ORIGINAL PAPER
Human Amniotic Fluid Mesenchymal Stem Cellsin Combination with Hyperbaric Oxygen AugmentPeripheral Nerve Regeneration
Hung-Chuan Pan Æ Chun-Shih Chin Æ Dar-Yu Yang ÆShu-Peng Ho Æ Chung-Jung Chen Æ Shiaw-Min Hwang ÆMing-Hong Chang Æ Fu-Chou Cheng
Accepted: 30 December 2008 / Published online: 17 January 2009
� Springer Science+Business Media, LLC 2009
Abstract Purpose Attenuation of pro-inflammatory
cytokines and associated inflammatory cell deposits res-
cues human amniotic fluid mesenchymal stem cells (AFS)
from apoptosis. Hyperbaric oxygen (HBO) suppressed
stimulus-induced pro-inflammatory cytokine production in
blood-derived monocyte-macrophages. Herein, we evalu-
ate the beneficial effect of hyperbaric oxygen on
transplanted AFS in a sciatic nerve injury model. Methods
Peripheral nerve injury was produced in Sprague-Dawley
rats by crushing the left sciatic nerve using a vessel clamp.
The AFS were embedded in fibrin glue and delivered to the
injured site. Hyperbaric oxygen (100% oxygen, 2 ATA,
60 min/day) was administered 12 h after operation for
seven consecutive days. Transplanted cell apoptosis, oxi-
dative stress, inflammatory cell deposits and associated
chemokines, pro-inflammatory cytokines, motor function,
and nerve regeneration were evaluated 7 and 28 days after
injury. Results Crush injury induced an inflammatory
response, disrupted nerve integrity, and impaired nerve
function in the sciatic nerve. However, crush injury-pro-
voked inflammatory cytokines, deposits of inflammatory
cytokines, and associated macrophage migration chemo-
kines were attenuated in groups receiving hyperbaric
oxygen but not in the AFS-only group. No significant
increase in oxidative stress was observed after adminis-
tration of HBO. In transplanted AFS, marked apoptosis was
detected and this event was reduced by HBO treatment.
Increased nerve myelination and improved motor function
were observed in AFS-transplant, HBO-administrated, and
AFS/HBO-combined treatment groups. Significantly, the
AFS/HBO combined treatment showed the most beneficial
effect. Conclusion AFS in combination with HBO augment
peripheral nerve regeneration, which may involve the
suppression of apoptotic death in implanted AFS and the
attenuation of an inflammatory response detrimental to
peripheral nerve regeneration.
H.-C. Pan
Department of Neurosurgery, Taichung Veterans General
Hospital, Taichung, Taiwan
H.-C. Pan � S.-P. Ho
Department of Veterinary Medicine, National Chung-Hsing
University, Taichung, Taiwan
H.-C. Pan � C.-J. Chen
Institute of Medical Technology, National Chung-Hsing
University, Taichung, Taiwan
C.-S. Chin
Department of Chest Medicine, Taichung Veterans General
Hospital, Taichung, Taiwan
D.-Y. Yang
Department of Neurosurgery, Chang Bing Show Chwan
Memorial Hospital, Changhua, Taiwan
S.-M. Hwang
Bioresource Collection and Research Center, Food Industry
Research and Development Institute, Hsinchu, Taiwan
M.-H. Chang
Department of Neurology, Taichung Veterans General Hospital,
Taichung, Taiwan
F.-C. Cheng (&)
Stem Cell Center, Department of Medical Research, Taichung
Veterans General Hospital, No. 160, Sec. 3, Taichung-Kang
Road, Taichung 407, Taiwan, R.O.C.
e-mail: [email protected]
123
Neurochem Res (2009) 34:1304–1316
DOI 10.1007/s11064-008-9910-7
Keywords Apoptosis � Amniotic fluid mesenchymal
stem cells � Hyperbaric oxygen � Sciatic nerve injury �Inflammatory cytokines
Introduction
In the past few decades there have been significant
advances in peripheral nerve repair. These have included
the introduction of the microscope, tension-free repair by
epineural or perineural suture and accurate nerve apposi-
tion by means of anatomic features, and histochemical or
immunohistochemical methods for motor and sensory
fiber. For complete lesions, primary end-to-end repair is
preferable when possible and has the best prognosis [1]. If
the gap between the proximal and distal stumps cannot be
made up in order to perform a tension-free end-to-end
neurorrhaphy, grafting or transfer is used. When a graft is
used, a portion of the regenerative axons is lost across the
suture line. Hence the preference for direct repair when the
gap can be made up and a tension-free neurorrhaphy per-
formed [2]. Despite early diagnosis and the use of modern
surgical techniques, no matter how accurate the nerve
repair, function recovery can never reach the pre-injury
level. Poor outcome may result from many factors, both
intrinsic and extrinsic to the nervous system, such as the
type and level of injury, the presence of associated injury,
timing of surgery, or change in spinal cord neurons and end
organs [3].
Several alternative approaches have been proposed to
have beneficial effects on peripheral nerve regeneration,
including application of an electric field, transplantation of
stem cells, and administration of neurotrophic factors [4–
7]. Recently, cell transplantation has become the focus of
research. The implantation of embryonic stem cells, neural
stem cells, and mesenchymal stem cells has been shown to
exert beneficial effects on peripheral nerve regeneration.
Cell replacement, trophic factor production, extracellular
matrix molecule synthesis, guidance, remyelination,
microenvironmental stabilization, and immune modulation
have been proposed as beneficial mechanisms after cell
implantation [5, 8, 9].
