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19improvement of cervical vertebra disease, make it more targeted, the subject of massage new basic theories, push forward the theory innovation massage science, and its application in clinical, provide clinical basis for the mechanism of action the further research of massage cervical area. References
Cervical vertigo He Ji Fan Dongsheng Sun Yu, Chinese Journal of practical Department of Internal Medicine, In 2011 06 period.
Study on change of endogenous neural stem cells after spinal cord transection lesion treated by Jiaji electroacupuncture
Tengxiuying, ,Zhangxiaomei, Laizengjiao, Jianghaixia, Wujianli,Qihuan,Yuqian, Yaoshun,Songwenbo,LiangqingYangjingjie,Li 'aimu
( HeilongjiangUniversity of Chinese Medicine,Harbin,China)
Abstract
AIM: To investigate the effect of Jiajielectroacupuncture on endogenous neural stem cells after spinal cord injury(SCI)in rats and the mechanism .METHOD:96 female Wister rats were randomly divided into four groups:® sham operated group©the control group :spinal cord injury at T10 level by complete transection lesion method, ©Jiajielectroacupuncture treatment group after spinal cord injury, ©Jiaji electroacupuncture pre-conditioning treatment group, in which the rats were treat one week by Jiaji electroacupuncture before SCI. Judging the motor function of rats after SCI at 3,7,14dtime point refer to BBB score.The rats were killed at 3d,7d and 14d after SCI , the tissues were analyzed using immunohistochemical and real-time quantitative PCR detection to study the response of endogenous neural stem cells. RESULT: The changes of BBB score show that the treatment groups were better than control group at 7,14d time points(P<0.05). The number of Brdu, Nestin and GFAP positive cellsare different between four groups(P<0.05) at 3d,7d and 14d. Nestin, GFAPmRNA expression are different between four groups(P<0.05) at 3d,7d and 14d. CONCLUSION: The research verifiesthatJiajielectroacupuncture and Jiaji electroacupuncture preconditioning can promote proliferation of endogenous neural stem cells after spinal cord injury.
Key words:Jiajielectroacupuncture; Spinal transection lesion; Rats; endogenous neural stem
cells
Introduction
Spinal cord injury is a serious damage to the central nervous system, it often causes disability and even death, the traditional opinion think that adult mammalian central nervous system does not have the ability to update, and dose not renew after damage. In recent years, many studies found in adult mammals including humans the central nervous system has many neural stem cells (NSCs) which can renew and split apart[1]. Normally these NSCs are dormant,however they can proliferate and migrate to replace the necrotic nerve cell, repair necrotic lesions and restore neural function under the stimulus such as damage and ischemia .can activate such a series of changes, This is after spinal cord injury treatment brings new hope. As is known to all, the curative effect of Jiajielectroacupuncture after spinal cord injury(SCI) is good in clinical practice[2]. This experiment observe the proliferation of endogenous neural stem cells by using the treatment of Jiajielectroacupunctureafter spinal cord injury(SCI)in rats and search the mechanism of this treatment. Theresearch providestheoreticalandexperimentalfunctionfor clinicaltreatmenttoSCI.
Materials and methods
1.1 Experimental animals and groups
96 adult female Wister rats(animal experiment center, heilongjiang university of trditional Chinese medicine, China) weighing 200-220g were randomly divided into four groups: the sham operated group,the control group, Jiaji electroacupuncture group andJiaji electroacupuncture preconditioning group. The rats of each group were randomly killed at 3d,7d and 14d ,and there are eight rats in every time point.Foursamples were studied by immunohistochemical method, another fourwere by real-time quantitative polymerase chain reaction method.
1.2 Preparation of animal models
Rats with 10% of chloral hydrateintraperitoneal injection (400mg/kg )have a successful anesthesia ,then were fixed in the prone position on the operating table. After disinfection in back skin, skin and subcutaneous tissue was layered cut, and spine was exposed at T8 - T10 scute . The neural scute was cut with surgical forceps, and the spinal of T9-T11 was exposed sufficiently. Spinal cord of T10 was scissor by microscissors,and was covered with gelatin sponge. Stitching up the wound, and intraperitoneally infusing with penicillin.Pressingbladder timing,hypodermicly infusingwith neostigmine to promote gastrointestinal peristalsis, and supplying enough water and diets.
