[Effect of "nape seven needles" on expression of hypoxia inducible factor-1α and Notch1 in cervical intervertebral disc degeneration rats].

OBJECTIVE: To observe the effect of "nape seven needles" on the expressions of hypoxia inducible factor-1α (HIF-1α) and Notch1 in cervical intervertebral disc of cervical disc degeneration (CDD) rats, so as to reveal its underlying mecha-nisms in improving CDD. METHODS: SD male rats were randomly divided into normal, model, non-acupoint and nape seven needles groups, with 10 rats in each group. Staticdynamic imbalance method was used to establish CDD model. Rats in the nape se-ven needles group were treated with acupuncture at "Fengfu"(GV16), and bilateral "Fengchi"(GB20), "Wangu"(GB12) and "Tianzhu"(BL10), and rats in the non-acupoint group received acupuncture at the sham acupoints at the caudal tip and armpit, both for 20 min, 6 days a week for 4 weeks. After intervention, tilted plane test and spiral CT were used to assess the neck movement function and cervical degeneration degree of rats; immunohistochemistry was used to observe the protein expression levels of HIF-1α and Notch1 in cervical discs tissue; Western blot and real-time quantitative PCR were used to detect the protein and mRNA expression levels of HIF-1α and Notch1 in cervical discs tissue, respectively. RESULTS: After modeling, the cervical curvature was straightened, with narrowed intervertebral space, rough and hardened articular surface, osteophytes, and blurred articular space and articular process, which was relatively milder in the nape seven needles group. Compared with the normal group, the angle of tilted plane was significantly reduced (P<0.05), while cervical scores, HIF-1α mRNA expression level in cervical intervertebral disc tissue were significantly increased (P<0.05) in the model group. Compared with the model and the non-acupoint groups, cervical scores were significantly reduced (P<0.05), while the angle of tilted plane, HIF-1α and Notch1 positive expressions, HIF-1α mRNA expression level, Notch1 protein and mRNA expression levels in cervical intervertebral disc tissue were significantly increased (P<0.05) in the nape seven needles group. CONCLUSION: Acupuncture of "nape seven needles" can reduce the degree of cervical degeneration in rats, which was possibly associated with its effects in up-regulating the expressions of HIF-1α and Notch1 in cervical intervertebral disc tissue, promoting the proliferation and recovery of endogenous cells in nucleus pulposus.

Quantitative analysis of near-implant magnesium accumulation for a Si-containing coated AZ31 cage from a goat cervical spine fusion model.

BACKGROUND: Magnesium (Mg) released from Mg-based implants degradation is believed to be effective in improving osteogenesis, however, studies focusing on Mg-based interbody cages are limited and fusion success was never reported. As excessive Mg accumulation can inhibit new bone formation, this study is designed to explain the possible reasons for the fusion failure of Mg-based cages by analyzing the relationships between the intervertebral Mg accumulation and the resulting interbody fusion. METHODS: The experimental cage was consisted of magnesium alloy (AZ31) substrate and Silicon (Si) -containing coating. C3/C4 and C5/C6 of 24 goats were implanted with cage or autologous iliac crest bone graft (Control group), which were analyzed at 3, 6, 12, and 24 weeks post-operatively. Intervertebral Mg concentrations, Mg-related Calcium (Ca)/ Phosphorus (P) ratios, radiological evaluations and histological findings were recorded for analyzing the relationships between the three of cage corrosion, Mg accumulation, and interbody fusion. RESULTS: Intervertebral Mg levels were significantly increased after cage implantation, especially in the areas that were closer to the cages at 3 weeks post-operatively, and these increased concentrations could persist up to 12 weeks post-operatively, indicating a relatively rapid corrosion process. Significantly lower Mg levels were only found at 24 weeks post-operatively, but these levels were still higher than those of the control group. In addition, Mg was found to be widely distributed at the intervertebral space since high Mg concentrations could even be detected at the posterior boundary of the vertebral body. Under this Mg accumulation profile, interbody fusion was not achieved, as indicated by the decreased Ca/P ratios, low CT fusion scores and negative histological results. CONCLUSIONS: Intervertebral excessive Mg accumulation might be the primary reason for interbody fusion failure. Quantitative Mg analysis can offer insight into the association between cage degeneration and biological response.

