Neurochem Res (2015) 40:98–108 DOI 10.1007/s11064-014-1470-4
ORIGINAL PAPER
Hyaluronan Tetrasaccharide Exerts Neuroprotective Effect and Promotes Functional Recovery After Acute Spinal Cord Injury in Rats Jun Wang • Xiaofang Wang • Jie Wei Manyi Wang
•
Received: 25 June 2014 / Revised: 11 September 2014 / Accepted: 30 October 2014 / Published online: 6 November 2014 Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to explore the therapeutic efficiency of hyaluronan tetrasaccharide (HA4) treatment after spinal cord injury (SCI) in rats and to investigate the underlying mechanism. Locomotor functional and electrophysiological evaluations revealed that the behavioral function of rats in the HA4-treated group was significantly improved compared with the vehicletreated group. The expression of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), cluster determinant (CD44) and Toll-like receptor-4 (TLR-4) was obviously upregulated in the HA4treated group than that in the sham and vehicle-treated group. Furthermore, HA4 could induce BDNF and VEGF expression in the astrocytes in vitro. In addition, the high expression of BDNF and VEGF could be inhibited by blocking CD44 and TLR-4. These findings indicate that HA4 could be useful as a promising therapeutic agent for SCI and might exert the effect by interaction with the CD44 and TLR-4. Keywords Spinal cord injury Hyaluronan tetrasaccharide Astrocytes Neurotrophic factor
J. Wang J. Wei M. Wang (&) Traumatology Department,Beijing Jishuitan Hospital, No. 31 East Street of Xin Jie Kou Beijing, Beijing 100035, People’s Republic of China e-mail: [email protected] J. Wang Medical Center Tsinghua University, No. 30 Shuangqing Rd, Haidian District, Beijing 100084, People’s Republic of China X. Wang Department of Pharmacy, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, People’s Republic of China
123
Introduction Spinal cord injuries (SCI) are highly disabling and deadly injuries. With the modern traffic development, the incidence and prevalence of spinal injuries have been increasing, with the incidence rate of traumatic SCI estimated at 15–40 cases per million worldwide, although injury prevention initiatives have tried to reduce the occurrence of SCIs [1]. The adult central nervous system (CNS) has limited regenerative capacity and is unable to achieve full functional recovery after traumatic injury. Thus, the patients with complete SCI always have poor outcomes and the problem causes a huge economic burden to society and family. Hyaluronan (HA) was first described by Meyer and Palmer and composed of repeating units of disaccharides [-Dglucuronic-acid-b1, 3-N-acetyl-D-glucosamine b1, 4-]n [2]. It has been reported that different molecular weight HA has distinct biological function [2–4]. In the literature, Many studies have showed that low molecular weight (LMW)-HA has the neuroprotective effect on the nervous system. The previous study has demonstrated that NMDA-induced neuronal cell death was partially blocked by hyaluronan tetrasaccharide (HA4) in vitro, and HA4 promotes motor function recovery after SCI in vivo [5]. It has been reported that, at an optimal dose, HA4 promotes neural regeneration and axonal outgrowth [6]. In our previous study, it has been reported that HA4 in the cerebrospinal fluid was associated with self-repair of rats after chronic spinal cord compression. Furthermore, HA4 could induce the neurotrophic factor expression in the astrocytes in vitro and these phenomenons illustrated that LMW-HA4 could exert positive influence on the SCI [7]. Thus, we performed this experiment to assess the neuroprotective effect of HA4 on the injured spinal cord in the rats.
