Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats
Using immunohistochemical analysis, we investigated the time profile of neuronal nitric oxide synthase (nNOS) expression in the lumbar spinal cord up to day 28 after transection of the L₅ spinal root. On day 14 after injury, we also evaluated the effect of intrathecal application of 7-nitroinda...
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Інститут фізіології ім. О.О. Богомольця НАН України
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| Cite this: | Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats / Z. Bahari, H. Manaheji, L. Dargahi, S. Daniali, M. Norozian, G.H. Meftahi, M. Sadeghi // Нейрофизиология. — 2015. — Т. 47, № 4. — С. 341-348. — Бібліогр.: 22 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1482092025-02-09T15:10:57Z Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats Часовий профіль експресії nNOS у дорсальному розі спинного мозку щурів після перерізання спінального корінця L₅ Bahari, Z. Manaheji, H. Dargahi, L. Daniali, S. Norozian, M. Meftahi, G.H. Sadeghi, M. Using immunohistochemical analysis, we investigated the time profile of neuronal nitric oxide synthase (nNOS) expression in the lumbar spinal cord up to day 28 after transection of the L₅ spinal root. On day 14 after injury, we also evaluated the effect of intrathecal application of 7-nitroindazole (7-NI), a selective nNOS inhibitor (8.15 µg in 5 µl), on thermal hyperalgesia. Our results indicated that nerve transection increased the intensity of nNOSimmunoreactivity in superficial and deep laminae of the dorsal horn within a late stage (days 7 to 28) of the neuropathy model used. Furthermore, 7-NI attenuated nerve injury-evoked thermal hypersensitivity on day 14 but did not reduce it between days 2 and 5 after transection. These data suggest that nNOS overexpression is more involved in the development than in the initiation of thermal hyperalgesia in L₅ -transected rats. Ми досліджували часовий профіль експресії нейронної NOсинтази (nNOS) у люмбальному відділі спинного мозку щурів протягом 28 діб після перерізання спінального корінця L₅ , використовуючи імуногістохімічну методику. Ми також оцінювали впливи інтратекальних аплікацій 7-нітроіндазолу (7-NI) – селективного інгібітора nNOS (8.15 мкг у 5 мкл) на термічну гіпералгезію через 14 діб після ушкодження. В результаті секції корінця кількість nNOS-імунореактивних клітин у поверхневих та глибоких пластинах дорсального рога зростала на відносно пізніх етапах (із сьомої по 28-му добу) використаної моделі нейропатії. Аплікації 7-NI зменшували термічну гіперсенситивність, викликану пошкодженням нервових волокон, на 14-ту добу, але не впливали на цей феномен протягом другої–п’ятої діб після індукції нейропатії. Подібні дані вказують на те, що після перетину корінця L₅ у щурів підвищена експресія nNOS більшою мірою залучена в процес розвитку, ніж в ініціацію термічної гіпералгезії. This study was conducted as a part of the PhD student thesis project in the Department of Neurophysiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2015 Article Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats / Z. Bahari, H. Manaheji, L. Dargahi, S. Daniali, M. Norozian, G.H. Meftahi, M. Sadeghi // Нейрофизиология. — 2015. — Т. 47, № 4. — С. 341-348. — Бібліогр.: 22 назв. — англ. 0028-2561 https://nasplib.isofts.kiev.ua/handle/123456789/148209 612.83:616.85 en Нейрофизиология application/pdf Інститут фізіології ім. О.О. Богомольця НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Using immunohistochemical analysis, we investigated the time profile of neuronal nitric
oxide synthase (nNOS) expression in the lumbar spinal cord up to day 28 after transection
of the L₅
spinal root. On day 14 after injury, we also evaluated the effect of intrathecal
application of 7-nitroindazole (7-NI), a selective nNOS inhibitor (8.15 µg in 5 µl), on thermal
hyperalgesia. Our results indicated that nerve transection increased the intensity of nNOSimmunoreactivity in superficial and deep laminae of the dorsal horn within a late stage (days
7 to 28) of the neuropathy model used. Furthermore, 7-NI attenuated nerve injury-evoked
thermal hypersensitivity on day 14 but did not reduce it between days 2 and 5 after transection.
