Effect of Arecoline on Regeneration of Injured Peripheral Nerves

Effect of Arecoline on Regenerationof Injured Peripheral Nerves

Sheng-Chi Lee,*,† Chin-Chuan Tsai,‡,§ Chun-Hsu Yao,¶,|| Yuan-Man Hsu,**

Yueh-Sheng Chen¶,|| and Ming-Chang Wu*

*Department of Food Science

National Pingtung University of Science and Technology

Pingtung, Taiwan†Department of Orthopaedics

Pingtung Branch, Kaohsiung Veterans General Hospital

Kaohsiung, Taiwan‡School of Chinese Medicine for Post-Baccalaureate

I-Shou University, Kaohsiung, Taiwan

§Chinese Medicine Department

E-DA Hospital, Kaohsiung, Taiwan¶Lab of Biomaterials, School of Chinese Medicine

||Department of Biomedical Imaging and Radiological Science**Department of Biological Science and Technology

China Medical University, Taichung, Taiwan

Abstract: The present study provides in vitro and in vivo evaluation of arecoline on peripheralnerve regeneration. In the in vitro study, we found that arecoline at 50�g=ml could sig-nificantly promote the survival and outgrowth of cultured Schwann cells as compared to thecontrols treated with culture medium only. In the in vivo study, we evaluated peripheral nerveregeneration across a 10-mm gap in the sciatic nerve of the rat, using a silicone rubber nervechamber filled with the arecoline solution. In the control group, the chambers were filled withnormal saline only. At the end of the fourth week, morphometric data revealed that thearecoline-treated group at 5�g=ml significantly increased the number and the density ofmyelinated axons as compared to the controls. Immunohistochemical staining in thearecoline-treated animals at 5�g=ml also showed their neural cells in the L4 and L5 dorsalroot ganglia ipsilateral to the injury were strongly retrograde-labeled with fluorogold andlamina I–II regions in the dorsal horn ipsilateral to the injury were significantly calcitoningene-related peptide-immunolabeled compared with the controls. In addition, we found thatthe number of macrophages recruited in the distal sciatic nerve was increased as the

Correspondence to: Dr. Ming-Chang Wu, Department of Food Science, National Pingtung University of Scienceand Technology, #1, Shuefu Rd., Neipu, Pingtung 91201, Taiwan. Tel: (þ886) 8-774-0240, E-mail: mcwu@mail.

npust.edu.tw

The American Journal of Chinese Medicine, Vol. 41, No. 4, 865–885© 2013 World Scientific Publishing Company

Institute for Advanced Research in Asian Science and MedicineDOI: 10.1142/S0192415X13500584

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http://dx.doi.org/10.1142/S0192415X13500584

concentration of arecoline was increased. Electrophysiological measurements showed thearecoline-treated groups at 5 and 50�g=ml had a relatively larger nerve conductive velocityof the evoked muscle action potentials compared to the controls. These results indicate thatarecoline could stimulate local inflammatory conditions, improving the recovery of a severeperipheral nerve injury.

Keywords: Arecoline; TCM; Peripheral Nerve Regeneration; Nerve Conduit; RegenerativeMedicine; Calcitonin Gene-Related Peptide; Macrophage.

Introduction

The incidence of peripheral nerve injury in developed countries is dramatically increasing.Therefore, the development for better regenerative therapies is necessary for diminishingthe disability of patients with peripheral nerve injuries. When the nerve is injured, theaxons in the distal segment die and their myelin sheaths are phagocytosed by Schwanncells and macrophages. Proliferating Schwann cells assist regenerating axons in reachingtheir target organs (Chen et al., 2010b). In the literature, tissue engineering has successfullyprovided nerve conduits, which function as guides for axonal growth. Studies have shownthat higher nerve repair rates in rats with peripheral nerve injury treated with nerve conduitsassociated with nerve growth factor (Sun et al., 2012), brain derived neurotrophic factor(Wilhelm et al., 2012), fibronectin (Ding et al., 2011), laminin (Huang et al., 2011), andcollagen (Swindle-Reilly et al., 2012). In addition, studies have employed well-establishednerve conduit models of neuro-enhancement using traditional Chinese medicine (TCM),such as extraction of Chungpa-Juhn (Kim et al., 2011), Dilong (Chen et al., 2010a),Chishao (Fang et al., 2010), Lumbricus (Wei et al., 2009), and Sanyak (Lee et al., 2009).Our group also found that components in TCM could promote neuro-regeneration, such asquercetin (Wang et al., 2011), puerarin (Hsiang et al., 2011), astragaloside (Cheng et al.,2006), and bilobalide (Chen et al., 2004). Arecoline, an alkaloid-type natural product foundin the areca nut, the fruit of the areca palm (Areca catechu), has raised our attentionrecently since it has been successfully used to treat cerebral ischemia for its capability toimprove peripheral arterial occlusion (Terranova et al., 1994). It has been also reported thatthe arecoline has neuroprotective effects and is efficient in neurodegenerative diseases, suchas Alzheimer’s disease (Chandra et al., 2008). However, in vitro and in vivo analyses of thearecoline effect and nerve regeneration have never been evaluated.

