- Email: [email protected]
Electro-acupuncture upregulates CGRP expression after rat spinal cord transection
Neurochemistry International 61 (2012) 1397–1403
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Electro-acupuncture upregulates CGRP expression after rat spinal cord transection Wen-Jie Li a,1, Shu-Min Li b,1, Ying Ding a, Bing He a, Jack Keegan d, Hongxin Dong d, Jing-Wen Ruan b,⇑, Yuan-Shan Zeng a,c,⇑ a
Division of Neuroscience, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Department of Acupuncture, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China c Institute of Spinal Cord Injury, Sun Yat-sen University, Guangzhou 510120, China d Department of Psychiatry and Behavioral Sciences, Northwestern University, Feinberg School of Medicine, Chicago, USA b
a r t i c l e
i n f o
Article history: Received 26 March 2012 Received in revised form 28 September 2012 Accepted 5 October 2012 Available online 13 October 2012 Keywords: Electro-acupuncture Calcitonin gene-related peptide Neuroprotection Spinal cord injury Cerebellar granule neurons
a b s t r a c t Calcitonin gene-related peptide (CGRP) plays a variety of important roles within the nervous system. Increasing CGRP expression could improve the survival of injured neurons and prevent neuronal loss. In this study, we first evaluated in vitro the neuroprotective function of CGRP on mechanically injured cerebellar granule neurons (CGNs) of rats. We then verified this result through exogenous administration of CGRP in a spinal cord transected completely in rats. Finally, we investigated the effect of electro-acupuncture (EA) on CGRP expression following the spinal cord transected completely in rats. We found that EA can improve CGRP expression, and exogenous CGRP may promote the survival of injured neurons, both in vivo and in vitro. Our results suggest that CGRP may be a specific neuropeptide expressed in GV-EA treatment of spinal cord injuries (SCI), and that CGRP may play a neuroprotective role in survival of neurons injured mechanically. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Calcitonin gene-related peptide (CGRP) is a widely distributed 37 amino-acid peptide (Van Rossum et al., 1997; Wimalawansa, 1997), that is known to play a critical role in regulating the inflammatory response in peripheral sensory neurons (Li et al., 2004). CGRP is synthesized in cell bodies of the dorsal root ganglion (DRG) and transported axonally to the peripheral and central endings of sensory nerve fibers (Kashihara et al., 1989). CGRP has been recognized as a nerve regeneration-promoting peptide in vivo (Blesch and Tuszynski, 2001) and has also been shown to inhibit apoptosis of cells in vitro (Schaeffer et al., 2003; Chan et al., 2009). Although some studies have linked CGRP with spinal cord injury (SCI) progression (Christensen and Hulsebosch, 1997; Ondarza et al., 2003; Zinck et al., 2007; Ackery et al., 2007; Wu et al., 2009), the neuroprotective role of CGRP is not yet fully understood. SCI is a devastating neurological injury that often ⇑ Corresponding authors. Addresses: Department of Acupuncture, The 1st Affiliated Hospital, Sun Yat-sen University, #58 Zhongshan 2nd Road, Guangzhou 510080, China. Tel.: +86 20 87755766 8390 (J.-W. Ruan), Division of Neuroscience, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yatsen University, #74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel./fax: +86 20 87332698 (Y.-S. Zeng). E-mail addresses: [email protected] (J.-W. Ruan), [email protected] (Y.-S. Zeng). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.10.002
results in profound functional deficits and is a frequent cause of mortality around the world (Ackery et al., 2004; Thuret et al., 2006). As SCI primarily affects young adults (between 16 and 30 years old), the personal and economic burden associated with this type of injury is often very high (Sekhon and Fehlings, 2001; Calancie et al., 2005). While the effects of spinal cord injuries are far reaching, recovery and treatment options remain limited. Acupuncture and electro-acupuncture (EA) are traditional Chinese medical methods that have been practiced in East Asia for more than 2500 years and are now widely used for a range of neurological disorders (Wong et al., 2003; Lee et al., 2007). EA has been successfully used to treat a number of SCI-related conditions, including motor deficits, pain, spasticity and syringomyelia (Cheng et al., 1998; Paola and Arnold, 2003; Dyson-Hudson et al., 2007). In previous studies, we found that EA treatment could induce the regeneration of CGRP positive nerve fibers in the injured spinal cord (Ding et al., 2009; Yan et al., 2011). In the present study, we first examined the neuroprotective properties of CGRP in injured cerebellar granule neurons (CGNs) of rats in vitro. Here, we referred to a new model with injured CGNs (Ma et al., 2012) which is feasible to investigate the precise effect of mechanical damage on neurons. We then confirmed this neuroprotective function by directly administering CGRP to the injured spinal cord. Finally, we investigated whether electro-acupuncture (EA) could stimulate CGRP expression in the injured spinal cord of rats.
1398
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
2. Materials and methods 2.1. Cell culture and scratch-wound model performed with CGNs CGNs were isolated and cultured from 7-day-old Sprague–Dawley rat pups (15–19 g) as previously described (Yan et al., 1995; Liu et al., 2007; Ma et al., 2012). The study protocol was in accordance with the guidelines for animal research and was approved by the Experimental Animal Center of Sun Yat-sen University. Scratch wounds were performed with CGNs at 8 days in vitro. At the time of the experiments, neurons with mature cell bodies and abundant processes composed 95% of each culture. CGNs on glass cover slips were placed in 6 hole culture plates and wounded in a ‘‘#’’ design every 6 mm with a cataract knife (Fig. 1). Some cultures received simultaneous 10 6M or 10 7M CGRP treatment with the performance of a scratch wound. All cultures were stained with calcein-AM and EthD-1. Viable cells exhibited green fluorescence that was generated by calcein-AM, while dead cells were marked by red fluorescence from EthD-1.
2.2. Exogenous CGRP acting on the injured spinal cord 2.2.1. Experimental groups A total of 14 adult female Sprague–Dawley (SD) rats (200–220 g) were used for this study:
Group 1: PBS group (n = 7). The spinal cord of each rat was transected completely, and a piece of gelfoam (2 2 2 mm3) preloaded with 5 ll 0.01 M PBS was implanted between the rostral and caudal cord stumps. Group 2: CGRP group (n = 7). The spinal cord of each rat was transected completely, and a piece of gelfoam (2 2 2 mm3) preloaded with 5 ll 10 7 M CGRP was implanted between the rostral and caudal spinal cord stumps. 2.2.2. Quantitative analysis of neuronal survival in RN and CN One month after transection surgery, brains and L1 spinal segments were removed, postfixed and cryoprotected. After brain samples were serially cut into 30 lm sections, one out of every six sections containing red nucleus (RN) was selected and placed on a gelatin coated glass slide. Each L1 spinal segment containing Clarke’s nucleus (CN) was also serially cut into 30 lm sections, and 50 sections from each spinal segment were placed on gelatin-coated glass slides. All sections were then soaked in 1% neutral red stain for 20 min, rinsed in de-ionized water for 10 s, and successively dehydrated in 70%, 80%, 90%, 95% and 100% alcohol. Finally, the sections were cleared with dimethylbenzene and observed under the microscope. After neutral red staining, RN and CN neurons with a visible nucleus were counted. We calculated the amount of surviving neurons in 50 transverse sections from each L1 spinal segment, representing the total number of neurons in the L1 CN of each rat. The neuronal densities of CN of L1 spinal cord segments and RN of midbrain samples were calculated using grid-equipped microscopy based on stereological principles. 2.3. Electro-acupuncture (EA) effect on CGRP expression in the injured spinal cord 2.3.1. Experimental groups A total of 40 adult female SD rats (200–220 g) were used for this study. Thirty of them were divided into five groups (n = 6 in each group): the normal group (normal), the SCI group (SCI), the SCI + Governor Vessel EA-treated group (GV-EA), the SCI + Jiaji points Governor Vessel EA-treated group (Jiaji-EA) and the SCI + non point area Governor Vessel EA-treated group (npa-EA). For partial dorsal rhizotomy, ten adult female SD rats (200–220 g) were divided into two groups (n = 5 in each group): the SCI + unilateral dorsal rhizotomy + Governor Vessel EA-treated group (GV-EA⁄) and the SCI + unilateral dorsal rhizotomy + Governor Vessel EA + point nerve blocking group (GV-NB⁄). All animals were provided by the Experimental Animal Center of Sun Yat-sen University and received humane care in compliance with the Public Health Service Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the number of animals used and the pain experienced during the course of the procedure. 2.3.2. Spinal cord injury mode Rats for spinal cord injury were deeply anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). A laminectomy was carried out at the T9 level to expose the T10 spinal segment. The dura was cut using ophthalmic scissors and the T10 spinal segment was completely transected. Immediately after the procedure, both hind limbs were completely paralyzed, while the front limbs remained functional. Following surgery, the incisions were closed and the rats were returned to their respective cages. Animals received intensive care including intramuscular injection of penicillin (50,000 U/kg/day) for 3 days and manual emission of urine twice a day before being sacrificed.
