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study supports the role of cell cycle activation in the pathophysiology of SCI and, by using a clinically relevant treatment
model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.
Cell Cycle 11:9, 1782-1795; May 1, 2012; © 2012 Landes Bioscience
Delayed cell cycle pathway modulation facilitates
recovery after spinal cord injury
1782 Cell Cycle Volume 11 Issue 9
*Correspondence to: Junfang Wu; Email: email@example.com
Submitted: 02/18/12; Revised: 03/24/12; Accepted: 03/26/12
Traumatic spinal cord injury (SCI) causes tissue loss and asso-
ciated neurological dysfunction due to both mechanical dam-
age and secondary biochemical and physiological responses.1-6
Mechanisms of secondary injury include a complex cellular
response through the activation and/or suppression of a large
number of transcriptional pathways in a heterogeneous cell
population. These mechanisms include neuronal cell death, loss
of oligodendrocytes, inflammation and reactive astrogliosis.1,7
Effective clinical strategies will likely include either combination
of selective inhibitors of secondary injury factors or single drugs
that modulate multiple injury components.
Our comprehensive gene profiling analysis of rat spinal cord
after contusion injury has demonstrated upregulation of a cluster
of cell cycle-related genes, including c-myc, Gadd45, cyclin D1,
proliferating cell nuclear antigen (PCNA), cyclin G, cyclin-
dependent kinase 4 (CDK4), E2 promoter binding factor 5
(E2F5) and retinoblastoma protein (Rb) at 4 h and 24 h after SCI.8
The cascade of molecular events linking activation of cell cycle to
traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage
and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell
cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment
initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following
SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-
dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis
demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, e2F1 and pCNA, at
various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. treatment with
flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional
recovery was significantly improved after SCI from day 7 through day 28. treatment significantly reduced lesion volume
and the number of Iba-1+ microglia in the preserved tissue and increased the myelinated area of spared white matter as
well as the number of CC1+ oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3,
associated with microglial activation and astrocytic reactivity by reduction of GFAp, NG2 and CHL1 expression. our current
Junfang Wu,1,* Bogdan A. Stoica,1 Michael Dinizo,1 Ahdeah pajoohesh-Ganji,2 Chunshu piao1 and Alan I. Faden1
1Department of Anesthesiology; and Center for Shock, trauma and Anesthesiology Research (StAR); University of Maryland School of Medicine; Baltimore, MD USA;
2Department of Anatomy and Regenerative Biology; George Washington University Medical School; Washington DC, USA
Key words: spinal cord injury, cyclin-dependent kinases, cell cycle pathway, flavopiridol, oligodendrocytes, myelination,
astrocytes, inflammation, neuron
Abbreviations: CDKIs, cyclin-dependent kinase inhibitors; CDKs, cyclin-activated kinases; pRb, phosphorylated retinoblastoma
protein; SCI, spinal cord injury; BBB, basso, beattie and bresnahan locomotor rating scale; CBS, combination behavioral scores
neuronal apoptosis involves formation of the cyclin D1-CDK4/6
complex, activation of CDK4/6, phosphorylation of Rb, dissocia-
tion of Rb-E2F complex and activation of E2F transcriptional
activity.9 The latter can contribute to increased transcription of
pro-apoptotic molecules such as caspases 3, 9 and 8 and Apaf-1 or
pro-apoptotic Bcl-2 family members.10,11 Aberrant cell cycle acti-
vation not only induces apoptosis of post-mitotic cells (neurons
and mature oligodendrocytes), but also initiates proliferation
and activation of mitotic cells (microglia/macrophages, astro-
cytes, precursor cells) in multiple experimental models, including
SCI,8,12-14 brain injury15-17 and cerebral ischemia.18,19 Thus, iden-
tification of common molecular pathways, including the role of
cell cycle proteins involved in both neuronal death and reactive
gliosis, may help to clarify the pathobiology of CNS injury and
lead to the elucidation of novel therapeutic targets.
CDK inhibitors have been widely studied as cancer thera-
peutics due to their potential role in restoring control of the cell
cycle.20 Flavopiridol is a potent competitive CDK inhibitor, act-
ing on all CDKs. We have reported previously in reference 13
that flavopiridol, when administered centrally by intrathecal
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to 10% of control levels at 1–3 d post-injury and remained at
30–40% of control values during 1–4 weeks (Fig. 1).
Western blot analysis demonstrated that flavopiridol treat-
ment significantly reduced the SCI-induced increase in Rb phos-
phorylation (ser780, Fig. 2A and B) and expression of cyclin D1
(Fig. 2C and D) at 3 d post-injury. Expression levels of CDK4
were also markedly attenuated by flavopiridol at 4 weeks after
SCI (Fig. 2E and F). In contrast, inhibition of p27 after SCI
was reversed by flavopiridol treatment beginning at 7 d (data not
shown) and significantly at 4 weeks post-injury (Fig. 2G–H).
Delayed systemic treatment with flavopiridol improves
recovery of locomotor function and reduces secondary tissue
damage. At day 1 after SCI, all rats had a BBB score of 0 or 1,
indicating nearly complete loss of motor function. Between 1
and 4 weeks, flavopiridol-treated rats showed improved neuro-
logical function compared with vehicle treatment as indicated
by BBB scores (Fig. 3A). By day 7 after injury, flavopiridol-
treated rats had significantly improved BBB scores (7.41 ± 0.61,
n = 11) compared with vehicle-treated animals (4.88 ± 0.34, n
= 13). Significant differences between groups remained through
28 d after injury (11.09 ± 0.60 for flavopiridol vs. 8.5 ± 0.32 for
vehicle, p < 0.05 with two-way ANOVA followed by repeated-
measures). Moreover, 82% of the flavopiridol-treated rats had
achieved a score of 8 or higher by 14 d, as compared with only
23% in the vehicle-treated group.
