes functional recovery. Resting-state functional connectivity (RS-FC) within and across hemispheres has been shown to correlate with behavioral recovery after stroke. However, the relationship between PNNs and RS-FC has not been examined. Here we studied a quadruple knock-out mouse (Q4) that lacks four ECM components: brevican, neurocan, tenascin-C and tenascin-R. We applied functional connectivity optical intrinsic signal (fcOIS) imaging in Q4 mice and wild-type (129S1 mice) before and 14 days after photothrombotic stroke (PT) to understand how the lack of crucial ECM components affects neuronal networks and functional recovery after stroke. Limb-placement ability was evaluated at 2, 7 and 14 days of recovery through the paw-placement test. Q4 mice exhibited significantly impaired homotopic RS-FC compared to wild-type mice, especially in the sensory and parietal regions. Changes in RS-FC were significantly correlated with the number of interhemispheric callosal crossings in those same regions. PT caused unilateral damage to the sensorimotor cortex and deficits of tactile-proprioceptive placing ability in contralesional fore- and hindlimbs, but the two experimental groups did not present significant differences in infarct size. Two weeks after PT, a general down-scaling of regional RS-FC as well as the number of regional functional connections was visible for all cortical regions and most notable in the somatosensory areas of both Q4 and wild-type mice. Q4 mice exhibited higher intrahemispheric RS-FC in contralesional sensory and motor cortices compared to control mice. We propose that the lack of growth inhibiting ECM components in the Q4 mice potentially worsen behavioral outcome in the early phase after stroke, but subsequently facilitates modulation of contralesional RS-FC which is relevant for recovery of sensory motor function. We conclude that Q4 mice represent a valuable model to study how the elimination of ECM genes compromises neuronal function and plasticity mechanisms after stroke

Neuroscore.

<p>The normal neuroscore before stroke is 28. Values at different times after PT in rats subsequently housed in standard (STD) and enriched environment (EE) are presented in box plot: median; 1^st quartile to 3^rd quartile, minimum and maximum value. (n = 7 for each housing condition) (*: p<0.05 when STD compared to EE; #: p<0.05 when STD and EE where compared to the respective values before stroke).</p

Wharton\u2019s jelly derived mesenchymal stromal cells: Biological properties, induction of neuronal phenotype and current applications in neurodegeneration research

Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from bone marrow or other tissues, including fat, muscle and umbilical cord. It has been shown that MSC behave in vitro as stem cells: they self-renew and are able to differentiate into mature cells typical of several mesenchymal tissues. Moreover, the differentiation toward non-mesenchymal cell lineages (e.g. neurons) has been reported as well. The clinical relevance of these cells is mainly related to their ability to spontaneously migrate to the site of inflammation/damage, to their safety profile thanks to their low immunogenicity and to their immunomodulation capacities. To date, MSCs isolated from the post- natal bone marrow have represented the most extensively studied population of adult MSCs, in view of their possible use in various therapeutical applications. However, the bone marrow-derived MSCs exhibit a series of limitations, mainly related to their problematic isolation, culturing and use. In recent years, umbilical cord (UC) matrix (i.e. Wharton\u2019s jelly, WJ) stromal cells have therefore emerged as a more suitable alternative source of MSCs, thanks to their primitive nature and the easy isolation without relevant ethical concerns. This review seeks to provide an overview of the main biological properties of WJ-derived MSCs. Moreover, the potential application of these cells for the treatment of some known dysfunctions in the central and peripheral nervous system will also be discussed

Experimental design.

<p>Rats were trained, and baseline values set for the different functional tests. Rats were then subjected to PT and the functional deficits assessed 2 days later. Subsequently, rats were placed either in standard (STD) or enriched environment (EE). Functional tests were regularly performed during 9 weeks following the onset of the stroke (the time of the different tests are represented by color dots). At 9 weeks, rats were perfusion fixed and brains were analyzed by immunohistochemistry.</p

Skilled reaching test.

<p>Skilled reaching test at different times of recovery after PT stroke for animals housed in standard (STD) and enriched environment (EE). (A) The total number of pasta pieces reached during 20 minutes prior to PT (pre) and at different time points after PT. (#: p<0.05 when compared to the respective group before stroke (pre). (B) Map of the reaching area presenting the number of animals (as indicated by colors) that reached the pasta pieces (n = 7 for each housing condition).</p

Effect of stroke on gait.

