Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats
We analyzed the homology of nerve growth factor (NGF) in the nerve regenerationconditioned fluid (NRCF) obtained from New Zealand white rabbits and SD rats. A nerve regeneration chamber bridged distal and proximal ends of the severed rabbit sciatic nerve, and the NRCF was extracted from such chamb...
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Інститут фізіології ім. О.О. Богомольця НАН України
2015
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| Цитувати: | Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats / Y. Ye, Q.F. Li, L. Li // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 26-30. — Бібліогр.: 23 назв. — англ. |
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irk-123456789-1481452019-02-18T01:23:51Z Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats Ye, Y. Li, Q.F. Li, L. We analyzed the homology of nerve growth factor (NGF) in the nerve regenerationconditioned fluid (NRCF) obtained from New Zealand white rabbits and SD rats. A nerve regeneration chamber bridged distal and proximal ends of the severed rabbit sciatic nerve, and the NRCF was extracted from such chamber. The supernatants and precipitates of nerve tissue of rabbits and rats were extracted from the sciatic nerves by grinding and centrifugation. An antigen-antibody reaction was detected by the ELISA technique using rabbit anti-mouse NGF as primary antibody and goat anti-rabbit IgG as secondary antibody. Antigen-antibody reactions were detected in all the above-mentioned samples obtained from rabbits and rats. The intensity rank of an antigen-antibody response for different samples was the following: rabbit NRCF > rabbit nerve tissue precipitation fluid > rabbit nerve tissue fluid supernatant > > rat nerve tissue fluid supernatant. Thus, there is high homology of the molecular structure of NGF in the NRCFs obtained from rabbits and rats (i.e., animals belonging to different taxonomic groups of mammals). Ми аналізували молекулярну гомологію росту нервів (NGF) у кондиційованій процесом нервової регенерації рідині (NRCF), котру отримували від новозеландських білих кролів та щурів лінії SD. Нейрорегенеративна камера з’єднувала дистальний та проксимальний кінці перерізаного сідничного нерва у кролів, і NRCF відбирали з такої камери. За допомогою гомогенізації та центрифугування з тканини сідничних нервів кролів та щурів готували супернатанти та преципітати. Реакцію антиген–антитіло виявляли за допомогою методики ELISA, використовуючи первинні антитіла щодо мишачого NGF та вторинні козячі антитіла щодо кролячого імуноглобуліну. Реакції антиген–антитіло виявлялися в усіх вищезгаданих зразках, отриманих від кролів та щурів. Послідовність інтенсивностей відповідей антиген-антитіло у різних зразках була такою: кроляча NRCF > преципітат із нервової тканини кроля > супернатант із нервової тканини кроля > супернатант із нервової тканини щура. Отже, молекулярна структура зразків NGF, що знаходяться в NRCF, отриманій від кролів та щурів (тобто тварин, котрі належать до різних таксономічних груп ссавців), демонструє високий рівень гомології. 2015 Article Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats / Y. Ye, Q.F. Li, L. Li // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 26-30. — Бібліогр.: 23 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148145 57.017.35:612.8.01 en Нейрофизиология 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 |
We analyzed the homology of nerve growth factor (NGF) in the nerve regenerationconditioned fluid (NRCF) obtained from New Zealand white rabbits and SD rats. A nerve
regeneration chamber bridged distal and proximal ends of the severed rabbit sciatic nerve,
and the NRCF was extracted from such chamber. The supernatants and precipitates of nerve
tissue of rabbits and rats were extracted from the sciatic nerves by grinding and centrifugation.
An antigen-antibody reaction was detected by the ELISA technique using rabbit anti-mouse
NGF as primary antibody and goat anti-rabbit IgG as secondary antibody. Antigen-antibody
reactions were detected in all the above-mentioned samples obtained from rabbits and rats.
