PLASTICITY MECHANISMS UNDERLYING MOTOR RECOVERY AFTER DEVELOPMENTAL AND ADULT ISCHEMIC STROKE IN RATS

UNIVERSITY OF PISA

PhD Course in Basic and Developmental Neuroscience

PLASTICITY MECHANISMS UNDERLYING

MOTOR RECOVERY AFTER DEVELOPMENTAL

AND ADULT ISCHEMIC STROKE IN RATS

Tutor: Prof Tommaso Pizzorusso Candidate: Mariangela Gennaro

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INDEX

1.1.1 The Role of Experience During Critical Period ... 10

1.1.2 Molecular “Players” of Critical Period Timecourse ... 12

1.1.3 Plasticity in Adult Brain: Beyond the Critical Period ... 15

1.1.4 The voluntary motor system: development, structure and function ... 18

1.1.4.1 Comparative Development of voluntary motor pathway, corticospinal tract (CST) ... 18

1.1.4.2 Factors contributing to CST development ... 19

1.1.5 Functional and Anatomical organization of Mature Voluntary Motor System ... 21

1.1.5.1 Corticospinal tract CST ... 21

1.1.5.2 Cortico-Rubrospinal Tract CRT ... 22

1.1.6 Stroke Pathophysiology ... 23

1.1.7 Animal Models of Ischemic Stroke ... 25

1.1.7.1 Preclinical Models of Adult Stroke ... 25

1.1.7.2 Preclinical Models Of Developmental Stroke ... 26

1.1.8 Rodents Corticospinal tract ... 27

1.1.9 Motor Outcome Greatly Differ after Young versus Adult Ischemic Stroke ... 28

1.1.9.1 Developmental Ischemic Stroke ... 28

1.1.9.2 Adult Ischemic Stroke ... 29

1.1.10 Post-Stroke Recovery ... 30

1.1.10.1 Timing of lesion occurrence establishes the pattern of Recovery ... 31

1.1.10.2 “Maladaptive Plasticity” after Developmental CST Injury ... 31

1.1.11 General Plasticity Rules in Stroke Recovery ... 34

1.1.11.1 Molecular substrates in stroke recovery ... 34

1.1.11.2 Cortical Remapping ... 36

1.1.11.3 Axonal reorganization after CNS injury ... 37

1.1.11.4 Dendritic changes after stroke ... 38

1.1.12 Treatment Options and Challenges ... 39

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1.1.12.2 Stem cell therapy ... 41

1.1.12.3 Neuro-restorative therapy ... 41

Aims of the Thesis ... 46

Chapter 2 ... 48

Modeling pediatric ischemic stroke in rats focus on plasticity mechanisms underlying motor recovery after early rehabilitative and / or pharmacological interventions ... 48

2.1 Introduction ... 49

2.2 Materials and Methods ... 51

2.2.1 Animals ... 51

2.2.2 Induction of juvenile focal stroke ... 51

2.2.3 Lesion size measurement ... 52

2.2.4 Profiling of motor impairments ... 52

2.2.5 Motor training ... 53

2.2.6 Kinematic analysis of reaching movement ... 53

2.2.7 Corticospinal tracing and axonal sprouting analysis ... 53

2.2.8 Pharmacological treatment with 7,8 DHF (dihydroxyflavone) ... 54

2.2.9 Behavioural assessment of 7,8 DHF effectiveness on fine motor outcome ... 54

2.2.10 Western blot analysis ... 54

2.2.11 Statistical analysis ... 55

2.3 Results ... 56

2.3.1 ET-1 injection into the forelimb representation of motor cortex generates a reproducible focal damage in P21 ... 56

2.3.2 Long term behavioral assessment of motor deficits following juvenile ET-1 stroke ... 57

2.3.3 ET-1 injury permanently alters walking behavior ... 57

2.3.4 ET-1 injury impairs vertical ladder climbing ... 58

2.3.5 ET-1 injury impairs grip strength ... 58

2.3.6 ET-1 injury causes permanent deficits in forelimb Skilled Reaching task ... 59

2.3.7 Early intervention with a specific Motor Training fully restores fine selective skilled abilities 60 2.3.8 Early specific motor training promotes partial recovery of normal reaching motor schemes62 2.3.9 Early specific training does not ameliorate unskilled general motor behavior ... 63

2.3.10 Rehabilitative training promotes sprouting of spared Corticospinal tract ... 64

2.3.11 Acute pharmacological Treatment with a BDNF mimetic, 7,8 Dihydrossyflavone ameliorates motor deficits after ET-1 injury induced at P21 ... 66

2.3.12 Molecular mechanisms underlying recovery of fine motor control in ET-1 injured animals treated with flavone ... 68

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2.4 Discussion ... 70

2.4.1 ET-1 intracortical injection in M1 forelimb area in P21 rats induce a focal ischemic stroke with long-lasting impairments ... 70

2.4.2 Early specific training promotes functional recovery in a task-dependent way ... 71

2.4.3 Training-induced plasticity of spared corticospinal tract CST ... 72

2.4.4 Acute Treatment with 7,8-Dihydroxyflavone, a TrkB Receptor Agonist, Attenuates skilled motor deficits after Experimental Juvenile Ischemic Stroke via PI3K/Akt Signaling ... 73

Chapter 3 ... 76

Scaling the model: perinatal ET-1 Ischemic Injury induced at perinatal age in rats (p14) ... 76

3.1 Introduction ... 77

3.2 Material and Methods ... 79

3.2.1 Perinatal Ischemic Stroke induction ... 79

3.2.2 Behavioral characterization of perinatal stroke model (P14) ... 79

3.2.3 Statistical analysis ... 79

3.3 Results: preliminary data... 80

3.3.1 Long-term motor impairments are more prominent when lesion occurs at P14 ... 80

3.4 Discussion ... 83

Chapter 4 ... 84

Perilesional Treatment with Chondroitinase ABC and Motor Training Promote Functional Recovery After Stroke in Rats ... 84

4.1 Introduction ... 85

4.2 Material and methods ... 87

4.2.1 Animals ... 87

4.2.2 Focal Ischemic Lesion and Treatment ... 87

4.2.3 Behavioral Testing ... 88

4.2.4 Training ... 88

4.2.5 Histological Procedure ... 88

4.2.6 Volume Measurement ... 89

4.2.7 Expression of Synaptic Markers ... 89

4.2.8 Anterograde Tracing of Thalamocortical Projection and Colocalization Analysis ... 90

4.2.9 Wisteria Floribunda Agglutinin and CSPG Stubs Staining ... 90

4.2.10 Analysis of Reactive Astrogliosis ... 91

4.3 Results ... 92

4.3.1 Skilled Reaching Task Learning ... 92

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4.3.3 Rats Treated with ChABC Show Motor Impairment at the Beginning of the Recall Phase . 92