<p>Presentation of gait parameters and the changes induced by PT. (A) Principle of the Catwalk. Rats run on a glass plate illuminated from the side. When the paws press the plate, light is diffracted and captured by a camera below the glass plate. The calculation of the gait parameters require the following steps: 1) the runs are recorded, 2) the light spots are associated with their respective paws, 3) the sequence of paw contact with the glass plate over time is determined and 4) the different gait parameters are calculated. (B) Stroke induces changes in different gait parameters. Here are summarized the general effect of stroke on gait two days after stroke. (C) Stroke also induces an asymmetry between left and right side in some gait parameters. The colored boxes represent time fractions when the paws are in contact with the surface of the glass plate. The black lines represent the values before stroke and the red lines the direction as well as increase or decrease of the values at 2 days after stroke (LH: left hind limb, RH: right hind limb). For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093121#pone.0093121.s003" target="_blank">Table S3</a>.</p

Environmental stimulation decreases the number of AB1031⁺ PV/GABA neurons in the rat cerebral cortex after PT.

<p>(A) Confocal images of parvalbumin expressing cells (green) enwrapped by AB1031⁺ PNNs (red) in the somatosensory cortex of a representative STD animal, scale bar 20 μm. (B and C) Representative bright-field micrographs of AB1031⁺ PNNs in the rat cerebral cortex ipsilateral to the lesion, scale bar 100 μm; (B1 and C1) higher magnification, scale bar 20 μm. AB1031 immunoreactivity in (B) STD and (C) EE conditions. Quantification of cortical neurons bearing AB1031⁺ PNNs in the peri-infarct cortex (D), the corresponding area contralateral to the lesion (E), the ipsilateral somatosensory cortex (F) and the contralateral somatosensory cortex (G). (n = 7 for each housing condition; 9 weeks after stroke; D with *p = 0.04; E with p = 0.3613; F with *p = 0.007; G with p = 0.0963; STD: standard environment, EE: enriched environment, PI: peri-infarct cortex, SS: somatosensory cortex, IPSI: ipsilateral, CONTRA: contralateral).</p

Enriched housing decreases the number of Cat-315⁺ PV/GABA neurons in the rat cerebral cortex after PT.

<p>(A) Confocal images of a parvalbumin (PV) expressing cell (green) enwrapped by a Cat-315⁺ PNN (red) in the somatosensory cortex of a representative STD animal; Z-stack demonstrates close proximity of the Cat-315 aggrecan antibody and PV, scale bar 20 μm. (B and C) Representative bright-field micrographs of Cat-315⁺ PNNs in the rat cerebral cortex ipsilateral to the lesion, scale bar 100 μm; (B1 and C1) higher magnification, scale bar 20 μm. Cat-315 immunoreactivity denotes a critical difference between (B) STD and (C) EE conditions. Quantification of cortical neurons bearing Cat-315⁺ PNNs in the peri-infarct cortex (D), the corresponding area contralateral to the lesion (E), the ipsilateral somatosensory cortex (F) and the contralateral somatosensory cortex (G). (n = 7 for each housing condition; 9 weeks after stroke; D with *p = 0.04; E with p = 0.06; F with *p = 0.0007; G with p = 0.1; STD: standard environment, EE: enriched environment, PI: peri-infarct cortex, SS: somatosensory cortex, IPSI: ipsilateral, CONTRA: contralateral).</p

Infarct Volume.

<p>Infarct volume was measured at 9 weeks after PT, and estimated by integration of the lesioned area on brain slices taken every 1-caudal brain axis. NeuN staining was performed to discriminate between intact and infarcted tissue. (A) Successive coronal slides of a representative brain infarct with their respective distances from the bregma for the STD housed animals, and (B) EE housed animals. (C) Infarct volume expressed as percentage of the hemisphere. Values are presented in box plots: median; 1^st quartile to 3^rd quartile; minimum and maximum value. (n = 7 for each housing condition; STD: standard environment, EE: enriched environment).</p