The intensity rank of an antigen-antibody response for different samples was the following:
rabbit NRCF > rabbit nerve tissue precipitation fluid > rabbit nerve tissue fluid supernatant >
> rat nerve tissue fluid supernatant. Thus, there is high homology of the molecular structure
of NGF in the NRCFs obtained from rabbits and rats (i.e., animals belonging to different
taxonomic groups of mammals). |
| format |
Article |
| author |
Ye, Y. Li, Q.F. Li, L. |
| spellingShingle |
Ye, Y. Li, Q.F. Li, L. Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats Нейрофизиология |
| author_facet |
Ye, Y. Li, Q.F. Li, L. |
| author_sort |
Ye, Y. |
| title |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats |
| title_short |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats |
| title_full |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats |
| title_fullStr |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats |
| title_full_unstemmed |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats |
| title_sort |
nerve growth factor homology in the nerve regeneration-conditioned fluid of rabbits and rats |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2015 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/148145 |
| citation_txt |
Nerve Growth Factor Homology in the Nerve Regeneration-Conditioned Fluid of Rabbits and Rats / Y. Ye, Q.F. Li, L. Li // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 26-30. — Бібліогр.: 23 назв. — англ. |
| series |
Нейрофизиология |
| work_keys_str_mv |
AT yey nervegrowthfactorhomologyinthenerveregenerationconditionedfluidofrabbitsandrats AT liqf nervegrowthfactorhomologyinthenerveregenerationconditionedfluidofrabbitsandrats AT lil nervegrowthfactorhomologyinthenerveregenerationconditionedfluidofrabbitsandrats |
| first_indexed |
2025-07-12T18:27:03Z |
| last_indexed |
2025-07-12T18:27:03Z |
| _version_ |
1837466732450545664 |
| fulltext |
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 126
UDC 57.017.35:612.8.01
Y. YE,1 Q. F. LI,2 and L. LI3
NERVE GROWTH FACTOR HOMOLOGY IN THE NERVE REGENERATION-
CONDITIONED FLUID OF RABBITS AND RATS
Received October 13, 2013
We analyzed the homology of nerve growth factor (NGF) in the nerve regeneration-
conditioned fluid (NRCF) obtained from New Zealand white rabbits and SD rats. A nerve
regeneration chamber bridged distal and proximal ends of the severed rabbit sciatic nerve,
and the NRCF was extracted from such chamber. The supernatants and precipitates of nerve
tissue of rabbits and rats were extracted from the sciatic nerves by grinding and centrifugation.
An antigen-antibody reaction was detected by the ELISA technique using rabbit anti-mouse
NGF as primary antibody and goat anti-rabbit IgG as secondary antibody. Antigen-antibody
reactions were detected in all the above-mentioned samples obtained from rabbits and rats.
The intensity rank of an antigen-antibody response for different samples was the following:
rabbit NRCF > rabbit nerve tissue precipitation fluid > rabbit nerve tissue fluid supernatant >
> rat nerve tissue fluid supernatant. Thus, there is high homology of the molecular structure
of NGF in the NRCFs obtained from rabbits and rats (i.e., animals belonging to different
taxonomic groups of mammals).
Keywords: nerve growth factor, nerve regeneration-conditioned fluid (NRCF), peripheral
nerve, homology of proteins.
1Department of Medical Cosmetology, Anhui University of Traditional
Chinese Medicine, Anhui, China.
2Affiliated Ninth People’s Hospital of the Shanghai Jiao Tong University,
Shanghai, China.
3Department of Pharmacology, Anhui University of Traditional Chinese
Medicine, Anhui, China.
Correspondence should be addressed to L. Li
(e-mail: lilill123@hotmail.com).
INTRODUCTION
A nerve regeneration chamber, NRC, consisting of a
silicone tube bridging distal and proximal ends of the
transected sciatic nerve, has been proposed as a device
that helps to study the sequence of events accompanying
axonal regeneration in vivo following nerve injury [1, 2].
The chamber provides an appropriate microenvironment
for survival of all types of fibers found in the sciatic
nerve, including axons of sympathetic neurons,
sensory fibers, and motor fibers (axons of spinal
motoneurons) [3, 4]. The microenvironment within
the nerve regeneration chamber contains regeneration-
conditioned fluid, a fibrous matrix, macrophages,
fibroblasts, Schwann cells, and endothelial cells.
Nerve regeneration-conditioned fluid (NRCF) contains
many proteins related to neural regeneration, and
research of these active proteins in NRCF is important
to understand the mechanism of neural regeneration
[5-7]. Thus, selection and functional studies of these
proteins are promising research directions [6, 8]. In
a number of previous studies of the NRCF, rats were
extensively used as donors in research with protein
purification and structural analysis; rats, however,
cannot provide sifficiently large NRCF sample sizes. It
seems that using bigger experimental animals (rabbits)
as donors in the respective studies is more practical, but
there is a problem needing to be considered: What is the
level of homology of proteins in the NRCF obtained
from rats and rabbits?
