Development/Plasticity/Repair
Exogenous Leukemia Inhibitory Factor Stimulates
Oligodendrocyte Progenitor Cell Proliferation and Enhances
Hippocampal Remyelination
Benjamin E. Deverman and Paul H. Patterson
Division of Biology, California Institute of Technology, Pasadena, California 91125
New CNS neurons and glia are generated throughout adulthood from endogenous neural stem and progenitor cells. These progenitors
can respond to injury, but their ability to proliferate, migrate, differentiate, and survive is usually insufficient to replace lost cells and
restore normal function. Potentiating the progenitor response with exogenous factors is an attractive strategy for the treatment of
nervous system injuries and neurodegenerative and demyelinating disorders. Previously, we reported that delivery of leukemia inhibi-
tory factor (LIF) to the CNS stimulates the self-renewal of neural stem cells and the proliferation of parenchymal glial progenitors. Here
we identify these parenchymal glia as oligodendrocyte (OL) progenitor cells (OPCs) and show that LIF delivery stimulates their prolifer-
ation through the activation of gp130 receptor signaling within these cells. Importantly, this effect of LIF on OPC proliferation can be
harnessed to enhance the generation of OLs that express myelin proteins and reform nodes of Ranvier in the context of chronic demy-
elination in the adult mouse hippocampus. Our findings, considered together with the known beneficial effects of LIF on OL and neuron
survival, suggest that LIF has both reparative and protective activities that make it a promising potential therapy for CNS demyelinating
disorders and injuries.
Introduction
Oligodendrocytes (OLs) wrap axons in myelin, coordinate ion
channel clustering at nodes of Ranvier, and provide trophic sup-
port for axons. Consequently, the OL loss that occurs in the le-
sions of demyelinating disorders such as multiple sclerosis (MS)
and after traumatic CNS injury contributes to ongoing disability.
Remyelination of these lesions by endogenous OL progenitor
cells (OPCs) occurs in animal models and some patients but is
variable and insufficient. One approach to enhance remyelina-
tion is to administer factors that can mobilize endogenous OPCs
and neural stem cells (NSCs) and direct their differentiation into
OLs. In this regard, several of the neuropoietic, gp130 cytokines,
most notably leukemia inhibitory factor (LIF) and ciliary neu-
rotrophic factor (CNTF), have multiple activities that make them
attractive therapeutic candidates. First, in culture, LIF and CNTF
enhance the proliferation of OPCs (Barres et al., 1996). Second,
LIF and CNTF promote the maturation of cultured OPCs into
OLs (Mayer et al., 1994), and in mixed hippocampal cultures, the
release of LIF by astrocytes stimulates myelination (Ishibashi et
al., 2006). Third, after injury, LIF activates astrocytes and micro-
glia (Sugiura et al., 2000; Kerr and Patterson, 2004), cell types that
can modulate disease by limiting the spread of the lesion and
clearing myelin debris, respectively, and produce cytokines and
growth factors critical for remyelination (Arnett et al., 2001; Ma-
son et al., 2001; Hendriks et al., 2008; Haroon et al., 2011).
Fourth, axon regrowth after optic nerve crush is enhanced by
inflammation through a process that requires CNTF and LIF
(Leibinger et al., 2009). Fifth, LIF stimulates the self-renewal of
adult NSCs in the subventricular zone (SVZ), which may expand
this population to facilitate repair (Bauer and Patterson, 2006).
This finding has relevance for the repair of demyelination since
NSCs can generate migratory OPCs that differentiate into OLs
and contribute to remyelination (Menn et al., 2006). Finally, sev-
eral gp130 cytokines also enhance the survival of OLs in culture
(Louis et al., 1993; Mayer et al., 1994; Barres et al., 1996; Zhang et
al., 2006) and in models of spinal cord injury and MS (Butzkue-
ven et al., 2002; Kerr et al., 2005; Marriott et al., 2008).
