BYHWD rescues axotomized neurons and promotes functional recovery after spinal cord injury in rats

BYHWD rescues axotomized neurons and promotes functional recovery after spinal cord injury in rats

Journal of Ethnopharmacology 117 (2008) 451–456 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevie...

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Journal of Ethnopharmacology 117 (2008) 451–456

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage:

BYHWD rescues axotomized neurons and promotes functional recovery after spinal cord injury in rats An Chen a,b , Hui Wang a , Jianwei Zhang a , Xiaoqiong Wu a , Jun Liao b , Hua Li a , Weijun Cai a , Xuegang Luo a,∗ , Gong Ju c,∗∗ a b c

Department of Anatomy & Neurobiology, Xiangya School of Medicine, Central South University, Changsha 410013, China Department of Anatomy, Hunan Traditional Chinese Medicine University, Changsha 410007, China Institute of Neurosciences, The Fourth Military Medical University, Xi’an 710032, China

a r t i c l e

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Article history: Received 18 October 2007 Received in revised form 13 February 2008 Accepted 19 February 2008 Available online 4 March 2008 Keywords: Buyang Huanwu Decoction Axotomized neuron Axon regeneration Functional recovery Spinal cord injury

a b s t r a c t Ethnopharmacological relevance: Buyang Huanwu Decoction (BYHWD), a Chinese prescription that has been used for hundreds of years to treat paralysis, has gained attention recently due to its significant neuroprotective properties. Aim of the study: This study was to investigate whether BYHWD treatment protected axotomized rubrospinal neurons (RN) following spinal cord injury (SCI) in rats. Materials and methods: Adult rats received a right lateral funiculus transection at the level between C3 and C4, and were either treated with BYHWD or with distilled water (DW) via gastrogavage. Effects on RN viability and atrophy were determined by Nissl staining, axon regeneration was examined by biotinylated dextran amine (BDA) tracing techniques and functional recovery was studied by a test of forelimb usage during spontaneous vertical exploration. Results: RN cell number and mean somal size were 20% and 29% higher, respectively, in BYHWD-treated rats relative to DW-treated rats. There were a small number of BDA-labeled axons in the caudal of injury site in BYHWD-treated rats, whereas no caudal axonal regeneration was detected in DW-treated rats. BYHWD-treated rats used the injured forelimb more often than rats treated with DW. Conclusions: These results indicate that administration of BYHWD following SCI protects injured neurons, promotes regeneration, and enhances functional recovery. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Damage to the mammalian spinal cord often results in permanent functional deficits due to failed axonal regeneration at the injury site and to the atrophy and/or retrograde death of axotomized neurons (Mori et al., 1997; Himes et al., 2001; Profyris et al., 2004). Treatments for promoting axon regeneration would aim to: (1) increase the regenerative potential of severed axons (Plunet et al., 2002; Lu et al., 2004); (2) alter the environment of the central nervous system (CNS) to establish conditions permissive to regeneration (Pearse et al., 2007; Kamei et al., 2007); (3) neutralize the inhibitory effects of the myelin-related inhibitory molecules, including NogoA, MAG, and versican (Li et al., 2004; Nie et al., 2007); and (4) prevent the inhibitory effects of glial scar-related chondroitin sulfate proteoglycans (CSPGs), including NG2, neuro-

∗ Corresponding author. Tel.: +86 731 2650426; fax: +86 731 2650426. ∗∗ Corresponding author. Tel.: +86 29 83374557; fax: +86 29 83246270. E-mail addresses: [email protected] (X. Luo), [email protected] (G. Ju). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.02.029

can, versican, brevican, and phosphacan (Jones et al., 2003; Tan et al., 2006; Cafferty et al., 2007). All of these strategies have been attempted and all have yielded significant regeneration in rodent spinal cord injury (SCI) models. However, translating these experimental therapies to humans is enormously challenging: to date, no human patient has benefited from a regenerative therapy following SCI (Fawcett, 2002), whether due to injury or disease (Bradbury and McMahon, 2006). Due to abundant resources, unique curative properties, and fewer adverse effects, traditional Chinese medicinal herbs have captured the attention of medical professionals and researchers worldwide in recent years. The identification of effective agents from traditional Chinese medicinal herbs, used for hundreds of years to treat neurological symptoms, may enable us to uncover novel treatments for SCI. The red nucleus is a distinct and readily recognizable population of rubrospinal neuron (RN) bodies within the brainstem. The rubrospinal tract (RST) emerges from the red nucleus, crosses over nearly completely (99%), and descends into the dorsolateral aspect of the spinal cord (Brown, 1974; Kwon et al., 2002a). This lateral position allows for selective cutting on one side of the RST


