Cell-Based Therapies in Neonatal Stroke

Fig. 17.1

Neonatal stroke account for up to 5–10 % of neonatal encephalopathy cases. Some neonatal stroke cases present with seizures without encephalopathy during the neonatal period. Approximately, 40 % of neonatal stroke cases do not present symptoms in the neonatal period

Incidence

Neonatal stroke is not a rare condition. The estimated incidence of ischemic perinatal arterial ischemic stroke ranges between 1 in 2300 and 1 in 5000 births, places the perinatal period second only to elderly age groups with respect to the incidence of stroke [2]. The estimated incidence of neonatal cerebral sinus venous thrombosis ranges between 1 and 2.7 in 100,000 births [1]. The incidence of neonatal encephalopathy in developed countries is 1–6 per 1000 live births [6]. From 50 to 80 % of neonatal encephalopathy cases are considered to have HIE, and up to 5–10 % of such cases are considered to have stroke [6] (Fig. 17.1).

Pediatric strokes occurring beyond the neonatal period are a serious health problem as well. The incidence of pediatric stroke is approximately 10–13 per 100,000 children per year, and cerebrovascular disorders are among the top ten causes of death in children in the USA [9]. This chapter, however, mostly focuses on neonatal stroke because of almost lack of publications in cell therapies in pediatric stroke.

Clinical Presentation

The cardinal clinical feature of neonatal encephalopathy is depressed level of consciousness, often associated with seizures [6]. Approximately, 60 % of the infants with perinatal/neonatal stroke present early symptoms, of which clonic and/or tonic seizures are most frequent (approximately 90 %) [4]. Other early symptoms include recurrent apnea/desaturation, persistently altered tone, and decreased level of consciousness. The remaining 40 % of the children with perinatal stroke do not present specific symptoms in the neonatal period, and are only recognized later with the emergence of the symptoms such as hemiplegia and seizures [4].

Treatment

The current treatment for infants with stroke is predominantly supportive, as there is no evidence-based specific treatment available [4, 10]. The onset of neonatal stroke is antenatal in some cases and is unknown in others. Hence, treatments that have a narrow therapeutic window, such as tissue plasminogen activator (tPA), are not feasible for perinatal/neonatal stroke. Cell-based-therapy has attracted attention as a novel treatment for a number of neurological diseases, including neonatal encephalopathy [11]. This is not only because of its possible regenerative properties but also because of the long therapeutic time window for the effect of stem cells. More than 1000 of therapeutic treatments for ischemic brain injury have reported neuroprotective in studies in neonatal and adult animals [12]. Although more than 100 treatments among more than 1000 candidates have been tested in clinical trials, as few as two treatments have proven clinical efficacy: tPA for adult stroke and therapeutic hypothermia for neonatal encephalopathy. Even in well-controlled animal studies, therapeutic time windows of those treatments are disappointingly short from a clinical standpoint. Most treatments are neuroprotective only when started before the insult. Although some therapies are neuroprotective with posttreatment, the therapeutic time windows are confined to first several hours after the insult. In contrast, cell therapies have been shown to have a neuroprotective effect in animal studies even when administered days after the insult [13, 14].

Preclinical Studies on Cell Therapies

Overview

More than 50 research articles on cell-based therapies for perinatal/neonatal brain injury have been published in English literature to date since the first report in this field by Elsayed et al. in 1996 [15] (Table 17.1, 17.2, 17.3). The vast majority of those studies have used rodent models of neonatal HIE. Only four studies in rodent models of neonatal stroke have been published [16–19] (Table 17.2). No study on the effects of cell-based therapy for perinatal/neonatal brain injury in large animal models has been reported, although some groups are doing research on it (abstracts of conferences and personal communications). No study in animal models of childhood stroke has been reported.