In addition to the BBB score, neurological functional defi-
cits were estimated using a battery of behavioral tests (the com-
bined behavioral score, CBS) for evaluation of overall hind limb
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sensory-motor deficits.22 By day 1, rats with nearly complete
injury had a score of 100. By day 7 after injury, animals in the
vehicle group had reached 82.9% ± 1.8, while flavopiridol treat-
ment resulted in a significantly greater reduction to 63.3% ± 3.8
(Fig. 3B). This improvement remained through 28 d after injury
(44.8% ± 3.4 for flavopiridol vs. 62.3 ± 1.9 for vehicle, p < 0.05
with two-way ANOVA followed by repeated-measures).
SCI-induced lesion volume/cavity formation was measured
with GFAP/DAB staining at 4 weeks after SCI and analyzed
by unbiased stereological techniques. The average lesion volume
assessed for the vehicle group was 12.98 ± 0.63 mm3, and flavo-
piridol treatment significantly reduced lesion cavity with an aver-
age lesion volume of 7.21 ± 0.64 mm3 (Fig. 3D). This reduction
occurred in both white and gray matter, with an overall decrease
in cavity formation and tissue loss (Fig. 3C).
Flavopiridol treatment increases the number of oligodendro-
cytes and myelinated white matter area at 4 weeks after SCI.
To determine if the observed behavioral improvement may relate
to increased remyelination of spared axons, spinal cord sections
from injured rats perfused at 28 d were stained with erichrome-
cyanine, and WM area was quantified at 1-mm intervals rostral
and caudal to the injury epicenter (Fig. 4). Flavopiridol treatment
significantly increased WM area at the injury epicenter as well as
caudal and rostral to the epicenter (Fig. 4B). Representative erio-
chrome stained sections at 1 mm caudal to the epicenter of each
subject illustrate the differences in myelinated WM area between
vehicle- and flavopiridol-treated animals (Fig. 4A).
To assess whether increased myelination of axons in the fla-
vopiridol-treated animals might relate to increased oligodendro-
cyte number, we quantified total mature oligodendrocyte (CC1+
cells) using the optical fractionator method of unbiased stereol-
ogy. SCI caused a 41% loss of mature CC1+ cells from 5 mm
rostral to 5 mm caudal the lesion site at 4 weeks after injury (Fig.
4C). Treatment with flavopiridol attenuated SCI-induced loss of
CC1+ cells in the preserved tissue. Figure 4D shows representa-
tive images of CC1/DAB staining located in ventromedial white
matter from sham, vehicle- and flavopiridol-treated spinal cord
sections. There was no difference in CC1+ cells between vehi-
cle- and flavopiridol-treated sham groups. There was a positive
correlation (r2 = 0.9079) between reduction of CBS scores and
increased CC1+ cells, due to flavopiridol treatment following SCI
We also studied the expression of myelin basic protein (MBP)
to assess possible treatment-related alterations in myelination in
the injured spinal cord. The 21.5 kDa isomer of MBP was signifi-
cantly upregulated in the flavopiridol-treated group (Fig. 4F). No
apparent differences between the groups were found for the other
MBP isomers (18.5, 17.0 and 14.0 kDa, Fig. 4G).
Flavopiridol reduces post-mitotic cellular apoptosis after
SCI. We have shown that central administration of flavopiridol
beginning 30 min after SCI reduces apoptosis, evidenced by
qualitative assessment of TUNEL and cleaved-caspase 3 stain-
ing.13 To investigate the effects of systemic delayed treatment of
flavopiridol on neuronal and oligodendrocyte cell death after
SCI, 5-mm segments of spinal cord tissue representing the injury
epicenter were assessed at 3 d post-injury for markers of apoptosis
injection 30 min post-injury and continuing for 7 d, significantly
improves motor recovery and reduces lesion volume at 28 d.
In the present study, we examined the effects of injury on cell
cycle expression and whether the delayed systemic administra-
tion of flavopiridol can promote recovery after SCI. Delayed
treatment is a more clinically relevant paradigm and may pro-
vide additional advantages to current clinical options, as methyl-
prednisolone has not proven effective when administrated more
than 8 h after injury.21 To test this hypothesis we investigated the
effects of systemic flavopiridol administration, beginning 24 h
Cell cycle activation is induced after SCI and attenuated by
systemic administration of flavopiridol. To evaluate the effect
of SCI on cell cycle activation, we first systematically examined
cell cycle pathway changes from 1 to 28 d after contusion injury.
