doi:10.1093/brain/awp147
Brain 2009: 132; 2206–2218
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BRAIN
A JOURNAL OF NEUROLOGY
The P2Y-like receptor GPR17 as a sensor of
damage and a new potential target in spinal
cord injury
Stefania Ceruti,1,2,* Giovanni Villa,1,2,* Tiziana Genovese,2,3,* Emanuela Mazzon,2,3
Renato Longhi,4 Patrizia Rosa,5 Placido Bramanti,2 Salvatore Cuzzocrea2,3 and
Maria P. Abbracchio1
1 Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, University of Milan,
Milan, Italy
2 IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Messina, Italy
3 Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy
4 CNR Institute of Biocatalysis and Molecular Recognition, Milan, Italy
5 CNR Institute of Neuroscience, Milan, Italy
*These authors contributed equally to this work.
Correspondence to: Maria P. Abbracchio, PhD,
Department of Pharmacological Sciences,
University of Milan,
Via Balzaretti 9,
20133 Milan,
Italy
E-mail: [email protected]
Upon central nervous system injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes, two unrelated
families of endogenous signalling molecules, are markedly increased at the site of damage, suggesting that they may act as
‘danger signals’ to alert responses to tissue damage and start repair. Here we show that, in non-injured spinal cord parenchyma,
GPR17, a P2Y-like receptor responding to both uracil nucleotides (e.g. UDP-glucose) and cysteinyl-leukotrienes (e.g. LTD4 and
LTC4), is present on a subset of neurons and of oligodendrocytes at different stages of maturation, whereas it is not expressed
by astrocytes. GPR17 immunoreactivity was also found on ependymal cells lining the central canal that still retain some of the
characteristics of stem/progenitor cells during adulthood. Induction of spinal cord injury (SCI) by acute compression resulted in
marked cell death of GPR17+ neurons and oligodendrocytes inside the lesion followed by the appearance of proliferating
GPR17+ microglia/macrophages migrating to and infiltrating into the lesioned area. Moreover, 72 h after SCI, GPR17+ ependymal cells started to proliferate and to express GFAP, suggesting their activation and ‘de-differentiation’ to pluripotent progenitor
cells. The in vivo knock down of GPR17 by an antisense oligonucleotide strategy during SCI induction markedly reduced tissue
damage and related histological and motor deficits, thus confirming the crucial role played by this receptor in the early phases of
tissue damage development. Taken together, our findings suggest a dual and spatiotemporal-dependent role for GPR17 in SCI.
At very early times after injury, GPR17 mediates neuronal and oligodendrocyte death inside the lesioned area. At later times,
GPR17+ microglia/macrophages are recruited from distal parenchymal areas and move toward the lesioned zone, to suggest a
role in orchestrating local remodelling responses. At the same time, the induction of the stem cell marker GFAP in GPR17+
ependymal cells suggests initiation of repair mechanisms. Thus, GPR17 may act as a ‘sensor’ of damage that is activated by
Received December 19, 2008. Revised and Accepted April 30, 2009. Advance Access publication June 15, 2009
ß The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
GPR17 in spinal cord injury
Brain 2009: 132; 2206–2218
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nucleotides and cysteinyl-leukotrienes released in the lesioned area, and could also participate in post-injury responses.
Moreover, its presence on spinal cord pre-oligodendrocytes and precursor-like cells suggests GPR17 as a novel target for
therapeutic manipulation to foster remyelination and functional repair in SCI.
Keywords: GPR17; spinal cord injury; antisense oligonucleotides; precursor cells; ependymal cells
Introduction
Traumatic spinal cord injury (SCI) is a devastating condition
primarily affecting young males with an annual incidence of
15–40 cases per million (Wyndaele and Wyndaele, 2006) and
enormous social and health costs.
Currently, there is no drug treatment that effectively improves
outcome after SCI. Notably, studies in animals suggest that even
small anatomical gains can produce disproportionate functional
benefits (Blight, 1983), raising the possibility that early drug
treatments, which may reduce SCI-associated damage could
significantly improve the neurological outcome.
Secondary injury, including spinal cord oedema, ischaemia, free
radical damage, electrolyte imbalance, excitotoxicity, inflammatory
injury and apoptosis (Kwon et al., 2004), exacerbates the extent
of traumatic spinal cord insults. Yet, the exact mechanistic basis
of this phenomenon has largely been unexplored.
Extracellular adenine (ATP, ADP), uracil (UTP, UDP) and sugar
nucleotides (e.g. UDP-glucose and UDP-galactose) are universal
signalling molecules involved in many biological processes
(Burnstock and Knight, 2004) acting via specific membrane receptors: the seven ligand-gated purinergic P2X channels and the eight
G protein-coupled P2Y receptors (the P2Y1,2,4,6,11,12,13,14 receptors) (Burnstock and Knight, 2004; Abbracchio et al., 2006).
Recently, ATP has been shown to act as a key player in the
local response to SCI induced by weight drop impact (Wang
et al., 2004), where broad peritraumatic zones around the lesion
core showed high ATP release for hours after SCI. In line with
these findings, during laser-induced damage to the brain, rapidly
released ATP from astrocyte induced responses in microglial cells
(Davalos et al., 2005), whose processes autonomously converged
on the site of injury, establishing a potential barrier between the
healthy and injured tissue. Therefore, as in the immune system
(Ferrari et al., 2006), nucleotides may also function as ‘danger
signals’ in the injured brain and spinal cord, initially alerting the
surrounding healthy tissue and initiating responses; but these
signals may become detrimental when inappropriately sustained
(Marchetti and Abbracchio, 2005; Ferrari et al., 2006). Insights
into the dynamics and molecular mechanisms of these responses
as a function of time after injury and of distance from the
traumatic core may help developing new neuroprotective strategies for both brain and spinal cord damage.
