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Temporal changes in the expression of some neurotrophins in spinal cord transected adult rats
Neuropeptides Neuropeptides 41 (2007) 135–143 www.elsevier.com/locate/npep
Temporal changes in the expression of some neurotrophins in spinal cord transected adult rats Xiao-Li Li a, Wei Zhang b, Xue Zhou b, Xu-Yang Wang a, Hong-Tian Zhang a, Dan-Xia Qin a, Han Zhang a, Qun Li a, Min Li a, Ting-Hua Wang a,b,* b
a Institute of Neuroscience, Kunming Medical College, Kunming 650031, China Department of Histology, Embryology and Neurobiology, College of Preclinical Forensic Medicine, Sichuan University, Chengdu 610065, China
Received 20 July 2006; accepted 15 February 2007 Available online 24 April 2007
Abstract Functional recovery of neurons in the spinal cord after physical injury is essentially abortive in clinical cases. As neurotrophins had been reported to be responsible, at least partially, for the lesion-induced recovery of spinal cord, it is not surprising that they have become the focus of numerous studies. Studies on endogenous neurotrophins, especially the three more important ones, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in injured spinal cord might provide some important clues in clinical treatment. Here we investigate the immunohistological expression of the above three factors at lower thoracic levels of the spinal cord as well as changes in the motor functions of the adult rat hindlimbs after cord transection. The injured rats were allowed to survive 3, 7, 14 and 21 days post operation (dpo). Flaccid paralysis was seen at 3 dpo following cord transection, however, hindlimb function showed partial recovery from 7 dpo to 21 dpo. The numbers of NGF, BDNF and NT-3 immunopositive neurons and their optical densities all increased in the lesion-induced cord. The immuno-expression of NGF and BDNF peaked at 7 dpo, while that of NT-3 peaked at 7 dpo and remained so at least up to 14 dpo. These results suggested that neurotrophins might play essential roles in functional recovery of after spinal cord injury, but the time points for the expression of the three factors differed somewhat. 2007 Elsevier Ltd. All rights reserved. Keywords: NGF; BDNF; NT-3; Expression; Transection; Immunohistochemistry; Rat
1. Introduction Spinal cord injuries in the form of crush injury, contusion, hemisection, and transection are commonly encountered in clinical practice. Of these spinal cord transection, which has the high incidence and mortality. Recovery, should there be any, is usually less than satisfactory. This prompted numerous studies to seek effective measures to * Corresponding author. Address: Institute of Neuroscience, Kunming Medical College, Kunming 650031, China. Tel.: +86 871 5329245; fax: +86 871 5316883. E-mail address: [email protected] (T.-H. Wang).
0143-4179/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.02.001
improve motor functions after spinal cord injury. Among these, the administration of neurotrophins appeared to offer some promises (Bregman et al., 1997; Namiki et al., 2000; Zhou and Shine, 2003; Scott et al., 2005). Neurotrophins are a group of small molecule polypeptides involved in both the development and maintenance of neurons, as well as non-neuronal cells (Seki et al., 2002; Jones et al., 2003; Gulino et al., 2004; Lambert et al., 2004; Feng et al., 2005). Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) as well as neurotrophin-3 (NT-3) are three important members of the neurotrophin family. They most probably play a crucial role in the lesion-induced regeneration
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of the spinal cord (Sariola et al., 1994; Jakeman et al., 1998; Markus et al., 2002). Although exogenous neurotrophins have been shown to rescue injured neurons in spinal cord, it is still not clear when and how much of exogenous neurotrophins need to be administered to promote clinically significant recovery. Before this, it is important to trace changes in the endogenous neurotrophins in the injured spinal cord. In the present study, we investigated the changes in the expression of NGF, BDNF, and NT-3 in spinal cord of adult rats after spinal cord transections. Hindlimb motor functions were also observed during their survival period. As it was the hindlimbs that were most affected after cord transection, this study was designed to focus on the caudal part of injured spinal cord and hindlimb motor functions.
2. Materials and methods 2.1. Animals and experimental procedures Thirty-seven young adult female Sprague–Dawley (SD) rats weighing 180–220 g, obtained from the Experimental Animal Center of Kunming Medical College were used in this study. They were individually housed in a 12/12 h light/dark, quiet and non-strong-light vivarium with free access to water and food. Two of them were used for Western Blot analysis. Seven of them, in the sham-operated group, were subjected to laminectomy without any injury to the spinal cord. The others were subjected to spinal cord transection. Prior to operation, the animals were anaesthetized by intraperitoneal injection of 3.6% chloral hydrate (CCl3CH(OH)2) (0.36 g/kg). A 2 cm incision was made on the skin of the back, centering at T10 vertebral level. The supraspinal ligaments were removed, and the spinous processes of T9 and T10 were clipped off with a pair of strong artery forceps, exposing the ligamentum flavum and about 1 cm of the spinal cord. A small piece of cord tissue measuring about 1 mm thickness, in the middle of the exposed spinal cord, was removed with a pair of iris scissors. The wound was then closed with sutures. All the operated animals were allowed to recover spontaneously, without the administration of any drugs. They were divided into four experimental groups (seven in each group) according to their survival periods (Table 1). 2.2. Assessment of motor functions We used the method of BBB Locomotor Rating Scale (Basso et al., 1995) for the post surgical analysis of the animal’s hindlimb motor functions, including frequency and quality of hindlimb movement as well as forelimb/ hindlimb coordination. The values of BBB Locomotor
Table 1 The survival periods post operation and the number of experimental animals used Survival periods (dpo) Number of animals
3 7
7 7
14 7
21 7
dpo: Days post operation.
