Pediatric Stroke and Cell-Based Treatment – Pivotal Role of Brain Plasticity

Review Article

J Stem Cell Res Transplant. 2019; 6(1): 1029.

Pediatric Stroke and Cell-Based Treatment – Pivotal Role of Brain Plasticity

Arne Jensen*

Campus Clinic Gynecology, Ruhr-University Bochum, Germany

*Corresponding author: Arne Jensen, Campus Clinic Gynecology, Ruhr-University Bochum, Universitätsstr. 140, 44799 Bochum, Germany

Received: March 15, 2019; Accepted: April 27, 2019; Published: May 04, 2019

Abstract

Pediatric cerebrovascular disorders like stroke are among the top 10 causes of death in children, with rates highest in the first year of life, while survivors may face lifelong sequelae, disability, and/or cerebral palsy for which there is no cure at present. Individual treatments using human autologous cord blood Mononuclear Cells (hucbMNC) containing stem cells have yielded promising results. However, stroke is an entity with heterogenic etiologies including arterial ischemic stroke, hemorrhagic stroke, hemorrhagic infarction, and sinus venous thrombosis in newborns, infants, children, and adults. Hence, any attempt to examine care, non-cellular or cell-based therapies has to merit the specific characteristics of this heterogeneity as far as age, symptoms, diagnostics, pathophysiology, histopathology, clinical course and prevalence are concerned. This review describes the various etiologies of stroke for the Neonatal/Perinatal and childhood subsets of the pediatric population as a basis for established non-cellular and novel cell-based therapeutic approaches using cord blood mononuclear cells in the preclinical and clinical setting. In addition, due to its fundamental importance for cell-based therapeutic strategies for stroke, an account of brain plasticity along with blood-brain barrier function and the distinctions between the developing brain and the adult brain is provided. It is concluded that on the present balance of evidence the pathophysiological characteristics associated with plasticity of the developing brain are closely linked to the pharmacological action of hucbMNC in such a way that cell-based treatment might be more efficacious in pediatric stroke than in adult stroke.

Keywords: Pediatric stroke; Autologous cord blood stem cells; Prevalence; Brain plasticity; Brain development; Blood-brain barrier; Cerebral palsy; Perinatal brain damage; Neuroregeneration; Adult stroke

Abbreviations

AIS: Arterial Ischemic Stroke; CP: Cerebral Palsy; CPISR: Canadian Pediatric Ischemic Stroke Registry; CSVT: Cerebral Sinus Venous Thrombosis; CVA: Cerebral Vascular Accident; DMSO: Dimethylsufoxid; FA: Fractional Anisotropy; FLAIR: Fluid-Attenuated Inversion Recovery; FVL: Factor V Leiden; EGF/FGF: Endothelial Growth Factor/Fibroblast Growth Factor; G-CSF: Granulocyte-Colony Stimulating Factor; GMFCS: Gross Motor Function Classification System; HIE: Hypoxic–Ischemic Encephalopathy; Hucb: Human Umbilical Cord Blood; Hucbmnc: Human Umbilical Cord Blood Mononuclear Cells; ICA: Intra Cerebral Artery; ICH: Intracranial Hemorrhage; IP: Intra Peritoneally; IV: Intra Venous; MCA: Middle Cerebral Artery; MoA: Mode of Action; MRA: Magnetic Resonance Angiography; MRI: Magnetic Resonance Imaging; MRI-DTI: Magnetic Resonance Imaging-Diffusion-Tensor Imaging; MTHFR: 5,10 Methylenetetrahydrofolate Reductase; NGF/RA: Nerve Growth Factor/Retinoic Acid; NHDS: National Hospital Discharge Survey; PAIS: Perinatal Arterial Ischemic Stroke; PFO: Patent Foramen Ovale; PVHI: Periventricular Hemorrhagic Infarction; ROI: Regions Of Interest; SCD: Sickle Cell Disease; SPECT: Single Photon Emission Computed Tomography; SVT: Sinus Venous Thrombosis; TIA: Transient Ischemic Attack; Tpa: Tissue Plasmin Activator; UFH: Unfractionated Heparin and WMD: White matter damage.

Introduction

Pediatric brain damage of various etiologies, including stroke, and subsequent Cerebral Palsy (CP) are devastating and debilitating ailments with lifelong sequelae and high mortality. Unfortunately, there is no causative treatment beyond symptomatic and supportive care and the estimated lifetime costs per affected child amount to 800,000 Euro. The enormous number of 17 million people worldwide that live with cerebral palsy reflects the magnitude of unmet needs and the personal, medical, and global socioeconomic burden of this brain disorder.

But there is hope. Only recently the first successful cell-based treatment of arterial ischemic stroke in a child using autologous cord blood mononuclear cells (Jensen and Hamelmann, 2016 [1]) and of adult stroke using allogeneic cord blood mononuclear cells have been reported (Laskowitz et al., 2018 [2]). This renders a comprehensive review of the literature mandatory to explore safety and efficacy of cell-based treatment options for young and elderly patients alike.

Stroke is an entity with heterogenic etiologies including arterial ischemic stroke, hemorrhagic stroke, hemorrhagic infarction, and sinus venous thrombosis in newborns, infants, children, and adults. Hence, any attempt to examine care and non-cellular or cell-based therapies has to merit the specific characteristics of this heterogeneity as far as age, symptoms, diagnostics, pathophysiology, histopathology, and clinical course are concerned. This review describes the various etiologies of stroke stratified by age, i.e. Neonatal/Perinatal and childhood subsets of the pediatric population, as a basis for established non-cell-based and novel cell-based therapeutic approaches using cord blood mononuclear cells in the preclinical and clinical setting. Due to its fundamental importance for cell-based therapeutic strategies for stroke, an account of brain plasticity along with bloodbrain barrier function and the distinctions between the developing and the adult brain is provided that might explain in part as to why the efficacy of cell-based treatment may be different in childhood from that in adulthood.