Nucleotides have been shown to report tissue injury by directly
activating some type of P2Y receptors (e.g. P2Y12) on microglial
cells in the brain (Haynes et al., 2006). However, blockade
of nucleotide-induced responses by broad-spectrum purinergic
inhibitors (Davalos et al., 2005) suggests that more than one
receptor subtype may be involved. In this respect, we have
recently deorphanized the P2Y-like receptor GPR17 (Blasius
et al., 1998), and showed that it can be specifically activated by
both uracil nucleotides (UDP, UDP-glucose and UDP-galactose)
and cysteinyl-leukotrienes (cysLTs). Cysteinyl-leukotrienes are arachidonic acid-derived pro-inflammatory molecules (Samuelsson,
2000), which are also massively released in the brain after ischaemia (Ciceri et al., 2001) and in the spinal cord after traumatic
injury (Nishisho et al., 1996). GPR17 is highly expressed in
organs typically undergoing ischaemic damage (i.e. brain, heart
and kidney; Ciana et al., 2006). In an established rodent model
of focal brain ischaemia, blockade of GPR17 significantly prevented the progression of brain damage suggesting its contribution to the evolution of neurodegeneration (Ciana et al., 2006;
Lecca et al., 2008). On the other hand, GPR17 was recently
identified as one of the three genes that were expressed in
adult hippocampal neuroprogenitor cells (NPCs) but not in foetal
progenitors (Maisel et al., 2007), highlighting its potential role in
brain repair. Thus, in the central nervous system (CNS), GPR17
might have a differential role in response to injury, depending
upon whether it is already constitutively expressed by the cell or
induced as a result of the insult. Moreover, a differential role of
GPR17 might also depend on specific phases of tissue response
after the insult (sequentially, death of irreversibly damaged cells in
the traumatic/ischaemic core, clearance of dead cells, remodelling
of damaged circuitries and repair).
At present, nothing is known on the presence and role(s) of this
receptor in spinal cord. On this basis, the present study has been
specifically aimed at assessing (i) the localization of GPR17 on cells
of different lineages (neurons, all types of glia and NPCs) in the
intact mouse spinal cord; and (ii) the spatio-temporal changes
of GPR17 expression following SCI.
Our data indicate GPR17 as a common regulatory gene mediating response to injury in several embryonically distinct cell types
and as a new possible target for intervention in SCI.
Materials and Methods
SCI model
SCI was induced in male adult CD1 mice by the clip compression
technique, as recently described (Genovese et al., 2008a). SHAMoperated animals were taken as control. Animal care was in compliance with Italian regulations on protection of animals used for
experimental and other scientific purpose (D.M. 116192) and with
the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). Animals
were sacrificed at 24, 72 h or 1 week post-injury (p.i.), and at least
five animals/experimental group were analysed.
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Bromodeoxyuridine treatment
In both SHAM-operated and SCI mice cell proliferation was evaluated
by multiple intraperitoneal (i.p.) injections of the thymidine analog
bromodeoxyuridine (BrdU) (70 mg/kg), 1 h before SCI, and twice a
day up to 7 days after SCI (one injection in the morning and one in
the afternoon). BrdU incorporation into cell nuclei was then assessed
by immunohistochemistry (see below).
Tissue staining
Spinal cord tissues were taken at 24 h, 72 h or 1 week after trauma.
Tissue segments containing the lesion (1 cm on each side of the lesion)
were paraffin embedded and cut into 5 mm-thick sections. Tissue sections were deparaffinized with xylene, stained with haematoxylin/
eosin, luxol fast blue (to check for the integrity of the myelin structure)
or methyl green pyronin (to detect DNA and RNA simultaneously in
intact cells) and studied using light microscopy (Dialux 22; Leitz,
Wetzlar, Germany), as previously described (Genovese et al.,
2008a,b). The evaluation of the histological score was performed
as described in Supplementary Methods.
Immunohistochemistry
The excised spinal cord was fixed in 10% (w/v) PBS-buffered formaldehyde, embedded in Paraplast (Sigma-Aldrich, Milan, Italy), and 8 mm
sections were cut on a microtome. After deparaffinization, sections
were incubated for 45 min in PBS containing 10% normal goat
serum (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich). The
rabbit anti-GPR17 polyclonal antibody (1:150), generated as
previously described (Ciana et al., 2006), was utilized in combination
with the following primary antibodies: mouse anti-b-TubulinIII
(b-TubIII; 1:1000; Promega, Milan, Italy), mouse anti-glial fibrillary
acidic protein (GFAP; 1:500, Cell Signalling, Danvers, MA, USA),
mouse anti-adenomatous polyposis coli (APC) clone CC1 (1:100;
Calbiochem, Merk, Beeston Nottingham, UK), mouse anti-Nestin
(1:100; Chemicon, Millipore, Vimodrone, Italy), rat anti-myelin basic
protein (MBP, 1:200; Chemicon, Millipore) and rat anti-BrdU (1:150;
AbCam, Cambridge, UK). For BrdU detection, sections were pretreated with 2N HCl for 30 min at 37 C. To identify activated
microglia/macrophages, Bandeireae simplicifolia isolectin B4 directly
conjugated to fluorescein isothiocyanate (IB4-FITC, 1:50; Sigma–
Aldrich) was utilized. After an incubation overnight at 4 C with
primary antibodies, sections were rinsed three times with PBS, and
incubated for 1-h RT with the AlexaFluorÕ 488- or AlexaFluorÕ
555-conjugated anti-mouse, anti-rabbit or anti-rat antibodies, or
with the AlexaFluorÕ 350-conjugated anti-rat secondary antibody
(1:600; Molecular Probes, Invitrogen, Milan, Italy). All antibodies
were diluted in PBS containing 0.1% Triton X-100.
To detect Iba1 expression by microglial cells, a rabbit anti-Iba1
antibody (Biocare Medical, Prodotti Gianni, Milan, Italy) was utilized.