Cervical Spondylotic Myelopathy: Metabolite Changes in the Primary Motor Cortex after Surgery.

Purpose To characterize longitudinal metabolite alterations in the motor cortex of patients with cervical spondylotic myelopathy (CSM) by using proton magnetic resonance (MR) spectroscopy and to evaluate white matter integrity with diffusion-tensor imaging in patients who are recovering neurologic function after decompression surgery. Materials and Methods Informed written consent was obtained for all procedures and the study was approved by Western University's Health Sciences Research Ethics Board. Twenty-eight patients with CSM and 10 healthy control subjects were prospectively recruited and underwent two separate 3-T MR imaging examinations 6 months apart. Patients with CSM underwent surgery after the first examination. N-acetylaspartate (NAA), an indicator of neuronal mitochondrial function, normalized to creatine (Cr) levels were measured from the motor cortex contralateral to the greater functional deficit side in the patient group and on both sides in the control group. Fractional anisotropy and mean diffusivity were measured by means of diffusion-tensor imaging in the white matter adjacent to the motor and sensory cortices of the hand and the entire cerebral white matter. Clinical data were analyzed by using Student t tests. Results In patients with CSM, NAA normalized to Cr (NAA/Cr) levels were significantly lower 6 months after surgery (1.48 ± 0.08; P < .03) compared with preoperative levels (1.73 ± 0.09), despite significant improvement in clinical questionnaire scores. Fractional anisotropy and mean diffusivity were the same (P > .05) between the patient and control groups in all measured regions at all time points. Conclusion NAA/Cr levels decreased in the motor cortex in patients with CSM 6 months after successful surgery. Intact white matter integrity with decreased NAA/Cr levels suggests that mitochondrial metabolic dysfunction persists after surgery. © RSNA, 2016 Online supplemental material is available for this article.

[Effect of Acupotomy Lysis at Cervical Acupoints on Expression of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinase 1 and Changes of Pulpiform Nucleus Ultrastructure in Rats with Degenerated Cervical Intervertebral Discs].

OBJECTIVE: To observe the effect of acupotomy lysis at acupoints around the neck on expression of matrix metalloproteinase 1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinase 1 (TIMP-1) genes and ultrastructure of pulpiform nucleus in cervical intervertebral disc degeneration (IVDD) rats, so as to explore its mechanism underlying easing IVDD. METHODS: SD rats were randomly allocated to control (n = 15), model (n = 14), Jiaji (EX-B 2, n = 13), cervico-acupoint (n = 14) and medication groups (n = 14). The cervical IVDD model was established by using static-dynamic imbalance method. For rats of the Jiaji (EX-B 2) and cervico-acupoint groups, EX-B 2-points of the cervical 2-7 segments, and peri-cervical acupoints: bilateral "Naokong" (GB 19) , "Naohu" (GV 17), "Dazhui" (GV 14), bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11) were separately punctured with a needle-knife, once every 5 days for 3 times, and for rats of the medication group, Brufen Capsules (15 mg · kg(-1) · d(-1)) and Jingfukang Granule (0.5 mg · kg(-1) · d(-1)) were given by intragastric administration, once daily for 10 days. The expression levels of MMP-1, MMP-3 and TIMP-1 genes in the pulpiform nucleus of cervical intervertebral discs were detected by RT-PCR and changes of the ultrastructure of the pulpiform nucleus observed under transmission electron microscope. RESULTS: Compared to the control group, the expression levels of MMP-1 mRNA and MMP-3 mRNA of the cervical intervertebral disc tissues were significantly up-regulated in the model group (P < 0.05), and that of TIMP-1 mRNA was obviously down-regulated in the model group (P < 0.05). After the treatment, the increased expression of MMP-1 mRNA and MMP-3 mRNA and the decreased expression of TIMP-1 mRNA were reversed by acupotomy lysis and medication (P < 0.05) except TIMP-1 mRNA in the medication group (P > 0.05). No significant differences were found between the Jiaji (EX-B 2) and cervico-acupoint groups in down-regulating MMP-1 mRNA and MMP-3 mRNA expression and up-regulating TIMP-1 mRNA expression (P > 0.05). Results of electron microscope examinations showed that the ultrastructural injury changes of cells of the pulpiform nucleus were relatively milder in the Jiaji (EX-B 2) and cervico-acupoint groups, followed by the medication group in comparison with those of the model group. CONCLUSION: Acupotomy lysis at acupoints around the neck can improve the ultrastructural changes of cells of the pulpiform nucleus of cervical intervertebral discs in IVDD rats, which is possibly by regulating the expression of MMP-1, MMP-3 and TIMP-1 genes.