HBO has several diverse functions such as cell prolif-
eration, vascular angiogenesis, regulation of stem cells, and
immunomodulation. It is well known that hyperbaric
oxygen (HBO) can stimulate endothelial cell and fibroblast
proliferation and neovascularization [10, 11] as well as
matrix biosynthesis [11]. HBO increased circulating stem
cell factor by 50% and increased the number of circulating
cells expressing stem cell antigen-1 and CD34 by 3.4-fold
and doubled the number of colony-forming cells [12]. It
has been hypothesized that during the release of endothelial
progenitor cells HBO increases the nitric oxide level in
perivascular tissues via the stimulation of nitric oxide
synthase (NOS) [13]. HBO also exerts a trophic effect on
vasculogenic stem cells through the CD34 vascular channel
[14]. HBO significantly increases the migration and the
tube formation of bone marrow-derived mesenchymal stem
cells as well as the expression of placental growth factors,
which may play an important role in HBO-induced vas-
culogenesis [15]. Recently, HBO has been applied in
neurological disorders such as brain ischemia or a nerve
crush injury model, which revealed that HBO generated
endogenous neurogenesis either through direct effects,
anti-inflammatory effects, or immunomodulation [16–18].
Furthermore, HBO has a potent anti-inflammatory tissue
effect [19, 20], and this effect has been postulated to occur
through the inhibition of pro-inflammatory cytokine syn-
thesis in monocyte-macrophages [21].
Recent evidence has shown amniotic fluid to be a novel
source of stem cells for therapeutic transplantation.
Amniotic fluid-derived stem cells express characteristics of
both mesenchymal and neural stem cells [22]. In our pre-
vious studies, we have demonstrated that transplantation of
amniotic fluid mesenchymal stem cells (AFS) promoted
peripheral nerve regeneration [6, 7]. The short-term sur-
vival of implanted stem cells might diminish cell
transplantation-mediated beneficial effects. The increased
survival of implanted cells could be augmented by the
suppression of inflammatory cytokines through the inhibi-
tion of inflammatory cell deposits [23]. Therefore, the
present study was designed to evaluate whether a combi-
nation of HBO and AFS transplantation could augment
peripheral nerve regeneration. The potential contribution of
the anti-apoptotic and anti-inflammatory effects of HBO
was also investigated.
Materials and Methods
Animal Model
Sprague-Dawley rats weighing from 250 to 300 g were
used in this study; permission was obtained from the Ethics
Committee of Taichung Veterans General Hospital. The
rats were anesthetized with 4% isoflurane in induction
followed by a maintenance dose (1–2%) [2% (v/v%) in
70% N2O/30% O2 at flow rate 0.5 l/min] (A.S.D. 1000).
The left sciatic nerve was exposed under a microscope
using the gluteal muscle splitting method. A vessel clamp
(B-3, pressure 1.5 gm/mm2; S&T Marketing LTD, Swit-
zerland) was applied 10 mm from the internal obturator
canal for 20 min [7]. The animals were categorized into
four groups: Group I (n = 30): The crushed nerve was
wrapped with fibrin glue; Group II (n = 30): AFS was
embedded in fibrin glue and delivered to the injured nerve;
Neurochem Res (2009) 34:1304–1316 1305
123
Group III (n = 30): The crushed nerve was wrapped with
fibrin glue. The rats received HBO (100%, 2 ATA) for
60 min/day 12 h after operation for seven consecutive
days; Group IV (n = 33): AFS were embedded in fibrin
glue and delivered to the injured nerve. The rats received
HBO (100% oxygen, 2 ATA) for 60 min/day 12 h after
operation for seven consecutive days. To avoid the rejection
of cell transplantation, cyclosporine was used in this study.
Six animals without injury were used for assay of pro-
inflammatory cytokines. It is known that macrophage
migration and inflammatory cytokine expression are influ-
enced by the administration of cyclosporine. To lessen these
effects, all animals in the experimental and control groups
were allowed free access to food and water supplemented
with cyclosporine (Novartis, USA) (12.5 mg cyclosporine
in 125 ml drinking water with daily intake of at least
50 ml). The administration of cyclosporin A began the first
day after injury and continued until sacrifice [23, 24].
Preparation and Culture of Human Amniotic
Mesenchymal Stem Cells (AFS)
Amniotic fluid samples (20 ml) were obtained by amnio-
centesis performed between 16 and 20 weeks of gestation
for fetal karyotyping. For culturing amniocytes, four pri-
mary in situ cultures were set up in 35 mm tissue culture-
grade dishes using Chang medium (Irvine Scientific, Santa
Ana, CA, USA). Microscopic analysis of Giemsa-stained
chromosome banding was performed, and the rules for
metaphase selection and colony definition were based on
the basic requirements for prenatal cytogenetic diagnosis in
amniocytes [25]. For culturing AFS, non-adhering amniotic
fluid cells in the supernatant medium were collected on the
fifth day after primary amniocyte culture and maintained
until completion of fetal chromosome analysis. The cells
were then centrifuged and plated in 5 ml of b-minimum
essential medium (b-MEM; Gibco-BRL) supplemented
with 20% fetal bovine serum (FBS; Hyclone, Logan, UT,
USA) and 4 ng/ml basic fibroblast growth factor (BFGF;
R&D Systems, Minneapolis, MN, USA) in a 25 cm flask
and incubated at 37% with 5% humidified CO2 [6]. This
protocol was approved by the Institutional Review Board
(IRB) of Taichung Veterans General Hospital, and written
informed consent was obtained from all patients.
Grafting Procedure
AFS were labeled with Hoechst 33342 before grafting. A
volume of 25 ll of AFS with a total amount of 106 cells
was suspended in 25 ll of Fibrin glue (Aventis Behring,
Germany) containing the woven Surgicel (Johnson &
Johnson, USA) and transplanted into the injured site
immediately after crush [7].