Therefore, this workfirst uses the Schwann cell line, which has been extensivelyadopted to study cell differentiation and neurite outgrowth (Sango et al., 2012) to study itsneuronal characteristics upon exposure to arecoline solution. In the in vivo study, weanalyzed the effects of arecoline on peripheral nerve regeneration across a critical defectgap in a rat sciatic nerve injury model. In addition to the commonly used morphologicaland the electrophysiological techniques to evaluate the recovery of regenerating nerves, weused retrograde labeling with fluorogold in the L4 and L5 dorsal root ganglia (DRGs),calcitonin gene-related peptide (CGRP) in the lumbar spinal cord, macrophage influx in thedistal nerve end, and changes in NGF-mRNA levels of rat sciatic nerve segments

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conditioned by IL-1β. This was done to understand mechanisms and estimate the thera-peutic benefit of the arecoline on peripheral nerve regeneration.

Materials and Methods

Cell Culture and Treatment

Schwann cells (RSC96) were maintained at 37�C in a humidified atmosphere of 5% CO2

and 95% air in DMEM medium supplemented with 5% fetal calf serum, 2 mM HEPES and2 mM L-glutamine. The Schwann cells (7� 103 cells/cm2) were then induced to undergoneuronal differentiation by treatment with 5, 50, and 500�g/ml of arecoline solution(Product 10980, Sigma-Aldrich, St. Louis, MO, USA) for two days. Cells that had beenexposed to the vehicle alone (culture medium only) were the control. Six replicates wereused in each study.

Analysis of Cell Viability

After 48 h of cell incubation, the medium was removed, replaced with 110�L/well of5mg/ml of MTT solution in 1� PBS and further incubated in an incubator at 37�C for 4 h.Then, the MTT solution was removed and replaced with 50�L of DMSO to dissolve theformazan. The color intensity was measured using a microplate reader (ELx800TM, Bio-Tek Instrument, Inc., Winoski, VT, USA) at the absorbance of 550 nm (Zakaria et al.,2011). Data were then expressed as a percent of control level of the optical density withinan individual experiment.

Analysis of Cell Apoptosis

We also detected the expression levels of apoptosis-related marker proteins in the RSC96cells as they were treated with arecoline solution at its highest concentration of 500�g/mlused in the present study (Xu et al., 2012). Cultured RSC96 cells were scraped and washedonce with PBS. The cell suspension was then spun down, and cell pellets were lysed for30min in the lysis buffer (0.5M Tris-HCl, pH 7.4, 10% SDS, 0.5M DTT). Protein con-centration was determined by Bradford method (Bio-Rad, Hercules, CA, USA). Proteinsample (50�g) was loaded and separated on SDS-PAGE using the Hoefer mini VE system(Amersham Biosciences, Piscataway, NJ, USA). Proteins were transferred to a PVDFmembrane (Hybond-P, Amersham) according to the manufacturer’s instructions. Follow-ing the transfer, the membrane was washed with PBS and blocked for 1 h at 37�C with 5%fat-free milk in PBS and 0.1% of Tween-20 (PBST). The primary antibody (Bax, Bcl-2,cytochrome c, caspase-3, caspase-9 and -actin (Santa Cruz Biotechnology, USA) wasadded at a dilution of 1/100. Blots were incubated with the peroxidase conjugated sec-ondary antibodies (horseradish peroxidase conjugated goat anti-mouse IgG and goat anti-rabbit IgG; Santa Cruz Biotechnology) at a dilution of 1/5000. Following removal of thesecondary antibody, blots were washed by PBST and developed by ECL-Western blotting

ARECOLINE ON REGENERATION OF PERIPHERAL NERVES 867

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system (Pierce, Rockford, IL, USA). Densities of the obtained immunoblots were quan-tified by Kodak digital science 1D (ver. 2.03) (Kodak, Rochester, NY, USA).