Fig. 1. CGNs scratch graph. CGNs in glass coverslips of 6 holes culture plates were wounded by ‘‘#’’ scratching every 6 mm with an iris blade (A). ⁄Represents the scratch injury region (B). Scale bar = 80 lm.
2.3.3. Electro-acupuncture stimulation procedure Two Ashi points on the Governor Vessel, ‘‘Jizhong’’ (GV6) and ‘‘Zhiyang’’ (GV9), were selected for the treatment of SCI rats in
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
the GV-EA group according to our previous studies (Chen et al., 2008; Ding et al., 2009; Li et al., 2010; Huang et al., 2011; Ding et al., 2011). These points were also targeted because they are the key needling points in acupuncture treatment following a clinical SCI in humans. In addition, we used the SCI + Jiaji points EA-treated group (Jiaji-EA), and the SCI + non point area EA-treated group (npa-EA) as controls. For the GV-EA group, Jizhong (GV6) and ‘Zhiyang’ (GV9) were located at the interval of T7-T8 and T11-T12 processus spinosus, respectively. A pair of stainless needles of 0.35 mm diameter was inserted into ‘Jizhong’ (GV6) and ‘Zhiyang’ (GV9) directing to the spinal dura mater (Li et al., 2010), but outside vertebral canal. In the Jiaji-EA group, two pairs of stainless needles of 0.35 mm diameter were inserted to a depth of 4–5 mm into the bilateral ‘Jiaji’ points (T7, T11), located 3–4 mm lateral to posterior median line. For the npa-EA group, the non point area was located at the interval of the T7–T8 and T11–T12 processus spinosus, 10– 13 mm lateral to posterior median line. This area was treated using oblique insertion to a depth of about 10 mm subcutaneously. For EA treatment, the rats were immobilized in wooden holders. Two needles were inserted into the rostral-caudal sites of the injured spinal cord and were then connected to the output terminals of an electro-acupuncture apparatus (Model G6805-2A, Shanghai Huayi Medical Electronic Apparatus Company, China). Alternating strings of dense-sparse frequencies (60 Hz for 1.05 s and 2 Hz for 2.85 s alternately) were selected as reported previously (Kim et al., 2004). The intensity was adjusted to induce a slight twitch of the hindlimbs (61 mA) with the entire procedure lasting 20 min. The GV-EA, Jiaji-EA and npa-EA groups began EA stimulation seven days post-SCI, since the physical condition of SCI animals began to stabilize at this time. EA treatments were performed at selected acupoints and the non point area once a day for a week. 2.3.4. Unilateral dorsal rhizotomy and point nerve blocking treatment Rats of the GV-EA⁄ group were deeply anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). A laminectomy was carried out at the T7–T12 level to expose the spinal segments on the right side. The dura was then cut with a pair of fine scissors to isolate and expose the T7–T12 spinal dorsal roots. The dorsal root ganglia (DRG) together with a 5 mm segment of the associated dorsal roots was then removed at the intervertebral foramina on the right side. And then, spinal cord transection was performed to these experimental rats immediately. For point nerve blocking treatment, rats of the GV-NB⁄ group received 50 ll lidocaine (a common local anesthetic drug) via microinjection in the GV6 and GV9 points 5 min before EA stimulation. Following treatment with lidocaine, EA treatment was conducted same as above. 2.3.5. Tissue preparation Following 1 week of EA stimulation, animals from the normal, SCI, GV-EA, Jiaji-EA, and npa-EA groups were sacrificed for tissue collection. The spinal cord was immediately removed, and the spinal cord segments containing the injury sites (T8–T12) were dissected and stored at 80 °C for Western blot (T9–T11) and immunofluorescence analysis (T8 and T12). The manner in which tissue samples were obtained following EA treatment was the same for the normal and SCI groups. Following 3 days of EA stimulation, rats of the GV-EA⁄ and GVNB⁄ groups were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS. Spinal cord segments T8 and T12 were dissected, placed into 4% paraformaldehyde for 24 h, and then placed in 0.1 M PBS containing 30% buffered sucrose at 4 °C until the tissue sank to the bottom of the container. Transverse sections (15 lm) of the spinal segments (T8 and T12) were cut on a freezing microtom.
1399
2.3.6. Western blot Protein extracts (40 lg) were separated by 12% SDS–PAGE and then transferred to a PVDF membrane. After blocking for nonspecific binding with 5% skim milk in TBST (0.5% Tween 20 in TBS) for 1 h at room temperature, the membranes were incubated with anti-CGRP (1:8000, Chemico) overnight at 4 °C. After washing with TBST, the membranes were treated with an HRP-conjugated secondary antibody (anti-rabbit, 1:5000, Jackson, USA) for 2 h at room temperature, and again washed with TBST. The protein bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) as described in the manufacturer’s instructions. Relative band intensities were determined by densitometry using Scion image software (version 4.0). 2.3.7. Immunofluorescence analysis of CGRP expression in the spinal dorsal horn Sections were removed from storage at 20 °C and warmed for 30 min at 37 °C. Following three 5 min rinses in 0.01 M PBS, the sections were preincubated and blocked with 10% normal goat serum in 0.01 M PBS with 0.3% Triton X-100 (NGST) for 30 min at room temperature. The sections were then incubated in the primary rabbit polyclonal anti-CGRP (1:8000, Chemico) overnight at 4 °C. The following day, sections were washed three times in 0.01 M PBS and incubated with Cy3-conjugated goat anti-rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, INC.) for 1 h at 37 °C. After three 5 min rinses in 0.01 M PBS, sections were sealed with 30% glycerol. The results were analyzed using a fluorescence microscope (Leica microsystem AG, Switzerland). 2.3.8. Determination of CGRP positive reaction area ratio In this study, we used ipp6.0 image analysis software to analyze the pixel ratio of the CGRP reaction area and 1/2 spinal cord crosssectional area above the central canal in the T12 spinal segment. 2.4. Statistical analysis All data were presented as the means ± SEM. A statistical analysis was performed using an ANOVA, and the statistical significance of the difference between groups was calculated using a Student–Newman–Keuls’ test (p < 0.05). 3. Results 3.1. Protective effect of CGRP on injured neurons in vitro To examine the role of CGRP in injured neurons, we measured the effect of CGRP on the survival rate of CGNs following a scratch wound. Our results show that compared to normal CGNs, the survival rate of neurons in the scratch-wound groups (1, 6, and 12 h after injury) was significantly lower. However, the survival rate of neurons in the scratch-wound groups (6 and 12 h after injury) treated with 10 6M or 10 7M CGRP was significantly higher than the scratch-wound groups that did not receive CGRP treatment There was no statistical difference in survival rate between the two doses of CGRP (10 6M and 10 7M) (Fig. 2). 3.2. Protective effect of CGRP on injured neurons of spinal cord To further investigate the importance of CGRP in injured neurons in vivo, the effect of exogenous CGRP on neuronal survival following an SCI was observed. We performed a Nissl’s staining (neutral red staining) to CN of L1 spinal segments and RN of midbrain samples to compare the condition of injured neurons in rats receiving either CGRP or PBS treatment. Results of cell counting show that the number of CN and RN neurons in the injured spinal
1400
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
Fig. 2. Fluorescence microscopic image of CGNs labeled by calcein-AM and EthD-1. The survival condition of CGNs in the normal (A), injury 6 h (B) and injury 6 h + CGRP (C) groups respectively. (A–C) ⁄Represents survival cells. Arrow (A–C) shows dead cells. Scale bar = 80 lm. (D) ⁄p < 0.05, ⁄⁄p < 0.01, compared with corresponding injury group.
cord was significantly higher in rats treated with CGRP as compared to those treated with PBS (Fig. 3).