Pediatric Stroke

Pediatric stroke is a subset of the general condition stroke. “The two periods of life in which the risk of having a stroke is highest are in the elderly and in the fetus or newborn infant. In both these periods, stroke is associated with heightened risk of death or, in survivors, of persisting neurological disability” (Nelson, 2007 [3]).

The condition Pediatric stroke, including Neonatal/Perinatal and Childhood stroke, is a cerebrovascular disorder in children which can be treated by autologous human mononuclear cells derived from cord blood (Jensen and Hamelmann, 2016 [1]). The condition is chronically debilitating and life threatening with a low prevalence. However, disorders of the cerebrovascular system are among the top 10 causes of death in children, with rates highest in the first year of life, the US mortality in 1998 attributable to stroke being 7.8/100,000 in children 0 to 1 year of age (Lynch et al., 2002 [4]).

Stroke in the pediatric population encompasses Neonatal/ Perinatal stroke (age 0 to <28 days) and Childhood stroke (age 28 days/1 month to <18 years). The reported outcome for stroke in children varies, but >50% of survivors develop neurologic or cognitive problems, about 33% have a recurrence of the disease, and 5% to 10% of affected children are bound to die (Lynch et al., 2002 [4]). Due to underlying differences in incidence, cause, and outcome, Neonatal/ Perinatal stroke is discussed separately from Childhood stroke.

Neonatal/Perinatal stroke

Neonatal/Perinatal stroke presented in the neonatal period includes a range of focal and multifocal ischemic, hemorrhagic, or venous sinus thrombosis cerebral tissue injuries, and to avoid confusion the terms Neonatal stroke and Perinatal stroke that are sometimes used interchangeably, the condition is from now onwards referred to as Neonatal/Perinatal stroke (Rutherford et al., 2011 [5]).

Definition: Neonatal/Perinatal stroke is defined as “a group of heterogeneous conditions in which there is focal disruption of cerebral blood flow secondary to arterial (Perinatal Arterial Ischemic Stroke (PAIS)) or cerebral venous thrombosis or embolization (Cerebral Sinus Venous Thrombosis (CSVT)), between 20 weeks of fetal life through the 28th postnatal day, confirmed by neuroimaging or neuropathologic studies” (Raju et al., 2007 [6]). This definition separates Neonatal/Perinatal stroke from neonatal Hypoxic-Ischemic Encephalopathy (HIE) (Tsuji et al., 2015 [7]; Rutherford et al., 2011 [4]).

Etiology:

Ischemic stroke: Risk factors for the development of Neonatal/ Perinatal PAIS and CSVT may be maternal and/or fetal and neonatal but both conditions are likely to result from a combination of predisposing factors (Rutherford et al., 2011 [5]). The reported causes of Neonatal/Perinatal stroke have included cardiac disorders, infection, blood abnormalities, and/or perinatal events, however, in a large number of cases the cause remains undetermined.

In Neonatal/Perinatal stroke prothrombotic factors are frequently found in studies of PAIS (range 42-78%) and CSVT (Chabrier et al., 2011 [8]). In 64% (27/42) of neonates with CSVT, not one had antithrombin, protein C or protein S deficiency but higher than expected rates of Factor V Leiden (FVL) and 5,10 Methylenetetrahydrofolate Reductase (MTHFR) mutations were found (Fitzgerald et al., 2006 [9]).

Mutation of FVL has been associated with thrombosis and other pregnancy disorders, including severe gestosis, fetal growth retardation, placental abruption, preterm delivery, placental infarction, spontaneous miscarriage, and fetal mortality (Lynch et al., 2002 [4]).

The most common discharge diagnoses reported in conjunction with Neonatal/Perinatal stroke included infection, cardiac disorders, and blood disorders, with <5% associated with “birth asphyxia”. Coagulation disorders, including the Factor V Leiden (FVL) and prothrombin mutation, protein C, protein S, and antithrombin III deficiencies have been identified in up to half of infants and children with cerebral thromboembolism (Bonduel et al., 1999 [10]; deVeber et al., 1998 [11]; Golomb et al., 2001 [12]). Phospholipids are important in the activation of protein C and the coagulation pathway. Antiphospholipid antibodies, including lupus anticoagulant and anticardiolipin, are directed against anticoagulant proteins, interfere with normal coagulation, and may be a risk factor for Neonatal/ Perinatal stroke. A study by Nelson et al. (1998) found elevated titers of antiphospholipid antibodies in children with cerebral palsy versus controls (Nelson et al., 1998 [13]).

Hereditary coagulation abnormalities may lead to adverse events via thrombosis at the maternal-placental interface. Maternal thrombosis may lead to preeclampsia, fetal growth restriction, abortion, or fetal mortality. Thrombosis in the fetal circulation is a possible source of emboli that can bypass the liver and the lungs to reach the fetal brain. Within the fetus or neonate thrombosis or embolism based on FVL mutation are always possible (Lynch et al., 2002 [4]).

Hemorrhagic stroke: A population-based study revealed that perinatal hemorrhagic stroke was typically unifocal (74%) and unilateral (83%) yielding a prevalence for perinatal hemorrhagic stroke of 6.2/100,000 live births while etiologies included thrombocytopenia and cavernous malformation; 75% were idiopathic, with evidence of encephalopathy (100%) and/or seizures (65%). Unlike birth weight, predictors of perinatal hemorrhagic stroke were male gender, fetal distress, emergency cesarean delivery, prematurity, and postmaturity (Armstrong-Wells et al., 2009 [14]). In a recent study, idiopathic hemorrhagic stroke was related to young maternal age, primiparity, difficult fetal transition, bradycardia, low Apgar scores, and fetal SGA. Recurrence is rare event, however, outcomes are often poor (Cole et al., 2017 [15]).

Sinus Venous Thrombosis (SVT): Neonatal sinovenous thrombosis is a multifactorial disease. An incidence of 1.4 to 12 per 100,000 term newborns has been reported. In term-born neonates, assisted or complicated delivery occurred in 62% of the cases and the majority presented seizures, some prothrombin mutation (FII G20210A). At follow-up, moderate to severe neurological sequelae were diagnosed in over one-third of the cases while 19% died (Berfelo et al., 2010 [16]).