1.3 treatment
The acupuncture place is away from the vertebral column for 3-4mm,the two upper needle are beside the clearance of the two vertebral processus spinosus which are above the damage,the other two nether needle are beside the clearance of the two vertebral processus spinosus which are below the damage, with an inch needle penetrating vertically ,making the tip of needle touch the lamino .Then join the pulse current to needle handle, the positive pole is joint to the upper needle handle , and the negative pole is nether needle handle. Using condensation wave ,the frequency was 100Hz , output current strength was refered to the twitch of back muscles. for mild twitch. Electric acupuncture group were treated for 15min in half an hour, four hours, eight hours after spinal cord injury, then once a day . Jiaji electroacupuncture pre-conditioning treatment group were treat one week by Jiaji electroacupuncture before SCI,once a day .After SCI ,the treatment was the same as Electric acupuncture group .
1.4major instruments and reagents
BIO-RAD fluorescence quantitative polymerase chain reaction (PCR) apparatus (USA), highspeed centrifuge, ultralow temperature refrigerator, optic microscope (Nikon), automatic photographic apparatus, electric acupuncture apparatus (CHINA). Phosphate buffer solution (PBS), BrdU (Sigma company ,USA),BrdU monoclonal antibodies in rats (NeoMarkers ),Nestin polyclonal antibody (ABcam), GFAP (wuhan,CHINA), PV6001/6002 two-step immunohistochemical immuosorbent (Beijing,CHINA), SYBR Premix Ex Taq kit (TaKaRa), reverse transcription pcr kit (TaKaRa), Total RNA extraction reagent (TaKaRa).
1.5 BBB score
Referd to BBB(Basso, Beattie, and Bresnahan open-field locomotion score)score suggested by Basso etc which is used to judge the motor function of rats after SCI, there are two fixed people who master the standard for evaluation well to get the score at 3,7,14d time point after SCI, then average their scores.
1.6 Sample preparation
©sample for immunohistochemical test is prepared as follows: Preparing 1% of brdu solution with physiologic saline solution. Giving rats intraperitoneal injection with 1% ofbrdu(150mg/kg) solutionthree days before they were killed.In rat executed , twice a day. Rats were injected with 10% of chloral hydrate (400mg/kg weight), then wereaffused with 4% paraformaldehyde.Rapidly taking out spinal cord whose impairedlociwere the center,thentakingthem into 4% paraformaldehyde for 24h for immunohistochemical staining. ® sample for real-time fluorescence quantitative PCR test is prepared as follows: The rats were killed with decapitation . Rapidly the damaged spinal cord were taked out,then were stored at -80°C.
1.7 immunohistochemistry
Each paraffin sectionwas dewax ,then wasmarinated in 3% hydrogen peroxide for 10minto blockade endogenous peroxidase.Highly compressed steam repaired antigen, The tissue sections were incubated overnight with BrdU antibody (1:200) or Nestin (1=400) ,GFAP (1=400) at 4°C,then were incubated with immunohistochemical immuosorbent for 30min at room. Dropwising add DAB Developer, and observing sections for 10~30min under the optic microscope. The cell with yellowish-brown dyeing was positive. Then counterstain with haematin, washing,dehydration with alcohol, transparent with xylene, lastly, finalize. Counting the total number of positive cells in 10x40 times microscope, and chose randomly five view of each paraffin section.
1 .8 Extraction of total RNA and cDNA synthesis
Total RNA was isolated using RNAiso Plus reagent .Reverse transcription (RT) was carried out at a
volume of 10^L, containing 5xPrimeScript Buffer 2^L, PrimeScript RT Enzyme Mix 0.5^L, Oligo Dt Primer 0.5^L, Random 6 mers 0.5^L, Total RNA 4^L, RNasefree H2O 2.5^L0 It required a 15-min ute at 37° then 5 seconds of 85°C.The products of amplification were stored at-80°C until used for PCR .
1.9 real-time fluorescence quantitative PCR
Specific primers were designed by TaKaRa Biotechnology(Dalian)Co.,Ltd. Relative quantification of GFAP and NESTIN mRNA expression were calculated using the threshold cycle method,using GAPDH for internal control reference.Total volume of the PCR reaction was 25^l ,consisting of RT-PCRmix and 0.5 ¡ul of each primer. Each reaction required a 30-second incubation at95°C. After the incubation, each cycle underwent 5 seconds of 95°C-denaturation, 30 seconds of 60°C-annealing,and elongation toamplify the DNA fragment. Dissociation curve analysis was performed after PCR.The agarose gel electrophoresis was also carried out in experiments to verify the successful DNA amplification.