To perform double immunostaining with GPR17, since both primary
antibodies were from rabbit, the High Sensitivity Tyramide Signal
Amplification kit was utilized (TSATM; Perkin Elmer, Monza, Milano,
Italy), as described in Supplementary Methods. Anti-Iba1 antibody was
therefore diluted 1:35 000.
To detect NG2 expression, a rabbit anti-NG2 primary antibody was
utilized (1:200; Chemicon, Millipore) on frozen sections. Briefly, after
mice perfusion with 4% paraformaldehyde, tissues were post-fixed
and cryoprotected for 24 h in 30% sucrose, embedded in mounting
medium (OCT; Tissue Tek, Sakura Finetek, Zoeterwoude,
The Netherlands), and cut on a cryostat at 10 mm thickness.
S. Ceruti et al.
To perform double immunostaining with GPR17, the high sensitivity
tiramide signal amplification kit was utilized (see above and
Supplementary Methods), and the anti-GPR17 antibody was here
diluted 1:20 000.
Staining of nuclei was obtained by a 20 min incubation at room
temperature with the Hoechst 33258 dye (1:10 000; Molecular
Probes, Invitrogen). Sections were finally analysed by using an inverted
fluorescence microscope (200M; Zeiss, Arese, Milano) equipped with
a CCD camera (AxioCam HRm; Zeiss). In each tissue section, the
number of positive cells in 20 optical fields (corresponding to
1.5 mm2) was counted under a 40 magnification following
the scheme reported in Supplementary Methods.
Antisense oligonucleotide injection
The antisense oligonucleotide for the in vivo knock down of GPR17
(named oligo616) was selected and synthesized as previously
described (Ciana et al., 2006). A ‘scrambled’ oligonucleotide was
randomly generated on the basis of the sequence of oligo616. We
chose to use unmodified oligonucleotides to avoid possible toxicity,
while the stability issue was faced by a multiple delivery experimental
design. The specificity of the selected oligos was established as
explained in Supplementary Methods. Oligo616 and the ‘scrambled’
oligo were administered to animals 48 and 24 h before and 10 min
after SCI. Each administration consisted of a total dose of 400 ng
oligo/animal subdivided in two local injections 2.5 mm rostrally and
caudally to the lesion site, respectively.
Grading of motor disturbance
The motor function of mice subjected to SCI was assessed once a day
for 7 days after injury. Recovery from motor disturbance was graded
using the modified murine Basso, Beattie and Bresnahan (BBB) (Basso
et al., 1995) hind limb locomotor rating scale, as described in detail
in Supplementary Methods.
Results
In the adult spinal cord, GPR17 is
expressed by neurons, mature and
immature oligodendrocytes and
ependymal cells
As a first step to the characterization of the role of GPR17 in SCI,
we determined its cellular localization in control mouse spinal cord.
Specific immunoreactivity to GPR17 (red in Fig. 1) was found
on several types of cells of spinal cord parenchyma. To identify
these cells, we utilized specific markers for neurons, astrocytes
or oligodendrocytes (all reported in green in Fig. 1). Several
GPR17-positive (GPR17+) cells were indeed neurons as confirmed
by co-staining with the neuronal-marker b-TubIII (see orangeyellowish cells in Fig. 1A and A0 ); in contrast, astroglial cells, as
identified with the astrocytic marker GFAP, were never found to
co-express GPR17 (Fig. 1B and B0 ). Several other GPR17+ cells also
co-expressed the marker of mature oligodendrocytes CC1 (Fig. 1C
and C0 ). Most of the b-TubIII+ neurons and of the CC1+ oligodendrocytes express GPR17. Expression of GPR17 by cells belonging
to the oligodendrocyte lineage was also confirmed by co-staining
GPR17 in spinal cord injury
Brain 2009: 132; 2206–2218
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with the specific pre-oligodendrocyte precursor marker proteoglycan NG2 (Fig. 1D and D0 ). Quantification analysis showed
that 29.8 2.1% of NG2+ cells also expressed GPR17. Marked
GPR17 immunoreactivity was also found in the ependymal
cells lining the central canal, which are in direct contact with the
cerebrospinal fluid (Fig. 1E) and have been shown to exhibit stem/
progenitor cell properties following injury (Mothe and Tator, 2005;
Meletis et al., 2008). Specificity of the staining was assessed by
pre-adsorption of the primary antibody with the peptide used for
rabbit immunization (Fig. 1F). Thus, in non-injured spinal cord
parenchyma, different cell populations express GPR17, namely
neurons, oligodendrocytes at different stages of maturation (as
shown by colocalization with CC1 and NG2; De Castro and
Bribian, 2005), and ependymal cells, whereas GPR17 immunoreactivity was never found on astrocytes. Finally, 6% of the
total number of GPR17+ cells was represented by Iba1+ resting
microglial cells (see below).
Immunohistochemical and cellular
evaluation of tissue damage after SCI
In line with previous findings, significant tissue damage progressing over time was observed following SCI (Cuzzocrea et al.,
2006). Figure 2A reports a series of representative haematoxylin/eosin fields, showing the structure of healthy spinal cord
(SHAM animals), and increasing tissue disruption 24, 72 h and
1 week p.i. Figure 2B shows progressive deterioration of the
myelin structure in SCI animals at different times p.i., as detected
by staining with an antibody for the MBP, in comparison with
SHAM-operated mice. As expected, the number of neuronal
cells was also markedly reduced inside the lesion as a function
of time (see below; Cuzzocrea et al., 2006).
The cellular changes occurring in both the perilesioned zone
(namely, at the boundary between the core necrotic area
and the penumbra zone) and in the areas distal to the lesion
(conventionally identified by counting five optical fields on both
sides from the core lesion; see Supplementary Methods) were
quantified by counting the various cell populations labelled
with antibodies against GPR17 and specific glial/precursor cell
markers. In line with previous literature data, a significant and
time-dependent increase in the number of Nestin+ and GFAP+
cells was found in the perilesioned area of SCI animals with
respect to SHAM-operated animals (Fig. 3A). Nestin+ cells were
Figure 1 Localization of GPR17 in non-injured spinal cord.