3.3. Effect of EA on CGRP expression in injured spinal cord Western blot analysis was used to detect CGRP expression in spinal segments (T9–T11) from each of the five groups. The results show lower levels of CGRP expression in the SCI group (14 days post-injury) as compared to the normal group. However, the three groups receiving EA all displayed higher than normal levels of CGRP expression following 7 days of treatment, and compared with two EA control groups, CGRP expression in the GV-EA group was significantly higher (Fig. 4). CGRP immunoreaction product was mainly confined to laminae I and II within the dorsal horn of the spinal cord bilaterally (Fig. 4).
3.4. Effect of unilateral dorsal rhizotomy on CGRP expression in injured spinal cord To assess whether the stimulation information from GV-EA treatment might have been transmitted through some neural pathway, we measured the CGRP immunoreaction region after performing a unilateral dorsal rhizotomy. Following GV-EA treatment the CGRP immunoreaction region at the right spinal dorsal horn was significantly less than at the left spinal dorsal horn in unilaterally dorsal rhizotomied SCI rats, regardless of whether point nerve blocking was performed (Fig. 5). Although the GV-EA⁄ group and GV-NB⁄ group displayed no significant difference in the CGRP immunoreaction region the left spinal dorsal horns, the GV-NB⁄ group did display a significantly smaller immunoreaction region at the right spinal dorsal horns.
Fig. 3. Comparison of the number of survival neurons in CN and RN between the PBS and CGRP groups (n = 7 in each group). (A, B, D, and E): Showing neutral red staining of CN and RN between the PBS and CGRP groups. (A and B) The labeled area points to CN area. Arrows show survival cells. (D and E) The labeled area points to RN area. Arrows show survival cells. (C and F) ⁄p < 0.05, ⁄⁄p < 0.01, compared with the PBS group.
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
1401
Fig. 4. Western blot analysis of protein extracts from spinal cord with antibody against CGRP (A). The results are presented as mean ± SEM (n = 6) (B). Student–Newman– Keuls’ test was performed to evaluate statistical significance. ⁄p < 0.05, compared with the normal group; #p < 0.05, compared with the SCI group. (C–E) Immunofluorescence labeling shows CGRP product in laminae I and II of T12 spinal dorsal horns. Showing positive staining area of CGRP in T12 spinal dorsal horn of the normal (C), SCI (D) and GVEA (E) groups, respectively. Scale bar = 80 lm.
4. Discussion In this study, we showed that the neuropeptide CGRP could play a neuroprotective role in cerebellar granule neurons of rats. We further demonstrated this function through exogenous CGRP treatment on the injured spinal cord. In addition, our study found that EA treatment can increase CGRP expression in the injured spinal cord of rats. CGRP has been shown to act as a survival factor, inhibiting apoptosis in liver cells (Song et al., 2009), cultured smooth muscle and gubemacular cells (Schaeffer et al., 2003; Chan et al., 2009). And its anti-apoptotic action was confirmed partly through ERK signaling pathway (Schaeffer et al., 2003; Uzan et al., 2008), which can provide further understanding on protective mechanism of CGRP. However, the protective role of CGRP in injured neurons, both in vitro and in vivo, has not yet been fully elucidated. Using the scratch wound model, we showed that CGRP can promote the survival of injured cerebellar granule neurons (CGNs) in vitro, thus raising the possibility that CGRP could function as a survival factor within neurons. CN neurons are known to undergo degenerative changes after axotomy (Schmitt et al., 2003). If injured neurons, including local interneurons at the site of injury as well as axotomized projection neurons, could be prevented from dying, chances of functional recovery could be greatly increased (Himes et al., 2001; Yick et al., 2003). Our results show that CGRP can increase the survival rate of CN and RN neurons. This not only demonstrates a possible neuroprotective role for endogenous CGRP, but also raises the possibility of CGRP contributing to recovery in SCI. This last finding has obvious clinical significance. Results from our study indicate that CGRP expression was increased in the spinal cord following EA treatment. Immunohistochemical expression of CGRP was clearly observed in the superficial layers of the dorsal horns of the spinal cord. As previously stated, expression of CGRP was higher in the injured spinal cord after GV-EA treatment compared with Jiaji-EA and npa-EA treatments. Since Jiaji-EA was used as an acupuncture control and npa-EA was selected as a non-acupuncture control, the results
could suggest that CGRP is an acupuncture-specific neuropeptide expressed during the course of GV-EA treatment of SCI. Because the T7–T12 dorsal roots were removed, peripheral stimulation information could not have been sent to the spinal cord by the central branches of the DRG. While stimulation information could not have been efficiently transmitted through a neural pathway after point nerve blocking we believe, based on our experimental results, that the stimulation information from GV-EA might have been first transmitted to the DRG by the meningeal branch and finally transmitted to the spinal cord by through the central branches of the DRG (thus increasing expression of CGRP). . In our previous study, we also found that acupuncture-specific proteins annexin A5 (ANXA5) and collapsin response mediator protein 2 (CRMP2) can exert at least a partial neuroprotective effect on the survival of CN neurons in SCI rats after GV-EA treatment (Li et al., 2010). We are currently investigating the possible relationship between the acupuncture-specific neuropeptide CGRP and acupuncture-specific proteins ANXA5 and CRMP2. CGRP released from primary sensory neurons is known to play an important role in nociception and nociceptive transmission (Hiruma et al., 2000; Yu et al., 2009). In addition, administration of CGRP may ameliorate spinal cord injury by significantly reducing motor disturbances and inhibiting increases in spinal cord tissue tumor necrosis factor (TNF) levels (Kitamura et al., 2007). In conclusion, this study attempted to clarify the role of CGRP in regulating the survival of injured neurons, because we found that exogenic CGRP might improve the survival of injured cerebellar granule neurons (CGNs) of rats in vitro and injured CN neurons in SCI rats. Thus, we speculate that endogenic CGRP specifically expressed in GV-EA treatment might play a neuroprotective role in injured neurons of rats following a spinal cord injury. Future work needs to verify if GV-EA treatment upregulating CGRP expression could improve the survival of injured neurons in the injured spinal cord of rats. Specific inhibitor (such as siRNA or microRNA) will be used to prevent the up-regulation of CGRP and explore if this effect could block the neuroprotection induced by GV-EA treatment. Taken together, the results of our study suggest that CGRP might
1402
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
References
Fig. 5. Immunofluorescence labeling shows CGRP expression in laminae I and II of T12 spinal dorsal horn. (A–F) Showing positive staining area of CGRP in T12 spinal dorsal horn of the normal group (n = 6) (A), GV-EA group (n = 6) (B), GV-EA⁄1 group (SCI + GV-EA + contralateral side of dorsal rhizotomy, n = 5) (C), GV-EA⁄2 group (SCI + GV-EA + ipsilateral side of dorsal rhizotomy, n = 5) (D), GV-NB⁄1 group (SCI + GV-EA + contralateral side of dorsal rhizotomy + Lidocaine blocking, n = 5) (E) and GV-NB⁄2 group (SCI + GV-EA + ipsilateral side of dorsal rhizotomy + Lidocaine blocking, n = 5) (F) respectively. Scale bar = 20 lm. (G) Pixel ratio of CGRP reaction area and 1/2 spinal cord cross-sectional area above central canal in T12 spinal segment. ⁄Represents p < 0.05; NS represents p > 0.05.
be an acupuncture-specific neuropeptide that plays a neuoroprotective role spinal cord injury progression. Furthermore, our results raise the possibility that electrical stimulation of sensory neurons in Governor Vessel might contribute to reduce spinal cord injury. More importantly, the results of our study provide a neuropeptide and neural pathway for the mechanism of GV-EA treatment of SCI.
Acknowledgments This work was supported by Natural Science Foundation of Guangdong Province, (No. 07001682) and Social Developmental Foundation of Guangdong Province (No. 2010B030700008) to J.W. Ruan, and Chinese National Natural Science Foundation (No. 30973721) and National 863 Project (No.2011A030300004) to Y.S. Zeng, and Chinese National Natural Science Foundation (No. 81102646) and Natural Science Foundation of Guangdong Province (No. S2011040004895) to Y. Ding.