These data suggest that nNOS overexpression is more involved in the development than in the
initiation of thermal hyperalgesia in L₅
-transected rats. |
| format |
Article |
| author |
Bahari, Z. Manaheji, H. Dargahi, L. Daniali, S. Norozian, M. Meftahi, G.H. Sadeghi, M. |
| spellingShingle |
Bahari, Z. Manaheji, H. Dargahi, L. Daniali, S. Norozian, M. Meftahi, G.H. Sadeghi, M. Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats Нейрофизиология |
| author_facet |
Bahari, Z. Manaheji, H. Dargahi, L. Daniali, S. Norozian, M. Meftahi, G.H. Sadeghi, M. |
| author_sort |
Bahari, Z. |
| title |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats |
| title_short |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats |
| title_full |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats |
| title_fullStr |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats |
| title_full_unstemmed |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats |
| title_sort |
time profile of nnos expression in the spinal dorsal horn after l₅ spinal root transection in rats |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2015 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/148209 |
| citation_txt |
Time Profile of nNOS Expression in the Spinal Dorsal Horn After L₅ Spinal Root Transection in Rats / Z. Bahari, H. Manaheji, L. Dargahi, S. Daniali, M. Norozian, G.H. Meftahi, M. Sadeghi // Нейрофизиология. — 2015. — Т. 47, № 4. — С. 341-348. — Бібліогр.: 22 назв. — англ. |
| series |
Нейрофизиология |
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NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4 341
UDC 612.83:616.85
Z. BAHARI,1 H. MANAHEJI,2,3 L. DARGAHI,3 S. DANIALI,1 M. NOROZIAN,4 G. H. MEFTAHI,5 and M. SADEGHI6
TIME PROFILE OF nNOS EXPRESSION IN THE SPINAL DORSAL HORN
AFTER L5 SPINAL ROOT TRANSECTION IN RATS
Received April 20, 2014
Using immunohistochemical analysis, we investigated the time profile of neuronal nitric
oxide synthase (nNOS) expression in the lumbar spinal cord up to day 28 after transection
of the L5 spinal root. On day 14 after injury, we also evaluated the effect of intrathecal
application of 7-nitroindazole (7-NI), a selective nNOS inhibitor (8.15 µg in 5 µl), on thermal
hyperalgesia. Our results indicated that nerve transection increased the intensity of nNOS-
immunoreactivity in superficial and deep laminae of the dorsal horn within a late stage (days
7 to 28) of the neuropathy model used. Furthermore, 7-NI attenuated nerve injury-evoked
thermal hypersensitivity on day 14 but did not reduce it between days 2 and 5 after transection.
These data suggest that nNOS overexpression is more involved in the development than in the
initiation of thermal hyperalgesia in L5-transected rats.
Keywords: neuropathic pain, spinal dorsal horn, L5 segment, nNOS, 7-nitroindazole,
thermal hyperalgesia.
Shahid Beheshti University of Medical Sciences, Tehran, Iran (1 Department
of Neurophysiology, 2 Department of Physiology, 3 Neuroscience Research
Center, 4Department of Anatomy).
5 Neuroscience Research Center, Baqiyatallah (a.s.) University of Medical
Sciences, Tehran, Iran.
6 Department of Physiology, Bushehr University of Medical Sciences,
Bushehr, Iran.
Correspondence should be addressed to H. Manaheji
(e-mail: hshardimanaheji@yahoo.com) or
Z. Bahari (e-mail: bahari_441@yahoo.com).
INTRODUCTION
At present, central mechanisms of neuropathic pain are
not fully understood. There is no sufficiently effective
treatment for this debilitating disorder [1]. Neuropathic
pain is characterized by exaggerated responses to
painful stimuli (hyperalgesia), pain sensation in
response to normally non-noxious stimuli (allodynia),
and spontaneous pain [1-3]. It is accepted that nNOS
is significantly involved in the regulation of several
cellular processes, probably including neuropathic pain.