Specific pathophysiological, Histopathological, Clinical characteristics:

Neonatal/Perinatal ischemic stroke: In Neonatal/Perinatal ischemic stroke the most common cause of arterial occlusion is septic embolisation associated with disseminated intravascular coagulation (Barmada et al., 1979 [17]).

Focal and multifocal ischemic brain necrosis of all cellular elements occur within the distribution of single or multiple cerebral vessel or vessels (Volpe, 2008 [18]). The cellular changes depend primarily on the time passed after the insult and anoxic neuronal change can be visualised by light microscopy after 18-24 hours. Then cells of the monocyte-macrophage type migrate from vessels entering the lesion as pleomorphic activated microglia, followed by astroglial proliferation over weeks and months forming a dense layer of glial fibrillary processes, i.e., a glial scar (Volpe, 2008 [18]; Jensen, 2014 [19]).

Neuropathology of 592 infants revealed an incidence of 5.4% for cerebral infarction that was related to gestational age (0 <28 weeks, 5% 28-32 weeks, 10% 32-37 weeks, and 15% between 37- 40 weeks gestation (Barmada et al., 1979 [17]). Involvement of the middle cerebral artery occurred in 50%. The topography of perinatal ischemic stroke/infarction is 75% unilateral, of these 65% involve the left middle cerebral artery, and 25% bilateral (Volpe, 2008 [18]).

Ischemic lesions of the preterm and term brain are of considerable importance to the clinician as they account for a high proportion of infants who present with mental retardation, seizures, or various forms of cerebral palsy, e.g., spasticity, choreoathetosis, and ataxia. In the term infant, ante partum and intra partum asphyxia account for 90% of the cases.

Neonatal/Perinatal hemorrhagic stroke: Hemorrhagic cerebral infarction results when the injured capillaries in an ischemic infarct are ruptured by release of an arterial obstruction (e.g., embolus) or an increase in venous pressure, or when small amounts of bleeding from injured capillaries are not controlled by an intact clotting system. Thus, hemorrhagic infarction is observed in the newborn primarily with [1] embolic arterial occlusion because of distal movement of an embolus, [2] venous thrombosis because of the increase in venous pressure proximally, and [3] arterial thrombosis (or perhaps vasospasm) that is partial (or intermittent) or accompanied by a disturbance of coagulation. Indeed, most examples of “intracerebral hemorrhages” or “grade IV” intraventricular hemorrhage (PIVH) in the term newborn probably represent hemorrhagic venous infarction (Volpe, 2008 [18]).

Neonatal/Perinatal cerebral venous thromboses involve in ~65% the superior sagittal sinus, and infarction is present in 40% to 60%. The infarcts characteristically are hemorrhagic and MRI often reveals hemorrhagic infarction of the parasagittal cortical regions bilaterally with sagittal sinus thrombosis. Intraventricular hemorrhage is present in 20% to 35% of the cases. When focal and multifocal necroses are associated with dissolution of tissue and cavitation then Porencephaly is used to describe the lesion (Volpe, 2008 [18]).

Primary venous infarction usually involves obstruction of the deep vein system. There is dilatation of the deep vein tributaries with surrounding hemorrhages and necrosis. There is no recognizable clinical pattern in live born infants with primary venous infarction; rupture of blood into the ventricles will produce the clinical picture of intraventricular hemorrhage (Pape et al., 1979 [20]; Berger et al., 1997 [21]; Jensen and Holmer et al., 2018 [22]). If an infant were to survive a significant venous infarct, severe mental retardation and a high incidence of cerebral palsy would be expected as a result of destruction of long fiber tracts in the paraventricular white matter (Jensen, Neuhäuser, Jensen, 2018 [23]). Cortical hemorrhage is seen in association with cortical necrosis, and it has been suggested that these lesions are often hemorrhagic arterial infarcts (Friede, 1975 [24]).

Histological changes in the early stages include shrinkage of neurons, with acidophilic cytoplasm and pyknosis or karyorrhexis of nuclei. Sometimes there is ballooning and vacuolation of the cytoplasm of the degenerating neurons. At a later stage, there is loss of neurons and infiltration with lipid-laden macrophages and development of gliosis with capillary proliferation. The changes are similar in all areas of cortical or nuclear damage and resemble those seen in ischemic lesions in the adult brain (Pape et al., 1979 [20]). The time period involved in the propensity to cavitation (e.g., Porencephaly) is from approximately the second trimester of gestation to the first postnatal week or months. The high water content (90%) of unmyelinated brain tissue predisposes to cavitation (Volpe, 2008 [18]).

There is no specific clinical sign with cortical or white matter hemorrhagic infarction. If the lesion develops in newborns with birth asphyxia, coma, seizures, abnormalities of tone and/or neonatal reflexes may occur alone or in combination. Unilateral cortical infarcts secondary to focal compression necrosis or emboli may produce contralateral neurological signs and ipsilateral EEG changes (Pape et al., 1979 [20]; Jensen and Hamelmann, 2016 [1]).

Neonatal/Perinatal sinus venous thrombosis stroke: Sinus Venous Thrombosis (SVT) in children affects primarily neonates and results in neurologic impairment or death in approximately half of the cases. SVT is diagnosed when venous blood flow in at least one major sinus is impaired. Parenchymal infarction secondary to impaired venous drainage, and therefore not in an arterial distribution, results in approximately 60% of the cases. Perinatal complications (51%), e.g., hypoxic encephalopathy, dehydration (30%), and prothrombotic state (20%) are most common (deVeber et al., 2001 [25]).

In neonates, cerebral parenchymal infarcts are present in 42%, and 35% are hemorrhagic infarcts. These lesions are adjacent to the thrombosed sinus, e.g., hemorrhagic infarction in the cortex and subcortical white matter with superior sagittal sinus thrombosis and thalamic hemorrhage from thrombus in the internal cerebral veins and straight sinus. There is dilation of the deep vein tributaries with surrounding hemorrhages and necrosis. Rupture of blood into the ventricles may cause internal hydrocephalus. If capillary veins rupture, fibrin thrombi are seen within the veins. In cases of massive hemorrhage, the branches of the terminal vein may be grossly congested and there may be hemorrhage or infarction affecting the related white matter. Neuropathologic changes are similar to those described above (Pape et al., 1979 [20]).

