J Neurosurg Spine 20:578584, 2014

578
AANS, 2014
J Neurosurg: Spine / Volume 20 / May 2014
E
PIDEMIOLOGICAL data indicate that various risk
factors predispose individuals to low-back pain
(LBP). The mechanisms of the injury may be con-
fusing, and the studies supporting these fndings are vari-
able and conficting regarding most environmental risks.
Furthermore, there are some factors, including emotional
circumstances, that heavily infuence back disability, yet
the cause of back disability is still unknown.
37,40
Stress is a state of the body, or it is the mental tension
that results from factors that tend to alter an existing equi-
librium. Stress is an unavoidable effect of daily life and
is an especially complex phenomenon in modern societ-
ies. Emotional stress causes biochemical changes in the
blood that affect different systems in the body. Stress has
been linked to coronary artery disease, immune system
suppression, psychosomatic disorders, and various other
mental and physical problems (http://encyclopedia2.the
freedictionary.com/Stress). Signs of stress may be cogni-
tive, emotional, physical, or behavioral. Stress is a predic-
tor of low-back disability for persons with previous LBP
but not for those without previous LBP.
2
Patients with acute stress usually have high levels of
catecholamines.
5
Stress increases proinfammatory cyto-
kine levels (that is, tumor necrosis factor and interleukin-
1a and -1b) in the periphery and brain.
22
Elevated levels
of infammatory mediators have been described in patho-
logical intervertebral disc (IVD) tissue, and these levels
increase with the severity of the degeneration.
39
There-
fore, emotional stress may also infuence IVD.
Because the effects of chronic stress on disc degen-
Induction of intervertebral disc cell apoptosis and
degeneration by chronic unpredictable stress
Laboratory investigation
HAMED REIHANI KERMANI, M.D.,
1
HAMID HOBOUBATI, M.D.,
1

SAEED ESMAEILI-MAHANI, PH.D.,
2
AND MAJID ASADI-SHEKAARI, PH.D.
3
Departments of
1
Neurosurgery and
3
Electron Microscopy, Neuroscience Research Center,
Neuropharmacology Institute, Kerman University of Medical Sciences; and
2
Department of Biology,
Faculty of Science, Shahid Bahonar University of Kerman, Iran
Object. The purpose of this study was to evaluate the effects of chronic unpredictable stress on the intervertebral
discs of rats.
Methods. The cellular events involved in injury- and stress-induced disc degeneration were investigated in male
Wistar rats. Disc degeneration and apoptosis were evaluated using microscopic (light and electron) and molecular
(immunoblotting and immunohistochemistry) methods. Corticosterone levels were used as markers of stress and
measured by radioimmunoassay.
Results. The data gathered in this study showed that chronic unpredictable stress can signifcantly increase
corticosterone levels. Furthermore, biochemical markers of apoptosis (that is, increases in the Bax/Bcl2 ratio and
TUNEL reactivity [p < 0.05]) were observed in the stressed animals. Electron and light microscopy also showed disc
degeneration and apoptotic cells in the experimental groups.
Conclusions. Taken together, these data demonstrated that chronic stress is most likely to be a risk factor for
creating intervertebral disc degeneration and that programmed cell death may be one of the mechanisms of stress-
induced disc degeneration.
(http://thejns.org/doi/abs/10.3171/2014.1.SPINE13466)
KEY WORDS apoptosis chronic unpredictable stress degenerative disc
intervertebral disc
This article contains some figures that are displayed in color
on line but in black-and-white in the print edition.
Abbreviations used in this paper: CUS = chronic unpredictable
stress; IVD = intervertebral disc; LBP = low-back pain; TBS-T =
Tris-buffered saline with Tween 20; TdT = terminal deoxynucleo-
tidyl transferase.
Chronic unpredictable stress and intervertebral disc
579 J Neurosurg: Spine / Volume 20 / May 2014
eration have not yet been clarifed, the present study was
designed to investigate the degenerative effects of chronic
unpredictable stress (CUS) on the IVDs of rats.
Methods
Experimental Animals
All experiments were performed on male adult Wis-
tar rats that weighed 300350 g and were housed 4 per
cage on a 12-hour light/dark cycle in a temperature-con-
trolled room (22 1C). Food and water were available.