[Effects of electroacupuncture of "Futu" (LI 18), etc. on pain behavior and expression of GABA receptor subunit genes in cervical spinal cord in rats with thyroid regional pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at different acupoints on the expression of gamma aminobutyric (GABA) receptor (R) subunit genes in the cervical spinal cord in rats with thyroid regional inflammatory pain so as to analyze its analgesic mechanism for thyroid surgery. METHODS: A total of 50 Wistar rats were randomized into control, model, Futu (LI 18), Hegu (LI 4)-Neiguan (PC 6, LI 4-PC 6), Zusanli (ST 36)-Yanglingquan (GB 34, ST 36-GB 34) groups, with 10 rats in each group. Thyroid regional pain model was established by subcutaneous injection of 2.5% formalin (100 microL). Ten minutes after modeling, EA (2 Hz/15 Hz, 1 mA) was applied to LI 18, LI 4-PC 6 and ST 36-GB 34 for 30 min, respectively. The animals' face-grooming (FG) times in 5 min and thermal pain threshold (paw withdrawal latency, PWL) were recorded. The expression of GABA(A) R, GABA(B) R1, and GABA(B) R2 genes in the cervical 1-3 segments of the spinal cord 80 min after modeling was detected by using reversed transcription-polymerase chain reaction (RT-PCR). Histological changes of the tissues of the thyroid region were observed by using H. E. staining. RESULTS: After subcutaneous injection of formalin, the animals' FG times in 5 min were increased considerably and the thermal pain threshold was decreased obviously in the model group (P < 0.05). Concomitantly, the expression levels of GABA(B) R1, and GABA(B) R2 genes of the cervical spinal cord were upregulated significantly in the model group (P < 0.05), and GABA(A) R expression was increased slightly (P > 0.05). Compared with the model group, the FG times in 5 min in the LI 18 and LI 4-PC 6 groups at 40 min and 70 min after modeling were decreased significantly (P < 0.05), and their thermal pain threshold values were increased markedly (P < 0.05). The expression levels of GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABAA R mRNA were significantly higher in the LI 18 and LI 4-PC 6 groups than in the model group (P < 0.05). The expression level of GABA(B) R1 mRNA of the ST 36-GB 34 group was obviously higher than that of the model group (P < 0.05). The inflammatory cells in the formalin-injected thyroid region were relatively fewer in the LI 18 and LI 4-PC 6 groups than in the model group. CONCLUSION: Both EA of LI 18 and LI 4-PC 6 can significantly suppress formalin-injection induced pain reactions in the thyroid region, which may be closely associated with its effects in upregulating expression levels of cervico-spinal GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABA(A) R mRNA, and reduce regional inflammatory reactions in the thyroid region.