Ackery, A., Tator, C., Krassioukov, A., 2004. A global perspective on spinal cord injury epidemiology. J. Neurotrauma 21, 1355–1370. Ackery, A.D., Norenberg, M.D., Krassioukov, A., 2007. Calcitonin gene-related peptide immunoreactivity in chronic human spinal cord injury. Spinal Cord 45, 678–686. Blesch, A., Tuszynski, M.H., 2001. GDNF gene delivery to injured adult CNS motor neurons promotes axonal growth, expression of the trophic neuropeptide CGRP, and cellular protection. J. Comp. Neurol. 436, 399–410. Calancie, B., Molano, M.R., Broton, J.G., 2005. Epidemiology and demography of acute spinal cord injury in a large urban setting. J. Spinal Cord Med. 28, 92–96. Chan, J.J., Farmer, P.J., Southwell, B.R., Sourial, M., Hutson, J.M., 2009. Calcitonin gene-related peptide is a survival factor, inhibiting apoptosis in neonatal rat gubernaculum in vitro. J. Pediatr. Surg. 44, 1497–1501. Chen, Y.Y., Zhang, W., Chen, Y.L., Chen, S.J., Dong, H.X., Zeng, Y.S., 2008. Electroacupuncture improves survival and migration of transplanted neural stem cells in injured spinal cord in rats. Acupunct. Electrother. Res. 33, 19–31. Cheng, P.T., Wong, M.K., Chang, P.L., 1998. A therapeutic trial of acupuncture in neurogenic bladder of spinal cord injured patients – a preliminary report. Spinal Cord 36, 476–480. Christensen, M.D., Hulsebosch, C.E., 1997. Spinal cord injury and anti-NGF treatment results in changes in CGRP density and distribution in the dorsal horn in the rat. Exp. Neurol. 147, 463–475. Ding, Y., Yan, Q., Ruan, J.W., Zhang, Y.Q., Li, W.J., Zhang, Y.J., Li, Y., Dong, H., Zeng, Y.S., 2009. Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cord-transected rats. BMC Neurosci. 10, 35. Ding, Y., Yan, Q., Ruan, J.W., Zhang, Y.Q., Li, W.J., Zeng, X., Huang, S.F., Zhang, Y.J., Wang, S., Dong, H., Zeng, Y.S., 2011. Bone marrow mesenchymal stem cells and electro-acupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats. Cell Transplant. 20, 475–491. Dyson-Hudson, T.A., Kadar, P., LaFountaine, M., 2007. Acupuncture for chronic shoulder pain in persons with spinal cord injury: a small-scale clinical trial. Arch Phys. Med. Rehabil. 88, 1276–1283. Himes, B.T., Liu, Y., Solowska, J.M., Snyder, E.Y., Fischer, I., Tessler, A., 2001. Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke’s nucleus neurons after spinal cord hemisection in adult rats. J. Neurosci. Res. 65, 549–564. Hiruma, H., Saito, A., Ichikawa, T., Kiriyama, Y., Hoka, S., Kusakabe, T., Kobayashi, H., Kawakami, T., 2000. Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons. Brain Res. 883, 184–191. Huang, S.F., Ding, Y., Ruan, J.W., Zhang, W., Wu, J.L., He, B., Zhang, Y.J., Li, Y., Zeng, Y.S., 2011. An experimental electro-acupuncture study in treatment of the rat demyelinated spinal cord injury induced by ethidium bromide. Neurosci. Res. 70, 294–304. Kashihara, Y., Sakaguchi, M., Kuno, M., 1989. Axonal transport and distribution of endogenous calcitonin gene-related peptide in rat peripheral nerve. J. Neurosci. 9, 3796–3802. Kim, J.H., Min, B.I., Na, H.S., Park, D.S., 2004. Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors. Brain Res. 998, 230–236. Kitamura, T., Harada, N., Goto, E., Tanaka, K., Arai, M., Shimada, S., Okajima, K., 2007. Activation of sensory neurons contributes to reduce spinal cord injury in rats. Neuropharmacology 52, 506–514. Lee, H., Park, H.J., Park, J., Kim, M.J., Hong, M., Yang, J.S., Choi, S., Lee, H., 2007. Acupuncture application for neurological disorders. Neurol. Res. 29, S49–S54. Li, X.Q., Verge, V.M.K., Johnston, J.M., Zochodne, D.W., 2004. CGRP peptide and regenerating sensory axons. J. Neuropathol. Exp. Neurol. 63, 1092–1103. Li, W.J., Pan, S.Q., Zeng, Y.S., Su, B.G., Li, S.M., Ding, Y., Li, Y., Ruan, J.W., 2010. Identification of acupuncture-specific proteins in the process of electroacupuncture after spinal cord injury. Neurosci. Res. 67, 307–316. Liu, A.L., Wang, X.W., Liu, A.H., Su, X.W., Jiang, W.J., Qiu, P.X., Yan, G.M., 2007. JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons. Exp. Toxicol. Pathol. 61, 131–143. Ma, Y.H., Zeng, X., Zhang, K., Zeng, Y.S., 2012. A new in vitro injury model of mouse neurons induced by mechanical scratching. Neurosci. Lett. 510, 14–19. Ondarza, A.B., Ye, Z.M., Hulsebosch, C.E., 2003. Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP. Exp. Neurol. 184, 373–380. Paola, F.A., Arnold, M., 2003. Acupuncture and spinal cord medicine. J. Spinal Cord Med. 26, 12–20. Schaeffer, C., Vandroux, D., Thomassin, L., Athias, P., Rochette, L., Connat, J.L., 2003. Calcitonin gene-related peptide partly protects cultured smooth muscle cells from apoptosis induced by an oxidative stress via activation of ERK1/2 MAPK. Biochim. Biophys. Acta, Mol. Cell Res. 1643, 65–73. Schmitt, A.B., Breuer, S., Polat, L., Pech, K., Kakulas, B., Love, S., Martin, D., Schoenen, J., Noth, J., Brook, G.A., 2003. Retrograde reactions of Clarke’s nucleus neurons after human spinal cord injury. Ann. Neurol. 54, 534–539. Sekhon, L.H., Fehlings, M.G., 2001. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26, S2–S12. Song, S., Liu, N., Liu, W., Shi, R., Guo, K.J., Liu, Y.F., 2009. The effect of pretreatment with calcitonin gene-related peptide on attenuation of liver ischemia and
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403 reperfusion injury due to oxygen free radicals and apoptosis. Hepatogastroenterology 56, 1724–1729. Thuret, S., Moon, L.D., Gage, F.H., 2006. Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7, 628–643. Uzan, B., Villemin, A., Garel, J.M., Cressent, M., 2008. Adrenomedullin is antiapoptotic in osteoblasts through CGRP1 receptors and MEK–ERK pathway. J. Cell. Physiol. 215, 122–128. Van Rossum, D., Hanisch, U.K., Quirion, R., 1997. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci. Biobehav. Rev. 21, 649–678. Wimalawansa, S.J., 1997. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit. Rev. Neurobiol. 11, 167–239. Wong, A.M., Leong, C.P., Su, T.Y., Yu, S.W., Tsai, W.C., Chen, C.P., 2003. Clinical trial of acupuncture for patients with spinal cord injuries. Am. J. Phys. Med. Rehabil. 82, 21–27. Wu, Y.C., Mi, Y.Q., Zhang, P., Zhang, J.F., Chen, W., 2009. Effect of warm acupuncture on nitric oxide synthase and calcitonin gene-related peptide in a rat model of lumbar nerve root compression. Neural Regen. Res. 4, 449–454.
1403
Yan, G.M., Irwin, R.P., Lin, S.Z., Weller, M., Wood, K.A., Paul, S.M., 1995. Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons. J. Pharmacol. Exp. Ther. 274, 983–990. Yan, Q., Ruan, J.W., Ding, Y., Li, W.J., Li, Y., Zeng, Y.S., 2011. Electro-acupuncture promotes differentiation of the mesenchymal stem cells, regeneration of nerve fibers and partial functional recovery after spinal cord injury. Exp. Toxicol. Pathol. 63, 151–156. Yick, L.W., Cheung, P.T., So, K.F., Wu, W.T., 2003. Axonal regeneration of Clarke’s neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp. Neurol. 182, 160–168. Yu, L.C., Hou, J.F., Fu, F.H., Zhang, Y.X., 2009. Roles of calcitonin gene-related peptide and its receptors in pain-related behavioral responses in the central nervous system. Neurosci. Biobehav. Rev. 33, 1185–1191. Zinck, N.D.T., Rafuse, V.F., Downie, J.W., 2007. Sprouting of CGRP primary afferents in lumbosacral spinal cord precedes emergence of bladder activity after spinal injury. Exp. Neurol. 204, 777–790.
