Human Hepatocyte Growth Factor Promotes Functional
Recovery in Primates after Spinal Cord Injury
Kazuya Kitamura1,2,3, Kanehiro Fujiyoshi1, Jun-ichi Yamane1, Fumika Toyota4, Keigo Hikishima2,4,
Tatsuji Nomura4, Hiroshi Funakoshi5, Toshikazu Nakamura6, Masashi Aoki7, Yoshiaki Toyama1,
Hideyuki Okano2*, Masaya Nakamura1*
1 Department of Orthopedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 2 Department of Physiology, Keio University School of Medicine,
Shinjuku-ku, Tokyo, Japan, 3 Department of Orthopedic Surgery, Hiratsuka City Hospital, Hiratsuka, Kanagawa, Japan, 4 Central Institute for Experimental Animals,
Miyamae-ku, Kawasaki, Kanagawa Japan, 5 Center for Advanced Research and Education, Asahikawa Medical University, Midorigaoka, Asahikawa, Japan, 6 Kringle Pharma
Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, Osaka, Japan, 7 Department of Neurology,
Tohoku University School of Medicine, Sendai, Miyagi, Japan

Abstract
Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area
damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which isa potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently
highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing
exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area ofdamaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects ofadministering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to humantreatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinanthuman HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on
manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved
the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging
showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal
outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment
did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-humanprimates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in nonhuman primates after contusive cervical SCI.
Citation: Kitamura K, Fujiyoshi K, Yamane J-i, Toyota F, Hikishima K, et al. (2011) Human Hepatocyte Growth Factor Promotes Functional Recovery in Primates
after Spinal Cord Injury. PLoS ONE 6(11): e27706. doi:10.1371/journal.pone.0027706
Editor: Cesario V. Borlongan, University of South Florida, United States of America
Received June 30, 2011; Accepted October 23, 2011; Published November 29, 2011
Copyright: ß 2011 Kitamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from Grants-in-Aid for Scientific Research from JSPS and the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT), the Project for Realization of Regenerative Medicine and Support for the Core Institutes for iPS Cell Research from MEXT, and by agrant-in-aid for the Global COE program from MEXT to Keio University. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: masa@a8.keio.jp (MN); hidokano@a2.keio.jp (HO)treatments to improve angiogenesis after SCI have shown arelationship between blood flow and functional recovery [11,12].
Hepatocyte growth factor (HGF) was first identified as a potent
mitogen for mature hepatocytes [13,14] and a natural ligand forthe c-Met proto-oncogene product [15]. Recent studies haverevealed that HGF acts as a neurotrophic factor for a variety ofneuron types [16,17,18,19,20], and that administering HGF
enhances angiogenesis, improves microcirculation, inhibits destruction of the blood-brain barrier [21], and exerts a neuroprotective effect after cerebral ischemia [22,23] and in the transgenic
amyotrophic lateral sclerosis (ALS) rat model [24]. We previously
reported that introducing exogenous HGF into the spinal cord
significantly reduces the damaged area and promotes functional
recovery in adult rats [25]. However, this strategy, which involved
injecting a herpes simplex virus-1 vector into the spinal cord prior
to SCI, can never be applied to clinical treatment. Furthermore,rodent SCI models are limited in their ability to ensure the efficacy

Introduction
Spinal cord injury (SCI) is followed by secondary degeneration,
which is characterized by progressive tissue necrosis. Manyexperimental interventions have focused on this posttraumatic
inflammatory process, using neurotrophic factors to reduce thedamaged area and to promote axonal regeneration through thelesion epicenter. Neurotrophins such as nerve growth factor (NGF)[1,2], brain-derived neurotrophic factor (BDNF) [3], neurotrophin-3 (NT-3) [4,5], and glial cell line-derived neurotrophic factor(GDNF) [6,7] have been reported to enhance axonal growth in the
injured spinal cord; some of these studies also showed thatneurotrophins promoted behavioral recovery after SCI [3,7]. Bothneurotrophic support and angiogenesis are critical to the endogenous regenerative response to trauma after SCI [8,9]. The initialdamage to local blood vessels is decisive for the progression ofdestructive events during secondary degeneration [10], and strategic
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rhHGF for SCI in Non-Human Primates

Figure 1. Retrograde labeling of motoneurons in the intact spinal cord by CTB injection. The motoneurons innervating the wrist andfinger flexors and extensors were mainly distributed in lamina IX of the C4–C7 segments and the C6-Th1 segments, respectively. Almost all of these
motoneurons were located at sites caudal to the lesion epicenter at the C5 level. Scale bar, 500 mm.
doi:10.1371/journal.pone.0027706.g001

calmodulin-dependent kinase IIa (CaMK IIa) antibody [29]. We
previously found that CaMK IIa-immunoreactivity (IR) labels the
CST in the marmoset spinal cord [28,30,31], although its specificity
remains to be demonstrated. In the present study, we made thefollowing observations. First, in the intact spinal cord, CaMK IIaIR was detected in the lateral column of white matter, the dorsal
horn, and the intermediate zone (IMZ) of the gray matter (Fig. 2A),
suggesting that the CST in the common marmoset is located inlateral columns, unlike in rodents, in which the CST projects mainly
to dorsal horn neurons and premotor spinal circuits [32]. Second,
we did not detect CaMK IIa-IR in either the ventral white mattercolumn or the ventral gray matter horn, suggesting that, as inrodents, the proportion of the ventral CST extending into the whitematter is small, and that the CST does not project directly tocervical motoneurons in the ventral horns.
Whereas CaMK II a-IR in the lateral column was obviously
reduced in segments caudal to the lesion site in the PBS groupcompared with the intact spinal cord control, the CaMK II a-IR
was well maintained, even caudal to the lesion site, in the rhHGF
group (Fig. 2B arrowheads). Stereological quantification of the
CaMK II a-positive area in the lateral column showed significant
differences between the two groups at the segments caudal to thelesion site (Fig. 2D). CaMK II a-IR was detected in the IMZ(laminae VI–VII) cell bodies, which could include segmental
interneurons (sINs) and propriospinal neurons (PNs). CaMK II apositive neurons in the IMZ at C5 had disappeared at 12 weeks
after SCI in both groups (Fig. 2B and C). The rhHGF group had
obviously greater numbers of these neurons around the lesion
epicenter, as compared with the PBS group (Fig. 2C). Quantitative
analysis of these neurons showed significant differences between
the two groups at the C4 and C6-Th1 segments (Fig. 2D). Notethat in some segments, the number of neurons was greater in therhHGF and PBS groups than in the intact spinal cord (Fig. 2D).
To examine rhHGF’s tissue-sparing effects, we stained axial
sections with Luxol fast blue (LFB) 12 weeks after SCI. There wasa significant rim of spared myelinated white matter, even at thelesion epicenter, in the rhHGF group (Fig. 3A). Stereologicalquantitative analysis of the LFB-positive myelinated areas revealed
significant differences around the lesion epicenter between the twogroups (Fig. 3B). Significant differences were observed within one
vertebral segment from the lesion site at the C5 level.