Nevertheless, the exact contribution of nNOS to the
development of pain-related behaviors remains unknown;
new information in this respect may provide a novel
treatment avenue. There are several controversial studies
on the time profile and location of nNOS expression in
the dorsal root ganglia (DRG) and spinal dorsal horn
under neuropathy conditions. According to some authors,
peripheral nerve lesion results in increased expression of
nNOS in the DRGs but not in the spinal cord laminae [4].
Other communications, however, reported that axotomy
in rats reduces the number of NOS-positive neurons in
the dorsal horn [5, 6]. The above discrepancies may be
related to different time intervals examined and type
of neuropathy models. The first aim of our study was
to further evaluate the time profile of nNOS expression
in superficial and deep laminae of the rat lumbar spinal
cord of rats with the spinal L5 root transected.
In addition, pharmacological studies regarding
the role of spinal nNOS in the development of pain-
related behaviors are conflicting. In some studies,
administration of nNOS inhibitors reduced the
exaggerated pain in rodents after nerve injury [7-10].
In contrast, other studies showed that administration
of nNOS inhibitors exerted no effect on diabetes-
related or nerve injury-induced behaviors [4, 11, 12].
Therefore, our subsequent aim was to evaluate the
contribution of spinal nNOS to the development of
nerve injury-evoked thermal hyperalgesia, using an
nNOS selective inhibitor.
METHODS
Animals. Male Wistar rats (Pasteur Institute, Tehran,
Iran) weighting 180-200 g were used. The rats were
housed in separate cages and kept in a temperature-
controlled colony room under a 12/12 h light-dark
cycle with free access to food and water.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4342
Z. BAHARI, H. MANAHEJI, L. DARGAHI, et al.
Induction of Neuropathic Pain. Neuropathic pain
was induced according to the method described by
Kim and Chung [13]. Briefly, animals were anesthe-
tized with pentobarbital sodium (60 mg/kg, i.p.). The
left paraspinal muscles were separated at the L4-S2 lev-
el, and the left transverse process of the L6 vertebrum
was removed. The L5 spinal root was tightly ligated
using a silk thread (6-0) and transected just distal to
the ligature, with all fibers surely interrupted. Then,
the wound was closed with 3-0 silk threads. Special
care was taken to avoid any damage to the L4 root.
In the control sham group, the surgical procedure was
identical to that described above, except for ligation
and transection of the left L5 spinal root. Only animals
showing minimum signs of motor deficiency were in-
cluded in further experimentation.
Immunohistochemistry of nNOS. Tissue samples
were collected from the lumbar enlargement of ani-
mals of the sham-operated (n = 5) and neuropathic
rats (6 groups, n = 6 in each). Rats were deeply an-
aesthetized with pentobarbital sodium (80 mg/kg, i.p.)
and perfused transcardially with heparinized 0.9% sa-
line followed by cold 4% paraformaldehyde in 0.1 M
phosphate buffer saline (PBS, pH 7.4). The lumbar en-
largement of the spinal cord was quickly removed and
post-fixed in the same fixative for 24 h at 4°C. Fixed
tissues were processed by graded alcohols and xylenes
and paraffin-embedded the next day. Five-µm-thick
transverse spinal; cord sections were cut on a micro-
tome and mounted onto slides.
The sections were deparaffinized, rehydrated seq-
uentially in graded alcohols, treated with freshly pre-
pared 0.3% hydrogen peroxide for 30 min (to eliminate
intrinsic peroxidase activity), and washed out three
times in PBS (pH 7.4). Microwave antigen retrieval
was performed in 0.01 M citrate buffer (pH 6.0) at
85°C for 3 min, cooled in the same solution for 2 min
(replicated three times), and rinsed three times in PBS.
Non-specific binding was reduced by using a blocking
solution of 5% normal goat serum (NGS) for 30 min.