Quantitative analysis of western blots showed that Rb was rapidly
phosphorylated by day 1 and subsequently declined after 3 d post-
lesion. The expression of cyclin D1 and CDK4 was highly upreg-
ulated, beginning at day 1 (2- to 1.5-fold) and remaining elevated
for at least 4 weeks after SCI (Fig. 1B). E2F1 expression levels
were rapidly increased by day 1, declined by 3 d and remained
1.5–2.8-fold that of controls from 1–4 weeks post-injury. Levels
of PCNA protein in spinal cord tissue was approximately 4–5-
fold that of controls by 3 d post-injury (Fig. 1B). In contrast,
the endogenous CDK inhibitor p27 was robustly downregulated
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1784 Cell Cycle Volume 11 Issue 9
Figure 1. Cell cycle activation changes over time following spinal cord contusion in rats. (A) Representative immunoblots for cell cycle-related
proteins (pRb, cyclin D1, e2F1, CDK4, pCNA and p27) and the loading control (GApDH). (B) expression levels of cell cycle proteins were normalized by
GApDH, as estimated by optical density measurements and expressed as a percentage of sham spinal cord. Quantitative analysis of western blots
showed that e2F1 and pRb expression levels were rapidly increased by day 1 and declined after 3 d post lesion. protein expression of cyclin D1 and
CDK4 was highly upregulated starting at 1 d (2- to 1.5-fold) and continuing for at least 4 weeks after SCI. the quantity of pCNA protein in spinal cord
tissue was approximately 4-fold that of controls by 3 d and then declined after 1 week post-injury, whereas the endogenous CDK inhibitor p27 was
downregulated. *p < 0.05 compared with sham group. n = 4 rats per time point.
by western blots. As shown in Figure 5A and B, SCI significantly
increased cleavage of fodrin as demonstrated by increased levels
of the 150/145 kDa cleavage product of fodrin. Notably, flavo-
piridol treatment markedly reduced the expression levels of the
cleavage products of fodrin when compared with vehicle-treated
samples (p < 0.05, vs. vehicle).
Neurons, as well as mature oligodendrocytes undergo apop-
tosis following SCI. We have shown increased oligodendrocyte
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with glial scar formation.23 Western blot analysis showed a sig-
nificant reduction of GFAP protein expression at 4 weeks post-
injury in flavopiridol-treated tissues (Fig. 6A and B, p < 0.05
vs. vehicle treated controls). Immunohistochemistry with GFAP
staining (Fig. 6Fb, e and h) confirmed the attenuation of GFAP
immunoreactivity with flavopiridol treatment (Fig. 6Fh).
NG2 proteoglycan and the Ig superfamily adhesion molecule
the close homolog of L1 (CHL1) are highly expressed by hypertro-
phic astrocytes at the glial scar, restricting posttraumatic axonal
growth.24-27 As shown by western blot analysis, NG2 glycopro-
tein and CHL1 levels in spinal cord tissue robustly increased at
4 weeks after our moderate SCI in rats (Fig. 6C). Flavopiridol
treatment significantly attenuated the expression of both NG2
number following flavopiridol treatment (Fig. 4C). Stereological
assessment of surviving neurons was also examined at 4 weeks
after SCI (Fig. 5C). SCI resulted in 50% of neuronal cell loss
(476,506.2 ± 36,060.4 vs. 966,968.5 ± 23,196.0 NeuN+ cells
for vehicle and sham samples, respectively), whereas flavopiridol
significantly improved neuronal survival at 4 weeks after injury
when compared with vehicle-treated samples (Fig. 5C, 586,324.5
± 72,282.2 vs. 476,506.2 ± 36,060.4 NeuN+ cells for flavopiridol-
treated and vehicle-treated samples, respectively). There was no
difference in NeuN+ cells between sham and flavopiridol-treated
Flavopiridol inhibits astrocyte reactivity and microglial acti-
vation. GFAP is an indicator of astrocyte reactivity associated
Figure 2. Systemic administration of flavopiridol attenuates cell cycle activation after SCI. Western blot analysis showed that flavopiridol treatment
significantly reduced the SCI-induced increase in phosphorylation of Rb (ser780, A and B) and expression of cyclin D1 (C and D) at 3 d post-injury.
expression levels of CDK4 were also markedly attenuated by flavopiridol at 4 weeks after SCI (e and F). Inhibition of endogenous CDK inhibitor p27
was significantly rescued by flavopiridol administration at 4 weeks post-injury (G and H). Representative western blots are shown in the left part. Mean
values and SeM of protein expression levels normalized to GApDH are shown in the right part. *p < 0.05 compared with sham group. n = 3–5/group.
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in preserved white matter and gray matter,
were morphologically characterized as oli-
godendrocyte precursor cells (OPC, stars
in Fig. 6G). However, NG2+-OPC cells
peak at 3 and 7 d after SCI.32-34 In agree-
ment with previous studies in references 32,
34–36, we also found that a subset of OX42+
macrophages in the central lesion area are
transiently immunopositive for NG2 after
insult (arrows in Fig. 6I). However, large
numbers of OX42+ cells in the field do not
express NG2 proteoglycan (arrowheads in
Fig. 6I). OX42+ cells have a distinct mor-
phology, whether co-labeled with NG2 or
not.34,36 NG2+/fibronectin+-meninges have
been observed at the section edges. However,
none of NG2-expressing cells inside the spi-
nal cord tissues appear fibronectin-positive
(arrows in Fig. 6J).