Table 17.1

Review of reported studies in models with neonatal hypoxic–ischemic brain damage

	Research group	Animal	Cell			Timing^a	Delivery route	Follow-up	Improvement		Author
			Source	Type	Dose				Histology	Behavior
1		P7 rat	Fetal brain	Neocortical tissue	block	7d	i.c.	6wk	no	NA	Elsayed et al. [15]
2		Neonatal rat	Fetal brain	Neocortical tissue	block	3d	i.c.	12wk	NA	yes	Jansen et al. [20]
3	A	P7 mouse	Mouse	NSC	1 × 107	7d	i.c.	12wk	yes	NA	Park et al. [21]
4	A	P7 mouse	Mouse	NSC	0.4 − 1.6 × 105	3d	i.c.	4wk	NA	NA	Park et al. [25]
5	A	P7 mouse	Mouse	NT3-NSC	~ 3 × 105	3d	i.c.		NA	NA	Park et al. [26]
6	A	P7 mouse	Human, mouse	NSC	~ 3 × 105	3d	i.c.	10wk	NA	NA	Imitola et al. [27]
7		P7 rat	Neonatal mouse	MASC	5 × 104	24 h or 5d	i.c.	3wk	NA	NA	Zheng et al. [28]
8	B	P7 rat	Rat BM	MAPC	2 × 105	7d	i.c.	3wk	NA	yes	Yasuhara et al. [29]
9	B	P7 rat	Mouse BM	MAPC	2 × 105	7d	i.c. or i.v.	3wk	yes	yes	Yasuhara et al. [24]
10	B	P7 rat	hUCB	MNC +mannitol	1.5 × 104	7d	i.v.	3wk	NA	yes	Yasuhara et al. [13]
11	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p.	2wk	NA	yes	Meier et al. [23]
12	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p.	2wk	NA	NA	Rosenkranz et al. [30]
13	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p.	6wk	yes	NA	Geißler et al. [31]
14	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p. or i.t.	7wk	yes	yes	Wasielewski et al. [32]
15	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p.	2wk	yes	NA	Rosenkranz et al. [33]
16	C	P7 rat	hUCB	MNC	1 × 107	24 h	i.p.	2wk	NA	NA	Rosenkranz et al. [34]
17		P7 mouse	Mouse ES cell	ES cell-derived cell	1 × 104	2–3d	i.c.	8m	yes	yes	Ma et al. [35]
18		P7 rat	Fetal rat brain	NSC +ChABC	2 × 105	24 h	i.c.	8d	yes	NA	Sato et al. [36]
19	D	P7 rat	hUCB	MNC	1 × 107	24 h	i.v.	3wk	no	no	de Paula et al. [37]
20	D	P7 rat	hUCB	MNC	1 × 106, 107, 108	24 h	i.v.	8wk	yes	yes	de Paula et al. [38]
21	D	P7 rat	hUCB	MNC	1 × 106, 107	24 h	Intraarterial	9wk	no	yes	Greggio et al. [39]
22		P7 rat	Human BM	MSC	1 × 106	72 h	Intracardiac	6wk	no	yes	Lee et al. [40
23		P7 rat	Human ES	NSC	1.5 × 105	24 h	i.c.	4wk	no	yes	Daadi et al. [41]
24		P7 rat	hUCB	MSC	1 × 105	3d	i.c.	4wk	yes	yes	Xia et al. [42]
25		P7 rat	hUCB	MNC	2 × 106	3h	i.p.	7d	yes	yes	Pimentel-Coelho et al. [43]
26	E	P9 mouse	Mouse BM	MSC	1 × 10⁵	3 or 10d 3 + 10d	i.c.	4wk	yes	yes	van Velthoven et al. [44]
27	E	P9 mouse	mouse BM	MSC	5 × 105	10d	Intranasal	4wk	yes	yes	van Velthoven et al. [45]
28	E	P9 mouse	Mouse BM	MSC	1 × 105	3, 10, or 3 + 10d	i.c.	4wk	yes	yes	van Velthoven et al. [46]
29	E	P9 mouse	Mouse BM	MSC	1 × 105	3, 10, or 3 + 10d	i.c.	4wk	yes	NA	van Velthoven et al. [47]
30	E	P9 mouse	Mouse BM	MSC	1 × 105	3d + 10d	i.c.	4wk	yes	yes	van Velthoven et al. [48]
31	E	P9 mouse	Mouse BM	MSC	0.25, 0.5, 1 × 106	3, 10, 17, or 3 + 10d	Intranasal	9wk	yes	yes	Donega et al. [14]
32	E	P9 mouse	Mouse BM	GM-MSC	5 × 105	10d	Intranasal	4wk	yes	yes	van Velthoven et al. [49]
33	E	P9 mouse	Mouse BM	MSC	1 × 106	10d	Intranasal	2wk	yes	NA	Donega et al. [50]
34	F	P10 rat	Mouse	NSC	5 × 105	3d	i.c.	58wk	NA	NA	Obenaus et al. [51]
35	F	P10 rat	Human	NSC	~ 2.5 × 105	3d	i.c.	13wk	yes	yes	Ashwal et al. [52
36		P7 rat	Rat embryo	NSC, VEGF-NSC	1 × 105	3d	i.c.	5wk	yes	yes	Zheng et al. [53]
37		P7 rat	hUCB	MNC	1 × 107	24 h	i.v.	10wk	yes	yes	Bae et al. [54]
38		P2 mouse	Mouse ES	NPC	2 × 105	48 h	i.c.	3wk	NA	yes	Shinoyama et al. [55]
39		P5 mouse	hDP	SHED	2 × 105	24 h	i.c.	8wk	yes	yes	Yamagata et al. [56]
40		P7 rat	hDP	hDP stem cell	1 × 105	3d	i.c.	5wk	yes	yes	Fang et al. [57]
41	G	P7 rat	hUCB	MNC	3 × 106	24h	i.c.	2wk	yes	NA	Wang et al. [58]
42	G	P7 rat	hUCB	MNC	3 × 106	24h	i.c.	4wk	yes	NA	Wang et al. [59]
43		P3 rat	hUCB	MSC	1 × 106	0, 1, and 2d	i.p.	4wk	yes	yes	Zhu et al. [60]
44		P7 mouse	Newborn mouse	splenocyte	5 × 106	3wk	i.v.	3wk	NA	no	Wang et al. [61]
45		P8 mouse	Rat BM	MSC, MNC	1 × 105	48 h	i.p. or i.v.	24h	NA	NA	Ohshima et al. [62]