Analysis of Functional Recovery
A technical assistant who was blinded to treatment alloca-
tion evaluated sciatic nerve function weekly after the
surgery. The evaluation method included ankle kinematics
and sciatic function index (SFI) [6, 7]. In the sagittal plane
analysis, the following formula was used in the mechanical
analysis of the rat ankle: h ankle = h foot-leg 90. Several
measurements were taken from the footprint by red ink
print: (1) distance from the heel to the third toe, the print
length (PL); (2) distance from the first to fifth toe, the toe
spread (TS); and (3) distance from the second to the fourth
toe, the intermediary toe spread (ITS). All three measure-
ments were taken from the experimental (E) and normal (N)
sides. The SFI was calculated according to the equation:
SFI = -38.3(EPL – NPL / NPL) ? 109.5(ETS – NTS /
NTS) ? 13.3(EIT – NIT / NIT) - 8.8. The SFI oscillates
around 0 for normal nerve function, whereas SFI around -
100 represents total dysfunction.
Electrophysiology Study
Six left sciatic nerves from each group were exposed
4 weeks after operation. Electric stimulation was applied to
the proximal side of the injured site; the conduction latency,
and the compound muscle action potential (CMAP) were
recorded with an active electrode needle 10 mm below the
tibia tubercle and a reference needle 20 mm from the active
electrode. The mean length from the stimulation from the
active recording electrode was 52.4 ± 0.4 mm. The stimu-
lation intensity and filtration ranges were 5 mA and 20–
2,000 Hz, respectively. The CMAP data and conduction
latency were converted to ratios of the injured side divided
by the normal side to adjust for the effect of anesthesia [6, 7].
Measurement of Lipid Peroxidation
A thiobarbituric acid reactive substances (TBARS) assay
kit (ZeptoMetrix) was used to measure lipid peroxidation.
In brief, 10 mm of sciatic nerve (marked ‘‘crush site’’ in
the middle of the nerve) was homogenized with 0.1 M
sodium phosphate buffer (pH 7.4). A volume of 100 ll of
homogenate was mixed with 2.5 ml reaction buffer (pro-
vided in the TBARS assay kit) and heated at 95�C for
60 min. After the mixture cooled, the absorbance of the
supernatant was measured at 532 nm using a spectropho-
tometer. The TBARS were expressed in terms of
malondialdehyde (MDA) equivalents.
Myeloperoxidase (MPO) Assay
Protein extracts (100 ll) from 10 mm of crushed nerve
(marked ‘‘crush site’’ in the middle of the nerve) were
1306 Neurochem Res (2009) 34:1304–1316
123
mixed with 2.9 ml of the assay solution. The optic absor-
bance was determined at 470 nm for 1 min with a
spectrophotometer. Arbitrary activity was expressed as the
value of OD 470 per amount of protein. The assay solution
consisted of H2O, 26.9 ml; 0.1 M sodium phosphate buffer
(pH 7.0), 3.0 ml; 0.1 M H2O2, 0.1 ml; guaiacol, 0.048 ml.
Isolation of RNA and Reverse Transcriptase-
Polymerase Chain Reaction (RT-PCR)
The isolation of RNA, synthesis of cDNA, and PCR were
carried out as in our previous report [7]. DNA fragments of
specific genes and internal controls were co-amplified in
one tune. The PCR reaction was carried out under the
following condition: one cycle of 94�C for 3 min, 28 cycles
of (94�C for 50 s, 58�C for 40 s, and 72�C for 45 s), and
then 72�C for 5 min. The amplified DNA fragments were
resolved by 1.5% agarose gel electrophoresis and stained
with ethidium bromide. The intensity of each signal was
determined using a computer image analysis system
(IS1000; Alpha Innotech Corporation). The primer sets
used in the study were: 50-CACCGTCATCCTCGTTGC
and 50-CACTTGGCGGTTCTTTCG for RANTES; 50-CA
GGTCTCTGTCACGCTTCT and 50-AGTATTCATGGA
AGGGAATAG for MCP-1; and 50-TCCTGTGGCATC
CATGAAACT and 50-GGAGCAATGATCTTGATCTTC
for b-actin.
Quantification of Pro-inflammatory Cytokines
Six nerve tissues from each group for every single
parameter were removed 7 days after the operation. The
regenerating tissues (10 mm in length; marked ‘‘crush site’’
in the middle of the nerve) were retrieved and the samples
were stored at -80�C. Subsequently, each tissue sample
was homogenized with Laemmli SDS buffer. The
homogenate was centrifuged for 10 min at 12,000g at 4�C.
The tissue homogenate, 100 ll in triplicate, was applied to
a microtiter plate and allowed to adhere overnight at 4�C.
The microtiter plates were washed with phosphate-buffered
saline (PBS)-Tween-20 and blocked with 1% BSA in PBS
(200 ll) for 1 h. The plates were then treated with
respective primary antibodies and allowed to incubate for
6 h at 37�C. A volume of 100 ll of the respective poly-
clonal antibodies against TNF-a, IL-1b, Il-6 (R&D
Systems, Inc.) and INF-c (Chemicon, Inc.) were applied
overnight to microtiter plates. After further washing in
PBS-Tween-20, the plates were incubated with the
respective second antibody conjugate to alkaline phosphate
(100 ll) for 1 h. The reaction was developed using p-
nitrophenyl phosphate, disodium (3 mM) in carbonate
buffer, pH 9.6 (100 mM Na2CO3 and 5 mM MgCl2)
(150 ll), and the reaction was terminated after 30 min
using 0.5 N NaOH (50 ll). The absorbance of colored
product was read at 450 nm using a microplate reader (Bio-
Tek Instruments). The relative amount of antigens present
was measured from the densitometric reading against a
standard curve.