The experimental procedure was approved by the Animal
Experimentation Ethics Committee of the Kerman Neu-
roscience Research Center. The animals were randomly
categorized into 4 groups. Each group included 10 rats, of
which 6 were studied with molecular methods and 4 were
studied with microscopic methods.
Study Design
Group 1 underwent both disc injury (induced degen-
eration) and CUS (Group 1: degenerated/CUS); Group
2 experienced only CUS (Group 2: CUS); Group 3 was
only subjected to disc injury (Group 3: degenerated); and
Group 4 was used as a control (Group 4: neither CUS
nor disc injury). Both normal and degenerated discs were
investigated to study the impact of stress on IVD degen-
eration. Thus, we also assessed the trends of expedition
and enhancement of degeneration. Intervertebral disc de-
generation was induced in Groups 1 and 3 in the frst 3
levels of the rats tails according to the method of Han et
al.
13
Briefy, the skin corresponding to the tail vertebrae
was marked with a permanent marker. Next, the rats were
weighed and injected intraperitoneally with 10% chlo-
ral hydrate (Sigma Aldrich) at a dose of 3.5 ml/kg body
weight. Tail skin was sterilized with iodinated polyvinyl-
pyrrolidone, and the tail vertebral discs were punctured
through the skin with a 20-gauge sterile needle that was
inserted into the tail in the dorsal-to-ventral direction
through the center of the disc until the skin on the oppo-
site side of the tail was ruptured. Extreme care was taken
to ensure that the needles were perpendicular with the
skin. Palpation of the adjacent vertebrae ensured that the
needle was inserted into the centers of the discs. The nee-
dle punctured the entire disc and was then rotated 360
and held in position for 30 seconds before extraction.
Then, the discs were examined after exposure to CUS.
The animals were exposed to CUS by using a modifed
version of the method recommended by Munhoz et al.
25

and Ortiz et al.
27
Over the next 14 days the procedures listed in Table
1 were repeated; thus, the rats were exposed to CUS over
a 28-day period.
On experiment days, 30 minutes after stress induc-
tion, blood samples were collected in tubes containing 5%
EDTA. Plasma samples were obtained by centrifuging
the blood at 2500 rpm for 10 minutes. The samples were
frozen immediately at -20C and stored until the time of
the corticosterone assay. Plasma corticosterone levels were
measured using a commercial radioimmunoassay kit for
rats (DRG International, Inc.). The sensitivity of the assay
was 0.25 ng/ml, and the antibody cross-reacted 100% with
corticosterone, 0.34% with desoxycorticosterone, and less
than 0.10% with other steroids. At the end of the CUS pe-
riod, all rats were killed, and discs were separated from
vertebral endplates under microscopic magnifcation.
Immunoblot Analysis
Discs were homogenized in ice-cold buffer contain-
ing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1% sodium
dodecyl sulfate, 0.1% sodium deoxycholate, 1% NP-40
with protease inhibitors (1 mM phenylmethylsulfonyl
fuoride, 2.5 g/ml leupeptin, 10 g/ml aprotinin), and 1
mM sodium orthovanadate. The homogenate was centri-
fuged at 14,000 rpm for 15 minutes at 4C. The result-
ing supernatant was retained as the whole cell fraction.
Protein concentrations were measured using the Brad-
ford method (Bio-Rad Laboratories). Equal amounts of
protein were resolved electrophoretically on 9% sodium
dodecyl sulfatepolyacrylamide gel electrophoresis gels
and transferred to nitrocellulose membranes (Hybond
ECL, GE Healthcare Bio-Sciences Corp.). After block-
ing overnight at 4C with 5% nonfat dried milk in Tris-
buffered saline with Tween 20 (TBS-T) (blocking buf-
fer, TBS-T, 150 mM NaCl, 20 mM Tris-HCl [pH 7.5],
0.1% Tween 20), the membranes were probed with rabbit
monoclonal antibodies for Bax ( 21: sc-6236) and Bcl-2
(C-2: sc-7382, Santa Cruz Biotechnology, Inc.) at 1:1000
for 3 hours at room temperature. After washing in TBS-
T (3 times, 5 minutes each), the blots and a horseradish
peroxidaseconjugated secondary antibody (1:15,000,
GE Healthcare Bio-Sciences Corp.) were incubated for
60 minutes at room temperature. All the antibodies were
diluted in blocking buffer. The antibody-antigen com-
plexes were detected using an ECL system and exposed
to Lumi-Film chemiluminescent detection flm (Roche).