4.3.4 Acute and Delayed ChABC Treatment Promotes Motor Learning After Cortical Stroke .... 94

4.3.5 ChABC Treatment Does Not Affect Damage Size in Ischemic Rats ... 96

4.3.6 ChABC and Behavioral Training Altered Expression of Synaptic Markers ... 97

4.3.7 Lack of Synaptic Marker Alteration in Untrained Rats Treated with ChABC ... 100

4.4 Discussion ... 101

4.4.1 Mechanisms of Action of ChABC ... 101

4.4.2 Site of Action of ChABC ... 102

Conclusions and future directions ... 105

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LIST OF ABBREVIATIONS

AKT protein kinase B

BDA biotinylated dextran amine

BDNF brain derived neurotrophic factor

CC cortico-cortico

ChABC chondroitinase ABC

CIMT constraint induced movement therapy

CNS central nervous system

CP critical period

CRT corticorubralspinal tract

CS corticospinal

CSPGs chondroitin sulphate proteoglycans

CST corticospinal tract

DBA 3,3'-diaminobenzidine

DHF 7,8 dihydroxyflavone

DMSO dimethyl sulfoxide

ERK extracellular signal-regulated kinase

ET-1 endothelin-1

GFAP glial fibrillary acid protein

M1 primary motor cortex

MCAO middle cerebral artery occlusion

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PI propidium iodide

PNNs perineuronal nets

SRT skilled reaching task

TrkB tropomyosin receptor kinase B

vGAT vesicular GABA transporter

vGLUT1 vesicular glutamate transporter 1

vGLUT2 vesicular glutamate transporter 2

VL/VM ventrolateral/ventromedial

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SUMMARY

Brain plasticity after Injury is one of the most innovative and promising targets in the field of restorative medicine. Neuroscientists have embraced the challenge of studying structural and molecular plasticity events occurring after damage to promote and direct functional recovery. However, in case of stroke in the motor area of the brain, plastic reorganization is often not sufficient to promote a complete recovery of motor deficits. It is well accepted in fact that many factors influence the extent of post injury functional outcome. Among these, age of lesion occurrence, size of the lesion, and location of specific cortical areas affected are crucial factors.

Stroke timing is particularly relevant in stroke recovery, as brain plastic potential declines with age. It is well established that after adult stroke it could be possible to achieve neurological recovery by blocking molecular factors that are known to limit neuronal plasticity in adulthood. On the other hand, promoting recovery after injury in a young brain is not a simple challenge: although brain plasticity is high during development, early stroke seems to also promote “mistaken” mechanisms of circuits rewiring that might account for long-lasting poor motor outcome. Thus, a better understanding of the mechanisms underlying the onset and process of acute and long lasting motor deficits after developmental stroke would correct the onset of eventually maladaptive pathways. In this context, the use of reliable animal models of developmental stroke that extensively recapitulate young brain stroke would be extremely useful. Indeed, that would provide the possibility to test pharmacological and rehabilitation treatments aimed at promoting recovery and in turn, to design more effective therapeutic protocols.

Here I characterised a model of early focal ischemia by injecting the vasoconstrictor Endothelin-1 in the motor cortex forelimb representation of young (P21) animals. This resulted in permanent motor deficits affecting the contralesional forelimb. We demonstrated that enduring motor deficits are prominent both for general and fine motor functions. We applied our model to demonstrate that behavioural outcome could be improved by physical intervention: by administering a skilled reaching training early after lesion. This rehabilitative treatment produced a complete recovery of skilled paw reaching ability. Moreover recovery is associated with sprouting of the uninjured corticospinal axons across the midline into the territory of the lesioned pathway, at red nucleus level. Early

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rehabilitative treatment however did not impinge on unskilled behavioral tasks, prompting the idea that early specific training promotes recovery of those “task-engaged” motor circuits in a use-dependent way. From this derived the concept of ameliorating function through specific training and not any training, already present in several pioneer work in men. The model was also instrumental for the study of a potentially relevant pharmacological intervention of neuroprotection. The effect of a 7,8 dihydroxyflavone (DHF), a specific Tropomyosin-related receptor kinase B (TrkB) agonist, on fine motor recovery was also investigated in our juvenile focal ET-1 stroke model. Our behavioral data in fact indicated that systemic treatment with DHF ameliorates skilled motor deficit. DHF administration induced an increased PI3K/Akt signaling in injured animals to sustain survival pathway activation against the effect of ET-1 lesion.

Timing in the occurrence of injury was also tested. The correlation between motor outcome and time of ischemia was demonstrated comparing the effect of a lesion at P21 with those induced by a lesion performed at P14: the former age correlates with the maximal expression of pruning and remodelling of ipsilateral corticospinal axons, whereas the latter corresponds to the developmental stage when pruning of ipsilateral corticospinal axons begins. Behavioral data indicate that ET-1 injected in rats at perinatal age causes long lasting behavioral impairments that resemble those induced after stroke in P21 animals. However lesion caused a prominent deficit in motor learning in perinatal injured animals versus P21 injured ones as P14 injured animals displayed a delay skilled reaching task.

The importance of the lesion size emerged as an important factor impinging on plasticity mechanisms after stroke. This issue was better investigated in a model we recently optimized , the ET-1 stroke induced in adult rat motor cortex. In the case of adult in fact, only size may play a role in the extent of plastic rearrangement, ruling out intrinsic young brain plasticity. Others and we have hypothesized that perilesional areas that survived the damage may undergo plastic remodeling. This evidence opens a question about whether treatments acting exclusively on cortical plasticity of perilesional areas, namely the penumbra, may result in behavioral amelioration. Here we pharmacologically enhanced plasticity in the ipsilesional cortex using local injections of chondroitinase ABC (ChABC). The extracellular matrix loses its tight grip on neurons once Chondroitinase ABC lysates the proteoglycans. This would result into a more amenable space for axon to sprout and remodel. I explored how this enhanced permissive nature of the extracellular matrix would

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result into a better recovery of skilled motor function in a focal cortical ischemia of forelimb motor cortex in adult rats. Moreover, it seems that the therapeutic time window of ChABC is not so strict as other anti-stroke agents as I found that acute as well as delayed ChABC treatment are able to induced recovery of impaired motor skills in treated rats. Moreover the combined effect of a matrix facilitator, ChABC and a physical training such as single pellet reaching test (SRT) applied after injury promotes local plasticity activation. The excitatory thalamo-cortical circuitry was involved in this functional recovery as vGLUT1, vGLUT2, and vGAT immune staining investigation indicated an enhanced activation of vGLUT2 (vesicular glutamate transporter 2) into the perilesional ChABC treated area just in trained rats. Thus, we conclude that combining a pharmacological treatment with physical intervention can restore and preserve functionality in fine movement after cortical ischemic lesion.

We developed a new reliable protocol for the study of ET-1 induced behavioral impairment in perinatal and juvenile age. We showed the enduring effectiveness up to adulthood of early specific training in ameliorating persistent fine paw motor impairment in juvenile stroke model. Moreover through this juvenile ischemic model we were able to demonstrate beneficial neuroprotective effect of DHF on fine motor functions. Finally, we also show that the combination of treatments targeting the CSPG component of the extracellular matrix in perilesional areas with rehabilitation could be sufficient to enhance functional recovery from a focal stroke induced at adult age. This might pave the way to the possibility of considering this combination therapy as a potential clinical rehabilitation approach.

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CHAPTER 1

GENERAL INTRODUCTION

1.1.1 THE ROLE OF EXPERIENCE DURING CRITICAL PERIOD

A major component of the development and maturation of the central nervous system (CNS) occurs through individual interaction and experience with the environment. During development, within a temporal window known as “critical period” CP, early environmental experience can markedly affect the subsequent properties and function of the adult brain. At this time the nervous system is able to functionally and structurally reorganize its connections in response to changes in environmental experience by a process called “plasticity”.

