Neuroscience Letters 561 (2014) 198–202

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Triptolide improves nerve regeneration and functional recovery following crush injury to rat sciatic nerve Yong-Guang Zhang a,1 , Qing-Song Sheng b,1 , Hong-Kun Wang c,1 , Li Lv a , Jun Zhang d , Jian-Mei Chen a,∗ , Hao Xu a,∗ a

Department of Orthopaedics, Fuzhou General Hospital, Fuzhou 350025, China Department of Obstetrics and Gynecology, Fuzhou General Hospital, Fuzhou 350025, China c Department of Burns and Plastic Surgery, Fuzhou General Hospital, Fuzhou 350025, China d Department of Pharmacology, Fourth Military Medical University, Xi’an 710032, China b

h i g h l i g h t s • Triptolide improves nerve regeneration after nerve crush injuries. • Triptolide promotes motor functional recovery after nerve crush injuries. • Triptolide may exert neuroprotective effects via its anti-inflammatory properties.

a r t i c l e

i n f o

Article history: Received 27 September 2013 Received in revised form 28 December 2013 Accepted 30 December 2013 Keywords: Triptolide Peripheral nerve injury Nerve regeneration Pro-inflammatory cytokines

a b s t r a c t Recently, accumulating data have demonstrated that triptolide exhibits neurotrophic and neuroprotective properties. However, the role of triptolide in repair and regeneration of peripheral nerve injury (PNI) has rarely been performed. The current study was designed to observe the possible beneficial effect of triptolide on promoting peripheral nerve regeneration in rats. Rats with sciatic nerve crush injury were administered daily with triptolide for 7 days. Axonal regeneration was evaluated by morphometric analysis and Fluoro-gold retrograde tracing. Motor functional recovery was evaluated by walking track analysis, electrophysiological assessment and histological appearance of target muscles. Levels of pro-inflammatory cytokines within injured nerves were also determined. The results demonstrated that triptolide was capable of promoting peripheral nerve regeneration. Additionally, triptolide significantly decreased the levels of pro-inflammatory cytokines within injured nerves. These findings indicate the possibility of developing triptolide as a therapeutic agent for PNI. The neuroprotective effects of triptolide might be associated with its anti-inflammatory properties. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Peripheral nerve injury (PNI) is a worldwide problem, with an incidence of 2.8% in multi-trauma victims [1]. Over the past century, a series of therapeutic strategies have been developed for the treatment of PNI, including microsurgical repair, pharmacotherapy, and physical rehabilitation. Among these strategies, pharmacotherapy is a promising approach for neurorehabilitation. Therefore, exploration of exogenous agents that can improve the efficacy of

∗ Corresponding authors at: Institution of Orthopaedics, Fuzhou General Hospital, 156 West Ring Road, Fuzhou 350025, China. Tel.: +86 0591 22859382; fax: +86 0591 22859382. E-mail addresses: [email protected] (J.-M. Chen), xuhao [email protected] (H. Xu). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.068

nerve regeneration has been attracting considerable research interest. Tripterygium wilfordii Hook.F. has been used for hundreds of years in treating inflammatory diseases. Triptolide is the major bioactive compound isolated from this plant and possesses a broad spectrum of pharmacological properties. During recent years, accumulating data have demonstrated that triptolide exhibits neurotrophic and neuroprotective potentials. In vitro studies show that triptolide protects dopaminergic neurons from inflammationmediated damage [2], and protects neural cells through the inhibition of CXCR2 activity [3]. In vivo studies show that triptolide is capable of promoting nerve regeneration and functional recovery after spinal cord and brain injuries [4,5]. All these studies indicate the potential use of triptolide in treating PNI. However, the role of triptolide in treating PNI has rarely been performed. Therefore, the main objective of the present study was to examine whether triptolide treatment would lead to improved nerve regeneration

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submitted to walk across a narrow wooden track (100 cm long and 7 cm wide) with a dark box at one end. The hind feet were dipped in red ink and changes of footprints were recorded on white papers. For normal footprints, the SFI value is near 0, while an SFI value of approximately −100 reflects complete loss of function. 2.3. Electrophysiological analysis After walking tests, rats were subjected to electrophysiological tests (Fig. 1). Under anesthesia, the sciatic nerve was exposed. A bipolar stimulating electrode was placed to the nerve trunk at its proximal portion, and a recording electrode was placed in the gastrocnemius muscle to record the Compound muscle action potentials (CMAPs). The CMAP peak amplitude and nerve conduction velocity (NCV) values were calculated [8]. 2.4. Retrograde tracing

Fig. 1. Schematic diagram of surgical procedures.