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while leaving most of the remaining cord (and the contralateral RST) intact, thereby allowing unambiguous interpretation of axonal tracing studies (Liu et al., 1999). In the rat, the rubrospinal system is believed to mediate fine motor skills, particularly of the forelimbs (Muir and Whishaw, 2000). Thus, the rat RST transection model is useful for evaluating the ability of treatment to protect axotomized RN, to facilitate the regeneration of rubrospinal axons, and to accelerate the recovery of forelimb function. Buyang Huanwu Decoction (BYHWD), a famous traditional Chinese prescription, has been employed clinically to treat paralysis in China for hundreds of years. Recent studies have reported neuroprotective effects associated with BYHWD, including improved neurological function in an animal model of CNS ischaemic injury (Li et al., 2003; Fan et al., 2006; Cai et al., 2007). BYHWD may also exhibit growth-promoting effects during peripheral nerve regeneration (Cheng et al., 2001). However, the effects of BYHWD in response to SCI have not been reported. In the present study, we employed the rat model of RST transection to determine whether administration of BYHWD promoted functional recovery after SCI. Furthermore, to investigate the possible mechanisms underlying the therapeutic effects of BYHWD, we evaluated the survival and regeneration of axotomized neurons following SCI. 2. Materials and methods 2.1. Preparation of BYHWD According to the original prescription from The Yi Lin Gai Cuo, BYHWD is composed of Radix Astragali (root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao) 120 g, Radix Angelicae Sinensis (root of Angelica sinensis (Oliv.) Diels) 6 g, Radix Paeoniae Rubra (root of Paeonia lactiflora Pall.) 4.5 g, Rhizoma Chuanxiong (root of Ligusticum chuanxiong Hort.) 3 g, Pheretima (Pheretima aspergillum (E. Perrier)) 3 g, Semen Persicae (Prunus persica (L.) Batsch) 3 g, and Flos Carthami (Carthamus tinctorius L.) 3 g. All herbs were purchased from Hunan Province Pharmacy Company (Changsha, China), and were decocted by boiling in water to yield a final concentration of 2 g crude drug/ml. 2.2. Experimental design Forty adult female Sprague-Dawley rats (200–220 g, obtained from the Experimental Animal Center of Xiangya School of Medicine, Central South University) were divided into three groups: (1) an untreated group that was not subjected to SCI (normal, n = 8); (2) a group that was treated with BYHWD following SCI (BYHWD, n = 16); and (3) a group that was treated with distilled water (DW) following SCI (DW, n = 16). The normal animals were used to establish baseline information concerning the size and number of cells within both the left red nucleus and the right red nucleus in the absence of SCI. All the rats in the BYHWD and DW groups received a right lateral funiculus transection at the level between C3 and C4 and were treated with BYHWD (25.6 g/kg per day) or with DW via gastrogavage for 8 weeks. Following treatment with BYHWD or DW, eight rats in each group were sacrificed for RN cell counting and to determine somal size of red nucleus neurons; the remaining eight rats in each group were injected with biotinylated dextran amine (BDA) in the left red nucleus 15 days prior to sacrificing them at 8 weeks post-SCI, and BDA anterograde tracing studies were performed. Behavioral studies were performed using all of the BYHWD- and DW-treated animals (n = 16) before surgery to induce SCI, during treatment, and prior to sacrificing the animals after 8 weeks of treatment. All the experiments were carried out in accor-

