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Cell therapy has emerged as a promising approach to
improve recovery after stroke. However, progress toward the development of
efficient cell based therapies for ischemic stroke has been so far
disappointing. The main problem remains the poor understanding of the dynamics
of cellular interactions between host cells and administered cells for
therapeutic purposes. The pathophysiological evolution of stroke events is
driven by complex cellular interactions between many different cell types whose
sequential recruitments have been insufficiently documented due to the lack of
non-invasive imaging modalities. Specifically, the interplay between host
neuroinflammation, which is considered to be a major obstacle to
exogenous-mediated neuronal precursor cells, and exogenously administered stem
cells, remains virtually unknown. Phagocytosis of dead and dying neurons and
neuronal debris is beneficial in part because it reduces inflammation. However,
microglia can also phagocytose live neurons, live neuronal progenitors or live
stressed-but-viable neurons like those presumably occurring after
transplantation causing death of the engulfed cell and compromising cell
therapy using neuronal precursor cells. This lack of basic knowledge critically
limits the optimization of timing and route of administration of stem cells for
therapeutic purpose. In this review, we aim to
decipherthe dynamic interplay between host neuroinflammation and therapeutic
stem cells for regeneration after stroke by two non-invasive molecular imaging
approaches (two photon microscopy and magnetic resonance imaging at high
magnetic field) and their impact on behavioural outcome measures.
Keywords: Aging,
Cell therapy, Imaging, Two photon microscopy, Magnetic resonance imaging
INTRODUCTION
Stroke is the third most common cause of death and the leading cause of
disabilities worldwide. The damages after stroke generally have two different
causes, the primary and secondary insult. The primary insult is mediated by the
ischemic event itself and leads to oxygen and glucose deprivation, crucial
substrates for the survival of the brain tissue, whereas the secondary insult
is caused by the inflammatory response after stroke and mainly affects the
penumbra, a brain area with a slightly compromised blood-supply that surrounds
the ischemic core. Up to date, tPA (tissue plasminogen activator) is the only
FDA approved therapeutic treatment for ischemic stroke [1]. But due to severe
side effects and because of its very limited time window it is only applicable
to less than 10 % of all stroke patients [1], thus indicating an urgent need
for alternative treatments.
Up to
now, studies of many brain diseases have limited their attention on the focal
lesion, ignoring the wider impact of the directly affected territory on other
brain regions. In this review we
approach the brain as a rather horizontally organized, complex network where
focal lesions will have far ranging effects on distal brain regions and their
corresponding functions. This approach provides new insight into the mechanisms
and reasons underlying functional disturbances and opens new avenues for future
diagnosis. Specifically, through this novel approach we expect to make predictions
with regard of the efficacy of stem cell therapy of ischemic stroke by gaining
insight into of the time window of application and route of administration of
stem cells on a background of neuroinflammation in aged subjects and to develop
an experimental, pre-clinical rationale for the development of cell based
therapeutic strategies for the treatment of stroke and neurodegenerative
diseases.
Cell Therapy has
Emerged as a Promising Approach to Improve Recovery after Stroke
However, progress toward the development of efficient cell based
therapies for ischemic stroke has been so far disappointing. The main problem
remains the poor understanding of the dynamics of cellular interactions between
host cells and administered cells for therapeutic purposes. The
pathophysiological evolution of stroke events is driven by complex cellular
interactions between many different cell types whose sequential recruitments
have been insufficiently documented due to the lack of non-invasive imaging
modalities. Specifically, the interplay between host neuroinflammation, which
is considered to be a major obstacle to exogenous-mediated neuronal precursor
cells, and exogenously administered stem cells, remains virtually unknown.