Double immunofluorescence staining of mouse non-injured
spinal cord revealed GPR17 expression on different cell types.
(A) Representative field showing colocalization of GPR17 (red)
with the neuronal marker b-TubIII (green). Inset showing two
cells that clearly express both markers (orange) is reported in
(A0 ) at a higher magnification. (B) and higher magnification of
inset in (B0 ): GPR17 (red) never colocalized with the specific
astrocytic marker glial fibrillary acidic protein (GFAP) (green).
(C), (D) and corresponding higher magnifications in (C0 ), (D0 ):
GPR17 (red) is expressed by oligodendrocytes at different
stages of differentiation, as shown by co-staining with the
mature oligodendrocyte marker CC1 (green in C and C0 ), and
with the oligodendrocyte precursor cell marker NG2 (green in
D and D0 ). (E) Representative field showing expression of
GPR17 (red) in the ependymal cell layer. Specificity of immunostaining is shown in (F), where GPR17 labelling is abolished
by pre-adsorption of the primary antibody with the peptide
used for rabbit immunization. Nuclei were labelled with
Hoechst 33258 (blue). Scale bars: 15 mm.
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S. Ceruti et al.
(Fig. 1). The majority of these cells underwent early cell death in
the lesioned area as a consequence of mechanical damage, as
shown by double immunohistochemistry with the anti-GPR17
antibody and either the neuronal marker b-TubIII (Fig. 3A–C) or
the specific oligodendroglial marker CC1 (Fig. 3D and E). Thus, the
observed increased number of GPR17+ cells has to be due to the
appearance of a new population of cells expressing the receptor
(see below).
The number of cells staining for Nestin, GFAP, IB4, GPR17 (but
not NG2) was also increased in spinal cord areas distal to the
lesion, although to a much smaller extent (data not shown),
suggesting the existence of a spatial gradient in the cellular
response to damage.
After SCI, GPR17 expression is induced
in a population of proliferating
microglial/macrophage cells migrating
towards and infiltrating the
lesioned area
Figure 2 SCI-induced disruption of tissue organization.
(A) Series of representative haematoxylin/eosin fields, showing
the structure of spinal cord from non-injured (SHAM) and SCI
mice at different times (24 h, 72 h and 1 week) p.i. (B) Series
of immunofluorescence fields showing staining for the MBP
in tissues from SHAM and SCI mice at the indicated times
p.i. Nuclei were labelled with Hoechst 33258 (blue). No
histological alterations were observed in SHAM-operated
mice. A significant tissue damage progressing over time is
observed following SCI, especially in myelin structure
and organization. Scale bars: 100 mm.
virtually absent in the parenchyma of SHAM animals (see below).
Furthermore, the number of cells expressing the oligodendroglial
precursor marker NG2 and the number of microglia/macrophages
expressing the marker of activation IB4 were also significantly
increased with time after injury (Fig. 3A). This latter finding is
consistent with previous literature data suggesting that, upon
CNS damage, both parenchymal resting microglial cells and monocyte/macrophages coming from the bloodstream are activated,
start proliferating and move towards the lesioned zone (Davalos
et al., 2005; Haynes et al., 2006; Stirling and Yong, 2008). Finally,
a marked increase in the number of GPR17+ cells was also
detected (Fig. 3A). As mentioned above, in the non-injured
spinal cord, GPR17 is expressed by neurons and oligodendrocytes
As shown above (Fig. 3A), despite massive death of neurons and
oligodendrocytes in the lesion core, the number of GPR17+ cells
progressively increased in the peritraumatic zone as a function of
time. To identify the nature of these cells, and, specifically, to
assess if GPR17 was expressed by activated IB4+ macrophages/
microglia (which are also markedly increased in SCI mice, Fig. 3A),
we quantified the number of GPR17+/IB4+ double-stained cells at
various time intervals after SCI. In SHAM-operated animals,
no activated IB4+, but only resting Iba1+ microglial cells
were found, and 57.4 4.2% of this small cell population also
co-expressed GPR17 (Fig. 4A). Twenty-four h p.i., a small
number of IB4+ cells was detected near the borders (but not
within) the lesioned area (green in Fig. 4B and higher magnification inset), and they all express GPR17 (see black histogram).
These cells embody a very low percentage of the total number
of GPR17+ cells (red in Fig. 4B and grey histogram) since at this
time point the vast majority of these cells is still represented by
neurons and oligodendrocytes. The number of GPR17+/IB4+
double-stained cells was dramatically increased 72 h p.i., when
many globose cells were found at the boundary of or within the
lesioned zone (Fig. 4C and higher magnification inset). Again, the
number of GPR17+/IB4+ double-labelled cells almost coincided
with the total number of IB4+ cells (see black histogram on
the right), suggesting that almost all macrophages/microglial
cells infiltrating the lesion express GPR17. At this stage,
IB4-expressing cells represent almost the totality of GPR17+ cells
(which were significantly increased with respect to 24 h p.i.; Fig. 3)
due to the massive death of neurons and oligodendrocytes within
the lesion area (Fig. 4C and corresponding grey histogram).