Nerve growth factor (NGF) is one of the main
active proteins in the NRCF. It is a potent growth
and survival factor for sympathetic neurons [9, 10].
Previous studies showed that NGF is essential for
enhanced post-infarct sympathetic sprouting and other
events related to nerve regeneration [5, 11-13]. So, it
is reasonable and important to detect the homology of
NGF in the NRCF obtained from rabbits and rats.
In our study, we carried out a comparative analysis
of the homology of NGF in the NRCF between New
Zealand white rabbits and SD rats through detecting an
antigen-antibody reaction using the ELISA technique.
The results showed that homology of this factor in the
NRCF of these two species is rather high.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 1 27
NERVE GROWTH FACTOR HOMOLOGY IN THE NERVE REGENERATION
METHODS
Experimental Animals and Groups. Five New
Zealand white rabbits (body mass 1.8 to 2.5 kg)
were chosen for NRCF extraction. The animals were
obtained from the Department of Laboratory Animal
Science (Medical College of Shanghai Jiao Tong
University, Shanghai, Chaina). Albino SD rats were
also used.
Extraction of the Sciatic Nerve NRCF from Rab-
bits. NRCF samples of New Zealand white rabbits were
extracted using a nerve regeneration chamber model.
Rabbits were anesthetized by i.m. injection of xyl-
azine-sailaqin (500 mg/kg) and ketamine (0.2-0.3 ml
per rabbit). Skin preparation was performed at the out-
sides of two hindlimbs, and the skin outside of the
right hindlimb was cut about 5 cm after disinfection
three times by benzalkonium. We separated muscle
fibers of the m. gluteus and exposed the sciatic nerve.
Then, the nerve was transected at its middle level, and
a 20-mm-long fragment of the nerve was removed. Fi-
nally, two ends of the sciatic nerve were sutured to a
silicone tube (length 4 cm, diameter 3 cm) by an atrau-
matic needle with 8/0 nylon to form the nerve regener-
ation chamber model, and then muscles and skin were
sutured gradually. After one week, the rabbits were
dissected in the same way, and the liquid (NRCF) was
collected into a silicone tube (using a micro-injector)
and stored at –20°C.
Extraction of the Sciatic Nerve Tissue Fluid from
Rabbits and Rats. After anesthesia, fragments of the
sciatic nerves of rabbits and rats (length 2.5 cm and
2.0 cm, respectively) were cut off from the hindlimbs
and stored in normal saline. The tissue fluid of the sci-
atic nerves was obtained (by triturating and homog-
enizing using a homogenizer after washing off with
PBS, 0.05 M, pH 7.2) and stored at 4°C.
Comparison of NGF Homology between Rabbits
and Rats. We studied the homology of NGF under the
following three conditions by the method of ELISA ac-
cording to the respective manual:
Supernatant samples of the sciatic nerve tissue flu-
id of rabbits and rats were obtained by centrifugation
and diluted in carbonate buffer (pH 9.5) to 1:5, 1:10,
1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. The primary
antibody of rabbit anti-mouse NGF (M-20, Santa Cruz
Biotech, USA; 100 ml in each well) was diluted to 1:20
and 1:50 in 0.3% BSA-PBS buffer, and the secondary
antibody of goat anti-rabbit IgG was diluted to 1:1000.
The ammonium sulfate technique was applied to
separate the sciatic nerve tissue fluid of rabbits and
rats using a 50% saturated ammonium sulfate over-
night at 4○C, and then the supernatant and precipitate
were extracted by centrifugation. These samples were
diluted to 1:2, 1:4, 1:8, and 1:16 in carbonate buffer
(pH 9.5) for ELISA detection, and primary antibody
(rabbit anti-mouse NGF, E-Ab) was diluted to 1:1000.
The ELISA detection was performed again for
samples of the rabbit NRCF and for supernatants and
precipitates of the rabbit and rat sciatic nerve tissue
fluids. The coating buffer was diluted separately
to 1:10, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, and
control. Primary antibody (rabbit to mouse NGF) was
diluted to 1:500, and secondary antibody (HRP-labeled
goat to rabbit IgG) was diluted to 1:1000.
RESULTS
A total of 500 µl NRCF from five rabbits (50-150 µl
per rabbit) was extracted, and ELISA experiments
were performed with different samples from New
Zealand white rabbits and SD rats to detect their
antigen-antibody reactions to NGF antibody under
three different conditions. Results can be summarized
in the following two paragraphs:
(i) Antigen-antibody reactions could be observed
in all samples (nerve tissue fluid, supernatants, and
precipitates treated by saturated ammonium sulfate) of
rabbits and rats, which indicated that there is rather
high homology between nerve tissue fluids of these
animals (Tables 1 and 2).
