Download From Cell Death to Neuronal Regeneration: Building a New Brain

Journal of Neuropathology and Experimental Neurology
Copyright q 2003 by the American Association of Neuropathologists
Vol. 62, No. 8
August, 2003
pp. 801 811
From Cell Death to Neuronal Regeneration: Building a New Brain after Traumatic Brain Injury
NICOLAS C. ROYO, PHD, JOOST W. SCHOUTEN, MD, CARL T. FULP, BA, BS, SAORI SHIMIZU, MD, PHD,
NIKLAS MARKLUND, MD, PHD, DAVID I. GRAHAM, MB, PHD, AND TRACY K. MCINTOSH, PHD
Abstract. During the past decade, there has been accumulating evidence of the involvement of passive and active cell death
mechanisms in both the clinical setting and in experimental models of traumatic brain injury (TBI). Traditionally, research
for a treatment of TBI consists of strategies to prevent cell death using acute pharmacological therapy. However, to date,
encouraging experimental work has not been translated into successful clinical trials. The development of cell replacement
therapies may offer an alternative or a complementary strategy for the treatment of TBI. Recent experimental studies have
identified a variety of candidate cell lines for transplantation into the injured CNS. Additionally, the characterization of the
neurogenic potential of specific regions of the adult mammalian brain and the elucidation of the molecular controls underlying
regeneration may allow for the development of neuronal replacement therapies that do not require transplantation of exogenous
cells. These novel strategies may represent a new opportunity of great interest for delayed intervention in patients with TBI.
Key Words:
Apoptosis; Cell death; Necrosis; Neurogenesis; Replacement therapy; Transplantation; Traumatic brain injury.
INTRODUCTION
Ongoing and progressive cell death after a single initial
mechanical injury is one of the hallmark features of the
pathophysiology of traumatic brain injury (TBI). Evidence for the involvement of both passive and active cell
death mechanisms has been documented in both the clinical setting and in experimental models of TBI developed
to understand the mechanisms of this progressive and diffuse cell loss. Traditionally, treatment of TBI consists of
the prevention of cell death using acute pharmacological
therapy, but to date, this strategy remains only partially
effective. In addition to the prevention of cell death, a
more novel therapeutic approach may be to replace lost
cells in order to restore function. Cell replacement therapies, including cellular transplantation and manipulation
of the endogenous neurogenic potential of the brain, may
offer an opportunity for delayed treatment of TBI.
NECROTIC CELL DEATH FOLLOWING
EXPERIMENTAL TBI
Neurodegenerative cells exhibiting necrotic features
under light and electron microscopy are observed within
the first hour after fluid percussion (FP) brain injury in
rats (1). Morphologically, necrotic cell death associated
From the Head Injury Center, Department of Neurosurgery (NCR,
JWS, CTF, NM, TKM), University of Pennsylvania, and the Veterans
Administration Medical Center (TKM), Philadelphia, Pennsylvania;
University Department of Neuropathology, Institute of Neurological
Sciences (DIG), Southern General Hospital, Glasgow, United Kingdom.
Correspondence to: Tracy K. McIntosh, PhD, Department of Neurosurgery, 105C Hayden Hall, 3320 Smith Walk, Philadelphia, PA 191046074. E-mail: [email protected]
This work was supported, in part, by grants from the National Institute of Health National Institute of Neurological Disorders and Stroke
(P50-NS08803 and RO1-NS40978), the National Institute of General
Medical Sciences (RO1-GM34690), a Merit Review grant from the Veterans Administration and a Veterans Administration-DOD consortium
Merit Review grant.
with TBI can be differentiated from apoptosis and is generally characterized by a loss of membrane integrity, early organelle damage, cellular swelling, mitochondrial
swelling, and uncontrolled cell lysis (Fig. 1A). In addition, necrosis induces an inflammatory response in brain
tissue, resulting in secondary damage. Neuronal cell
death has been demonstrated in a variety of experimental
models of TBI using Nissl (1, 2), acid fuchsin (1, 2),
silver staining (1), TUNEL (3), and Fluoro-Jade staining
(4) after TBI. Following experimental TBI, Cortez et al
(2) observed necrotic neurons in the injured cortex, CA1,
CA2, and CA3 regions of the hippocampus and granule
cells in the dentate gyrus (DG) using acid fuchsin staining at acute time points (between 10 min and 24 h), followed by a significant reduction of neurons in the injured
pyramidal cell layer of the hippocampus and in the ventral and lateral posterior thalamus in the more chronic
postinjury phase (between 1 and 4 weeks) (2). Intensely
acidophilic neurons were observed that appeared to have
undergone necrosis, that is, cells were shrunken and eosinophilic. Others have observed dystrophic neurons after
controlled cortical impact (CCI) injury in the rat localized
to the DG, hippocampal CA1 and CA3 regions, amygdala, entorhinal and piriform cortices, and thalamic and
hypothalamic regions (5). Following experimental TBI,
neuronal cell body and dendrites are often stained by silver stains and appear as dark or argyrophilic neurons (6).