Many reports indicate that neuronal plasticity is particularly prominent in the developing brain. For example, a similar “critical period” was first described for filial imprinting in several bird species (Lorenz, 1935) that closely resemble the critical period also shown in human infants for first language acquisition (Kuhl, 2010).

Classically the study of the critical periods and neuronal plasticity in the mammalian concerns the development of the visual system. In the ’60, David Hubel and Thorsten Wiesel provided a groundbreaking contribution to the understanding of the role of experience during CP in visual system. Their studies demonstrated that neuronal signals coming from both eyes are combined at the level of individual neurons in the primary visual cortex, rendering many cells in the visual cortex sensitive to stimulation of either eye. For most of these cells, one eye exerts a stronger influence than the other one, a property for which Hubel and Wiesel (1962) coined the term ocular dominance (OD). They also found that, in higher mammals, the visual cortex is tessellated into alternating bands or patches (of cells) dominated by one or the other eye, forming what is known as OD columns (Wiesel et al., 1974). Hubel and Wiesel were the first to show that closing one eye (by suturing animal’s eye lid, a paradigm called monocular deprivation, MD) for several weeks had dramatic consequences for OD: the non-deprived eye gained control over cortical neurons at the cost of the temporarily closed eye (Wiesel and Hubel, 1963).

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These consequences arose from anatomical and physiological changes in visual cortex circuitry in response to unbalanced inputs from the two eyes (Tropea et al., 2009). This phenomenon, known as OD shift (Antonini and Stryker, 1996; Antonini et al., 1999) resembles the physiological alterations of human amblyopia, which results from an imbalanced binocular vision during childhood. Interestingly, this ocular dominance shift results as a consequence of the heightened plasticity present in cortical circuits during early postnatal development (i.e., during the CP). Indeed, susceptibility to MD changes with age: it occurs only if MD is performed during CP, and not in the adult (Hubel and Wiesel, 1963; Hubel and Wiesel, 1970). Thereafter, others experimental evidences suggested that MD effect occur only if only one eye is deprived of vision, whereas binocular deprivation has no effect (Antonini and Stryker, 1998). Thus, during visual system development competition between sensory inputs occurs, because the two eyes compete for the functional possess of cortical territories. MD unravels this competition, as only thalamo-cortical fibers with sustained electrical activity (triggered by the open eye) are able to maintain synaptic connection while the fibers coming from the deprived eye are retracted (Antonini and Stryker, 1996). Therefore, this finding provided the first evidence of the existence of a limited temporal window of heightened brain plasticity early in postnatal life, wherein neural circuitry can be sculpted and changed by experience through activity-dependent mechanisms.

Understanding the mechanisms involved in the development and plasticity of neural connections is also important for specifying possible deviations from the proper developmental plan, and hence the aetiology of developmental brain disorders. In this context, visual cortex studies of amblyopia can help to a better understanding of the mechanisms of plasticity occurring after a brain injury affecting the voluntary motor system. Many similarities between visual and motor system exist. For example, as visual cortex neurons, voluntary motor system, represented by corticospinal tract (CST) is initially immature and does not attain adult properties until later in life (Chakrabarty and Martin, 2000). Accordingly, early experience shapes the development of the motor circuitry at level of topography, morphology and spinal circuits (Goodman and Shatz 1993). Furthermore, as with amblyopia, a developmental brain injury causing a “deprivation” of activity of CST can also have long-term functional consequences (Eyre et al., 2007; Martin, 2005). Thus, similar activity-dependent mechanisms and consequences as those argued by Hubel and Wiesel

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more than 40 years ago may be also applied to the development of efferent pathways from the motor cortex.

However, in contrast to visual system, corticospinal development appears to last longer and it shows a protracted vulnerability to developmental insults later into postnatal life (Kirton and deVeber, 2015). Accordingly, the higher vulnerability of developing motor system makes this more susceptible to undergo injury-derived changes. Thus, given the presence of a protracted experience-dependent development of motor system during the critical period characterized by heightened plasticity, the scientific challenge of many investigators is represented by the possibility to embody this highly experience-sensitive period into a therapeutic window. This may have great implications because activity- and experience-dependent plasticity mechanisms might be harnessed to promote motor recovery.

1.1.2 MOLECULAR “PLAYERS” OF CRITICAL PERIOD TIMECOURSE

Great efforts have been made by neuroscientists to shed light on the mechanisms responsible for the opening and closure of CP, in order to find an explanation to why once the CP is closed, during adulthood, the brain becomes less plastic. Experimental visual system studies of amblyopia have provided great insights about the mechanisms underlying CP plasticity and thanks to these efforts a better understanding of the molecular determinants that regulate the timing (opening and closure) of CP during development has been currently achieved.

One of the most important factors underpinning the timecourse of neuroplasticity is the balance between excitation and inhibition (Hensch, 2005; Hensch and Fagiolini, 2005). The first evidence revealing that the balance between excitation and inhibition is important for the modulation of ocular dominance plasticity was the discovery that the development of GABAergic inhibition was necessary to trigger the onset of the sensitive period. Hensch et al. (1998) demonstrated that ocular dominance plasticity could not be induced in transgenic mice in which the GABA-synthesizing enzyme glutamic decarboxylase (GAD665) had been deleted, reducing therefore the synthesis of GABA. Interestingly, in these mice, independently of their age (so even during adulthood) the critical period for ocular dominance could not be restored until they were treated with benzodiazepines that are GABAA receptors agonists (Hensch et al., 1998). Similarly, in naive mice, the treatment with benzodiazepines can anticipate the onset of the critical period, supporting the hypothesis that an inhibitory threshold must be reached to initiate the sensitive period (Fagiolini &

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Hensch, 2000). The evidence listed above clearly demonstrates that the onset of the critical period depends on the development of GABAergic inhibition.

Accordingly, in the last decades the mechanisms modulating the development of inhibitory neurons have been long explored and many experimental works revealed various signaling pathways laying at the basis of maturation of inhibitory circuitry. These pathways include activity-induced BDNF (brain derived neurotrophic factor) release driving GABAergic synapse formation (Gianfranceschi et al. 2003; Hanover al. 1999); IGF-1 signals (Ciucci et al., 2007) and removal of the PSA moiety from the adhesion molecule NCAM (Di Cristo et al., 2007). Concerning with the role of neurotrophins in CP plasticity, a conspicuous number of observations have further suggested that BDNF play a pivotal role in regulating circuits development by exerting both a fast control on neuron electrical activity (Berardi and Maffei, 1999) and a slow actions, by modulating gene expression (Poo, 2001). BDNF is a member of the neurotrophin growth factor family that plays fundamental roles in neuronal growth, differentiation, and survival during development and adulthood (Nikolaou et al. 2006; Numakawa et al. 2010). After binding its tropomyosin receptor kinase B (TrkB), BDNF elicits TrkB dimerization and autophoshorylation, thereby leading to neurotrophin-downstream intracellular signaling Fig.1.

Experiments using transgenic mice and/or pharmacological manipulations have identified three signaling kinases that can modulate synaptic strength and that play a major role in regulating the CP plasticity: extracellular signal-regulated kinase 1,2 (ERK-1,2), cAMP-dependent protein kinase (PKA), and calcium/calmodulincAMP-dependent protein kinase II alpha (CaMKIIα) (Ratto and Pizzorusso, 2006; Di Cristo et al., 2001; Taha and Stryker, 2005).