One week p.i., many double-labelled cells were found inside the
lesion, but expression of GPR17 on microglia/macrophages started
decreasing (Fig. 4D). The latter findings suggest that expression
of the receptor might be maximal in these cells during their
migration/infiltration to the lesioned area; when these processes
are completed, GPR17 expression is down-regulated. The number
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Figure 3 SCI-induced changes in the cellular composition of the tissue at the borders of the lesioned area. (A) Quantification of
the number of Nestin+, GFAP+, NG2+, IB4+, GPR17+ and b-tubIII+ cells near the lesion borders in SCI animals 24 h, 72 h and 1 week
p.i. In SHAM-operated animals, the central area of each tissue section was taken as a reference point and an identical number of optical
fields was counted (see Supplementary Methods for details). Data are reported as mean SE. For each staining, sections taken from
three different animals were counted. **P50.01 with respect to corresponding SHAM-operated animals, one way ANOVA followed by
Scheffe’s F-test. (B)–(E) Expression of GPR17 (red) in SHAM- (B and D) and in SCI-operated mice (C and E) 72 h p.i. on neurons
(b-TubIII+ cells, green in B and C) and oligodendrocytes (CC1+ cells, green in D and E). In SCI-animals, pictures were taken at the
borders of the lesion site, showing that almost all the neuronal (C) and oligodendroglial cells (E) were dead.
of GPR17+/IB4+ cells also significantly increased distally to the
lesion site (not shown), suggesting their migration from the
healthy tissue. Thus, GPR17 could represent one of the molecules
driving the migration of phagocytes to the site of injury, where
they are activated by nucleotides and cysteinyl-leukotrienes
massively released into the lesion core.
Characterization of cell proliferation
after SCI
Diffuse cellular proliferative responses throughout the grey and
white matter have been observed in several models of SCI
(Kozlova, 2003; Zai and Wrathall, 2005; for review, see
Kulbatski et al., 2005). On this basis, to determine which cell
populations undergo proliferation in our experimental model and
to assess whether they migrate as a result of injury at different
times p.i., SHAM or SCI animals were treated with the thymidine
analog BrdU, which is selectively incorporated by dividing cells in
the S phase of the cell cycle.
At 24 h p.i., there were no significant differences in the very
small number of BrdU-positive nuclei between SHAM- and SCIoperated mice. The number of proliferating cells was strongly
increased 72 h and, to a greater extent, 1 week p.i. (Fig. 5A).
To fully identify the nature of proliferating cells, we stained sections with the anti-BrdU antibody and antibodies against GPR17,
IB4, Nestin or NG2 and counted the number of double-positive
cells near the lesion borders. 72 h and 1 week after SCI, a significant percentage of GPR17+, IB4+, Nestin+ and NG2+ cells showed
BrdU incorporation (Fig. 5B). A representative picture showing the
increase of proliferating GPR17+ cells at different times (24 h, 72 h,
and 1 week) p.i. compared to SHAM animals is shown in Fig. 5C.
Triple staining experiments showed that 72 h p.i., a significant
percentage of the BrdU+ proliferating cells are IB4+/GPR17+
microglia/macrophages (Fig. 5D), i.e. the cell population already
described in Fig. 4B–D migrating towards and infiltrating the
lesioned area. Following SCI, an increased BrdU incorporation
was also detected in ependymal cells (see below).
Induction of markers of multi-potential
progenitors in different cell populations
following SCI
It is now well accepted that astrocytes represent a reservoir of
precursor cells in the adult brain since they can de-differentiate
upon injury, eventually giving rise to various cell lineages (Buffo
et al., 2008). In line with these findings, in our SCI model,
we found that many astrocytes started to express Nestin, as
shown by its colocalization with GFAP, which increased over
time p.i. (Fig. 6C; representative micrographs from SHAM and
SCI animals 72 h p.i. are shown in Figs 6A and B, respectively).
As already shown in Fig. 5B, a significant percentage of Nestin+
cells incorporate BrdU, consistent with literature data suggesting
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Figure 4 Changes in the pattern of GPR17 expression in parallel with microglia activation. (A) In SHAM-operated mice, 57.4 4.2%
of the very few resting Iba1+ microglial cells (green arrow) co-express GPR17 (yellow arrow). The vast majority of GPR17+ cells are
represented by neurons and oligodendrocytes (Fig. 1). (B–D) Following SCI, activated IB4+ microglia/macrophages (green) recruited
from the healthy tissue and expressing GPR17 (red) progressively infiltrate the lesioned area. (B) Twenty-four hour p.i., very few IB4+
cells, which also stained for GPR17, were present in the perilesioned area. (C) Seventy-two hour p.i., the number of GPR17/IB4 double
positive cells was greatly increased, and almost the totality of GPR17+ cells were now microglia/macrophages. (D) One week p.i.,
the lesioned area was completely filled with IB4+ cells. Noteworthy, the percentage of colocalization of IB4 with GPR17 was slightly
decreased with respect to 72 h. In each Panel, quantification of data is shown by histograms on the right. Insets: higher magnification
of the selected areas. Lesion borders are indicated with dotted lines, with the lesion being on the right. Nuclei were labelled with
Hoechst 33258 (blue). Scale bars: 15 mm.
that, upon SCI, precursor-like multi-potent stem cells are activated
and start proliferating (Kulbatski et al., 2005). Thus, these data
indicate that, also in the spinal cord, astrocytes become reactive as
a response to damage, start proliferating and re-acquire some of
the typical features of multi-potent stem cells (Buffo et al., 2008).
In a similar way to the non-injured spinal cord, where GPR17
never colocalized with GFAP (Fig. 1), GPR17 was never found
to be expressed by Nestin+ astrocytes in the injured spinal cord
(data not shown).
Although spinal cord central canal was not routinely analysed in
our experiments due to the massive tissue disruption, when possible, SCI-induced cellular changes in this area were also investigated. In SHAM animals, ependymal cells lining the central
canal and expressing GPR17 (Fig. 1 and red in Fig. 6D)
never co-express GFAP, taken here as a marker of cell
de-differentiation and pluripotency (Meletis et al., 2008; Fig. 6D0
and D00 ). Noteworthy, the number of GFAP+ cells significantly
increased upon induction SCI, not only due to astrocytes proliferation around the central canal (compare Fig. 6D0 with E0 ), but
also to the fact that GPR17+ ependymal cells started expressing
GFAP (yellowish-orange cells in Fig. 6E00 and G). This indicates the
reacquisition of a pluripotent phenotype by ependymal cells as a
tentative to counteract trauma-induced cell damage. In line with
literature data (Meletis et al., 2008), a subset of these central
canal reacting ependymal cells actively proliferate, as shown by
incorporation of BrdU (green arrows in Fig. 6F and F0 ); at
least some of these proliferating cells were GPR17/GFAP
double-positive (light blue arrow in Fig. 6G). Taken together,
these data further demonstrate the reaction to injury of ependymal cells, and suggest that GPR17 may be involved.