Although necrotic neurons detected by silver staining appeared in a similar spatial pattern as those detected by
acid fuchsin, silver staining also detected microglia, astrocytes, and cellular debris. More recently, Fluoro-Jade,
an acidic dye that is thought to stain the same substrates
as acid fuchsin, has been used as a marker of degenerating neuronal cell bodies and their processes. After FP
brain injury in rat, the regional distribution of FluoroJade-labeled neurons corresponded to a similar pattern of
silver and TUNEL staining (4). Severely injured neurons
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characteristics inconsistent with the morphology they exhibit at a given time point (e.g. DNA laddering patterns
suggestive of apoptosis), while having ultrastructural features consistent with a necrotic phenotype. It has been
suggested that when sufficient sources of cellular energy
are available, cell death will occur via a process consistent with the apoptotic morphology, but when energy is
lacking, necrotic cell death will occur (7). Thus, the constant balance between energy supply and consumption
during the death continuum is a critical factor in determining whether cell death ultimately becomes an active
(apoptotic) or passive (necrotic) process.
APOPTOTIC CELL DEATH FOLLOWING
EXPERIMENTAL TBI
Fig. 1. Two types of TUNEL-positive cells were detected
under electron microscopy after FP brain injury in rat. Type I:
necrotic cell death is characterized by a loss of membrane integrity, early organelle damage, cellular swelling, mitochondrial
swelling, and uncontrolled cell lysis (A). Type II: apoptotic cell
death is characterized by a condensation and margination of
chromatin, activation of an endonuclease that degrades nuclear
DNA, and cellular fragments containing intact organelles (B).
Reprinted with permission from the American Society for Investigative Pathology (8).
appeared in the cortex and hippocampus within 3 h after
injury and were most evident at 24 h postinjury. In the
thalamus, a delayed peak of necrotic neurons was observed at 7 days after injury and remained until 28 days.
At later time points, these Fluoro-jade-positive cells exhibited deterioration of the neuronal cell body and processes.
Traditionally, necrosis was believed to be the primary
cause of cell death following TBI. However, distinguishing between apoptosis and necrosis within a particular
group of cells at a particular time point after injury is
often misleading since cells appear to move freely between the two morphologies over the continuum of the
death processes. Cells may, in fact, assume biochemical
J Neuropathol Exp Neurol, Vol 62, August, 2003
Apoptosis is the morphological manifestation of cells
that die by programmed cell death during CNS development. The defining characteristics of apoptosis include
condensation and margination of the chromatin, activation of an endonuclease that degrades nuclear DNA, and
the formation of cellular fragments containing intact organelles (Fig. 1B). Using combined in situ DNA nick end
labeling and DNA gel electrophoresis, numerous studies
have detected internucleosomal DNA fragmentation following experimental TBI. After FP brain injury in rats,
TUNEL staining at acute time points (12–72 h) yielded
both type I (necrotic) and type II (apoptotic) populations
of TUNEL(1) cells localized to the subcortical white
matter, DG, and hippocampal CA3 region, in addition to
the cortical site of injury (8). Consistent with these results, an apoptotic-like internucleosomal DNA gel electrophoresis fragmentation pattern (185–200 bp) was observed within tissue isolated from the injured cortex and
hippocampus and this degree of fragmentation directly
correlated with the severity of the injury (8). Colicos et
al (5) subsequently observed that at 24 h following CCI
injury in rats, a subpopulation of cells within the DG,
CA1, and CA3 subfields exhibited apoptotic-like morphology. Conti et al (9) observed a biphasic increase of
apoptotic cells in the injured cortex, with an initial peak
at 24 h that colabeled with markers for neurons and oligodendrocytes, and a secondary peak at 1 week following
injury. Increased numbers of apoptotic cells were observed up to 8 weeks postinjury. Within the hippocampus, apoptotic neurons were significantly increased as
early as 12 h, with a peak at 48 h, and returning to control
levels by 4 weeks. By 1 week following injury, apoptotic
neurons were observed in the thalamus, with a significant
peak at 2 weeks postinjury. Additional biochemical evidence exists to support the observation that DNA fragmentation is a result of the activation of endonucleases.
The stress-activated kinases, including the p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal
kinase (JNK), are activated in response to DNA damage
(10). However, the question of whether JNK or p38 plays
CELL REPLACEMENT THERAPY FOR BRAIN TRAUMA
Fig. 2.