Activation of microglia/macrophages peaks at 7 d after SCI
during the early phase of cellular inflammation.37-39 In order to
evaluate the effects of systemic delayed administration of flavo-
piridol on SCI-induced inflammation, we examined activation
of resident microglia/infiltrating macrophages using either west-
ern blot analysis or unbiased stereological assessment of Iba-1+
cells. As shown in Figure 7B, flavopiridol treatment significantly
reduced the SCI-induced upregulation of Iba-1 protein at 7 d
post-injury. Representative immunoblots are shown in left part of
1786 Cell Cycle Volume 11 Issue 9
A. Flavopiridol treatment did not significantly affect the number
of Iba-1+ microglial/macrophages (Fig. 7C, p = 0.122 vs. vehi-
cle). Interestingly, flavopiridol treatment significantly reduced
numbers of Iba-1+ cells in the preserved tissue at 7 d post-injury
(p = 0.028 vs. vehicle) but not in the central lesion area (Fig. 7C,
p = 0.345 vs. vehicle), suggesting inhibition of proliferation of
microglial but no significant effect on infiltration/proliferation of
macrophages. Figure 7D shows lesion boundary between spared
tissue and lesion cavity as indicated by GFAP staining. GFAP+
astrocytes are absent in the lesion cavity after rat contusion SCI,
glycoprotein and CHL1 (p < 0.05, vs. vehi-
cle, Fig. 6D and E). We also performed an
additional immunohistochemistry study to
examine NG2-expressing cell types in our
rat SCI contusion model chronically 4 weeks
after injury. Immunohistochemistry with
NG2 staining (Fig. 6Fa, d and g) confirmed
the attenuation of NG2 immunoreactivity
with flavopiridol treatment (Fig. 6Fg). NG2-
immunostained cells present in the border
zone are double immunostained for GFAP
(arrows in Fig. 6Fd–f and G). Note that
immunostainings for GFAP also detected
NG2-negative cells (arrowheads in Fig. 6G).
Thus, in agreement with prior studies,25,27,28
we show here that a subset of GFAP+ astro-
cytes concentrated at the boundary between
residual spinal cord tissue and the central
lesion are immunopositive for NG2 pro-
teoglycan. In addition, we found using in
vitro cultures that hypertrophic astrocytes
induced by TGFβ1 upregulate expression of
NG2 (unpublished results). The second type
of cells expressing NG2 after chronic injury
were p75+ Schwann cells observed inside the
lesion (arrows in Fig. 6H), consistent with
reports by others.27,29-31 The third type of
NG2+ cells, observed at 4 weeks after injury
Figure 3. Delayed systemic treatment of flavopiridol improves functional recovery and reduces
lesion volume after SCI. Both BBB (A) and CBS (B) behavioral tests showed greater functional
recovery in flavopiridol treated rats. the BBB (0 = paralysis; 21 = normal), which evaluates open
field locomotion, showed a difference in recovery by the first week after moderate SCI. the
CBS (100 = paralysis; 0 = normal), which evaluates overall hind limb sensory-motor deficits,
indicated a significant reduction in functional deficits in the flavopiridol treated group by the
first week after injury. *p < 0.05 vs. vehicle group. n = 11–13/group. (C) Unbiased stereological
assessment of lesion volume at 28 d post-injury was performed on GFAp/DAB stained coronal
sections. Representative images showed lesion cavity at 2 mm caudal to the injury center at
4 weeks after injury. Scale bars = 500 μm. (D) Quantification analysis showed significantly
reduced lesion volume in flavopiridol treated group. *p < 0.05 vs. vehicle group. n = 6–8/group.
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the spared tissue are CNS resident microglia. Expression levels
of Iba-1 and galectin-3 remained high at 28 d post-injury, but
with significantly reduced levels in flavopiridol-treated tissue in
comparison to vehicle-treated tissue (Fig. 7E–G).
but instead, these cells surround the lesion. Circulating macro-
phages infiltrate the lesion and its core, and the area with the
high concentration of macrophages was labeled as lesion area,
indicated in Figure 7C and D. The majority of Iba-1+ cells in
Figure 4. For figure legend, see page 1788.
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1788 Cell Cycle Volume 11 Issue 9
Our results show that delayed systemic administration of the
CDK inhibitor flavopiridol, beginning at 24 h and once daily for
7 d after injury can significantly improve motor function recov-
ery and reduce histopathological changes after SCI. Our data
also include detailed temporal measurements of cell cycle protein
expression (1, 3, 7, 14 and 28 d) after SCI.
Many studies have suggested that cell cycle activation plays
a pathophysiological role in CNS injury.12-17,40 Cyclin D1 eleva-
tion in neurons leads to activation of CDK4/6, which then phos-
phorylates Rb.41 Phosphorylation of Rb increases transcription
To clarify the effect of systemic flavopiridol treatment on
Iba-1+ cells in the central lesion area, where circulating mac-
rophages infiltrate after SCI, complete blood cell counts and
differentials were performed on day 7 after flavopiridol or
vehicle treatment in both injured and laminectomy-only rats.
Flavopiridol administration did not affect the number of periph-
eral circulating white blood cells, including monocytes, lym-
phocytes, neurotrophils, basophils and eosinophils (data not
shown). Overall, these data suggest that systemic administration
of flavoripidol at a dose of 1 mg/kg inhibits microglial activation
without affecting the invasion of inflammatory monocytes into
the lesion site.
Figure 4 (See previous page). Flavopiridol treatment increases number of oligodendrocytes and myelinated area in residual tissue after SCI.