Then, the sections were incubated with rabbit poly-
clonal anti-nNOS primary antibody (abcam, ab95436,
1:1000 dilution using distilled water) at 4°C overnight.
After rinsing three times with PBS, the sections were
exposed with a mouse- and rabbit-specific HRP/DAB
(ABC) detection kit (abcam, ab64264) for 60 min at
room temperature and washed out three times in PBS.
After this, the sections were exposed to diaminoben-
zidine tetrahydrochloride for 10 min. Immunostained
sections were counterstained with hematoxylin, dehyd-
rated by increasing alcohol concentrations, rinsed in
xylene, and coverslipped with Entellan. Control sec-
tions were treated in the same way except for the ex-
posure to primary antibody.
Counting Procedure and Image Analysis. Labeled
cell bodies were viewed and digitized under a light mi-
croscope (Olympus, Japan). At least 80 sections from
the L5 spinal segment of both sides (two randomly se-
lected sections per animal) were photographed and
counted. The number of nNOS-ir cells in laminae I-V
of the dorsal horn was counted within a test-frame
of 500 µm × 500 µm in each section. The number of
nNOS-ir cells in lamina X was counted within a circle
500 µm in diameter around the center of the central
canal in each section. The analyses were performed by
an observer masked to the experiment.
Behavioral Study. We used the plantar test to
confirm the successful induction of neuropathy
before immunohistochemical study. Sham-operated
(n = 8) and neuropathic rats (n = 8 in each group) were
examined for the development of thermal hyperalgesia
one day before and 2, 5, 7, 14, 21, and 28 days after
section of the L5 spinal root. Immunohistochemical
analyses showed that nerve damage produced
maximum increases in the number of nNOS positive
neurons on day 14 after the neuropathy-inducting
operation. Therefore, the intrathecal effect of 7-NI on
thermal hyperalgesia was evaluated on day 14. The
neuropathic rats (n = 8) received intrathecal infusion
of 7-NI (8.15 µg in 5 µl) or of an equal volume of
0.9% NaCl saline. 7-NI was injected over a period
of 60 sec, and introduction was followed by 5 µl of
0.9% saline to flush the catheter. Thermal hyperalgesia
was evaluated just before administration and 30,
60, 90, 120,150 and 180 min after 7-NI or saline
administration. The dose of drug administration was
based on the data of the earlier study [9].
Thermal Hyperalgesia. The paw withdrawal
latency, PWL, in response to application of radiant
heat was assessed using the plantar test apparatus
(Ugo Basile, Italy). Briefly, the animal was placed
in a Plexiglas box with a glass floor and allowed to
acclimate 30 min before the experiment. A mobile
radiant heat source was positioned under the plantar
surface of the left hind paw, and the PWL value was
measured. The injured paw was tested three times
with 5-min-long intervals, and the average value of
the consecutive testes was calculated. The cut-off time
was 33 sec.
Implantation of an Intrathechal Catheter. Under
pentobarbital sodium anesthesia (60 mg/kg, i.p.), an
intrathecal cannula was implanted according to the
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4 343
TIME PROFILE OF nNOS EXPRESSION IN THE SPINAL DORSAL
method of Storkson et al. [14]. Concisely, a PE-10
polyethylene tube was inserted between the L5
and L6 vertebrae and carefully threaded into the
subarachnoid space in the cranial direction to reach
the lumbar enlargement. The outer part of the catheter
was plugged and fixed onto the skin. The correct
subarachnoid position was assured by a typical tail
or hindpaw flick. Also, the catheter placement was
verified by observing transient hindpaw paralysis
induced by intrathecal injection of 10 μl 2% lidocaine.
Only the rats showing complete paralysis of both
hindlimbs and tail after lidocaine administration
were used for the subsequent experiments. At the end
of each experiment, the position of the PE tubing in
the intrathecal space at the lumbar enlargement was
visually verified by exposing the lumbar spinal cord.