and functional recovery in the rat model of sciatic nerve crush injury. 2. Materials and methods 2.1. Surgical procedures Sprague-Dawley rats (weighing 220–250 g) were anesthetized by an intraperitoneal injection of 1.5% sodium pentobarbital solution (40 mg/kg) and the model of nerve crush injury was established (Fig. 1). In brief, the right sciatic nerve was exposed and compressed at the proximal segment 5 mm from the bifurcation using a pair of forceps for 3 times (10 s each time) with an interval of 10 s. The skin was closed with 6–0 stitches. The rats were then divided randomly into three groups (Table 1): (1) sham-operative group, in which the sciatic nerve was exposed but not compressed; (2) vehicle group, which received crush injury and were intraperitoneally administrated with 25 ␮l of 5% dimethylsulfoxide (DMSO, Sigma, St. Louis, MO) in saline solution; (3) triptolide group, which received crush injury and were intraperitoneally administrated with triptolide (100 ␮g/kg, Sigma, St. Louis, MO). Triptolide was dissolved in 5% DMSO/saline at a concentration of 1 ␮g/␮l as described previously [6]. Administration of triptolide or DMSO was performed once a day for successive 7 days. All experimental protocols were carried out in accordance with NIH guidelines for care and use of animals with scientific purposes, and were approved by the Animal Experimentation Ethics Committee of Fourth Military Medical University. 2.2. Behavioral analysis Walking track analysis was performed weekly and sciatic functional index (SFI) was calculated [7]. In brief, trained rats were Table 1 Number of rats allocated to different assessments in all groups. Sham-operative group Pro-inflammatory cytokines levels determination Retrograde tracing Behavioral, electrophysiological and histomorphometric analysis Total number

Vehicle group

Triptolide group

4

4

4

4 6

4 6

4 6

14

14

14

Retrograde tracing was performed at 3 weeks post-injury. Briefly, the sciatic nerve was exposed and 5 ␮l of 4% Fluoro-gold (Biotium, Hayward, CA) solution was intraneurally injected into the nerve trunk at the site of bifurcation (Fig. 1). The incision was then sutured and the rats were back to their cages. 5 days later, the rats were intracardially perfused with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer under deep anesthesia. The lumbar spinal cord was exposed and L4, L5 and L6 segments were harvested, postfixed in buffered 4% paraformaldehyde for 4 h, cryoprotected in 30% sucrose overnight at 4 ◦ C, and then sectioned on a cryostat. Transverse sections (25 ␮m thick) were mounted on glass slides, viewed and photographed under a fluorescent microscope (BX-60; Olympus). The number of FG-labeled motoneurons was counted. 2.5. Morphometric analysis Four weeks post-injury, the regenerative nerve was harvested and fixed with 3 wt% glutaraldehyde. The sample was then postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH = 7.3), dehydrated and embedded in resin according to standard protocols. From the distal portion of the sample, 1.0 ␮m thick transverse semithin sections (stained with 1% toluidine blue/1% borax solution) and 50.0 nm thick ultrathin sections (stained with uranyl acetate and lead citrate) were prepared. The semithin sections were observed under light microscopes and the ultrathin sections were observed with transmission electron microscopes. Morphometric evaluations included total number of myelinated axons, mean diameter of nerve fibers and the degree of myelination (g-ratio) were performed. Then, the gastrocnemius muscles were removed, fixed in 4% paraformaldehyde, and subjected to hematoxylin and eosin staining. Photographs were taken under light microscopes. For each sample, the cross-sectional area of muscle fibers was measured by photographs taken from 4 random fields, analyzed with a Leica software package, and evaluated by calculating the ratio between the area of muscle fibers in each field and the total area in the same field (magnification 200×). 2.6. Determination of TNF-˛, IL-1ˇ and IL-6 levels in the nerve tissues Three days post-injury, the levels of TNF-␣, IL-1␤ and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). Briefly, the injured nerve was harvested, frozen in liquid nitrogen, and stored at −80 ◦ C. The samples were centrifuged for 30 min (14,000 rpm, 4 ◦ C). Supernatants were transferred to Eppendorf tubes and assayed in

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Fig. 2. Representative micrographs following Fluoro-gold retrograde tracing (A–C), transmission electron microscopy (D–F), and target muscles (G–I) in sham-operative group (A, D, and G), vehicle group (B, E, and H), and triptolide group (C, F, and I) at 4 weeks post-injury. Original magnification: (A–C) 100×; (G–I) 200×. Scale bar (D–F) = 2 ␮m.

duplicate using TNF-␣, IL-1␤ and IL-6 assay kits according to the manufacturer’s procedures. 2.7. Statistical analysis For statistical analysis, the SPSS13.0 software package (SPSS Inc., Chicago, IL) was used. All data are expressed as mean ± standard error of the mean (SEM), and analyzed using one-way analysis of variance with Bonferroni test for pairwise comparisons. p < 0.05 were considered statistically significant.

myelinated axons and mean diameter of the nerve fibers in triptolide group were significantly higher than those in vehicle group (p < 0.05, Fig. 2D–F, Table 2). To examine the degree of myelination, the relation of axon diameter to total fiber diameter was calculated as g-ratio. As shown in Table 2, g-ratio in triptolide group was significantly lower than that in vehicle group (p < 0.05), suggesting that better degree of myelination was achieved in triptolide group. Additionally, these three parameters in sham-operative group was the best in comparison with the remaining two groups (p < 0.05).