and safety of treatments for humans. It is critical to examine potential
treatments in non-human primates before proceeding to clinical trials.
In this study, a total of 400 mg of recombinant human HGF(rhHGF) was infused intrathecally for four weeks immediately afterC5-level contusive SCI in adult common marmosets, as a preclinical
trial. Contusive injury is considered the most relevant to human SCI
when evaluating therapies and predicting their translation to humantreatment. However, to assess hand dexterity recovery, we needed
an open field rating scale for a primate cervical SCI model thatwould be comparable to the BBB scale in rodents [26]. Therefore,
we established an original open field rating scale to evaluate hand
neurological performance after contusive cervical SCI. We foundthat intrathecal rhHGF infusion significantly reduced the areas ofdamaged tissue in the spinal cord and promoted functional
recovery, consistent with our previous study using rats. This is thefirst study to report the efficacy and safety of rhHGF for SCI, and topresent a novel scale for assessing neurological motor performanceafter contusive SCI in non-human primates.

Results
Distribution of the spinal motoneurons regulating wrist
and finger motion in common marmosets
To precisely evaluate motor functions in the marmoset SCI model,
we focused on the wrist and finger extension movements, because
these motions are the most impaired in incomplete cervical SCI [27].
We first investigated the distribution of spinal motoneurons thatinnervate the hand muscles in marmoset forelimbs, by injecting
cholera toxin B subunit (CTB) into the forearm flexor and extensor
muscles. The wrist and finger extensor motoneurons were mainly
located in lamina IX of the cervical (C)4-C7 segments, and wrist
flexor motoneurons were in lamina IX of the C6-thoracic (Th)1
segments (Fig. 1). Based on these findings, we quantified the numberof ChAT-positive motoneurons at these segments in marmosets
treated with rhHGF or with sterile phosphate-buffered saline (PBS)
after SCI. Twelve weeks after injury, the ChAT-positive motoneurons around the lesion epicenter had almost disappeared, consistent
with our previous report [28], and we did not detect any significant
differences in the number of surviving C2-Th1 ChAT-positive
motoneurons between the two groups (data not shown).

rhHGF significantly preserved the corticospinal tract
pathway and LFB-positive myelinated areas

In vivo MRI and DTT findings reflected the histologicalseverity of the spinal cord injury

We next investigated the pattern of the corticospinal tract (CST)
pathway and its terminations in common marmosets. Axial sectionsof intact and injured spinal cords were immunostained with an antiPLoS ONE | www.plosone.org

In vivo MRI and diffusion tensor tractography (DTT) [30] wereconducted at multiple time points (1, 3, and 12 weeks after SCI) in2

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rhHGF for SCI in Non-Human Primates

Figure 2. CaMK II a-immunoreactivity in the marmoset spinal cord. CaMK II a-immunoreactivity (IR) in the lateral column of the white matterand in the intermediate zone (IMZ) of the gray matter in an intact spinal cord (A) and injured spinal cord 12 weeks after SCI (B, C). (A) In the intact
spinal cord, CaMK II a-IR was detected in the lateral column, dorsal horn and intermediate zone of the gray matter. No CaMK II a-IR was detected in
the ventral horn of the gray matter. Scale bar, 1 mm. (B) CaMK II a-IR in the lateral column, suggesting the corticospinal tract (CST) pathway, wassignificantly preserved even at sites caudal to the lesion epicenter (arrowheads) in the rhHGF group compared with the PBS group. Scale bar, 1 mm.
(C) CaMK II a-IR was detected in cell bodies at the IMZ (laminae VI–VII), suggested to be segmental interneurons and propriospinal neurons. There
were significantly more IMZ CaMK II a-positive neurons at the C4 and C6-Th1 segments in the rhHGF group than in the PBS group. IMZ CaMK2-apositive neurons were absent from the C5 segment in both groups. Scale bar, 500 mm. (D) Quantitative analyses of the CaMK II a-IR showedsignificant differences between the two groups. *P,0.05, **P,0.01. (n = 6 for the rhHGF group; n = 5 for the PBS group; n = 1 for the intact spinalcord).
doi:10.1371/journal.pone.0027706.g002

recovery beginning 3 weeks after SCI; until that point there were
no obvious differences in the MRIs between the two animals(Fig. 4A and B), corresponding with the time of functional
recovery as assessed by our original open field rating scale. The
open field scores for the upper limbs of the rhHGF-treated animal
and the vehicle control animal were, respectively, 3 and 6 at 3
weeks, and 14.5 and 6.5 at 12 weeks after SCI.
In vivo diffusion tensor analyses were performed at the same time
points, and no obvious differences were observed between the twogroups until 3 weeks after SCI (data not shown). At 12 weeks after
SCI, DTT showed a larger number of spinal tract fibers aroundthe lesion epicenter in the rhHGF group than in the PBS group,
especially in the ventral white matter, which may have been a
precise reflection of the spared rim of myelinated white matter
detected by LFB staining and axial T2WIs (Fig. 5).