Terminal Dexonucleotidyl Transferase-Mediated
Biotinylated UTP Nick-End Labeling (TUNEL) Assay
Serial 8 lm-thick sections of sciatic nerve (7 days after
surgery) were cut on a cryostat and mounted on superfrost/
plus slides (Menzel-Glaser, Braunschweig, Germany). The
TUNEL assay (Roche Molecular Biochemicals, Mann-
heim, Germany) was carried out as previously described
[26]. Apoptotic cells were defined as those cells with
TUNEL-positive nuclei that were condensed and frag-
mented, as assayed by DAPI (Molecular Probes, Eugene,
OR; 1:2,000 dilutions). The number of apoptotic trans-
planted cells over the crush site was expressed as a
percentage of the total number of nuclei counted, with at
least 25,000 nuclei for each condition (n = 6 for each
group).
Immunohistochemistry
Serial 8 lm-thick sections of sciatic nerve were cut on a
cryostat and mounted on superfrost/plus slides (Menzel-
Glaser, Braunschweig, Germany), and were subjected to
immunohistochemistry with antibodies against CD68
(Chemicon, 1:200 dilution) (7 days after surgery); CD8
(Serotec 1:200 dilution); CD19 (Thermo, 1:200 dilution);
neutrophils (Abcam, 1:200 dilution); S-100 (Neomarkers,
1:400 dilution) (4 weeks after surgery); and neurofilament
(Chemicon, 1:300 dilution) (7 days after surgery) for the
detection of inflammatory cells, Schwann cells, and nerve
fibers, respectively. The immunoreactive signals were
observed by goat anti-mouse IgG (FITC) (Jackson, 1:200
dilution); anti-mouse IgG (Rhodamine) (Jackson, 1:200
dilution); or 3, 30-diaminobenzidine brown color. Among
longitudinal consecutive resections, five consecutive
resections contiguous to a maximum diameter were chosen
to be measured. Of 100 squares with a surface area of
0.01 mm2 each, 20 were randomly selected in an ocular
grid and used to count the number of inflammatory cells.
For the determination of neurofilament and S-100, six
nerves in each group were cut longitudinally into 8 lm-
thick sections and stained with each antibody. The maxi-
mum diameter of the resected nerve tissue with crush mark
was chosen to be examined. Areas of activity (0.2 mm2)
appeared as density against the background and were
measured by a computer image analysis system (Alpha
Innotech Corporation, IS 1000) [23].
Neurochem Res (2009) 34:1304–1316 1307
123
Histological Examination
After the neurobehavioral and electrophysiological testing,
six rats in each group underwent transcardial perfusion with
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
after being reanesthetized. The sciatic nerve was harvested
from the animals after the electrophysiological testing and
the nerve tissue was fixed on a plastic plate by the stay sutures
to keep the nerve straight [6]. The nerve was embedded, cut
longitudinally into sections 8 lm-thick and stained with
haematoxylin–eosin (H&E) for the measurement of vacuole
number and vascular staining. Among longitudinal consec-
utive resections, five consecutive resections contiguous to a
maximum diameter were chosen and used to collect the data
for comparison. Of 100 squares with a surface area of
0.01 mm2 each, 20 were randomly selected in an ocular grid
and used to count the vacuole number and vascular staining.
Statistical Analysis
Data are expressed as the mean ± SE (standard error). The
statistical significance of differences between groups was
determined by one-way analysis of variance (ANOVA)
followed by Dunnett’s test. In SFI and angle of ankle study,
the results were analyzed by repeated-measures ANOVA
followed by the multiple comparison method of Bonfer-
roni. A P value less than 0.05 was considered significant.
Results
Increase of Motor Function and Electrophysiological
Function by AFS ? HBO
SFI was significantly different among groups (F = 700,
P \ 0.001). SFI was significantly higher in groups II, III,
IV as compared with group I (P \ 0.01, P \ 0.01, and
P \ 0.01, respectively). In addition, a significant discrep-
ancy also existed between groups IV and III and groups IV
and II (P \ 0.01 and P \ 0.01, respectively). No signifi-
cant difference existed between groups II and III (Fig. 1a).
The presentation of angle of ankle also showed the same
trends (F = 1,400, P \ 0.001). Angle of ankle was sig-
nificantly increased in groups II, III, and IV as compared
with group I (P \ 0.01, P \ 0.01, and P \ 0.01, respec-
tively). A significant discrepancy also existed between
groups IV and III and groups IV and II (P \ 0.01 and
P \ 0.01, respectively). No significant difference existed
between groups II and III (Fig. 1b).
The average percentages of CMAP in the four groups
were 23.7 ± 3.8 (group I), 52.1 ± 3.1 (group II),
52.3 ± 2.3 (group III), and 76.9 ± 6.8% (group IV),
respectively, (Fig. 2a). There were significant differences
between groups I and II (P \ 0.001), I and III (P \ 0.001),
I and IV (P \ 0.001), II and IV (P \ 0.008), and III and IV
(P \ 0.006), respectively. However, no significant differ-
ence existed between groups II and III (P = 0.95).
The ratios of conduction latency in the four groups were
2.54 ± 0.058 (group I), 1.66 ± 0.085 (group II),
1.75 ± 0.076 (group III), and 1.31 ± 0.075 (group IV),
respectively, (Fig. 2b). There were significant differences
between groups I and II (P \ 0.001), I and III (P \ 0.001),
I and IV (P \ 0.001), II and III (P = 0.011), and III and IV
(P = 0.002), respectively. However, no significant differ-
ence existed between groups II and III (P = 0.45).
The above findings reveal that treatment with either AFS
or HBO alone promoted nerve regeneration better than the
control. AFS/HBO combined treatment showed the most
beneficial effects.