Rating Scale for evaluation of hindlimb function ranged from 0, which corresponds to flaccid paralysis, to 21, which is normal gait. Rats were allowed to walk around freely in a spacious field for about 4 min while movements of the hindlimbs were closely observed. As the grading could be biased by subjectivity, three colleagues of ours who had no knowledge of the operative procedure and survival time were requested to assess the functions of both hindlimbs in all the operated and normal animals. The scores of these three were equated to get the average. All the behavior evaluations were performed at 8–9 am at various periods after operation. 2.3. Characterization of antibodies The specificities of the three antibodies against NGF, BDNF and NT-3 (received as gifts from Professor Xin-Fu Zhou, the Department of Human Physiology and Centre for Neuroscience, Flinders University of South Australia) (Deng et al., 2000; Zhou et al., 2000) were confirmed by Western Blot analysis, using spinal cord homogenates of rat in our lab. The polyclonal neurotrophin antisera for NGF, BDNF, and NT-3 were specific for the appropriate neurotrophins and no cross-reaction with other neurotrophins was detected. As little as 10 ng of each neurotrophin could be visualized, using these antisera. Control of immunostaining specificity was performed by omitting the primary antibody and antibodies pre-adsorbed with the appropriate neurotrophins. These controls did not exhibit any specific immunostaining. 2.4. Tissue preparation For immunohistochemistry, the animals were perfused through intracardiac administration of 250 ml of isotonic saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS), at 4 C. After perfusion, the vertebral canal in each animal was exposed to dislodge the spinal cord, which was then immersed in 4% paraformaldehyde at 4 C for further fixation. For Western Blot analysis, the vertebral canals of the two normal animals were anesthetised and rapidly opened. About 1 cm segment of spinal cord centering T10 vertebral level was obtained. The removal of spinal pia mater was performed on ice. All these samples
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freshly obtained were stored at 80 C until use for the detection of neurotrophins. 2.5. Western Blot The procedure of Western Blot used in this study has been introduced previously (Zhang et al., 2007). Briefly, the frozen spinal cord samples of the rats were homogenized on ice in 10 mM Tris–HCl buffer (pH 7.4), 10 mM EDTA, 30% Triton-1000, 10% SDS and NaCl using a homogenizer. Homogenates were centrifuged at 12,000g for 30 min at 4 C. The supernatant was phase collected in aliquots and stored at 80 C. The protein concentration was assayed with BCA reagent (Sigma, USA). Samples were loaded, electrophoresed on 5% stacking gel at 80 V and 15% resolving gel at 120 V. After electrophoresis, the gels were transferred to nitrocellulose membranes (Schleicher and Schuell, USA) for 6 h at 16 V. To reduced background staining, the filters were incubated with 5% non-fat dry milk in Tris buffered saline (TBS) containing 0.05% Tween 20 (TBST) overnight at 4 C, sequentially incubated with primary antisera (NGF 1:400, BDNF 1:200, and NT-3 1:200) in the same additive at 4 C, the membrane was rinsed in TBST before incubation in the peroxidase conjugated goat anti-rabbit IgG for 1 h at room temperature. The membrane was developed in ECL kit (Amersham, USA) and exposed against X-ray film in dark room. 2.6. Immunohistochemistry On the night before sectioning, about 1.5 cm segment of the spinal cord below the site of transection was obtained and placed in 20% sucrose solution in 0.1 M phosphate buffer (PB). After the specimen had sunken to the bottom of the bottle, it was placed on a freezing microtome (Leica CM1900, Germany) and horizontal sections were cut at 20 lm thickness. For accurate representation of the data, five sections that are designated as 10th, 20th, 30th, 40th, and 50th of each animal were processed for immunohistochemistry. Briefly, after washing three times (5 min each) in 0.1 M PBS, the sections were incubated free-floating in 3% hydrogen peroxide for 30 min, and soaked in 5% normal goat serum containing 0.3% Triton X-100 for 30 min at 37 C. They were then transferred to a solution of the respective primary antibodies for 48 h at 4 C (polyclonal neurotrophin antisera for NGF, BDNF, or NT-3, diluted 1:1000) containing 2% normal goat serum and 0.3% Triton X-100. After immersion in the primary antibodies or PBS, the sections were incubated in Reagents I and II from the Reagent Kit (Chemicon, Anti-Rabbit/Mouse Poly-HRP IHC Detection Kit, USA), each for 30 min at 37 C. This was followed by three times of washing, each 5 min in 0.1 M PBS. The immunoreactive products were visual-
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ized by placing the sections in a staining solution containing 0.05% 3,3 0 -diaminobenzidine, 0.1% nickel sulphate and 0.01% hydrogen peroxide for 10 min. Sections were observed in a light microscope (Leica DMI 6000 B inverted microscope, Germany) coupled with a computer assisted video camera (Leica, Germany). 2.7. Morphological analysis In order to ensure consistency in the analysis, all the sections obtained for immunohistochemistry were stained under the same conditions. Two individuals, who had no knowledge of the experiment, examined the sections separately. The Leica QWin image analysis software (Leica Qwin, Germany) was used to count the number of NGF, BDNF and NT-3 immunoreactive (IR) neurons in spinal laminae VIII–IX and I–VII on both sides of each section. Only clearly stained immunopositive profiles were included. Briefly, the number of positive neurons was counted and the area of lamina I–VII and VIII–IX were measured at a magnification of 100·. All the number were calculated in an equal area of (2812.5 lm2) to enable the investigators to compare the number of cells in different sections. In order to analyze intensity of immunoreactive staining, we measured the optical density (OD) of the immunopositive neuronal cell bodies, outlined manually by the experimenter from grey scale images (400· magnification). The method has been detailed in a former study (Gulino et al., 2004). All the processed sections of each animal were used to measure the average grey scale of NGF-, BDNF- and NT-3 IR products manually of both sides in laminae VIII–IX and I–VII. 2.8. Statistical analysis We used the SPSS 12.0 statistical analysis software package to analyze the data. Mean and standard error of the mean (SEM) of positively stained neurons and intra-neuron OD, including the transected and shamoperated control groups were evaluated. One-way AVOVA and Least Significant Difference were adopted in this study.