(A and B) eriochrome staining for myelin was performed on spinal cord sections at 4 weeks post-injury to quantify residual white matter. Representa-
tive images at 1 mm caudal to the injury center revealed a central core lesion with spared peripheral white matter [middle part in (A)] and an increased
area of spared white matter in flavopiridol treated cord [bottom part in (A)]. Quantification analysis (B) showed that flavopiridol treated subjects dis-
play significantly more remaining white matter around the lesion area than vehicle treated group at 1–3 mm rostral or 1 mm caudal to the epicenter. n
= 6–8 in vehicle or flavopiridol groups, n = 3 in sham or sham + flavopiridol group. (C and D) Unbiased stereological assessment of oligodendrocytes
loss at 28 d post-injury was performed on CC1/DAB stained coronal sections. treatment with flavopiridol attenuated SCI-induced loss of CC1+ mature
oligodendrocytes in the preserved tissue (C). Representative images (D) of CC1/DAB staining located in both of ventromedial white matter from Sham,
Vehicle- and flavopiridol-treated spinal cord. (e) Linear regression analysis comparing SCI-induced oligodendrocytes loss with CBS scores at 4 weeks
post-SCI. R2 = 0.9079. (F and G) Western blot analysis showed that one of the MBp isoforms (21.5 kDa) was significantly upregulated in the flavopiridol-
treated group at 4 weeks post-injury. No apparent differences between the groups were found for the other MBp isomers (18.5, 17.0 and 14.0 kDa, in
H). n = 4 in vehicle or flavopiridol groups, n = 2 in sham or sham + flavopiridol group. *p < 0.05 vs. vehicle group. Scale bars = 500 μm in (A and D).
Figure 5. Delayed administration of flavopiridol reduces SCI-induced apoptotis and rescues neuronal cell loss. (A and B) Western blot analysis for
markers of apoptotis following SCI. Flavopiridol significantly reduced the SCI-induced increases in the cleavage products of fodrin at 3 d post-injury.
Representative immunoblots are shown in right part of (A). n = 3 in vehicle or flavopiridol groups, n = 2 in sham or sham + flavopiridol group. (C) Unbi-
ased stereological assessment of neuronal loss at 28 d post-injury was performed on NeuN/DAB stained coronal sections. treatment with flavopiridol
attenuated SCI-induced loss of NeuN+ neurons in the preserved gray matter (C). *p < 0.05 vs. vehicle group. n = 6–8 in vehicle or flavopiridol groups,
n = 4 in sham or sham + flavopiridol group.
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Figure 6. For figure legend, see page 1790.
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tides that block cyclin/CDKs activity, either by forming an inac-
tive complex or by acting as a competitive ligand for CDKs. Both
endogenous and exogenous CDKIs can block cell cycle progres-
sion. Flavopiridol, a semi-synthetic flavonoid derived from the
bark of rohitukin,47 inhibits all CDKs, reduces cyclin D1 mRNA
transcription and leads to cell cycle arrest in G1 or at the G2/M
transition.48,49 We have previously reported attenuation of Rb
phosphorylation and expression of cyclin D1 by intrathecal injec-
tion of flavopiridol after SCI.13 The present study confirms simi-
lar results by systemic administration. Moreover, we demonstrate
that flavopiridol treatment reduces CDK4 and cyclin D1 expres-
sion chronically after SCI, further confirming the capacity of
flavopiridol to inhibit cell cycle pathways in this model, and sug-
gesting that persistent cell cycle activation after injury may reflect
a positive feedback loop that can be broken with subacute cell
cycle inhibitor administration. Consistent with this view, expres-
sion of the endogenous inhibitor p27 is restored toward normal
levels in injured spinal cord after flavopiridol treatment.
In our prior study, flavopiridol administered centrally begin-
ning 30 min after SCI significantly improves functional recov-
ery.13 Delayed systemic administration with a therapeutic window
of 24 h post-injury in our current study represents a more clini-
cally relevant treatment paradigm. Using the BBB scale and a
battery of tests of hindlimb reflexes as well as coordinated motor
activity to evaluate different motor abilities, we found significant
overall improvements in flavopiridol-treated rats compared with
control animals. The flavopiridol-treated group demonstrated
approximately 2-point improvement in the BBB score compared
1790 Cell Cycle Volume 11 Issue 9
with the vehicle-treated group, reflecting the ability of rats to
place their paws in plantar positions or sweep hind-limbs in
walking motions. This 2-point improvement is both statistically
significant and functionally meaningful.
Cavity volume assessed by GFAP staining serves as a reli-
able marker for CNS tissue loss in rat contusion SCI model.
Flavopridol treatment significantly reduced lesion volume. This
was accompanied by a significant increase in the area of spared
white matter, an effect that was associated with significantly
improved hind limb function. Increased white matter sparing
resulting from flavopiridol treatment was found both rostral and
caudal to the lesion site, indicating higher levels of myelinated
residual white matter area. Flavopiridol-treated animals had a
significantly higher number of oligodendrocytes than the vehicle
group, and this effect was correlated with significantly improved
CBS scores (r2 = 0.9079). Associated with increased oligoden-
drocyte number was a significant upregulation of the 21.5 kDa
isoform of MBP, which correlates with remyelination in multiple
sclerosis.50 Flavopiridol has been shown to reduce apoptosis of
oligodendrocytes induced by SCI.13 Thus, it is possible that the
increased number of oligodendrocytes observed in the current
study is due to, at least partially, attenuation of apoptosis follow-
ing flavopiridol treatment.