3. Results

3.2. Triptolide promotes motor functional recovery

All the animals underwent the inoculation of triptolide successfully. No animal died, and no signs of inflammation or trophic ulcerations on the operated legs were seen after surgery.

At 1 and 2 weeks post-injury, SFI values in triptolide group were slightly higher than that in vehicle group with no significant differences. At 3 and 4 weeks post-injury, SFI values in triptolide group were significantly higher than those in vehicle group (p < 0.05, Fig. 3B), suggesting that triptolide is beneficial to promote motor functional recovery. Additionally, the motor function of rats in sham-operative group was barely affected, with the SFI values oscillating around 0 at each predefined time point. At 3 and 4 weeks post-injury, CMAP amplitude in triptolide group was significantly higher than that in vehicle group (p < 0.05, Fig. 3C), suggesting that more nerve fibers innervated target muscles in triptolide group in comparison with that in vehicle group. Also, the nerve conduction velocity in triptolide group was significantly higher than that in vehicle group at 3 and 4 weeks post-injury (p < 0.05, Fig. 3D), which was concomitant with the larger mean diameter of nerve fibers and better degree of myelination in triptolide group.

3.1. Triptolide improves nerve regeneration after crush injury The beneficial effects of triptolide on nerve regeneration were demonstrated by Fluoro-gold retrograde labeling. At 4 weeks post-injury, the number of Fluoro-gold-labeled motoneurons in triptolide group was higher than that in vehicle group with significant difference (p < 0.05, Figs. 2A–C and 3A), indicating that more motoneurons successfully regenerated into the distal stump after triptolide administration. Additionally, the number of Fluoro-goldlabeled motoneurons in sham-operative group were significantly higher than that in the remaining two groups (p < 0.05). Morphometric analysis also proved that triptolide is capable of enhancing nerve regeneration. 4 weeks post-injury, the number of

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Fig. 3. The total number of Fluoro-gold-positive motoneurons (A), SFI (B), CMAP amplitude and NCV (C and D), and the average percentage of muscle fiber area (E) for each group. All data were expressed as mean ± SEM. *p < 0.05 for the comparison with vehicle group. **p < 0.05 for comparison with sham-operative group. Table 2 Morphometric values in all groups at 4 weeks post-injury. Morphometric values 4

Total number of myelinated axons (10 ) Mean diameter of myelinated axons (␮m) Axon to fiber diameter ratio (g-ratio) a b

Sham-operative group

Vehicle group

Triptolide group

0.537 ± 0.038 4.681 ± 0.593 0.558 ± 0.091

0.265 ± 0.043 3.471 ± 0.237a 0.719 ± 0.126a a

0.341 ± 0.022a , b 4.015 ± 0.386a , b 0.676 ± 0.132a , b

p < 0.05 for comparison with sham-operative group. p < 0.05 for comparison with vehicle group.

The atrophy of gastrocnemius muscles post-injury was partially alleviated by administration of triptolide. The average percentage of muscle fiber area in triptolide group was significantly higher than that in vehicle group at 4 weeks post-injury (p < 0.05, Fig. 3E). Additionally, the average percentage of muscle fiber area in shamoperative group was the highest in comparison with the remaining two groups (p < 0.05). 3.3. Triptolide decreases pro-inflammatory cytokines levels The levels of TNF-␣, IL-1␤ and IL-6 were measured at 3 days post-injury. As shown in Table 3, crush-injured animals had a significant increase of TNF-␣, IL-1␤ and IL-6 levels. However, triptolide treatment significantly reduced the levels of TNF-␣, IL-1␤ and IL-6 when compared with vehicle group (p < 0.05). 4. Discussion In the current study, we analyzed the efficacy of triptolide treatment in promoting nerve regeneration and functional recovery after sciatic nerve crush injury in rats. The results showed that triptolide treatment led to better nerve regeneration and faster functional recovery after crush injury during the study period. Additionally, triptolide treatment significantly decreased the levels of pro-inflammatory cytokines at the predefined time point, which might play important roles in the morphological and functional recovery of the sciatic nerve. All these findings indicated the beneficial effect of applying triptolide for PNI therapy.