[Effects of electroacupuncture at Jiaji (EX-B 2) on extracellular matrix MMP-1, MMP-3 and TIMP-1 levels in the degenerated cervical intervertebral disc in rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of Jiaji (EX-B 2) on the contents of extracellular matrix metalloproteinase (MMP)-1, MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the cervical intervertebral disc (OIVD) tissue in rats with CIVD degeneration so as to explore its mechanism in relieving intervertebral disc degeneration. METHODS: SD rats were randomized into control (n = 15), model (n = 14), EA (n = 13) and medication (n = 14) groups. CIVO model was established according to Wang's and Shi's method. EA (10/20 Hz, 1 - 3 mA) was applied to Jiaji (EX-B 2) for 10 min, once daily for 30 days. The rats of medication group were fed with Fenbid (15 mg/kg/d) and Jingfukang (0.5 g/kg/d) for 30 days. Extracellular matrix MMP-1, MMP-3 and TIMP-1 levels of the intervertebral disc tissue (C4-6) were detected by ELISA. RESULTS: The contents of MMP-1 and MMP-3 in model group were increased significantly compared with control group (P < 0.05), while TIMP-1 in model group was markedly lower than that of control group (P < 0.05). Compared with model group, both MMP-1 and MMP-3 levels in EA group and MMP-1 in medication group were reduced considerably (P < 0.05), while TIMP-1 of EA group increased significantly (P < 0.05). No significant differences were found between medication and model groups in MMP-3 and TIMP-1 levels (P > 0.05). Under optical microscope, the injured degrees of the fibrous ring and nucleus pulposus, and the outline of the intervertebral disc of both EA and medication groups were evidently lighter than those of model group, but the degeneration severity of the intervertebral disc of EA group was lighter than that of medication group. CONCLUSION: EA of Jiaji (EX-B 2) can effectively relieve the degeneration degree of cervical intervertebral disc in the rat, which is probably related to its effect in regulating extracellular matrix system.

[Effects of electroacupuncture at different acupoints on the pain behavior and NMDA receptor 2 B subunit mRNA and protein expression and phosphorylation level in the cervical spinal cord in rats with thyroid regional pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of different acupoint groups on the expression of NR 2 B subunit of N-methyl-D-aspartate (NMDA) receptor in the cervical spinal cord in rats with thyroid regional pain so as to analyze the analgesic mechanism of acupuncture for performing thyroid surgery. METHODS: A total of 50 Wistar rats were randomized into control, model, EA-Futu (ST 32), EA-Hegu (LI 4)-Neiguan (PC 6, EA-LI 4-PC 6), EA-Zusanli (ST 36)-Yanglingquan (GB 34, EA-ST 36-GB 34) groups, with 10 cases in each. Thyroid regional pain model was established by subcutaneous injection of 2.5% formaldehyde. Ten minutes after modeling, EA (2 Hz/100 Hz, 1 mA) was applied to ST 32, LI 4-PC 6 and ST 36-GB 34 for 30 min respectively. The animals' face-grooming (FG) times and paw withdrawal latency (PWL) of radiant heat irradiation were recorded. The spinal cord tissues of the cervical 1-3 segments were removed for detecting the expression of NR 2 B mRNA and protein of NMDA receptor with reversed transcription-polymerase chain reaction and Western blotting techniques separately. RESULTS: After subcutaneous injection of 2.5% formaldehyde, the animals' FG times increased considerably and the PWL was shortened obviously (P<0.05). Compared with model group, the FG times of EA-ST 32 and EA-LI 4-PC 6 groups at 40-45 min and 70-75 min after modeling decreased significantly, and their PWL prolonged markedly (P<0.05). The effect of EA-ST 32 was apparently superior to that of EA-LI 4-PC 6 group in reducing FG times. No significant differences were found between EA-ST 32 and EA-LI 4-PC 6 groups in PWL, and between model and EA-ST 36-GB 34 groups (P>0.05). Results of NR 2 B mRNA and protein detections showed no significant differences among the 5 groups in their expression (P>0.05). Comparison of the phosphorylated NR 2 B among 5 groups showed that EA-ST 32 and EA-LI 4-PC 6 could effectively down-regulate 2.5% formaldehyde induced increase of NR 2 B phosphorylation levels (P<0.05), while EA-ST 36-GB 34 had no obvious effect on it (P>0.05). CONCLUSION: Both EA-ST 32 and EA-LI 4-PC 6 can significantly suppress thyroid regional pain induced behavior reactions, which may be closely associated with their effects in down-regulating NR 2 B phosphorylated level. The analgesic effect of EA-ST 32 and EA-LI 4-PC 6 for thyroid surgery is relatively more effective in comparison with EA-ST 36-GB 34.

[A rabbit model of cervical spondylosis established by stimulation of wind, cold and dampness].