Sub toxic insults such as inflammation, stressed but viable neurons may
reversibly expose, eat-me signals, may expose the “eat-me” signal
phosphatidylserine (PS) on neuronal surface. Activated microglia detect exposed
‘eat‑me’ signals and engulfment of neurons or parts of neurons exposing such
signals follows. This process has been coined primary phagocytosis or “phagoptosis”
[2,3]. Toxic neuronal insults, such as dying neurons after stroke, irreversibly
expose the “eat-me” signal recognized by primed microglia resulting in the
phagocytosis if dead neurons or the so-called secondary phagocytosis [4]. There
are “eat me” signals other than phosphatidylserine. During brain development
and normal functioning in the adult, immune molecules, including complement
proteins, C1q and C3, have emerged as critical mediators of synaptic refinement
and plasticity via C3-dependent microglial phagocytosis of synapses. Following
an acute injury such as stroke the apoptotic neurons release a chemotactic
signal such as fractalkine/CX3CL1 [5,6] and microglia expressing the
fractalkine receptor(CX3CR1), promotes phagocytosis of apoptotic cells
expressing CX3CL1 [5,7]. The complement components C1q and C3, which are
produced by microglia and astrocytes, may induce phagocytosis by binding
opsonized/altered neuronal surfaces. In this process C1q promotes the
conversion of C3, expressed by microglia, to C3b. C3b then opsonizes neurons
and is recognized through complement receptor 3 (CR3) expressed by activated
microglia.
Phagocytosis of dead and dying neurons and neuronal debris is
beneficial in part because it reduces inflammation. However, microglia can also
phagocytose live neurons, live neuronal progenitors or live stressed-but-viable
neurons like those presumably occurring after transplantation causing death of
the engulfed cell and compromising cell therapy using neuronal precursor cells.
Mild neuroinflammation can be beneficial for regenerative events aimed
at functional restoration after stroke [8]. Cell therapy itself can be used
during the first week post-stroke to limit neuroinflammation in animal models
[9-11]. However, persistent post-stroke neuroinflammation results in decreased
proliferation of the newly born NPCs and ineffective integration into the
circuitry of the re-organized brain area [12]. Moreover, a number of studies
have demonstrated that neuroinflammatory processes can induce apoptosis in NPCs
and immature neurons [13,14] and decrease the efficacy of both stroke-induced
neurogenesis and exogenously supported neurogenesis. This hypothesis is
supported by findings from studies using anti-inflammatory drugs such as
indomethacin or minocyclin block microglia-induced apoptosis of NPCs in a
pro-inflammatory milieu [15-17]. More recently studies directly implicated TNFa
produced by lipopolysaccharide-activated microglia is as a key determinant in
microglia induced-apoptosis in mouse NPCs in vitro and in vivo [18].
This lack of basic knowledge critically limits the optimization of
timing and route of administration of stem cells for therapeutic purpose. In the
present review, we aim to investigate the dynamic interplay between host
neuroinflammation and therapeutic stem cells for regeneration after stroke by
combining non-invasive molecular imaging modalities (two photon microscopy
(2P-LSM) and magnetic resonance imaging (MRI) at high magnetic field) with in
vivo neurophysiological and behavioural outcome measures. Up to now, studies of many brain
diseases have limited their attention on the focal lesion, ignoring the wider
impact of the directly affected territory on other brain regions. In the
proposed project we approach the brain as a rather horizontally organized, complex
network where focal lesions will have far ranging effects on distal brain
regions and their corresponding functions. This approach provides new insight
into the mechanisms and reasons underlying functional disturbances and opens
new avenues for future diagnosis. Specifically, through this novel approach we
expect to make significant improvements in the efficacy of stem cell therapy of
ischemic stroke by optimizing the time window of application and route of
administration of stem cells on a background of neuroinflammation in aged
subjects. Finally, we expect to decipher experimental, pre-clinical rationale
for the development of cell based therapeutic strategies for the treatment of
stroke and neurodegenerative diseases.
Role of the Immune
Response on Tissue Protection and Recovery
We have explored the role of the polarization phenotype of microglia
and macrophages for the neuronal survival and functional deficit, functional
improvement respectively. Injecting the microRNA 124 (mir-124), we found a substantial
shift from the pro- to the anti-inflammatory phenotype, combined with a
significantly higher neuronal survival. In parallel, there was a tight
correlation between decrease of neurological deficit and increase of
anti-inflammatory (M2) phenotype [19,20].