Neuronal apoptosis is often detected in acute injured spinal
cord tissue for days to months after SCI. Apoptosis evidenced
by TUNEL and cleaved-caspase 3 staining increased at 3 d after
SCI, with many of these apoptotic cells being neurons.13 Here we
show elevated levels of 150/145 kDa fodrin cleavage product at
3 d post-injury. Fodrin, also known as spectrin, is a high molecu-
lar weight (240 kDa) cytoskeletal protein that undergoes degra-
dation catalyzed by activated proteases during apoptosis.51,52 The
cleavage of the 150/145 kDa fragment is both calpain-(145 kDa)
and caspase-(150 kDa) mediated.53 Flavopiridol treatment sig-
nificantly reduced products of fodrin at 72 h after injury.
Cell cycle activation induces proliferation in mitotic cells such
as astrocytes and microglia. Administration of cell cycle inhibi-
tors—including flavopiridol, roscovitine and olomoucine—
inhibits microglial and astrocyte proliferation both in vitro and
in vivo.9 Here we confirm similar results by quantitative west-
ern blot analysis and qualitative immunostaining. The NG2
proteoglycan, one of the core proteins of chondroitin sulfate
proteoglycans (CSPGs), are the major axon growth inhibitory
component of the glial scar in SCI.25-27,54,55 CHL1 has been dem-
onstrated as a glial scar component that restricts posttraumatic
axonal growth.24,27 Both of these molecules are highly expressed
of E2F pro-apoptotic targets and promotes progression of the
cell to S phase of the cell cycle in both mitotic and post-mitotic
cells.42-44 Consistent with our previous studies in references 8
and 13, we show here that phosphorylation of Rb and expres-
sion of cyclin D1 increases at 24 h post-injury, followed by eleva-
tion of CDK4 expression at 3 d, indicating general activation of
CDKs. Activation of CDK1 results in phosphorylation of numer-
ous targets in the nucleus and mitochondria, for instance, early
and robust upregulation of the E2F1 transcription factor. We
also show that CDK4, as well as cyclin D1 are persistently ele-
vated during a later phase (1–4 weeks) following injury PCNA is
increased during day 3–7 post-injury, likely reflecting expression
by activated microglia/macrophages. In addition, the endogenous
CDK inhibitor p27, normally highly expressed in neurons and
mature oligodendrocytes11,45,46 is downregulated and remained at
30–40% of control during weeks 1–4.
Cyclin-dependent kinase inhibitors (CDKIs) are small pep-
Figure 6 (See previous page). Flavopiridol decreases astrocytic reactivity after SCI. (A and B) Western blot analysis showed that flavopiridol treatment
significantly reduced the SCI-induced upregulation of GFAp at 4 weeks post-injury. Representative immunoblots are shown in (A). (C–e) Increases of
NG2 and CHL1 expression at 4 weeks after SCI were significantly attenuated by treatment of flavopiridol. Representative immunoblots are shown in
(C). *p < 0.05 vs. vehicle group. n = 4–5 in vehicle or flavopiridol groups, n = 2 in sham or sham + flavopiridol group. (F) Wide-field high-resolution con-
focal images of a complete transversal section of the injured spinal cord (d–f) revealed that NG2 (green) and GFAp (red) immunoreactivity is increased
throughout the cord at 4 weeks after SCI. this was attenuated by treatment with flavopiridol (g–i). Scale bars = 500 μm, (G) NG2-immunostained cells
present in the border zone were double immunostained for GFAp (arrows). Note that immunostainings for GFAp also detected NG2-negative cells
(arrowheads). NG2+-oligodendrocyte precursor cells cells were negative for GFAp (stars). (H) NG2+/p75+ Schwann cells were observed inside the lesion
(arrows). (I) A subset of oX42+ macrophages in the central lesion area were transiently immunopositive for NG2 after insult (arrows). However, large
numbers of oX42+ cells in the field did not express NG2 proteoglycan (arrowheads). (J) None of NG2-expressing cells inside the spinal cord tissues ap-
peared fibronectin positive (arrows). Scale bars for (G–J) = 50 μm.
© 2012 Landes Bioscience.
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www.landesbioscience.com Cell Cycle 1791
Microglial activation includes both proliferation and induc-
tion of specific markers in microglia. In agreement with our
previous findings in references 13 and 40, we detected signifi-
cant reduction of inflammation in the SCI rats treated with fla-
vopiridol, including decreased expression of Iba-1 and galectin 3.
by hypertrophic astrocytes after SCI. The significant reduction of
NG2 and CHL1 expression observed in the present study could
be the consequence of suppression of astrocytic activation by fla-
vopiridol treatment. Thus, the ability of flavopiridol to limit scar
formation may facilitate endogenous restorative potential.
Figure 7. Flavopiridol inhibits the microglial/macrophage activation after SCI. (A) Western blot analysis showed that flavopiridol treatment significant-
ly reduced the SCI-induced upregulation of Iba-1 protein at 7 d post-injury. Representative immunoblots are shown in left part of (A). (B) treatment
of flavopiridol significantly reduced numbers of Iba-1+ microglia/macrophages in the preserved tissue at 7 d post-injury, but not in the central lesion
area. Right image shows lesion boundary between spared tissue and lesion cavity as indicated by GFAp staining. (C) Increases of Iba-1 and galectin 3
protein expression at 4 weeks after SCI were significantly attenuated by treatment of flavopiridol. Representative immunoblots are shown in left part
of (C). *p < 0.05 vs. vehicle group. n = 3–5 in vehicle or flavopiridol groups, n = 2 in sham or sham + flavopiridol group.
© 2012 Landes Bioscience.