Neurochem Res (2015) 40:98–108
Materials and Methods A total of 36 Sprague–Dawley rats, aged 12 weeks (250–300 g), were used for the experiment. Anesthesia was induced with pentobarbital sodium (50 mg/kg). All protocols were approved by the animal care committee of the Peking University Health Science Center. We have minimized the number of animals used and their suffering. A laminectomy was carried out on the T8 vertebra. SCI was induced using the weight-drop A moderate SCI (force, 40 g/cm) was induced by dropping a weight of 10 g from a height of 4 cm onto an impounder (diameter, 0.2 cm) gently placed on the spinal cord. Sham animals received the same surgical procedure but sustained no impact injury. Muscle and skin were sutured in layers, and an antibacterial Baytril (3 mg/kg) was applied subcutaneously each morning for 3 days. After the operation, animals were maintained in the same postoperative condition of lying on a heated bed. At 24 h after SCI, a short behavior test was conducted. Only the rats with a Basso, Beattie, and Bresnahan (BBB) locomotor score of \1 were included in the study. The SCI animals were randomly divided into three groups (n = 12): sham group; vehicle-treated group; HA4treated group. HA4 or vehicle was administered to the injured site by the ALZET mini-osmotic pump with a catheter at 1 day after SCI for 4 weeks. Saline was applied to the vehicle-treated group. Neurobehavioral Evaluations Locomotor activity was evaluated in the rats (n = 12, per group) before surgery and at days 1, 3 postoperation, and then weekly until the rats were killed, using the BBB scoring system that measures locomotor ability during 5 min [8]. Two independent examiners who were blinded to experiment observed the hind limbs movements and scored the locomotor function according to the Basso, Beattie and Bresnahan (BBB) scale, wherein 0 indicates no motor activity and 21 indicates a normal performance. The score of each animal was obtained by averaging the values from both examiners. Electrophysiological Evaluations Animals (n = 12, per group) were anesthetized with pentobarbital sodium and placed in a prone position. At 10 weeks after the operation, electrophysiological tests were performed on both hind limbs using the Cadwell Cascade system (Cadwell Laboratories Inc., Kennewick, WA, USA). To assess the motor function after injury, motor evoked potentials (MEPs) were elicited by transcranial stimulation with two needle electrodes placed over the skull. The anode was placed over the sensorimotor
99
cortex and the cathode was placed on the hard palate. Single electrical pulses (30 V intensity, 100 ls duration, and train of four pulses) were delivered. MEPs were recorded from the tibialis anterior muscle and the reference electrode inserted into the footpad. The signals were amplified and filtered (amplifier gain of 10; bandpass frequency, 10–3,000 Hz). To ensure reproducibility, five consecutive responses were recorded, with a time interval of 30 s between stimuli. The recording with the average amplitude and latency was used for analysis. All signals of MEP were obtained on a computer by measuring the peak latency and peak-to-peak amplitude of the evoked potentials. Tissue Processing for Electron Microscopy Tissue (n = 3, per group) was post-fixed with osmium tetroxide and potassium ferrocyanide. Following five 10-min sodium cacodylate washes and five quick washes with 0.1 M acetate buffer, the tissue was stained with 1 % uranylacetate overnight at 4 °C. Five 10-min washes with acetate buffer were followed by progressive dehydration in 50, 70, 80, 90, 95, and 100 % ethanol, each with a 10-min duration. Three additional 10-min washes with 100 % ethanol were followed by two short-duration washes with propylene oxide and rotation overnight with 1:1 propylene oxide and epon. The tissue was then embedded in inverted polyethylene beam capsules with the tips removed and allowed to cure for 48 h at 60 °C under a vacuum of 20 mm Hg. Immunofluorescent Double-Labeling For tissue staining, 8 lm of spinal cord frozen sections (n = 4, per group) were permeabilized with 1 % Triton X-100 and 10 % normal goat serum at room temperature for 15 min. A monoclonal mouse anti-GFAP antibody (1:250, Santa Cruz Biotechnology) with either a polyclonal rabbit anti-BDNF antibody (1:500; Abcam) or a rabbit vascular endothelial growth factor (VEGF) antibody (1:500; Abcam) were applied to the sections at 4 °C overnight. Next, sections were incubated with an FITCconjugated goat anti-rabbit IgG antibody (1:200; SigmaAldrich) and a Texas Red-conjugated goat anti-mouse antibody (1:200; Sigma-Aldrich). Following immunostaining, all of the sections were coverslipped with Mowiol containing DAPI to counterstain the nuclei. Images were taken using a Leica DM3000. Western Blot Analysis For Western blotting, 1.5-cm-long spinal cord samples centered at the injured site from the sham, vehicle-treated,