dance with the National Institute of Health Criteria for the Care of Laboratory Animals. 2.3. Surgical procedures Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 ml/kg body weight). Under an operating microscope, the yellow ligament between C3 and C4 was transected to expose the spinal cord segment. After identification of the dorsal root entry zone and the midline of the spinal cord, a small incision was made through the dura mater, and the right dorsolateral funiculus of the spinal cord was transected using a sharp knife. Such a lesion completely transected the lateral funiculus (containing the RST) and partially lesioned the ipsilateral ventral funiculus and gray matter, but left the dorsal columns intact. The dura was then closed with interrupted 10-O silk sutures, and the muscle and skin were closed in layers. After the surgery, animals were maintained on heating pads, observed closely until waking, and then returned to their home cages. 2.4. Anterograde tracing of the RST with BDA Labeling of the RST was achieved by injecting BDA (Molecular Probes) into the left red nucleus. Fifteen days prior to sacrifice, rats were anesthetized and placed into a stereotaxic apparatus, using Bregma as the zero point, anterior–posterior (AP) 5.8 mm; medial–lateral (ML) 0.7 mm; and dorsal–ventral (DV, from dural surface) 7.0 mm (Tobias et al., 2003), and 0.5 ␮l of 10% BDA was injected slowly over 2–3 min using a 1 ␮l Hamilton syringe after a burr hole was made in the skull using a dental drill. After injection, the needle was left in place for an additional 15 min to allow for diffusion of the BDA and was gradually withdrawn over 2–3 min. 2.5. Behavioral testing The use of forelimbs during spontaneous vertical exploration was examined as described (Liu et al., 1999; Yick et al., 2004); this test is highly sensitive for determining the asymmetrical use of forelimbs, and repeated testing does not influence the asymmetry score because weight-shifting movements initiated by the forelimbs are typically used by the animal in its home cage. Briefly, rats were placed in a transparent glass cylinder (18 cm in diameter and 24 cm high) for 5 min. The cylinder was used to encourage the use of forelimbs for vertical exploration. The following behaviors were scored: independent use of the left (unimpaired) or right (impaired) forelimb for contacting the wall of the cylinder, and simultaneous use of both forelimbs to contact the wall of the cylinder. The data were presented as the percent use of the contralateral forelimb (left, unimpaired), the ipsilateral forelimb (right, impaired), and both forelimbs, relative to the total number of forelimb usages. The assessments were made by two researchers who were blinded to the treatments. 2.6. Tissue preparation Animals were deeply anesthetized with an overdose of 10% chloral hydrate, perfused through the heart with 0.9% saline solution (200 ml), followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 at 4 ◦ C (500 ml). The midbrain plus cervical and thoracic segments of the spinal cord were dissected out and post-fixed at 4 ◦ C for 2 h, followed by cryoprotection in a graded series of phosphate-buffered sucrose solutions (10–30%). Spinal cord and brain tissue were embedded in OCT compound, and 30 ␮m sections were cut using a cryostat.

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Table 1 Neuronal cell counts and mean soma size in the left (injured) red nucleus and right (uninjured) red nucleus (cresyl violet staining, mean ± S.D., n = 8 animals for each group) Mean cell size (␮m2 )


Neuron counts Right


Ratio (L/R)



Ratio (L/R)


1338 ± 56 1380 ± 58 1360 ± 43

1350 ± 64 1076 ± 50 788 ± 60

1.01 ± 0.02 0.78 ± 0.05* 0.58 ± 0.04* , #

412 ± 21 408 ± 19 415 ± 20

410 ± 18 265 ± 36 149 ± 28

1.00 ± 0.01 0.65 ± 0.02* 0.36 ± 0.02* , #

* #

p < 0.01 compared with Normal. p < 0.01 compared with BYHWD.

2.7. Detection of BDA-labeled fibers For BDA microscopic processing, rostral sections of C2 and caudal sections of C6 were stained using an avidin–biotin–peroxidase complex kit (ABC Kit, Vector) and visualized using nickelintensified diaminobenzidine (Kuchler et al., 2002). Briefly, free-floating sections were rinsed three times for 30 min each in TBST (50 mM Tris-buffered saline, pH 7.4), followed by overnight incubation with the ABC complex at room temperature. On the

second day, after three 30 min rinses in TBST, the sections were visualized with nickel-intensified diaminobenzidine (Sigma) for 10–15 min. Following TBST rinses, the sections were mounted on gelatin-coated slides for light microscopic examination. 2.8. Cell counting and somal size analysis of the red nucleus For analysis of total cell number and somal size of red nucleus neurons on both sides, a series of midbrain sections (40–45) was

Fig. 1. Photomicrographs showing RN neurons with Nissl staining. (A and B) Normal group. (C and D) BYHWD group. (E and F) DW group. Panels A, C and E are from the right or control side. Panels B, D and F are from the left or injury side. (All sections are approximately 210 ␮m from the caudal pole. Bar: 50 ␮m).