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Apoptotic cell death pathways.
a role in cell death following TBI remains highly controversial. Following FP injury in rats, immunoreactivity for
phospho-JNK, but not phospho-p38, has been observed
to significantly increase within intact pyramidal cells of
the injured hippocampus as early as 5 min and return to
baseline expression levels at 1 h, while phospho-ERK
was also significantly increased in hippocampal regions
vulnerable to TBI (11). Recently, a more prolonged activation (up to 72 h) of ERK1/2 and JNK was reported
in a similar model (12). In contrast, following CCI injury,
phospho-ERK and p-p38, but not p-JNK, are transiently
increased within the injured cortex at acute time points,
and administration of an ERK kinase inhibitor, but not a
p38/JNK kinase inhibitor, resulted in decreased traumatic
lesion volume (13). However, Dash et al (14) reported a
significant enhancement of loss of MAP-2-immunoreactivity within the CA3 subfield of the hippocampus upon
administration of an ERK kinase inhibitor.
The caspase family of proteins, of which 14 are now
known in mammals, are interleukin-1b-converting enzyme (ICE)-like, aspartate-specific proteases, synthesized
as procaspases that are cleaved at active Asp-X sites, generating a large and small subunit, which together constitute the active protease. Caspases can be categorized into
2 broad groups: initiator caspases, which generally contain large prodomains, autoactivate, and are thought to be
involved in the initiation of the apoptotic response, and
executioner caspases, which generally contain short prodomains and appear to be activated by initiator caspases.
The activation of effector caspases, particularly caspase3, appears to represent the cell’s commitment to die and
the convergence of numerous signaling pathways (Fig.
2). Activation of caspase-3 has been observed after TBI,
and intracerebroventricular administration of the caspase3 inhibitor z-DEVD-fmk was shown to reduce DNA
cleavage and TUNEL staining and to improve neurological outcome after FP injury (15). Cytochrome
c-mediated pathways such as cytochrome c and activated
caspase-3 are readily detected in damaged axons following the impact acceleration model of TBI in rats (16).
Following experimental TBI, caspase-8 mRNA and protein rapidly increase within neurons, astrocytes, and oligodendrocytes in the cortex and hippocampus. It is now
clear that the homeostatic mechanisms that are involved
in the activation of caspases are far more complex than
the simple regulation of their cleavage.
The Bcl-2 family of protein plays a critical role in the
intracellular apoptotic signaling transduction cascade.
Following CCI injury with induced hypoxia in rats, bcl-2
expression has been shown to increase primarily within
TUNEL(2) neurons, and to a lesser extent in the glia,
the ipsilateral cortex, DG, and CA3 region, beginning at
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8 h and continuing up to 7 days (17). Increased expression of Bcl-2 protein, but not Bcl-XL or Bax, within neurons was also observed in the cortical regions of excised
tissue from patients sustaining severe TBI (18). Recent
evidence has suggested the existence of a caspase-independent pathway of apoptosis involving the release of
apoptosis-inducing factor (AIF) from the mitochondria.
In dying cells, AIF relocates from the mitochondria to
the nucleus where its DNA binding activity is thought to
mediate chromatin condensation and large-scale DNA
fragmentation (19).
CELL DEATH FOLLOWING HUMAN TBI
The nature and distribution of the pathological cell
death after blunt head injury in man has been partially
determined (20). These include surface contusions, a focal form of TBI that occurs at the moment of injury,
traumatic axonal injury (TAI), and subsequently hypoxicischemic damage, which are diffuse (multifocal) forms of
TBI. Microscopic studies of patients dying after TBI have
identified necrosis as the principal type of death by which
cells die in the acute phase following injury (21), although apoptosis, initiated early in the course of clinical
TBI, can persist for many weeks or months postinjury
(22). In man, initial studies were undertaken on surface
contusions either from material that had been surgically
excised from head-injured patients admitted to a neurosurgical intensive trauma unit (18) or sampled at autopsy
(23). In the latter study, frontal lobe contusions from 18
patients who survived 6 h to 10 days were stained by the
TUNEL histochemical technique for evidence of DNA
fragmentation. TUNEL(1) cells were found in both the
neocortex and in the associated white matter. First seen
in grey and white matter at 5 h postinjury, TUNEL(1)
cells peaked in number between 25 h and 48 h and were
still identifiable at 10 days postinjury. Quantitation
showed that there were fewer TUNEL(1) cells in grey
than in white matter and that most of these were neurons
with the morphological features of necrosis, i.e. type I or
nonapoptotic cells (8). However, the microscopic features
of some of the TUNEL(1) neurons and of glial cells in
both grey and white matter were those of apoptosis—the
type II cell (8). Apoptosis was not observed in age-sexmatched uninjured controls. The morphology and immunohistochemical profile of the TUNEL(1) cells indicated that in the acute phase after TBI, some of these
strongly resembled oligodendroglia, whereas by 5 to 10
days after injury, many appeared as macrophages. Further
studies on the hippocampus, the cingulate, parasagittal
and the insular cortex, and the associated white matter
from patients who had died 5 h to 10 days after blunt
TBI showed that TUNEL(1) cells were present in both
grey and white matter, and that their numbers increased
between 5 h and 24 h before peaking between 24 h and
48 h and were still present at 10 days postinjury (23).