Stem Cell Therapies
in Preclinical Models of Stroke Associated with Aging
Stroke has limited treatment options, demanding a vigorous search for
new therapeutic strategies. Initial enthusiasm to stimulate restorative
processes in the ischemic brain by means of cell-based therapies has meanwhile
converted into a more balanced view recognizing impediments related to
unfavorable environments that are in part related to aging processes. Since
stroke afflicts mostly the elderly, it is highly desirable and clinically
important to test the efficacy of cell therapies in aged brain
microenvironments. Although widely believed to be refractory to regeneration,
recent studies done by us using both neural precursor cells and bone
marrow-derived mesenchymal stem cells for stroke therapy suggest that the aged
rat brain is not refractory to cell-based therapy, and that it also supports
plasticity and remodeling [21,22]. Yet, important differences exist in the aged
compared with young brain, i.e., the accelerated progression of ischemic injury
to brain infarction, the reduced rate of endogenous neurogenesis and the
delayed initiation of neurological recovery. Pitfalls in the development of
cell-based therapies may also be related to age-associated comorbidities, e.g.,
diabetes or hyperlipidemia, which may result in maladaptive or compromised
brain remodeling, respectively. These age-related aspects should be carefully
considered in the clinical translation of restorative therapies [23]. In the
last 3 years, upon acquiring a 2P-LSM microscope, now the gold standard of
microglia reaction [24-26], our focus has been on in vivo monitoring of the
efficacy of anti-inflammatory therapies by quantifying microglia reaction to
focal (Figure 1) or global lesions
[27].
Functional Dynamics
of Stem Cell Grafts
For almost 20 years, our research has focused on stem cell based
regeneration of cerebral disorders. Based on the long-standing extensive work
on stroke pathophysiology we have concentrated on various aspects of stem cell
dynamics such as migration, proliferation, vitality, and neuronal
differentiation. Using high resolution high field in vivo MRI at 7Tesla, we
were the first to monitor directed stem cell migration towards the ischemic
target in noninvasive longitudinal imaging studies [28]. Optimization of the
bioluminescence imaging (BLI) for brain studies and identification of maximally
sensitive luciferases allowed the in vivo assessment of stem cell graft
vitality during longitudinal investigations [29,30]. Combination of this BLI
protocol with 19F-MRI solved the controversial debate over the best suited
location for graft vitality: it has been shown that the vitality of grafts in
the peri-infarct area is equivalent to that of grafts in healthy tissue, thus
allowing to safely implant stem cells close to but outside of the ischemic
target zone [31]. From further studies, it was concluded that the graft size,
but not the immune response to the grafting, influences the vitality. This
resulted in recommendation of maximal graft size [32-34].
Differentiation and
Integration of Neural Stem Cell Grafts
In a major project the temporal differentiation profile of human neural
stem cells (NSCs) after grafting was identified. Stem cells were transduced to
express imaging reporters under cell-specific control for the in vivo
monitoring of selected stages of differentiation. For this purpose, luciferases
for detection by BLI and fluorescent markers for immunohistological validation
of in vivo observations by immunohistochemistry were chosen as imaging
reporters. Setting the imaging reporters under gene control of early
(Doublecortin) and late (Synapsin) neuronal differentiation, a time line was
generated which permits for the first time the direct correlation between
functional improvement, assessed by behavioral test, and the neuronal
differentiation of the graft and its capacity to integrate. This time line
further allows a reliable discrimination between discussed modes of action of
stem cells for a functional improvement: bystander effect (during early phase
before neuronal differentiation) and tissue replacement (after neuronal
differentiation and during cell integration) [32]. In a further study the graft
innervation by the host tissue was unraveled. Whole-brain light sheet
microscopy of a translucent brain permitted at cellular resolution the afferent
connection from far-ranging neuronal sites to the graft. The corresponding
anatomical assignment of the connecting host tissue regions was achieved by
coregistration of the whole-brain light sheet data set with high-resolution
anatomical MRI and mouse brain atlas [35].
Neuronal Networks
and Post-Stroke Recovery
The understanding of cerebral diseases has moved from the focal attention on the lesion itself to more global concepts of far ranging effects in neuronal networks during the past years. Invasive approaches of experimental investigations are either limited to a few local recordings such as electrophysiology or permit only a description of a single time point because of invasiveness of the method. Modern noninvasive molecular imaging modalities not only allow the investigation of dynamic cellular processes (see above) but have recently begun to open new doors to generate temporal profiles of dynamic processes relating to both structural and functional connectivity networks in the brain. Animal studies in rodent models can now be investigated with impressive sensitivity and resolution, thus unravelling mechanisms and interactions between various components such as different cell types during lesion development and (therapeutically intervened) outcome. Thus, investigations by Dijkhuizen and colleagues have shown the disturbance of the functional networks after stroke induction, affecting the ischemic hemisphere but also extending the deficits into the transhemispheric connections of homotopic areas of the motor and sensory cortex [36,37].
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