Whereas the protective effects of LIF on OLs are well de-
scribed, it is not clear that LIF delivery can enhance the endoge-
nous remyelination response. Since LIF not only stimulates OPC
proliferation and OL generation in vitro, but also acts on micro-
glia and astrocytes, we hypothesized that supplying exogenous
LIF could provide the necessary signals to expand the population
of OPCs in demyelinated lesions and promote their differentia-
tion into myelinating OLs. To test this hypothesis, we chose to use
the cuprizone model of demyelination in a proof-of-concept ex-
Received July 25, 2011; revised Dec. 1, 2011; accepted Dec. 16, 2011.
Author contributions: B.E.D. and P.H.P. designed research; B.E.D. performed research; B.E.D. and P.H.P. analyzed
data; B.E.D. and P.H.P. wrote the paper.
The authors declare no competing financial interests.
This work was supported by a fellowship to B.E.D. from the California Institute for Regenerative Medicine and by
grants from the National Institute of Neurological Disorders and Stroke (NS045744 and ARRANS45744) and the
McGrath Foundation. We thank Sylvian Bauer for thoughtful discussions and assistance initiating this study; Tania
Banerji for technical assistance; Elaine Hsiao, Jan Ko, Natalia Malkova, and Puja Saluja for critical reading of this
manuscript; and members of the California Institute of Technology Office of Laboratory Animal Resources, especially
N. Miyamoto, for consistent excellent animal care. We also thank W. Muller and W. Richardson for the generous gifts
of transgenic mouse lines.
Correspondence should be addressed to Paul H. Patterson, Division of Biology M/C 216-76, California Institute of
Technology, Pasadena, CA 91125. E-mail: php@caltech.edu.
DOI:10.1523/JNEUROSCI.3803-11.2012
Copyright © 2012 the authors 0270-6474/12/322100-10$15.00/0
2100
•
The Journal of Neuroscience, February 8, 2012
•
32(6):2100 –2109
periment. Cuprizone induces a time course of demyelination and
remyelination that is well characterized and reproducible in re-
gional impact and severity, and it allows for testing potential
remyelination-promoting paradigms at times when OLs have
been almost completely ablated. This provides for a clear inter-
pretation of the mechanism underlying any observed therapeutic
benefit (i.e., that it is attributable to enhanced remyelination
rather than protection of existing OLs), a distinction that is crit-
ical for evaluating the potential remyelination-promoting effects
of LIF and dissociating these effects from its pro-survival effects
on OLs.
Materials and Methods
Animals.
C57BL/6J, Rosa-YFP (Srinivas et al., 2001), and Ng2-Cre BAC
(Zhu et al., 2008) transgenic mice were obtained from The Jackson Lab-
oratory. The gp130
fl
mice (Betz et al., 1998) and PDGFR
-CreER BAC
transgenic mice (Rivers et al., 2008) were generous gifts from W. Muller
(University of Manchester, Manchester, UK) and W. Richardson (Wolfson
Institute for Biomedical Research, University College London, London,
UK), respectively. The gp130
fl
;Ng2-Cre and PDGFR
-CreER;Rosa-YFP
micewereonmixedbackgrounds:C57BL/6;FVBandCBA;C57BL/6,respec-
tively. Littermates, both male and female, were used for all studies in genet-
ically modified mice. Primer sequences for genotyping are available on
request. All procedures were approved by the California Institute of Tech-
nology Institutional Animal Care and Use Committee.
Adenovirus injection.
Recombinant adenovirus,
2.7
10
6
pfu in 3
l
(Zhu et al., 2001), encoding mouse LIF (Ad-LIF) or LacZ (Ad-LacZ) was
injected into the lateral ventricle as described (Bauer and Patterson,
2006). This method primarily infects ependymal cells (Doetsch et al.,
1999; Bauer and Patterson, 2006). Five to six mice were used per virus-
injected group for cuprizone experiments, and two to three mice were
used per group for Ad-LIF-induced proliferation assays.