Early and Late Nerve Regeneration by Concomitant
Treatment with AFS ? HBO
Treatment with either AFS (974.83 ± 32.53 relative den-
sity/mm2) or HBO (863.33 ± 40.22 relative density/mm2)
Fig. 1 Neurobehavioral evaluation. A Representative illustrations of
SFI (a) and angle of ankle (b) in the four treatment groups are
depicted. ** P \ 0.01 and *** P \ 0.001 versus crush control, n = 6
1308 Neurochem Res (2009) 34:1304–1316
123
enhanced significant expression of neurofilament as com-
pared to non-treatment (211.3 ± 12.01 relative density/
mms2) (P \ 0.001 and P \ 0.001, respectively).
Furthermore, treatment with AFS ? HBO
(127,833 ± 30.57 relative density/mm2) produced higher
expression of neurofilament than either AFS (P \ 0.001) or
HBO (P \ 0.001) alone (Fig. 3).
The parameters of late nerve regeneration such as vac-
uole number, vascular staining, and myelination as
evidenced by the expression of S-100 are presented in
Table 1 and Fig. 4. Crush induced vacuole formation and
this event was reduced by the administration of AFS
(P \ 0.01) and HBO (P \ 0.01). The combined treatment
enhanced the results (P \ 0.001). The vascular density was
also attenuated by crush and the restoration was augmented
by AFS (P \ 0.01) and HBO (P \ 0.01). However, the
combined treatment reinforced the effect (P \ 0.001).
Crush induced less expression of S-100 and a significant
amount of S-100 expression was observed in AFS
(P \ 0.01) and HBO (P \ 0.01). The most beneficial res-
toration was observed in the combined treatment
(P \ 0.001). Based on the early expression of neurofila-
ment and late regeneration markers, treatment with either
AFS or HBO alone promoted greater nerve regeneration
than non-treatment; however, the combined treatment
stimulated more regeneration than either of the single
treatments.
Reduction of Apoptosis of AFS by HBO
Hoechst 33342-positive implanted AFS were found in the
retrieved nerve tissues 7 days after grafting. Apoptotic
AFS (8.43 ± 0.34%) were detected by the TUNEL-
Fig. 2 Electrophysiological evaluation. Electrophysiological exami-
nation, including CMAP (a) and conduction latency (b), was
conducted 4 weeks after injury in the four treatment groups. P values
for AFS, HBO, AFS ? HBO were determined relative to the crush
group. * P \ 0.05, ** P \ 0.01, and *** P \ 0.001, n = 6
Fig. 3 Determination of
neurofilament. The nerve tissues
were retrieved 7 days after
injury and were subjected to
immunohistochemistry with
antibodies against neurofilament
in the four treatment groups,
a Crush, b AFS, c HBO,
d AFS ? HBO. The relative
density of neurofilament is
depicted in e. P values for AFS,
HBO, and AFS ? HBO were
determined relative to the crush
group. ** P \ 0.01 and
*** P \ 0.001; n = 6. Barlength = 50 lm
Neurochem Res (2009) 34:1304–1316 1309
123
positive nuclei. The apoptosis of implanted AFS
(3.01 ± 0.27%) was attenuated by HBO treatment
(P \ 0.001) (Fig. 5). These findings indicate that one of
the beneficial effects of HBO may be strengthening the
viability of implanted AFS so as to prevent apoptosis.
No Increase of Lipid Peroxidation After HBO
Therapy
HBO has the potential to enhance the survival of tissue
graft. However, enhanced oxygen delivery may promote
the formation of oxygen-derived free radicals and cellular
lipid peroxidation. Such free radical damage could com-
promise AFS graft benefits. The activities of lipid
peroxidation presented as (MDA) equivalents in the four
groups were 1.36 ± 0.11, 1.05 ± 0.03, 1.34 ± 0.06, and
1.01 ± 0.02, respectively. No significant increase of lipid
peroxidation was observed in those groups treated with
HBO and HBO combined with AFS. This finding indicates
that HBO administration did not aggravate the oxidative
stress detrimental to AFS transplantation.
Attenuation of Inflammation by Concomitant
Treatment with AFS ? HBO
Over-activated inflammatory response is a detrimental
stress on the nerve tissues and is a potential cytotoxic factor
in the survival of implanted cells. Macrophages play the
central role of the pathogen in the peripheral nerves.
Clearance of macrophage deposits and inhibition of
inflammatory cytokines augmented the regeneration in
peripheral nerve crush injury. The immunohistochemical
results showed an accumulation of inflammatory cells in
the injured nerve tissues (32.33 ± 1.02/0.05 mm2). The
accumulation of these cells was not altered in the AFS
group (32.81 ± 1.89/0.05 mm2) (P = 0.8), whereas it was
markedly reduced in the HBO (15.11 ± 1.01/0.05 mm2)
(P \ 0.001) (Fig. 6c) and AFS ? HBO (13.01 ± 0.79/
0.05 mm2) (P \ 0.001) (Fig. 6d) groups.
Table 1 Distribution of inflammatory cells and results of histology
Time 7 days (counts/0.05 mm2) 28 days (counts/0.05 mm2)
Group M N T B Vacuoles Vascularity
Crush 32.33 ± 1.02 3.01 ± 0.41 5.56 ± 1.21 6.21 ± 1.41 211.01 ± 9.81 11.41 ± 1.41
AFS 32.81 ± 1.89*** 2.89 ± 0.39 4.98 ± 1.51 7.05 ± 2.1 132.1 ± 9.81** 22.1 ± 2.21**
HBO 15.01 ± 1.01*** 3.25 ± 0.85 5.49 ± 0.98 6.89 ± 1.41 157.01 ± 6.81** 19.23 ± 3.01**
AFS ? HBO 13.12 ± 0.79*** 2.79 ± 0.48 5.01 ± 0.89 7.54 ± 2.12 89.71 ± 6.45*** 32.01 ± 4.52***
M Macrophages; N neutrophils; T T cells; B B cells; Vacuoles vacuole counts; Vascularity vascularity counts
P value in AFS, HBO, and AFS ? HBO was relative to the crush group; ** P \ 0.01; *** P \ 0.001
Fig. 4 Expression of S-100.