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Quantitation showed that in short surviving cases there
were more TUNEL(1) cells in grey than white matter
corresponding to necrotic cell change, whereas with increasing survival, fewer TUNEL(1) cells in grey matter
were visible and more were seen in the corpus callosum,
parasagittal white matter, the external and extreme capsules, and white matter of the hippocampus. Type I cells
again predominated in the acute post-traumatic period
and were strongly associated with ischemic neurons,
whereas type II cells with the morphology of neurons and
glia were seen at all survival periods. By 5 to 10 days
postinjury, most of the type II cells were in the white
matter and resembled macrophages.
The experimental literature suggests that cellular
changes initiated by acute head injury might persist and
indeed be progressive. Such evidence combined with
analogous clinical observations indicates that in addition
to the necrosis induced at the time of acute TBI, there
may also be continuing or progressive loss of cells via
apoptosis over the ensuing months. Additional studies using the TUNEL technique for evidence of DNA fragmentation were performed in multiple brain areas in 18
patients who survived for up to 12 months after head
injury. Compared with matched controls, more TUNEL(1) cells were observed in the TBI patients and
quantitative studies showed that these were predominantly localized to the white matter. Some of these cells
had the morphological features of apoptosis and routine
H&E staining and double labeling immunohistochemical
studies indicated that most of these cells were microglia
or macrophages (22). Similar changes were reported in
patients who survived in a vegetative state for many
months or years after TBI (Wilson et al, personal communication), and age at time of injury did not appear to
influence the amount of DNA fragmentation in the brains
of fatal cases of TBI aged 4 to 89 years compared with
controls (24).
Taken together, these studies in autopsy material from
patients after TBI suggest that TUNEL(1) cells are present in both acutely injured material, but also in the white
matter of patients whose TBI was months or years earlier.
Most of the TUNEL(1) cells appeared as microglia/macrophages and were thought to reflect the prolonged time
course of the macrophage response of Wallerian degeneration. Evidence that oligodendrocytes can undergo apoptosis days or even weeks after injury raises the possibility that the death of oligodendroglia by apoptosis may
contribute to demyelination of intact axons (25).
CELL REPLACEMENT STRATEGIES
Cell Transplantation after TBI
Restorative and regenerative strategies that have focused on replacement or repair of dysfunctional and dead
cells have received little attention in the treatment of TBI.
CELL REPLACEMENT THERAPY FOR BRAIN TRAUMA
Cellular transplantation is one option to repair the injured
CNS, aiming at replacing the function of cells lost to
injury. Initially, neural transplantation was developed as
a potential therapy for neurodegenerative diseases such
as Parkinson disease (PD) and Huntington disease (HD),
and although cellular transplantation has begun to be
evaluated in models of traumatic CNS injury, the variability in anatomical structures involved and cell types to
be replaced is one of the major challenges of replacement
therapy for TBI compared to other neurodegenerative diseases.
Early studies initially evaluated the use of fetal tissue
in models of PD, where grafts of fetal mesencephalic
tissue, rich in dopaminergic neurons, were shown to reinnervate the denervated rat striatum, synthesize and release dopamine, and ameliorate motor symptoms. Fetal
cortical tissue that was transplanted into the injured cortex of nonimmunosuppressed adult rats subjected to FP
brain injury survived best when transplanted between 2
days and 2 weeks postinjury (26), and was shown to ameliorate post-traumatic hippocampal cell death (27). Transplantation at 4 weeks postinjury resulted in reduced graft
integration as measured by acetylcholinergic fibers crossing the graft-host interface, most likely due to increased
glial scar formation at later postinjury time-points. Recovery of both cognitive and motor function was observed subsequently after transplantation of fetal rat cortical tissue into the injured cortex in combination with
nerve growth factor (NGF) infusion after lateral FP TBI
in nonimmunosuppressed adult rats (28).
Although promising results have been obtained transplanting fetal tissue in clinical trials for PD, technical and
ethical concerns in the use of fetal tissue as source for
transplantation have been raised, and alternative sources
for cellular transplantation are currently under investigation. While multipotent stem and progenitor cells have
been shown to differentiate into the 3 major neural cell
types in vitro, their fate after transplantation in vivo in
the adult brain depends on both cell intrinsic features and
the host environment. The age of the host, the location
of transplantation, and the factors and signals generated
by the injured CNS all seem to influence transplanted
stem and progenitor cells as well as endogenous neural
stem cells, possibly limiting their multipotentiality and
forcing them to differentiate into predominantly glial lineages (29). When the goal of cellular transplantation is
strictly neuronal replacement, one option is the transplantation of a homogenous population of terminally differentiated postmitotic neurons such as NT2N cells, commercially known as hNT cells or LBS neurons. These
cells are derived from a human embryonal teratocarcinoma line (NT2 cell line) by in vitro retinoic acid treatment and are a safe and well-characterized source of human neurons (30). These cells resemble region-specific
neuronal morphology at the location of transplantation
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and possess many of the key features of mature human
neurons, including the development of appropriate axonal
and dendritic polarity, expression of cytoskeletal proteins,
synaptophysin expression, and synaptic vesicle formation. Positive behavioral and histological results in experimental models of focal ischemia recently resulted in
the initiation of the first phase I neuronal transplantation
trial in 12 patients with chronic basal ganglia infarction
(31). The efficacy of the transplantation to improve
stroke-related neurological deficits was suggestive of a
trend towards improved scores in 4 patients receiving the
highest number of cells (6 million), and improved metabolism in the area of the stroke as shown by fluorodeoxyglucose positron emission tomography (32).
