Delayed Paraplegia After Spinal Cord Ischemic Injury
Requires Caspase-3 Activation in Mice
Manabu Kakinohana, MD, PhD; Kotaro Kida, MD, PhD;
Shizuka Minamishima, MD; Dmitriy N. Atochin, MD, PhD; Paul L. Huang, MD, PhD;
Masao Kaneki, MD, PhD; Fumito Ichinose, MD, PhD
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Background and Purpose—Delayed paraplegia remains a devastating complication after ischemic spinal cord injury
associated with aortic surgery and trauma. Although apoptosis has been implicated in the pathogenesis of delayed
neurodegeneration, mechanisms responsible for the delayed paraplegia remain incompletely understood. The aim of this
study was to elucidate the role of apoptosis in delayed motor neuron degeneration after spinal cord ischemia.
Methods—Mice were subjected to spinal cord ischemia induced by occlusion of the aortic arch and left subclavian artery
for 5 or 9 minutes. Motor function in the hind limb was evaluated up to 72 hours after spinal cord ischemia. Histological
studies were performed to detect caspase-3 activation, glial activation, and motor neuron survival in the serial spinal cord
sections. To investigate the impact of caspase-3 activation on spinal cord ischemia, outcome of the spinal cord ischemia
was examined in mice deficient for caspase-3.
Results—In wild-type mice, 9 minutes of spinal cord ischemia caused immediate paraplegia, whereas 5 minutes of
ischemia caused delayed paraplegia. Delayed paraplegia after 5 minutes of spinal cord ischemia was associated with
histological evidence of caspase-3 activation, reactive astrogliosis, microglial activation, and motor neuron loss starting
at approximately 24 to 48 hours after spinal cord ischemia. Caspase-3 deficiency prevented delayed paraplegia and
motor neuron loss after 5 minutes of spinal cord ischemia, but not immediate paraplegia after 9 minutes of ischemia.
Conclusions—The present results suggest that caspase-3 activation is required for delayed paraplegia and motor neuron
degeneration after spinal cord ischemia. (Stroke. 2011;42:2302-2307.)
Key Words: apoptosis 䡲 cleaved caspase-3 䡲 delayed neuronal death 䡲 delayed paraplegia 䡲 spinal cord ischemia
D
elayed paraplegia is a devastating complication of spinal
cord ischemia (SCI), which can occur after thoracic and
abdominal aortic surgery for a variety of aortic pathologies,
including aneurysm and trauma.1 Rates of immediate and
delayed-onset neurological deficits after major thoracic aortic
repairs range between 4% and 11%.1 Of all incidences of
paraplegia (immediate and delayed combined) associated
with aortic surgery, the reported incidence of delayed paraplegia varied from 12% to 73%.1 Although introduction of
several adjunct procedures, including cerebrospinal fluid
drainage, reduced the incidence of immediate paraplegia after
aortic surgery, the incidence of delayed paraplegia has not
changed.2
Although immediate paraplegia is thought to be caused by
an irreversible ischemic neuronal injury in the spinal cord, the
mechanism responsible for delayed paraplegia is incompletely understood.3 Several potential mechanisms responsi-
ble for the development of delayed paraplegia have been
proposed, including delayed apoptotic neuronal death executed by caspase-3 activation.4 – 6 Nonetheless, the role of
motor neuron apoptosis and caspase-3 activation in the
pathogenesis of delayed paraplegia remains controversial;
some studies show the presence of apoptosis in the spinal
cord of animals exhibiting delayed paraplegia, whereas others
do not.7,8 Because the majority of these studies examined the
role of apoptosis using immunohistochemical detection of
caspase-3 and/or DNA fragmentation, no causal relationship
between caspase-3 activation and delayed motor neuron death
has been established to date. Furthermore, elucidation of the
role of apoptosis in delayed paraplegia has been hindered by
the lack of reproducible animal models in which genetic
modification can be exploited to determine the molecular
mechanisms.
To define the molecular mechanisms responsible for the
delayed paraplegia after SCI, we have recently developed a
Received August 19, 2010; final revision received February 8, 2011; accepted February 23, 2011.