Statistics. All data are presented below as means ±
± s.e.m. In the immunohistochemistry study; one-
way ANOVA followed by the Tukey post-hoc test was
used. In the behavioral study, the data were analyzed
by one-way repeated-measures and two-way ANOVA
followed by the Bonferroni post-hoc test. Graphics and
statistical analysis were performed using Graphpad
Prism, version 5.0 (Graphpad Prism software, USA).
RESULTS
Time Profile of nNOS-Immunoreactivity in
Superficial and Deep Laminae of the Dorsal Horn
in Neuropathy. We examined the time profile of
nNOS expression in the L5 segment of the spinal
dorsal horn in neuropathic rats. The nNOS-ir neurons
at the ipsilateral (left) and contralateral (right) sides
were calculated in neuropathic rats, sham rats, and
control (naive) animals. In the latter (naive and sham)
rats taken as the controls for neuropathic animals,
immunohistochemical nNOS staining did not reveal
significant differences in the number of positive
neurons on both sides (ipsilateral and contralateral)
of the dorsal horn. After transection of the L5 spinal
root, significant increases in the number of nNOS-ir
cells on both ipsilateral and contralateral sides of the
L5 segment were observed, as compared to the control.
This increase was observed in the superficial laminae
(I-II), deep laminae (III-V) of the dorsal horn (Figs.
1-3), and also around the central canal (lamina X; Figs.
4 and 5). This elevation became significant on day 7,
reached the maximum level on day 14, and stayed at a
high level up to 28 days after induction of neuropathy,
as compared to the control (Table 1).
Effect of Intrathecal Infusion of the nNOS
Inhibitor on Thermal Hyperalgesia. Thermal
hyperalgesia after transection of the L5 spinal nerve
became obvious on day 2, reached the maximum on
day 7, and then remained relatively steady up to 28
days, as compared to the pre-injury baseline latencies
(Fig. 6A). Noxious thermal stimulation before spinal
nerve transection elicited lifting of the hindpaw
with the mean latency of 17.03 ± 0.48 sec, and the
development of neuropathy produced a dramatic
decline in the withdrawal latency (8.97 ± 0.26 sec) on
day 14 after the above transection. As expected, sham
operation did not produce any reliable change in the
baseline values within the entire observation period.
To define the effect of inhibition of nNOS on thermal
sensitivity at the spinal level, we examined the effect
of intrathecal injection of 7-NI, a selective nNOS
inhibitor, on nerve injury-related behavioral changes
and induction of thermal hyperalgesia on day 14 after
Normalized numbers (%) of nNOS-ir cells at the ipsilateral and contralateral sides of laminae I-II, laminae III-V, and also lamina X
of the L5 segment in neuropathic rats
Нормовані кількості (%) nNOS-імунореактивних клітин у різних пластинах сегмента L5 щурів із нейропатією
Groups (days
after root
transection)
Laminae
I-II III-V X
ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral
D-2 85.71±20.82 14.28±13.36 107.69±27.19 76.92±19.61 27.73±18.24 8.76±18.24
D-5 92.85±18.21 21.42±18.21 115.38±22.42* 15.38±12.16 82.48±36.49 64.23±34.94
D-7 185.71±15.97*** 71.42±20.82 161.53±14.39*** 115.38±15.38* 173.72±34.94*** 9.48±21.07
D-14 428.57±24.22*** 264.28±18.21** 507.69±33.08*** 176.92±22.42*** 301.45±36.49*** 137.22±18.24**
D-21 200.00±26.24*** 128.57±30.72*** 323.07±19.61*** 300.00±27.19*** 283.21±34.94*** 155.47±36.49**
D-28 192±85±20.82*** 128.57±26.72** 169.23±27.19*** 92.30±27.19 210.21±18.24*** 137.22±18.24**
Footnotes: Indices in the naive animals are taken as the control (100%). Values represent means ± s.e.m. (eight rats per group). Statistical
analysis was performed using one-way ANOVA and Tukey’s post-hoc test.*P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant
differences between neuropathy and sham-operated animals.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4344
Z. BAHARI, H. MANAHEJI, L. DARGAHI, et al.
F i g. 1. Representative photomicrographs showing nNOS-ir neurons (arrows) at the ipsilateral and contralateral sides of the dorsal horn of
the L5 segment in neuropathic rats. A) In control animals. B1-G2) Images made at different times after transection of the L5 spinal root (2,
5, 7, 14, 21, and 28 days, respectively).