The most common symptoms at presentation in the neonates are seizures (57%) (Grunt et al., 2010 [26]), but sinus venous thrombosis can also present in all ages with fever, lethargy, or coma or in younger infants with a history of decreased oral intake or respiratory distress. Even dilated scalp veins, eyelid swelling, or a large anterior fontanel may be revealed by physical examination (Tsze et al., 2011[27]). The best available clinical estimate of outcome is that after a mean of 2.1 years, 77% percent of neonates are neurologically normal. The primary neurologic manifestations in the neonates are seizures and diffuse neurologic signs. Adverse neurologic outcomes include seizures (20%), and the presence of infarcts (non-hemorrhagic or hemorrhagic) (deVeber et al., 2001 [25]).

Incidence and prevalence: Neonatal/Perinatal stroke affects between 1 in 2,330 – 5,000 newborns. In the National Hospital Discharge Survey (NHDS) from 1980 through 1998, the prevalence of stroke for term infants <30 days of age was 26.4/100,000, that of hemorrhagic stroke was 6.7/100,000, and that of ischemic stroke was 17.8/100,000 live births per year. This results in a rate of Neonatal/ Perinatal of approx. 1/4,000 live births per year (Lynch et al., 2002 [4]).

In Europe unilateral Neonatal/Perinatal Arterial Ischemic Stroke (PAIS) was recognized in the neonatal period in 1/2,300 term infants (Schulzke et al., 2005 [28]) and also bilateral stroke is not uncommon (Nelson, 2007 [3]). In general, Neonatal/Perinatal stroke is underrecognized clinically, because some infants are neurologically asymptomatic in the newborn period, and motor impairment is recognized only at about 4 to 5 months of age (Lynch et al., 2002 [4]).

In one study of infants greater than 31 weeks gestation with neonatal seizures, 12% had underlying cerebral infarction (Estan et al., 1997 [29]). These results suggest a prevalence of Neonatal/ Perinatal stroke of 24.7/100,000 per year in infants greater than 31 weeks gestation and are similar to a published report of 28.6/100,000 by Perlman and coworkers (Perlman et al., 1994 [30]).

Arterial ischemic stroke: In the Swiss Registry, neonates with a diagnosis of Neonatal Arterial Ischemic Stroke (NAIS) were included. Follow-up was performed 2 years (mean 23.3 months, SD 4.3) after birth (Grunt et al., 2015 [31]) and 100 neonates (two-third boys) had a diagnosis of NAIS with seizures being the most common symptom (95%). Furthermore, 81% had unilateral (80% left-sided) and 19% had bilateral lesions, respectively. There were maternal risk conditions (32%), birth complications (68%), and neonatal comorbidities (54%). Two years after birth, 39% presented with cerebral palsy and 31% with mental delay (Grunt et al., 2015 [31]). Given the cases of symptomatic NAIS, the prevalence is 12.5/100,000 live births. In addition, the fraction of 40% of total NAIS that is asymptomatic has to be accounted for (Grunt et al., 2015 [31]).

Hemorrhagic stroke: There is no specific prevalence data for hemorrhagic Neonatal/Perinatal stroke in Europe. In the Northern California Kaiser Permanente Medical Care Program 17 cases of symptomatic Neonatal/Perinatal hemorrhagic stroke were identified, which yielded a population prevalence of 8.14/100,000 live births per year (Agrawal et al., 2009 [32]).

Venous sinus thrombosis: In the Swiss Registry, neonates with a diagnosis of Cerebral Sinus Venous Thrombosis (CSVT), symptomatic at presentation were included. Follow-up was performed 2 years after birth. The prevalence of symptomatic CSVT was 3.2/100,000 live births (Grunt et al., 2010 [26]).

Thus, the total prevalence of Neonatal/Perinatal stroke, i.e., stroke caused by arterial ischemia, hemorrhages, and venous thrombosis combined, is 32.1/100,000 live births. This is largely in agreement with data from the Canadian Pediatric Ischemic Stroke Registry (CPISR).

Management of Neonatal/Perinatal stroke: As mentioned, risk factors for the development of Neonatal/Perinatal PAIS and CSVT may be maternal and/or fetal and neonatal but both conditions are likely to result from a combination of predisposing factors (Rutherford et al., 2011 [5]). The reported causes of Neonatal/Perinatal stroke have included infection, blood abnormalities, perinatal events, and or cardiac disorders.

Prevention of infection (early intervention, antibiotics), blood abnormalties (treatment of thrombophilia), trauma (prospective risk management during labour, including large term-born infants (Jensen and Holmer, 2018 [22]), and perinatal events during birth (early intervention during asphyxia (Berger et al., 1997 [21]) are the most important means of preventing Neonatal/Perinatal stroke. Secondly, if prevention fails, follow professional management of perhaps infected, traumatized, and asphyxiated newborns along the guidelines (BNCM, 2009 [33]). Furthermore sentinel events during parturition particularly when combined with neonatal cephalic molding during birth and head deformity require further examination of the head by imaging for early diagnosis of neonatal brain damage (Jensen and Holmer, 2018 [22]). If Neonatal/Perinatal stroke is inevitable, symptomatic, and diagnosed by appropriate imaging techniques (Cranial ultrasound, CT, MRI), therapeutic cooling procedure within 6 hours after birth has become standard of care for more mature preterm (>=36 weeks gestation) and term newborn infants to reduce the adverse effects of encephalopathy (Gluckman et al., 2005 [34]).

Symptoms: Due to the fact that 40% of infants are clinically normal in spite of having a cerebral infarction, makes imaging, i.e., cranial ultrasound, CT, and particularly MRI imaging indispensable for a proper and timely diagnosis. As stated above, only 60% of the infants presenting with cerebral infarction are symptomatic, generally showing seizures, apnoe, or abnormal tone. Depending on the location and extent of the lesion the clinical signs vary considerably between inapparent, asymptomatic, and coma.