From the Department of Anesthesia, Critical Care and Pain Medicine (M.K., K.K., S.M., S.M., M.K., F.I.) and the Cardiovascular Research Center
of the Department of Medicine (D.N.A., P.L.H.), Massachusetts General Hospital, Boston, MA; and the Department of Anesthesiology (M.K.), Faculty
of Medicine, University of the Ryukyus, Okinawa, Japan.
The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.600429/DC1.
Costantino Iadecola, MD, was the Consulting Editor for this paper.
Correspondence to Fumito Ichinose, MD, PhD, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 149 13th Street, 4315,
Charlestown, MA 02129. E-mail [email protected]
© 2011 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.110.600429
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Caspase-3 and Ischemic Spinal Cord Injury
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Figure 2. Percentage of paraplegic mice as a function of the
duration of SCI in minutes. The curves labeled “delayed” (solid
line) and “immediate” (dashed line) show the probabilities of
developing delayed and immediate paraplegia, respectively, calculated by quantal bioasay. The horizontal bars represent SD at
the probability of producing delayed and immediate paraplegia
in 50% of mice (P50d and P50i, respectively). **P⬍0.01 vs
P50d. SCI indicates spinal cord ischemia.
deficit is indicated by a score of 0. Although BMS score ⬍6 (0 to 5)
indicates paraplegia, BMS score ⱖ6 (6 to 9) indicates ability to walk.
Statistics
Figure 1. Time course of Basso Mouse Scale (BMS) in mice
subjected to 9 minutes SCI (n⫽6), 5 minutes SCI (n⫽8), or sham
operation (n⫽3). *P⬍0.05 vs sham and preischemia (pre-SCI).
#P⬍0.05 vs 9 minutes SCI at corresponding time point.
†P⬍0.05 vs 8 hours after 5 minutes SCI. SCI indicates spinal
cord ischemia.
mouse model of delayed paraplegia by modifying a previously reported mouse model of SCI.9 This model is unique in
which mice that are subjected to SCI initially recover from
surgery and anesthesia and exhibit ability to walk for approximately 24 hours. Subsequently, however, all mice develop
delayed paraplegia starting at approximately 30 to 36 hours
after surgery. Using this robust model, we sought to determine the role of apoptotic neuronal death in immediate and
delayed paraplegia after SCI. We report that caspase-3
activation is required for delayed paraplegia but not for
immediate paraplegia in mice.
Materials and Methods
Mouse Model of SCI
After approval by the Massachusetts General Hospital Subcommittee
on Research Animal Care, male wild-type mice (WT, C57BL/6J, 8 to
10 weeks old; Jackson Laboratory, Bar Harbor, ME) and male mice
deficient for caspase-3 backcrossed onto C57BL/6 background ⬎10
generations (caspase-3⫺/⫺, 8 to 10 weeks old)10 were anesthetized
with isoflurane and subjected to SCI according to the method
described by Lang-Lazdunski and colleagues with modifications.9
See http://stroke.ahajournals.org for the details of surgical procedures, measurements of physiological parameters, quantal bioassay,
and histological studies.
Assessment of Motor Neuron Function
Motor function was quantified serially at pre-SCI, 8, 24, 48, and 72
hours after SCI by the Basso Mouse Scale (BMS).11 The maximum
Parametric data were presented as mean⫾SD. Analysis of variance
followed by Bonferroni and Tukey-Kramer tests or Student t test was
used to compare parametric data. The quantal bioassay was based on
logistic analysis.12 The difference between the probability of producing delayed and immediate paraplegia in 50% of mice (P50d and
P50i, respectively) was graphically demonstrated by computer construction of an ischemic duration probability of paraplegia curve for
each group.13 Changes in BMS were analyzed by 2-way repeatedmeasures analysis of variance followed by Tukey test. The number
of viable neurons between experimental groups at individual time
points was compared with Mann-Whitney U test. Probability values
⬍0.05 were considered significant.