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Electro-acupuncture upregulates CGRP expression after rat spinal cord transection Wen-Jie Li a,1, Shu-Min Li b,1, Ying Ding a, Bing He a, Jack Keegan d, Hongxin Dong d, Jing-Wen Ruan b,⇑, Yuan-Shan Zeng a,c,⇑ a
Division of Neuroscience, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Department of Acupuncture, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China c Institute of Spinal Cord Injury, Sun Yat-sen University, Guangzhou 510120, China d Department of Psychiatry and Behavioral Sciences, Northwestern University, Feinberg School of Medicine, Chicago, USA b
a r t i c l e
i n f o
Article history: Received 26 March 2012 Received in revised form 28 September 2012 Accepted 5 October 2012 Available online 13 October 2012 Keywords: Electro-acupuncture Calcitonin gene-related peptide Neuroprotection Spinal cord injury Cerebellar granule neurons
a b s t r a c t Calcitonin gene-related peptide (CGRP) plays a variety of important roles within the nervous system. Increasing CGRP expression could improve the survival of injured neurons and prevent neuronal loss. In this study, we first evaluated in vitro the neuroprotective function of CGRP on mechanically injured cerebellar granule neurons (CGNs) of rats. We then verified this result through exogenous administration of CGRP in a spinal cord transected completely in rats. Finally, we investigated the effect of electro-acupuncture (EA) on CGRP expression following the spinal cord transected completely in rats. We found that EA can improve CGRP expression, and exogenous CGRP may promote the survival of injured neurons, both in vivo and in vitro. Our results suggest that CGRP may be a specific neuropeptide expressed in GV-EA treatment of spinal cord injuries (SCI), and that CGRP may play a neuroprotective role in survival of neurons injured mechanically. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Calcitonin gene-related peptide (CGRP) is a widely distributed 37 amino-acid peptide (Van Rossum et al., 1997; Wimalawansa, 1997), that is known to play a critical role in regulating the inflammatory response in peripheral sensory neurons (Li et al., 2004). CGRP is synthesized in cell bodies of the dorsal root ganglion (DRG) and transported axonally to the peripheral and central endings of sensory nerve fibers (Kashihara et al., 1989). CGRP has been recognized as a nerve regeneration-promoting peptide in vivo (Blesch and Tuszynski, 2001) and has also been shown to inhibit apoptosis of cells in vitro (Schaeffer et al., 2003; Chan et al., 2009). Although some studies have linked CGRP with spinal cord injury (SCI) progression (Christensen and Hulsebosch, 1997; Ondarza et al., 2003; Zinck et al., 2007; Ackery et al., 2007; Wu et al., 2009), the neuroprotective role of CGRP is not yet fully understood. SCI is a devastating neurological injury that often ⇑ Corresponding authors. Addresses: Department of Acupuncture, The 1st Affiliated Hospital, Sun Yat-sen University, #58 Zhongshan 2nd Road, Guangzhou 510080, China. Tel.: +86 20 87755766 8390 (J.-W. Ruan), Division of Neuroscience, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yatsen University, #74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel./fax: +86 20 87332698 (Y.-S. Zeng). E-mail addresses: [email protected] (J.-W. Ruan), [email protected] (Y.-S. Zeng). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.10.002
results in profound functional deficits and is a frequent cause of mortality around the world (Ackery et al., 2004; Thuret et al., 2006). As SCI primarily affects young adults (between 16 and 30 years old), the personal and economic burden associated with this type of injury is often very high (Sekhon and Fehlings, 2001; Calancie et al., 2005). While the effects of spinal cord injuries are far reaching, recovery and treatment options remain limited. Acupuncture and electro-acupuncture (EA) are traditional Chinese medical methods that have been practiced in East Asia for more than 2500 years and are now widely used for a range of neurological disorders (Wong et al., 2003; Lee et al., 2007). EA has been successfully used to treat a number of SCI-related conditions, including motor deficits, pain, spasticity and syringomyelia (Cheng et al., 1998; Paola and Arnold, 2003; Dyson-Hudson et al., 2007). In previous studies, we found that EA treatment could induce the regeneration of CGRP positive nerve fibers in the injured spinal cord (Ding et al., 2009; Yan et al., 2011). In the present study, we first examined the neuroprotective properties of CGRP in injured cerebellar granule neurons (CGNs) of rats in vitro. Here, we referred to a new model with injured CGNs (Ma et al., 2012) which is feasible to investigate the precise effect of mechanical damage on neurons. We then confirmed this neuroprotective function by directly administering CGRP to the injured spinal cord. Finally, we investigated whether electro-acupuncture (EA) could stimulate CGRP expression in the injured spinal cord of rats.
1398
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
2. Materials and methods 2.1. Cell culture and scratch-wound model performed with CGNs CGNs were isolated and cultured from 7-day-old Sprague–Dawley rat pups (15–19 g) as previously described (Yan et al., 1995; Liu et al., 2007; Ma et al., 2012). The study protocol was in accordance with the guidelines for animal research and was approved by the Experimental Animal Center of Sun Yat-sen University. Scratch wounds were performed with CGNs at 8 days in vitro. At the time of the experiments, neurons with mature cell bodies and abundant processes composed 95% of each culture. CGNs on glass cover slips were placed in 6 hole culture plates and wounded in a ‘‘#’’ design every 6 mm with a cataract knife (Fig. 1). Some cultures received simultaneous 10 6M or 10 7M CGRP treatment with the performance of a scratch wound. All cultures were stained with calcein-AM and EthD-1. Viable cells exhibited green fluorescence that was generated by calcein-AM, while dead cells were marked by red fluorescence from EthD-1.
2.2. Exogenous CGRP acting on the injured spinal cord 2.2.1. Experimental groups A total of 14 adult female Sprague–Dawley (SD) rats (200–220 g) were used for this study:
Group 1: PBS group (n = 7). The spinal cord of each rat was transected completely, and a piece of gelfoam (2 2 2 mm3) preloaded with 5 ll 0.01 M PBS was implanted between the rostral and caudal cord stumps. Group 2: CGRP group (n = 7). The spinal cord of each rat was transected completely, and a piece of gelfoam (2 2 2 mm3) preloaded with 5 ll 10 7 M CGRP was implanted between the rostral and caudal spinal cord stumps. 2.2.2. Quantitative analysis of neuronal survival in RN and CN One month after transection surgery, brains and L1 spinal segments were removed, postfixed and cryoprotected. After brain samples were serially cut into 30 lm sections, one out of every six sections containing red nucleus (RN) was selected and placed on a gelatin coated glass slide. Each L1 spinal segment containing Clarke’s nucleus (CN) was also serially cut into 30 lm sections, and 50 sections from each spinal segment were placed on gelatin-coated glass slides. All sections were then soaked in 1% neutral red stain for 20 min, rinsed in de-ionized water for 10 s, and successively dehydrated in 70%, 80%, 90%, 95% and 100% alcohol. Finally, the sections were cleared with dimethylbenzene and observed under the microscope. After neutral red staining, RN and CN neurons with a visible nucleus were counted. We calculated the amount of surviving neurons in 50 transverse sections from each L1 spinal segment, representing the total number of neurons in the L1 CN of each rat. The neuronal densities of CN of L1 spinal cord segments and RN of midbrain samples were calculated using grid-equipped microscopy based on stereological principles. 2.3. Electro-acupuncture (EA) effect on CGRP expression in the injured spinal cord 2.3.1. Experimental groups A total of 40 adult female SD rats (200–220 g) were used for this study. Thirty of them were divided into five groups (n = 6 in each group): the normal group (normal), the SCI group (SCI), the SCI + Governor Vessel EA-treated group (GV-EA), the SCI + Jiaji points Governor Vessel EA-treated group (Jiaji-EA) and the SCI + non point area Governor Vessel EA-treated group (npa-EA). For partial dorsal rhizotomy, ten adult female SD rats (200–220 g) were divided into two groups (n = 5 in each group): the SCI + unilateral dorsal rhizotomy + Governor Vessel EA-treated group (GV-EA⁄) and the SCI + unilateral dorsal rhizotomy + Governor Vessel EA + point nerve blocking group (GV-NB⁄). All animals were provided by the Experimental Animal Center of Sun Yat-sen University and received humane care in compliance with the Public Health Service Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the number of animals used and the pain experienced during the course of the procedure. 2.3.2. Spinal cord injury mode Rats for spinal cord injury were deeply anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). A laminectomy was carried out at the T9 level to expose the T10 spinal segment. The dura was cut using ophthalmic scissors and the T10 spinal segment was completely transected. Immediately after the procedure, both hind limbs were completely paralyzed, while the front limbs remained functional. Following surgery, the incisions were closed and the rats were returned to their respective cages. Animals received intensive care including intramuscular injection of penicillin (50,000 U/kg/day) for 3 days and manual emission of urine twice a day before being sacrificed.