Bromodeoxyuridine and tamoxifen administration.
Bromodeoxyuri-
dine (BrdU; Sigma-Aldrich) for injection was made at 10 mg/ml in 0.9%
NaCl and sterile filtered. Mice were given intraperitoneal injections of 50
mg/kg BrdU at the indicated times before they were killed. For long-term
BrdU labeling, BrdU was dissolved in H
2
O at a concentration of 0.8
mg/ml, sterile filtered, aliquoted, and stored at
20°C. Water containing
BrdU was changed daily. Tamoxifen was dissolved by sonication in sun-
flower oil. Mice were given 4 mg of tamoxifen by a single daily intraperi-
toneal injection for 3 consecutive days.
Cuprizone treatment.
Eight-week-old C57BL/6J mice from The Jack-
son Laboratory were fed a diet containing 0.2% cuprizone (Sigma-
Aldrich), which was freshly prepared and mixed into milled chow (5001
Rodent Diet; Lab Diet) three times per week. After cuprizone treatment,
mice were returned to a standard pellet chow.
Tissue processing and immunostaining.
Mice were anesthetized with
Nembutal and transcardially perfused first with 0.1
M
phosphate buffer
(PB), pH 7.4 (room temperature) and then with freshly prepared 4%
paraformaldehyde in PB (4°C). Brains were postfixed for 4–5 h for cry-
ostat sectioning (cuprizone experiments) or overnight for vibratome sec-
tioning (experiments in Fig. 1
E
–
H
, 4). For cryostat sectioning, brains
were placed in 4°C PB
overnight followed by 24 h of immersion in 20%
sucrose in PB. The brains were then frozen in OCT (Sakura Tissue-
Tek) and stored at
80°C until sectioning. Fourteen-micrometer cor-
onal sections were cut on a cryostat (Leica), dried overnight at room
temperature, and stored at
20°C. Floating coronal vibratome sec-
tions were cut at 50
m, collected in PB containing 0.02% sodium
azide, and stored at 4°C.
Immunostaining was performed by diluting primary and secondary
antibodies in PBS containing 10% goat or donkey serum and 0.1% Triton
X-100 (frozen sections) or 0.5% Triton X-100 (floating sections). Pri-
mary antibodies used were rat anti-BrdU* (1:250; Oxford Biotechnology
or Abcam), mouse anti-CC1 (1:200; Calbiochem), rabbit anti-GFAP (1:
1000; Dako), rabbit anti-GFP (1:1000; Invitrogen), chicken anti-GFP
(1:2000; Abcam), rabbit anti-Iba1 (1:500; Biocare Medical), mouse anti-
K
V
1.2* (1:1000; NeuromAb), mouse anti-Na
V
1.6* (1:500; Alamone
Labs), mouse anti-neurofilament (NF)* (1:50, clone 2H3; Developmen-
tal Studies Hybridoma Bank), rabbit anti-Ng2 (1:300; Millipore), goat
anti-Olig2 (1:500
;R&D
Systems), mouse anti-proteolipid protein
(PLP)* (1:500; Millipore), mouse anti-Rip (1:50; Developmental Studies
Hybridoma Bank), and rabbit anti-pSTAT3-Y705* (1:500; Cell Signaling
Technology). Primary antibody incubations were performed at room
temperature overnight (slide-mounted sections) and for 2–3 d at 4°C
(floating sections). Several antigens (marked above with an asterisk) re-
quired an antigen unmasking pretreatment. For frozen sections, this was
performed by incubating slides at 100°C for 30 min with a pH 6 unmask-
ing agent (Dako) before proceeding with primary antibody incubation.