Studies in which cells are administered before the brain injury are not included here. Non-English literatures are not included.

A–G each alphabet indicates the same research group, P postnatal day, BM bone marrow, hUCB human umbilical cord blood, ES embryonic stem, hDP human dental pulp, NSC neural stem cell, NT3-NSC neurotrophin-3-expressing NSC, MASC multipotent astrocytic stem cell, MAPC multipotent adult progenitor cell, MNC mononuclear cell, ChABC chondroitinase ABC, MSC mesenchymal stem cell, GM-MSC genetically modified MSC, VEGF-NSC vascular endothelial growth factor transfected NSC, SHED stem cell from human exfoliated deciduous teeth, i.c. intracranial, i.t. intrathecal, i.v. intravenous, i.p. intraperitoneal, NA not assessed, yes, at least one test among several tests examined is improved by the cell therapy, or at least one treatment protocol among several protocols tested is beneficial

^atiming of administration after injury

Table 17.2

Review of reported studies in models with neonatal stroke

	Animal	Model	Cell			Timing^a	Delivery route	Follow-up	Improvement		Author
	Animal	Model	source	type	dose	Timing^a	Delivery route	Follow-up	Histology	Behavior	Author
1	P12 mouse	permanent CCAO	mouse ES cell	NSC	1 × 10⁵	2d or 7d	into striatum	4wk	yes	no	Comi et al. [16]
2	P10 rat	permanent MCAO	hUCB	MSC	1 × 10⁵	6 h	intraventricular	4wk	yes	yes	Kim et al. [17]
3	P10 rat	transient MCAO	rat BM	MSC^b	1 × 10⁶	3d	intranasal	4wk	yes	yes	van Velthoven et al. [18]
4	P12 mouse	permanent MCAO	hUCB	CD34⁺ cell	1 × 10⁵	48 h	i.v.	7wk	yes	no	Tsuji et al. [19]

P postnatal day, CCAO common carotid artery occlusion, MCAO middle cerebral artery occlusion, ES embryonic stem, hUCB human umbilical cord blood, BM bone marrow, NSC neural stem cell, MSC mesenchymal stem cell, i.v. intravenous, yes, at least one test among several tests examined is improved by the cell therapy, or at least one treatment protocol among several protocols tested is beneficial

^aTiming of administration after injury

^bMSC, or MSC overexpressing brain-derived neurotrophic factor

Table 17.3

Review of reported studies in models with excitotoxic brain damage

	Research group	Animal	Cell			Timing^a	Delivery route	Follow-up	Improvement		Author
	Research group	Animal	Source	Type	Dose		Delivery route	Follow-up	Histology	Behavior	Author
1		P5–7 mouse	Human EG cell	NSC	1.5 × 10⁵	3d	brain parenchymal, intraventricular	10d	yes	NA	Mueller et al. [63]
2		P7 rat	Mouse ES cell	ES cell	1 × 10⁶	8d	into injured striatum	2wk	NA	NA	Vadivelu et al. [64]
3		P5 rat	Neonatal rat	MSC		1d	near the lesion		yes	yes	Chen et al. [65]
4	A	P5 rat	Mouse embryo’s brain	NDP	3 × 10⁵	4 h or 72 h	into contralateral hemisphere	5m	yes	yes	Titomanlio et al. [66]
5	A	P5 rat	hUCB	MNC	1, 3 × 10⁶, 1 × 10⁷	0 h or 24 h	i.p. or i.v.	5d	no	NA	Dalous et al. [67]

A same research group, P postnatal day, EG embryonic germ, ES embryonic stem, hUCB human umbilical cord blood, NSC neural stem cell, MSC mesenchymal stem cell, NDP neurosphere-derived precursor, MNC mononuclear cell, i.p. intraperitoneal, i.v. intravenous, NA not assessed, yes, at least one test among several tests examined is improved by the cell therapy, or at least one treatment protocol among several protocols tested is beneficial