Results
SCI Causes Immediate or Delayed Paraplegia
All mice subjected to 9 minutes SCI showed immediate
paraplegia that persisted up to 72 hours after SCI (P⬍0.01
versus sham and pre-SCI; Figure 1). All mice subjected to 5
minutes SCI showed modest and transient motor weakness
(BMS⫽6 to 9) at 30 minutes after reperfusion followed by
gradual recovery over the next 24 to 30 hours (Figure 1). Up
to approximately 8 hours after 5 minutes SCI, motor deficit
manifested as ataxia and partial weakness in place-stepping
reflex with preserved ability to walk (P⬍0.05 versus 9
minutes SCI at 8 and 24 hours after SCI; Figure 1). The motor
function of lower extremities of mice subjected to 5 minutes
SCI gradually worsened starting at approximately 30 hours
after reperfusion and all mice exhibited complete paraplegia
by 48 hours after reperfusion (Figure 1). Long-term outcomes
after SCI (6 weeks) were examined in subgroups of mice that
were subjected to 9 or 5 minutes SCI. Although all mice that
were subjected to 9 minutes SCI died within 7 days, 20% of
mice survived up to 6 weeks after 5 minutes of SCI
(Supplemental Figure I). Once they became paraplegic, no
mice showed any recovery of hind limb motor function until
death or up to 6 weeks after SCI of either duration. Sham-
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Figure 3. A, Representative photomicrographs of the lumber spinal cord sections of mice subjected to 9 or 5 minutes SCI showing
cresyl violet (Nissl staining)-positive neurons. Yellow arrows indicate viable spinal ventral neurons. B, Time-dependent changes in the
number of viable spinal ventral neurons after 9 or 5 minutes SCI. N⫽4 to 7. *P⬍0.01 vs sham-operated mice. ##P⬍0.01 vs 9 minutes
SCI. #P⬍0.05 vs 9 minutes SCI. C, Representative photomicrographs of lumber spinal cord sections of mice showing NeuN-positive
neurons (green) 72 hours after 9 or 5 minutes SCI. Size bars⫽100 ␮m. SCI indicates spinal cord ischemia.
operated mice did not show any neurological deficits
throughout the experiments (Figure 1).
Quantal Bioassay for the Relationship Between the
Duration of SCI and Neurological Function
The incidence of paraplegia (BMS⫽0 to 5) increased as the
duration of SCI increased (Supplemental Table I; Figure 2).
In the current model, ⬍3 minutes of SCI appeared to be well
tolerated that did not cause motor deficit. Although many
mice exhibited ability to walk (BMS⫽6 to 9) up to 24 hours
after SCI after shorter durations of aortic clamping (from 3.5
to 7.5 minutes), the majority of the mice developed complete
paraplegia by 48 hours of reperfusion (delayed paraplegia).
All mice subjected to ⬎8 minutes of SCI showed motor
deficit in the hind limb immediately after recovering from
anesthesia without any recovery up to 72 hours after SCI
(immediate paraplegia). Quantal bioassay analysis revealed
that the P50i and P50d were 6.6⫾0.1 minutes and 3.5⫾0.1
minutes, respectively (Figure 2, P⬍0.01).
Time Course of Neurodegeneration After SCI
In the spinal cord of mice subjected to 9 minutes SCI, neurons
in the ventral horn started to degenerate at 8 hours after SCI.
Extensive neuronal loss appeared to have completed by 24
hours of reperfusion without further changes up to 72 hours
after 9 minutes SCI (Figure 3A–B). In contrast, 5 minutes
SCI did not affect the number and appearance of neurons in
the ventral horn at 8 and 24 hours after reperfusion (Figure
3A–B). However, the ventral horns of mice subjected to 5
minutes SCI exhibited extensive cellular loss with marked
cavitation at 48 and 72 hours after SCI. Although the
structure of the ventral horn appeared to be better preserved
at 72 hours after 9 minutes of SCI than after 5 minutes of SCI,
extensive neuronal loss after SCI of both durations was
confirmed by the absence of NeuN-positive neurons at 72
hours after 9 or 5 minutes of SCI (Figure 3C).
SCI Activates Astrocytes and Microglia
To examine the mechanisms responsible for the delayed
extensive cavitation observed in the spinal cord of mice
Kakinohana et al
Caspase-3 and Ischemic Spinal Cord Injury
2305
after reperfusion (Figure 4A). These results suggest that
delayed inflammatory reaction associated with glial activation may have contributed to the extensive spinal cord
damage and delayed paraplegia after 5 minutes SCI.