Cell Imaging

After treating with the arecoline solution for 48 h, the Schwann cells were washed withPBS twice, fixed in 2% paraformaldehyde for 30min and then permeabilized with 0.1%Triton X-100/PBS for 30min at room temperature. After washing with PBS, TUNEL assaywas performed according to the manufacturer’s instructions (Boehringer Mannheim). Cellswere incubated in TUNEL reaction buffer in a 37�C humidified chamber for 1 h in the dark,then rinsed twice with PBS and incubated with DAPI (1mg/ml) at 37�C for 10min, stainedcells were visualized using a fluorescence microscope (Olympus DP70/U-RFLT50,Olympus Optical Co., Ltd., Japan). DAPI-positive and TUNEL-positive cells were countedas live and apoptotic cells, respectively.

Surgical Preparation of Animals

Forty adult Sprague-Dawley rats underwent placement of silicone chambers. The animalswere anesthetized with an inhalational anesthetic technique (AErrane®, Baxter, USA).Following the skin incision, fascia and muscle groups were separated using blunt dissec-tion, and the right sciatic nerve was severed into proximal and distal segments. Theproximal stump was then secured with a single 9-0 nylon suture through the epineuriumand the outer wall of the silicone rubber chamber (1.47mm ID, 1.96mm OD; HelixMedical, Inc., Carpinteria, CA, USA). Animals were divided into four groups. In group A(n ¼ 10), the chambers were filled with normal saline as the controls. In group B (n ¼ 10),5�g/ml of arecoline solution was filled in the chambers. Similarly, chambers in groups C(n ¼ 10) and D (n ¼ 10) were filled with 50�g/ml and 500�g/ml of arecoline solution,respectively.

The volume of the chamber lumen was approximately 25.5�l. These fillings, whichwere in the liquid state, were injected through a micropipette into the lumens by passing thetip of the needle inside the silicone rubber chambers as slowly as possible to preventleakage. As we secured the proximal stump into the end of the silicone rubber chamber, theswollen nerve end blocked the opening of the chamber. The chamber was then tilted andthe distal stump secured into the other end of the chamber. Both the proximal and distalstumps were secured to a depth of 1mm into the chamber, leaving a 10-mm gap betweenthe stumps. The muscle layer was re-approximated with 4-0 chromic gut sutures, and theskin was closed with 2-0 silk sutures. All animals were then returned to plastic cages wherea sufficient amount of sawdust was present to decrease direct contact of the paralyzed limbof the rat with the floor. These animals were housed in temperature (22�C) and humidity(45%) controlled rooms with 12-hour light cycles, and they had access to food and waterad libitum. All animals were maintained in facilities approved by the China MedicalUniversity for Accreditation of Laboratory Animal Care and in accordance with currentROC National Science Council of health regulations and standards.


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Electrophysiological Techniques

After four weeks of regeneration, the animals were re-anaesthetized and the silicone rubberconduit was removed to expose their sciatic nerve. The stimulating cathode was a stainless-steel monopolar needle, which was placed directly on the sciatic nerve trunk, 5-mmproximal to the transection site. The anode was another stainless-steel monopolar needleplaced 3-mm proximally to the cathode. Amplitude, area, and nerve conductive velocity(NCV) of the evoked muscle action potentials (MAP) were recorded from gastrocnemiusmuscles with micro-needle electrodes linked to a computer system (Biopac Systems, Inc.,USA). The amplitude and the area under the MAP curve were calculated from the baselineto the maximal negative peak. The NCV was carried out by placing the recording elec-trodes in the gastrocnemius muscles and stimulating the sciatic nerve proximally anddistally to the silicone rubber conduit. The NCV was then calculated by dividing thedistance between the stimulating sites by the difference in latency time. All data areexpressed as mean�standard deviation (S.D.) Statistical comparisons between groups weremade by the one-way analysis of variance.