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was established (10–14 d after SCI). Sham-injured rats received
a laminectomy without weight drop. The experimental protocols
were approved by the University of Maryland School of Medicine
Animal Care and Use Committee and met all NIH guidelines.
To verify injury, animals were blindly tested for hind limb motor
function on day 1 after SCI. Only rats with a motor score of 1
or lower for both hind limbs (no movement or only occasional,
slight movement of hind limbs) were accepted for the study.
Drug administration. After SCI, rats were assigned to a
treatment group according to a randomized block experimental
design. Flavopiridol was dissolved completely in DMSO. The
concentrated solution was then diluted in sterile saline to a final
concentration of 2.28% DMSO and administered intraperitone-
ally once daily beginning 24 h post-injury and continuing for
7 d. Rats received 1 mg/kg flavopiridol or the equivalent vol-
ume of saline with 2.28% DMSO. This regimen of flavopiridol
administration was adopted due to its proven effectiveness in
experimental cancer models47,57 and based on our pilot studies.
Complete blood cell count and differential assessment were per-
formed by Antech diagnostics on day 7 after systemic flavopiridol
or vehicle administration.
Western blot analysis. Rat spinal cord tissue (5 mm) centered
on the injury site was obtained at 1, 3, 7, 14 and 28 d after injury,
with n = 4 rats per time point with an additional four laminec-
tomy-only controls. The samples were lysed in radioimmunopre-
cipitation assay (RIPA) buffer (Sigma), then homogenized and
sonicated.27 Supernatants were collected after centrifugation at
20,600x g for 20 min at 4°C. Protein concentration was measured
1792 Cell Cycle Volume 11 Issue 9
by the Pierce BCA Protein Assay kit (Thermo Scientific). Equal
amounts of protein were electrophoretically separated on 4 to
12% NuPAGE Novex Bis-Tris gradient gels (Invitrogen) and
then transferred to nitrocellulose membranes (Invitrogen). After
blocking in 5% nonfat milk for 1 h at room temperature, mem-
branes were probed with primary antibodies (Table 1) overnight
at 4°C followed by horseradish peroxidase-conjugated second-
ary antibodies (GE Healthcare) for 1 h at room temperature.
The blots were then visualized using SuperSignal West Dura
Extended Duration Substrate (Thermo Scientific), and quanti-
fied by band densitometry of scanned films using the Gel-Pro
Analyzer program (Media Cybernetics, Inc.).
Functional assessment. Rats were assessed for hind limb
function in open field locomotion on day 1 post-injury and
weekly thereafter for up to 4 weeks using the Basso, Beattie
and Bresnahan (BBB) open field expanded locomotor score.58
Neurological functional deficits were also estimated with a com-
bined behavioral score (CBS),22 which included open field loco-
motion (motor score); withdrawal reflex to hind limb extension,
pain and pressure; foot placing, toe spread and righting reflexes;
maintenance of position on an inclined plane and swimming
Tissue processing and histopathology. At specific times after
injury, rats were anesthetized and transcardially perfused with
saline followed by 4% paraformaldehyde (PFA) in PBS. Spinal
cords were removed and post-fixed in 4% PFA overnight before
cryoprotecting in 30% sucrose at 4°C. A 2.0-cm segment of spi-
nal cord centered at the injury epicenter was sectioned at 20-μm
thickness and thaw-mounted onto Superfrost Plus slides (Fisher
Scientific). Every twentieth section was stained with Eriochrome-
cyanine RC for myelinated white matter (blue), hematoxylin for
cell nuclei and eosin/phloxine (cytoplasm) to visualize cells and
gray matter neuropil. The lesion epicenter was defined as the sec-
tion with the least amount of spared white matter.59
Estimation of lesion volume. Lesion volume was assessed using
the Stereologer 2000 software (Systems Planning and Analysis).
Sections spaced 1 mm apart from 5 mm caudal to 5 mm rostral
the injury epicenter were stained with GFAP and DAB as the
chromogen for lesion volume assessment based on the Cavalieri
method of unbiased stereology with a grid spacing of 200 μm.
Assessment of white matter sparing. Representative slides from
each set were stained with eriochrome cyanine (ECRC) and
residual white matter (WM) area was calculated at the injury
epicenter as well as at points rostral and caudal to the epicenter by
quantifying the total area stained by ECRC.50 Images were taken
at 2.5x magnification and analyzed using NIH ImageJ software.
The threshold level of each 8-bit image was set to display only
ECRC-positive pixels, and total ECRC-positive area was calcu-
lated for each section.
Immunohistochemistry. Immunohistochemistry was per-
formed on spinal cord sections at specified distances rostral and
caudal to the injury epicenter.27 Water-bath antigen demasking
was performed in 0.01 M sodium citrate solution, pH 9.0, for 30
min at 80°C for all antigens.24 Sections were fixed with 10% buff-
ered formalin after pre-warm at room temperature and blocked
for 1 h with 10% normal goat serum in PBS + 0.3% Triton
We also found that flavopiridol significantly reduced numbers of
Iba-1+ cells in the preserved tissue at 7 d post-injury but not in
the central lesion area. Combined with our results from complete
blood cell count and differential assessment, these data suggest
that, although systemic macrophage is not reduced by flavopiri-
dol, microglial activation is decreased.