four animals, two from each group that underwent SCI, toinvestigate in vivo images that might reflect the pathologicalchanges after SCI. One animal from each group was subjected to
MRI and DTT analysis on the same day, and the recovery of
motor function was assessed over time using our open field rating
scale. Sagittal T2-weighted images (T2WIs) from each individual
showed a diffusely expanded high-intensity area around the lesion
epicenter 1 week after SCI, which was gradually compacted
thereafter with the low-intensity area inside (Fig. 4A). At 12 weeks
after SCI, the area with abnormal intensity (low-intensity areasurrounded by high-intensity area), suggesting a region ofdamaged parenchyma, was markedly reduced in the rhHGFtreated animal compared with the vehicle control animal (Fig. 4A).
The axial T2WIs also showed marked reduction in the area withabnormal-intensity area. Note that the ventral and dorsal parts of
the white matter in the rhHGF-treated animal showed normal
intensity (Fig. 4B arrowheads) even at the lesion epicenter, whereas
the abnormal T2-high intensity area in both the gray and whitematter was greatly expanded in the vehicle control animal. The
rhHGF-treated animal showed remarkable motor function
PLoS ONE | www.plosone.org

rhHGF did not give rise to abnormal CGRP-positive fiber
outgrowth
Although allodynia-like withdrawal responses to mechanicalstimulation of the four extremities were not observed after SCI in
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rhHGF for SCI in Non-Human Primates

Figure 3. Significant reduction in the size of damaged parenchyma in the rhHGF group. Quantification of the LFB-positive myelinatedarea 12 weeks after SCI (A) showed a significant difference between the two groups around the lesion epicenter (B). Scale bar, 2 mm. *P,0.05,**P,0.01. (n = 6 for the rhHGF group; n = 5 for the PBS group).
doi:10.1371/journal.pone.0027706.g003

fiber length in both groups compared to the intact spinal cord, but
did not show significant differences between the two groups
rostrocaudal to the lesion epicenter at any site examined (Fig. 6B),
suggesting that rhHGF treatment did not give rise to the abnormal
sprouting of CGRP-positive nerve fibers.

either group, we quantified the calcitonin gene-related peptide(CGRP)-immunoreactive sensory axons in the Rexed lamina III
12 weeks after SCI to determine whether rhHGF treatment
induced histological changes that could cause allodynia. Dense
CGRP-positive innervation of lamina I and the outer part oflamina II was observed before and after SCI, and abnormal
innervations of lamina III were detected after SCI in both groups(Fig. 6A). Stereological quantification of the total length of CGRPpositive fibers in lamina III revealed increased CGRP-positive

Motor function recovery over time, original scale
development, and category definitions
All animals showed severe quadriplegia 1 day after injury; they
lay on the floor in a prone position, with little limb movement, andcould not roll over or move forward by themselves. After C5-level
SCI, irrespective of therapeutic intervention, the animals showed
progressive recovery of motor function as assessed in three maincategories: upper limbs, lower limbs, and trunk stability (Table 1).
This study’s injury model, cervical SCI, requires a detailed analysis
of upper limb function to precisely evaluate motor function
recovery. Thus, the upper limb category was divided into threesubdivisions: weight bearing, reach and grip performance, andsomatosensory function (Table 1). Because there were various
combinations of improvement in multiple subdivisions andattributes, we adopted a point-addition scoring system in thisoriginal open field rating scale, unlike the system used in the BBB
scale [26].
Weight bearing. The early phase of recovery wascharacterized initially by the animals’ movement and placing of
weight on the lower limbs. As the animals recovered, they showedslight to extensive movements of the three lower limb joints, andbegan to lift their upper body from the floor by bearing weight on
the upper limbs in stance only. They also began to move forward
by placing weight on the lower limbs, dragging the anterior cheston the floor without other observable movements, and placing
weight on the upper limbs with the hands below shoulder level.
The next phase of recovery was characterized by lifting the upper

Figure 4. Representative MR images around the lesion
epicenter after SCI, and correlation with functional recovery.
(A) S Sagittal T2-weighted images around the lesion epicenter 1, 3, and12 weeks after SCI. A marked reduction in the T2 low-signal-intensity
area surrounded by a T2 high-signal-intensity area, suggesting cavity
and glial scar formations, was detected in the rhHGF-treated animalcompared with the vehicle control animal 12 weeks after SCI, eventhough there were no obvious differences between them up to 3 weeks
after SCI. (B) Axial MR images around the lesion epicenter 12 weeks
after SCI. The damaged T2 high-signal-intensity areas (arrowheads)
were obviously reduced in the rhHGF-treated animal.
doi:10.1371/journal.pone.0027706.g004

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rhHGF for SCI in Non-Human Primates

Figure 6. Quantitative analysis of CGRP-positive fibers at thedorsal horns. (A) Abnormal sprouting into lamina III at the dorsal
horns was detected in both groups 12 weeks after SCI. Scale bar,
200 mm upper, 100 mm lower. (B) Quantitative analysis of the total
length of these abnormal fibers showed no significant differences
between the two groups in any segment examined (n = 6 for the rhHGF
group; n = 5 for the PBS group; n = 1 for intact spinal cord).
doi:10.1371/journal.pone.0027706.g006

Figure 5. Diffusion tensor tractography (DTT) showing spinal
fibers around the lesion epicenter, correlating with thehistology and MR images. The region of interest (ROI) was placedin the upper cervical spinal cord, and full-width DTT of the spinal cord
was traced in the caudal direction. Spinal fibers revealed by DTT in theventral white matter (arrowheads) 12 weeks after SCI were correlatedwith the spared rim of LFB-positive myelinated area and the intact
signal-intensity area in the axial T2-weighted images. Scale bar, 2 mm.
doi:10.1371/journal.pone.0027706.g005

to head height, and to reach and grasp a pen perpendicular to thebody plane, with incomplete extension of the fingers and wrist
joint. Many animals did not recover sufficiently to grasp a pen
parallel to the body plane, even at head height (Fig. 7A), because it
required full forearm pronation. They were also unable to elevatetheir hands to 3 cm above head height or to grasp pens in both
directions, as this required active full shoulder joint flexion and full
finger and wrist extension (Fig. 7B). In this assessment, the best
performance was determined to be those attributes that wereclosest to normal behavior, even if they occurred only once.
Somatosensory function. Whereas intact animals neverdropped their limbs through the gaps in the cage bars, animals
after SCI often dropped the limbs through the floor gaps. We
evaluated the distance the limb was dropped, as follows: lower
limbs up to the thigh, knee, or foot only, and upper limbs up to thehumerus, elbow, or hand only. Initially, the animals dropped their
upper limbs up to the humerus and lower limbs up to the thigh,and were not able to raise the limbs at all (Fig. 7F). Over thecourse of motor recovery, they began to drop their upper limbs up
to the elbow or only the hand, and lower limbs to the knee or only
the foot (Fig. 7E). They also started pulling up the dropped limbs
more quickly. Pulling up dropped limbs, which suggests
somatosensory function recovery, was more likely to be observedin the lower than upper limbs. Somatosensory function was judged
by the worst performance, even if it occurred only once.