Retrograde Labeling with Fluorogold

Fluorogold (FG; Fluorochrome, Denver, CO, USA) was dissolved in distilled water tomake a 2% suspension and stored at 4�C in the dark. Immediately after the recording ofmuscle action potential, 1�l and 2�l of 2% FG solution were directly injected via a 10-�lHamilton microsyringe into the common peroneal nerve and posterior tibial nerve.Following the injection, the site was wiped with a swab, flushed with sterile 0.9% saline,and the intraneural injection site closed with 10-0 nylon sutures. Then 5-mm distal to theinjection site, the common peroneal nerve was ligated with 4-0 silk and 5-mm-long seg-ments just distal to the ligation were harvested for histological assessment. The wound wasthen closed by 4-0 silk sutures. After allowing five days for retrograde transport, theanimals were perfused transcardially with 200ml of 0.9% saline, followed by 500ml ofice-cold 4% paraformaldehyde in 0.1M phosphate buffer. After perfusion, the L4 and L5DRGs ipsilateral to the injury were dissected and removed, placed overnight in 4% par-aformaldehyde in 0.1M phosphate buffer for post-fixation and stored overnight in 30%phosphate-buffered sucrose solution. Frozen longitudinal sections of the spinal cord andDRGs, 40�m thick, were made using a cryostat. After drying for 30min, the sections weremounted and examined with an ultraviolet fluorescence microscope (Olympus ckx41,Center Valley, PA, USA).

Histological Techniques

After perfusion, as was done for the retrograde labeling, the L4 spinal cord and the distalstump outside the nerve gap were quickly removed and post-fixed in the same fixative for3–4 h. Tissue samples were placed overnight in 30% sucrose for cryoprotection at 4�C,followed by embedding in optimal cutting temperature solution. Samples were the kept at


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�20�C until preparation of 18�m sections was performed using a cryostat, with samplesplaced upon poly-L-lysine-coated slide. Immunohistochemistry of frozen sections wascarried out using a two-step protocol according to the manufacturer’s instructions(Novolink Polymer Detection System, Novocastra). Briefly, frozen sections were requiredendogenous peroxidase activity was blocked with incubation of the slides in 0.3% H2O2,and nonspecific binding sites were blocked with Protein Block (RE7102; Novocastra).After serial incubation with rabbit- anti-CGRP polyclonal antibody 1:1000 (Calbiochem,Germany), Post Primary Block (RE7111; Novocastra), and secondary antibody (NovolinkPolymer RE7112), the L4 spinal cord sections were developed in diaminobenzidinesolution under a microscope and counter stained with hematoxylin. Similar protocolswere applied in the sections from the distal stump except they were incubated with anti-rat CD68 1:100 (AbD Serotec, Kidlington, UK). Sciatic nerve sections were taken fromthe middle regions of the regenerated nerve in the chamber. After the fixation, the nervetissue was post-fixed in 0.5% osmium tetroxide, dehydrated, and embedded in Spurr’sresin. The tissue was then cut to 5-�m thickness by using a microtome (Leica EM UC6,Leica Biosystems, Mount Waverley, Australia) with a dry glass knife, stained withtoluidine blue.

Changes in NGF-mRNA Levels of Rat Sciatic Nerve SegmentsConditioned by IL-1ß

Sciatic nerve segments (3 cm) of adult Sprague-Dawley rats were cultured in 1mlDulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. After threedays culture, medium conditioned by recombinant rat IL-1β (PeproTech, Rocky Hill, NJ)was added. Total RNAs were extracted from nerve tissues with TRIzol reagent and theamount of RNA estimated by spectrophotometry at 260 nm. For real-time (RT)-PCRanalysis, two-step RT-PCR was carried out using a high-capacity cDNA reverse tran-scription kit (Applied Biosystems, USA), and a 16S rRNA gene PCR assay was used as ahousekeeping gene control assay. The reactions were performed in 20�l (total volume)mixtures containing primers at a concentration of 400 nM. The reaction conditions con-sisted of 2min at 50�C, 10min at 95�C, then 40 cycles of 15 s at 95�C, followed by 1minat 60�C. Melting curve analysis was used to determine the PCR specificity and wasperformed using 80 10-s cycles, with the first cycle at 60�C and the temperature increasingby 0:5�C for each succeeding cycle. All reactions were carried out in triplicate from threecultures. Each assay was run on an Applied Biosystems 7300 Real-Time PCR system. Thethreshold cycle (Ct) is defined as the fractional cycle number at which the fluorescencepasses the fixed threshold data, and was determined using the default threshold settings.Relative quantification of mRNA expression was calculated using the 2-��Ct method(Applied Biosystems User Bulletin N�2 (P/N 4303859)). Data were presented as therelative expression of target mRNA, normalized with respect to GAPDH mRNA andrelative to a calibrator sample that was collected at 0 min of infection. The primers used inthis study are shown in Table 1.