Inhibition of GPR17 by administration
of a specific antisense oligonucleotide
during SCI markedly reduces tissue
damage and the related motor deficit
We next asked whether GPR17 plays any role in the induction of
cell death inside the lesioned area. We have previously shown
that, in both rats and mice with brain focal ischaemia, GPR17 is
up-regulated in neurons undergoing death inside the ischaemic
infarct. We also showed that blockade of GPR17 in these animals
results in a marked attenuation of ischaemia-associated neuronal
death (Ciana et al., 2006; Lecca et al., 2008), suggesting a
causal relationship between pathological GPR17 activation and
excitotoxicity-based neuronal degeneration.
On this basis, we determined if the inhibition of this receptor
during induction of SCI can protect against cell death. To this
purpose, an antisense oligonucleotide specifically designed to
knock down rodent GPR17 (oligo616; Ciana et al., 2006)
GPR17 in spinal cord injury
Brain 2009: 132; 2206–2218
| 2213
Figure 5 Proliferative responses after SCI. (A) At 24 h p.i. very few BrdU-positive nuclei were observed in both SHAM- and SCIoperated mice. In SCI-operated animals, the number of proliferating cells strongly increased over time p.i. **P50.01 with respect
to SHAM-operated mice, one way ANOVA followed by Scheffe’s F-test. (B) Identification of BrdU+ cells near the lesion borders by
co-staining with different cell markers (i.e. GPR17, IB4, nestin and NG2). Results represent the percentage of proliferating cells over the
total number of cells expressing the selected marker. (C) Representative pictures showing BrdU (green) and GPR17 (red) staining in
the spinal cord from SHAM and SCI-operated mice at different times (24 h, 72 h and 1 week) p.i. (D) Triple immunofluorescence
staining showing that at 72 h p.i. the same cells were positive for IB4 (green), GPR17 (red) and BrdU (blue; see also ‘merged’).
Scale bars: 15 mm.
was injected directly into the spinal cord 48 and 24 h before and
10 min after induction of SCI. Animals treated with a ‘scrambled’
oligonucleotide randomly generated on the basis of oligo616
sequence were analysed in parallel. In animals treated with
oligo616, a marked protection against tissue damage was
observed already 24 h after SCI, in comparison to mice receiving
the corresponding scrambled oligonucleotide that showed massive
cell death in the injured area (haematoxylin/eosin staining;
Fig. 7A), disruption of myelin integrity (as determined by Luxol
Fast Blue staining; Fig. 7B), and a significant loss of DNA and RNA
presence in lateral and dorsal funiculi (as shown by methyl green
pyronin staining; Fig. 7C). Protection was even more evident 72 h
and 1 week p.i., when SCI-induced massive disruption of the
myelin structure was prevented by oligo616 administration
(Fig. 7D and E), and a significant amelioration of the histological
score (determined by counting damaged neurons and evaluating
the extent of tissue infarction; see Supplementary Methods)
was observed in oligo616-treated animals (Fig. 7F).
Tissue preservation was paralleled by a significant, and increasing over time, amelioration of the motor score in oligo616-treated
animals with respect to animals exposed to the scrambled oligo
(Fig. 7G). Taken together these results suggest a crucial role for
GPR17 in the induction of cell death following SCI and in the
consequent loss of locomotor ability.
Discussion
Primary injury to the adult spinal cord is irreversible, whereas
secondary degeneration is delayed and therefore amenable to
intervention. Accordingly, several studies have shown that
therapies targeting various factors involved in the secondary
degeneration cascade lead to tissue sparing and improved
behavioural outcomes in SCI animals (Bao et al., 2003;
Cuzzocrea et al., 2006; Genovese et al., 2006; Glaser et al.,
2006).
The current management of SCI consists of supportive care and
stabilization of the spine (Samadikuchaksaraei, 2007). Numerous
pharmacological approaches have been evaluated, although none
have met substantial success. High-dose corticosteroids, given
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| Brain 2009: 132; 2206–2218
S. Ceruti et al.
Figure 6 Induced expression of markers of multipotent progenitors in astrocytes and ependymal cells, and increased incorporation of
BrdU in ependymal cells after SCI. (A) In SHAM-operated mice, GFAP+ parenchymal astrocytes (green) never expressed the stem cell
marker Nestin; (B) and (C) After SCI, the percentage of GFAP+ cells (green) co-expressing nestin (red) significantly increased over time
p.i., suggesting injury-induced de-differentiation to precursor-like cells. (D), (D0 ) and (D00 ) In SHAM-operated animals, GPR17+ ependymal cells never co-express GFAP (taken here as a marker of pluripotency; Meletis et al., 2008), whereas a diffuse colocalization in
specific subsets of cells could be detected 72 h after SCI (E), (E0 ) and (E00 ). (F) and (F0 ) An increased number of GPR17+ ependymal cells
that had incorporated BrdU was detected 72 h after SCI (arrows). Triple immunostaining showed that some of these GPR17-expressing
proliferating cells are also positive for GFAP (arrow in G). From (A) to (F0 ) nuclei were labelled with Hoechst 33258 (blue). Scale bars:
15 mm.
within the first 8 h after injury and continued for 24–48 h, were
regarded as part of the standard treatment regimen (Bracken
et al., 1998), although recently the results of studies, which
supported the efficacy of corticosteroids in SCI, have come
under scrutiny.