In conclusion, we provide evidence that cell cycle activation
occurs in both early and later phases after SCI, and that systemic
flavopiridol treatment administered daily for one week beginning
24 h following SCI enhances functional recovery. We attribute
these effects to the ability of flavopiridol to reduce the loss of
neurons and oligodendrocytes through reduction of apoptosis,
to improve the local microenvironment through limitation of
inflammation and to attenuate reactive astrogliosis with increased
Materials and Methods
Spinal cord injury and post-surgical care. Adult male Sprague-
Dawley rats weighing 275–325 g were subjected to an incomplete
contusive SCI.13,56 Rats were deeply anesthetized with sodium pen-
tobarbital (65 mg/kg i.p.), while a laminectomy was performed
at the level of T8 to expose a circle of dura mater. Rats were then
subjected to a moderate spinal cord contusion injury by drop-
ping a 10-g weight from a height of 2.5 cm onto an impounder
positioned on the exposed dura. After SCI surgery, rats were kept
on a heating pad until fully alert, and their bladders were manu-
ally expressed twice a day until a reliable bladder emptying reflex
© 2012 Landes Bioscience.
Do not distribute.
perature for fluorescent-conjugated secondary antibody (Alexa
488-conjugated goat anti-mouse, 1:400, Molecular Probes).
Cell nuclei were labeled with bis-benzimide solution (Hoechst
33258 dye, 5 ug/ml in PBS, Sigma). Finally, slides were washed
and mounted with an anti-fading medium. Immunofluorescence
was visualized by tile scan using a Leica TCS SP5 II Tunable
Spectral Confocal microscope (Leica Microsystems Inc.). The
images were processed using Adobe Photoshop 7.0 software
(Adobe Systems). All immunohistological staining experiments
were performed with appropriate positive control tissue as well as
primary/secondary-only negative controls.
Cell counts by unbiased stereology. CC1+ and NeuN+ cells.
6–8 representative subjects (based on BBB score) from the 28-d
rat study were selected from each treatment group for quanti-
fication of mature oligodendrocytes (CC1+ cells) and neurons
(NeuN+ cells) using unbiased stereology using the optical frac-
tionators method with the aid of StereoInvestigator Software
(MBF Biosciences). Sections spaced 1 mm apart from 5 mm cau-
dal to 5 mm rostral the injury epicenter were included for count-
ing. A sampling grid comprised of 250 μm by 250 μm squares
www.landesbioscience.com Cell Cycle 1793
(150 μm by 150 μm squares in the gray matter for NeuN+ cells)
were laid over each section. Cells were counted in a 50 μm by
50 μm counting frame within each square of the counting grid
with a height of 10 μm and a guard zone of 4 μm from the top
of the section. CC1+ or NeuN+ cells were counted throughout the
Iba-1+ cells. StereoInvestigator Software (MBF Biosciences)
was used to count the number of microglia in both the lesion and
spared tissue using the optical fractionators method of unbiased
stereology. Sections from the 7 d rat study spaced 1 mm apart
from 5 mm caudal to 5 mm rostral the injury epicenter were
stained for Iba-1 and DAB. Contours outlining spared and lesion
tissue were traced onto each section based on the GFAP staining
of adjacent sections. The optical dissector had a size of 50 μm by
50 μm in the x and y axis respectively with a height of 10 μm
and a guard zone of 4 μm from the top of the section. A grid
spacing of 250 μm in the x axis and 250 μm in the y axis was
used. Microglia were counted throughout both spared and lesion
Sampling and statistical analysis. All data are plotted as mean
± SEM, where “n” is the number of individual animals. Behavioral
scores, lesion volume, white matter sparring and unbiased ste-
reological analysis were performed by an investigator blinded
to treatment group. All statistical analyses were conducted by
using the GraphPad Prism Program, Version 3.02 for Windows
(GraphPad Software). BBB and CBS scores were analyzed with
two-way ANOVA and repeated measures. To determine signifi-
cance between two groups, a two-tailed unpaired Student t-test
was used. Statistical significance between multiple groups was
conducted by one-way ANOVA with Tukey or Newman-Keuls
post-hoc test. Regression analysis between behavioral scores and
stereological assessment was performed by linear regression and
correlation coefficient, with r2 determined. The threshold value
for significance was p < 0.05.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conceived and designed the experiments: J.W., B.A.S., A.I.F.
Performed the experiments: J.W., B.A.S., M.D., A.P.G., C.P.
Analyzed the data: J.W., M.D. Wrote the paper: J.W., A.I.F.
Funding the personnel: A.I.
Flavopiridol was provided by the NCI/Aventis. This work was
supported by a National Institute of Health contract NIH-
X-100. After washing, sections were incubated overnight at 4°C
with primary antibodies (Table 1) and then 1 h at room tem-
Table 1. primary antibodies used
AntibodySpeciesManufacturer Catalog no.
ApC (CC1) MouseAbcamAb 16794
CDK4 Rabbit Santa Cruz Bio.Sc-260
CHL1 Goat R and D Systems 2147-CH
Cyclin D1Rabbit Neomarkers RM 9104 S
Fibronectin RabbitAbcam ab 2413
FodrinMouseAffinity Research products FG 6090
Galectin 3 MouseAbcamAb 2785
Mouse StressgenCsa 335
Iba-1 RabbitWAKo 16–20001
MBp (SMI94)Mouse CovanceSMI-94R
NeuNMouseChemicon MAB 377
p27 RabbitSanta Cruz Bio.Sc-528
p75(NGFR)MouseChemicon MAB 365
Rabbit Santa Cruz Bio.Sc-7907
pRb ser780Rabbit Cell Signaling tech.9307S
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