Р и с. 1. Мікрофотографії розподілу nNOS-імунореактивних нейронів (вказані стрілками) в іпси- та контралатеральному дорсаль-
них рогах спінального сегмента L5 щурів із нейропатією.
Control Day 2-IPSI
Day 7-IPSI
Day 2-CONTRA
A B1
C1
E1
G1 G2F
C2
E2D2
B2
D1
100 μm 100 μm 100 μm
Day 5-IPSI
Day 14-IPSI
Day 28-IPSI
Day 5-CONTRA
Day 14-CONTRA
Day 28-CONTRA
Day 7-CONTRA
Day 21
neuropathy. Intrathecal injection of 7-NI significantly
decreased thermal hyperalgesia in the ipsilateral
hindpaw 60 min after administration (the mean
paw lifting latency increased to 12.43 ± 0.60 sec),
which persisted for 120 min after treatment (13.74 ±
± 0.56 sec) and became closer to the pre-drug
administration level after 150 min (12.04 ± 0.50 sec);
the mean pre-drug administration value in these series
was 9.96 ± 0.68 sec (B).
DISCUSSION
We demonstrated that L5 root transection is
accompanied by profound alterations of nNOS
expression in the spinal dorsal horn. Such transection
increased the number of nNOS-ir cells in the
superficial and deep laminae of the L5 segment of
the dorsal horn and also in the lamina X, but this
occurred mostly within the late stage (days 7 to 28) of
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4 345
TIME PROFILE OF nNOS EXPRESSION IN THE SPINAL DORSAL
0
2 5 7 14
***
**** *
*** ***
***
******
***
***
***
***
**
**
21 28 daysControl
100
200
300
400
500
600
F i g. 2. Numbers of nNOS-ir neurons in laminae I-II at the
contralateral and ipsilateral sides of the L5 spinal segment in
neuropathic rats. Means ± s.e.m. are shown (calculations were
performed for five rats per group). Data are expressed as percentage
of the control. **P < 0.01 and ***P < 0.001 indicate significant
differences with respect to control rats.
Р и с. 2. Кількість nNOS-імунореактивних нейронів у пластинах
І та ІІ дорсального рога спінального сегмента L5 контра- та
іпсилатерально перерізці дорсального корінця.
2 5 7 14 21 28Control
0
100
200
300
400
500
600
700
F i g. 3. Numbers of nNOS-ir neurons in laminae III-V. *P < 0.05;
other indications are similar to those in Fig. 2.
Р и с. 3. Кількість nNOS-імунореактивних нейронів у плас-
тинах III–V.
A
E
I
O P Q
J K L M
B
F
C
G
D
H
Control CONTRAIPSI IPSI IPSI
IPSI
IPSIIPSIIPSIIPSI
CONTRA CONTRA CONTRA
CONTRACONTRACONTRACONTRA
Sham Day 2
Day 7 Day 14 Day 21 Day 28
Day 5
100 μm
10 μm10 μm10 μm10 μm
100 μm 100 μm 100 μm
I
J
K
L
M N O P
Q
F i g. 4. Representative photomicrographs showing nNOS-ir neurons (arrows) in lamina X of the L5 segment in neuropathic rats. A and B)
In control and sham-operated animals. C-H) Images at different terms after root transection (2, 5, 7, 14, 21, and 28 days, respectively). I-Q)
Images at higher magnification.