3. Results 3.1. Specificity of antisera Using the rat homogenates, the antibodies specifically recognized appropriate bands at a molecular weight of approximately 14.7 kDa, 14.2 kDa and 13.6 kDa, for NGF, BDNF, and NT-3 respectively (Fig. 1). These positions coincided with the molecular weights of the neurotrophins studied (Catania et al., 2007; Zhang et al., 2007).
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ber of NGF and BDNF peaked at 7 dpo, while that of NT-3 peaked at 7 dpo and remained so up to 14 dpo (Table 2). 3.4. Changes in intracellular optical density (OD)
Fig. 1. The specificities of NGF (B), BDNF (C), and NT-3 (D) were analysed by Western Bolt analysis (A) showed the position of b-actin at 43 kDa approximately.
After cord transection, intracellular OD values of the three neurotrophins in the spinal cord neurons showed different degrees of increases at 3, 7, 14 and 21 dpo, similar to the changes in the numbers of immunopositive neurons. Interestingly, the OD values of NGF and BDNF IR neurons peaked at 7 dpo and those of NT-3 IR neurons, at 14 dpo (Fig. 4). Though lower thereafter, those of NGF and NT-3 IR neurons were still higher than those in the control rats, while those of BDNF IR neurons declined to control value at 14 and 21 dpo.
3.2. BBB scores in control and operated animals 4. Discussion In the control group, each rat had a BBB score of 21. The mean of BBB scores for the cord transected rats was recorded in Fig. 2. The score was 0 at 3 dpo (p < 0.05). It was increased (p < 0.05) at 7 dpo, further increased (p < 0.05) at 14 dpo, and then plateaued up till at least 21 dpo. 3.3. NGF, BDNF and NT-3 immunostaining and numbers of stained profiles The immunohistochemical products of NGF, BDNF and NT-3 were seen in the neurons located both in laminae VIII–IX and I–VII of the spinal cord. Both the cytoplasm and the nucleus of NGF and NT-3 IR neurons displayed intense staining, with the former showing greater intensity, while BDNF positive neurons displayed mainly cytoplamic staining (Fig. 3). The numbers of NGF, BDNF and NT-3 immunopositive neurons increased in the lesion-induced cord. Such increase was, however, transient for BDNF. The num-
4.1. Specificity of antibodies Analysis by Western Blot showed that each antibody recognized specific domains of the neurotrophin proteins NGF, BDNF and NT-3 respectively in homogenates of the spinal cord of adult SD rats. Immunohistochemical controls demonstrated no immunostaining. These showed that the antibodies were highly specific and could recognize native proteins in adult rat CNS. 4.2. Recovery of locomotor function After cord transection, the hindlimbs of the rats showed flaccid paralysis, indicating that motoneuron activity was dramatically reduced. There was, however, gradual restoration of hindlimb functions from 7 dpo to 21 dpo. The scores of BBB Locomotor Rating Scale increased, beginning at 7 dpo, further increased at 14 dpo, and remained so up to at least 21 dpo. The results indicated spontaneous, howbeit incomplete, recovery of the rats’ hindlimbs after spinal cord transection. 4.3. Neurotrophins expression in motoneurons
Fig. 2. Mean values of BBB scores in cord transected rats. *p < 0.05, compared with the values of sham-operated control group. #p < 0.05, compared with the values of its former group. The error bars represented the standard error of the mean (SEM). dpo: Days post operation.
The expression and up-regulation of NGF, BDNF and NT-3 in the spinal neurons indicated that these neurotrophins could play essential roles in neuroplasticity following cord transection. Numerous studies have demonstrated that neurotrophins were involved in both physiological functions and pathological condition (Lindsay et al., 1985; Alderson et al., 1990; Risling et al., 1992; Sariola et al., 1994; Novikova et al., 1996; Vischer, 1997; Zhou and Shine, 2003; Gulino et al., 2004; Gurok et al., 2004; Pinzon-Duarte et al., 2004). The up-regulation of neurotrophin expression could
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Fig. 3. Note the expression of NGF-IR (A), BDNF-IR (B) and NT-3-IR (C) neurons in the spinal cord of a sham-operated rat. The OD values of NGF-immunoreactive (IR) neurons peaked at 7 dpo (D), BDNF at 7 dpo (E), and NT-3 at 14 dpo (F). BDNF-IR neurons in laminae VIII–IX are shown in (B) and (E), and NT-3-IR neurons in (C, F). Control cord sections with PBS replacing the primary antibodies are shown in (G), (H), and (I), for NGF, BDNF, and NT-3 antibodies respectively. Sections subjected to pre-adsorption blocking controls for NGF, BDNF and NT-3 are shown in (J), (K), and (L) respectively. Arrows indicated neurotrophins-IR motoneurons in laminae VIII–IX. Bars in (A), (D), (G), (I), (J), and (L) = 100 lm; bars in (B), (C), (E), (F), (H), and (K) = 50 lm.
underline the observed functional plasticity. In the present study, the BBB scores in cord transected rats showed a gradual increase from 7 dpo to 14 dpo, through 21 dpo. The expression of NGF peaked at 7 dpo, thereafter declined but still appeared higher than that of controls at 21 dpo. Increased NGF at the caudal part of the lesion site might in some way contribute to the recovery
of the injured motoneurons. Previous studies showed that NGF could prevent the death of motoneurons after injury both in newborn and adult rats (Diener and Bregman, 1994; Murakami et al., 2002). Also, NGF receptor mRNA increased in adult motoneurons after axonal injury, indicating the protective role of NGF (Koliatsos et al., 1991).
