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: fucheng@vghtc.gov.tw

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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