body by placing weight on the upper limbs, even when moving
forward. When the animals progressed to raising the upper bodyfrom the floor in walking, they began to move their upper limbs tothe rhythm of the walk, with their hands above shoulder level.
However, more typically, the forearms were dragged on the floor,
with the wrist joints dropped and the forearms not pronated. They
were eventually able to bear weight on the upper limbs while
walking, dragging only the hands, and finally recovered to walking
without dragging the hands, in the most-recovered cases in therhHGF group. The hand position also gradually recovered frombeing between the shoulder and head level to above the head. The
most remarkable category, hand placement in walking, wasassessed when the hands were above shoulder level, and wascharacterized by wrist extension, forearm pronation, and finger
extension. Initially, the wrist joints were completely dropped, andthe forearm was not pronated; thus, the animals supported theirweight on the dorsal side of the hands (Fig. 7C) or on the ulnar sideof the upper limbs. They eventually were able to extend the wrist
joints, pronate the forearms, extend the fingers, and reestablish
palmar contact with weight support (Fig. 7D); however no animals
in the PBS group attained this level of recovery. The best
performance was judged by the animal’s ability to maintain aparticular pattern throughout the testing session, not by its ability
to exhibit a behavior once.
Reach-and-grasp performance. The assessment of reachand-grasp performance highlighted finger flexion and extension,
wrist extension, forearm pronation, and shoulder flexion. For
several days after SCI, the animals could not raise their upper
limbs, and could not actively flex their fingers even when a pen
was placed in their hands. The animals eventually began to grasp apen passively placed in their hands, and to lift their upper limbs toreach and grasp a pen that was suddenly presented in front of theireyes. They typically recovered the ability to elevate their hands up
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rhHGF promoted significant motor function recovery
The bar grip (Fig. 8A) and spontaneous motor activity (Fig. 8B)[28,31,33] revealed significant motor function recovery in therhHGF group. After SCI, the bar grip strength and spontaneous
movements decreased sharply, and then recovered gradually.
Spontaneous motor activity tests showed greater than 100%
recovery in some animals in the rhHGF group around 8 weeks
after SCI. At 12 weeks after SCI, the animals had recovered
62.462.6% and 38.964.3% of their bar-grip strength, and77.8612.7% and 34.5610.9% of their spontaneous motor
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Table 1. Original open field rating scale.

UPPER LIMBS maximum 20 points
1. WEIGHT BEARING
weight bearing in stanceweight bearing in walking

anterior chest on the floor, no weight support of upperlimbs+0

anterior chest lifted up from the floor with weight support of upperlimbs+1

anterior chest on the floor, no weight support of upperlimbs+0

anterior chest lifted up from the floor with weight support of upperlimbs,

hand position in walking

hand placement in walking

with forearm dragged+1

with hand dragged+1

stepping with hand clearance from the floor+1

coordinated steps with lowerlimbs+1

below shoulder+0

between shoulder and head

+1

above head+1

dorsal placement (dropped wrist)+0

ulnar placement, not pronated+1

pronated, but no palmar placement+1

pronated with palmar placement+1

2. REACH AND GRASP PERFORMANCE
elevation below head-height+0

cannot grasp a pen+0grasp a pen+1elevation up to head-height+1

cannot grasp a pen at head-height+0

grasp a pen perpendicular to the body plane+1grasp a pen parellel to the body plane+1elevation up to 3cm above head-height+1

cannot grasp a pen at 3 cm above head-height+0

grasp a pen perpendicular to the body plane+1grasp a pen parellel to the body plane+1

3. SOMATOSENSORY FUNCTION
somatosensory

drop upper limbs through gaps in the cage bars up to
humerus

+0

elbow

+1

hand only+1

never drop

+1

TRUNK STABILITY maximum 3 points
cannot roll-over from supine to prone position+0roll-over from supine to prone position+1keep sitting position+1keep standing position+1

LOWER LIMBS maximum 7 points
ROM & weight bearing

somatosensory

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slight movement of lowerlimbs+0

extensive movement of lowerlimbs+1

move forward with weight bearing of lowerlimbs

+1

jump

+1

jump successively+1drop lower limbs through gaps in the cage bars up to
thigh

+0

knee

+1

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Table 1. Cont.

LOWER LIMBS maximum 7 points
foot only+1

never drop

+1

Definitions and abbreviations.
,UPPERLIMBS. dorsal placement: wrist joint is dropped and dorsal side of the hand is placed on the floor; ulnar placement: pronation of forearm is insufficientand little finger of the hand (ulnar side of the hand) is placed on the floor; pronated, but no palmar placement: forearm is pronated, wrist joint is extended, but nopalmar placement due to limitted fingrs extension; pronated with palmar placement: palmar placement with forearm pronated and wrist and fingers extended,TRUNK STABILITY. keep sitting or standing position: keep sitting or standing position more than 5 seconds in the cage ,LOWER LIMBS. ROM: range of
motion; slight movement of lowerlimbs: partial joint movement through less than half of the range of the three joints motion; extensive movement oflowerlimbs: movement through more than half of the range of the three joints motion.
doi:10.1371/journal.pone.0027706.t001

activity, in the rhHGF and PBS groups, respectively. The
evaluation of motor function by the original open field scale also
showed significant differences between the two groups (Fig. 8C–E).
Animals in both groups gradually recovered, reaching a plateau
around 8 weeks after SCI, and the original score of the upper
limbs (pre-injury score = 20) recovered to 15.960.8 and 7.861.8
in the rhHGF and PBS groups, respectively, 12 weeks after SCI(Fig. 8C). Significant differences between the two groups wereobserved at most time points from 12 days after SCI in the bar grip
test, from 21 days after SCI in spontaneous motor activity, andfrom 14 days onward after SCI in the open field upper limb scores.