123
100
and HA4-treated groups (n = 5, per group) were washed twice in ice-cold PBS. The samples were individually homogenized in 5 mM Tris–HCI (4 mM EDTA, pH 7.4, containing 1 lM pepstatin, 100 lM leupeptin, 100 lM phenylmethyl sulfonylfluoride, and 10 lg/ml aprotinin) and cleared by centrifugation at 14,000g for 10 min at 4 °C. Protein concentrations were determined by the Lowry method. Approximately 100 lg of protein were run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5 % skim milk in TBS containing 0.05 % Tween 20 and were incubated with the following primary antibodies: a polyclonal rabbit anti-VEGF antibody (1:1000; Abcam); and a rabbit anti-BDNF antibody (1:100; Abcam). The optical density (OD) of the signals was quantified and expressed as the ratio of OD of the tested proteins to that of b-actin. Astrocytic Primary Culture and Treatment For astrocytic cultures, the cortex was homogenized by mechanical fragmentation, and the cell suspension passed sequentially through steel screens of 230-, 104-, and 73.3lm pore size. Cells were subsequently collected by centrifugation at 700 g for 10 min and resuspended in DMEM supplemented with 10 % fetal calf serum. Finally, 2.0 9 105 cells/cm2 were plated on tin plates and maintained at 37 °C in a humidified atmosphere of 5 % CO2. The culture medium was replaced at day 7, and the confluent cultures were used at day 10. The detailed information was followed by the previous study [9]. The experimental cells were incubated in a medium containing 1,000 lg/ml of HA4 for 24 h. The control cells were cultured without HA4. A separate set of plates were also first treated with the specific antibody against either TLR-4 or CD44 alone or with both antibodies. Immunofluorescent double-labeling is performed for the cells with the procedure described previously using the following antibodies: a polyclonal mouse anti-GFAP antibody (1:250, Santa Cruz Biotechnology); a polyclonal rabbit anti-VEGF antibody (1:500; Abcam); and a rabbit antiBDNF antibody (1:500; Abcam). The control group, group treated with 1,000 lg/ml of HA4 and groups treated with the specific antibody against either TLR-4 or CD44 alone or with both antibodies were analyzed by the western blot analysis. Statistical Analysis Comparisons of data with more than two variables were analyzed by two-way repeated measure-ANOVAs with post hoc Tukey’s analysis. All data are plotted as the mean ± standard error.
123
Neurochem Res (2015) 40:98–108
Results Hyaluronan Tetrasaccharide Promotes Hindlimb Locomotion Recovery After SCI To assess if HA4 promoted the hindlimb locomotion recovery, we performed the BBB scores test. All animals had an initial BBB score of 21, and the injury resulted in a score of 0, with complete hindlimb paralysis. In the following weeks, 1000 lg/ml HA4-treated group (from 5 weeks post-SCI) showed more significant improvements compared with the vehicle-treated group (P \ 0.05), and sustained throughout the survival period. At 10 weeks after SCI, the average BBB scores for the HA4-treated rats was 16.51 ± 2.12. In comparison, although the vehicle-treated rats also regained some motor functional recovery due to the self-repair mechanism, their BBB score was significantly lower (11.13 ± 2.05) compared with HA4-treated rats (P \ 0.05; Fig. 1a). Electrophysiological changes were evaluated based on the amplitude and latency of MEPs. The representative MEP wave forms of sham, vehicle-treated, HA4-treated rats were showed in Fig. 1b. Interestingly, HA4-treated rats showed significantly higher amplitudes and shorter latencies than vehicle-treated at 10 weeks (P \ 0.05). Furthermore, there was no significant difference between the sham and HA4-treated rats (Fig. 1c). Hyaluronan Tetrasaccharide Induces BDNF and VEGF Expression in Astrocytes In Vivo After SCI To determine whether BDNF and VEGF were upregulated under HA4 treatment in astrocytes, we performed an immunofluorescent double-labeling and Western blot analysis at the epicenter of the injured sites. The results showed that the expression of BDNF and VEGF in the astrocytes in the HA4-treated group was significantly higher than that in the vehicle-treated group (P \ 0.05) (Fig. 2). Hyaluronan Tetrasaccharide Induces CD44 and TLR-4 Expression at the Injured Spinal Cord To investigate the expression of CD44 and TLR-4 under HA4 treatment in the astrocytes, we performed an immunofluorescent double-labeling analysis at the epicenter of the injured sites. We found that HA4 treatment could stimulate the CD44 and TLR-4 expression in the astrocytes. The expression of CD44 and TLR-4 in the HA4treated and vehicle-treated group was obviously increased compared with that in the sham group (P \ 0.05). Furthermore, There was significant difference between the HA4-treated and vehicle-treated group (P \ 0.05; Fig. 3).