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mounted onto gelatin-coated slides in sequence and stained using cresyl violet (Sigma). Images were captured using a Motic brand microscope equipped with a video camera system (Motic, Xiamen, China). The number of cresyl violet-stained red nucleus neurons were counted in every third serial section throughout the rostrocaudal extent of the red nucleus, only those cells with identifiable nuclei, nucleoli, and characteristic neuronal morphology were counted. The cross-sectional area of neurons was measured using the Motic Images Advanced 3.2 software application.

of 1076 RN remaining on the injured side, which represented 78% of the cell number found on the uninjured side and corresponded to a 22% RN cell loss on the injured side (Table 1, Fig. 1C and D). In contrast, control animals treated with DW showed an average of 788 neurons remaining in the injured red nucleus, which represented 58% of the cell number found on the uninjured side and corresponded to a 42% RN cell loss on the injured side (Table 1, Fig. 1E and F). Therefore, BYHWD treatment reduced the loss of RN from 42% to 22%, and led to a 20% rescue of RN following SCI.

2.9. Statistical analysis

3.2. Effects of BYHWD on RN atrophy

The data corresponding to cell numbers, cross-sectional areas of cell soma, numbers of BDA-labeled fibers, and the percentage of forelimb usage were expressed as mean ± S.D. A one-way ANOVA followed by a Student–Newman–Keuls test was used for statistical evaluation. Statistically significant differences were considered at p < 0.05.

The cross-sectional areas of RN cell soma were measured and compared to determine whether BYHWD prevented atrophy following axotomy. In normal control animals, the left and right mean soma size was 410 ␮m2 , with a left/right ratio of 1.0 (Table 1, Fig. 1A and B). Eight weeks after SCI, the mean soma size in animals treated with BYHWD was 265 ␮m2 on the injured side and 408 ␮m2 on the uninjured side; the left/right ratio was 0.65, indicating a 35% decrease in mean soma size (Table 1, Fig. 1C and D). In animals treated with DW, the mean soma size was 149 ␮m2 on the injured side and 415 ␮m2 on the uninjured side; the left/right ratio was reduced to 0.36, indicating a 64% decrease in mean soma size (Table 1, Fig. 1E and F). Therefore, BYHWD treatment led to a 29% decrease in atrophy in surviving RN.

3. Results 3.1. Effects of BYHWD on RN survival We studied the effects of BYHWD on the survival of axotomized RN by comparing the number of neurons remaining in the injured red nucleus to the number of neurons in the uninjured red nucleus. In normal control animals, the left red nucleus and the right red nucleus contained roughly equal numbers of neurons: there were approximately 1300 neurons on each side and the left/right ratio was approximately 1.0 (Table 1, Fig. 1A and B). Eight weeks after SCI, cell counts in animals treated with BYHWD showed an average

3.3. Effects of BYHWD on regeneration of RN The effect of BYHWD on the regeneration of axotomized RN was determined using the BDA anterograde tracing technique. Cross

Fig. 2. Photomicrographs of spinal cord sections showing BDA-labeled RST axons (arrows). (A and B) BYHWD group. (C and D) DW group. Panels A and C are from the rostral segments of C2. Panels B and D are from the caudal segments of C6. A1, B1, C1, and D1 (Bar: 100 ␮m) are high magnification images of A, B, C and D (Bar: 200 ␮m); asterisks indicate the same points as reference.

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caudal injury; in contrast, no axon regeneration was detected in the caudal region of DW-treated control mice. Finally, rats treated with BYHWD after SCI exhibited significantly enhanced functional recovery of injured forelimb use in a spontaneous vertical exploration behavioral test; this effect may be partially mediated by the RST regeneration. 4.1. Effects of BYHWD on the axotomized RN

Fig. 3. Behavioral test of forelimb use during spontaneous vertical exploration. Normal (before SCI) rats used a single or both forelimbs to explore the wall of cylinder, but they did not use the right/injured forelimb after SCI. Eight weeks after SCI, rats treated with BYHWD used their right forelimbs to explore the wall of cylinder, whereas rats treated with DW rarely did so (* and #, p < 0.01 compared with DW).

sections were used to visualize the presence of BDA-labeled fibers in the rostral segments of C2 and in the caudal segments of C6. In rostral sections of C2 (Fig. 2A), the number of BDA-labeled RST axons was 78 ± 16 in BYHWD-treated animals, and 43 ± 12 in DW-treated animals (Fig. 2C). There was a statistically significant difference between these two groups (p < 0.01). In caudal sections of C6, a small number of BDA-labeled RST axons were seen in animals treated with BYHWD (Fig. 2B), whereas no BDA-labeled RST axons were detected in caudal sections of C6 in DW-treated animals (Fig. 2D).