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Figure 1. Brain-derived neurotrophic factor (BDNF)–tropomyosin-related kinase B (TrkB) signaling pathways.

BDNF binding to the extracellular domain of TrkB induces dimerization and activation of the intracellular tyrosine kinase domain. This results in autophosphorylation of tyrosine residues that then serve as sites for interaction with adaptor proteins and activation of intracellular signaling cascades, including the Ras– microtubule-associated protein kinase (MAPK), phosphatidyl inositol-3 kinase (PI3K)/serine threonine kinase (Akt) and phospholipase C (PLC)-g pathways. Adapted by Duman and Voleti, 2012.

These kinases may rapidly promote plasticity by directly phosphorylating plasticity-regulating molecules at the synapse (such as glutamate or GABA receptors) or cytoplasmatic substrates crucial for synaptic transmission, neuronal excitability and morphological stabilization (e.g. synapsin I, potassium channels, MAP2), or they may signal to the nucleus to mediate changes in gene transcription (Berardi et al., 2003).

In contrast, the end of the CP arises from different mechanisms that relate with the emergence of molecular ‘‘brakes’’ on plasticity. These molecular brakes make more difficult for new synapses to form and play an important role in diminishing plasticity but at the same time they stabilize existing connections. One type of brake is the maturation of specific components of ECM, such as chondroitin sulphate proteoglycans (CSPGs).CSPGs are widely expressed in the brain, but their specific role in limiting plasticity in the adult visual cortex is tightly linked to their organization into so called perineuronal nets (PNNs), dense structures sheathing the somata and proximal dendrites of some classes of cortical neurons, among them parvalbumin (PV)-positive GABAergic neurons (Pizzorusso et al., 2002). PNNs regulate per se experience-dependent plasticity in the visual cortex acting as a structural brake for plasticity (Pizzorusso et al., 2002). Indeed, PNNs gradually increases during development in the visual cortex until adulthood (Ye and Miao, 2013), and limit plasticity in the adult.

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Accordingly, PNNs removal by enzymatic digestion leads to reopening of the CP in adult animals (Pizzorusso et al., 2002) and mice lacking a protein involved in PNNs condensation, the cartilage-link protein 1 , Crtl1, still retain plasticity in the adult visual cortex (Carulli et al., 2010).

A second type of brake that leads to closure of the critical period is represented by the receptors for myelin associated growth inhibitors Nogo-66 receptor (McGee et al. 2005) and PirB (paired immunoglobulin-like receptor B) (Syken et al. 2006, Atwal et al. 2008). The myelin sheath that forms around axons is replete with ligands that actively prevent axonal outgrowth after binding these receptors. Indeed, it has been shown that in mice deficient for either receptor (Nogo-66 or PirB), plasticity can be readily induced in adulthood after a period of monocular deprivation, MD. Thus, all these evidences suggest that inhibition of structural plasticity through changes in the ECM and myelin-based factors determines the closure of the critical period.

However, others mechanisms involved with CP closure have been further demonstrated. Among these, reduced epigenetic regulation of CREB-mediated transcription (Putignano et al., 2007; Silingardi et al., 2010; Tognini et al., 2011) and decreased neuro modulatory activity, such as reduction in cholinergic and serotoninergic inputs (Morishita, et al., 2010; Maya-Vetencourt et al., 2008) play a key role in the closure of CP and accordingly in the decline of plasticity at adulthood.

Importantly, all the above mentioned CP plasticity “players” are tightly regulated by experience because each of these can be manipulated by either decreasing sensory experience (such as by monocular deprivation, MD or “dark rearing”, DR) (Fagiolini et al., 1994), as well as by increasing sensory experience on brain and behavior (Sale et al., 2007) (for example by housing animals in complex environment, an experimental paradigm known as “enriched environment”, EE). However, whereas the former promotes a delay in critical period occurrence and maintains the brain immature, the latter leads to a conspicuous acceleration of visual system development in rodents by steering a precious maturation of GABAergic transmission (Cancedda et al., 2004; Heimel et al., 2011; Levelt and Hubener, 2012).

1.1.3 PLASTICITY IN ADULT BRAIN: BEYOND THE CRITICAL PERIOD

It is widely accepted that experience-dependent plasticity is a prominent feature of the developing brain and that this property declines with age. However, decades of

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experimentation in the cerebral cortex have demonstrated many physiological and anatomical examples of adult cortical plasticity. With the advent of functional magnetic resonance imaging (fMRI), a noninvasive imaging method that assesses activity in regions of the brain based on measurement of blood flow, cognitive neuroscience research highlighted the plasticity of the adult brain, with changes and reorganization occurring throughout the life span (Gutchess, 2014). Indeed, virtually any experience can change the brain, especially if there is an associated behavioral change. We learn and remember, create new thoughts, and behavior changes throughout our lifetime. Changing behavior requires changes in the neural circuits that underlie it. To confirm this issue, there are many studies showing that either learning neuropsychological tasks (Kolb et al., 2008) or being exposed to environmental enriched experience (Rosenzweig et al., 1962), axonal sprouting, dendritic morphology changes and alterations in synaptic connectivity take place. Accordingly, dendritic and synaptic morphology of adult motor cortex neurons can be altered by specific motor learning tasks (Jones et al., 1999; Kleim et al., 2002) and in turn the ability of the motor cortex to encode acquired motor skills relies on reorganization of motor maps (Monfils et al., 2005). Interestingly, the adult central nervous system retains to a certain extent still the ability to undergo functional and structural plasticity after injury (Nudo et al., 2013; Overman and Carmichael, 2013; Biernaskie and Corbett, 2001). More specifically it seems that a crucial time window opens in the post-acute ischemic phase that is characterized by intense neuronal remodeling (Carmichael, 2006; Murphy and Corbett, 2009). Additionally, brain capillaries sprout, and glial cells are activated to create a favorable cerebral environment for neuronal growth and plasticity (Zhang and Chopp, 2009; Hermann and Zechariah, 2009). However, these changes that relate with spontaneous recovery and adaptation, are pathological consequences of injury (e.g., edema) and often this endogenous remodeling of the CNS is not sufficient to restore neurological function. Therefore, the current goal of many scientists is to take advantage of these spontaneous recovery events to further stimulate and amplify endogenous restorative mechanisms by means of pharmacological, physical therapy or by a combination of both.

Experimental and clinical evidences suggested that the mechanisms involved in the recovery from stroke follow similar rules to those hold during CP plasticity of the developing brain (Murphy and Corbett, 2009). Interestingly, once again visual cortex studies on amblyopia have been instrumental in addressing the question of adult cortical plasticity in a systematic

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manner, due to the fact that the critical period had most extensively been studied in the visual system. In this way, the background knowledge in the visual system helped the interpretation of the functional and structural changes observed for adult plasticity.