Neurochem Res (2015) 40:98–108
101
Fig. 1 a HA4 improves behavioral recovery after SCI. We performed the BBB score test before and after SCI. The rats in the HA4-treated group showed significantly higher scores than that in the vehicletreated group from 5 weeks and sustained until sacrifice. b The representative waveforms of the MEPs in the sham, vehicle-treated
and HA4-treated groups. c Plots show the mean latency and amplitude of the MEPs. HA4-treated rats showed significantly higher amplitudes and shorter latencies than vehicle-treated at 10 weeks. *P \ 0.05, as compared with vehicle group; error bars represent SD
Hyaluronan Tetrasaccharide Promotes Axons Survival After SCI
the morphological change of astrocytes in vitro. The result showed that HA4 promoted dramatical stellation of astrocytes and it transformed the cell body of astrocytes into a thin cell body like a stella (Fig. 5).
To determine the condition of axons under HA4 treatment after SCI, we performed an electron micrograph analysis at the epicenter of the injured sites. The white matter from the sham tissue contained many axons encircled by compact myelin laminations. After SCI, ruptured axons were observed, with expelling their axoplasmic contents into the extracellular space. The conditions of the axons in the HA4-treated group were better than those in vehicle-treated group. Furthermore, the density of axons in the HA4-treated group was higher than that in the vehicle-treated group (Fig. 4).
Hyaluronan Tetrasaccharide Induces BDNF and VEGF Expression in Astrocytes In Vitro To observe whether HA4 could stimulate the BDNF and VEGF expression in astrocytes in vitro, we performed an immunofluorescent double-labeling analysis. We found that 1,000 lg/ml of HA4 obviously stimulated the BDNF and VEGF expression in astrocytes in vitro (Fig. 6).
Hyaluronan Tetrasaccharide Stimulates the Morphological Change of Astrocytes In Vitro
The BDNF and VEGF Expression Induced by Hyaluronan Tetrasaccharide was Blocked by the Antibody Against TLR-4 or CD44 In Vitro
To assess the effect of HA4 on the condition of astrocytes, we performed the immunofluorescent labeling to observe
To investigate whether the BDNF and VEGF expression induced by HA4 was blocked by the antibody
123
102 Fig. 2 a The upregulation of BDNF and VEGF expression in the immunofluorescent doublelabeling experiment. Scale bar 1,000 and 100 lm. b Western blot analysis also showed that BDNF and VEGF expression was increased under the HA4 treatment after SCI. #P \ 0.05, as compared with vehicle group, error bars represent SD
123
Neurochem Res (2015) 40:98–108
Neurochem Res (2015) 40:98–108
103
Fig. 3 The induction effect of HA4 treatment on the expression of CD44 and TLR-4 in vivo. The expression of CD44 and TLR-4 in the HA4-treated group was significantly increased compared with that in
the vehicle-treated group. *P \ 0.05, as compared with sham group; # P \ 0.05, as compared with vehicle group, error bars represent SD. Scale bar 100 lm
against TLR-4 or CD44, we performed the immunofluorescent double-labeling and western blot analysis of cultured astrocytes. In the immunofluorescent double-labeling analysis, the astrocyte didn’t expressed the BDNF without HA4 treatment (Fig. 7A1, A2, A). The BDNF expression in the astrocytes was upregulated by either blocking TLR-4 or CD44 (Fig. 7B1, B2, B and C1, C2, C). When both of TLR-4 and CD44 were blocked, the BDNF didn’t expressed in the astrocytes (Fig. 7D1, D2, D). Meanwhile, in the Western blot analysis, the results were similar to that in the immunofluorescent double-labeling analysis. Overall, the result showed that HA4 could stimulate the BDNF expression in astrocytes by interaction with either TLR-4 or CD44 (Fig. 8).