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Table 2 Numbers of immunopositive neurons in Laminae (Lam) VIII–IX and I–VII in control and rats sacrificed at different times after transection NGF IR neurons
Control 3 dpo 7 dpo 14 dpo 21 dpo
BDNF IR neurons
NT-3 IR neurons
Lam VIII–IX
Lam I–VII
Lam VIII–IX
Lam I–VII
Lam I–VII
Lam VIII–IX
45.53 ± 2.83 65.01 ± 2.11* 83.41 ± 4.32* 75.40 ± 2.51* 71.02 ± 6.13*
14.53 ± 3.00 27.66 ± 2.18* 41.94 ± 5.63* 37.40 ± 4.48* 26.89 ± 2.22*
73.64 ± 5.76 86.70 ± 7.02* 120.70 ± 8.95* 71.21 ± 9.23 69.35 ± 9.85
30.20 ± 9.40 66.42 ± 5.34* 87.36 ± 8.54* 33.56 ± 5.62 29.69 ± 6.35
60.50 ± 7.21 86.23 ± 3.34* 97.35 ± 2.56* 78.40 ± 5.91* 74.56 ± 3.22*
22.54 ± 3.28 40.12 ± 2.48* 60.45 ± 9.12* 35.55 ± 4.26* 30.46 ± 3.45*
Numbers refer to mean numbers (M) per equal area (2812.5 lm2) ± standard error of mean (SEM). dpo: Days post operation. * p < 0.05, compared with normal control ones.
Fig. 4. (A–C) Changes in the optical densities of NGF, BDNF and NT-3 IR cells in different groups. The mean optical density of every neurotrophin in the sham-operated control group was arbitrarily considered as 100%. *p < 0.05, compared with sham-operated rats. The error bars represented the standard error of the mean (SEM). dpo: Days post operation.
The expression of BDNF increased and peaked at 7 dpo but returned to control level at 14 dpo. Although the increase of BDNF expression was transient, it could also in some way play an important role in the functional plasticity of injured motoneurons. That its role could not be ignored during recovery of injured spinal cord is also emphasized by several authors (Diener and Bregman, 1994; Namiki et al., 2000; Blits et al., 2003; Zvarova et al., 2004). In addition, BDNF has been
known to be involved in synaptic plasticity (McAllister et al., 1999; Gomez-Pinilla et al., 2002), neuroprotection and sprouting of spared pathways after injury (Henderson et al., 1993; Jakeman et al., 1998). The expression of NT-3 after cord transection peaked at 7th and 14th dpo. This indicated NT-3 could also exert its effects on spinal cord plasticity after transection. It is well known that NT-3 plays an important role in supporting the development and survival of motoneu-
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rons (Yan et al., 1993). It could also regulate the excitability of spinal cord cells and motoneuron spindle physiology (Mendell et al., 1999) as well as spinal cord activity-dependent plasticity (Gomez-Pinilla et al., 2001, 2004). Neuromuscular activity has specific effects on the BDNF and NT-3 systems (Gomez-Pinilla et al., 2001). Evidences indicated that NT-3 might be of benefit in preventing the secondary cell loss that occurs following spinal injury (Bradbury et al., 1998). Additionally, the up-regulation of NGF and NT-3 lasted longer than that of BDNF, which indicated that the three proteins might play different roles at different times after spinal cord injury. 4.4. Neurotrophins expression in sensory neurons In the present study, the expression of NGF and BDNF in laminae I–VII peaked at 7 dpo, while that of NT-3 at 7 dpo and 14 dpo. These suggested that the three neurotrophins might affect not only motoneurons but also sensory neurons following cord transection. Although NGF had been shown to be crucial for the development of sensory neurons (Diener and Bregman, 1994), recent studies indicated such dependency declined with the animal’s growth. The main function of NGF in adult animals may be related to nociception (Lewin and Mendell, 1993; Krenz and Weaver, 2000), and some studies have shown that NGF may regulate pain sensation in the human (Anand, 1995). Correlated with this observation is the finding that NGF stimulates sprouting of small-diameter afferent fibers and its concentration increases after spinal cord injury (Anand, 1995; Krenz and Weaver, 2000). Thus the increased expression of NGF in spinal sensory neurons in this study may not only be able to promote the survival of sensory neurons, but also could be responsible for the development of central pain. Former study has shown that BDNF and NT-3 could promote the outgrowth of sensory neurites in vitro (Blits et al., 2003). Besides contributing to the survival and recovery of injured sensory neurons, BDNF might also participate in the transmission of central pain with NGF after spinal cord injury (Pezet and McMahon, 2006). NT-3 was said to be important in the development and maintenance of sensory neurons (Zhou and Rush, 1995). Previous reports showed that following spinal cord hemisection both in newborn and adult rats, administration of exogenous NT-3, but not NGF or BDNF, rescued neurons in nucleus dorsalis (Diener and Bregman, 1994; Shibayama et al., 1998; Novikova et al., 2000). Himes et al. (2001) reported that NT-3 could be of benefit in the long-time survival of nucleus dorsalis. In the present study, the increased expression of BDNF and NT-3 might be crucial for the recovery of injured spinal neurons. Further work is being done to explore the mechanism of action.
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4.5. Use of female rats Young adult female rats were chosen for this study because they were easier to feed and less prone to urinary tract infection than the male rats when subjected to spinal cord transection. Female rats also had a better survival rate after surgery. The benefits of choosing female rats for studies of spinal cord injury have been discussed previously (Talmadge et al., 2002). As we were not aware of any work done to study the influence of estrous cycle to neurotrophins expressed in spinal cord, we did not observe the estrous cycle of the rats. In conclusion, based on the increase in the number of neurotrophic positive neurons and in the intracellular OD values as well as the results of behavior test throughout the experimental period, we conjectured that the partial recovery of functions of the rat hindlimbs could at least partly be related to the increased expression of NGF, BDNF and NT-3. Moreover, the numbers of NGF-positive and NT-3-positive neurons were significantly increased at all post-operative periods in the experimental group, when compared with that in the sham-operated group. The increased expression of BDNF was only transient. Further studies need to be done to explore the exact roles of these three neurotrophins and their different expressions in this model of neuroplasticity.
Acknowledgements We thank Dr. S. K. Leong for his invaluable comments in the writing of this manuscript. This research was supported by a grant from the National Science Foundation (No. 30260125), the New York–China Medical Grant (CMB00-722) and the Development Grants of the Kunming Medical College (Nos. 2005DG03, 2005DG01).