Philips et al (33) transplanted hNT cells into the injured cortex of nonimmunosuppressed adult rats 24 h after FP brain injury and observed viable hNT transplants
up to 4 weeks, without attenuation of post-traumatic neuromotor deficits. The presence of activated macrophages
and microglia at 4 weeks post-transplantation suggested
a variable degree of graft rejection. More recently, transplantation of hNT cells transduced ex vivo to express
NGF into the medial septum following CCI injury in
mice attenuated long-term cognitive dysfunction and protected the NGF responsive cholinergic neurons of the
septo-hippocampal pathway (34). As another source of
cells for transplantation, multipotential immortalized
stem and progenitor cell lines combine the advantages of
cultured cells (unlimited expansion in cultures and the
potential to be genetically manipulated in vitro) with the
ability to give rise to multiple lineages. Three immortalized cell lines (i.e. HiB5, MHP36, and C17.2) have been
transplanted in experimental models of TBI. The HiB5
cells are conditionally immortalized progenitor cells derived from an embryonic (E16) rat hippocampus and
have been shown to differentiate into neurons and glia
when transplanted into the neonatal hippocampus, cerebellum, and cortex, or into the adult striatum. In rats subjected to FP brain injury, HiB5 cells transduced to secrete
NGF (HiB5-NGF) were transplanted into 3 sites surrounding the injured cortex 24 h after injury (35). Braininjured animals receiving either the HiB5-NGF cells or
untransduced HiB5 cells showed a similar improvement
in neuromotor function and spatial learning. However,
hippocampal cell death was significantly reduced only in
the HiB5-NGF-engrafted group (35). Although engrafted
progenitor or stem cells are likely to secrete various trophic factors that can contribute to plasticity, regeneration,
and neuroprotection, the transplantation of cells transduced to produce NGF (e.g. hNT cells and the HiB5 cells
described above or fetal tissue transplanted in combination with NGF infusion [28]) seems to have distinct advantages over cell engraftment alone.
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The use of transplanted migrating cells will allow for
long-term expression and diffuse delivery of neuroprotective factors and may have a greater potential to integrate with the host tissue. In addition, autocrine and paracrine effects of the expressed neurotrophic factors might
be important in increasing graft survival. The MHP36
cells are conditionally immortalized cells derived from
E14 hippocampal neuro-epithelium of the H-2Kb-tsA58
transgenic mouse and have the ability to self-renew and
differentiate into neurons, astrocytes, and oligodendrocytes. When transplanted contralateral into a focal ischemic infarct, cells migrated to the cortex, striatum, and
corpus callosum of the injured hemisphere, resulting in
recovery of motor function (36). Transplantation of
MHP-36 cells into the injured cortex of adult immunosuppressed rats 48 h after FP TBI significantly improved
cognition by 16 weeks postinjury (P. M. Lenzlinger et al,
personal communication). Finally, the clonal multi-potent
C17.2 progenitor is derived from the external germinal
layer of the neonatal murine cerebellum immortalized by
retroviral transduction of the avian myc gene. Transplantation of C17.2 cells in the newborn mouse brain resulted
in widespread engraftment in a cytoarchitecturally appropriate, nontumorigenic manner and differentiated into
neuronal and glial cells (37). In mice subjected to CCI
injury, transplantation of C17–2 cells 3 days after injury
significantly improved motor function over a 12-week period. By 12 weeks postinjury, C17–2 cells expressed 60%
neuronal and 40% astrocytic markers when transplanted
ipsilateral to the lesion, whereas after contralateral transplantation only neuronal markers were observed (38).
A stem cell is an undifferentiated cell (i.e. lacking antigenic markers typical of mature cells), displaying significant proliferation potential in combination with the
potential to give rise to differentiated cells through asymmetric division. Embryonic stem cells are totipotent cells
derived from the inner cell mass of the early blastocyst
phase and can be propagated in vitro before transplantation. Following intracerebroventricular transplantation
into the naı
¨ve E16-E18 embryonic brain, embryonic stem
cells can enter and integrate throughout the neuraxis and
differentiate into neurons, astrocytes, and oligodendrocytes, suggesting that they retain the entire developmental
program of neural stem cells. In adult animals, neural
stem cells exist throughout life and can be isolated from
the walls of the ventricular system of the adult CNS, as
well as from the hippocampus. Embryo-derived neural
stem cells have been transplanted into unilateral striatum
or the injury cavity, with or without an injectable fibronectin-based scaffold, 1 week after CCI injury in mice
(39). Cells seeded in the scaffold and transplanted survived better and after 1 week were found lining the injury
cavity. By 3 months postinjury, the majority of the cells
had migrated towards the hippocampus, suggesting that
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transplant location and environment may play an important role in neural stem cell graft survival and integration
after experimental TBI (39). To date, studies involving
transplantation of adult-derived neural stem cells or embryonic stem cells into the traumatically injured brain
have not been performed.