Fig. 1. CGNs scratch graph. CGNs in glass coverslips of 6 holes culture plates were wounded by ‘‘#’’ scratching every 6 mm with an iris blade (A). ⁄Represents the scratch injury region (B). Scale bar = 80 lm.
2.3.3. Electro-acupuncture stimulation procedure Two Ashi points on the Governor Vessel, ‘‘Jizhong’’ (GV6) and ‘‘Zhiyang’’ (GV9), were selected for the treatment of SCI rats in
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
the GV-EA group according to our previous studies (Chen et al., 2008; Ding et al., 2009; Li et al., 2010; Huang et al., 2011; Ding et al., 2011). These points were also targeted because they are the key needling points in acupuncture treatment following a clinical SCI in humans. In addition, we used the SCI + Jiaji points EA-treated group (Jiaji-EA), and the SCI + non point area EA-treated group (npa-EA) as controls. For the GV-EA group, Jizhong (GV6) and ‘Zhiyang’ (GV9) were located at the interval of T7-T8 and T11-T12 processus spinosus, respectively. A pair of stainless needles of 0.35 mm diameter was inserted into ‘Jizhong’ (GV6) and ‘Zhiyang’ (GV9) directing to the spinal dura mater (Li et al., 2010), but outside vertebral canal. In the Jiaji-EA group, two pairs of stainless needles of 0.35 mm diameter were inserted to a depth of 4–5 mm into the bilateral ‘Jiaji’ points (T7, T11), located 3–4 mm lateral to posterior median line. For the npa-EA group, the non point area was located at the interval of the T7–T8 and T11–T12 processus spinosus, 10– 13 mm lateral to posterior median line. This area was treated using oblique insertion to a depth of about 10 mm subcutaneously. For EA treatment, the rats were immobilized in wooden holders. Two needles were inserted into the rostral-caudal sites of the injured spinal cord and were then connected to the output terminals of an electro-acupuncture apparatus (Model G6805-2A, Shanghai Huayi Medical Electronic Apparatus Company, China). Alternating strings of dense-sparse frequencies (60 Hz for 1.05 s and 2 Hz for 2.85 s alternately) were selected as reported previously (Kim et al., 2004). The intensity was adjusted to induce a slight twitch of the hindlimbs (61 mA) with the entire procedure lasting 20 min. The GV-EA, Jiaji-EA and npa-EA groups began EA stimulation seven days post-SCI, since the physical condition of SCI animals began to stabilize at this time. EA treatments were performed at selected acupoints and the non point area once a day for a week. 2.3.4. Unilateral dorsal rhizotomy and point nerve blocking treatment Rats of the GV-EA⁄ group were deeply anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). A laminectomy was carried out at the T7–T12 level to expose the spinal segments on the right side. The dura was then cut with a pair of fine scissors to isolate and expose the T7–T12 spinal dorsal roots. The dorsal root ganglia (DRG) together with a 5 mm segment of the associated dorsal roots was then removed at the intervertebral foramina on the right side. And then, spinal cord transection was performed to these experimental rats immediately. For point nerve blocking treatment, rats of the GV-NB⁄ group received 50 ll lidocaine (a common local anesthetic drug) via microinjection in the GV6 and GV9 points 5 min before EA stimulation. Following treatment with lidocaine, EA treatment was conducted same as above. 2.3.5. Tissue preparation Following 1 week of EA stimulation, animals from the normal, SCI, GV-EA, Jiaji-EA, and npa-EA groups were sacrificed for tissue collection. The spinal cord was immediately removed, and the spinal cord segments containing the injury sites (T8–T12) were dissected and stored at 80 °C for Western blot (T9–T11) and immunofluorescence analysis (T8 and T12). The manner in which tissue samples were obtained following EA treatment was the same for the normal and SCI groups. Following 3 days of EA stimulation, rats of the GV-EA⁄ and GVNB⁄ groups were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS. Spinal cord segments T8 and T12 were dissected, placed into 4% paraformaldehyde for 24 h, and then placed in 0.1 M PBS containing 30% buffered sucrose at 4 °C until the tissue sank to the bottom of the container. Transverse sections (15 lm) of the spinal segments (T8 and T12) were cut on a freezing microtom.
1399
2.3.6. Western blot Protein extracts (40 lg) were separated by 12% SDS–PAGE and then transferred to a PVDF membrane. After blocking for nonspecific binding with 5% skim milk in TBST (0.5% Tween 20 in TBS) for 1 h at room temperature, the membranes were incubated with anti-CGRP (1:8000, Chemico) overnight at 4 °C. After washing with TBST, the membranes were treated with an HRP-conjugated secondary antibody (anti-rabbit, 1:5000, Jackson, USA) for 2 h at room temperature, and again washed with TBST. The protein bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) as described in the manufacturer’s instructions. Relative band intensities were determined by densitometry using Scion image software (version 4.0). 2.3.7. Immunofluorescence analysis of CGRP expression in the spinal dorsal horn Sections were removed from storage at 20 °C and warmed for 30 min at 37 °C. Following three 5 min rinses in 0.01 M PBS, the sections were preincubated and blocked with 10% normal goat serum in 0.01 M PBS with 0.3% Triton X-100 (NGST) for 30 min at room temperature. The sections were then incubated in the primary rabbit polyclonal anti-CGRP (1:8000, Chemico) overnight at 4 °C. The following day, sections were washed three times in 0.01 M PBS and incubated with Cy3-conjugated goat anti-rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, INC.) for 1 h at 37 °C. After three 5 min rinses in 0.01 M PBS, sections were sealed with 30% glycerol. The results were analyzed using a fluorescence microscope (Leica microsystem AG, Switzerland). 2.3.8. Determination of CGRP positive reaction area ratio In this study, we used ipp6.0 image analysis software to analyze the pixel ratio of the CGRP reaction area and 1/2 spinal cord crosssectional area above the central canal in the T12 spinal segment. 2.4. Statistical analysis All data were presented as the means ± SEM. A statistical analysis was performed using an ANOVA, and the statistical significance of the difference between groups was calculated using a Student–Newman–Keuls’ test (p < 0.05). 3. Results 3.1. Protective effect of CGRP on injured neurons in vitro To examine the role of CGRP in injured neurons, we measured the effect of CGRP on the survival rate of CGNs following a scratch wound. Our results show that compared to normal CGNs, the survival rate of neurons in the scratch-wound groups (1, 6, and 12 h after injury) was significantly lower. However, the survival rate of neurons in the scratch-wound groups (6 and 12 h after injury) treated with 10 6M or 10 7M CGRP was significantly higher than the scratch-wound groups that did not receive CGRP treatment There was no statistical difference in survival rate between the two doses of CGRP (10 6M and 10 7M) (Fig. 2). 3.2. Protective effect of CGRP on injured neurons of spinal cord To further investigate the importance of CGRP in injured neurons in vivo, the effect of exogenous CGRP on neuronal survival following an SCI was observed. We performed a Nissl’s staining (neutral red staining) to CN of L1 spinal segments and RN of midbrain samples to compare the condition of injured neurons in rats receiving either CGRP or PBS treatment. Results of cell counting show that the number of CN and RN neurons in the injured spinal
1400
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
Fig. 2. Fluorescence microscopic image of CGNs labeled by calcein-AM and EthD-1. The survival condition of CGNs in the normal (A), injury 6 h (B) and injury 6 h + CGRP (C) groups respectively. (A–C) ⁄Represents survival cells. Arrow (A–C) shows dead cells. Scale bar = 80 lm. (D) ⁄p < 0.05, ⁄⁄p < 0.01, compared with corresponding injury group.
cord was significantly higher in rats treated with CGRP as compared to those treated with PBS (Fig. 3).