Р и с. 4. Мікрофотографії розподілу nNOS-імунореактивних нейронів (вказані стрілками) в пластині X сегмента L5.
days
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4346
Z. BAHARI, H. MANAHEJI, L. DARGAHI, et al.
28, similarly to what was observed in a previous study
[15]. To study the involvement of spinal nNOS in the
development of neuropathic pain, we used the nNOS
inhibitor 7-NI. Our results showed that 14 days after
nerve transection, intrathecal administration of 7-NI
significantly attenuated thermal hyperalgesia 60 min
after injection, and such a shift persisted up to 120-
150 min. We did not observe any significant behavioral
effect of administration of the nNOS inhibitor on days
2 and 5 (data not shown). Our pharmacological results
are consistent with reports that inhibition of nNOS
attenuated neuropathic pain-related behaviors [7].
However, there is some evidence that nNOS inhibition
did not reduce the pain sensitivity in neuropathic rats
[4, 6, 12]. Our quantitative analyses demonstrated
that a significant increase in the number of nNOS-ir
neurons was observed within both ipsilateral and
contralateral sides of laminae I-V and even in a region
around the central canal (lamina X) of the spinal cord
on day 7 after L5 nerve transection. The respective
changes reached a maximum level on day 14 and
remained elevated up to 28 days.
Our immunohistochemical results to a certain
extent contradict those of our behavioral studies.
We observed significant appearance of thermal
hyperalgesia on day 2 after spinal nerve transection,
but immunohistochemical results showed that the
maximum number of nNOS-ir neurons was found
on day 14. Furthermore, as was mentioned, our
pharmacological data did not reveal any analgesic
effect of application of the nNOS inhibitor on
postoperation days 2 and 5. Therefore, it should
be suggested that nNOS may be involved within
relatively late stages (days 7-28) more than within
early stages (days 2-5) in our neuropathy model. So, it
seems that nNOS overexpression is more involved in
the development than in the initiation of hyperalgesia.
Some studies showed that nerve injury increased the
number of nNOS-positive neurons in the spinal cord
[17-19]. However, there is controversial evidence
concerning nNOS expression during the development
of neuropathy. For example, Zhang et al. [5] showed
that spinal axotomy in the rat reduced the number of
nNOS-positive neurons in lamina II of the ipsilateral
dorsal horn but increased the respective indices in DRG
neurons at the L4-L6 level [5]. Several pathological
conditions occur after nerve injuries that may induce
nNOS overexpression in the spinal cord. Among
those, there are: (i) abnormal spontaneous ectopic
action potentials (APs) in injured nerve fibers, which
come to the spinal cord, and (ii) different signaling
***
*** ***
***
0
100
200
300
400
500
2 5 7 14 21 28 daysControl
** **
**
F i g. 5. Numbers of nNOS-ir in neurons lamina X of the L5 segment
in control and neuropathic rats. Indications are similar to those in
Figs. 2 and 3.
Р и с. 5. Кількість nNOS-імунореактивних нейронів у плас-
тині X сегмента L5.
F i g. 6. Thermal hyperalgesia induced by L5 root transection. A)
Time profile of thermal hyperalgesia measured by the plantar test.
Thermal thresholds were measured on day 0 (one day before nerve
injury) up to day 28. B) Time profile ot the effect of interathecal
injection of the nNOS inhibitor 7-NI on thermal hyperalgesia
(day 14). Designations are similar to those in Figs. 2, 3, and 5.
Р и с. 6. Термічна гіпералгезія, індукована перерізкою ко-
рінця L5.
0
0
0 30 60 90 120 150 180
2 5 7 14 21 28
2
4
6
8
10
12
14
16
18
20
sec A
B
*** ***
*** *** ***
***
0
2
4
6
8
10
12
14
16
18
20
the development of transection-induced neuropathy.