One of the reasons why most of the treatments utilized in
preclinical studies (Leker and Shohami, 2002) have had limited
success in clinical trials is the complexity of the secondary degenerative response. In fact, many treatments affect only one aspect
of this response, and a successful treatment should probably target
several of these mechanisms (King et al., 2006). In this respect,
the P2Y-like receptor GPR17 represents an intriguing possible
target in the management of SCI-associated damage, due
to its dualistic pattern of activation (by both nucleotides
GPR17 in spinal cord injury
Brain 2009: 132; 2206–2218
| 2215
Figure 7 The in vivo knock-down of GPR17 markedly attenuated SCI-associated tissue damage and related motor deficit.
Immunohistochemical evaluation of tissue structure and integrity at various times p.i. by haematoxylin/eosin (A), Luxol Fast Blue
(B, D and E) and methyl green pyronin staining (C) in SHAM-operated animals and in mice subjected to SCI and treated with a
scrambled or with the oligo 616 antisense oligonucleotide (see Methods section for details). A massive and increasing over time
disruption of tissue architecture and of the myelin layer can be observed after induction of SCI (see Scrambled). The administration of
oligo 616 counteracted tissue degeneration at any of the evaluated time points. Scale bars: 100 mm except for (A): 200 mm. In parallel,
a marked amelioration of the histological score was found in SCI animals treated with oligo 616 with respect to mice receiving the
corresponding oligonucleotide (five animals/group) (F). (G) Evaluation of motor disturbance after induction of SCI and administration of
antisense oligonucleotides. Five animals/group were evaluated for their motor skills once a day for up to 7 days p.i. A significant
amelioration of the SCI-related motor disturbance can be detected starting from Day 3 in animals receiving oligo 616 with respect to
animals treated with the Scrambled oligonucleotide. *P50.05 with respect to Scrambled-treated animals, one-way ANOVA followed
by Scheffe’s F-test. See Supplementary Methods for details on the scoring procedures.
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| Brain 2009: 132; 2206–2218
and cysteinyl-leukotrienes), to its biphasic activity during the
development of ischaemic brain damage (see Introduction and
below) and to its presence on various embryonically distinct cells
involved in different phases of repair.
Here, we characterize for the first time the presence of GPR17
in mouse spinal cord and its changes after SCI. Under physiological
conditions, GPR17 is expressed by neurons and oligodendrocytes
at different stages of maturation and by ependymal cells lining the
central canal (Kulbatski et al., 2005; Mothe and Tator, 2005), but
never by astrocytes. Consistent with these data, in the intact
rodent brain, GPR17 is expressed by neurons (Ciana et al.,
2006; Lecca et al., 2008) and by a subset of precursor cells in
both parenchyma and neurogenic areas (Lecca et al., 2008).
Moreover, in brain parenchyma GPR17 segregated to a subset
of quiescent pre-oligodendrocytes. Globally, these data suggest
very similar expression profiles of GPR17 in both intact brain
and spinal cord and support a role for this receptor in both
neurons and oligodendrocytes.
The physiological role played by this receptor is still largely
unknown. GPR17 is activated by both uracil nucleotides and
cysteinyl-leukotrienes (Ciana et al., 2006; Lecca et al., 2008;
Parravicini et al., 2008). Significant concentrations of the former
are extracellularly released also under physiological conditions
(Lecca and Ceruti, 2008), and could therefore account for the
basal activation of the receptor.
It is well known that both nucleotides and cysteinyl-leukotrienes
are massively released during trauma and ischaemia (Nishisho
et al., 1996; Ciceri et al., 2001; Wang et al., 2004; Davalos
et al., 2005), suggesting that GPR17 might acquire a prominent
role upon stressful and pathological conditions. In agreement with
this hypothesis, currently available data suggest that GPR17 might
act as a sensor of warning signs after trauma, and might be also
involved in remodelling/repair, in agreement with the emerging
role of endogenous nucleotides and their specific receptors as
danger signals in both spinal cord and brain injury (Wang et al.,
2004; Davalos et al., 2005; Haynes et al., 2006). In line with this
hypothesis, here we show that SCI induces a dramatic and rapid
death of GPR17+ neurons and oligodendrocytes inside the lesioned
area. These data are also highly consistent with what has been
found in rodent brain in the Middle Cerebral Artery occlusion
(MCAo) ischaemia model. An initial up-regulation of GPR17 on
neuronal cells within the lesioned area, followed by a dramatic
decrease of the number of neurons inside the ischaemic core
(Lecca et al., 2008) were found, indicating a causal relationship
between ischaemia-induced receptor up-regulation and commitment to cell death. Similar data have been obtained in a model
of brain trauma (data not shown). Due to the much more
severe and immediate cellular damage associated to SCI, in the
present study it was not possible to assess if GPR17 undergoes
up-regulation in cells inside the lesioned area. However, a causal
relationship between GPR17 expression and cell death under this
pathological condition is strongly suggested by our demonstration
that injection of a specific GPR17 antisense oligonucleotide into
the spinal cord significantly ameliorates SCI-induced tissue damage
and the related histological and motor deficits. Bioinformatic
analysis showed that the antisense oligonucleotide utilized here
to effectively prevent SCI was not homologous to any other
S. Ceruti et al.
G protein-coupled receptor (Ciana et al., 2006), and a scrambled
oligonucleotide injected in parallel in mice undergoing SCI was
ineffective. Thus, these data strongly indicate that the protective
effect observed with oligo616 is actually due to GPR17 inhibition.
Interestingly, only oligodendrocytes expressing GPR17 undergo
massive cell death when exposed to high ATP concentrations
in vitro (Ceruti et al., unpublished results), thus confirming
the role of this receptor in ‘sensing’ pathological situations and
in conferring sensitivity to cell death.