Indeed, many ways to bring back plasticity of adult brain to CP levels have been investigated in visual system. Manipulation of the balance between intracortical excitation and inhibition (Maya Vetencourt et al., 2008; Morishita et al., 2010; Harauzov et al., 2010), as well as removal of the structural brakes that are responsible for the restrained plasticity in adulthood have been shown all successful in experimental models of amblyopia. More specifically, dissolving the perineuronal nets with ChABC injections into adult rat binocular visual cortex renders the system plastic again and ocular dominance shifts can be re-induced in normal adult rats by combining ChABC with monocular deprivation. Moreover, amblyopic rats can recover normal acuity, ocular dominance, and spine density by ChABC treatment combined with concurrent reverse suturing of the initially non deprived eye (Pizzorusso et al., 2006).

Similar results have been achieved when myelin-related signaling through the Nogo receptor has been targeted (McGee et al., 2005). Deleting or blocking the Nogo receptor in adulthood can restore acuity after earlier monocular deprivation (Sengpiel, 2005). Interestingly, it has been also demonstrated that PirB receptor plays a crucial role in restricting structural plasticity in adult brain and a link between these molecules has also been found. PirB seems to act as a receptor for Nogo-A (Atwal et al., 2008), and CSPG components bind to Nogo-66 receptor subtypes (Dickendesher et al., 2012), thereby mediating growth inhibition. Importantly, these receptors accomplish the business end of axonal growth inhibition through RhoA and its downstream Rho kinase (ROCK) signaling. In this way these “brakes” of plasticity are linked to the cytoskeleton by promoting microtubule depolymerization and actin contraction, resulting thus in changes at both pre- and post-synaptic level.

It has also been shown that plasticity can be restored to the adult brain by a variety of less invasive interventions aimed at reducing intracortical inhibition: enriched environment EE (Sale et al., 2007; Baroncelli et al., 2011) or chronic administration of fluoxetine (Maya Vetencourt et al., 2008). Thus, taking these evidences together, it seems that blocking molecular factors that contribute to synapse maturation and that normally limit neuronal plasticity in the adult brain may help recovery of neural function.

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In the last decades, the approaches used in amblyopia to counteract molecular ‘‘brakes on plasticity” are beginning to be translated into clinical field of brain lesion. For example, blocking Nogo-A with a specific antibody was shown to promote axonal regeneration in the rat spinal cord (Schnell and Schwab, 1990; Buchli and Schwab 2005), as well asagents that block signaling through the Nogo receptor(Papadopoulos et al. 2006; Kilic et al. 2010; Wahl et al., 2014) promoted functional recovery in animal models of stroke. Accordingly, by digesting CSPG side chains, chondroitinase ABC (ChABC) modifies extracellular matrix and allows axonal sprouting (Bruckner et al. 1998; Crespo et al. 2007). For example, following spinal cord lesions in rats, local CSPG degradation promotes axonal growth and improves sensory and motor function in behavioral essays (Bradbury et al., 2002), an effect that is probably largely due to enabling growing axons to cross the lesion-induced glial scar, which contains high levels of CSPGs (Asher et al., 2001). Furthermore, recently, ChABC has been tested in models of brain trauma (Harris et al. 2010), however the therapeutic potential of ChABC after stroke is only partially explored (Soleman et al. 2012). Thus, given that recovery crucially depends on the adult brain’s capacity for plasticity, the above-described approaches may yield further permissive role for recovery after stroke, spinal cord or traumatic brain injury. Although a number of molecular factors interfering with these brakes have been identified and have proven their principal usefulness in animal studies, human clinical trial data are still very scarce. It is plausible to assume that combining alterations of sensory input or sensory-motor training with direct molecular interventions act synergistically to enhance plasticity and thereby promote recovery of function after injury.

1.1.4 THE VOLUNTARY MOTOR SYSTEM: DEVELOPMENT, STRUCTURE AND FUNCTION

1.1.4.1 Comparative Development of voluntary motor pathway, corticospinal tract (CST) Some animal species are born with extraordinary capabilities. Considering, for example, a wildebeest with precocious motor skills, it is able to maintain a quadrupedal posture within moments after birth and can run with the herd soon thereafter. By contrast, many mammals including rats, cats, and humans are born with the capacity only to express motor behaviors that are necessary for survival, such as respiration and feeding-related behaviors (Muir 2000) and with no developed skillful motor functions.

Whereas CST development begins prenatally, rats develop mature motor skills during the first month (Terashima T, 1995), cats during the first 2 to 3 months (Martin et al., 2007) and

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human motor development is incomplete until 13 to 15 years (Nezu et al., 1997). Thus, the most adaptive and skillful motor functions, like reaching and manipulative behaviors, complete their development after birth, during CP (Lawrence and Hopkins 1976; Westerga and Gramsbergen 1993; Galea and Darian-Smith 1995). This suggests that late motor development and late development of the corticospinal system correlate significantly because as the corticospinal system matures, adaptive motor behaviors begin to be expressed (Lawrence and Hopkins 1976), and damage to this system severely impairs adaptive control (Armand and Kably 1993).

1.1.4.2 Factors contributing to CST development

Across many species and as for other neural system, the pattern of maturation of corticospinal axons termination involve both intrinsic genetic mechanisms, whereby guidance cues are sensed by receptors on the growth cones of developing CST axons (Dottori et al.,1998), and activity-dependent processes (Martin et al., 2004), involving the N-methyl-D-aspartate (NMDA)-2B receptor (Ohno T et al., 2010). In this way, the interplay between genetics, neural activity, and experience allow appropriate circuit formation and performance. Genetic mechanisms specify first laterality of CST spinal projection because they play a key role in guiding CST axons to their targets in the brainstem and spinal cord. Recently, it has been suggested a principal role of the guidance cue Ephrin B3 and its receptor tyrosine kinase Eph A4 in ensuring the correct CST pathfinding (Serradj et al., 2014). For example, a selective elimination of Eph A4 gene in mice forebrain led to a strong CST bilateral projection to the spinal cord that persisted up to adulthood (Paixao et al., 2013) that resulted in permanent motor deficit in skilled reaching in mice.

Activity-and use-dependent processes subsequently shape the pattern initially established by genetic mechanisms and lead to the refinement of CST projections by means the elimination of less active CS synapses. By this mechanism, most of the ipsilateral terminations are withdrawn, by “pruning”, on behalf of the contralateral ones that instead will result reinforced (Eyre et al., 2001). This mechanism also explains the reason why at early stage during development, CST termination pattern is more extensive than the one later in development and in maturity (Eyre JA, 2007; Martin JH, 2005; Joonsten et al., 1992). In summary, across different species, three CST developmental stages can be identified Fig.2.

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Figure 2. Comparative corticospinal tract development: timecourse of corticospinal tract development across

different species. Adapted by Martin JH, 2005.

The first is growth of axons of cortical lamina 5 neurons to the spinal cord gray matter, whereby the growing corticospinal axons descend within specific regions of the subcortical, brain stem, and spinal white matter through a small contingent of “pioneer” axons, which lead the way into the cord. Afterward, later waves of axons will further populate the corticospinal tract (Joosten and others 1987, 1989). Pathfinding is organized by tissue molecular cues diffused into the local environment and can be either attractants and/or repellants, in guiding growing corticospinal axons (Joosten, 1999). After a variable delay period, gray matter innervation is mediated by target-specific chemotropic factors that induce branching.