Caspase-3 Activation Precedes the Onset of
Delayed Paraplegia
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A small number of cleaved caspase-3-positive neurons was
observed in the ventral horn of the spinal cord of mice at 8
hours after 9 minutes SCI (Figure 4B). However, only a few
cleaved caspase-3-positive neurons were found at 24 and 48
hours after 9 minutes SCI, presumably because of the
extensive necrosis and disappearance of the spinal ventral
neurons. In contrast, the number of ventral horn neurons with
positive cleaved caspase-3 immunoreactivity markedly increased at 24 and 48 hours after 5 minutes SCI (Figure 4).
These results suggest caspase-3 activation precedes the onset
of extensive neuroinflammation, neurodegeneration, and delayed paraplegia after 5 minutes SCI.
Caspase-3 Deficiency Prevents Delayed Paraplegia
in Mice
Figure 4. A, Representative photomicrographs of the lumber
spinal cord sections of mice subjected to 9 or 5 minutes SCI
showing Iba-1 or GFAP-immunoreactive cells at 8, 24, or 48
hours after reperfusion. Size bar⫽100 ␮m for low and 200 ␮m
for high magnification pictures. B, Representative photomicrographs of the ventral horn of the lumber spinal cord sections of
mice subjected to 9 or 5 minutes SCI showing cleaved
caspase-3-immunoreactive neurons. Yellow arrows indicate
cleaved caspase3-immunoreactive neurons. Size bar⫽100 ␮m.
SCI indicates spinal cord ischemia; Iba-1, ionized calcium binding adaptor molecule 1; GFAP, glial fibrillary acidic protein.
subjected to 5 minutes SCI, activation of astrocytes and
microglia was assessed by immunohistochemical staining of
glial fibrillary acidic protein and ionized calcium binding
adaptor molecule 1, respectively. Although only few reactive
astrocytes and activated microglia were observed at 8 and 24
hours after 5 minutes SCI (Figure 4A), the number of reactive
astrocytes and activated microglia markedly increased at 48
hours after 5 minutes SCI (Figure 4A). In contrast, in the
spinal cord of mice subjected to 9 minutes SCI, no reactive
astrocyte or activated microglia were observed up to 48 hours
To determine the role of caspase-3 on the immediate and
delayed onset paraplegia after SCI, we examined outcomes of
SCI in caspase-3⫺/⫺ mice. There was no difference in
physiological parameters, including regional spinal cord
blood flow during SCI between WT and caspase-3⫺/⫺ mice
(Supplemental Tables II, III, and IV). Similar to WT mice,
caspase-3⫺/⫺ mice that were subjected to 9 minutes SCI
exhibit flaccid paraplegia immediately after recovery from
anesthesia (Figure 5). Histopathologic analysis revealed extensive neuronal loss in the spinal cord of caspase-3⫺/⫺ mice
harvested at 72 hours after 9 minutes SCI (data not shown). In
contrast to WT mice that developed delayed paraplegia after
5 minutes SCI, caspase-3⫺/⫺ mice that were subjected to 5
minutes SCI exhibited near normal motor function of lower
extremity up to 72 hours after reperfusion (P⬍0.01 versus
WT 48 and 72 hours after 5 minutes SCI; Figure 5).
Histopathologic analysis of the spinal cord of caspase-3⫺/⫺
mice subjected to 5 minutes SCI revealed normal appearances
of the spinal ventral gray matter at 72 hours after reperfusion
(Figure 6A). There was a statistically significant difference
(P⬍0.01) in the number of Nissl-positive spinal cord neurons
at 72 hours after reperfusion between WT and caspase-3⫺/⫺
mice subjected to 5 minutes SCI (Figure 6B).
Figure 5. Time-dependent changes of
motor function indicated by Basso
Mouse Scale (BMS) after 9 or 5 minutes
SCI in wild type (WT) and caspase-3⫺/⫺
mice. *P⬍0.05 vs pre-SCI. #P⬍0.05 vs 9
minutes SCI at corresponding time point.
†P⬍0.05 vs WT at 48 and 72 hours after
5 minutes SCI. SCI indicates spinal cord
ischemia.
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Figure 6. A, Spinal cord histopathology at the lumbar spinal
cord at 72 hours after 5 minutes SCI in wild type (WT) and
caspase-3⫺/⫺ mice. Size bar⫽100 ␮m. B, The number of viable
spinal ventral neurons at 72 hours after 5 minutes SCI in WT
and caspase-3⫺/⫺ mice (n⫽3 each). *P⬍0.01 vs WT mice. SCI
indicates spinal cord ischemia.