major advantage of using non-human primates in translational
research is the ability to assess fine motor control recovery in theforelimbs. Another advantage of using marmosets is the similarityof their neuroanatomy to that of human beings. Some features ofmotor projections have undergone pronounced changes fromrodents to primates.
The CST is a major pathway for voluntary movement control.
It is a phylogenetically new system that appeared first in mammals
and developed predominantly in primates [35,36]. Over thecourse of the evolution of human and non-human primates, theCST has changed its location from the dorsal to lateral columns of
the spinal cord [37] and changed its terminations. Whereas inrodents the CST axons project mainly to dorsal horn neurons andpremotor spinal circuits, in primates, a significant proportion of
the CST axons project to the ventral horn, and some synapse
directly onto motoneurons innervating hand and forearm muscles,
as direct cortico-motoneuronal (CM) connections [38].
Direct CM connections predominantly developed in higher
primates such as macaque monkeys, apes, and humans [39,40,41].
Moreover, previous studies have suggested a strong correlation
between the number of direct CM connections and the level ofmanual dexterity of non-human primate species [38,39,42,43]; theappearance of direct CM connections is correlated with the
emergence of precision grip between the thumb and the index
finger, which is observed in macaque monkeys but not inmarmosets. Thus, macaque monkeys have been used in many
experiments because they have more advanced finger dexterity
than marmosets or squirrels. In previous experiments in macaquemonkeys, a unilateral transection of the spinal cord was made tointerrupt the CST, including direct CM connections, at thedesignated cervical segment, and the recovery of precision grip
was assessed after SCI [44,45,46,47]. However, to evaluate andpredict the therapeutic effects of interventions as humantreatments, contusive injury is considered most relevant to human
SCI. Furthermore, for precise assessments, the monkeys used in atranslational study should be the same age, sex and body weight,
which is problematic in experiments using macaque monkeys, but
feasible in marmosets [48].
In this study, as shown in Figure 2, the CaMK II a-positive
putative CST axons in marmosets were located in the lateralcolumn, unlike in rodents, but projected mainly to the dorsal hornand IMZ without a visible CM connection, as in rodents,
indicating the possibility that the marmoset CST is intermediatein the course of evolution from rodents to macaque monkeys andhumans. Marmosets cannot perform a precise finger-thumb grip,
but can be evaluated by reach-and-grasp prehensile tasks using a
pen and by hand placement while walking. However, we could notevaluate these precise tasks using the previously reported bar grip

Validation of the original open field rating scale
Of the three methods assessing motor function (bar grip test,
spontaneous motor activity, and open field scoring), our original
open field rating scale yielded the smoothest recovery curveafter SCI. Using this scale, the rating for the upper limbs
showed a smoother recovery curve than that for lower limbs,
reflecting the fact that the cervical SCI model used in this study
is not a complete injury model, but rather a severe central cord
syndrome model, which shows better and earlier functional
recovery in the lower limbs than in the upper ones (Fig. 8C and
D). To assess the validity of our open field scale, we evaluated its
relationship to the anatomical lesions. Simple linear regression
analyses showed that the original scale had more positive
correlation with the quantitative histological analyses (Fig. 9A–
C) than did the spontaneous motor activity test (Fig. 9D–F); thecorrelation was especially good with the CaMK II a-positive
area [29] in the lateral column at Th1 (Fig. 8A; R2 = 0.785),
suggesting that the scale closely reflected the involvement of the
CST pathway.

Discussion
We recently demonstrated that applying exogenous HGF by
HSV-1 vector into the injured spinal cord of adult rats after SCI
exerts significant neuroprotective and anti-apoptotic effects onneurons and oligodendrocytes to promote their survival, andenhances angiogenesis around the lesion epicenter. These multiple
effects significantly reduce the area of damage and provide a better
scaffold for axonal regeneration, thereby promoting functional
recovery after thoracic SCI in rodents [25].
To begin to translate this HGF strategy to the treatment of
human patients, here we performed intrathecal rhHGF infusion
during the acute phase of cervical SCI in marmosets. Althoughprevious clinical studies revealed that cervical SCI, which is more
frequent in humans than SCI at other levels, is the main cause ofmanual dysfunction [34], a detailed examination of manualfunction following cervical SCI is difficult to obtain in rodents. A
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Figure 1, almost all of the wrist extensor and flexor motoneurons
were located at a site caudal to the lesion epicenter, suggesting theusefulness of evaluating wrist and finger extension and forearm
pronation, which are important for performing reach-and-grasp
prehensile tasks with a pen, and for hand placement while walking.
Intrathecal rhHGF infusion significantly reduced the damaged
areas and promoted functional recovery after SCI, consistent with
our previous report [25]. Although HGF’s anti-apoptotic,
neurotrophic, and angiogenic effects were not investigated during
the acute phase of SCI [25] due to the small number of animals
involved in this study, the LFB-positive myelinated areas and
CaMK II a-positive fibers were significantly preserved in therhHGF group. These effects resulted in significantly betterneurological hand performance, which was successfully assessedby our original open field rating scale. Notably, our original open
field scale had a better positive correlation with the CaMK II apositive area in the lateral column at the Th1 level (Fig. 9A) andwith the number of CaMK II a-positive IMZ neurons at T1(Fig. 9B) than with the LFB-positive area at the C5 level (Fig. 9C),
suggesting that it primarily reflects the neurological hand
performance controlled by the CST. Furthermore, compared tothe spontaneous motor recovery test (Fig. 9D–F) and bar grip test,
our original open field scale (Fig. 9A–C) had more a greater
positive correlation with the CaMK II a-positive area, CaMK II
a-positive IMZ neurons and LFB-positive areas, suggesting that
this open field rating scale more precisely reflects the neurologicaland pathological status after cervical SCI. However, the numberof surviving ChAT-positive motoneurons, which innervate forearm muscles, showed no significant differences between the HGFtreated and control groups, in contrast with our previous reports.
Based on this finding, it was necessary to reexamine the direct andindirect CM pathways after cervical SCI.
Compared with the direct CM pathway, the role of indirect
pathways from the cerebral cortex to motoneurons, via subcorticalor spinal interneuronal systems, has received much less attention,even though such pathways contribute the majority of motoneuron
input [49]. In primates, many neuroanatomical studies haveshown that the majority of corticospinal fiber terminations are notdistributed in lamina IX, but in the IMZ of the spinal gray matter[50,51], where various types of interneurons are located.
Intracellular recordings from motoneurons to investigate theeffects of electrically stimulating the contralateral medullary
pyramid (Pyr) in macaque monkeys suggest that PNs located in
the C3–C4 segments can mediate the disynaptic excitation to
motoneurons [52,53]. Furthermore, an evaluation of finger
dexterity (precision grip with the tips of the index finger andthumb) suggests that the C3–C4 PNs play a significant role inrecovering precision grip after a CST lesion at the border between
C4 and C5, in which direct CM connections to forelimb
motoneurons are interrupted, while a major portion of the of
the C3–C4 PN descending axons (indirect CM connections)remain intact [44]. In contrast, precision grip does not recoverafter a CST lesion at C1/C2, which interrupts corticospinal input
to the C3–C4 PNs [54]. These findings suggest that after a CST
lesion at C4/C5, indirect CM connections can be used to control
the reach-and-grasp hand movements requiring a precision grip in
macaque monkeys.
In the present study, we induced a C5-level contusive injury as
shown in Figure 2B; that is, caudal to the C3–C4 PNs and rostral
to almost all forelimb motoneurons, as shown in Figure 1. This
interrupted almost all the direct CM connections to forelimb
motoneurons, whereas the majority of corticospinal input to theC3–C4 PNs remained intact, as shown in Figure 2. Therefore, thiscervical SCI model is appropriate for investigating the involve-