Primer sequences for quantitative real-time PCRand the length of amplification GA 5' -GGCACAGTCAAGGCTGAGAATG- 143bp
PDH 3'
5'-ATGGTGGTGAAGACGCCAGTA-3' GF 5' -AGTGGCCACCAGTAACATGCAA- 163bp
AP 3'
5' -GGACTCAAGGTCGCAGGTCAA-3' Nest 5' -CAGCAACTGGCACACCTCAAG-3' 101bp
in 5' -CCTCGTCCAGGTGTCTGCAA-3'
1.10 amplification efficiency
Taking a series of diluted cDNA (20,10,5,2.5 ,1.25 ng)of each target for fluorescent real-time quantitatie (PCR), with the concentration gradient for the y axis, the corresponding CT for the x axis , establish standard curve, standard curve equation, according to the slope of the standard curve equation to compute the amplification efficiency: E=10- -1.
1.11 Data analysis
Data are presented as mean ± standard deviation (SD) for normally distributed continuous variables and tested by 1-way analysis of variance (ANOVA). Tukey test or Dunnet test was then utilized for post-hoc analysis of continuous variables with or without equal variance between groups. BBB score is presented as median (interquartile range) owing to non-normal distribution. The difference in BBB score among groups was verified by Kruskal-Wallis test, and the MannWhitney U test was applied for post-hoc analysis of BBB scores. A 2-sided p < 0.05 was considered statistically significant, and the significance level was adjusted to 0.005 (= 0.05/number of post-hoc tests) when the Mann-Whitney U test was implemented. Statistical analyses were performed with SPSS statistical software (v. 15.0; SPSS Inc., Chicago, IL, USA).
Result and discussion
According to statistical results, The changes of BBB score show that the treatment groups were better than control group at 7,14d time points(P<0.05). Brdu, Nestin, GFAP positive cell count
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in treatment groups is higher than control group(P<0.05),brdu,Nestin,GFAPpositive cell count in electroacupuncture pre-conditioning group is higher than electroacupuncture group(P<0.05). Treatment groups' relative expression of Nestin,GFAPmRNA is higher than control group(P<0.05), electroacupuncture pre-conditioning group' relative expression of Nestin, GFAPmRNA is higher than electroacupuncture group at 7 day(P<0.05).
The aim of the present study was to assess the effect of Jiaji electroacupuncture on cell proliferation and the expression of nestin and GFAP, as markers of neural stem cells, in transected rat spinal cord. Results showed that Jiaji electroacupuncture, applied either after transection or both before and after transection, increased cell proliferation in the spinal cord, as assessed by BrdU staining and cell quantification, and increased the number of cells positive for nestin and GFAP immunostaining compared to the control group.
We assessed 3 markers of neural stem cells in this study. BrdU intercalates into the DNA and is used as a marker of cell division and proliferation. Nestin, also termed nidogen, is an intermediate filament protein expressed in neuroepithelial precursor cells and also in adult pluripotent stem cells[3]. GFAP is commonly used as a marker of glial response to injury, and astrocytes can differentiate along multiple lines and exert various effects in response to spinal cord injury[4'5]. In fact, Lang and colleagues reported that astrocytes can revert to neural stem cells after spinal cord injury in rats[6]. Therefore, we feel the combination of these 3 markers is indicative of neural stem cell activation.
Results of motor function assessments were mirrored by neural conduction experiments, showing a lack of spinal cord EPs after transection, followed by partial recovery within7 to 14 days.This improvement was enhanced by Jiaji electroacupuncture, and particularly by preconditioning.We did not perform a quantitative comparison of wave amplitude height, but visual assessment showed no marked differences between groups. EP conduction in the spinal cord was shorter and SSCV was elevated in the electroacupuncture groups (more so in the preconditioning group) compared to the control groups at 7 and 14 days after transection.
Many studies have shown that acupuncture preconditioning can exert favorable protective effects on hypoxia and ischemia in the heart, brain, gastrointestinal tract, and spinal cord, and it has been suggested that preconditioning generates some trigger factor to induce a protective effect[7]. Our present results showed a greater effect in the Jiaji electroacupuncture preconditioning group compared to the postinjury electroacupuncture treatment group. Thus, preconditioning may also promoteendogenous neural stem cell activation. Future studies are necessary to confirm the present findings and to further elucidate the mechanisms underlying the effects of Jiaji electroacupuncture on endogenous neural stem cells.