3.3. Effect of EA on CGRP expression in injured spinal cord Western blot analysis was used to detect CGRP expression in spinal segments (T9–T11) from each of the five groups. The results show lower levels of CGRP expression in the SCI group (14 days post-injury) as compared to the normal group. However, the three groups receiving EA all displayed higher than normal levels of CGRP expression following 7 days of treatment, and compared with two EA control groups, CGRP expression in the GV-EA group was significantly higher (Fig. 4). CGRP immunoreaction product was mainly confined to laminae I and II within the dorsal horn of the spinal cord bilaterally (Fig. 4).
3.4. Effect of unilateral dorsal rhizotomy on CGRP expression in injured spinal cord To assess whether the stimulation information from GV-EA treatment might have been transmitted through some neural pathway, we measured the CGRP immunoreaction region after performing a unilateral dorsal rhizotomy. Following GV-EA treatment the CGRP immunoreaction region at the right spinal dorsal horn was significantly less than at the left spinal dorsal horn in unilaterally dorsal rhizotomied SCI rats, regardless of whether point nerve blocking was performed (Fig. 5). Although the GV-EA⁄ group and GV-NB⁄ group displayed no significant difference in the CGRP immunoreaction region the left spinal dorsal horns, the GV-NB⁄ group did display a significantly smaller immunoreaction region at the right spinal dorsal horns.
Fig. 3. Comparison of the number of survival neurons in CN and RN between the PBS and CGRP groups (n = 7 in each group). (A, B, D, and E): Showing neutral red staining of CN and RN between the PBS and CGRP groups. (A and B) The labeled area points to CN area. Arrows show survival cells. (D and E) The labeled area points to RN area. Arrows show survival cells. (C and F) ⁄p < 0.05, ⁄⁄p < 0.01, compared with the PBS group.
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
1401
Fig. 4. Western blot analysis of protein extracts from spinal cord with antibody against CGRP (A). The results are presented as mean ± SEM (n = 6) (B). Student–Newman– Keuls’ test was performed to evaluate statistical significance. ⁄p < 0.05, compared with the normal group; #p < 0.05, compared with the SCI group. (C–E) Immunofluorescence labeling shows CGRP product in laminae I and II of T12 spinal dorsal horns. Showing positive staining area of CGRP in T12 spinal dorsal horn of the normal (C), SCI (D) and GVEA (E) groups, respectively. Scale bar = 80 lm.
4. Discussion In this study, we showed that the neuropeptide CGRP could play a neuroprotective role in cerebellar granule neurons of rats. We further demonstrated this function through exogenous CGRP treatment on the injured spinal cord. In addition, our study found that EA treatment can increase CGRP expression in the injured spinal cord of rats. CGRP has been shown to act as a survival factor, inhibiting apoptosis in liver cells (Song et al., 2009), cultured smooth muscle and gubemacular cells (Schaeffer et al., 2003; Chan et al., 2009). And its anti-apoptotic action was confirmed partly through ERK signaling pathway (Schaeffer et al., 2003; Uzan et al., 2008), which can provide further understanding on protective mechanism of CGRP. However, the protective role of CGRP in injured neurons, both in vitro and in vivo, has not yet been fully elucidated. Using the scratch wound model, we showed that CGRP can promote the survival of injured cerebellar granule neurons (CGNs) in vitro, thus raising the possibility that CGRP could function as a survival factor within neurons. CN neurons are known to undergo degenerative changes after axotomy (Schmitt et al., 2003). If injured neurons, including local interneurons at the site of injury as well as axotomized projection neurons, could be prevented from dying, chances of functional recovery could be greatly increased (Himes et al., 2001; Yick et al., 2003). Our results show that CGRP can increase the survival rate of CN and RN neurons. This not only demonstrates a possible neuroprotective role for endogenous CGRP, but also raises the possibility of CGRP contributing to recovery in SCI. This last finding has obvious clinical significance. Results from our study indicate that CGRP expression was increased in the spinal cord following EA treatment. Immunohistochemical expression of CGRP was clearly observed in the superficial layers of the dorsal horns of the spinal cord. As previously stated, expression of CGRP was higher in the injured spinal cord after GV-EA treatment compared with Jiaji-EA and npa-EA treatments. Since Jiaji-EA was used as an acupuncture control and npa-EA was selected as a non-acupuncture control, the results
could suggest that CGRP is an acupuncture-specific neuropeptide expressed during the course of GV-EA treatment of SCI. Because the T7–T12 dorsal roots were removed, peripheral stimulation information could not have been sent to the spinal cord by the central branches of the DRG. While stimulation information could not have been efficiently transmitted through a neural pathway after point nerve blocking we believe, based on our experimental results, that the stimulation information from GV-EA might have been first transmitted to the DRG by the meningeal branch and finally transmitted to the spinal cord by through the central branches of the DRG (thus increasing expression of CGRP). . In our previous study, we also found that acupuncture-specific proteins annexin A5 (ANXA5) and collapsin response mediator protein 2 (CRMP2) can exert at least a partial neuroprotective effect on the survival of CN neurons in SCI rats after GV-EA treatment (Li et al., 2010). We are currently investigating the possible relationship between the acupuncture-specific neuropeptide CGRP and acupuncture-specific proteins ANXA5 and CRMP2. CGRP released from primary sensory neurons is known to play an important role in nociception and nociceptive transmission (Hiruma et al., 2000; Yu et al., 2009). In addition, administration of CGRP may ameliorate spinal cord injury by significantly reducing motor disturbances and inhibiting increases in spinal cord tissue tumor necrosis factor (TNF) levels (Kitamura et al., 2007). In conclusion, this study attempted to clarify the role of CGRP in regulating the survival of injured neurons, because we found that exogenic CGRP might improve the survival of injured cerebellar granule neurons (CGNs) of rats in vitro and injured CN neurons in SCI rats. Thus, we speculate that endogenic CGRP specifically expressed in GV-EA treatment might play a neuroprotective role in injured neurons of rats following a spinal cord injury. Future work needs to verify if GV-EA treatment upregulating CGRP expression could improve the survival of injured neurons in the injured spinal cord of rats. Specific inhibitor (such as siRNA or microRNA) will be used to prevent the up-regulation of CGRP and explore if this effect could block the neuroprotection induced by GV-EA treatment. Taken together, the results of our study suggest that CGRP might
1402
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403
References
Fig. 5. Immunofluorescence labeling shows CGRP expression in laminae I and II of T12 spinal dorsal horn. (A–F) Showing positive staining area of CGRP in T12 spinal dorsal horn of the normal group (n = 6) (A), GV-EA group (n = 6) (B), GV-EA⁄1 group (SCI + GV-EA + contralateral side of dorsal rhizotomy, n = 5) (C), GV-EA⁄2 group (SCI + GV-EA + ipsilateral side of dorsal rhizotomy, n = 5) (D), GV-NB⁄1 group (SCI + GV-EA + contralateral side of dorsal rhizotomy + Lidocaine blocking, n = 5) (E) and GV-NB⁄2 group (SCI + GV-EA + ipsilateral side of dorsal rhizotomy + Lidocaine blocking, n = 5) (F) respectively. Scale bar = 20 lm. (G) Pixel ratio of CGRP reaction area and 1/2 spinal cord cross-sectional area above central canal in T12 spinal segment. ⁄Represents p < 0.05; NS represents p > 0.05.
be an acupuncture-specific neuropeptide that plays a neuoroprotective role spinal cord injury progression. Furthermore, our results raise the possibility that electrical stimulation of sensory neurons in Governor Vessel might contribute to reduce spinal cord injury. More importantly, the results of our study provide a neuropeptide and neural pathway for the mechanism of GV-EA treatment of SCI.
Acknowledgments This work was supported by Natural Science Foundation of Guangdong Province, (No. 07001682) and Social Developmental Foundation of Guangdong Province (No. 2010B030700008) to J.W. Ruan, and Chinese National Natural Science Foundation (No. 30973721) and National 863 Project (No.2011A030300004) to Y.S. Zeng, and Chinese National Natural Science Foundation (No. 81102646) and Natural Science Foundation of Guangdong Province (No. S2011040004895) to Y. Ding.