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Temporal changes in the expression of some neurotrophins in spinal cord transected adult rats Xiao-Li Li a, Wei Zhang b, Xue Zhou b, Xu-Yang Wang a, Hong-Tian Zhang a, Dan-Xia Qin a, Han Zhang a, Qun Li a, Min Li a, Ting-Hua Wang a,b,* b
a Institute of Neuroscience, Kunming Medical College, Kunming 650031, China Department of Histology, Embryology and Neurobiology, College of Preclinical Forensic Medicine, Sichuan University, Chengdu 610065, China
Received 20 July 2006; accepted 15 February 2007 Available online 24 April 2007
Abstract Functional recovery of neurons in the spinal cord after physical injury is essentially abortive in clinical cases. As neurotrophins had been reported to be responsible, at least partially, for the lesion-induced recovery of spinal cord, it is not surprising that they have become the focus of numerous studies. Studies on endogenous neurotrophins, especially the three more important ones, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in injured spinal cord might provide some important clues in clinical treatment. Here we investigate the immunohistological expression of the above three factors at lower thoracic levels of the spinal cord as well as changes in the motor functions of the adult rat hindlimbs after cord transection. The injured rats were allowed to survive 3, 7, 14 and 21 days post operation (dpo). Flaccid paralysis was seen at 3 dpo following cord transection, however, hindlimb function showed partial recovery from 7 dpo to 21 dpo. The numbers of NGF, BDNF and NT-3 immunopositive neurons and their optical densities all increased in the lesion-induced cord. The immuno-expression of NGF and BDNF peaked at 7 dpo, while that of NT-3 peaked at 7 dpo and remained so at least up to 14 dpo. These results suggested that neurotrophins might play essential roles in functional recovery of after spinal cord injury, but the time points for the expression of the three factors differed somewhat. 2007 Elsevier Ltd. All rights reserved. Keywords: NGF; BDNF; NT-3; Expression; Transection; Immunohistochemistry; Rat
1. Introduction Spinal cord injuries in the form of crush injury, contusion, hemisection, and transection are commonly encountered in clinical practice. Of these spinal cord transection, which has the high incidence and mortality. Recovery, should there be any, is usually less than satisfactory. This prompted numerous studies to seek effective measures to * Corresponding author. Address: Institute of Neuroscience, Kunming Medical College, Kunming 650031, China. Tel.: +86 871 5329245; fax: +86 871 5316883. E-mail address: [email protected] (T.-H. Wang).
0143-4179/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.02.001
improve motor functions after spinal cord injury. Among these, the administration of neurotrophins appeared to offer some promises (Bregman et al., 1997; Namiki et al., 2000; Zhou and Shine, 2003; Scott et al., 2005). Neurotrophins are a group of small molecule polypeptides involved in both the development and maintenance of neurons, as well as non-neuronal cells (Seki et al., 2002; Jones et al., 2003; Gulino et al., 2004; Lambert et al., 2004; Feng et al., 2005). Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) as well as neurotrophin-3 (NT-3) are three important members of the neurotrophin family. They most probably play a crucial role in the lesion-induced regeneration
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of the spinal cord (Sariola et al., 1994; Jakeman et al., 1998; Markus et al., 2002). Although exogenous neurotrophins have been shown to rescue injured neurons in spinal cord, it is still not clear when and how much of exogenous neurotrophins need to be administered to promote clinically significant recovery. Before this, it is important to trace changes in the endogenous neurotrophins in the injured spinal cord. In the present study, we investigated the changes in the expression of NGF, BDNF, and NT-3 in spinal cord of adult rats after spinal cord transections. Hindlimb motor functions were also observed during their survival period. As it was the hindlimbs that were most affected after cord transection, this study was designed to focus on the caudal part of injured spinal cord and hindlimb motor functions.
2. Materials and methods 2.1. Animals and experimental procedures Thirty-seven young adult female Sprague–Dawley (SD) rats weighing 180–220 g, obtained from the Experimental Animal Center of Kunming Medical College were used in this study. They were individually housed in a 12/12 h light/dark, quiet and non-strong-light vivarium with free access to water and food. Two of them were used for Western Blot analysis. Seven of them, in the sham-operated group, were subjected to laminectomy without any injury to the spinal cord. The others were subjected to spinal cord transection. Prior to operation, the animals were anaesthetized by intraperitoneal injection of 3.6% chloral hydrate (CCl3CH(OH)2) (0.36 g/kg). A 2 cm incision was made on the skin of the back, centering at T10 vertebral level. The supraspinal ligaments were removed, and the spinous processes of T9 and T10 were clipped off with a pair of strong artery forceps, exposing the ligamentum flavum and about 1 cm of the spinal cord. A small piece of cord tissue measuring about 1 mm thickness, in the middle of the exposed spinal cord, was removed with a pair of iris scissors. The wound was then closed with sutures. All the operated animals were allowed to recover spontaneously, without the administration of any drugs. They were divided into four experimental groups (seven in each group) according to their survival periods (Table 1). 2.2. Assessment of motor functions We used the method of BBB Locomotor Rating Scale (Basso et al., 1995) for the post surgical analysis of the animal’s hindlimb motor functions, including frequency and quality of hindlimb movement as well as forelimb/ hindlimb coordination. The values of BBB Locomotor
Table 1 The survival periods post operation and the number of experimental animals used Survival periods (dpo) Number of animals
3 7
7 7
14 7
21 7
dpo: Days post operation.