Lab Work software (UVP, LLC) was used to analyze the
TABLE 1: Chronic unpredictable stress performed in
experimental groups (20 rats) over two 14-day periods*
Day Experiment Duration
1 (2 p.m.) restraint 60 min
2 (9 a.m.) forced swim 15 min
3 (3 p.m.) cold isolation 90 min
4 (7 p.m.) lights on overnight
5 (10 a.m.) forced swim 5 min
6 (7 p.m.) water/food deprivation overnight
7 (2 p.m.) restraint 120 min
8 (3 p.m.) light off 120 min
9 (9 a.m.) forced swim 5 min
10 (7 p.m.) lights on overnight
11 (2 p.m.) cold isolation 90 min
12 (9 a.m.) restraint 60 min
13 (7 p.m.) water/food deprivation overnight
14 (9 a.m.) restraint 60 min
* Animals were exposed to CUS over 28 days (14 2 = 28).
H. Reihani Kermani et al.
580 J Neurosurg: Spine / Volume 20 / May 2014
expression intensities, and b-actin immunoblotting (the
antibody was obtained from Cell Signaling Technology,
Inc.; 1:1000) was used as a loading control.
Histopathological Examination
Staining With the TUNEL Method. Tissue sections
were dewaxed by heating at 60C and were then irrigat-
ed with xylenes and rehydrated using ethanol and deion-
ized water. This process was followed by 15 minutes of
incubation in proteinase K solution (10 g/ml, Boehringer
Mannheim) at room temperature. Endogenous peroxidase
was inactivated with 2% H
2
O
2
. After a short equilibration
in terminal deoxynucleotidyl transferase (TdT) buffer, the
sections were incubated for 60 minutes with TdT and bioti-
nylated deoxyuridine triphosphate in TdT buffer in a 37C
humidifed chamber. The slides were thoroughly washed,
and the sections were covered with ExtrAvidin peroxi-
dase. Color was developed by exposing the sections to a
33-diaminobenzidine solution containing H
2
O
2
. For nega-
tive staining controls, either TdT or ExtrAvidin peroxidase
was omitted. The slides were then dehydrated, cleared, and
mounted. The numbers of positively stained cells (dark
brown) were quantifed in 3 noncontiguous felds of the
nucleus pulposus for each sample (12 total felds).
Light Microscopy. The IVDs were fxed in 10% for-
malin, decalcifed using 10% standard decalcifying solu-
tion (10% EDTA for 48 hours), washed with running tap
water, and fxed again in the same fxative. The fxed, de-
calcifed discs were embedded in paraffn and sectioned
(5 mm thick). The sections were stained with H & E.
Electron Microscopy. The specimens were fxed se-
quentially in buffered 2.5% glutaraldehyde (Merck) for
24 hours, decalcifed using 10% standard decalcifying so-
lution (10% EDTA) for 48 hours, washed in 0.1 M phos-
phate-buffered saline, and postfxed in OsO
4
1% sodium
phosphate-buffered saline 0.1 M buffer (pH 7.4) at room
temperature for 1 hour. After dehydration in increasing
concentrations of ethanol, the specimens were embedded
in Epon 812 resin (TAAB Laboratories Equipment Ltd.).
Ultrathin sections were cut at 70 nm, stained with lead
citrate and uranyl acetate 2%, and viewed with a Philips
(EM300) electron microscope.
Statistical Analysis
The results are expressed as the mean SEM. The
Bax/Bcl-2 and b-actin band density values were obtained
by band densitometry. These values are expressed as the
tested protein/b-actin ratio for each sample. The apoptot-
ic cell values are expressed as the percentage of positive-
ly stained cells. Averages were compared across groups
with ANOVAs, followed by Newman-Keuls and post hoc
Tukey tests. A p value < 0.05 was considered signifcant.
Results
Corticosterone Assay Findings
The plasma corticosterone levels were 424.50 69.45
ng/ml after CUS and 249.86 42.46 ng/ml in the control
group (mean SEM); this difference was signifcant (p <
0.05; Fig. 1).