SCI causes disruption of descending pathways from the brainstem and cortex and initiates a neuronal response that can lead to atrophy or death of the injured cells (Himes et al., 1994; Giehl and Tetzlaff, 1996; Buffo et al., 1998). Transection of RST at C3–C4 in the rat model used in this study resulted in reduced RN cell number (42%) and somal atrophy (64%) in comparison to normal rats not subjected to SCI. These results are similar to previous reports of SCI on RST neurons (Mori et al., 1997; Liu et al., 2002; Zhang et al., 2003). The application of neural tissue transplants (Bregman and Reier, 1986; Bernstein-Goral and Bregman, 1997; Xiao et al., 2005) or neurotrophic factors (Kobayashi et al., 1997; Liu et al., 2002; Tobias et al., 2003; Kwon et al., 2007) has been shown to prevent some axotomized RN cell atrophy. In the current study, we demonstrate that BYHWD treatment after SCI led to reduced RN cell loss (22%), and decreased somal atrophy (35%) when compared to normal rats not subjected to SCI. Therefore, BYHWD treatment provided a 20% increase in RN cell number, and a 29% decrease in RN somal atrophy following SCI in this rat model. Taken together, these data provide evidence that BYHWD treatment can rescue axotomized RN from death and prevent RN atrophy. 4.2. Effects of BYHWD on functional recovery and RST axonal regeneration

3.4. Effects of BYHWD on functional recovery When placed in a cylinder, normal rats spontaneously reared and explored the wall of the cylinder using a single forelimb alone (50%) or using both forelimbs together (50%) (Fig. 3). Rats receiving the right lateral funiculus dissection at C3–C4 rarely used the forelimb ipsilateral to the injury to explore the cylinder wall, and prominent deficits were observed in elbow and digit extension of the right (injured) forelimb. At 1 week after SCI, rats treated with either BYHWD or DW used only the left (uninjured) forelimb to explore. At 2 weeks after SCI, rats treated with BYHWD began to use both forelimbs together to explore (5.3%), and after 3 weeks they began to use the right (injured) forelimb alone to explore (3.2%). Rats treated with DW began to use both forelimbs together at 4 weeks after SCI (4.8%), and at 6 weeks after surgery they began to use the right forelimb alone (2.5%). By 8 weeks, rats treated with BYHWD used the right forelimb alone 13% of the time, and their posture was closer to normal (Fig. 3). In contrast, rats treated with DW used the right forelimb alone only 3% of the time, and their posture remained abnormal in forepaw flexion at 8 weeks after SCI (Fig. 3). 4. Discussion In the present study, we report that BYHWD treatment led to enhanced regeneration of the RST in rats with transection of the right dorsolateral funiculus of the cervical spinal cord. In rats treated with BYHWD following SCI, RN cell number and mean somal size were 20% and 29% higher, respectively, compared to DW-treated control mice. Administration of BYHWD also led to enhanced regeneration of the RST as determined by the presence of a small number of axons that had regenerated to the site of the

In addition to the effects on RN survival and atrophy, BYHWD treatment also increased the functional recovery of injured forelimbs following SCI. At 8 weeks after SCI, the BYHWD-treated animals displayed better locomotor activity during spontaneous vertical exploration and significant improvements in forelimb usage ipsilateral to the lesion when exploring the wall of a cylinder. Importantly, the enhanced functions were consistent with RST axonal regeneration, and suggested that the increased function may be attributed at least partially to repair of the RN and RST pathway. Further support for this comes from studies of BDA anterograde tracing of RST axons, which showed axonal regeneration to the caudal injury site only in BYHWD-treated animals. It has been proposed that injured neurons in the CNS of adult mammals cannot regenerate spontaneously. However, these neurons have an intrinsic potential for axonal regeneration following injury, provided that they are supplied with a suitable environment. Many strategies have been reported to promote the axonal regeneration of injured RST. In a model of chronic SCI, continuous administration of BDNF at the midbrain reversed massive atrophy and promoted regeneration of rubrospinal axons into a peripheral nerve implanted at the lesion site (Kwon et al., 2002b). Following partial hemisection of the cervical spinal cord, transplants of neural progenitors obtained from adult human olfactory epithelium following engraftment into host spinal cord maintained axotomized RN, promoted RST axonal regeneration, and led to enhanced function (Xiao et al., 2005, 2007). Adult rats that received a unilateral hemisection at the seventh cervical spinal cord segment, followed by 4 weeks of a combined treatment of intraperitoneal injection of LiCl together with ChABC at the lesion site significantly increased the regeneration of RST axons and enhanced the recovery of forelimb function (Yick et al., 2004).


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