The second stage is refinement of the gray matter terminations driven by neural activity in the motor cortical areas and by limb motor experience. Axon branches of more active CST neurons are able to maintain, expand, and strengthen their spinal connections, whereas the axons of less active neurons are eliminated. Competition is evident in establishing the predominantly contralateral CST projection. In this way more active terminals are more competitive and are able to secure more synaptic space than their less active counterparts. In Fig.1 the major period of concurrent growth and elimination is shown by the red box. The third stage is motor control development, during which the corticospinal system’s role in distal limb control and other adaptive movements becomes expressed. This period is characterized by a mature pattern of corticospinal terminals: loss of transient terminations has occurred, growth to local targets is well under way, development of motor cortical

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motor maps is ongoing and finally myelination begins during this refinement period (Huttenlocher 1970).

1.1.5 FUNCTIONAL AND ANATOMICAL ORGANIZATION OF MATURE VOLUNTARY MOTOR

SYSTEM

1.1.5.1 Corticospinal tract CST

Voluntary movements are mediated by direct connection between the primary motor cortex (M1) and the spinal cord (Sanes and Donoghue, 2000). There are six layers in the cerebral cortex and layer five of M1 gives rise to the corticospinal tract (CST, or pyramidal tract) which is the main descending motor pathway in mammals. The CST is mainly responsible for the control of skilled limb movements, and is somatotopically organized. M1 somatotopic arrangement is conserved to various degrees the entire length of the descending corticospinal tract and the topography of corticospinal tract (CST) terminations in the spinal cord are related to the M1 map (Chakrabarty et al., 2009). Specific regions of M1 influence the activity of specific muscle groups.

In addition to being somatotopically organized and being the major motor efferent via layer five pyramidal neurons, M1 has a complex, local architecture of horizontal connections that extend to nearby cortical columns and cortical layers, up to approximately one centimeter in distance (Hess and Donoghue, 1994). The human CST consists of approximately one million axons, of which approximately 40% originate in the motor cortex. The rest of the axons have their origins mainly in the premotor and supplementary motor cortices, and in the parietal areas lying posterior to the pre central sulcus. These axons descend through the subcortical white matter, the internal capsule, and the cerebral peduncle in the midbrain. In the medulla the fibers form prominent protuberances on the ventral surface called the “medullary pyramids” at which level most of CST fibers cross the midline and descend in the contralateral white matter of the cord, as the lateral corticospinal tract. A small percentage, 10% of axons do not decussate in the pyramid and descend as the ventral corticospinal tract Fig.3.

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Figure 3. Organization of the Mature Corticospinal System: adapted by Martin JH, 2005.

Furthermore, the rubrospinal, reticulospinal, and vestibulospinal tracts are other three brain stem-spinal pathways that receive cortical input. The cortical motor areas and brain stem in turn receive input from both cerebellum and basal ganglia that provide essential feedback for the smooth execution of skilled movements and thus are important for motor learning. However spinal cord circuits are not solely regulated by descending commands. Reflex circuits and pattern generators within the spinal cord can also coordinate stereotyped movements such as stepping without descending signals. Descending system coordinate reflex and patterned movements generated by spinal motor circuits and can even create new patterns of muscle activation through direct action of motor neurons. This cortical control enables greater flexibility of movements than is possible through exclusively local coordination among the spinal motor circuits. Thus, motor systems organization reveals a dazzling number of alternate routes from supraspinal motor centers to the motoneuron that might be remodeled in their functional connections after injury.

1.1.5.2 Cortico-Rubrospinal Tract CRT

In addition to the corticospinal tract, the voluntary motor system contains indirect paths that project first to brain stem motor nuclei and from there to the spinal cord that also play a crucial role in controlling spinal motor neurons function, even those innervating distal muscles (Riddle and Baker, 2010). Among these indirect pathways, the rubrospinal tract,

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arising from Red Nucleus (RN), represents another major descending pathway for distal limb control (Martin and Ghez, 1988). Whereas the CST pathway is thought to play a greater role in more flexible and adaptive movements, the CRT system is supposed to be involved in more automatic limb movements. Furthermore, although the CRT system has been speculated to play a role in recovery after CST damage in maturity (Ruber et al., 2012) and after pyradotomy in rats during development (Z'Graggen et al., 2000), its role in motor development in health and in cortical stroke disease is not yet completely known. Recent evidences suggest that rubrospinal tract plays an active role in motor skilled behavior development and this is true even before the CST has accomplished its development (Williams et al., 2014). Specifically, it seems that Red Nucleus can actively take part to compensatory remodeling mechanisms underlying spontaneous recovery, which rarely occurs after early CST loss of function (for example following pharmacological inactivation of CST by a GABA A receptor antagonist, muscimol) (Williams et al., 2014). Interestingly this evidence suggest that the rubrospinal system may have even more anatomical and functional prominence early in human development than it does later in life (Ulfig et al., 2001).

1.1.6 STROKE PATHOPHYSIOLOGY

Interruptions in the blood supply restricted to the territory of a major brain artery lead to a debilitating neurological condition termed ischemic stroke. In most cases, ischemic stroke is caused by the occlusion of a cerebral artery either by an embolus or by local thrombosis. Depending on whether the interruption of blood flow is transient or permanent, focal or global, different pathophysiological scenarios and stroke related dysfunctions could spring. For instance, brain injury following transient or permanent focal cerebral ischemia develops from a complex series of pathophysiological events that evolve in time and space (Dirnagl et al., 1999) Fig.4.

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Figure 4. Putative cascade of damaging events in focal cerebral ischemia: very early after the onset of the

focal perfusion deficit, excitotoxic mechanisms can damage neurones and glia lethally. In addition, excitotoxicity triggers a number of events that can further contribute to the demise of the tissue. Such events include peri-infarct depolarizations and the more-delayed mechanisms of inflammation and programmed cell death. The x-axis reflects the evolution of the cascade over time, while the y-axis aims to illustrate the impact of each element of the cascade on final outcome. Adapted by Dirnagl et al., 1999.

Although the existence of different types of stroke, however for the purpose of the present thesis, focal permanent ischemia, which represents the most common type of stroke, will be taken into account. Briefly, after stroke-induced interruption of blood flow to an area of the brain, energy-dependent processes fail, and as consequence neurons and glia are unable to maintain their normal transmembrane ionic gradients, resulting in an ions and water imbalance that originate excitotoxic mechanisms. In addition, excitotoxicity triggers a number of events that can further contribute to the demise of the tissue, such as peri-infarct depolarization and the more-delayed mechanisms of inflammation and programmed cell death, by apoptosis (Dirnagl, 1999).

In light of this, as soon as two minutes after the onset of ischemia, necrosis represents the predominant mechanism that follows acute vascular occlusion and orchestrates the immediate loss of brain tissue, termed “core” of the lesion. Whereas, during stroke chronic phases, delayed cascade reactions involving excitotoxicity, acidosis, inflammation, oxidative stress, peri-infarct depolarizations, diaschisis and apoptosis (for further detail, see Doyle, Simon and Stenzel-Poore, 2008; Krnjevic, 2008; Carrera and Tononi, 2014; JP Dreier, 2011) are responsible for the slowly evolving secondary damage into the peri infarct area (Carmichael ST, 2012), the so called ischemic “penumbra”. The ischemic penumbra corresponds to a boundary but dynamic area between ischemic “core” and the unaffected tissue where surviving neurons undergo active structural and functional remodeling after stroke and sow the seeds for recovery (Murphy and Corbett, 2009), Fig.5.