Ackery, A., Tator, C., Krassioukov, A., 2004. A global perspective on spinal cord injury epidemiology. J. Neurotrauma 21, 1355–1370. Ackery, A.D., Norenberg, M.D., Krassioukov, A., 2007. Calcitonin gene-related peptide immunoreactivity in chronic human spinal cord injury. Spinal Cord 45, 678–686. Blesch, A., Tuszynski, M.H., 2001. GDNF gene delivery to injured adult CNS motor neurons promotes axonal growth, expression of the trophic neuropeptide CGRP, and cellular protection. J. Comp. Neurol. 436, 399–410. Calancie, B., Molano, M.R., Broton, J.G., 2005. Epidemiology and demography of acute spinal cord injury in a large urban setting. J. Spinal Cord Med. 28, 92–96. Chan, J.J., Farmer, P.J., Southwell, B.R., Sourial, M., Hutson, J.M., 2009. Calcitonin gene-related peptide is a survival factor, inhibiting apoptosis in neonatal rat gubernaculum in vitro. J. Pediatr. Surg. 44, 1497–1501. Chen, Y.Y., Zhang, W., Chen, Y.L., Chen, S.J., Dong, H.X., Zeng, Y.S., 2008. Electroacupuncture improves survival and migration of transplanted neural stem cells in injured spinal cord in rats. Acupunct. Electrother. Res. 33, 19–31. Cheng, P.T., Wong, M.K., Chang, P.L., 1998. A therapeutic trial of acupuncture in neurogenic bladder of spinal cord injured patients – a preliminary report. Spinal Cord 36, 476–480. Christensen, M.D., Hulsebosch, C.E., 1997. Spinal cord injury and anti-NGF treatment results in changes in CGRP density and distribution in the dorsal horn in the rat. Exp. Neurol. 147, 463–475. Ding, Y., Yan, Q., Ruan, J.W., Zhang, Y.Q., Li, W.J., Zhang, Y.J., Li, Y., Dong, H., Zeng, Y.S., 2009. Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cord-transected rats. BMC Neurosci. 10, 35. Ding, Y., Yan, Q., Ruan, J.W., Zhang, Y.Q., Li, W.J., Zeng, X., Huang, S.F., Zhang, Y.J., Wang, S., Dong, H., Zeng, Y.S., 2011. Bone marrow mesenchymal stem cells and electro-acupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats. Cell Transplant. 20, 475–491. Dyson-Hudson, T.A., Kadar, P., LaFountaine, M., 2007. Acupuncture for chronic shoulder pain in persons with spinal cord injury: a small-scale clinical trial. Arch Phys. Med. Rehabil. 88, 1276–1283. Himes, B.T., Liu, Y., Solowska, J.M., Snyder, E.Y., Fischer, I., Tessler, A., 2001. Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke’s nucleus neurons after spinal cord hemisection in adult rats. J. Neurosci. Res. 65, 549–564. Hiruma, H., Saito, A., Ichikawa, T., Kiriyama, Y., Hoka, S., Kusakabe, T., Kobayashi, H., Kawakami, T., 2000. Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons. Brain Res. 883, 184–191. Huang, S.F., Ding, Y., Ruan, J.W., Zhang, W., Wu, J.L., He, B., Zhang, Y.J., Li, Y., Zeng, Y.S., 2011. An experimental electro-acupuncture study in treatment of the rat demyelinated spinal cord injury induced by ethidium bromide. Neurosci. Res. 70, 294–304. Kashihara, Y., Sakaguchi, M., Kuno, M., 1989. Axonal transport and distribution of endogenous calcitonin gene-related peptide in rat peripheral nerve. J. Neurosci. 9, 3796–3802. Kim, J.H., Min, B.I., Na, H.S., Park, D.S., 2004. Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors. Brain Res. 998, 230–236. Kitamura, T., Harada, N., Goto, E., Tanaka, K., Arai, M., Shimada, S., Okajima, K., 2007. Activation of sensory neurons contributes to reduce spinal cord injury in rats. Neuropharmacology 52, 506–514. Lee, H., Park, H.J., Park, J., Kim, M.J., Hong, M., Yang, J.S., Choi, S., Lee, H., 2007. Acupuncture application for neurological disorders. Neurol. Res. 29, S49–S54. Li, X.Q., Verge, V.M.K., Johnston, J.M., Zochodne, D.W., 2004. CGRP peptide and regenerating sensory axons. J. Neuropathol. Exp. Neurol. 63, 1092–1103. Li, W.J., Pan, S.Q., Zeng, Y.S., Su, B.G., Li, S.M., Ding, Y., Li, Y., Ruan, J.W., 2010. Identification of acupuncture-specific proteins in the process of electroacupuncture after spinal cord injury. Neurosci. Res. 67, 307–316. Liu, A.L., Wang, X.W., Liu, A.H., Su, X.W., Jiang, W.J., Qiu, P.X., Yan, G.M., 2007. JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons. Exp. Toxicol. Pathol. 61, 131–143. Ma, Y.H., Zeng, X., Zhang, K., Zeng, Y.S., 2012. A new in vitro injury model of mouse neurons induced by mechanical scratching. Neurosci. Lett. 510, 14–19. Ondarza, A.B., Ye, Z.M., Hulsebosch, C.E., 2003. Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP. Exp. Neurol. 184, 373–380. Paola, F.A., Arnold, M., 2003. Acupuncture and spinal cord medicine. J. Spinal Cord Med. 26, 12–20. Schaeffer, C., Vandroux, D., Thomassin, L., Athias, P., Rochette, L., Connat, J.L., 2003. Calcitonin gene-related peptide partly protects cultured smooth muscle cells from apoptosis induced by an oxidative stress via activation of ERK1/2 MAPK. Biochim. Biophys. Acta, Mol. Cell Res. 1643, 65–73. Schmitt, A.B., Breuer, S., Polat, L., Pech, K., Kakulas, B., Love, S., Martin, D., Schoenen, J., Noth, J., Brook, G.A., 2003. Retrograde reactions of Clarke’s nucleus neurons after human spinal cord injury. Ann. Neurol. 54, 534–539. Sekhon, L.H., Fehlings, M.G., 2001. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26, S2–S12. Song, S., Liu, N., Liu, W., Shi, R., Guo, K.J., Liu, Y.F., 2009. The effect of pretreatment with calcitonin gene-related peptide on attenuation of liver ischemia and
W.-J. Li et al. / Neurochemistry International 61 (2012) 1397–1403 reperfusion injury due to oxygen free radicals and apoptosis. Hepatogastroenterology 56, 1724–1729. Thuret, S., Moon, L.D., Gage, F.H., 2006. Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7, 628–643. Uzan, B., Villemin, A., Garel, J.M., Cressent, M., 2008. Adrenomedullin is antiapoptotic in osteoblasts through CGRP1 receptors and MEK–ERK pathway. J. Cell. Physiol. 215, 122–128. Van Rossum, D., Hanisch, U.K., Quirion, R., 1997. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci. Biobehav. Rev. 21, 649–678. Wimalawansa, S.J., 1997. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit. Rev. Neurobiol. 11, 167–239. Wong, A.M., Leong, C.P., Su, T.Y., Yu, S.W., Tsai, W.C., Chen, C.P., 2003. Clinical trial of acupuncture for patients with spinal cord injuries. Am. J. Phys. Med. Rehabil. 82, 21–27. Wu, Y.C., Mi, Y.Q., Zhang, P., Zhang, J.F., Chen, W., 2009. Effect of warm acupuncture on nitric oxide synthase and calcitonin gene-related peptide in a rat model of lumbar nerve root compression. Neural Regen. Res. 4, 449–454.
1403
Yan, G.M., Irwin, R.P., Lin, S.Z., Weller, M., Wood, K.A., Paul, S.M., 1995. Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons. J. Pharmacol. Exp. Ther. 274, 983–990. Yan, Q., Ruan, J.W., Ding, Y., Li, W.J., Li, Y., Zeng, Y.S., 2011. Electro-acupuncture promotes differentiation of the mesenchymal stem cells, regeneration of nerve fibers and partial functional recovery after spinal cord injury. Exp. Toxicol. Pathol. 63, 151–156. Yick, L.W., Cheung, P.T., So, K.F., Wu, W.T., 2003. Axonal regeneration of Clarke’s neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp. Neurol. 182, 160–168. Yu, L.C., Hou, J.F., Fu, F.H., Zhang, Y.X., 2009. Roles of calcitonin gene-related peptide and its receptors in pain-related behavioral responses in the central nervous system. Neurosci. Biobehav. Rev. 33, 1185–1191. Zinck, N.D.T., Rafuse, V.F., Downie, J.W., 2007. Sprouting of CGRP primary afferents in lumbosacral spinal cord precedes emergence of bladder activity after spinal injury. Exp. Neurol. 204, 777–790.