The nerve tissues were retrieved
28 days after injury and were
subjected to
immunohistochemistry with
antibodies against S-100 in the
four treatment groups, a Crush,
b AFS, c HBO, d AFS ? HBO.
The relative density of S-100 is
depicted in e. P values for AFS,
HBO, and AFS ? HBO were
determined relative to the crush
group. ** P \ 0.01 and
*** P \ 0.001; n = 6. Barlength = 50 lm
1310 Neurochem Res (2009) 34:1304–1316
123
Monocyte chemoattractant protein 1 (MCP-1) and
RANTES are the important regulators of macrophage
response that lead to rapid myelin breakdown and clear-
ance in Wallerian degeneration. Inhibition of MCP-1 and
RANTES suppresses macrophage deposits and myelin
clearance. The expression of MCP-1 and RANTES was
down-regulated in those groups treated with HBO (groups
III and IV) (Fig. 6f, g), which was compatible with
decreased deposits of macrophages. No significant
expression of neutrophils or T and B cells was observed in
these four groups (Table 1). There was no significant dif-
ference in deposits of neutrophils among the four groups,
which was compatible with no discernable expression of
MPO activity (Fig. 6h).
On the other hand, crush injury triggered the production
of inflammatory cytokines, including IL-1b, IL-6, TNF-a,
and IFN-c (Fig. 7). The elevated production of IL-1b,
TNF-a, and IFN-c was attenuated in the HBO and
AFS ? HBO groups. Inducible inflammatory cytokines
were not abrogated by AFS transplantation alone. These
findings indicate that HBO but not AFS possesses an anti-
inflammatory effect.
Discussion
The delivery of AFS to the injured nerve is regarded as one
of the treatment strategies in peripheral nerve injury. Both
immunomodulation and neurotrophic factor secretion have
been postulated to exert effects on regeneration [6, 7].
However, the short-term survival of implanted cells
restricts their clinical application and reduces their efficacy
[26, 27]. Attenuation of inflammatory cell deposits and
associated inflammatory cytokines could rescue trans-
planted AFS from apoptosis and augment nerve
regeneration [23]. HBO has been shown to harbor anti-
apoptotic/anti-inflammatory effects [16–21]. In this study,
we found that the administration of HBO not only
decreased inflammatory cell infiltration and associated
chemokines, such as MCP-1 and RANTES, but also
attenuated the elevated production of inflammatory cyto-
kines, including TNF-a, IL-1b, and IFN-c, as well as
exerting an anti-apoptotic effect on implanted AFS.
Treatment with either AFS or HBO alone promoted better
nerve regeneration than that of control. Moreover, the
combination of HBO and AFS significantly augmented
peripheral nerve repair. The combined benefit has been
postulated to be due to the decreased production of cyto-
toxic inflammatory mediators and increased survival of
implanted AFS modulated by HBO.
A sciatic nerve crush injury is regarded as a hypoxic
injury model, as shown by the consistent improvement of
nerve regeneration by administration of HBO [28]. How-
ever, concerns have been raised that HBO may cause
increased oxidative stress through the production of reac-
tive oxygen species [29]. Several studies using HBO at 2.5
ATA or greater have found some beneficial of increased
oxidative stress [30–32]. Support for this higher pressure
effect was found in one study, which demonstrated that
HBO at 2.0 ATA increased SOD levels, whereas HBO at
3.0 ATA caused SOD levels to decrease, presumably
because SOD was able to neutralize more free radicals at
3.0 ATA pressure. Furthermore, oxidative stress appears to
be less of a concern at pressure under 2.0 ATA, which is
the setting normally used clinically [33]. Another point
concerning the oxidative stress is the treatment time. In an
Fig. 5 Determination of apoptosis. The nerve tissues were retrieved
7 days after injury and were subjected to apoptotic assay by TUNEL
in the AFS (a) and AFS ? HBO (c) groups. The implanted AFS
within the corresponding areas was demonstrated by the positivity of
Hoechst 33342 in the AFS (b) and AFS ? HBO (d) groups.
Quantitative analysis of TUNEL test is depicted in e.
*** P \ 0.001, n = 6. Bar length = 50 lm. The vertical axis
represents the percentage of positive TUNEL assay
Neurochem Res (2009) 34:1304–1316 1311
123
ischemic-perfusion rat model, there was no significant
increase of oxidative stress such as thiobarbituric acid-
reactive substance (TBARS), superoxide dismutase (SOD),
and glutathione peroxidase (GSH-Px) when the animals
were subjected to HBO therapy at 3.0 ATA with treatment
time less than 60 min [34]. A treatment window to enhance
the HBO effect is mandatory to conduct therapy after the
injury. The therapeutic window for effective HBO treat-
ment could be delayed up to 12 h after hypoxic-ischemic
brain damage, but the effectiveness decreases 24 h after
such damage [16]. The HBO treatment regimen for sciatic
nerve injury varies from 1 to 10 days with HBO being
administered one to four times per day. The pressure usu-
ally ranges from 1.5 to 3.5 ATA. Previous studies
attempted to balance the efficacy of treatment and possible
adverse effects by using an HBO treatment paradigm of
100% oxygen, 2 ATA, 60 min treatment/day, beginning
12 h after injury. Thus, no escalation of oxidative stress,
such as TBARS, was observed and enhanced nerve
regeneration was found, suggesting the viability of HBO
treatment as an adjuvant therapy in sciatic nerve injury
either alone or combined with stem cell therapy.