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Image Analysis

All tissue samples were observed under optical microscopy. CGRP-immunoreactivity (IR)in dorsal horn in the lumbar spinal cord was detected by immunohistochemistry as describedpreviously (Yao et al., 2012). The immuno-products were confirmed positive-labeled iftheir density level was over five times background levels. Under a 100� magnification, theratio of area occupied by positive CGRP-IR in dorsal horn ipsilateral to the injury followingneurorrhaphy relative to the lumbar spinal cord was measured using an image analyzersystem (Image-Pro Lite, Media Cybernetics, USA) coupled to the microscope.

As counting the myelinated axons, at least 30 to 50% of the sciatic nerve section arearandomly selected from each nerve specimen at a magnification of 400� was observed.The myelinated axon counts were extrapolated by using the area algorithm to estimate thetotal number of axons for each nerve. Axon density was then obtained by dividing the axoncounts by the total nerve areas. All data are expressed as mean�S.D. Statistical com-parisons between groups were made by one-way analysis of variance with Scheffe test.

Results

Cell Viability and Imaging

Spindle-shaped cellular morphology of Schwann cells cultured on the culture plate wasviable and there was no sign of infection (Fig. 1). The optical density of Schwann cellstreated with arecoline solution at 50�g/ml was significantly increased as compared to thatof the controls. This result is supported by the cell imaging that increasing DAPI positivecells and no TUNEL positive cells were seen in the groups treated with the arecolinesolution at both the concentrations. These results indicate that treatment of the arecolinesolution at a proper concentration could exert growth-promoting effects on the culturedSchwann cells. However, these positive effects of arecoline solution on Schwann cells weresharply declined as its concentration was boosted to 500�g/ml. The cultured cells wereobviously intoxicated by the high concentration of arecoline solution as nearly no live cellsadhering on the culture plates could be seen.

Analysis of Cell Apoptosis

As mentioned in the results above, we found that arecoline treatment at a high concen-tration could induce neuronal apoptotic death. The effect of arecoline at 500�g/ml onmediating apoptosis in the RSC96 cells was then studied. As a result, the expression of

Table 1. PCR Primers

rat NGF-F GTGGACCCCAAACTGTTTAAGAA

rat NGF-R AGTCTAAATCCAGAGTGTCCGAAGArat-GAPDH-F GGTGGACCTCATGGCCTACARat-GAPDH-R CAGCAACTGAGGGCCTCTCT


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(A)

0%

20%

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60%

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120%

140%

0 5 50 500 (μg/ml)

Viability (%)

* *

* * *

(B)

Figure 1. (A) Nuclei of Schwann cells characterized by DAPI and TUNEL assays and investigated via fluorescent

microscopy. (B) Quantification of viability of Schwann cells treated with arecoline solution relative to controls.Values are mean�S.D. *p < 0:05 indicates a significant difference from other groups.


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Bcl-2, Bax, cytochrome c, caspase-9, and caspase-3 in treated cells were monitored(Fig. 2). Compared with untreated cells, arecoline at 500�g/ml significantly induced theexpression of Bax, cytochrome c, caspase-9, and caspase-3. However, the expression of theanti-apoptotic protein Bcl-2 was down-regulated. These data indicate that the arecolinetreatment at 500�g/ml could induce apoptosis in the RSC96 cells.

Electrophysiological Measurements

Electrophysiological tests performed during follow-up after operation indicated successfulreinnervation of the gastrocnemius muscle by the regenerating sciatic nerve with obviousexcitability and conductivity in all the rats of the four groups evaluated (Fig. 3). Animalstreated with 5 and 50�g/ml of arecoline had a relatively larger NCV as compared to thecontrols, although there was no significant difference between the groups. Only thedifference of NCV between group B at 5�g/ml and group D at 500�g/ml of arecolinereached the significant level at p < 0:05 (Fig. 4). Large standard deviations were noted inthe other electrophysiological measurements, including latency, amplitude, and MAP areathat could be caused by atrophy of the muscle, which was still serious even after fourweeks of recovery.