One exciting, albeit controversial discovery is the possibility that bone marrow-derived cells, either hematopoietic stem cells or mesenchymal stem cells, may be
able to give rise to neural and glial cells when transplanted into the adult CNS (40). Recently, mesenchymal stem
cells have been shown to possess the ability to express
neuronal and glial markers in vitro. When transplanted
into the naive adult rat CNS, these cells migrate throughout the brain and lose their immunoreactivity for mesenchymal markers and express neuronal and glial markers at 12 and 45 days post-transplantation. In rodent
models of focal ischemia, intra-parenchymal, intravenous, and intra-arterial administration of bone marrowderived cells or whole bone marrow has been reported to
attenuate postischemic neurological deficits, with or without reduction of lesion volume. In rats subjected to CCI
injury, mesenchymal stem cells administered 24 h postinjury via the tail vein or internal carotid artery migrated
into the injured brain parenchyma and were observed to
express neuronal and astroglial markers at 2 weeks after
administration and improved the neuromotor function of
animals (33). Intra-parenchymal transplantation of whole
bone marrow into the pericontusional tissue in the CCI
model in rats, at 24 h after injury, also improved functional outcome and differentiation of transplanted cells
into neurons and glia (40).
Although the field is still in its infancy, transplantation
of a wide variety of cells into the CNS is currently under
laboratory investigation. Cells have been shown to survive and integrate after transplantation into the injured
brain, but mechanisms of graft function are still unclear.
In addition, results obtained using rodent-derived stem
and progenitor cells should be confirmed using human
equivalents to evaluate if positive results can be replicated.
Neurogenesis
The discovery of a population of endogenous progenitor cells and their association with continuing neurogenesis within the adult brain has engendered great interest
in the potential role of these cell populations for neuronal
replacement therapy in CNS injury and disease (41, 42).
Under physiological conditions, neurogenesis in adult animals is restricted to the subgranular layer of the dentate
gyrus (DG) and the subventricular zone (SVZ). For endogenous neural stem cells to yield therapeutic value in
models of CNS injury, they must proliferate at a rate
sufficient to replace dying cells, migrate to sites of pathology, survive within the site of pathology, differentiate
CELL REPLACEMENT THERAPY FOR BRAIN TRAUMA
into the appropriate cellular phenotypes, and in the case
of neurons, undergo a program of maturation that includes neuritogenesis, myelination, and synaptogenesis.
The hippocampal formation is selectively vulnerable to
experimental TBI (1). Bilateral loss of dentate hilar cells
projecting to the molecular layers of the DG following
FP TBI has been associated with persistent memory dysfunction and loss of both DG neurons and CA3 pyramidal
neurons has been observed (1, 27). Nonetheless, TBI appears to induce a transient increase in the regenerative
potential of injured hippocampal neurons, as evidenced
by enhanced expression of growth-related proteins, including polysialylated neural cell adhesion molecule
(PSA-NCAM) (43) and growth-associated protein 43
(GAP43) (44). Dash and colleagues (45) were the first to
document increased neurogenesis following CCI injury
and reported a bilateral increase in BrdU-immunopositive
cells within the granule cell layer of the hippocampus as
early as 24 h postinjury. This reactive proliferation peaked between 3 d and 7 d postinjury and returned to baseline levels by 14 d postinjury. Despite the fact that the
environment of the damaged CNS contains high levels of
molecules that inhibit neurite outgrowth (46), a population of endogenous cells within the DG in response to
brain injury has been shown to proliferate and project
axons to the correct hippocampal subfield, that is, from
the DG to the CA3 (D. L. Emery et al, personal communication). Within rodent and nonhuman primate brain,
cells from the SVZ normally traverse the brain to their
target, the olfactory bulb, via the rostral migratory
stream. Upon reaching the core of the olfactory bulb,
these neuroblasts move radially into the granular and periglomerular layers, where they differentiate into mature
neurons (47). Recently, an increase in cellular proliferation was observed 48 h after TBI in rats both in the
hippocampus and in the SVZ (45, 48). While these proliferating hippocampal cells were positive for immature
astrocyte and activated microglia markers, the proliferating cells in the SVZ seemed not to have begun to differentiate at this time point after injury. It has been suggested that neural stem cells exhibit an intrinsic tropism
CNS pathology (49), allowing them to migrate toward
the site of injury. Although an increase in the rate of
neurogenesis within the DG of the hippocampus and SVZ
has been observed in several models of experimental TBI
(45, 50) it is unclear whether these new cells are capable
of migrating towards the site of injury. Although recent
evidence suggests that newly born cells possess essential
properties of functional neurons in uninjured adult mice
(51), it remains to be unequivocally determined whether
these cells contribute to behavioral changes in normal or
pathological conditions.