The concept of danger signal is quite well established in the
immune system but is just now beginning to be translated to
the CNS (Di Virgilio et al., 2009). In the immune system,
stressed/injured cells release soluble signals, such as nucleotides,
that alert patrolling cells and start response (e.g. beneficial inflammation) by activating P2 receptors on immune cells. Therefore,
nucleotides fulfil all the requirements to be classified as ‘danger
signals’ (Ferrari et al., 2006). Based on the present data, we speculate that nucleotides may similarly report tissue injury in the
spinal cord by directly activating GPR17.
Danger signals are beneficial in principle, but may be detrimental if inappropriately sustained (Abbracchio and Ceruti, 2006;
Ferrari et al., 2006). Therefore, if damage is very severe, as is
the case of the core of the lesioned spinal cord after injury
(where nucleotides concentrations are likely to reach very high
toxic levels), receptors for danger signals (e.g. GPR17) could also
contribute to massive cell death (see above). This has been already
demonstrated for purinergic receptors in the brain, where following traumatic events massively released ATP mediates apoptotic
and necrotic neuronal and astrocytic death (for review, see Franke
et al., 2006; Abbracchio and Ceruti, 2006). The same receptors
may, however, participate to the beneficial remodelling and repair
response activated by danger signals through recruitment
and migration of microglia/macrophages from areas distal to the
traumatic core (Franke et al., 2006; Lecca et al., 2008).
In line with this hypothesis, in SHAM-operated animals, very
few resting microglial cells (here identified by positivity to the
microglial marker Iba1) were detected, partly co-expressing
GPR17. Starting from 24 h after SCI, activated microglia/
macrophages (expressing the specific marker of activation IB4
and whose totality co-expressed GPR17) migrate towards and
infiltrate into the lesioned area, with their number increasing
over time after injury. One week p.i., most of these double-positive cells were found inside the lesioned area and receptor expression started decreasing. This suggests that GPR17 expression
is increased along the first phases of microglia/macrophages
activation and might favour their migration and infiltration
to the lesioned area; when these processes are completed,
GPR17 expression is down-regulated. A similar phenomenon was
detected in mouse ischaemic brain where 72 h after MCAo, cells
co-expressing GPR17 and IB4 were found at the boundaries of the
ischaemic infarct; 1 week after MCAo, these cells were detected
within the damaged area (Lecca et al., 2008). In the injured spinal
cord, this behaviour for GPR17+ in microglia/macrophages is
even more striking.
Previous literature data have demonstrated an important role
for the P2Y12 nucleotide receptor in the phenotypic changes and
chemotactic response at very early stages of activation of resting
GPR17 in spinal cord injury
microglia after brain injury, and suggested that other purinergic
receptors might be involved at later times (Haynes et al., 2006).
In our experimental model, GPR17 expression is increased on
globose cells that have the morphological appearance of already
activated ameboid cells as early as 24 h p.i. and starts decreasing 1
week p.i. Thus, we speculate that P2Y12 and GPR17 could be
sequentially involved in the recruitment and chemotaxis of microglia at the site of injury, with a main involvement of P2Y12 in the
immediate microglia response and a role for GPR17 at later phases
of cell migration towards the lesioned area. In this respect, it is
worth noting that human GRP17 has been originally cloned in an
attempt to isolate new members of the chemokine receptor family
(Raport et al., 1996). Moreover, the P2Y6 receptor subtype has
recently been demonstrated to control microglia phagocytosis
(Koizumi et al., 2007), and could therefore be activated at later
stages of microglia activation, thus confirming the central role
played by the purinergic system in driving and modulating the
remodelling process after damage.
An additional interesting observation that further supports the
apparently opposite effects (i.e. cell death and damage repair)
mediated by GPR17 in the SCI model is represented by its expression on ependymal cells lining the central canal. These cells have
been recently demonstrated to represent the true stem cells in the
adult spinal cord, since they start proliferating after injury and
produce oligodendrocytes, and more abundantly, astrocytes that
migrate to the lesion site building a substantial part of the glial
scar (Meletis et al., 2008). Also in the brain, where they are normally quiescent, ependymal cells can generate neuroblasts and
astrocytes after stroke (Carlén et al., 2009). Here we demonstrate,
for the first time, that GPR17 is constitutively expressed by spinal
cord ependymal cells which start proliferating and expressing the
marker of pluripotency GFAP upon induction of SCI. It is worth
noting that GPR17 is one of the three genes that are exclusively
expressed by neuroprecursor cells in the adult human hippocampus (Maisel et al., 2007), thus suggesting its possible role in
driving precursor cell specification and differentiation after brain
and SCI.
Due to its dualistic pattern of activation, it can be further
hypothesized that GPR17 is also involved in other promising
pharmacological approaches to SCI. For example, the cysLT receptor antagonist Montelukast, which has recently proven effective in
preventing secondary damage in SCI (Genovese et al., 2008c),
also inhibits GPR17 activation (Ciana et al., 2006). Moreover,
two additional effective treatments (i.e. administration of corticosteroids and of the 5-lipooxygenase inhibitor Zileuton; Genovese
et al., 2007, 2008c) resulted in a reduction of cysteinylleukotrienes production that might in turn reduce GPR17
activation. It is worth noting that no significant difference was
found between the protection afforded by in vivo GPR17
knockdown observed here and the anti-inflammatory property of
dexamethasone treatment previously reported in the same experimental model (Genovese et al., 2007). Thus, taken together,
our data suggest that GPR17 represents an interesting target to
develop new approaches to the management of secondary
damage in SCI aimed at both reducing cell death and fostering
tissue repair.
Brain 2009: 132; 2206–2218
| 2217
Supplementary material
Supplementary material is available at Brain online.
Acknowledgements
Authors are deeply grateful to Dr Annalisa Buffo, University of
Turin and to Dr Paolo Ciana and Dr. Davide Lecca, University
of Milan, Italy for useful discussion and advice.
Funding
IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Messina, Italy; Italian
Ministry
of
Education
and
Research
(FIRB
project
RBNE03YA3L_009 and PRIN-COFIN project prot. 2006059022
to M.P.A.).
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