Figure 7. Representative images of marmosets after SCI. (A andB) An animal in the PBS group showed good elevation of upper limbs
up to the head level 8 weeks after SCI. However, this animal could not
grasp a pen parallel to the body plane because of insufficient forearm
pronation and wrist and finger extension (A, arrowheads). An animal in
the rhHGF group could reach for a pen at 3 cm above head height, andcould grasp a pen even when parallel to the body plane. Note that theforearm was well pronated, with the wrist joint fully extended (Barrowhead). (C and D) The same animal as in (A) walked with theanterior chest lifted up from the floor by bearing its weight on theupper limbs. However, the wrist joints dropped completely, and thedorsal surfaces of the hands were placed and dragged on the floor (Carrowheads). On the other hand, the same animal as in (B) showed
good palmar placement of the hands, with the wrist joints completely
extended (D arrowheads), and walked with its hands clear of the floor.
(E and F) One week after SCI, an animal in the PBS group dropped its
limbs through gaps in the cage floor as far as the humerus or thigh, andcould not raise the dropped limb quickly (F). The animal gradually
recovered, and at 6 weeks after SCI, rarely dropped its limbs to thehumerus or thigh level, but still often dropped them as far as the elbow
or knee (E).
doi:10.1371/journal.pone.0027706.g007

test, cage-climbing test, or monitoring of spontaneous motor
activity [31,33]. Furthermore, in the spontaneous motor activity
test, some animals of the rhHGF group around 8 weeks after SCI
showed activity that was more than 100% of the pre-injury level,
suggesting that this test does not necessarily reflect neurologicalrecovery. Therefore, we established an original open field rating
scale, which enabled us to the evaluate neurological hand
performance after cervical SCI in marmosets. As shown in
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rhHGF for SCI in Non-Human Primates

Figure 8. Functional recovery in rhHGF-treated marmoset SCI models. Behavioral analyses were performed by bar grip test (A), monitoringof spontaneous motor activity (B), and the original open field rating scale for the upper limbs (maximum, 20 points) (C), for the lower limbs and trunk(maximum, 10 points) (D) and combined (maximum 30 points) (E). All three methods showed significant recovery in the rhHGF group compared with
the PBS group. Note that the original open field rating scale shows the smoothest recovery curve compared with the others. *P,0.05, **,0.01. (n = 6
for the rhHGF group; n = 5 for the PBS group).
doi:10.1371/journal.pone.0027706.g008

Figure 9. Relationship of the original open field scale and spontaneous motor activity to anatomical lesions. The original open field
scale, with a maximum of 30 points, showed the greatest positive correlation with CaMK II a-positive areas in the lateral column at Th1 (A: R2 = 0.785)
and the next most positive correlation with CaMK II a-positive IMZ neurons at T1 (B: R2 = 0.5716) compared with the four other correlations: theoriginal open field scale and LFB-positive area at C5 (C: R2 = 0.525), the spontaneous motor activity and CaMK II a-positive area in the lateral column at
Th1 (D: R2 = 0.3046), the spontaneous motor activity and CaMK II a-positive IMZ neurons at T1 (E: R2 = 0.3274) and the spontaneous motor activity
and LFB-positive area at C5 (F: R2 = 0.3039).
doi:10.1371/journal.pone.0027706.g009

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rhHGF for SCI in Non-Human Primates

HGF were observed [25]. Therefore, we assume that theenhancement of HGF concentration in the spinal cord to no
more than 3-fold above the control level would be sufficient toshow therapeutic effects, and there is probably no need to increase
the HGF concentration to an extremely high level. The dosage tobe used in clinical applications of HGF by intrathecal administration needs to be carefully assessed. Regarding the safety of
intrathecal catheter insertion, infusion from the lumbar level has agreat advantage over that from the cervical or thoracic level.
Moreover, catheter insertion at the lumbar level might be usefulfor non-operated patients with SCI. This information needs to beassessed further to establish a therapeutic dose range and the
optimal administration site for use in future clinical studies in SCI
patients.
Overall, intrathecal rhHGF administration after SCI significantly reduced the extent of damage to the spinal cordparenchyma, preserved the CST pathway in the lateral column,
and promoted hand dexterity, presumably by involving indirect
CM pathways connected by interneurons. Furthermore, marmosets did not show abnormal behaviors or signs of discomfort
or pain when the examiner touched their bodies or manipulated
their limbs. These findings suggest that rhHGF treatment did
not induce allodynia; this was also indicated by immunohistological analysis with an anti-CGRP antibody [65]. Taken
together, the present study demonstrates the validity of ouroriginal open field behavior rating scale in a contusive cervical
SCI model in primates, the efficacy and safety of intrathecal
rhHGF treatment for SCI in adult non-human primates and thepossibility that this novel therapy may be suitable for clinicalapplication.

