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Acupuncture induced Nestin expression in the intervention of intracerebral
hemorrhage rats
Wei Zoua* ,Qiu-Xin Chenb,Xiao-Wei Sun a
aFirst Affiliated Hospital of Heilongjiang University of Chinese Medicine, Haerbin 150004, China, bHeilongjiang University of Chinese Medicine, Haerbin 150004, China
Abstract
This study evaluated the effect of acupuncture on the Nestin expression in the basal ganglia hemorrhage in rats, using immunostaining, Western Blot .The positive products of Nestin protein in the acupuncture trial increased greatly more than that of control group. Therefore, acupuncture has important implications in cerebral protection and could promote neural stem cell regeneration in cases of cerebral hemorrhage.
Keywords: Nestin,; Cerebral hemorrhage; Acupuncture
1. Introduction
Intracerebral hemorrhage is an important public health problem leading to high rates of death 1 2 and disability in adults1and no clinically proven treatment strategy is available to date2.
Cerebrovascular diseases seriously affect the patient's quality of life, to become a heavy burden to
the society and to their families.Acupuncture,has been applied to the clinical and experimental
cerebral hemorrhage3-5.Nowadays, this traditional Chinese medical technique has become very
popular worldwide as a complementary medicine. For example, using acupuncture therapy
neurological diseases, heart disease,neuropathic pains, Cancer, asthma6-9.Although Clinical studies
have shown that acupuncture can promote the recovery of neurological function in patients with
cerebral hemorrhage, reduce muscle tension, improve daily living skills10, acupuncture for acute
cerebral hemorrhage neural remodeling mechanism is still not clear.Nestin which is a intermediate
filament protein specifically expressed in neuroepithelial stem cells; may play a role in neural cell
differentiation.we observed the expression of the Nestin protein in rat brain tissue by scalp
acupuncture stimulation Baihui (GV20) and Qubin(GB7) of acute cerebral hemorrhage in rats, and
explored the mechanism of the head nerve function after brain hemorrhage remodeling.
2. Materials and methods
2.1 Animals
One hundred seventy-six healthy male Wistar rats, each weighing 300~350g, were used in
this study. The animals were provided by the Beijing Vital River Laboratory Animal Technology
Co., Ltd.These rats were randomly divided into three groups:Model group, acupuncture group
(model + acupuncture group), each groups of 80 rats (50 rats were used for light microscopy and
immunohistochemistry ,another 30 rats used for Western blot).Each group were randomly divided
into five subgroups: 6h, ld, 2d, 3d, 7d,and each subgroup contains 16 rats.
2.2 Screening successful animal model and model
According to the stereotactic atlas of the rat brain determines the rat right caudate putamen position. Reference to Rosenberg's method of making a rat model of intracerebral hemorrhage. After the rats left limb paralysis, pull tail left upper limb flexion appeared vacant, or when traveling tilted to the left, circling, suggesting that successful model.
2.3 Scalp acupuncture
2.3.1 Blank group: containing sixteen rats without any intervention. Ten rats after perfusion fixation, remove the hematoma surrounding brain tissue, for light microscopy and immunohistochemical detection and another six rats were killed directly, remove the hematoma surrounding brain tissue for Western blot detection.
2.3.2 Model group: Eighty rats were divided into the 6h, ld, 2d, 3d, 7d five subgroups, according to the point in time and each alkylene group contains 16 rats. After completion of the cerebral hemorrhage model, the rats were given the same bundled with the acupuncture group, 30 minutes a day. Detection methods were the same as the blank group.
2.3.3 Acupuncture group: Eighty rats were divided into the 6h, ld, 2d, 3d, 7d five subgroups, Acupuncture methods: The rats were bundled to the acupuncture board, using 1 inch needle acupuncture ipsilateral Baihui acupoint and penetration acupuncture to the Qubin acupoint.The acupoints Refer Huaxingbang's ((experimental animal acupuncture points map)) . Needling depth of 0.8 inches, and maintained for 30 minutes. With acupuncture practices twisting manipulation three times during this period, every 5 minutes, and a speed of 200 cycles per minute. Then remove the brain tissue at 6h, ld, 2d, 3d, 7d five time points. Detection methods were the same as the blank group.
2.4 Immunohistochemistry
After the end of treatment for each time point, the rats were given excessive anesthesia, and then the rats heart was perfused with phosphate buffered saline(PBS), taken after the end of the rat brain tissue detected by immunohistochemistry. The animals were weighted and then overdosed with Zoletil 50 (10 mg/kg, i.p.; Vibac Laboratories, Carros, France). After a complete lack of response was observed, the rats were transcardially perfused with 50 mM phosphate buffere sdaline (PBS) and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in 100 mM phosphate buffer (PB,pH 7.4). The brains were dissected and post-fixed in the same fixative overnight, and then the brains were transferred into a 30% sucrose solution for cryoprotection. Serial coronal sections of 40 mm thickness were made using a freezing microtome (Leica, Nussloch, Germany).