Discussion
In the present study, we found that 9 and 5 minutes of SCI
produced immediate- and delayed-onset paraplegia, respectively, in mice. Once they became paraplegic, no mice
showed any recovery of motor function in this model up to 6
weeks after SCI of either duration. Although degeneration of
the spinal cord neurons and motor deficit became apparent 8
hours after 9 minutes SCI, neurodegeneration and paralysis
did not manifest until 36 to 48 hours after 5 minutes SCI in
WT mice. The delayed neurodegeneration and paraplegia
after 5 minutes SCI were associated with reactive astrogliosis
and microglial activation as well as marked cavitation in the
ventral horn at 48 hours after reperfusion. Caspase-3 activation after 5 minutes SCI precedes the onset of glial activation
and extensive neuronal loss. Of note, although 9 minutes SCI
produced immediate paraplegia in caspase-3⫺/⫺ mice,
caspase-3 deficiency prevented delayed motor neuron loss
and paraplegia after 5 minutes SCI. These observations
suggest that caspase-3 activation is required for delayed
neurodegeneration and paraplegia after spinal cord ischemic
injury.
A variety of animal models including the dog,14 rabbit,4
baboon,15 pig,16 and rat17 have been used to study SCI. The
rabbit model of SCI has been particularly popular because it
can produce delayed and immediate paraplegia by temporarily occluding the infrarenal aorta. However, the arterial blood
supply of the spinal cord in rabbit is almost purely segmental
and different from that in humans.18,19 In rats and mice,9 1
anterior and 2 posterior spinal arteries supply the spinal cord,
which is similar to humans. Moreover, availability of the
variety of genetically altered mouse strains is a clear advantage of mouse models of human diseases compared with rats
and rabbit.
We modified a previously reported mouse model of SCI by
Lang-Lazdunski and colleagues9 by improving the stability of
the model using endotracheal intubation and mechanical
ventilation during anesthesia and surgery. The robustness of
our model enabled us to determine the relationship between
the duration of SCI and neurological outcomes (Figure 2).
The duration-dependent acceleration of the onset of paraplegia after SCI observed in this study is consistent with what
was reported in rabbits.20 Whether or not duration of ischemia
affects outcomes of SCI in humans is incompletely understood. An individual difference in the extent of collateral
blood supply and/or surgical techniques is likely to have a
significant impact on the outcome of SCI. Nonetheless, our
results suggest that severity of ischemia determines the
timing of onset of neurodegeneration and paraplegia after SCI
in mice. Furthermore, the high long-term mortality rate of
paraplegic mice observed in the current study is consistent
with a clinical report that showed a decreased survival rate in
patients who developed delayed paraplegia after thoracoabdominal aortic operations.21
Delayed paraplegia and neurodegeneration after 5 minutes
SCI were associated with marked cavitation in the ventral
horn at 48 and 72 hours after reperfusion. The extensive
cellular loss was associated with reactive astrogliosis and
microglial activation at 48 hours after 5 minutes SCI. Although the role of activated astrocytes and microglia after
SCI is incompletely understood, it is possible that necrosis
and/or apoptosis activates proinflammatory and phagocytotic
activity of glial cells promoting tissue damage.22 The current
observation that caspase-3 activation preceded the glial activation supports this hypothesis. In contrast to 5 minutes SCI,
no cavitation or glial activation was observed after 9 minutes
SCI. Although the reason for the differing impact of 5 and 9
minutes SCI may be multifactorial, it is conceivable that 9
minutes of ischemic insult is too severe for any cells to
survive in the ischemic spinal cord regions.
The role of caspase-3 activation and apoptosis in the
pathogenesis of delayed motor neuron death has been implicated primarily based on immunohistochemical detection of
cleaved caspase-3 in the spinal cord of rabbits that exhibited
delayed paraplegia after SCI.4 – 6 However, the role of
caspase-3 in delayed motor degeneration has been questioned
because of the failure of a conventional caspase inhibitor to
prevent delayed paraplegia7 and lack of caspase-3 activation
and apoptosis in the spinal cord of rabbits.8 These conflicting
results are at least in part due to the limited potency and/or
toxicity of chemical caspase inhibitors and lack of reproducible and standardizable animal models of SCI. In the current
study, using caspase-3⫺/⫺ mice, we demonstrated that
caspase-3 is required for the SCI-induced development of
delayed paraplegia, but not immediate paraplegia, in mice.