Rating Scale for evaluation of hindlimb function ranged from 0, which corresponds to flaccid paralysis, to 21, which is normal gait. Rats were allowed to walk around freely in a spacious field for about 4 min while movements of the hindlimbs were closely observed. As the grading could be biased by subjectivity, three colleagues of ours who had no knowledge of the operative procedure and survival time were requested to assess the functions of both hindlimbs in all the operated and normal animals. The scores of these three were equated to get the average. All the behavior evaluations were performed at 8–9 am at various periods after operation. 2.3. Characterization of antibodies The specificities of the three antibodies against NGF, BDNF and NT-3 (received as gifts from Professor Xin-Fu Zhou, the Department of Human Physiology and Centre for Neuroscience, Flinders University of South Australia) (Deng et al., 2000; Zhou et al., 2000) were confirmed by Western Blot analysis, using spinal cord homogenates of rat in our lab. The polyclonal neurotrophin antisera for NGF, BDNF, and NT-3 were specific for the appropriate neurotrophins and no cross-reaction with other neurotrophins was detected. As little as 10 ng of each neurotrophin could be visualized, using these antisera. Control of immunostaining specificity was performed by omitting the primary antibody and antibodies pre-adsorbed with the appropriate neurotrophins. These controls did not exhibit any specific immunostaining. 2.4. Tissue preparation For immunohistochemistry, the animals were perfused through intracardiac administration of 250 ml of isotonic saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS), at 4 C. After perfusion, the vertebral canal in each animal was exposed to dislodge the spinal cord, which was then immersed in 4% paraformaldehyde at 4 C for further fixation. For Western Blot analysis, the vertebral canals of the two normal animals were anesthetised and rapidly opened. About 1 cm segment of spinal cord centering T10 vertebral level was obtained. The removal of spinal pia mater was performed on ice. All these samples
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freshly obtained were stored at 80 C until use for the detection of neurotrophins. 2.5. Western Blot The procedure of Western Blot used in this study has been introduced previously (Zhang et al., 2007). Briefly, the frozen spinal cord samples of the rats were homogenized on ice in 10 mM Tris–HCl buffer (pH 7.4), 10 mM EDTA, 30% Triton-1000, 10% SDS and NaCl using a homogenizer. Homogenates were centrifuged at 12,000g for 30 min at 4 C. The supernatant was phase collected in aliquots and stored at 80 C. The protein concentration was assayed with BCA reagent (Sigma, USA). Samples were loaded, electrophoresed on 5% stacking gel at 80 V and 15% resolving gel at 120 V. After electrophoresis, the gels were transferred to nitrocellulose membranes (Schleicher and Schuell, USA) for 6 h at 16 V. To reduced background staining, the filters were incubated with 5% non-fat dry milk in Tris buffered saline (TBS) containing 0.05% Tween 20 (TBST) overnight at 4 C, sequentially incubated with primary antisera (NGF 1:400, BDNF 1:200, and NT-3 1:200) in the same additive at 4 C, the membrane was rinsed in TBST before incubation in the peroxidase conjugated goat anti-rabbit IgG for 1 h at room temperature. The membrane was developed in ECL kit (Amersham, USA) and exposed against X-ray film in dark room. 2.6. Immunohistochemistry On the night before sectioning, about 1.5 cm segment of the spinal cord below the site of transection was obtained and placed in 20% sucrose solution in 0.1 M phosphate buffer (PB). After the specimen had sunken to the bottom of the bottle, it was placed on a freezing microtome (Leica CM1900, Germany) and horizontal sections were cut at 20 lm thickness. For accurate representation of the data, five sections that are designated as 10th, 20th, 30th, 40th, and 50th of each animal were processed for immunohistochemistry. Briefly, after washing three times (5 min each) in 0.1 M PBS, the sections were incubated free-floating in 3% hydrogen peroxide for 30 min, and soaked in 5% normal goat serum containing 0.3% Triton X-100 for 30 min at 37 C. They were then transferred to a solution of the respective primary antibodies for 48 h at 4 C (polyclonal neurotrophin antisera for NGF, BDNF, or NT-3, diluted 1:1000) containing 2% normal goat serum and 0.3% Triton X-100. After immersion in the primary antibodies or PBS, the sections were incubated in Reagents I and II from the Reagent Kit (Chemicon, Anti-Rabbit/Mouse Poly-HRP IHC Detection Kit, USA), each for 30 min at 37 C. This was followed by three times of washing, each 5 min in 0.1 M PBS. The immunoreactive products were visual-
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ized by placing the sections in a staining solution containing 0.05% 3,3 0 -diaminobenzidine, 0.1% nickel sulphate and 0.01% hydrogen peroxide for 10 min. Sections were observed in a light microscope (Leica DMI 6000 B inverted microscope, Germany) coupled with a computer assisted video camera (Leica, Germany). 2.7. Morphological analysis In order to ensure consistency in the analysis, all the sections obtained for immunohistochemistry were stained under the same conditions. Two individuals, who had no knowledge of the experiment, examined the sections separately. The Leica QWin image analysis software (Leica Qwin, Germany) was used to count the number of NGF, BDNF and NT-3 immunoreactive (IR) neurons in spinal laminae VIII–IX and I–VII on both sides of each section. Only clearly stained immunopositive profiles were included. Briefly, the number of positive neurons was counted and the area of lamina I–VII and VIII–IX were measured at a magnification of 100·. All the number were calculated in an equal area of (2812.5 lm2) to enable the investigators to compare the number of cells in different sections. In order to analyze intensity of immunoreactive staining, we measured the optical density (OD) of the immunopositive neuronal cell bodies, outlined manually by the experimenter from grey scale images (400· magnification). The method has been detailed in a former study (Gulino et al., 2004). All the processed sections of each animal were used to measure the average grey scale of NGF-, BDNF- and NT-3 IR products manually of both sides in laminae VIII–IX and I–VII. 2.8. Statistical analysis We used the SPSS 12.0 statistical analysis software package to analyze the data. Mean and standard error of the mean (SEM) of positively stained neurons and intra-neuron OD, including the transected and shamoperated control groups were evaluated. One-way AVOVA and Least Significant Difference were adopted in this study.