Retrograde Labeling with Fluorogold

It was found that fluorogold labeling in the DRGs was dramatically increased in the groupwith 5�g/ml of arecoline as compared to control levels (Fig. 5). The labeled cell count then

0 15 30 60 Min

Bcl-2 26 kDa

Bax 23 kDa

Cyt c 15 kDa

Caspases-9 35 kDa

Caspases-3 11 kDa

Actin43 kDa

Figure 2. Effect of arecoline at 500�g=ml on the expression levels of c Bcl-2, Bax, cytochrome c, caspase-9, andcaspase-3 on RSC96 cells.


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decreased with the concentration of the arecoline. Little fluorogold labeling could be seenin the group of arecoline at 500�g/ml.

CGRP-IR in the Dorsal Horn Following Injury

Immunohistochemical staining showed that CGRP-labeled fibers were noted in the area oflamina III–V (Fig. 6). Lamina I-II regions in the dorsal horn ipsilateral to the injury werealso strongly CGRP-immunolabeled in all of the animals in which those in group B with5�g/ml of arecoline had a relatively higher CGRP expression (Fig. 7). In addition, a

Figure 3. Representative recordings of evoked MAPs in gastrocnemius muscles from groups A–D. Obvious

excitability and conductivity exists for all rats in all four groups.

0

5

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15

20

25

30

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0 5 50 500 (ug/ml)

NCV (m/s)

(A)

Figure 4. Analysis of evoked MAPs, including (A) NCV, (B) peak amplitude, and (C) area under the MAP

curves. *p < 0:05 indicates significant difference from other groups.


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Amplitude (mV)

(B)

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(C)

Figure 4. (Continued )

(A) (B)

Figure 5. Representative images of the retrograde axonal tracing with fluorogold (panel A, controls (group A);panel B, arecoline at 5�g=ml (group B); panel C, arecoline at 50�g=ml (group C); panel D, arecoline at 500�g=ml(group D)). The number of fluorogold-labeled cell in DRGs was increased with the concentration of arecoline.

However, only few fluorogold labeling could be seen in the group of arecoline at 500�g=ml. Scale bar ¼ 100�m.


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significant increase in the CGRP expression was noted at the significant level of 0.05 ingroup B (5�g/ml of arecoline) as compared to group D (500�g/ml of arecoline). Theseresults indicated CGRP expression dynamics in the lumbar spinal cord differed dependingupon the concentration of arecoline filled in the bridging conduit.

Macrophages Recruited in the Distal Nerve Ends

The filled arecoline could stimulate local inflammatory conditions in the bridging chamberssince the density of macrophage in the distal nerve stumps was increased as the concen-tration of arecoline was increased (Fig. 8). Specially, a significantly higher density of

(C) (D)


(A) (B)

Figure 6. Photo shows the area of lamina III-V examined for CGRP-labeled fibers (arrows). Shown in panel B isthe higher magnification of the boxed area in panel A. Scale bar ¼ 100�m for panel A and 20�m for panel B.


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(C) (D)

0.0%

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0 5 50 500 (ug/ml)

Area ratio (%)

(E)

Figure 7. Photomicrographs demonstrating CGRP-IR in dorsal horn in the lumbar spinal cord after injury of (A)controls (group A) and arecoline-treated animals at (B) 5�g=ml (group B), (C) 50�g=ml (group C), and (D)500�g=ml (group D). (E) Note the significantly decreased CGRP-IR area ratios in arecoline-treated animals at

500�g=ml compared to those at 5�g=ml. *p < 0:05. Scale bars ¼ 200�m.


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(A) (B)

(C) (D)

* *

(E)

Figure 8. Photomicrographs demonstrating anti-rat CD68 immunoreactivity in macrophages (arrows) from cross-

sections of distal nerve cables of (A) controls (group A) and arecoline-treated animals at (B) 5�g=ml (group B),(C) 50�g=ml (group C), and (D) 500�g=ml (group D). (E) Note the dramatically increased density of macro-phages with the concentration of arecoline. *p < 0:05. Scale bars ¼ 100�m.


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macrophages was recruited into the nerve stumps in the group D (500�g/ml of arecoline)compared to the controls and the group B (5�g/ml of arecoline) at p < 0:05. This resultindicated that deliberate superimposition of arecoline could dramatically enhance influx ofmacrophages in injured nerves.