Time-dependent changes of the SCI-induced caspase-3 activation in the spinal cord support the critical role of caspase-3
in the pathogenesis of delayed paraplegia (see Figure 4).
Although the precise mechanism whereby caspase-3 deficiency prevents SCI-induced delayed paraplegia remains to
be determined, our results suggest that inhibition of apoptotic
cell death and glial activation in caspase-3⫺/⫺ mice contributed to the preserved spinal cord neurons after 5 minutes of
Kakinohana et al
SCI. To elucidate the role of caspases in the pathogenesis of
delayed paraplegia, further studies using genetically altered
mouse models and new-generation caspase inhibitors are
warranted.
Conclusions
The current study demonstrates that SCI in mice can cause
immediate or delayed-onset motor neuron degeneration and
paraplegia depending on the duration of ischemia. Our results
also provide definitive evidence that caspase-3 activation is
required for the development of delayed paraplegia after 5
minutes of SCI in mice. These observations have important
clinical implications suggesting the preventive effects of
caspase-3 inhibition in SCI induced by surgery or trauma.
Furthermore, the unique mouse model of delayed paraplegia
described here provides an important experimental platform
to further examine the molecular mechanisms of delayed
motor neuron degeneration.
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Sources of Funding
This work was supported by grant-in-aid for scientific research from
the Ministry of Education of Japan (B) 22390299 to M.K. and
National Institutes of Health grant R01HL101930 to F.I.
Disclosures
None.
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Delayed Paraplegia After Spinal Cord Ischemic Injury Requires Caspase-3 Activation in
Mice
Manabu Kakinohana, Kotaro Kida, Shizuka Minamishima, Dmitriy N. Atochin, Paul L. Huang,
Masao Kaneki and Fumito Ichinose
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Stroke. 2011;42:2302-2307; originally published online June 23, 2011;
doi: 10.1161/STROKEAHA.110.600429
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Copyright © 2011 American Heart Association, Inc. All rights reserved.
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SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 1
SUPPLEMENTAL MATERIAL
Delayed paraplegia after spinal cord Ischemic injury requires
caspase-3 activation in mice
Manabu Kakinohana, MD, PhD, Kotaro Kida, MD, PhD, Shizuka Minamishima, MD,
Dmitriy N. Atochin, PhD, Paul L. Huang, MD, PhD, Masao Kaneki MD, PhD., Fumito
Ichinose, MD, PhD
From the Department of Anesthesia, Critical care and Pain medicine and the
Cardiovascular Research Center of the Department of Medicine Massachusetts General
Hospital, Boston, MA and Department of Anesthesiology, Faculty of Medicine,
University of the Ryukyus, Okinawa, JAPAN
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 2
Supplemental Methods
Mouse model of spinal cord ischemia
The operative procedures to produce transient SCI in mice were performed
according to a method described by Lang-Lazdunski and colleagues1 with some
modifications. After the animals were weighed, anesthesia was induced by 5 %
isoflurane, and trachea was intubated by 20 G custom made catheter for mechanical
ventilation. Mice were positioned in the spine position and anesthesia was maintained
by 1.5% to 2% isoflurane with 100% oxygen. Paravertebral muscle temperature was
measured by a thermocouple placed at the level of L1-L3 and maintained at 37.5 ±
0.2°C during surgery using a heating pad. Subsequently, PE-10 catheter was inserted
into the left femoral artery to monitor distal arterial pressure. A ventral midline
cervicothoracic incision was made, submaxillary glands were retracted, and the chest
wall was incised from the apex of the manubrium caudal along the left sternal border, to
the second rib. The thymus was retracted superiorly, and the aortic arch was gently
isolated between the brachiocephalic artery and the left subclavian artery (LSA),
avoiding the vagus nerve and the left recurrent laryngeal nerve. Heparin (1000 IU/kg)
was injected via the left femoral arterial catheter. Then, under direct vision, the first clip
was placed on the aortic arch between the left common carotid artery and the LSA, and
then the second clip was placed on the origin of the LSA (within 30 seconds). The
completeness of the occlusion was ascertained by an immediate and sustained loss of
any detectable pulse pressure in the femoral artery pressure tracing. After ischemia, the
clips were removed, and the chest was closed in layers. Protamine sulfate (1 mg) was
then administered subcutaneously. At 10 minutes of reperfusion, the arterial catheter
was removed, incisions were closed, and animals were allowed to recover from
anesthesia. In sham-operated mice, all surgical procedures were performed as
described, but no clips were placed. All mice were placed in a cage kept at 30 - 31°C for
the following 2 hours.