References
1.Lie D C, Song H, Colamrino S A, et al. Neurogenesis in the adult brain: new strategies for central nervous system diseases.Annu Rev Pharmacol Toxicol,2004,44: 399-421.
2.Taoyingli. efficacy of spinal cord injury treated by jiaji-electroaccupuncture . Shanghai Journal of Acupuncture and Moxibustion,2002,5:42.
3.Gritti A, Parati EA, Cova L, Frolichsthal P, Galli R, Wanke E, Faravelli L, Morassutti DJ, Roisen F, Nickel DD, Vescovi AL. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 1996, 16:1091-1100
4.Ao Q, Wang AJ, Chen GQ, Wang SJ, Zuo HC, Zhang XF. Combined transplantation of neural stem cells and olfactory ensheathing cells for the repair of spinal cord injuries. Med Hypotheses 2007, 69:1234-1237
5.Iriki A, Sakura O. The neuroscience of primate intellectual evolution: natural selection and passive and intentional niche construction. Philos Trans R Soc Lond B Biol Sci 2008, 363:22292241
6.Lang B, Liu HL, Liu R, Feng GD, Jiao XY, Ju G. Astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells. Neuroscience 2004, 128:775-783
7.Tatlisumak T, Takano K, Carano RA, Miller LP, Foster AC, Fisher M. Delayed treatment with an adenosine kinase inhibitor, GP683, attenuates infarct size in rats with temporary middle cerebral artery occlusion. Stroke 1998, 29:1952-1958
The Effect of Jingmaitong Granule on Vascular Endothelial Injury in Rat
Guo Weiguang,Tenglin,Wang Jing Thesecond hospital affiliated toHeilongjiang universityof traditional Chinesemedicine,Harbin,China
Abstract: Objective : to observe the effect of Jingmaitong granule on vascular endothelial injury in Rat. Methods: The vascular endothelial injury was induced, and the changes of thromboxane B2 (TXB2 ) and 6-keto-prostaglandinF1a (6-keto-PGF1a) were observed. Results: Jingmaitong granule could decrease the levels of TXB2, and increade the levels of 6-keto-PGFla , compared with control group(p<0.05). Conclusion: Jingmaitong granule has treatment effect on vascular endothelial injury.
Keywords: Jingmaitong granule Vascular endothelial injury Thromboxane B2 (TXB2 )
6-keto-prostaglandinF 1 a (6-keto-PGF1 a)
Jingmaitong granule, the extracts of ten kinds of Chinese medicine, including Beautiful Sweetgum Fruit, Cowherb Seed, Glabrous Greenbrier Rhizome, Dioscoreae Hypoglaucae Root,etc.which has the effects of warming Yan and enriching Qi and promoting blood circulation to remove blood stasis, can be used to treat the diseases caused by vascular endothelial injury, such as arteriosclerotic occlusion, lower extremity deep venous thrombosis, thromboangiitis obliterans. Presently, more studies are focused on its clinical effects, but the basis theories are relatively ignored. Thus this experiment mainly study the effects of Jingmaitong granule on vascular endothelial injury by observing the changes of TXB2 and 6-keto-PGF1a.
PART 1 ExperimentalMaterials
1.1 Experimental Animals
20 male Wistar rats(200±30g) were provided by experimental animal center of HarbinMedicalUniversity.
1.2Experimental Drug
Jingmaitong granule was provided by the preparation room of Heilongjiang University of Chinese Medicine Affiliated Hospital.
1.3 Experiment Reagents
The TXB2 and 6-keto-PGF1a kit, were provided by Beijing forest biological engineering co., LTD. The batch number of the kit is 20081201.
1.4 Experimental Apparatus
CNT automatic radioimmunity y counter, ELIASA, Desk Centrifuge,Surgical Scissors, Wire Scissors, Microsurgical Scissors, Microscope Forceps, Needle Forceps
Vascular Clamp, 1#fP4# Absorbable Surgical Thread, PVP, and Absorbable Gelatin Sponge.
PART 2 Methods
All male Wistar rats were devided into tow groups, including Jingmaitong group and control group, and there were 10 rats in each group. The vascular endothelial injury was induced by damnifying the femoral vein endothelial of rats with some kind of guide wire. Rats in Jingmaitong group were fed with Jingmaitong granule(25g/100gd for 15 days intragastric administration), while the rats in control group were fed with 0.9% Nacl for 15 days. Then all rats were fastinged 12 hours, and taken samples from femoral venous to observe the changes of TXB2 and 6-keto-PGF1a.
PART 3 Statistical Methods