3. Results 3.1. Specificity of antisera Using the rat homogenates, the antibodies specifically recognized appropriate bands at a molecular weight of approximately 14.7 kDa, 14.2 kDa and 13.6 kDa, for NGF, BDNF, and NT-3 respectively (Fig. 1). These positions coincided with the molecular weights of the neurotrophins studied (Catania et al., 2007; Zhang et al., 2007).
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ber of NGF and BDNF peaked at 7 dpo, while that of NT-3 peaked at 7 dpo and remained so up to 14 dpo (Table 2). 3.4. Changes in intracellular optical density (OD)
Fig. 1. The specificities of NGF (B), BDNF (C), and NT-3 (D) were analysed by Western Bolt analysis (A) showed the position of b-actin at 43 kDa approximately.
After cord transection, intracellular OD values of the three neurotrophins in the spinal cord neurons showed different degrees of increases at 3, 7, 14 and 21 dpo, similar to the changes in the numbers of immunopositive neurons. Interestingly, the OD values of NGF and BDNF IR neurons peaked at 7 dpo and those of NT-3 IR neurons, at 14 dpo (Fig. 4). Though lower thereafter, those of NGF and NT-3 IR neurons were still higher than those in the control rats, while those of BDNF IR neurons declined to control value at 14 and 21 dpo.
3.2. BBB scores in control and operated animals 4. Discussion In the control group, each rat had a BBB score of 21. The mean of BBB scores for the cord transected rats was recorded in Fig. 2. The score was 0 at 3 dpo (p < 0.05). It was increased (p < 0.05) at 7 dpo, further increased (p < 0.05) at 14 dpo, and then plateaued up till at least 21 dpo. 3.3. NGF, BDNF and NT-3 immunostaining and numbers of stained profiles The immunohistochemical products of NGF, BDNF and NT-3 were seen in the neurons located both in laminae VIII–IX and I–VII of the spinal cord. Both the cytoplasm and the nucleus of NGF and NT-3 IR neurons displayed intense staining, with the former showing greater intensity, while BDNF positive neurons displayed mainly cytoplamic staining (Fig. 3). The numbers of NGF, BDNF and NT-3 immunopositive neurons increased in the lesion-induced cord. Such increase was, however, transient for BDNF. The num-
4.1. Specificity of antibodies Analysis by Western Blot showed that each antibody recognized specific domains of the neurotrophin proteins NGF, BDNF and NT-3 respectively in homogenates of the spinal cord of adult SD rats. Immunohistochemical controls demonstrated no immunostaining. These showed that the antibodies were highly specific and could recognize native proteins in adult rat CNS. 4.2. Recovery of locomotor function After cord transection, the hindlimbs of the rats showed flaccid paralysis, indicating that motoneuron activity was dramatically reduced. There was, however, gradual restoration of hindlimb functions from 7 dpo to 21 dpo. The scores of BBB Locomotor Rating Scale increased, beginning at 7 dpo, further increased at 14 dpo, and remained so up to at least 21 dpo. The results indicated spontaneous, howbeit incomplete, recovery of the rats’ hindlimbs after spinal cord transection. 4.3. Neurotrophins expression in motoneurons
Fig. 2. Mean values of BBB scores in cord transected rats. *p < 0.05, compared with the values of sham-operated control group. #p < 0.05, compared with the values of its former group. The error bars represented the standard error of the mean (SEM). dpo: Days post operation.
The expression and up-regulation of NGF, BDNF and NT-3 in the spinal neurons indicated that these neurotrophins could play essential roles in neuroplasticity following cord transection. Numerous studies have demonstrated that neurotrophins were involved in both physiological functions and pathological condition (Lindsay et al., 1985; Alderson et al., 1990; Risling et al., 1992; Sariola et al., 1994; Novikova et al., 1996; Vischer, 1997; Zhou and Shine, 2003; Gulino et al., 2004; Gurok et al., 2004; Pinzon-Duarte et al., 2004). The up-regulation of neurotrophin expression could
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Fig. 3. Note the expression of NGF-IR (A), BDNF-IR (B) and NT-3-IR (C) neurons in the spinal cord of a sham-operated rat. The OD values of NGF-immunoreactive (IR) neurons peaked at 7 dpo (D), BDNF at 7 dpo (E), and NT-3 at 14 dpo (F). BDNF-IR neurons in laminae VIII–IX are shown in (B) and (E), and NT-3-IR neurons in (C, F). Control cord sections with PBS replacing the primary antibodies are shown in (G), (H), and (I), for NGF, BDNF, and NT-3 antibodies respectively. Sections subjected to pre-adsorption blocking controls for NGF, BDNF and NT-3 are shown in (J), (K), and (L) respectively. Arrows indicated neurotrophins-IR motoneurons in laminae VIII–IX. Bars in (A), (D), (G), (I), (J), and (L) = 100 lm; bars in (B), (C), (E), (F), (H), and (K) = 50 lm.
underline the observed functional plasticity. In the present study, the BBB scores in cord transected rats showed a gradual increase from 7 dpo to 14 dpo, through 21 dpo. The expression of NGF peaked at 7 dpo, thereafter declined but still appeared higher than that of controls at 21 dpo. Increased NGF at the caudal part of the lesion site might in some way contribute to the recovery
of the injured motoneurons. Previous studies showed that NGF could prevent the death of motoneurons after injury both in newborn and adult rats (Diener and Bregman, 1994; Murakami et al., 2002). Also, NGF receptor mRNA increased in adult motoneurons after axonal injury, indicating the protective role of NGF (Koliatsos et al., 1991).