ment and role of C3–C4 PNs in the recovery of hand dexterity.
Significantly greater numbers of CaMK II a-positive interneurons in the IMZ at C4 and C6-Th1 segments were observed in
the rhHGF group than the PBS group, suggesting that C3–C4
PNs played a major role in the recovery of hand movement after
SCI in marmosets, as also described in macaques [44,46,47]. In
spite of severe spinal cord damage around the lesion epicenter inthe rhHGF group, motor function in the four extremities wasrecovered to a considerable extent, presumably by the endogenous plasticity of motor pathways, including indirect CM
connections.
Beyond the CST, there are other possible mechanisms forfunctional recovery after SCI. First, the C3–C4 PNs could receive
convergent inputs from various descending tracts, such as the
rubro-, reticulo-, raphe-, and tectospinal tracts, as in rodents andcats [55,56,57]. Second, compared to rodents, primates engage
more complex neural circuits in the parietal and frontal lobes of
the cerebral cortex for even the simplest of skilled movements,suggesting that primates rely on the cortex for motor function[46,58,59,60,61,62]; this reliance could enable greater plasticity
and recovery after SCI. Third, the reticulospinal tract, located in
the ventral part of the spinal cord, controls both proximal anddistal arm and hand muscles [63,64]. Experiments combining
brain imaging with the pharmacological inactivation of motor
cortical regions revealed time-dependent central compensatory
mechanisms for finger dexterity after SCI in macaque monkeys;
the recovery of finger dexterity involves the bilateral primary
motor cortex during the early recovery stage, and more extensive
regions of the contralesional primary motor cortex and bilateral
premotor cortex during the late recovery stage [46]. In terms ofclinical implications, these findings suggest that even limited
sparing and regeneration of spinal projections after cervical SCI
can be extremely beneficial to humans, enabling the recovery ofsome aspects of fine motor control.
With regard to clinical trials, methods for evaluating the sparedand regenerating axons after SCI intervention are critical. If theextent and severity of the spinal cord lesion can be estimated by invivo MRI techniques, including DTT [30], these analyses could
provide strong predictive factors for functional recovery. In thepresent study, spinal tracts around the lesion site, revealed by
DTT and by the intact intensity area in axial T2WI images,
seemed to correlate with the spared rim of the LFB-positive
myelinated area representing motor function recovery at 12
weeks after SCI; no obvious relationship between the in vivo
images and motor function recovery was observed before 12
weeks. To improve the predictive and analytical value of thesetechniques, precise quantitative comparisons of in vivo MR
images, pathological findings, and motor function over time after
SCI will be necessary, especially during the acute phase of SCI(Konomi et al., submitted).
We believe that intrathecal administration would also be themost effective administration route in the clinical application ofrhHGF for SCI patients, because the blood-brain barriersignificantly limits the translocation of rhHGF from blood to thespinal cord. We observed that the subcutaneous injection ofrhHGF was ineffective in ALS rats (Funakoshi et al, unpublishedresult), while the intrathecal administration of rhHGF showedsignificant therapeutic effects in these animals [24]. In the present
study, we administered 400 mg of rhHGF intrathecally over 4
weeks in marmosets, which corresponds to 0.05 mg/kg/day.
Although we could not determine the HGF concentrations in the
CSF of marmosets, we previously reported that the HGF levels inthe spinal cord was elevated around 3-fold compared to thecontrol level in SCI rats, when therapeutic effects of exogenous
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Materials and Methods
Preparation of osmotic mini-pump containing rhHGF or
PBS
Osmotic mini pumps (Alzet model 2004; nominal pumping rate
0.25 ml/hr, nominal duration 4 weeks, nominal reservoir 200 ml,
Alzet, CA, USA) were filled with PBS, or with rhHGF (Kringle
Pharma, Inc., Osaka, Japan) diluted with PBS so that each pumpcontained 400 mg of rhHGF. Each pump was connected to an
intrathecal catheter (rat Intrathecal Catheter short; Alzet, CA,USA), and the apparatuses were incubated in sterile PBS at 37uC
for 48 hours before use.

Contusive SCI and intrathecal infusion of rhHGF in
common marmosets
Adult female common marmosets (295–350 g; Clea Japan,
Tokyo, Japan) were used (n = 6 for the rhHGF group; n = 5 for
the PBS group; n = 2 for CTB injection; n = 1 for intact spinalcord). All surgeries were performed under general anesthesia
induced by intramuscular injection of ketamine (50 mg/kg;
Sankyo, Tokyo, Japan) and xylazine (5 mg/kg; Bayer, Leverkusen, Germany) and maintained by isoflurane (Foren; Abbott,Tokyo, Japan). The animal’s pulse, arterial oxygen saturation,and rectal temperature were monitored during the surgicalprocedures. After a laminectomy at the C5 level, the dura mater
was exposed and a 20-g weight was dropped from a height of
50 mm onto the dura using a modified-NYU impactor asreported previously [28,31,33]. Right after contusive SCI at the
C5 level, a C7 laminectomy was carried out and an intrathecal
catheter was inserted from the C7 level. An osmotic mini pump,filled with 400 mg rhHGF (HGF group) or PBS (control group)
was connected to a catheter and placed in the subcutaneous spaceon the right side of the animal’s back. The pump was left in place
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rhHGF for SCI in Non-Human Primates

to deliver the total 400-mg dose of rhHGF for 4 weeks; dosage wasbased on previous results from intrathecal rhHGF administrationin ALS rats [24], which have about the same body weight as
marmosets. The animals were placed in a temperature-controlledchamber until thermoregulation was reestablished and received
disinfectant treatment once a day. Manual manipulation of thefingers and joints in the four extremities was carried out twice a
day and manual bladder expression was also performed twice a
day until voiding reflexes were reestablished. Paralyzed animals
were given adequate amounts of food and water until they
recovered their ability to ingest food and water without
assistance; thereafter, they had free access to food and water inthe cage.