Several animal studies have disclosed that HBO has a
potent anti-inflammatory tissue effect [19, 20]. One study
used HBO at 2.4 ATA and 100% oxygen, which is
equivalent to diclofenac 20 mg/kg [35]. HBO has also been
shown to decrease the symptoms of advanced arthritis in
rats [36], and it attenuated the inflammatory response in the
peritoneal cavity caused by injected meconium [37]. In
addition, one study using HBO at 2.5 ATA showed
increased survival and decreased proteinuria, anti-dsDNA
antibody titers, and immune-complex deposition in lupus-
prone autoimmune mice [38]. Hence, HBO could be used
as an adjuvant therapy for various inflammatory conditions,
including necrotizing soft tissue infection, gas gangrene,
refractory osteomyelitis, burns, and chronic wounds.
Fig. 6 Determination of inflammatory cells and associated cytokines.
The nerve tissues were retrieved 7 days after injury and were
subjected to immunohistochemistry with antibodies against CD68 and
RT-PRC for determination of MCP-1 and RANTES, and enzyme
activity of MPO in the four treatment groups. Deposits of macrophage
in a Crush, b AFS, c HBO, d AFS ? HBO. Results of quantitative
analysis of inflammatory cells, MCP-1, RANTES, and MPO activity
are depicted in e, f, g, and h, respectively. *** P \ 0.001 versus the
crush group, n = 6. Bar length = 50 lm
1312 Neurochem Res (2009) 34:1304–1316
123
Although HBO has been shown to enhance some aspects of
host defense, its overall effect appears to be immunosup-
pressive [39]. More specifically, HBO can impair
macrophage function [40]. In this study, we found
decreased deposits of macrophages over the injured nerve
treated with HBO alone or combined with AFS treatment.
Concerning the immune cell deposits such as T and B cells,
there was no significant discrepancy in immune cell
accumulation. This phenomenon was compatible with the
effect of HBO mainly on the alternation of macrophage
migration.
The monocyte-macrophage is an important source of IL-
1b and TNF-a. Several studies showed that monocyte-
macrophages isolated from HBO-exposed rodents pro-
duced fewer pro-inflammatory cytokines than exposed
control groups [41, 42]. Monocyte-macrophages obtained
from patients with Crohn’s disease secreted less IL-1, IL-6,
and TNF-a in response to LPS when isolated after HBO
therapy than cells obtained prior to the treatment [43].
Furthermore, the expression of pro-inflammatory cytokine
genetic profiles in macrophages was suppressed by HBO
therapy [21]. In this study, pro-inflammatory cytokines
such as TNF-a, IL-1b, and IFN-c were suppressed by the
administration of HBO therapy. The inhibition of inflam-
matory cytokines paralleled the decreased deposits of
macrophages in the injured nerve, which further confirmed
the anti-inflammatory effect of HBO through monocyte-
macrophage depletion.
After crush injury, recruitment of macrophages into the
endoneurium is essential for nerve regeneration. Monocyte
chemoattractant protein 1 (MCP-1) and RANTES are the
important regulators of macrophage response that lead to
rapid myelin breakdown and clearance in Wallerian
degeneration. Inhibition of MCP-1 and RANTES sup-
presses macrophage deposits and myelin clearance. The
expression of MCP-1 and RANTES occurs mainly in
Schwann cells in the early stage (less than 5 days after
injury) and in macrophages in the late stage (more than
5 days after injury [44]. In this study, there were two
possible reasons for this phenomenon. First, HBO inhibited
the expression of MCP-I and RANTES mainly in the
recruitment macrophages (7 days after injury), which
prohibited further deposits of macrophages. Second,
depletion of macrophage deposits by HBO reflected the
low titers of MCP-1 and RANTES detected in injured
tissue. Further studies are needed to determine which of the
two reasons is more plausible.
Nerve injury initiates inflammatory response and indu-
ces expression of pro-inflammatory cytokines such as TNF-
a, IL-1b, and IFN-c [25, 46]. Inflammatory cells and
inflammatory mediators not only cause tissue damage and
secondary injury but also play a role in the regenerative
process. In regard to cell transplantation, an alternative role
for inflammatory cytokines is to be an important determi-
nant of the survival and fate of implanted cells. In our
previous study, a significant accumulation of inflammatory
cells was detected in the injured sites after nerve crush
injury. The injured nerve tissues produced elevated levels
of pro-inflammatory cytokines, including TNF-a, IL-1b,
IL-6, and IFN-c. Attenuation of inflammatory cytokines
and associated inflammatory cells paralleled the decreased
apoptosis of transplanted stem cells in nerve crush injury
[23]. However, the expression of inflammatory cytokines is
a necessary response to nerve crush injury. In the early
stage, the production of inflammatory cytokines is detri-
mental to nerve regeneration. In the late stage, the
inflammatory cytokines are crucial in the recruitment of
immature Schwann cells and accelerated rapid migration of
Schwann cells [45]. It is argued that suppression of
inflammatory cytokines may hinder nerve regeneration in
the late stage. Even though inflammatory cytokines can be
suppressed, a significant amount can still be retrieved from
the crush site, which may support nerve regeneration in the
late stage [23]. In this study, decreased transplanted cell
apoptosis was in line with decreased inflammatory cell
deposits and pro-inflammatory cytokines. Thus, the anti-
inflammatory effect of HBO resulted in decreased trans-
planted AFS apoptosis, which in turn decreased
inflammatory cell deposits and subsequently inhibited the
secretion of inflammatory cytokines.