(ii) In accordance with the optic density, the rank of
the immune response intensity of antigen-antibody re-
actions was the following: rabbit NRCF > rabbit nerve
tissue precipitation fluid > rabbit nerve tissue fluid
supernatant > rat nerve tissue fluid supernatant. This
indicated that proteins in the NRCF of New Zealand
white rabbits demonstrate the highest activity in the
reaction to NGF antibody (Table 3).
DISCUSSION
We detected the antigen-antibody reaction of the
NRCF and nerve tissue fluids from rabbits and rats
with rabbit anti-mouse NGF antibody by ELISA under
different conditions in order to obtain more reliable
results. These results showed that all samples of the
supernatants and precipitates that were extracted by
centrifugation or 50% saturated ammonium sulfate
precipitation technique from the rabbit and rat nerve
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 128
Y. YE, Q. F. LI, and L. LI
TABLE 1. ELISA indices for the sciatic nerve tissue fluid of New Zealand white rabbits and SD rats
T a б л и ц я 1. Показники, отримані з використанням методики ELISA, для рідини з тканини сідничного нерва
новозеландських білих кролів та щурів лінії SD
Coating buffer dilution 1 2 3 4
1:5 1.952 1.501 1.515 0.866
1:10 1.859 1.050 0.852 0.882
1:20 1.890 1.416 1.465 0.895
1:40 1.938 2.047 1.724 1.072
1:80 1.851 1.718 1.958 1.490
1:160 1.569 2.123 1.966 2.417
1:320 1.386 2.024 1.577 1.974
1:640 1.351 1.691 1.205 1.459
Footnotes: Columns 1 and 2 are samples of the nerve tissue fluid of rabbits, while columns 3 and 4 are those of the fluid of rats. Primary
antibodies for samples 1 and 3 were diluted to 1:20, while those for samples 2 and 4 were dilute to 1:50; secondary antibody was diluted
to 1:1000.
TABLE 2. ELISA indices for the sciatic nerve tissue fluid of New Zealand white rabbits and SD rats treated with saturated
ammonium sulfate
T a б л и ц я 2. Показники, отримані з використанням методики ELISA, для рідини з тканини сідничного нерва кролів та
щурів, обробленої насиченим розчином амонію сульфату
Coating buffer dilution 1 2 3 4
1:2 2.830 0.350 3.304 0.727
1:4 2.918 0.497 3.394 0.712
1:8 2.932 0.530 3.808 0.746
1:16 2.703 0.325 3.900 0.838
Footnotes: Columns 1 and 3 are for samples of rabbits; column 1 is for the supernatant, and column 3 is for the precipitate; columns 2 and 4
are for samples of rats; column 2 is for the supernatant, and column 4 is for the precipitate.
TABLE 3. ELISA indices for the NRCF and sciatic nerve tissue fluid of New Zealand white rabbits and SD rats
T a б л и ц я 3. Показники, отримані з використанням методики ELISA, для рідини, кондиційованої процесом регенерації
нерва, та рідини з тканини сідничного нерва кролів та щурів
Coating buffer dilution 1 2 3 4
1:10 2.318 2.165 3.330 0.363
1:102 2.272 2.685 0.735 0.261
1:103 3.328 2.115 0.068 0.044
1:104 3.202 0.208 0.178 0.039
1:105 2.023 0.067 0.062 0.151
1:106 0.323 0.107 0.041 0.145
1:107 0.073 0.060 0.045 0.087
Control 0.018 0.126 0.009 0.134
Footnotes: Column 1 is for the NRCF of rabbits, column 2 is for the supernatant of the nerve tissue fluid of rabbits, column 3 is for the
precipitate of the rabbit nerve tissue fluid, and column 4 is for the precipitate of the rat nerve tissue fluid. Primary antibody was diluted to
1:500, and secondary antibody was diluteв ещ 1:1000.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 1 29
NERVE GROWTH FACTOR HOMOLOGY IN THE NERVE REGENERATION
tissue stock solution, the sciatic nerve tissue fluid of
rabbits and rats, and the NRCF of rabbits can react
with rabbit anti-mouse NGF, indicating that the NRCF
of rabbits demonstrates high homology with the rat
nerve fluid. The rank of the immune response intensity
indicated that the rabbit NRCF showed the strongest
protein activity in reacting with NGF antibody, which
might be related to the higher concentration of the
protein in rabbit samples.