Biochemical Findings
The mean Bax/Bcl2 ratios were compared across
groups (Fig. 2). The Bax/Bcl2 ratio of the degenerated/
CUS group (Group 1) was signifcantly greater than that
of the control group (p < 0.01). The Bax/Bcl2 ratios of the
CUS and degenerated groups (Groups 2 and 3, respective-
ly) were also elevated compared with that of the control
group (p < 0.05). The Bax/Bcl2 increase in the degener-
ated/CUS group was not signifcantly different from that
of the degenerated group. Thus, cell apoptosis in the discs
was signifcantly increased by CUS, but this process was
not intensifed in degenerated discs after CUS.
Histopathological Findings
Staining With the TUNEL Method. Apoptotic cells
were examined using the TUNEL method (Fig. 3). The
degenerated, CUS, and degenerated/CUS groups exhib-
ited positive cell rates of 43%, 52%, and 69%, respective-
ly (Fig. 4). The difference between the degenerated and
control groups was signifcant (p < 0.05). The difference
between the CUS and degenerated/CUS groups and the
control group was more obvious (p < 0.001), and the dif-
ference between the degenerated/CUS and CUS groups
was not statistically signifcant.
Light Microscopy. Light microscopic evaluation of
the tail discs revealed normal and well-organized anuli f-
brosi and nuclei pulposi in the control group. In the exper-
imental groups, the structures of the IVDs showed some
degenerative changes such as the dissociation of multilay-
ered structures, the loss of normal fbrillation of the carti-
lage, and vascular proliferation. The laminar structure of
the anuli fbrosi disappeared, and the fbers were disorga-
nized and fragmented. The cells of the nucleus pulposus
were sparse and reduced in number (Fig. 5).
Electron Microscopy. Normal ultrastructure was ob-
served in the cells of the control group on electron mi-
croscopic survey. The organelles were intact within the
cytoplasm. The nuclear membranes were smooth, and
FIG. 1. Bar graph showing plasma corticosterone concentration in
control and stressed rats (n = 10 rats per group). Each bar and whisker
represents the mean SEM, respectively (*p < 0.001 vs control).
Chronic unpredictable stress and intervertebral disc
581 J Neurosurg: Spine / Volume 20 / May 2014
chromatin was dispersed throughout the nuclei. Small
and irregular projections on the surfaces of the cells gave
these cells a scalloped appearance (Fig. 6A and B). In the
CUS group, the shapes of anuli and nuclei of the cells
were irregular and exhibited signs of apoptosis. Disconti-
nuities of the plasma membranes were not observed in the
bizarrely shaped cells, but the nuclear membranes were
disintegrated. The nuclei had irregular shapes, and chro-
matin margination was evident. The cellular organelles
showed some evidence of degeneration, including dilated
rough endoplasmic reticulum and Golgi cisterns. The f-
bers of the anuli fbrosi were not packed densely and were
also disorganized. The most noticeable fnding in the
CUS group was the presence of many bizarrely shaped
cells that were not observed in the other groups. Apop-
totic bodies were obvious within the cytoplasm (Fig. 6C
and D).
Discussion
In this study we found CUS-induced IVD degenera-
tion in rats and confrmed this fnding with molecular
and structural surveys. Because the molecular and im-
munohistochemical assays were the main methods of our
survey, the results of these assays were presented quanti-
tatively. Because complementary histopathological stud-
ies were also performed, the results of these studies were
presented descriptively and subjectively.
Intervertebral disc degeneration is caused by abnor-
malities in collagen, vascular ingrowths, abnormal pro-
teoglycans, local infammation, and ultimately apoptosis.
FIG. 5. Photomicrographs showing normal anulus fbrosus (B) and
nucleus pulposus (A) of the rat IVD, and an anulus (D) and a nucleus (C)
after CUS. Note the dissociation of the multilayered structure and the
loss of normal fbrillation. H & E, original magnifcation 400.
FIG. 4. Bar graph showing percentage of apoptotic cells in different
groups (*p < 0.05 vs control, ***p < 0.001 vs control).
FIG. 3. Photomicrographs of nucleus pulposus cells stained with the
TUNEL method. The number of TUNEL-positive cells increased after
CUS. Morphometric study showed signifcant differences in TUNEL-
positive cells in the stress groups (lower) compared with the control
group (upper). Original magnifcation 400.
FIG. 2. Bar graph showing results of Western blot analysis of Bax/
Bcl2 protein across experimental groups. Each value in the graph rep-
resents the mean SEM band density ratio for each group (*p < 0.05,
**p < 0.01 compared with control group).
H. Reihani Kermani et al.
582 J Neurosurg: Spine / Volume 20 / May 2014
Apoptosis is an active process that is associated with
programmed cell death.