For floating sections, unmasking the BrdU and pSTAT3 antigens re-
quired pretreatment with 0.1N HCl at 4°C for 30 min followed by im-
mersion in 2N HCl at 37°C for 30 min. Goat or donkey anti-species- and
isotype-specific antibodies conjugated to Alexa-488, 568, or 633 (Invit-
rogen) were incubated with tissue sections for 1–2 h (frozen sections) or
overnight (floating sections). The Rip and NF antibodies were developed
by S. Hockfield (Massachusetts Institute of Technology, Cambridge,
MA) and T. Jessell (Columbia University, New York, NY), respectively,
and were obtained from the Developmental Studies Hybridoma Bank
(dshb@uiowa.edu).
Image analysis and quantification.
Slide-mounted sections were im-
aged using a TCS SP confocal microscope (Leica) equipped with argon,
krypton, and He/Ne lasers. Care was taken to sample sections at similar
anatomical levels. For cuprizone studies, cell counts were made from
confocal images taken of coronal sections of the medial CC (
0.6 to 0.8
mm caudal to bregma) and the dorsal hippocampus (
1.5 to
1.9 mm
caudal to bregma). For the experiment shown in Figure 5, BrdU
cells
were counted in images of the fimbria between
0.7 and
1.6 mm.
PLP and NF staining quantification was performed on 63
,10
m
projection images of the CA3 stratum radiatum (medial to the lateral
extent of the mossy fibers) of the hippocampus. A manual threshold,
equivalent for all images, was set in ImageJ (http://rsb.info.nih.gov/ij/)
for each channel, and the area above that threshold was quantified. The
percentage area over threshold for PLP was then divided by the percent-
age area over threshold for NF and presented as a ratio. All values were
normalized to those of the untreated group. Nodes were counted from
images of the CA3 stratum radiatum. The number of Na
V
1.6
nodes,
flanked on both sides by Caspr
paranodes, was counted in 7
m con
-
focal projections images taken with a 63
objective. Adobe Photoshop
CS3 was used to adjust image contrast and brightness (any adjustments
made were made equally for all images within an experiment).
Statistical analysis.
Differences between group means were tested for
statistical significance using an unpaired, two-tailed Student’s t test (two
groups) or with a one-way ANOVA and Tukey’s multiple comparison
test (three or more comparisons). All data are presented as mean
SEM.
Analyses were performed using Prism 4.0b software.
Results
LIF promotes OPC proliferation in vivo
In previous work from our group, Bauer et al. (2006) demonstrated
that delivery of exogenous LIF using an adenovirus encoding a se-
creted form of mouse LIF (Ad-LIF) increases the number of prolif-
erating parenchymal glial progenitors expressing Olig2 or S100
threefold to fivefold over the controls that received a
-galactosidase
(LacZ)-encoding virus. Because the primary adult glial
progenitor
cell population that proliferates outside of the neurogenic niches
are Olig2
OPCs (Nishiyama, 2007), this finding suggested that
LIF delivery stimulates the proliferation and/or generation of this
population. However, whereas Olig2 expression is typically re-
stricted to the OL lineage, it is also expressed by a subset of astro-
cytes during development and after injury (Cai et al., 2007;
Tatsumi et al., 2008), and S100 can be expressed by astrocytes,
OPCs, and myelinating OLs (Rickmann, 1995; Hachem et al.,
2005). Therefore, we sought to further clarify the identity of the
proliferating glia.
Adult OPCs are typically defined and identified by their ex-
pression of Ng2 and platelet-derived growth factor receptor
Deverman and Patterson
•
LIF Stimulates OPC Proliferation In Vivo
J. Neurosci., February 8, 2012
•
32(6):2100 –2109
• 2101
(PDGFR
). Three days after intracerebroventricular injection of
Ad-LIF, we found that 41.0
2.7% (mean
SEM; n
2) of the
BrdU
cells proliferating outside of the SVZ are Ng2
(Fig. 1
A
).