Changes in NGF-mRNA Levels of Rat Sciatic Nerve Segments Conditioned by IL-1ß

As aforementioned results, we found that the arecoline loaded in the bridging conduit couldactivate the macrophages in the degenerating part of the nerves. To confirm the mechan-isms of how the activated macrophages promoted peripheral nerve regeneration, we studiedthe changes in NGF-mRNA levels of rat sciatic nerve segments by adding conditionedmedia of recombinant rat IL-1β. As a result, it was found that the NGF-mRNA could beincreased after addition of recombinant rat IL-1β (Table 2). The effect of rat IL-1β wasdiscernible at a concentration of 100 ng/ml. These results indicated the IL-1β could beimportant in the regulation of NGF synthesis in transected adult nerves.

Sciatic Nerve Regeneration

Dramatic differences were noted among the regenerated nerves treated with differentconcentrations of the arecoline. In the controls, the successfully regenerated nerves had astructure with circumferential fibroblasts surrounding the centrally located endoneurium. Asubstantial portion of the endoneurial area was occupied by connective tissue in whichloosely distributed axons and a few blood vessels and Schwann cells were seen (Fig. 9). Bycomparison, the regenerated nerves in the group B (5�g/ml of arecoline) and C (50�g/mlof arecoline) had a more mature structure composed of compact myelinated axons withtheir associated Schwann cells surrounded by perineurial sheaths. Morphometric data alsorevealed that the arecolineat 5�g/ml significantly increased the number and the density ofmyelinated axons that grew into the distal end of the chamber as compared with thecontrols (Fig. 10). However, the high dose arecolinein group D (500�g/ml) had begun toreverse this positive effect of growth-promoting capability and inhibit nerve regeneration.

Table 2. Changes in NGF-mRNA Levels of RatSciatic Nerve Segments by Adding ConditionedMedia of Recombinant Rat IL-1β

Group IL-1β Fold to Group A (control)

A 0 ng/ml 1:00� 0:03§¶

B 50 ng/ml 1:01� 0:03§¶

C 100 ng/ml 1:21� 0:03*þ¶

D 200 ng/ml 1:34� 0:04*þ§

Notes:*p < 0:05 compared with group A.þp < 0:05 compared with group B.§p < 0:05 compared with group C.¶p < 0:05 compared with group D.


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0

1000

2000

3000

4000

5000

6000

7000

0 5 50 500 (ug/ml)

* *

(A)

Figure 10. Morphometric analysis from the regenerated nerves, including (A) axon number, (B) axon density, and

(C) total nerve area. *p < 0:05, significant difference from other groups.

(A) (B)

(C) (D)

Figure 9. Photomicrographs demonstrating regenerated nerve cross-sections of (A) controls (group A) andarecoline-treated animals at (B) 5�g=ml (group B), (C) 50�g=ml (group C), and (D) 500�g=ml (group D). Notethe increased number and compact organization of myelinated axons in the arecoline-treated animals at 5 and

50�g=ml. Scale bars ¼ 100�m.


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Most of the successfully regenerated nerves in this group had no such aforementionedmature structure in which significantly fewer axons were noted as compared to those ingroup B at the significant level of 0.05.

Discussion

Regenerative medicine is the process of replacing or regenerating animal cells, tissues ororgans to restore or establish normal function. This field has the potential to solve theproblem of the shortage of organs available for donation and of organ transplant rejection.Recently, application of TCM as a means to accelerate the process of regeneration is a newapproach. Therapies using the TCM offer a natural and cost-effective intervention tochange the course of chronic disease and may regenerate failing organ systems (Kim et al.,2011; Zeng et al., 2011).

The arecoline is an alkaloid-type natural product found in the areca nut, which is one ofthe world’s most popular stimulants, which could directly stimulate cholinergic receptors(Coppola and Mondola, 2012; Yuan et al., 2012). It has been clinically shown to improvethe intellectual capabilities in the patients with Alzheimer’s disease (Asthana et al., 1996;

0.0

5000.0

10000.0

15000.0

20000.0

0 5 50 500 (ug/ml)

* *

(B)