Stereology
To quantify the CaMK II a-positive area, LFB-positive
myelinated area, and CGRP-positive fiber length, axial section
images were obtained and analyzed using grain counting with lightintensity by a Micro Computer Imaging Device (Imaging
Research Inc., St. Catharines, Ontario, Canada). Thresholdvalues were maintained at constant levels for all analyses. Afterobtaining images of axial sections stained with an anti- CaMK II aantibody, the number of CaMK II a-positive neurons in theintermediate zone (IMZ) of the gray matter was counted.

Behavioral analyses
Bar grip test. The bar grip test, which tests the animal’s
gripping reflex (the motion undertaken when attempting to grasp
an object placed in front of their eyes) and evaluates the grip
power, was carried out as previously reported [28,31,33]. The
same device (220-mm wide, 500-mm deep, and 400-mm high,with a bar diameter of 2.5 mm in a 163 [70 mm6100 mm] grid
pattern) was used in the present study. The test was carried out
three times a day. The percentage of the maximal grip strength
relative to the mean strength before the injury was calculated oneach day after the injury.
Spontaneous motor activity test. Spontaneous motor
activity tests were carried out as previously reported [28,31,33].
We used cages (350-mm wide, 500-mm deep, and 500-mm high)equipped with infrared sensors (Murata Manufacturing Corp.,
Nagaokakyo, Kyoto, Japan) on the ceiling to continually record
the marmosets’ 3D motion. Our system utilized a passive
thermographic infrared sensor to monitor heat emitted from theanimals. The 3D localization of the heat source was monitored,
and a change in this localization was recorded as movement. Eachanimal’s data were recorded and monitored hourly on a computer,and the activity measured after the SCI was calculated as apercentage relative to that before the injury.
Original open field rating scale. To develop an original
open field rating scale, the natural course of motor function
recovery after cervical SCI was precisely observed in 6 marmosets
before starting experiments using rhHGF. Two blinded examiners
participated in all the open field tests and were positioned acrossfrom each other to observe both sides of the marmoset. Marmosets
were tested alone for 5 minutes on the floor and in the cage 3 times
per week until 4 weeks after SCI, and once per week thereafter.
During the open field test, marmosets were encouraged by slighttapping when they remained stationary for longer than 15 seconds.
In cases of borderline locomotor performance or disagreement
between examiners, scores indicating the greater deficit wereassigned. All lower limb movements were counted except thosethat were obviously part of a reflex (i.e., spastic extensive bilateral
flexion, or extension of the hip, knee, or ankle joints). The bilateral
upper limbs were assessed separately, and the score was defined as
the mean of the bilateral scores for motor function.

Ethics statement
All interventions and animal care procedures were performedin accordance with the Laboratory Animal Welfare Act, the Guide
for the Care and Use of Laboratory Animals (National Institutes of
Health, USA), the Guidelines and Policies for Animal Surgery provided
by the Animal Study Committee of the Central Institute forExperimental Animals and Keio University and the guidelines
outlined by Weatherall Report,and were approved by the Animal
Study Committee of Keio University (IRB approval number
09091-8).

In vivo magnetic resonance imaging and diffusion tensor
analysis
In vivo MRI and diffusion tensor studies were carried out at 1, 3,
and 12 weeks after SCI in two animals from each group, fouranimals in total, under general anesthesia as described above.
MRI was performed using a 7.0 tesla MRI, PharmaScan 70/16(BioSpin; Bruker) with a coil dedicated for small animals; diffusion
tensor analyses were performed as previously described [30]. The
region of interest (ROI) was placed in the upper cervical spinal
cord, and full-width DTT of the spinal cord was traced in thecaudal direction.

Retrograde cervical motoneuron labeling by cholera
toxin B subunit
Two intact animals were anesthetized with intramuscular
injections of ketamine and xylazine as described above. A small
skin incision was made in the left forearm of each, and 5 ml of
CTB (List Biological Laboratories, CA, USA) was manually
injected into 4 points of the forearm flexor or extensor muscles.
The animals were sacrificed 5 days after the CTB injection. Spinal
cords were collected and immunohistological analysis performed
as described below.

Histology
Spinal cords were perfusion-fixed with 4% paraformaldehyde in
0.1 M phosphate-buffered saline (PBS), and post-fixed in the same
fixative (24 hr) with 10% sucrose in 0.1 M PBS (24 hr) and 30%
sucrose in 0.1 M PBS (24 hr). Spinal cord segments were embedded
in an optimal cutting-temperature compound and cut into 20-mmthick sections on a cryostat. For diaminobenzidine staining, thesections were incubated at 48uC with polyclonal anti- CaMK II a
(CaMK II a; 1:100; Zymed), polyclonal anti-CGRP (CGRP; 1:100;
Affinity), and polyclonal anti-CTB (CTB; 1:500; Serotec) antibodies, followed by biotinylated secondary antibodies (1:500; Jackson
Immunoresearch). Biotinylated antibodies were visualized using the
Vectastain Elite ABC kit (Vector Laboratories), followed bydiaminobenzidine (Sigma). All images were obtained by microscopy(Axioskop 2 Plus; Zeiss, Oberkochen, Germany).
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Statistical analyses
All data are reported as the mean6SEM. In all the histologicalexaminations, an unpaired two-tailed Student’s t-test was used forsingle comparisons between the rhHGF and PBS groups. The
results of the original open field rating scale, bar grip test, andspontaneous motor recovery test were analyzed using the MannWhitney U-test. To validate the original open field rating scale,correlations between the results of this scale and the spontaneous
motor activity or histological findings (CaMK II a-positive areas in
the lateral column at Th1, CaMK II a-positive neurons in IMZ
andLFB-positive myelinated areas at C5) were analyzed by simple
linear regression analyses.
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rhHGF for SCI in Non-Human Primates

FT KH. Contributed reagents/materials/analysis tools: T. Nomura HF T.
Nakamura YT MA HO MN. Wrote the paper: KK HO MN.

Author Contributions
Conceived and designed the experiments: KK KF JY HO YT MN.
Performed the experiments: KK KF FT KH. Analyzed the data: KK KF

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