^aTiming of administration after injury

During the first decade since 1996 in this research arena, intracerebral transplantation of either the neocortical block of fetal brain or neural stem cells (NSCs) was investigated [20, 21] (Table 17.1). Systemic administration by intraperitoneal injection of cells was first reported by Guan et al. in non-English literature in 2004 [22], and by Meier et al. in English literature in 2006 [23]. Also, Guan et al. was the first to report the effects of mesenchymal stem cells (MSCs), and Meier et al. was the first to report the effects of mononuclear cells (MNCs) in neonatal models. A clinically feasible route of systemic administration by intravenous injection, was first reported by Yasuhara et al. [24]. During the second decade of this research arena, a similar number of studies using intracranial transplantation or systemic administration of stem cells have been reported. With regard to the donor cells, the MNC fraction of human umbilical cord blood (hUCB) and MSCs derived from rodent bone marrow (BM) have been most extensively explored. In many of those studies, MNCs are transfused systematically and MSCs are transplanted intracranially.

hUCB-MNCs

The MNC fraction of hUCB contains many stem cell types, including hematopoietic stem cells, endothelial progenitor cells, and MSCs [68–70]. A subpopulation of cells within human UCB-MNC fraction has the potential to become neural cells [71]. hUCB-MNCs secrete a higher level of trophic factors, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, than adult peripheral blood MNCs [72].

MSCs

MSCs are present in BM, adipose tissue, amniotic tissue, and UCB. MSCs are easy to harvest, and are capable of differentiating into mesodermal cell lineages such as adipocytes, skeletal myoblasts, chondroblasts and osteoblasts, and neuroglial cells [69, 73]. MSCs have several favorable characteristics such as low immunogenicity in allogeneic (nonself) transplantation and no tumorigenicity [74].

Animal Models

There is no widely used model of neonatal stroke. Some rodent models of stroke subject animals to transient or permanent occlusion of unilateral middle cerebral artery (MCAO) [75–78]. Other stroke models use occlusion of the common carotid artery (CCA) only [79, 80] or a combination of CCA and MCA ligation [81]. Rodent models of neonatal brain injury use postnatal day 7–12 (P7–12) rat or mouse pups, as those pups are considered comparable to human term P0 newborns with regard to brain maturation [82].

This chapter focuses on neonatal stroke, but introduces a brief summary of study data obtained in rodent models of neonatal HIE. That is because rodent models of neonatal stroke and neonatal HIE form a continuum of hypoxia–ischemia ranging from a more hypoxic to an ischemic insult with respect to pathophysiology, which also occurs in clinical settings. An extensively used rodent model of neonatal HIE, the Rice–Vannucci model, has mixed histopathology and exhibits a focal stroke in approximately half of the pups with the HI insult [83]. The rodent model of HIE subjects animals to permanent unilateral CCA occlusion (CCAO) followed by transient exposure to systemic hypoxia (30 min to 4 h, 8–10 % O₂) [83, 84].

Studies in Neonatal Stoke Models: Four Reports

The four studies that examined cell therapies in rodent models of neonatal stroke are summarized in Table 17.2.

Comi et al. reported the effects of stem-cell-based therapy in a neonatal stroke model for the first time [16]. They used P12 CD1 mice with permanent unilateral CCAO. They prepared murine embryonic stem (ES) cell-derived NSCs, and injected a suspension of 1 × 10⁵ cells into ipsilateral striatum 2 or 7 days after the occlusion. Pups with the NSC treatment administered at 2 days, but not at 7 days, had less severe hemispheric brain atrophy compared with either nontreated or vehicle-treated pups 28 days after the occlusion. Three out of ten pups injected with the NSCs developed local tumors.

Kim et al. reported effects of MSC transplantation in P10 Sprague-Dawley rats with permanent MCAO [17]. hUCB-derived MSCs (1 × 10⁵ cells) were administered into the ipsilateral lateral ventricle at 6 h after the occlusion. MSC transplantation improved the survival rate as well as the body weight gain after MCAO. MSC transplantation attenuated infarct volume measured by MRI at three time points examined; 3, 7, and 28 days after MCAO. MRI demonstrated the presence of transplanted super-paramagnetic iron oxide-tagged MSCs until 28 days after the transplantation. Functional deficits measured by the rotarod and cylinder tests after MCAO were partially ameliorated by the transplantation. An increased number of cells with markers for apoptotic cell death, reactive microglia, and astrogliosis in the penumbra of MCAO were also reduced by the MSC transplantation. Only a few transplanted MSCs were labeled with markers for neurons or astrocytes. The authors consider that anti-inflammatory effects mediated by increased expression of trophic factors may be the primary mechanism underlying the effects of transplanted MSCs on injured brain.

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