6,38
In vivo and in vitro studies
have indicated that apoptosis has a central function in
the degenerative process.
4
The importance of apoptosis
in the diverse diseases that are associated with disc de-
generation has been extensively reviewed.
1
The Bcl2 gene
has antiapoptotic effects and is inversely related to the
Bax gene, which promotes apoptosis.
11,20
Hence, the Bax/
Bcl2 expression ratio can be strongly suggestive of cell
apoptosis.
36
In our study, the elevated Bax/Bcl2 ratio of
the CUS-only group compared with that of the control
group indicated the induction of apoptosis. The results of
TUNEL staining also confrmed apoptosis in the stress
groups. The electron microscopic fnding of bizarrely
shaped cells that were only observed in the stress groups
favors the molecular data. These fndings show that stress
can somehow induce disc degeneration.
As expected, the Bax/Bcl2 ratios and positive cell per-
centages were elevated in the degenerated group, but nei-
ther the Bax/Bcl2 ratios nor the percentages of apoptotic
cells were different between the degenerated/CUS group
and the degenerated group. This latter fnding indicates
that stress did not have signifcant degenerative effects on
discs that were already degenerated. Therefore, it seems
that the normal discs were more vulnerable to the effects
of chronic stress than the degenerated ones. It is possible
that the effects of needle puncture induced enough degen-
eration that the effects of stress could not be observed. An-
other presumable explanation for this fnding is that some
unknown defensive mechanisms may be activated to inhib-
it further degeneration after its induction, which prevents
stress from causing further degeneration.
The mechanism by which stress induced disc de-
generation is still unknown. Stress causes biochemical
changes in the blood that affect different systems in the
body. When the adaptive system is switched on and off ef-
fciently, the body is able to recover from imposed stress.
However, when the system is activated repeatedly or the
activity is sustained (for example, by chronic or exces-
sive stress), an allostatic load is generated that can lead to
disease over long periods of time.
30
In adult males, stress
decreases plasma testosterone and fertility.
9
Stress in rats
increases corticotropin-releasing hormone and arginine-
vasopressin levels in the plasma via the hypothalamus-
pituitary-adrenal axis. Elevated corticotropin-releasing
hormone increases the risk of osteoporosis, suppresses
the immune system, and increases blood sugar through
cortisol in humans and corticosterone in rats. Moreover,
elevated arginine-vasopressin elevates blood pressure and
disturbs the water/salt balance through peripheral vascu-
lar resistance.
21
Stress increases serum prolactin in rats.
15

Increases in growth hormone occur after stressful pro-
cedures.
3
Acute stress increases human blood catechol-
amines, which, in turn, causes myofbrillar degeneration,
necrosis of heart muscle, and dopaminergic neuron de-
generation.
5,16,26
Stress can change the structure and func-
tion of different brain regions.
29

Other various effects of stress include exacerbation
of liver, skin, gastrointestinal, pulmonary, oncological,
otological, and skeletal diseases.
7,14,17,23,24,28,32,33
Suscep-
tibility to infections, autoimmune disorders, and tumor
progression are strongly infuenced by the activities of
the endocrine and nervous systems in response to stress-
ful stimuli.
8
Chronic stress increases plasma melatonin
concentrations in rats, and serum melatonin levels are
affected by the disc degeneration process in patients
with IVD herniations.
34
Won et al.
41
investigated the ef-
fects of hyperglycemia on notochordal cell apoptosis and
IVD degeneration in diabetic rats. These authors suggest
that diabetes is associated with premature and excessive
apoptosis of notochordal cells of the nucleus pulposus,
which accelerates the transition from a notochordal to a
fbrocartilaginous nucleus pulposus, which in turn leads
to early IVD degeneration.
The biochemical changes that accompany IVD de-
generation include altered expression of both matrix me-
talloproteinases and proinfammatory cytokines.
35
Ele-
vated levels of molecular mediators of infammation have
been described in pathological IVD tissue, and these
levels increase with increasing grades of degeneration.
39

Such fndings have been observed for both interleukin-1
and tumor necrosis factora, both of which have estab-
lished roles in regulating nitric oxide and prostaglandin
production, metalloproteinase expression, and apopto-
sis. Metalloproteinases exert direct and indirect effects
through the promotion of neovascularization in the IVD.