2.5 Western blot
Animals were anesthetized before undergoing intracardiac perfusion with saline. The frozen tissue was extracted with RIPA lysis buffer protein BCA kit for determination of the concentration of total protein, the total protein of 50 / hole, the sample volume, gel electrophoresis and transferred to a membrane. The film with 5% skim milk-containing phosphate Tween buffer closed 4 °C overnight. Membranes were probed with a 1:1000 dilution of the primary antibody (mouse anti-nestin) and a 1:1500 dilution of the second antibody. When the end of the above steps with phosphate-Tween buffer, the membrane was washed for 10 min 3 times, chemiluminescence display purposes strip.
2.6 Statistical analysis
SPSS17.0 statistical software for statistical analysis, the data were presented as mean ± standard deviation (± SD) representation. Two sets of data were compared using the t test, multiple sets of data were compared using the SNK and LSD test. P <0.05 difference was statistically significant.
3^ Results
3.1 Immunoreactive expression of Nestin in surrounding tissue lesions in the cerebral hemorrhage
Nestin-positive cells express brownish yellow or brown, in the cerebral hemorrhage area.
Comparison of each group of rats Nestin positive cell numbers at different time points
Grouping
Quantity
h
1d
2d
3d
7d
Blank group 10 9.91±2.01 9.91±2.01 9.91±2.01 9.91±2.01 9.91±2.01
Model group 50 18.34±1.80*26.19±1.93* 34.18±2.26* 24.47±1.88* 21.26±2.09*
Acupuncture 50 21.91±1.51a 30.23±2.89a 40.33±1.88A 29.75±1.75a 28.47±1.91A group
AP < 0.01 Acupuncture group compared with model group
*P < 0.01 Model group compared with the control group
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Fig.1 A-E is positive cells the expression of Nestin five time points in the model group; F-J is positive cells the expression of Nestin five time points in the acupuncture group; K is blank group.
3.3 Western blot expression of Nestin in surrounding tissue lesions in the cerebral hemorrhage
Nestin-protein expression detected by western blot in the hematoma surrounding brain tissue results show:A small amount of blank group expression;At each time point, the acupuncture group and the model group comparison that Nestin-protein cells increased and the differences were statistically significant (P<0.01).
Nestin protein content in the brain tissue of rats at different time points in each group
Grouping Quantity 6h 1d 2d 3d 7d
Blank group 6 0.54±0.05 0.54±0.05 0.54±0.05 0.54±0.05 0.54±0.05
Model group 30 0.81±0.19* 1.55±0.20* 1.62±0.18* 1.39±0.17* 1.00±0.18*
Acupuncture 30 1.13±0.13A 1.93±0.14a 2.21±0.29a 1.86±0.23a1 ,43±0.14a
group
AP < 0.01 Acupuncture group compared with model group *P < 0.01 Model group compared with the control group
ABCD EF GH
J K I M N O P
Fig.2 Intracerebral hemorrhage in rats at each time point Nestin protein expressed strip. 4.Discussion
Scalp acupuncture therapy based on specific acupuncture scalp area lines, a therapy used to treat the disease, also known as "scalp acupuncture therapy" or "cranial acupuncture therapy". Cerebral hemorrhage is a high level of disability disease. For many years, many medical workers plunge into the treatment of cerebral hemorrhage, which studied the neurotrophic factor family and neural stem cells.
The Nestin is Class VI intermediate filament protein, first discovered in 1985 by Hockfield and McKay. It was a transient expression in embryonic neural precursor cells and neural stem cells. Nestin is mainly present in the central nervous system of the embryonic period, and it is one of the main markers of neural stem cells. Nestin in stem cell proliferation stage and expression of a large number of phenomena, suggesting its role in the differentiation of pluripotent stem cell self-renewal and maintain cannot be replaced by other intermediate filament. Therefore, it has been widely used in the identification of neural stem cells. Studies have shown that the expression of Nestin can be re-expressed in the adult animal brain and spinal cord traumatic injury to the nerve area. When the
central nervous system is injured, the re-expression of nestin may increase the ability of the cells
18
resistant to injury, help repair damaged foci .
The results of this experimental study showed that normal rat brain Nestin protein expression mild positive, cerebral hemorrhage model group 6h perihematoma organization the Nestin protein began to increase and reached a peak 2d. At 7d cerebral hemorrhage was still visible after the apparent expression of Nestin protein. Acupuncture group at each time point up-regulation of nestin protein expression, and compared with the model group, the obvious effect in the 2.3.7d time point. This evidence of raised Nestin protein expression in the brain may be the acupuncture treatment of cerebral hemorrhage effective mechanism. The early intervention and acupuncture treatment of cerebral hemorrhage can improve Nestin level. The raised Nestin, is conducive to the structure and function of neural tissue remodeling after cerebral hemorrhage and provides a new means of clinical treatment of cerebral hemorrhage.