Diagnosis: Neuro-imaging is required to confirm the diagnosis of Neonatal/Perinatal stroke or Cerebral sinus venous thrombosis. Available techniques include Ultrasonography (transfontanel, power Doppler), CT, and MRI. The use of CT is discouraged because of excessive radiation dosage. Also, intravenous contrast may be required for detection of sinus thrombosis. Conventional T1-weighted and T2-weighted MRI sequences, Diffusion-Weighted Imaging (DWI) or diffusion tensor imaging, MR venography, and MR Angiography (MRA) should be included if necessary (Rutherford et al., 2004 [4]).

Non-cellular treatment: There is no curative treatment for Neonatal/Perinatal stroke hitherto. Treatment is symptomatic and supportive. However, children with Neonatal/Perinatal stroke should receive regular medical screenings to determine appropriate interventions. Acute treatment of neonatal AIS includes supportive measures, e.g., maintenance of normal hydration, electrolytes, hemoglobin, clotting factors, glucose, oxygen, and pH levels are important. Beyond anticonvulsants, antibiotics and/or antivirals should be given if sepsis is suspected. At present, therapeutic hypothermia in neonates with cerebral arterial ischemic stroke or sinus, venous thrombosis has not yet to been validated but hyperthermia is to be avoided. There are no randomised controlled trials addressing the acute or chronic treatment of Neonatal/ Perinatal stroke and the majority of recommendations are based on case series and expert consensus. Administration of Unfractionated Heparin (UFH) or Low Molecular Weight Heparin (LMWH) in neonates with a first AIS and an ongoing documented cardioembolic source has been recommended by the American College of Chest Physicians (ACCP). However, anticoagulation or antiplatelet therapy is not recommended since the risk of recurrent stroke is minimal. Administration of anticoagulants in neonatal CSVT is controversial because recurrent CSVT is also rare (Rutherford et al., 2004 [4]).

Therapeutic hypothermia (cooling): Infants with symptomatic Neonatal/Perinatal stroke (accounting for 10% of all Neonatal Encephalopathies (NE)) and infants with evidence for encephalopathy on the basis of perinatal events (asphyxia, 85% of all NE), infection, blood abnormalties, trauma, and/or seizures are often very sick with multi-organ failure requiring intensive care. Current evidence shows that mild hypothermia (33-34°C) can improve neurological outcome at 2 years and is now recommended as the standard of care. However, cooling should be part of a package of neurointensive care that enables appropriate investigation, treatment, and imaging to be undertaken prior to long term follow-up being arranged for surviving infants. Infants with Neonatal/Perinatal stroke and suspected HIE who meet the inclusion criteria should be considered for treatment with cooling (Walston et al., 2012 [35]).

Seizures may be apparent on clinical examination (abnormal rhythmic movement of limbs, lip smacking, etc.) but can sometimes be difficult to diagnose. The amplitude integrated EEG (aEEG) as recorded by a Cerebral Function Monitor (CFM) is able to detect most seizures including many seizures not apparent clinically - known as subclinical seizures. Although an aEEG should ideally be performed on all infants with moderate-severe HIE, it is not mandatory prior to starting cooling and may not be available in all units. But there are other evidence based provisions to consider, too, as for example the Recommendations Class 1-3 for Neonatal/Perinatal Stroke (Roach et al., 2008 [36]).

Outcome and prognosis: The outcome of children with Neonatal/ Perinatal stroke varies among studies because of differences in stroke type, duration of follow-up, specific outcomes, and population studied. Electroencephalography and neuro-imaging studies provide measures of long-term cognitive and motor outcome. Children with an abnormal electroencephalogram during the first week after stroke typically develop hemiplegia. Mercuri and colleagues (1999) found that infarction involving the internal capsule predicts the development of hemiplegia, whereas involvement of other regions is less reliable (Mercuri et al., 1999 [37]). These results are similar to a previous study of preterm children with hemorrhagic infarction. In reference to language function, Trauner and colleagues (1996) have found that children with early unilateral brain damage exhibit prosodoic deficits when compared with controls, and the side of the lesion seems to be related to affective comprehension (Trauner et al., 1996 [38]). In contrast to the relationship between infarct location and language deficits or hemiplegia, the location of the lesion does not adequately predict visual abnormalities.

The recurrence rate of neonatal stroke is much less than in childhood stroke. In the Canadian Pediatric Ischemic Stroke Registry (CPISR), the largest cohort of stroke in children, the recurrence rate of children with neonatal stroke was 3% to 5%, and in survivors outcome was normal in 33% (deVeber et al., 2000 [39]).

Childhood stroke

Definition: Childhood stroke is defined as a cerebrovascular event that occurs between >28 days and <18 years of age (Lynch et al., 2002 [4]). The diagnosis is based on Magnetic Resonance Angiography (MRA) or conventional angiography abnormalities.

Etiology:

Ischemic stroke: There are no widely accepted classification systems for the cause of ischemic stroke in children. However, the mechanisms by which ischemic stroke occurs in children include the following: thromboembolism from an intracranial or extracranial vessel, or the heart; acute, transient, or progressive arteriopathy; and other rare causes, but in a large percentage of cases the cause is undetermined (Al Sulaiman et al., 1999 [40]). The diagnosis of stroke attributable to cardiac or transcardiac embolism has varied among cohort studies. Children with stroke attributable to cardiogenic embolism typically have some evidence of an underlying heart disorder, with or without a recognized thrombus. However, a recent study of Childhood stroke categorized children with cardiac or transcardiac embolism even without a history of cardiac disease (Chabrier et al., 2011 [8]). Children with stroke attributable to angiopathy typically have some evidence based on angiographic, non-invasive, or other studies of arterial dissection, vasculitis, Moyamoya disease, transient cerebral angiopathy, or other vasculopathy (Chabrier et al., 2011 [8]).

The most frequently reported risk factors for ischemic stroke in children include arteriopathy coagulation disorders, Sickle Cell Disease (SCD), cardiac disorders, infection, Moyamoya disease, arterial dissection, and other rare genetic disorders. But there are differences as far as the geographical region is concerned (Mackay et al., 2011 [41]).