FIG. 6. Electron photomicrographs showing IVD cells. A and B:
Normal ultrastructure in the control group. The nuclear membrane is
smooth, and chromatin is dispersed throughout the nucleus. Cytoplasm
organelles are intact. Small and irregular projections on the surface of
the cell produce a scalloped appearance. C and D: Nuclear deforma-
tion with chromatin condensation in the stressed group. Note the dilated
rough endoplasmic reticulum and Golgi cisterns. Original magnifcation
11,500 (A), 15,500 (B and C), and 19,000 (D).
Chronic unpredictable stress and intervertebral disc
583 J Neurosurg: Spine / Volume 20 / May 2014
This complex effect acts both in disc matrix degeneration
and in the pain generated by contact between the protrud-
ing disc and the nerve roots. All of these changes may
contribute to the progressive degeneration of the IVD.
31
Our data showed that CUS increases plasma cor-
ticosterone levels. It seems that the CUS induced IVD
programmed cell death and degeneration through corti-
costerone secretion. However, the actual linkage between
stress-induced elevation in corticosterone and disc degener-
ation is an important issue that needs to be considered. Nu-
merous scientifc reports have indicated that stress-induced
corticosterone secretion is responsible for the induction
of apoptosis in stressful situations in laboratory animals.
It has been reported that restraint stress results in signif-
cant attenuation of the phosphatidylinositol 3kinase/Akt
signaling pathway and increased apoptosis in rat skeletal
muscle.
10
Additionally, prolonged exposure to stress-level
glucocorticoids causes neuronal cell apoptosis through the
activity of caspase-9 and caspase-3.
18
Furthermore, chronic
isolation stress leads to cytochrome cmediated activation
of caspase-3 and apoptotic cell death in the prefrontal cor-
tex.
11
Li and colleagues reported that a single episode of
prolonged stress increased the plasma corticosterone level,
altered the Bcl-2/Bax ratio, and induced neuronal apopto-
sis in the rat medial prefrontal cortex.
19
The results of our study also showed that chronic
stress that is accompanied by corticosterone release pro-
motes the proapoptotic factor Bax and, eventually, IVD
apoptosis. Additionally, high circulating levels of cat-
echolamines, matrix metalloproteinases, proinfamma-
tory cytokines, and serum melatonin are linked to both
stress and degeneration.
12,34
According to our data and
the above-mentioned reports, we conclude that there is
a probable link between stress and disc degeneration.
Stress may infuence the discs through all of the above-
mentioned mechanisms.
The limitations of this study are as follows: we were
unable to evaluate symptomatic IVD degeneration in rats
(because we had no access to rats with spontaneously de-
generated discs with which to evaluate the effect of stress
on the discs), and there are no existing methods to induce
occupational stress in animals. Finally, it is diffcult to
extrapolate these nonprimate results to humans.
Conclusions
Overall, based on our results, stress is most likely
to be a risk factor for the degeneration of IVDs, but the
mechanism of this effect and its impact on the escalation
of IVD degeneration and LBP require further investiga-
tion. Future research is also required to clarify the corre-
lation between stress and apoptosis in disc degeneration,
to inhibit or eliminate the stress axis and the hormones
that are relevant to IVD degenerative processes.
Disclosure
This work was supported by funds received from the Kerman
Neuroscience Research Center (KNR/87-31). The authors report no
conflict of interest concerning the materials or methods used in this
study or the findings specified in this paper.
Author contributions to the study and manuscript preparation
include the following. Conception and design: Reihani Kermani.
Acquisition of data: Hoboubati, Esmaeili-Mahani, Asadi-Shekaari.
Analysis and interpretation of data: all authors. Critically revising
the article: all authors. Reviewed submitted version of manuscript:
all authors. Approved the final version of the manuscript on behalf of
all authors: Reihani Kermani. Statistical analysis: Esmaeili-Mahani.
Administrative/technical/material support: Reihani Kermani. Study
supervision: Reihani Kermani.
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Manuscript submitted May 18, 2013.
Accepted January 27, 2014.
Please include this information when citing this paper: pub-
lished online March 7, 2014; DOI: 10.3171/2014.1.SPINE13466.
Address correspondence to: Hamed Reihani Kermani, M.D.,
Hezaroyekshab St., Alley No. 5, 76186 53771 Kerman, Iran. email:
h_reihani@hotmail.com.