Although it is well-established that acupuncture could promote remodeling of neural function after cerebral hemorrhage, the mechanism is not fully understood. The present study proposed that acupuncture could induce an up-regulation of Nestin, show that acupuncture may have a role to promote the regeneration of neural stem cells, which play a cerebral protective effect. 4^ References
[1]Adnan, I ., A, D, M.,Daniel ,F,H.,2011.Intracerebral hemorrhage.J.Lancet. 373(9675),1632-1644.
[2]Zhe L,Ling G,Yan W, et al.,2012.History and Mechanism for Treatment of Intracerebral Hemorrhage with Scalp Acupuncture.J.Evidence-Based Complementary and Alternative Medicine.Published online 2012 February 22.:10.1155/2012/895032.
[3]Chang-Nam Ko, In-Whan Lee,Seung-Yeon Cho, et al. Acupuncture for Cerebral Vasospasm After Subarachnoid Hemorrhage: A Retrospective Case-Control Study.[J]. The journal of alternative and complementary medicine.2012.18.00.pp.1-3.
[4]Zheng GQ,Zhao ZM,Wang Y, et al. Meta-analysis of scalp acupuncture for acute hypertensive intracerebral hemorrhage.[J].Altern Complement Med.2011.17.4 :293-9
[5]Wang F,Lei HS,Zhao X, et al. Effect of penetrative needling of scalp- acupoints on cerebral N-acetyl- aspartate and choline levels in intracerebral hemorrhage rabbits.[J].Zhen Ci Yan Jiu.2011.36.4 :242-6.
[6]F. J. Zijlstra, I. Van Den Berg-De Lange, F. J. P. M. Huygen,and J. Klein, "Anti-inflammatory actions of acupuncture,"Mediators of Inflammation, vol. 12, no. 2, pp. 59-69, 2003.
[7]Y.-G. Choi, J.-H. Park, and S. Lim, "Acupuncture inhibits ferric iron deposition and ferritin-heavy chain reduction in an MPTP-induced parkinsonism model, "Neuroscience Letters, vol. 450, no. 2, pp. 92-96, 2009.
[8]J.-H. Lin, C.-H. Shih, K. Kaphle et al., "Acupuncture effects on cardiac functions measured by cardiac magnetic resonance imaging in a feline model," Evidence-Based Complementary and Alternative Medicine, vol. 7, no. 2, pp. 169-176, 2010.
[9]M. S. Lee, B.-C. Shin, S.-M. Choi, and J. Y. Kim, "Randomized clinical trials of constitutional acupuncture: a systematic review," Evidence-Based Complementary and Alternative Medicine, vol. 6, supplement 1, pp. 59-64, 2009.
[10]Zou Wei, Liu Fang, Kuo-Wei Chang Acupuncture Research Progress in the treatment of cerebral hemorrhage [J]. Information on Traditional Chinese Medicine 2007, 24 (2) :31-33
A study on the effect of needling baihui acupoint on heart rate variability in
patients with depression
Weiping Cheng1 Tianyu Zou2LuWang3Gerhard Litscher3 Yangyang Xu2
1.First Affiliated Hospital of HeilongjiangUniversity of Chinese Medicine, Harbin, China
2.HeilongjiangUniversity of Chinese Medicine, Harbin, China
3.MedicalUniversityof Graz, Graz, Austria
Abstracts: Objective: To observe the change in heart rate variability (HRV) at different time intervals during the needling of Bai Hui acupoint.The effect of acupuncture on the sympathetic nerve and vagus nerve is also studied, thereby providing theorical evidence for the treatment of depression. Methods: The Hamilton Depression Rating Scale (HAMD) was used to select 33 patients (6 men, 27 women, ages 22-72).Bai Hui acupoint was needled and an AR12 HRV device was used to measure the HRV total power (TP), high frequency (HF) power, low frequency (LF) power, LF/HF, and mean heart rate (HRmean) values at 10 minutes before needle insertion, 10 minutes after needle insertion, 10 minutes after needle manipulation, and 10 minutes after needle removal.The data was then exported from the device, and using short term recording method, the parameter changes were analyzed every 5 minutes.SPSS 18.0 was used to perform statistical analysis, with P<0.05 having statistical significance.Conclusion:(1)Acupuncture on Bai Hui acupoint can increase HRV in patients with depression, and its lasting effects are evident. (2) Acupuncture on Bai Hui acupoint can increase vagus nerve excitability in patients with depression. (3) The treatment effect of acupuncture on Bai Hui acupoint works mostly by influencing the vagus nerve in patients with depression.
Keywords: Needling; Bai Hui; Depression; Heart rate variability
Depression is a type of low mood-based mental illness. WHO reports that as of 2012, a total of 350 million people worldwide will suffer from depression. It is expected that by 2020, depression will be second only to heart disease in disease prevalence.