Thermal hyperalgesia was induced from day 2 to day
days
min
sec
* ******
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 4 347
TIME PROFILE OF nNOS EXPRESSION IN THE SPINAL DORSAL
molecules that are up- or down-regulated in the injured
axons [20-21]. So, these conditions may have either
a negative or a positive influence on spinal nNOS
expression. It seems that, under normal conditions,
primary afferent firing and retrogradely transported
signaling molecules regulate spinal nNOS expression
mostly by a negative feedback mechanism. Peripheral
nerve injury disrupts this feedback mechanism and
leads to nNOS overexpression. Our results showed that
there is no significant increase in the number of nNOS-
ir neurons within early stages of the development of
neuropathy (between days 2-5). It is suggested that
relatively slow nNOS expression after nerve injury is
not directly controlled by the arrival of initially evoked
ectopic APs. However, it is possible that ongoing
ectopic discharges could indirectly intensify de novo
synthesis of the nNOS protein. Such a supposition is
supported by the findings of Herdegen et al. [22]; these
authors demonstrated that transcription factors (such
as c-Jun and c-Fos) are overexpressed and colocalized
with NOS overexpression in spinal cord neurons after
noxious stimulation. Our results confirm that nNOS
plays an important role in the development of thermal
hyperalgesia within the late stages of our neuropathy
model.
In conclusion, our data suggest that nNOS
overexpression in the dorsal horn, as well as around
the central canal, is more involved in the development
than in the initiation of thermal hyperalgesia in our
neuropathy model (spinal root transection in rats).
Acknowledgements. This study was conducted as a part
of the PhD student thesis project in the Department of Neuro-
physiology, Faculty of Medicine, Shahid Beheshti University of
Medical Sciences, Tehran, Iran.
Animal experiments were performed according to the
Guidelines for the Care and Use of Laboratory Animals by the
National Institutes of Health (NIH) and were approved by the
Animal Ethics Committee of the Shahid Beheshti University of
Medical Sciences, Tehran, Iran. (194/-90/3/18; 2011).
The authors of this communication, Z. Bahari, H. Manaheji,
L. Dargahi, S. Daniali, M. Norozian, G. H. Meftahi, and
M. Sadeghi, confirm the absense of any conflict related
to comercial or financial interests, to interrelations with
organizations or persons in any way involved in the research,
and to interrelations of the co-authors.
З. Бахарі1, Х. Манахеджі2,2, Л. Даргані2, С. Даніалі1,
M. Нероз’ян1, Г. Х. Мефтахі3, M. Садегхі4
ЧАСОВИЙ ПРОФІЛЬ ЕКСПРЕСІЇ nNOS У ДОРСАЛЬ-
НОМУ РОЗІ СПИННОГО МОЗКУ ЩУРІВ ПІСЛЯ
ПЕРЕРІЗАННЯ СПІНАЛЬНОГО КОРІНЦЯ L5
1 Медичний університет Шахід Бехешті, Тегеран (Іран).
2 Центр досліджень у сфері нейронаук Медичного
університету Шахід Бехешті, Тегеран (Іран).
3 Центр досліджень у сфері нейронаук Медичного
університету Бакійяталлах (a.s.), Тегеран (Іран).
4 Бушерський медичний університет (Іран).
Р е з ю м е
Ми досліджували часовий профіль експресії нейронної NO-
синтази (nNOS) у люмбальному відділі спинного мозку щу-
рів протягом 28 діб після перерізання спінального корінця
L5, використовуючи імуногістохімічну методику. Ми також
оцінювали впливи інтратекальних аплікацій 7-нітроіндазо-
лу (7-NI) – селективного інгібітора nNOS (8.15 мкг у 5 мкл)
на термічну гіпералгезію через 14 діб після ушкодження. В
результаті секції корінця кількість nNOS-імунореактивних
клітин у поверхневих та глибоких пластинах дорсального
рога зростала на відносно пізніх етапах (із сьомої по 28-му
добу) використаної моделі нейропатії. Аплікації 7-NI змен-
шували термічну гіперсенситивність, викликану пошко-
дженням нервових волокон, на 14-ту добу, але не впливали
на цей феномен протягом другої–п’ятої діб після індукції
нейропатії. Подібні дані вказують на те, що після перетину
корінця L5 у щурів підвищена експресія nNOS більшою мі-
рою залучена в процес розвитку, ніж в ініціацію термічної
гіпералгезії.
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