Nearly all of the proliferating cells not labeled with antibodies di-
rected against Ng2 or Olig2 are Iba1
microglia (55.7
4.0% non-
SVZ BrdU
cells; mean
SEM; n
2) (Fig. 1
B
), a finding
consistent with the previous observation that LIF induces microglial
proliferation (Kerr and Patterson, 2004). LIF signaling activates the
transcription factor STAT3, and in periventricular areas where
STAT3 activation is greatest, we detect activated, phosphorylated
STAT3 in 86.5
2.5% of the proliferating Olig2
OPCs, suggesting
that these cells respond directly to LIF (Fig. 1
C
). Although LIF
strongly increases STAT3 activation and GFAP expression in astro-
cytes, GFAP
cells outside of the SVZ rarely proliferate in response
to acute LIF treatment (Fig. 1
D
). In line with this finding that most
oftheBrdU
neuralparenchymalcellsinLIF-treatedmiceareOPCs
andnotastrocytes,triplestainingforS100,Olig2,andBrdUcon
firms
that the BrdU
/S100
and BrdU
/Olig2
populations we previously
described are nearly completely overlapping (data not shown), suggest-
ingthatthesubsetof S100
cells that incorporate BrdU in response
to LIF are Olig2
OPCs.
We next asked whether the stimulation of OPC proliferation by
LIF expands the pool of OPCs. To test this, we used PDGFR
-
CreER;ROSA-YFP mice (Rivers et al., 2008), which allow for
tamoxifen-inducible cre-dependent expression of the yellow fluo-
rescent protein (YFP) reporter in PDGFR
OPCs and their prog
-
eny. YFP expression was induced in OPCs by tamoxifen
administration, and
mice were given injections of either Ad-LIF or
Ad-LacZ. Three weeks later, we found that the population of
YFP
cells is dramatically expanded in the periventricular re
-
gions of Ad-LIF-treated animals compared with control animals
receiving Ad-LacZ (Fig. 1
E
–
I
). The vast majority of the YFP
cells are Olig2
in both Ad-LIF- and Ad-LacZ-treated mice
(97.2
0.4 and 98.5
0.6%, respectively, in the dorsal forebrain;
n
3 per group; Fig. 1
G
,
H
). We observe occasional YFP
cells
with neuronal and pericyte morphology, as has been reported
after tamoxifen injection in this line (Rivers et al., 2008).
LIF restores OL number after demyelination
To examine whether exogenous LIF treatment can enhance the
generation of OLs after cuprizone-induced demyelination, we
treated 8-week-old adult C57BL/6 mice with a cuprizone-
supplemented diet for 5 weeks, delivered Ad-LIF or Ad-LacZ by
intracerebroventricular injection, and returned the mice to a
standard diet. To label cells that proliferate after treatment, we
administered BrdU in the drinking water for a total of 7 d, start-
ing 3 d after virus injection. Twenty-eight days after ceasing cu-
prizone treatment and injecting Ad-LIF or Ad-LacZ (18 d after
removing BrdU from the drinking water), we assessed the num-
ber of newly generated BrdU
OLs with and without LIF treat
-
ment (Fig. 2
B
). Remarkably, compared with controls, LIF
increases the number of mature BrdU
/CC1
OLs in both the
Figure 1.
Exogenous LIF stimulates OPC proliferation. To identify cells responding to acute LIF,
mice were treated with Ad-LIF and given injections of BrdU 3 an
d 6 h before they were killed. Images
show characterization of BrdU
cells (green) by immunostaining.
A
, Many BrdU
cells, highlighted
by arrows, also display staining for both Olig2 (blue) and Ng2 (red). Asterisks highlight BrdU
cells
unlabeled by either of these OPC markers.
B
, Olig2-negative BrdU
cells are mostly Iba1
(red)
microglia(arrows).
C
,Ad-LIFtreatmentinducesStat3activation(phospho-Y705Stat3-specificimmu-
nostaining; blue) in Olig2
/BrdU
OPCs (arrows). The cell highlighted in the dashed box is positive
for BrdU (green), Olig2 (red), and pSTAT3 (blue) immunostaining, as can be seen from the enlarged
individual channel images shown to the right of the tricolor image.