0.0

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Raffaele et al., 1996). These previous studies show that arecoline may exert positive effectson the nervous system. In the present study, we also found that arecoline shows growth-promoting effects on Schwann cells, exerting a promotion action upon the cell proliferationand viability. Therefore, we believe that the arecoline could play important roles in nerveregeneration since the Schwann cells can provide an essentially supportive activity forneuron regeneration (Fontana et al., 2012; Newbern and Snider, 2012). In addition, wefound that administration of an appropriate dosage of arecoline within the bridging conduitcould enhance axonal growth in the regenerated nerve cable, especially in the group ofarecoline at 5�g/ml. The mean value in this group, approximately two-fold axons (5431)successfully grew across the 10-mm gap as compared to the controls (3346). The histo-logical results were supported by the retrograde labeling with fluorogold in the DRGs andthe protein levels of CGRP in associated spinal cord segments, which were highlyexpressed in the group of 5�g/ml of arecoline. Fluorogold particles in the DRGs are takenup by primary sensory neurons (Zhang et al., 2006). The increased fluorogold-labeled cellsin the cryostat section revealed that the arecoline could promote more migrating axonsovercoming the bridging nerve tissue in the conduit and reaching the DRGs. In addition,the arecoline could also trigger more injury-related signals derived from the injured nervesretrogradely transported to neurons in the dorsal horn and synthesize more CGRP. As weknow, the CGRP is critical for interactions between axons and Schwann cells duringperipheral nerve regrowth (Toth et al., 2009; Chen et al., 2010c; Wang et al., 2010) andplays important roles in nerve function and repair when axons are severed (Li et al., 2004).Therefore, the increasing CGRP expression induced by the arecoline suggests that it mayaccelerate the recovery of regenerating axons.

Though those aforementioned findings are encouraging, the growth-promoting capa-bility of arecoline was suppressed as its concentration was increased to a high level of500�g/ml. We believe that local inflammatory conditions induced by the filled arecoline inthe bridging conduits may contribute to these events. In the present study, we found thatarecoline could enhance the function of the immune system, specifically the macrophages.The macrophages have been considered a vital role in peripheral nerve regeneration mainlybecause they are profoundly involved in regulating the expression of neurotrophic factorssuch as NGF by releasing interleukin-1 as seen in the present study (Rosenberg et al.,2012; Ydens et al., 2012). Therefore, the relatively low-dose of arecoline loaded in thebridging conduit (5 and 50�g/ml) could activate the macrophages in the degenerating partof the nerve, releasing neurotrophic factors to promote the growth of axons to cross gapsand grow into the distal segment. However, the deliberate superimposition of localinflammation induced by the high dose of arecoline at 500�g/ml dramatically provokedadverse responses to the recovery of regenerated nerves. In the present study, we found thatthe high dose of arecoline significantly induced the expression of Bax, cytochrome c,caspase-9, and caspase-3 but down-regulated the anti-apoptotic protein Bcl-2. Therefore,we believe the over-dosed arecoline treatment could induce apoptosis in the nervous cells.The over-dosed arecoline could also induce neuronal apoptotic death by attenuatingantioxidant defense and enhancing oxidative stress (Shih et al., 2010). In addition, it hasbeen reported that excessive nerve growth-promoting substances could saturate receptors


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on neuron, such as trkA and p75NTR, thereby blocking their functions in the regenerativeprocesses (Gallo et al., 1997; Boyd and Gordon, 2002). Excessive nerve growth-promotingsubstances could also directly suppress the axotomy-induced elevation of neurotrophicfactors, such as growth-associated protein 43 (GAP-43), resulting in inappropriate rees-tablishment of injured nerve (Mohiuddin et al., 1999; Hirata et al., 2002).

Although histological observations demonstrate nerve regeneration was affected posi-tively by arecoline administration, electrophysiological evaluations fail to show convincingevidence of such event. This result is similar to that seen in our previous studies (Lu et al.,2010) that we believe misdirected regeneration, i.e., a sensory fascicle anastomosed to amotor one or vice versa, and the serious muscle atrophy during the regenerative periods arethe main reasons to cause the inconsistent data (Hou and Zhu, 1998).

In summary, based on our findings in this experimental model, arecoline is an agent thathas beneficial effects on peripheral nerve regeneration as evidenced by the analysis ofSchwann cell viability, the biochemical imaging, the histomorphometric nerve analysis,and the electrophysiological analysis. Further development of the arecoline may lead to thegeneration of new drugs for the treatment of human peripheral nerve injuries.

Acknowledgments

The authors would like to thank Miss I-Ching Chen for her technical assistance. M.-C. Wuand Y.-S. Chen contributed equally to this work. Part of this work was supported byresearch grants from the China Medical University and Hospital (DMR-100-183;CMU100-S-37; CMU99-COL-20-2) and Taiwan Department of Health Clinical Trial andResearch Center of Excellence (DOH102-TD-B-111-004).

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Effect of Arecoline on Regeneration of Injured Peripheral Nerves

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