These modifications enabled us to enhance the completeness of the surgical
isolation and clamping of aortic arch that resulted in a highly reproducible reduction of
rSCBF down to less than 10 % of baseline compared to ~30% in the Lang-Lazdunski
study.1 While 11 min of aortic occlusion resulted in immediate paraplegia in ~80% of
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 3
mice in the Lang-Lazdunski study,1 9 min of SCI was sufficient to produce immediate
paraplegia in 100 % of mice at 72h after reperfusion in the current study with minimum
operative mortality.
Measurements of physiological parameters
In addition to paravertebral muscle temperature, rectal temperature was also
monitored and recorded periodically (before ischemia, at the end of ischemia, and 10
min of reperfusion) as core temperature during surgery. Blood gas analysis was
performed before aortic occlusion and at 10 min of reperfusion. Arterial blood gases and
pH were measured before ischemia and 10 minutes of reperfusion in 50µL samples
obtained from the left femoral arterial catheter by a blood gas /pH analyzer (IRMAs
TRUPOINTTM, ITC. USA).
We used a qualitative real-time measure of regional spinal cord blood flow
(rSCBF) by laser-Doppler flowmetry (PF2B, Perimed) with a 0.8-mm fiberoptic
extension. As described by Lang-Lazdunski et al.,1 the probe was affixed
perpendicularly as much as possible on the intervertebral ligament surface between
vertebra L1 and L2 through a limited skin incision.
Quantal bioassay for the relationship between the duration of SCI and
neurological function
For the quantal bioassay of the relationship between the duration of SCI and neurologic
function2,3, the duration of SCI was selected to span all grades of neurologic function
ranging from walking (BMS = 6 - 9) to paraplegia or paraparesis (BMS = 0 - 5). Based
on our pilot experiments, the duration of SCI for individual animals was varied from 1
min up to 10 min. The P50i and P50d represent the duration of ischemia (in minutes)
associated with 50% probability of immediate and delayed paraplegia, respectively. The
onset of neurologic deficit (BMS = 0 - 5) was considered immediate if it was present at
the initial examination (2 hrs after reperfusion) and delayed if the deficit occurred after a
period during which mice exhibited the ability to walk (BMS = 6 - 9).
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 4
Histological studies
After SCI, lumbar enlargement of the spinal cord was perfusion fixed and embedded in
paraffin. Spinal cord embedded in paraffin was sectioned to 5 μm thickness and 50
sections of each spinal cord were divided on ten slide glasses (five sections each on
one slide glass). For each histopathological analysis described below, one slide glass of
each spinal cord was chosen randomly by the investigator (KK) without the knowledge
of the identity of the sample.
Detection and quantification of viable neurons with Nissl staining—Viability of neurons in
paraffin-embedded spinal cord sections obtained from mice subjected to sham
operation or 9 or 5 min of SCI were evaluated with Nissl staining.4 Cells that contained
Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were
considered to be viable. For quantitative analysis, the number of Nissle-positive neuron
in the spinal ventral horn was counted in a randomly-chosen section under high-power
magnification (200x) by an investigator (KK) blinded as to the identity of mice. N=4 for
Sham, 8, 24, and 48h after 9 or 5 min SCI. N=6 for 72h after 5 min SCI. N=7 for 72h
after 9 min SCI.