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Figure 5. Relantionship between synaptic circuit damage and local blood flow: cross section through the

rodent cortex that shows the stroke core (darker brown) and penumbra (lighter brown) after occlusion of the middle cerebral artery. The core has less than 20% of baseline blood flow and fails to regain its fine dendritic structure after reperfusion. In the penumbra, blood flow increases towards the midline, as tissues in this region are supplied by other artery systems that were not blocked during the stroke, and some loss of dendrite structure will reverse when reperfusion occurs. This is where rewiring will occur to replace connectivity that has been lost because of the stroke. Adapted by Murphy and Corbett, 2009.

1.1.7 ANIMAL MODELS OF ISCHEMIC STROKE

It is widely accepted that research with animal models is crucial to developing and testing new therapies. Animal models are important tools because they may help to understand the molecular mechanisms that underlie the organism’s response to brain injury in the short and long term. Furthermore, it is assumed that at the cellular level all mammals share these responses. However, there are drawbacks to this approach. The choice of a reliable model is therefore essential for the study of functional and morphological aspects of stroke in order to draw more effective treatments for promoting recovery of the spared functions (Kolb B and Muhammad A, 2014; Nishibe, et al., 2014). In contrast to developmental stroke models, many models of adult focal ischemic stroke have been set thus far.

1.1.7.1 Preclinical Models of Adult Stroke

Intraluminal Suture A coated suture is advanced into the carotid artery until it lodges at the junction of the middle cerebral artery (MCA). The damage that results from the interruption of blood flow is mainly in the striatum and cortex (Longa, et al., 1989). The suture is withdrawn after 30–120 min, which results in the reperfusion of ischemic tissue. Occlusion durations of 90–120 min are required to achieve reproducible tissue damage and result in very large infarcts that occupy much of the hemisphere. These often include hypothalamic injury, which can complicate the interpretation of histological and behavioral outcomes owing to impaired motivation and temperature regulation. Such extensive damage is akin to a malignant infarct in humans, which is frequently fatal or untreatable (Carmichael, 2005).

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Proximal or distal middle cerebral artery occlusion The MCA is transiently occluded using microvascular clips, or permanently occluded by cauterization. Damage is restricted to the cortex if blood flow is interrupted distal to the striatal branches of the MCA, whereas occlusion proximal to these small arteries results in both striatal and cortical injury.

Middle cerebral artery embolism A blood clot is introduced through the internal carotid to occlude the MCA. This model closely resembles human ischemic stroke. Resulting strokes tend to be much smaller than those produced using the suture model. Clots can undergo spontaneous thrombolysis, thereby causing multiple infarcts and high variability and mortality (Hainsworth et al., 2008; Carmichael, 2005).

endothelin 1(ET-1) vasoconstriction Endothelin 1 (ET1) produces ischemia by constricting blood vessels. ET1 is stereotaxically injected into parenchymal regions of interest, to constrict local arterioles, or near the MCA (Windle et al., 2006). Reperfusion occurs, but at a much slower rate than with the intraluminal suture model. Lesion size can be adjusted by varying the concentration or volume of ET1 to achieve reproducible injury.

Photothrombosis A photosensitive dye is injected systemically into animals in which a section of skull has been removed or thinned (Schmidt et al., 2012). The underlying cortical blood vessels are exposed to a green laser or epifluorescent light source, generating singlet oxygen species that lead to platelet activation and microvascular occlusion. This model can be used to produce small infarcts in any cortical region without invasive surgery (Zhang and Murphy, 2007).

1.1.7.2 Preclinical Models Of Developmental Stroke

In contrast to the great number of adult ischemic stroke models, few animal models of perinatal and juvenile stroke that attempt to recapitulate the mechanisms underlying the onset and the evolution of acute and long lasting motor deficits in children are currently available. Among these, Levine-Rice model to study neonatal hypoxic-ischemic brain damage has been widely used for 3 decades for histological analysis as well as behavioral tests. Concerning with “Hypoxia-Ischemia Levine-Rice model” (Levine, 1960) a unilateral ligation of common carotid artery is practiced for one or more hours and after reperfusion and recovery, animals are placed into an hypoxic chamber containing humidified 8% hypoxic gas producing whole body hypoxia. This model causes hypoperfusion in the ligated side of the brain, while the non-ligated side is exposed to hypoxia alone. However Hypoxia-ischemia neonatal model generates a complicated condition, in which the cause, severity, magnitude,

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and deteriorating speed are different in each case, leading therefore to high variability in infarct size. Middle Cerebral Artery Occlusion (MCAO) in rodents (Vannucci RC et al.; 2004; Herson et al., 2013) and photo-trombotic stroke in piglets (Kuluz JW et al., 2007) are also used but they generate extensive lesions of variable size. Recently, the vasoconstrictor endothelin, ET-1 was injected into the striatal area of the juvenile brain to induce reproducible focal lesion (Saggu, 2013), but poor information about effect on motor outcome are thus far available for developmental brain injury. Except the work of Saggu et al., 2013, where authors use this method to induce a stroke into the striatal area of the juvenile brain, thus far this procedure has not been applied to induce a focal cortical lesion (involving M1) in young animals. Kittens have also been studied as they also have the advantage of being born earlier than the development of the motor system (Martin, 2005). However, the cat model of CP, produced by pharmacological inactivation of CST (performed by muscimol, a GABA A receptor antagonist) does not recapitulate ischemic stroke neurobiology.

1.1.8 RODENTS CORTICOSPINAL TRACT

The availability of rodent models of developmental focal ischemic stroke allow researchers to set therapeutic strategies in order to enhance recovery and to establish the best temporal window within which treatments are more effective. Furthermore, there are many similarities between humans and rodents CST. As with humans, rodents have a CST that projects the full length of the spinal cord (Bareyre et al., 2005) and is involved in fine movement control (Whishaw et al., 1993). Both in human and rodents, CST development is accomplished at postnatal stage Fig.6. Furthermore, in neonatal rat, the corticospinal projection originates from the whole neocortex including the visual cortex (O’Leary and Stanfield, 1986) and corticospinal projections also have transient ipsilateral projections that are predominantly pruned when maturity is reached (O’Leary and Stanfield, 1986). This makes rodents a good tool to test new therapeutic strategies and to reveal the related plastic mechanisms underlying recovery in response to such therapy.

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Figure 6. (A) Schematic representation of the rat spinal cord. (B) CST extension diagram. (C) Camera lucida

drawings of transverse sections of the dorsal funiculus with the labelled CST. Adapted by Joosten and Bar, 1999.

1.1.9 MOTOR OUTCOME GREATLY DIFFER AFTER YOUNG VERSUS ADULT ISCHEMIC STROKE

Stroke occurs throughout life with unique features in the fetus, neonate, and child with respect to adult stroke. Indeed, clinical considerations vary depending on the life stage. Although important commonalities probably exist between young adult and adolescent stroke, the consequences in the elderly are different from those in children: the younger the child, the more non-specific their symptoms may be (Kirton and deVeber, 2015).