OBJECTIVE: To make an animal model of cervical spondylosis (arthralgia syndrome type) with stimulation of wind, cold, and dampness. METHODS: Twenty-four 8 months old male New Zealand white rabbits were randomly allocated into four groups: normal control group, light stimulation group, moderate stimulation group and severe stimulation group. The wind speed was 10.8-13.8 m/s, the temperature was (5+/-0.5)degrees centigrade, and the humidity was 100%. The rabbits of light, moderate, and severe stimulation groups were kept in the above-mentioned environments for 4 hours everyday, and for a total of 32, 64, and 128 hours, respectively. The intervertebral discs were stained with HE method, and observed with a light microscope. Prostaglandin E(2) (PGE(2)), 6-ketone-prostaglandin F1alpha (6-K-PGF(1alpha)) and thromboxane B(2) (TXB(2)) contents were measured by ELISA. Fas and Bcl-2 expressions were examined by immunohistochemical avidin-biotin peroxidose complex technique. Interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta) mRNA expressions were examined by reverse transcription-polymerase chain reaction. RESULTS: The nucleus pulposus of rabbits in the light and moderate stimulation groups shrunken, and in the severe stimulation group, the anulus fibrosus loosed or ruptured, and the cartilage end-plate became proliferated. Compared with rabbits in the normal control group, the PGE(2) content rose in the light stimulation group, the contents of PGE(2), 6-K-PGF(1alpha), and TXB(2) increased, the expressions of IL-1beta and TNF-alpha mRNAs and Fas were up-regulated, and the expressions of TGF-beta mRNA and Bcl-2 were down-regulated in the moderate and severe stimulation groups. The expression of Fas was up-regulated mostly and Bcl-2 was down-regulated mostly in the severe group. CONCLUSION: Moderate and severe stimulations of wind, cold and dampness can lead to degeneration of cervical intervertebral discs of rabbits. The model corresponds to the theory of traditional Chinese medicine about arthralgia syndrome caused by wind, cold and dampness.

Brainstem and cervical spinal cord Fos immunoreactivity evoked by nerve growth factor injection into neck muscles in mice.

Although myofascial tenderness is thought to play a key role in the pathophysiology of tension-type headache, very few studies have addressed neck muscle nociception. The neuronal activation pattern following local nerve growth factor (NGF) administration into semispinal neck muscles in anaesthetized mice was investigated using Fos protein immunohistochemistry. In order to differentiate between the effects of NGF administration on c-fos expression and the effects of surgical preparation, needle insertion and intramuscular injection, the experiments were conducted in three groups. In the sham group (n=7) cannula needles were only inserted without any injection. In the saline (n=7) and NGF groups (n=7) 0.9% physiological saline solution or 0.8 microm NGF solution were injected in both muscles, respectively. In comparison with sham and saline conditions, NGF administration induced significantly stronger Fos immunoreactivity in the mesencephalic periaqueductal grey (PAG), the medullary lateral reticular nucleus (LRN), and superficial layers I and II of cervical spinal dorsal horns C1, C2 and C3. This activation pattern corresponds very well to central nervous system processing of deep noxious input. A knowledge of the central anatomical representation of neck muscle pain is an essential prerequisite for the investigation of neck muscle nociception in order to develop a future model of tension-type headache.

The effect of different dietary levels of Ca and P on the plasma Ca, inorganic P and Mg as well as the ash, Ca, P and Mg content of cervical vertebrae and total body of sheep.

Six 14 month old rams were divided equally into three groups and received either a high Ca (1,102% Ca : 0,192% P : 0,128% Mg), control (0,322% Ca : 0,311=P : 0,128% Mg) or high P (0,127% Ca : 1,043% P: 0,130% Mg) diet in pellected form for 150 days. 2. Dry mass and ash % of the third cervical vertebra decreased as Ca intake decreased while fat % increased. There was a highly significant negative correlation (= -0,992) between bone ash% and bone fat %. Bone P and Mg showed no particular trends. 3. Total body Ca and P as well as % body Ca and P all decreased with a decrease in dietary Ca and increase in dietary P intake. Body Mg showed no particular trend. 4. The results suggest that while plasma values may be useful in assessing the P intake of sheep on natural pastures, bone and total body P may not be.