Suggesting a potential role for endogenous neural stem
cells in ‘‘self-repair,’’ Macklis and colleagues (52) observed that the formation of mature neurons from endogenous precursors was restricted to the layer of cortex
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where synchronous apoptosis was induced, and these new
neurons survived at least 28 weeks. Retrograde labeling
from the thalamus revealed that BrdU-immunopositive
cells extended axons to the thalamus. However, this model lacked the immune response associated with more clinically relevant injury models and was limited to few cells.
Nonetheless, these data provide an important ‘‘proof of
principle,’’ i.e. a small number of cells from neurogenic
regions retain the ability to respond to injury by migrating toward the sites of injury, becoming mature neurons,
and express regional-specific markers consistent with
those normally expressed by cells lost as a result of the
injury. Although this phenomena remains to be evaluated
in models of TBI, it has recently been observed that the
dormant capacity of endogenous progenitor cells to participate in structural and functional recovery after transient forebrain ischemia in rats can be induced with infusion of basic fibroblast growth factor (FGF-2) and
epidermal growth factor (EGF) into the lateral ventricles
(53). Most interestingly, these new neurons appeared in
the pyramidal layer of the CA1, an area of brain exquisitely vulnerable to ischemia-induced cell death, and the
presence of these new neurons correlated with a significant amelioration of cognitive deficits. These observations suggest that exogenous augmentation of the neurogenic response to CNS injury will likely be required to
result in functional recovery.
The identification of the factors regulating adult neurogenesis has been intensively investigated during the
past decade. The birth and survival of new neurons in
the adult brain appears to be under dynamic regulation
and neurogenic regions are sensitive to both pharmacological manipulation and environmental stimuli. Adult
animals housed in an enriched environment exhibit increased dentate granule cell neurogenesis (54), as well as
improved spatial memory (55), while voluntary exercise
is sufficient for enhanced neurogenesis in the adult mouse
DG (56). Studies have demonstrated that chronic stress
elevates circulating adrenal steroids and stimulates glutamate release in the hippocampus (57). Subsequent activation of NMDA receptors decreases DG granule cell
proliferation, while administration of NMDA antagonists
leads to an increase in production of new granule cells.
In addition, the stress-induced suppression of LTP resembles the alterations in LTP that occur after TBI (58). Although a massive release of glutamate is thought to be
one of the major initial events in the pathophysiology of
TBI, whether this prolonged elevation in glutamate levels
compromises the neurogenic response following TBI remains to be elucidated. Interestingly, Riluzole, a sodiumchannel blocker with glutamate antagonist activity, previously shown to be neuroprotective in experimental
models of TBI (59), has recently been shown to enhance
expression of BDNF with consequent proliferation of
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ROYO ET AL
granule precursor cells in the rat hippocampus (60). Regulation of neurogenesis by environmental stimuli, stress,
and corticosteroids has led to the investigation of the effect of psychotropic drugs on neurogenesis. Serotonin
seems to play an important role in the regulation of neurogenesis through the activation of the 5HT–1A receptor.
Chronic treatment with selective serotonin reuptake inhibitors (SSRI) such as fluoxetine was shown to increase
rat hippocampal neurogenesis (61). Consistent with these
results, the administration of 5HT–1A receptor antagonists was shown to decrease cell proliferation in the DG
of the hippocampus of experimental animals (62). Several
studies suggest that the cAMP signaling cascade and
phosphorylation of the transcription factor CREB are implicated in hippocampal neurogenesis since phosphodiesterase (PDE) inhibitors such as Rolipram (PDE-4) (63)
and Sidenafil (PDE-5) have been shown to enhance neurogenesis and to be neuroprotective after experimental
cerebral ischemia (64).
Endogenous growth factors that affect neurogenesis in
vivo have also been identified. Peripheral administration
of IGF-1 has been shown to selectively induce neurogenesis in the adult rat hippocampus (65), and exercise-induced neurogenesis in the adult rat hippocampus has been
reported to be mediated by uptake of IGF-1 into the brain
parenchyma (66). Since peripheral administration IGF-1
has also been shown to be neuroprotective after experimental TBI (67), it would be interesting to evaluate its
ability to stimulate neurogenesis in the context of injury.