D
, LIF induces STAT3 phosphory-
lation (blue) in GFAP
astrocytes (red), but these cells rarely incorporate BrdU (green) in response to
4
LIF.
E–I
, Ad-LIF expands the OPC pool. PDGFR
-CreER
/
;ROSA-YFP
/
mice were induced
with tamoxifen, treated 1 week later with Ad-LacZ (
E
,
G
) or Ad-LIF (
F
,
H
), and killed 3 weeks
after adenovirus injection.
E
,
F
, Immunostaining for the YFP reporter is shown in green.
Dotted
lines outline the lateral ventricle (LV) walls. cc, Corpus callosum; st, striatum; sep, septum.
G
,
H
,
Representative images are shown of immunostaining for Olig2 (purple) and YFP (green) in the CC of
Ad-LacZ-treated (
G
) and Ad-LIF-treated (
H
) mice. Scale bars:
A
–
D
,
G
,
H
,20
m;
E
,
F
, 200
m.
I
,
Quantification of the fold increase in YFP
/Olig2
cells in periventricular regions of the corpus cal
-
losum (cc), striatum (str), hippocampus (hip), fimbria (fimb), and ventral cortex (v cx). **
p
0.01;
***
p
0.001. The effect of LIF is not significant in the ventral cortex.
2102
•
J. Neurosci., February 8, 2012
•
32(6):2100 –2109
Deverman and Patterson
•
LIF Stimulates OPC Proliferation In Vivo
hippocampus and the corpus callosum (CC) by 11.5-fold (105
6.05 BrdU
/CC1
cells/mm
2
in LIF-treated animals vs 9.2
2.6
in LacZ-treated animals;
p
0.001) and 2.5-fold (491
57.1
BrdU
/CC1
cells/mm
2
in LIF-treated animals vs 196
62.2 in
LacZ-treated animals;
p
0.01), respectively. LIF treatment also
increases the percentage of CC1
cells that are BrdU
by 4.6-fold
(44.4
3.5% in LIF-treated animals vs 9.6
2.4% in LacZ-
treated animals;
p
0.001) in the hippocampus and 2.3-fold
(16.5
1.9% in LIF-treated animals vs 7.1
2.1% in LacZ-
treated animals;
p
0.01) in the CC. This results in a recovery of
CC1
OLs in the hippocampus to a level that is significantly
greater than that seen in the control, LacZ-treated animals (Fig.
2
C
). In contrast, the number of CC1
OLs in the medial CC,
where the spontaneous regeneration of OLs is much greater, is
not significantly different between Ad-LIF- and Ad-LacZ-treated
animals (2998
114 vs 2673
164 CC1
cells/mm
2
, respec
-
tively;
p
0.05). In gray matter (GM) areas such as the hip-
pocampus and cortex, where individual myelinated axons are
more easily discernible, we observe Rip
OLs derived from
OPCs, which had proliferated early during LIF treatment, mak-
ing extended contact along axons (Fig. 2
D
).
These findings demonstrate that LIF dramatically enhances
the generation of OLs in both gray matter and white matter tracts
after demyelination. Assessing the effect of LIF on remyelination
in these mice is, however, complicated by the significant sponta-
neous remyelination that occurs in the CC and, to a lesser extent,
in the hippocampus, after 5 week cuprizone
treatment. In contrast to 5–6 week cuprizone
treatment, spontaneous remyelination has
been reported to be limited after long-term,
12–16 week treatment with cuprizone. The
lack of remyelination after long-term cupri-
zone treatment has been attributed to a re-
duction in OPCs (Mason et al., 2004) and
inhibition by fibroblast growth factor 2
(FGF2) (Armstrong et al., 2002; Armstrong
et al., 2006). Therefore, we next tested
whether LIF treatment would stimulate OPC
proliferation, OL generation, and remyelina-
tion under conditions where OL generation
and remyelination are otherwise impaired.