Immunohistochemical detection of glial activation and viable neurons—Sections were
incubated for 1h in blocking solution and incubated overnight at 4°C with primary
antibodies against markers of activated astrocytes (glial fibrillary acidic protein, GFAP,
1:500, Dako, Carpinteria, CA) or activated microglia (ionized calcium binding adaptor
molecule 1, Iba-1, 1:500, Wako Chemicals USA, Richmond, VA). After rinsing, sections
were incubated with secondary antibodies for 1h: Rhodamine RedTX-conjugated goat
anti-rabbit antibody (for GFAP, 1:200, Jackson ImmunoReasearch, West Grove, PA) or
Alexa Fluor 488 goat anti-rabbit antibody (for Iba-1, 1:200, Invitrogen, Carlsbad, CA).
Viable neurons were detected by mouse anti-neuronal nuclei (NeuN) conjugated to
Alexa Fluor 488 (1:100, Millipore, Billerica, MA). The fluorescence images were
captured using appropriate filters with a fluorescence microscope (Nikon ECLIPSE TE2000-S).
Detection of cleaved caspase 3—Activation of caspase-3 was assessed by
immunohistochemistry in paraffin-embedded spinal cord sections using a rabbit
polyclonal antibody against cleaved caspase-3 (1:50, Cell Signaling) according to the
protocol recommended by the manufacturer.
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 5
Supplemental Tables
Table S1. Comparison of the Effects of Spinal Cord Ischemic Time on Neurological
Outcome
Duration of SCI
(min)
1
2
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
9
10
Total number of
mice
3
4
5
5
5
5
7
7
8
10
8
6
6
6
5
Number of mice exhibiting
Normal motor
function
3
4
5
2
1
0
0
0
0
0
0
0
0
0
0
Delayed
paraplegia
0
0
0
3
4
5
7
7
6
6
2
1
0
0
0
Immediate
paraplegia
0
0
0
0
0
0
0
0
2
4
6
5
6
6
5
Table S2. Physiological variables –Immediate paraplegiaIschemic Time (min)
Femoral artery pressure (mmHg)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Paravertebral muscle temperature (ºC)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Rectal temperature (ºC)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Wild-type
9
Caspase-3-/9
79±5
8±1
75±19
82±6
8±1
87±26
37.4±0.1
37.4±0.1
37.4±0.1
37.4±0.1
37.4±0.1
37.5±0.2
36.6±0.3
36.8±0.4
36.9±0.2
36.9±0.3
36.3±0.5
36.8±0.2
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 6
Table S3. Physiological variables –Delayed paraplegiaIschemic Time (min)
Femoral artery pressure (mmHg)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Paravertebral muscle temperature (ºC)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Rectal temperature (ºC)
Pre-ischemia
Intra-ischemia
10 min of reperfusion
Wild-type
5
Caspase-3-/5
83±7
7±1
84±9
79±6
5±1
87±9
37.4±0.0
37.4±0.1
37.4±0.1
37.6±0.1
37.5±0.0
37.3±0.1
36.6±0.3
37.4±0.4
36.9±0.3
37.1±0.4
37.5±0.3
36.9±0.2
Table S4. Regional spinal cord blood flow in Wild-type and Caspase-3-/- mice
Wild-type
rSCBP (%)
Pre-ischemia
100
Intra-ischemia
8±4
10 min of reperfusion
91±14
rSCBF : regional spinal cord blood flow
Caspase-3-/100
7±3
101±13
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 7
Percent survival
Figure S1.
100
5 min SCI
9 min SCI
80
60
40
20
0
0
7
14
21
28
35
42
Days after SCI
Figure S1. Long-term survival rates of mice subjected to 5 or 9 min of spinal cord
ischemia (SCI). N=10 in each group.
SUPPLEMENTAL MATERIAL. Kakinohana et. al. STROKE/2010/600429 Page 8
Supplemental Reference List
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Dietrich WD. Spinal cord ischemia. Development of a model in the mouse. Stroke.
2000;31:208-213.
2. Zivin JA, Waud DR. Quantal bioassay and stroke. Stroke. 1992;23:767-773.
3. Zivin JA, DeGirolami U, Hurwitz EL. Spectrum of Neurological Deficits in
Experimental CNS Ischemia: A Quantitative Study. Arch Neurol. 1982;39:408-412.
4. Vogel P, Putten H, Popp E, Krumnikl JJ, Teschendorf P, Galmbacher R, Kisielow
M, Wiessner C, Schmitz A, Tomaselli KJ, Schmitz B, Martin E, Bottiger BW.
Improved resuscitation after cardiac arrest in rats expressing the baculovirus
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