During nerve regeneration, myelination of the nerve
fiber is a marker to determine the intensity of nerve
regeneration [46]. Neurofilament expression is the early
evidence of nerve regeneration potential [18, 47]. The
amount of S-100 immunoreactivity in myelinated fibers
appears to correlate directly with the thickness of the
Fig. 7 Determination of pro-inflammatory cytokines. The left sciatic
nerve of rats was injured by crushing. The injured nerves (10 mm) in
the different treatment groups were retrieved 7 days after injury and
subjected to ELISA for the determination of TNF-a, IL-1b, IL-6, and
IFN-c. *** P \ 0.001, n = 6. P value in the crush group was
determined relative to the normal group and P values in the AFS,
HBO, and AFS ? AFS groups were determined relative to the crush
group
Neurochem Res (2009) 34:1304–1316 1313
123
myelin sheath formed by Schwann cells [48]. Further,
vacuole formation and vascular staining reflect the inten-
sity of nerve regeneration [23]. Hence, in this study, we
utilized neurofilament and the expression of immunoreac-
tivity of S-100 as an early marker and vacuole counts or
vascular staining as a late marker to evaluate the intensity
of nerve regeneration. Based on the expression of nerve
regeneration markers, neurobehavioral studies, and elec-
trophysiological parameter alternation, we found that either
HBO or AFS transplantation alone had a similar effect, but
the combined treatment showed the most beneficial results.
There is some controversy about whether nerve regenera-
tion occurs solely through the proliferation of resident
Schwann cells or through the involvement of other cells.
According to a study that investigated the migration of
transplanted cells surrounding the injured nerve, there was
no evidence of stem cells migrating into the host nerve
tissue [7]. On the other hand, HBO may have the potential
to mobilize bone marrow progenitor cells, especially CD34
progenitor cells, which may be able to differentiate into
Schwann cells and become integrated into the nerve tissue
[12, 13]. Up to now, few studies have addressed the
potential of CD34 progenitor cells to differentiate into
Schwann lineage cells. Hence, the mobilization of CD34
progenitor cells differentiating into Schwann cells has not
been regarded as a potential mechanism in nerve regener-
ation. In this study, nerve regeneration following treatment
with HBO, AFS, or the combined regimen was attributed to
endogenously proliferated Schwann cells.
To date, the mechanism of AFS involved in nerve
regeneration has not been determined. However, there are
several possible candidates, including immunomodulation,
secretion of neurotrophic factors, and integration of AFS as
part of the host nerve tissue [6, 7, 49]. In our previous
study, amniotic fluid mesenchymal stem cells were found
to have a high mRNA expression of neurotrophic factors
such as GDNF, BDNF, CNTF, and NT-3. Expression of
NT-3 and CNTF was demonstrated in transplanted AFS as
well as in the nerve tissue [7]. Furthermore, there was no
evidence of S-100 positive cells in transplanted cells or
alternation of immune cells adjacent to the crushed nerve
(data not shown). Hence, the paracrine effect of AFS on
nerve regeneration was regarded as the most likely
mechanism.
Spontaneous recovery of motor function in a sciatic
nerve crush injury model was reported several decades ago
and again recently [46, 50]. These studies concluded that
there was a tendency toward spontaneous recovery in some
cases. If a model of serious nerve damage such as nerve
transaction is not used, the evaluation of nerve regeneration
after HBO, AFS, or combined treatment becomes sheer
speculation [51]. In our previous studies, we found that
nerve regeneration in a crush experimental model did not
achieve full recovery and serious neurological deficits
remained at 4 weeks after injury [7, 23]. Using a cut-off
point of 4 weeks for neurological deficits, electrophysio-
logical parameters, and even more importantly for nerve
myelination, we found a significant difference between
groups, which could be considered as a valuable indicator
to evaluate any treatment of nerve regeneration. Further-
more, transection model got involved in the suture
technique and materials regarded as an inflammatory pro-
voked agent, which may also interfere the interpretation the
inflammatory response [52]. For the sake of simplicity in
investigating the effects of HBO on the survival of trans-
planted AFS, the crush model was used in this study.
Hoechst 33342 has been used as exogenous marker for
cell transplantation. However, a major concern with the
extrinsic labeling method is that the label could be diluted
with cell division or could be redistributed from the
transplanted cells to host cells [7, 53, 54]. The dispropor-
tion of Hoechst 33342 among the transplanted cells and
host tissue found in our study should help to dispel this
concern. The characteristics of this abnormal uptake by
host tissue usually occurred in areas remote from or adja-
cent to the margin of the transplanted site. Furthermore, the
expression of this abnormal signal occurred in the late
stage (more than 2 weeks after injury), and this signal was
weaker than that in the transplanted cells.
In this study, we did not clearly define what percentages
of Hoechst 33342-positive cells were AFS 1 week after the
transplantation. But we found that Hoechst 33342-positive
cells were confined to the epineuria area without pene-
trating into the crushed nerve, and they expressed strong
immunoreactivity without decaying, which implied that
most of the Hoechst 33342-positive cells were AFS. For
more precise tracking of the fate of transplanted cells,
endogenous markers such as anti-human nuclear antigens
or anti-Y chromosome antigens are preferable.
Conclusion
Hyperbaric oxygen alleviated the apoptosis of transplanted
cells in sciatic nerve injury and further escalated the nerve
regeneration. The augmentation of nerve regeneration by
HBO was mainly through anti-inflammatory effects by
arresting the recruitment of macrophage deposits. The
decreased transplanted stem cell apoptosis justifies using
HBO as an adjuvant therapy in stem cell transplantation.
Acknowledgments This study was supported by grants from the
National Science Council (NSC 96-2314-B-075A-001) and Taichung
Veterans General Hospital (TCVGH-964906D and TCVGH-
PU968102), Taiwan, ROC. We also thank the Biostatistics Task
Force of Taichung Veterans General Hospital for help with statistical
analysis.
1314 Neurochem Res (2009) 34:1304–1316
123
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