Cardiac disease, as previously stated, is a common cause of stroke in children, accounting for up to 50% of strokes in case series (Chabrier et al., 2011 [8]). In the CPISR, cardiac disease was identified in 25% of children with ischemic stroke. Several cardiac disorders are associated with stroke in children, including congenital heart disease, intracardiac defects, cardiac procedures, and acquired heart disease. Several acquired and genetic coagulation factor abnormalities have been associated with ischemic stroke in children. A study of cerebral thromboembolism that included 92 children from the Hospital for Sick Children in Toronto found coagulation abnormalities in 38% of children studied. Antiphospholipid antibodies have been reported in a quarter of patients with a first stroke. In a study by Angelini and co-workers (1994), 75% of children with cerebral ischemia were positive for antiphospholipid antibodies (Angelini et al., 1994 [42]). Sickle cell-related stroke is associated with large vessel stenosis of the proximal middle cerebral or distal internal carotid artery. The risk of stroke in children with Sickle Cell Disease (SCD) is 200 to 400 times that of children without SCD, the recurrence rate is extremely high (50% by 3 years), and in children with stroke aged 6 months to 10 years, up to 30% have post varicella angiopathy. Moyamoya, a chronic non-inflammatory occlusive intracranial vasculopathy of unknown cause, is primarily seen in the Japanese population and accounts for 10% to 20% of arterial infarcts in children. Extracranial arterial dissection, mostly caused by trauma, is a common cause of stroke in young adults and children while intracranial arterial dissection occurs almost as frequently as extracranial dissection in children (see Lynch et al., 2002 [4] for review).

Hemorrhagic stroke: Hemorrhagic stroke is less common than ischemic stroke in children and several risk factors for hemorrhagic stroke have been identified in children, including vascular malformations, malignancy, and trauma. Furthermore, primary and secondary coagulation disorders including hemophilia, thrombocytopenia, liver failure, leukemia, and warfarin therapy are associated with hemorrhagic stroke in children (Lynch et al., 2002 [4]).

The most common cause of hemorrhagic stroke in children are Arteriovenous Malformations (AVM) (Menovsky et al., 1997 [43]) resulting from failure in the formation of the capillary bed between cerebral primitive arteries and veins during the first trimester of gestation. The incidence of AVM in children is 1/100,000, of these approximately 10% to 20% will become symptomatic during childhood (Menovsky et al., 1997 [43]). The average probability of a first hemorrhage is 2% to 4% per year, with a recurrence risk as high as 25% by 5 years. Approximately 1% to 2% of aneurysms become symptomatic during childhood and are typically associated with other vascular lesions or chronic disorders. Also, cavernous malformations can lead to hemorrhagic stroke in children, one third being related to family history and have recently been linked to abnormalities in the long arm of chromosome 7. Magnetic Resonance (MR) imaging and angiography (MRA) confirm the diagnosis of AVM (see Lynch et al., 2002 [4] for review).

Sinus venous thrombosis (SVT): Data from the CPISR reveal that the incidence of SVT in children is 0.6/100 000, children per year and is highest in the first year of life (deVeber et al., 2001 [25]). A number of risk factors are linked to the development of SVT, including head and neck infections, dehydration, perinatal complications, and coagulation disorders. In one study, prothrombotic abnormalities were present 50% of the children with SVT and many of these children had multiple risk factors (deVeber et al., 1998 [11]).

Specific pathophysiological, histopathological, clinical characteristics:

Childhood ischemic stroke: Childhood ischemic stroke results in focal and multifocal ischemic brain necrosis of all cellular elements that occur within the distribution of single or multiple cerebral vessel or vessels (Volpe, 2008 [18]). As already mentioned, the cellular changes relate primarily to the time after theinsult and are accompanied with migration of cells of the monocyte-macrophage type that enter the lesion as activated microglia, followed by astroglial proliferation eventually forming a glial scar (Volpe, 2008 [18]).

Ischemic lesions of the brain during childhood are of considerable importance to the clinician as they account for a high proportion of infants who present as a focal neurologic deficit, e.g. hemiplegia (94%), or seizures (50%). Older children demonstrate more specific neurological defects similar to adults, e.g., hemiparesis, language (e.g., aphasia) and speech difficulties, visual deficits, and headache (Tsze et al., 2011 [27]).

Childhood hemorrhagic stroke: Hemorrhagic stroke is less common than ischemic stroke in children. The histopathological changes are those described above, are similar in all areas of cortical or nuclear damage, and resemble those seen in ischemic lesions in the adult brain (Pape et al., 1979 [20]).

Childhood hemorrhagic strokes usually present as headaches (59%), reduced consciousness (88%), or seizures (29%) and are, unlike arterial ischemic stroke, more likely to cause vomiting (48%). A subarachnoid hemorrhage can also present as irritability and a bulging fontanel in infants, but should be suspected in older children complaining of sudden acute onset headache, neck pain, meningisms, or photophobia. Dysarthria and bulbar dysfunction suggest lower brainstem involvement while aphasia points to involvement of the basal ganglia, thalamus, or cerebral hemispheres. The eyes look towards the lesion if the hemispheres, however, away from the lesion if the brainstem is involved (Tsze et al., 2011 [27]).

Childhood cerebral sinus venous thrombosis stroke (SVT): Data from the CPISR reveal that the incidence of Sinus Venous Thrombosis (SVT) in children is 0.6/100,000 per year and is highest in the first year of life (deVeber et al., 2001 [25]). A number of risk factors are linked to the development of Sinus Venous Thrombosis stroke (SVT), including head and neck infections, dehydration, perinatal complications, and coagulation disorders. In one study, prothrombotic abnormalities were present in half of children with SVT and many of these children had multiple risk factors (deVeber et al., 1998 [11]).

Venous infarction involves obstruction of the deep vein system. There is dilation of the deep vein tributaries with surrounding hemorrhages and necrosis. Rupture of blood into the ventricles may cause internal hydrocephalus. The histological changes largely resemble those described above (Pape et al., 1979 [20]).