EGF and FGF-2 also appear to have differential effects
on neural progenitors. While intracerebroventricular administration of both growth factors has been shown to
expand the SVZ progenitor population, only FGF-2 was
able to induce an increase in newborn cells, mostly neurons, in the olfactory bulb (68). Following experimental
cerebral ischemia, it has been demonstrated that endogenously synthesized FGF-2 is necessary and sufficient to
stimulate proliferation and differentiation of neuroprecursor cells in the hippocampus (69). Heparin-bindingEGF-like growth factor, an endogenous mitogenic and
chemotactic glycoprotein containing an EGF-like domain, has also been shown to stimulate proliferation of
neuronal precursors in the SVZ and the DG (70), while
vascular endothelial growth factor (VEGF) has been
shown to stimulate neurogenesis in vitro and in vivo together with the proliferation of astrocytes and endothelial
cells (71). The angiogenic properties of VEGF together
with its ability to stimulate the proliferation of neuronal
progenitors may represent an interesting strategy to promote both angiogenesis and neurogenesis after TBI. It
has recently been shown that erythropoietin (EPO) is not
only a hematopoietic factor, but is also an intrinsic hypoxia-induced factor produced in the brain together with
J Neuropathol Exp Neurol, Vol 62, August, 2003
its receptor, capable of regulating the production of neuronal progenitor cells from the forebrain neurogenic region in mice (72). Although several members of the neurotrophin family have been shown to stimulate neuronal
proliferation and/or survival in vitro, only BDNF has
been demonstrated to stimulate neurogenesis in vivo. Administration of BDNF has been demonstrated to increase
the number of new neurons in the striatum, thalamus, and
hypothalamus of mature rats (73) as well as in the olfactory bulb (74). A decrease in DG cell proliferation has
also been observed in heterozygous BDNF knockout
mice (73). Conversely, long-term delivery via the transduction of BDNF gene in the hippocampus has been
shown to suppress ischemia-induced proliferation in the
DG of adult rats (75).
In many regions of the brain following TBI, neuronal
cell bodies are spared but their axonal connections are
severed. One crucial event for proper neuronal regeneration may be the ability to rebuild appropriate circuitry,
implicating proper growth and guidance of the newly
forming axons and dendrites towards the target cells, and
the degree of axonal regeneration may play a major role
in determining outcome after TBI. This process is under
the control of intrinsic molecules within the growth cones
and extrinsic guidance molecules (either attractant or repellent), sometimes acting over long distances in the CNS
(76). These molecules include netrins, semaphorins,
ephrins, slits, and cell adhesion molecules, all of which
are transcriptionally regulated, constituting a system able
to generate diverse and sometimes opposed outcomes
(77). Although adult mammalian CNS neurons exhibit a
capacity for axonal outgrowth given a permissive environment, anatomical and functional recovery is limited
by inhibitory factors including myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein
(OMgp), chondroitin sulphate proteoglycans (CSPG), and
Nogo-A, a myelin-associated neurite outgrowth inhibitor
expressed by oligodendrocytes (78, 79). MAG, Nogo,
and OMgp all bind with high affinity to the Nogo receptor complex, interacting with p75 as a co-receptor (80),
and may cause intracellular activation of the family of
Rho GTPases including Rho, Rac, and Cdc42. Rho activation, also by netrin and ephrin receptors and cell adhesion molecules, causes growth cone collapse, neurite
retraction, and cell rounding through its effect on the cytoskeleton (81). Whether this incomplete regeneration after CNS injury to the adult brain results from a lack of
intrinsic growth capacity or the presence of an extrinsic
growth inhibitory environment remains to be established.
Recently, a monoclonal antibody against Nogo-A (11C7)
was found to improve neurological and cognitive outcome following TBI in rats (P. M. Lenzlinger et al, personal communication), and inhibition of Rho was shown
to promote axonal growth and regeneration with rapid
neurological recovery after spinal cord injury in mice
CELL REPLACEMENT THERAPY FOR BRAIN TRAUMA
(82). Promoting axonal recovery through a combination
of neurogenesis and modulation of the factors mentioned
above may be a future treatment option in TBI.
CONCLUSION
The complete understanding of the mechanisms controlling the proliferation, migration, differentiation, and
axon extension of endogenous precursors will likely aid
in the development of efficient therapies for CNS injury
such as TBI. Pharmacological manipulation of this neurogenic potential, either by increasing the proliferation
rate or inducing neuronal differentiation, may represent a
new strategy with tremendous potential for the treatment
of TBI. These observations underscore the need for further exploration of the role of growth factors in mediating
neurogenesis in the adult CNS to advance the development of neuronal replacement strategies after brain damage by pharmacologically manipulating neural precursors
in situ. Despite improvements in the medical and surgical
treatment of TBI, there are still currently no neuroprotective agents available to counteract secondary or delayed damage to the injured brain or to stimulate its repair
and recovery. The development of cell replacement therapies may offer an alternative or a complementary strategy for the treatment of TBI. Recent advances both in
the identification of a variety of cell lines with a potential
for CNS transplantation and in the characterization of the
neurogenic regions of the brain represent a new opportunity of great interest for delayed intervention in TBI
patients.
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