Childhood stroke is a rare event. With an incidence ranging from 1.2 to 13 cases per 100,000 children under 18 years of age (Earley et al., 1998) pediatric stroke represent a devastating cause of morbidity and mortality. Roughly 10–25% of children with a stroke will die, up to 25% of children will have a recurrence, and up to 66% will have persistent neurological deficits or develop subsequent seizure disorders, learning, or developmental problems (Lanthier et al., 2000; deVeber et al., 2000; deVeber, 2005). Pediatric stroke, as perinatal stroke, is frequently undiagnosed or misdiagnosed. This may be due to the fact that in most of cases, functional deficits are difficult to predict because of the paucity of signs of motor handicaps that rather occur at later stages.

The majority of pediatric ischemic strokes occur in the distribution of the middle cerebral artery, with hemiplegia being the most common focal manifestation in up to 94% of cases (Earley et al., 1998; Eeg-Olofsson and Ringheim, 1983; Zenz et al., 1998). As result, a compromised use of the arm and hand to reach, grasp, release, and manipulate objects is the main deficit occurred after injury. A feature of corticospinal (CS) system damage during development is the gain of aberrant and debilitating functions that are key motor

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impairments in cerebral palsy (CP) (Sanger, 2008). These include hyperreflexia, spasticity and aberrant limb and postural coordination as well as mirror movements (Kuhtz-Buschbeck et al., 2000). Mirror movements are also a landmark in children with hemiplegic cerebral palsy (CP) and rely on the gain of aberrant functions

Thus, a developmental brain damage generates persistent specific effects and produces complex and often severe patterns of impairment that are different from those observed following lesions in adult brain. For this reason, a better understanding of the nature of the impairments and of plasticity mechanisms in response to damage is critical to augment recovery from neurological insults.

1.1.9.2 Adult Ischemic Stroke

Adult stroke is the third leading cause of death all over the world and also the leading cause of adult disability, because 76% of people survive their stroke. Of these survivors, 50% have a hemiparesis, 26% are dependent in activities of daily living, and 26% are forced into a nursing home. This long-term disability means that $30 billion of the $53.5 billion annual dollar cost of stroke is incurred in supporting long-term survivors (Carmichael ST, 2006). Chronic upper limb impairment (usually unilateral) is the most common deficits reported in stroke survivors, due to a loss of functional use of the hand or arm contralateral to the lesion locus (paretic body side; i.e., paretic limb). As result, there is a loss of motor skill and coordination accompanied by other symptoms such as weakness and paresis. A natural response to loss of function is the development of alternative strategies to circumvent the problem, a consequence known as “compensatory strategy” among stroke survivors (Schallert, 2006; Taub et al., 2006). However the degree of functional deficits tightly relates to the lesion size. Indeed, small stroke lesions where 10–15% of the cortex is unilaterally destroyed often show good spontaneous recovery within days and weeks (Liepert et al., 2000; Cramer, 2008) and is accompanied with a plastic reorganization of nearby areas. In contrast, larger stroke lesions, where 60% of the cortex is unilaterally destroyed, often result in permanent neurological deficits (Biernaskie et al., 2005). Such larger lesions are accompanied by a somatotopic reorganization of more remote areas, such as the contralesional motor- and premotor cortex in humans and in experimental animals (Johansen- Berg et al., 2002; Biernaskie et al., 2005; Bestmann et al., 2010; Rehme et al., 2011). Reliance on the non-paretic limb contributes to a learned nonuse of the paretic limb (Taub et al., 2006) and may limit long-term functional outcome following stroke.

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Interestingly, recent high-resolution imaging and stimulation techniques describe a correlation between functional recovery and somatotopic reorganization of the intact hemisphere (Johansen-Berg et al., 2002; Bestmann et al., 2010; Stoeckel and Binkofski, 2010; Rehme et al., 2011). However, this is still a matter of debate and the size and precise location of the lesion play crucial roles that require more detailed investigation.

1.1.10 POST-STROKE RECOVERY

Recovery after stroke represents both an exciting and controversial topic in neurology. With theories of recovery emerging only in the last decade, recovery after stroke is a relatively new field of medicine. Recovery is a complex process and may be heterogeneous across patients and the extent to which improved performance reflects true recovery, behavioral compensation or a combination of both, represents a matter of debate among neuroscientists.

Neurologist Bruce H. Dobkin defines recovery as “the complete return of identical functions that were impaired” (Dobkin, 2003). This is distinct from restitution, which is subtly different from recovery. With restitution, the neural network regains most of its activity. Finally, a stroke patient may make functional gains by developing compensation strategies, which are defined as behavioral adaptations. Each of these processes exhibits a spontaneous recovery that occurs unaided by therapeutic intervention (Dancause et al., 2005).

However, commonly used human and animal behavioral assessment protocols (relayed on detailed post-injury kinematic analysis) of the reaching movements have showed that postural adjustments and compensations often allow a spontaneous partial return to pre-stroke motor performance levels (Whishaw, 2000). Considering recovery itself as a process of both reinstatement and relearning of lost functions, as well as adaptation and compensation of spared, residual function, it follows that the neurophysiological mechanisms that support learning in the intact cortex should mediate motor relearning and adaptation in the injured brain. In this view, it has been shown that motor recovery rely on synapse-based learning rules involving either homeostatic (Turriggiano and Nelson, 2004) and Hebbian plasticity mechanisms (Hebb, 1949). This synaptic plasticity ensures that compensatory circuits can form after stroke. Studies in both animals and patients have demonstrated that stroke-induced neuronal plasticity drives the formation of new local circuits, intracortical connections, and descending projection remodeling.

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1.1.10.1 Timing of lesion occurrence establishes the pattern of Recovery

Clinical considerations vary depending on the life stage of lesion occurrence, with adult stroke inducing a type of neuronal reorganization, which seems to be tightly dependent on the location and size of lesion. In contrast, early developmental stroke seems to spring a type of brain remodeling, known as “contralesional reorganization”, which is more strongly influenced by the experience following damage than lesion size and brain area affected. Indeed, it has been suggested that early unilateral motor system injury impairs hemispheric ability to innervate contralateral spinal lower motor neuron pools, during its developmental refinement. The motor consequences rely on a complex interaction between residual motor output from the affected hemisphere and the pathological success of congenitally present ipsilateral corticospinal projections to ‘take-over’ the injured connections.

1.1.10.2 “Maladaptive Plasticity” after Developmental CST Injury

It has been well established that the brain is extraordinarily plastic in early development and that early stroke represents an ideal, although unfortunate situation in children to study developmental plasticity. This has led to consider motor system plasticity as one of the main focus for both human and animal research (Kirton, 2013; Martin et al., 2007). Thanks to this combined approach, researchers continue to shed light on motor system development after unilateral early lesion (Eyre, 2007; Staudt, 2007).

However, recent experimental and clinical evidences suggested that unilateral damage during the period of CST refinement has a particular signature: in fact not only development of the damaged side is affected, but also the developmental process of the spared hemisphere, Fig.7.

In particular, in contrast with the well-established Kennard’s principle, according to which the younger the brain the greater is the capability to recover from early injury, it seems that the enhanced plasticity during development can somehow disturb the normal functional refinement of cerebral pathways after early brain injury (Martin et al., 2007).

Indeed, it has been proposed that an aberrant mechanism of plasticity, called “maladaptive plasticity ”, takes place in case of developmental injury, and this often results in a miswiring of CS motor circuits: the affected corticospinal tract does not assume a primary role in movement control in the first few months after birth (Eyre et al.,2001) and as consequence, an abnormal bilateral pattern of innervation is established.