Recently, increasing data showed that triptolide exerts neuroprotective effects such as protecting dopaminergic neurons from inflammation-mediated damage and promoting nerve regeneration after spinal cord injury [2,4]. In the current study, triptolide exhibited beneficial effects on promoting axonal regeneration and functional recovery. The significantly improved histomorphologic appearance of gastrocnemius muscles in rats with triptolide treatment indicated that more regenerative axons might achieve and reinnervate the gastrocnemius muscles, which might also lead to the significantly higher CMAP amplitude in comparison with that in vehicle group. Additionally, the myelin sheath and mean diameter of regenerative axons in rats with triptolide treatment were superior to those in rats without, which might greatly contribute to the significantly higher NCV in the triptolide group. Furthermore, the motor functional performance assessed by walking track analysis was also significantly higher in rats with triptolide treatment than that in rats without. Wallerian degeneration distal to axonal injury involves clearance of myelin debris and production of a permissive environment for axon regrowth, which is characterized by vigorous inflammatory responses within the injured nerve. Removal of myelin debris is initially carried out by Schwann cells and resident macrophages. Then, an increased number of macrophages and phagocytic neutrophils are recruited to the injured nerve, anticipating in clearance of myelin debris and contributing to nerve regeneration. However, it has to be realized that excessive inflammatory responses may conversely hinder nerve regeneration. Previous studies show that interfering with the development of PNI-induced

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Table 3 Levels of pro-inflammatory cytokines in all groups. Protein levels (pg/mg)

Sham-operative group

Vehicle group

Triptolide group

TNF-␣ IL-1␤ IL-6

23.91 ± 1.75 69.13 ± 5.27 46.74 ± 6.15

64.34 ± 4.08a 161.24 ± 11.91a 97.89 ± 10.04a

49.39 ± 3.83a , b 125.37 ± 8.34a , b 76.31 ± 8.32a , b

a b

p < 0.05 for comparison with sham-operative group. p < 0.05 for comparison with vehicle group.

inflammation favors the morphological and functional recovery process [9]. In the current study, the levels of TNF-␣, IL-1␤ and IL-6 in vehicle group were significantly higher than those in shamoperative group. However, the levels of TNF-␣, IL-1␤ and IL-6 in triptolide group were significantly lower than those in vehicle group. These results indicated that triptolide might attenuate PNIinduced inflammatory responses, accordingly promoting nerve regeneration and functional recovery. It has been reported that triptolide has strong anti-inflammatory activities, which might inhibit excessive inflammatory cytokines produced by non-neuronal cells (such as Schwann cells and macrophages) and thus play essential roles in promoting nerve regeneration and functional recovery. Additionally, injured neurons are suggested to be one of the major sources of inflammatory cytokines in crushed nerves [10,11]. Triptolide might exert its neuroprotective effect on injured neurons and decreased neuronal death, which might contribute largely to modulate inflammatory responses at the injury site. For example, oxidative stress generated after PNI commonly induces apoptosis of neurons. Triptolide is reported to be capable of alleviating oxidative stress, which might protect injured neurons from death [12]. However, this remains to be investigated by additional work. Other mechanisms might be also involved in the improved nerve regeneration by triptolide treatment. For example, triptolide holds the potential to prolong the activation of c-Jun N-terminal kinase (JNK) in vitro [13]. JNK plays an important role in regulating neurite outgrowth during development [14,15], and possesses the ability to promote neurite outgrowth in PC12 cells and axonal regeneration in dorsal root ganglion neurons [16–18]. In the current study, more Fluoro-gold-labeled motoneurons were observed in rats with triptolide treatment than those without. This suggested that triptolide treatment facilitated the survival and neurite outgrowth of neurons, thereby more nerve fibers were generated by survived neurons and successfully achieved the distal stump. In conclusion, the current study demonstrated that triptolide is capable of promoting nerve regeneration and functional recovery, which raises the possibility of using triptolide as a potential neuroprotective agent for PNI therapy. However, the mechanism underlying the beneficial effects of triptolide on peripheral nerve regeneration remains to be elucidated by future work. Additionally, it is unclear whether the protective effect of triptolide on nerve injury is dose-dependent. Also, the optimal dose of triptolide and the duration of use for promoting nerve regeneration and functional recovery have not been identified yet. All these issues still need to be addressed by additional work. Conflicts of interest No conflicts of interest.

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Triptolide improves nerve regeneration and functional recovery following crush injury to rat sciatic nerve.

Recently, accumulating data have demonstrated that triptolide exhibits neurotrophic and neuroprotective properties. However, the role of triptolide in...
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