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Table 2 Numbers of immunopositive neurons in Laminae (Lam) VIII–IX and I–VII in control and rats sacrificed at different times after transection NGF IR neurons
Control 3 dpo 7 dpo 14 dpo 21 dpo
BDNF IR neurons
NT-3 IR neurons
Lam VIII–IX
Lam I–VII
Lam VIII–IX
Lam I–VII
Lam I–VII
Lam VIII–IX
45.53 ± 2.83 65.01 ± 2.11* 83.41 ± 4.32* 75.40 ± 2.51* 71.02 ± 6.13*
14.53 ± 3.00 27.66 ± 2.18* 41.94 ± 5.63* 37.40 ± 4.48* 26.89 ± 2.22*
73.64 ± 5.76 86.70 ± 7.02* 120.70 ± 8.95* 71.21 ± 9.23 69.35 ± 9.85
30.20 ± 9.40 66.42 ± 5.34* 87.36 ± 8.54* 33.56 ± 5.62 29.69 ± 6.35
60.50 ± 7.21 86.23 ± 3.34* 97.35 ± 2.56* 78.40 ± 5.91* 74.56 ± 3.22*
22.54 ± 3.28 40.12 ± 2.48* 60.45 ± 9.12* 35.55 ± 4.26* 30.46 ± 3.45*
Numbers refer to mean numbers (M) per equal area (2812.5 lm2) ± standard error of mean (SEM). dpo: Days post operation. * p < 0.05, compared with normal control ones.
Fig. 4. (A–C) Changes in the optical densities of NGF, BDNF and NT-3 IR cells in different groups. The mean optical density of every neurotrophin in the sham-operated control group was arbitrarily considered as 100%. *p < 0.05, compared with sham-operated rats. The error bars represented the standard error of the mean (SEM). dpo: Days post operation.
The expression of BDNF increased and peaked at 7 dpo but returned to control level at 14 dpo. Although the increase of BDNF expression was transient, it could also in some way play an important role in the functional plasticity of injured motoneurons. That its role could not be ignored during recovery of injured spinal cord is also emphasized by several authors (Diener and Bregman, 1994; Namiki et al., 2000; Blits et al., 2003; Zvarova et al., 2004). In addition, BDNF has been
known to be involved in synaptic plasticity (McAllister et al., 1999; Gomez-Pinilla et al., 2002), neuroprotection and sprouting of spared pathways after injury (Henderson et al., 1993; Jakeman et al., 1998). The expression of NT-3 after cord transection peaked at 7th and 14th dpo. This indicated NT-3 could also exert its effects on spinal cord plasticity after transection. It is well known that NT-3 plays an important role in supporting the development and survival of motoneu-
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rons (Yan et al., 1993). It could also regulate the excitability of spinal cord cells and motoneuron spindle physiology (Mendell et al., 1999) as well as spinal cord activity-dependent plasticity (Gomez-Pinilla et al., 2001, 2004). Neuromuscular activity has specific effects on the BDNF and NT-3 systems (Gomez-Pinilla et al., 2001). Evidences indicated that NT-3 might be of benefit in preventing the secondary cell loss that occurs following spinal injury (Bradbury et al., 1998). Additionally, the up-regulation of NGF and NT-3 lasted longer than that of BDNF, which indicated that the three proteins might play different roles at different times after spinal cord injury. 4.4. Neurotrophins expression in sensory neurons In the present study, the expression of NGF and BDNF in laminae I–VII peaked at 7 dpo, while that of NT-3 at 7 dpo and 14 dpo. These suggested that the three neurotrophins might affect not only motoneurons but also sensory neurons following cord transection. Although NGF had been shown to be crucial for the development of sensory neurons (Diener and Bregman, 1994), recent studies indicated such dependency declined with the animal’s growth. The main function of NGF in adult animals may be related to nociception (Lewin and Mendell, 1993; Krenz and Weaver, 2000), and some studies have shown that NGF may regulate pain sensation in the human (Anand, 1995). Correlated with this observation is the finding that NGF stimulates sprouting of small-diameter afferent fibers and its concentration increases after spinal cord injury (Anand, 1995; Krenz and Weaver, 2000). Thus the increased expression of NGF in spinal sensory neurons in this study may not only be able to promote the survival of sensory neurons, but also could be responsible for the development of central pain. Former study has shown that BDNF and NT-3 could promote the outgrowth of sensory neurites in vitro (Blits et al., 2003). Besides contributing to the survival and recovery of injured sensory neurons, BDNF might also participate in the transmission of central pain with NGF after spinal cord injury (Pezet and McMahon, 2006). NT-3 was said to be important in the development and maintenance of sensory neurons (Zhou and Rush, 1995). Previous reports showed that following spinal cord hemisection both in newborn and adult rats, administration of exogenous NT-3, but not NGF or BDNF, rescued neurons in nucleus dorsalis (Diener and Bregman, 1994; Shibayama et al., 1998; Novikova et al., 2000). Himes et al. (2001) reported that NT-3 could be of benefit in the long-time survival of nucleus dorsalis. In the present study, the increased expression of BDNF and NT-3 might be crucial for the recovery of injured spinal neurons. Further work is being done to explore the mechanism of action.
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4.5. Use of female rats Young adult female rats were chosen for this study because they were easier to feed and less prone to urinary tract infection than the male rats when subjected to spinal cord transection. Female rats also had a better survival rate after surgery. The benefits of choosing female rats for studies of spinal cord injury have been discussed previously (Talmadge et al., 2002). As we were not aware of any work done to study the influence of estrous cycle to neurotrophins expressed in spinal cord, we did not observe the estrous cycle of the rats. In conclusion, based on the increase in the number of neurotrophic positive neurons and in the intracellular OD values as well as the results of behavior test throughout the experimental period, we conjectured that the partial recovery of functions of the rat hindlimbs could at least partly be related to the increased expression of NGF, BDNF and NT-3. Moreover, the numbers of NGF-positive and NT-3-positive neurons were significantly increased at all post-operative periods in the experimental group, when compared with that in the sham-operated group. The increased expression of BDNF was only transient. Further studies need to be done to explore the exact roles of these three neurotrophins and their different expressions in this model of neuroplasticity.
Acknowledgements We thank Dr. S. K. Leong for his invaluable comments in the writing of this manuscript. This research was supported by a grant from the National Science Foundation (No. 30260125), the New York–China Medical Grant (CMB00-722) and the Development Grants of the Kunming Medical College (Nos. 2005DG03, 2005DG01).
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