Physical examination may reveal dilated scalp veins, eyelid swelling, or a large anterior fontanel whereas an older child would likely present with more slowly progressing signs, such as vomiting, headache, or any other signs of increased intracranial pressure. As mentioned above sinus venous thrombosis can present clinically in all ages with fever and lethargy or coma, but young infants can present with a history of decreased oral intake or respiratory distress (Tsze et al., 2011 [27]).

Incidence and prevalence: A stroke or Cerebral Vascular Accident (CVA) in children is considered to be a rare event. The prevalence of childhood stroke from population-based studies is estimated between 2-3/100,000 children in the United States and as high as 13/100,000 children in France (Kittner et al., 1996 [44]; Giroud et al., 1995 [45]). The CPISR reported ischemic stroke in 2.7/100,000 children <18 years of age per year, 40% being under 1 year of age, with a male to female (1.5:1) predominance. In the NHDS (1980 to 1998), for children 0 to 18 years, the prevalence of stroke was 13.5/100,000; that of hemorrhagic stroke was 2.9/100,000, and that of ischemic stroke was 7.8/100,000 children per year with a US mortality rate for stroke in children (1-15 years of age) of 0.6/100,000 children. Mortality was higher in males than females and in black compared with white individuals (see Lynch et al., 2002 [4] for review). About 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, develop subsequent seizure disorders, and learning or developmental problems (Tsze et al., 2011 [27]).

Arterial ischemic stroke: In the Swiss Registry, 116 children age 1 month to 16 years with a diagnosis of Arterial Ischemic childhood Stroke (AIS) confirmed by neuroimaging were included. Follow-up information was obtained in 95/116 children (Simonetti et al., et al., 2015 [46]). Mortality was 14% (13/95) and was higher than in young adults. In 56% of the children followed, the outcome was favorable. The study comprised all childhood AIS cases in Switzerland with a prevalence of AIS of 0.17/100,000.

Hemorrhagic stroke: Arteriovenous Malformations (AVM) are the most common cause of hemorrhagic stroke in childhood and become symptomatic in 10-20% whereas only 1-2% of cerebral vascular aneurysms do so (Menovsky et al., 1997 [43]). The incidence of the previous (AVM) is reported to be 1/100,000 per year that of the latter (aneurysm) is not known. However, of a series of patients with suspected genetic disposition to development of Familial Intracranial Aneurysm (FIA), in which mutations on chromosome 11 and 6 may be involved, an annual rupture rate of 1.2% in undiagnosed aneurysms was determined (n=113, adults and children) which is 17 times higher than in subjects without a family history. At any rate, rupture of intracerebral aneurysms tend to occur in older ages rather than in childhood.

Venous sinus thrombosis: The Canadian study group CPISR determined an incidence of 0.6/100,000 CVST per year (deVeber et al., 2001 [25]). This is in general agreement with other populationbased studies (Agrawal et al., 2009 [32]. Thus, ignoring the miniscule fraction of cerebral ruptured vascular aneurysms in childhood, the total prevalence of childhood stroke amounts to 0.97/100,000 population.

Management of Childhood stroke: Employment of appropriate imaging techniques is crucial for diagnosis and hence for successful care provided. Non-contrast head Computed Tomography (CT) is sensitive for acute bleeding and should be obtained emergently to exclude a hemorrhagic cause of stroke. If a hemorrhagic stroke is identified, Magnetic Resonance Venography (MRV) should follow as 10% of hemorrhages in children is due to cerebral sinus venous thrombosis. For diagnosing Arterial Ischemic Stroke (AIS), noncontrast CT is also the initial study, however, within 12 hours from the insult CT may appear normal while MRI is more sensitive for early detection of an infarction. The most precise detail of vascular anatomy of all imaging modalities is provided by Catheter Angiography (CA) which is superior to MRA and Computed Tomography Angiography (CTA) and can be performed along with endovascular therapy (Tsze et al., 2011 [27]). Once the type of stroke is identified, treatment depends on etiology. Hemorrhagic strokes may require prevention of re-bleeding by recombinant factor VIIa, though adult studies have not confirmed improvement of survival or functional outcome. Surgical management of hemorrhagic stroke is considered highly controversial.

AIS management in childhood includes preventing subsequent ischemic events, e.g., by anticoagulation. Thrombolytic therapy in children with ischemic strokes must be carried out with caution, tPA may be considered in a selected group of children with CVST while management of stroke in Sickle cell disease requires exchange blood transfusion (Tsze et al., 2011 [27]).

There are guidelines for the general management of pediatric stroke covering presentation and diagnosis, investigations, acute care, secondary prevention (RCP Guidelines, 2004 [47]), and recommendations for children with sickle cell disease (Class 1-2), hypercoagulable states (Class 1-2), evaluation and treatment of hemorrhage in children (Class 1), treatment of cerebral venous sinus thrombosis (Class 1-2), and for stroke and heart disease (Class 1-3) (Roach et al., 2008 [36]).

Symptoms: There are some generalizations that can be made as to how stroke presents in children (Tsze et al., 2011 [27]). Arterial Ischemic Stroke (AIS) in childhood most often presents as a focal neurologic deficit. Hemiplegia is the most common focal manifestation, occurring in up to 94% of cases. Hemorrhagic stroke most commonly presents as headaches or altered level of consciousness, and are more likely to cause vomiting than AIS. Seizures are common in both ischemic- and hemorrhagic strokes. They occur in up to 50% of children with strokes, are not restricted to any group, and are not limited to any specific seizure type. There can be significant differences in the clinical presentation based on the child’s age. The younger the child, the more non-specific their symptoms may be. Neonatal/Perinatal strokes are more likely to initially present with focal seizures or lethargy in the first few days after birth. Older children demonstrate more specific neurological defects similar to adults. These include hemiparesis, language (e.g., aphasia) and speech difficulties, visual deficits, and headache (see Tsze et al., 2011 [27] for review).