For this experiment, we treated mice with cu-
prizone for 12 weeks (Fig. 3
A
). Similar to a 5
week course of cuprizone, we found that a 12
week course of cuprizone induces a near-
complete loss of mature OLs and myelin
from the dorsal hippocampus (Fig. 3
E
and
data not shown). We first assessed whether
OPCs remaining after 12 weeks of cuprizone
treatment still respond to LIF with increased
proliferation. Ad-LIF or Ad-LacZ was deliv-
ered to mice after a 12 week course of cupri-
zone, and the mice were returned to their
standard diet and allowed to recover for 3
weeks. At the end of this recovery period, we
administered BrdU by intraperitoneal injec-
tion 2 and 4 h before the mice were killed to
label proliferating cells. The number of pro-
liferating Olig2
cells is increased by 3.9-fold
over controls after LIF treatment in the hip-
pocampus (Fig. 3
B
,
C)
as is the percentage of
Olig2
cells that are BrdU
(3.8
0.2% vs
1.3
0.3% in Ad-LIF- vs Ad-LacZ-treated
animals;
p
0.001). A similar trend is observed with LIF treat-
ment in the CC (Fig. 3
D
), but this does not reach significance.
Therefore, even in the context of chronic demyelination, LIF en-
hances the endogenous progenitor response, at least in the hip-
pocampus where there is less spontaneous OPC proliferation. We
next examined whether LIF treatment would promote the gener-
ation of mature OLs after long-term demyelination. Indeed, after
3 weeks of recovery, the number of CC1
OLs in the hippocam
-
pus is increased in LIF-treated mice compared with Ad-LacZ-
treated mice (Fig. 3
E
). Remarkably, after 6 weeks of recovery, the
number of OLs is restored to near-normal numbers in LIF-
treated mice, whereas in stark contrast, little recovery is observed
in mice that receive the control LacZ virus (Fig. 3
E
). Unlike what
we observe in the hippocampus, the number of OLs in the medial
CC recovers to near that seen in untreated mice even in the ab-
sence of LIF treatment (Fig. 3
F
). Thus, the spontaneous OL gen-
eration that occurs in the CC obscures any beneficial effect of LIF
that may occur in this region.
LIF treatment promotes remyelination
Based on our encouraging findings in the hippocampus, and our
finding that long-term, 12 week cuprizone treatment completely
abolishes myelin throughout the dorsal hippocampus, as judged
by the loss of PLP immunostaining (Fig. 4
B
,
I
), we next sought to
assess the extent of remyelination in the hippocampus. To do
this, we performed immunostaining for PLP together with a NF
Figure 2.
LIF enhances OL generation after acute demyelination. Male C57BL/6 mice were fed cuprizone for 5 weeks,
returned to a standard diet, and given an injection of Ad-LIF or Ad-LacZ. Three days after virus injection, BrdU was supplied in
the drinking water for 7 d, and newly generated OLs were assessed 4 weeks after virus injection.
A
, A schematic provides an
overview of the experimental design. i.c.v. inj., Intracerebroventricular injection.
B
, LIF delivery increases the number of newly
generated BrdU
/CC1
OLs in the hippocampus compared with Ad-LacZ controls. Representative images are shown of
immunostaining of the hippocampi (CA3 region) of mice treated as indicated. BrdU
(green)/CC1
(purple) OLs are high
-
lighted by arrows. Quantification of BrdU
/CC1
cells is provided in the text.
C
, Quantification of the total number of CC1
cells in the CA3 region of the hippocampus. ***
p
0.001. The difference between the 5
4w Ad-LacZ group and the 5 week
group is not significant (
p
0.05).
D
, An example of immunostaining for BrdU (green), the mature OL protein RIP (red), and
axons (NF; blue) is shown for the hippocampus of an Ad-LIF-treated mouse. Scale bars, 20
m.
Deverman and Patterson
•
LIF Stimulates OPC Proliferation In Vivo
J. Neurosci., February 8, 2012
•
32(6):2100 –2109
• 2103