The nerve regeneration chamber is implanted
between the transected ends of the nerve, and this is the
main convenient model to study the nerve regeneration
microenvironment [5, 8, 14]. Researchers found that
the chemotaxis and specificity of nerve regeneration
could be fully tapped when the renewable regeneration
chamber helped to repair the peripheral nerve, and
there was no (or the smallest) scar after the process of
nerve regeneration. This indicates that the regenerative
microenvironment (and NRCF as its main ingredient)
formed in the regeneration chamber played an important
role in self-regulation and self-improvement of the
process of nerve regeneration [15-17]. Thus, research
on the NRCF provides us with an important way to
study the nerve regeneration mechanisms [18, 19].
Our research has focused mainly on the extraction,
purification, and testing of active proteins in the NRCF
in order to understand the structures, sources, and
biological activity of these active ingredients. These
researches are mostly based on chromatography and
immunohistochemistry, thus requiring a sufficiently
large NRCF sample size [20, 21]. Naturally, the size
of NRCF sample extracted from the traditional rat
model is too small to be suitable for the requirements
of experimental research. Recently, the rabbit model
has been used to acquire larger NRCF sample sizes
[22, 23]. So, our data on the immune homology of
the NRCF between rabbits and rats are, probably,
rather significant for comparing and studying the
results obtained using different models. Our results
showed that NGF in the NRCF obtained from rabbits
demonstrated high homology with that in the rat
samples. Probably, it can be stated that the molecular
protein structures of NGFs of mammals belonging to
different taxonomic groups (rabbits as Lagomorpha
and rats as rodents) are rather similar. These data
suggest that the rabbit model can be successfully used
for further experimental studies of nerve regeneration
mechanisms.
Acknowledgment. The research was supported by the
National Natural Science Foundation of China (No. 30070776).
The authors thank Prof. Wenxiang Guan (China) for his
valuable assistance in the research.
The study was carried out in accordance with the statements
of the Council Directive regarding the protection of animals
used for experimental and other scientific purposes (86/609/
EEC, 1986, Strasbourg) and respective regulations of the local
Ethics Committee.
The authors of this study, Y. Ye, Q. F. Li, аnd L. Li, confirm
that the research and publication of the results were not
associated with any conflicts regarding commercial or financial
relations, relations with organizations and/or individuals who
may have been related to the study, and interrelations between
co-authors of the article.
Йі. Йє1, К. Ф. Лі2, Л. Лі1
ГОМОЛОГІЯ ФАКТОРА РОСТУ НЕРВІВ (NGF) У РІДИНІ,
КОНДИЦІЙОВАНІЙ ПРОЦЕСОМ РЕГЕНЕРАЦІЇ НЕРВА,
У КРОЛІВ ТА ЩУРІВ
1Університет традиційної китайської медицини, Аньху
(Китай).
2Дев’ята народна лікарня Шанхайського Університету Жіао
Тонг (Китай).
Р е з ю м е
Ми аналізували молекулярну гомологію росту нервів (NGF) у
кондиційованій процесом нервової регенерації рідині (NRCF),
котру отримували від новозеландських білих кролів та щурів
лінії SD. Нейрорегенеративна камера з’єднувала дистальний
та проксимальний кінці перерізаного сідничного нерва у кро-
лів, і NRCF відбирали з такої камери. За допомогою гомогені-
зації та центрифугування з тканини сідничних нервів кролів
та щурів готували супернатанти та преципітати. Реакцію ан-
тиген–антитіло виявляли за допомогою методики ELISA, ви-
користовуючи первинні антитіла щодо мишачого NGF та вто-
ринні козячі антитіла щодо кролячого імуноглобуліну. Реакції
антиген–антитіло виявлялися в усіх вищезгаданих зразках,
отриманих від кролів та щурів. Послідовність інтенсивностей
відповідей антиген-антитіло у різних зразках була такою: кро-
ляча NRCF > преципітат із нервової тканини кроля > супер-
натант із нервової тканини кроля > супернатант із нервової
тканини щура. Отже, молекулярна структура зразків NGF, що
знаходяться в NRCF, отриманій від кролів та щурів (тобто тва-
рин, котрі належать до різних таксономічних груп ссавців), де-
монструє високий рівень гомології.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 130
Y. YE, Q. F. LI, and L. LI
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