Diagnosis: There are no published consensus guidelines on the evaluation of stroke in children, but, the evaluation should rule out other nonvascular causes and identify the cause of the stroke. Cranial ultrasound imaging is particularly useful for intraventricular and germinal matrix hemorrhages, but is less suitable for diagnosing ischemic stroke, in particular cortical or posterior infarcts. Transcranial doppler is useful in sickle cell-related stroke while CT is instrumental for identifying acute hemorrhages. Various MR techniques, including diffusion, perfusion, gradient echo, and FLAIR (Fluid-attenuated inversion recovery) imaging, are used to detect ischemic stroke in children. MR arteriography is helpful to diagnose arteriopathies as is computed tomography and MR venography for Sinus Venous Thrombosis (SVT). For children with suspected extracranial arterial dissection MR imaging should be considered and ultrasonography can detect abnormal flow patterns in >90% of patients. Functional MR is useful in monitoring changes over time during recovery. Conventional angiography may be considered when MR scanning has failed to identify a cause (see Lynch et al., 2002 [4] for review).

Also hematologic, metabolic, and angiographic studies have to be included in the work-up procedure, as recent evidence suggests that the identification of multiple risk factors predicts worse longterm outcome. Also, any history of head or neck trauma, unexplained fever or recent infection, drug ingestion, developmental delay, family history of bleeding problems, and associated headache are of importance (Lynch et al., 2002 [4]).

Treatment

Non-cellular treatment: There is no specific curative treatment for Childhood stroke hitherto. Treatment is symptomatic and supportive; however, children with Childhood stroke should receive regular medical screenings to determine appropriate interventions. In adults, Tissue Plasminogen Activator (TPA) is the only approved treatment for acute ischemic stroke, and has not been tested in children (Lynch et al., 2002 [4]). Once the type of stroke is identified, treatment depends on etiology. A goal specific to AIS management includes preventing a subsequent ischemic event, e.g., by anticoagulation. Thrombolytic therapy in children with ischemic strokes must be carried out carefully. Published guidelines suggest that tPA may be considered in a selected group of children with CVST. Management of stroke in Sickle Cell Disease (SCD) requires exchange blood transfusion (Tsze et al., 2011[27]).

The treatment for hemorrhagic stroke in children depends on the cause and the condition of the patient. In general, hemorrhagic strokes may require medical management beyond supportive measure, e.g., prevention of re-bleeding by recombinant factor VIIa, though adult studies lack demonstration of benefit. Although, surgical management of hemorrhagic stroke continues to be controversial, treatments for vascular malformations include surgery, endovascular embolization, and radiosurgery (Menovsky et al., 1997 [43]).

Outcome and prognosis: Depending on differences in functional measures, stroke type, and population studied, the outcome of children after stroke varies among studies. According to CPISR data including children with arterial ischemic stroke (n=402) and children with sinus venous thrombosis (n=160), revealed that 27% of children were neurologically normal, 61% were abnormal, 21.6% recurred, and 12% died (Lynch et al., 2002 [4]). Poor outcome in children with stroke is associated with certain neuroimaging abnormalities and seizures at presentation. Children with infarct volumes greater than 10% of intracranial volume have a worse outcome than children with less than 10% infarct volume and the recurrence rate is 20% to 40% (Lynch et al., 2002 [4]).

Cell-Based Therapy of Pediatric Stroke

Cellular characteristics of human autologous cord blood mononuclear cells

The first documented cell-based treatment of a girl presenting Neonatal/Perinatal birth traumatic arterial ischemic stroke with autologous cord blood mononuclear cells (hucbMNC) performed at five years of age dates back to 2009 (Jensen and Hamelmann, 2016 [1]). We deliberately used the whole range of unmanipulated mononuclear cells derived from autologous human umbilical cord blood including progenitor and stem cells. These include among other leukocytes (CD45+), lymphcytes (CD3+ T-cells, CD19+ B-cells, CD56+ NKcells), Monocytes (CD14+), Haemopoietic progenitor cells (CD34+), Mesenchymal stem cells (CD73+, CD90+, CD105+, CD13+, CD44+, CD29+), and Unrestricted Somatic Stem Cells (USSC)(CD45-).

Preclinical studies - Mode of action

Initially, in preclinical studies the mode of action was unclear. However, in cell culture, analysis of medium conditioned by human umbilical cord blood-derived Mononuclear Cells (hucbMNC) revealed substantial secretion of a number of cytokines, chemokines, and growth factors, induced by EGF/FGF-2 or NGF/RA treatment. Many of the secreted factors are well-known for their beneficial effects under inflammatory or brain-damaging conditions (Neuhoff et al., 2007 [49]) (Figure 1). “Thus, one potential mode of interaction was considered to be the release of neuroprotective, anti-inflammatory, or angiogenic factors by transplanted cells, thereby providing the basis for an indirect effect on host tissue” (Neuhoff et al., 2007 [49]). Hence, it was proposed that secondary mechanisms might be responsible for the beneficial outcome upon hucb mononuclear cell transplantation in a stroke-like lesion model (Meier et al., 2006 [52]). We used the Levine model in neonatal rats at PN7 (post-natal day 7) inducing hypoxic ischemia in the periventricular region by unilateral ligation of the (left) carotid artery in combination with 80 minutes respiration of hypoxic ambient air (8% O2) resulting in periventricular white matter damage, a porencephalic cyst, and contralateral (right) spastic hemiparesis, the key symptom of cerebral palsy subsequent to stroke affecting the internal capsule where long corticospinal tracts pass (Levine et al., 1960 [50]; Meier et al., 2006 [52]). In recent years, ischemic and hemorrhagic brain lesions have been successfully treated in juvenile and adult animal models of various species using human cord blood mononuclear cells (containing among other mesenchymal stem cells, MSC), i.e., in fetal sheep (Garnier et al., 2002 [51]), in newborn rats (Meier et al., 2006 [52]; Pimentel-Coehlo, 2010 [53]; Rosenkranz et al., 2010 [54]; Ahn et al., 2013 [55]), in newborn rabbits (Drobyshevsky et al., 2015 [56]), and in adult rats (Chen et al., 2001 [48]); Liao et al., 2009 [57]) (see Li et al., 2014 [58] for review).