Discussion This study demonstrates that treatment with HA4 could improve the functional recovery by upregulating BDNF and VEGF expression after SCI in the rats. Spinal cord injuries (SCIs) can be divided into two different groups: traumatic spinal cord injuries (TSCI) and non-traumatic spinal cord injuries (NTSCI). Traumatic SCI evolves through three phases: the acute, mostly due to the direct mechanical damage, the secondary, and the chronic stages [10]. Mounting evidence indicates that glial scars constitute a major component of the post-injury environment that discourages spontaneous axonal regeneration [11–16]. Astrocytes expressing glial fibrillary acidic protein (GFAP) become hypertrophic and highly proliferative
123
104
Neurochem Res (2015) 40:98–108
Fig. 4 The condition of axons under HA4 treatment after SCI by the electron micrograph analysis. The density of axons in the HA4-treated group was higher than that in the vehicletreated group. Scale bar 10 lm. *P \ 0.05, as compared with sham group; #P \ 0.05, as compared with vehicle group, error bars represent SD
Fig. 5 The condition of astrocytes under HA4 treatment. HA4 promoted the morphological change of astrocytes in vitro
to form eventually a dense network of glial scars around the cavity [17–19]. Thus, modulating the condition of astrocytes is an important direction of the therapy.
123
It has been reported that different molecular weight HA has distinct biological effect. High molecular weight hyaluronic acid (HA) is an essential organizational and structural
Neurochem Res (2015) 40:98–108
105
Fig. 6 The effect of HA4 on the BDNF and VEGF expression in astrocytes in vitro. The intensity of BDNF and VEGF expression in astrocytes under 1,000 lg/ml of HA4 treatment was significantly higher than that under 0 lg/ml of HA4 treatment
component in the ECM of native tissues [20, 21]. High molecular weight hyaluronan (HMW-HA) surrounds neuronal cell bodies in the nervous system and maintains astrocytes in a state of quiescence. Furthermore, the degradation of HA following SCI relieves the astrocyte growth inhibition, resulting in increased astrocyte proliferation [22]. Seidlits et al. [23] reported that hyaluronic acid hydrogels could induce the neural progenitor cell differentiation. Austin et al. [24] revealed that intrathecal injection of
hyaluronic acid hydrogels reduced the extent of fibrosis and inflammation in the subarachnoid space, promoted neurobehavioral recovery, enhanced axonal conduction and reduced the extent of post-traumatic parenchymal fibrous scar formation. Wei et al. [25] reported that hyaluronic acid hydrogel modified with nogo-66 receptor antibody and polyL-lysine promoted axon regrowth after SCI. The previous studies illustrated that HMW-HA had the positive and negative effects on the functional recovery in the acute SCI.
123
106
Neurochem Res (2015) 40:98–108
Fig. 7 The immunofluorescent double-labeling analysis showed the expression of BDNF in astrocyte blocked by the antibody against TLR-4 or CD44 receptors. (A1, A2, A) the astrocytes without HA4 treatment. (B1, B2, B and C1, C2, C) Under HA4 treatment, the
astrocytes treated by the specific antibody against either TLR-4 or CD44 alone. (D1, D2, D) Under HA4 treatment, the astrocytes treated by both of specific antibodies against TLR-4 and CD44. Scale bar 10 lm
In the literature, many studies have revealed that low molecular weight (LMW)-HA could exert a profound neuroprotective effect on the injured nervous system [6, 26, 27]. It has been reported that hyaluronan tetrasaccharide (HA4) could induce astrocytes to express neurotrophic factor [7, 27]. In the previous study, we found that HA4 could induce astrocytes to express more BDNF and VEGF in vitro and was involved in the self-repair process of chronic spinal cord compression injury [7]. In this study, the results showed that HA4 could improve the motor function and induce the neurotrophic factor expression in the astrocytes. It illustrated that HA4 has the neuroprotective effect on the injured spinal cord. In our study, we determined the effects of hyaluronan tetrasaccharide treatment on the expression of BDNF and VEGF. Brain-derived neurotrophic factor (BDNF) is an important neurotrophic factor. It exerts an positive
influence on the survival of neurons and enhances the remyelination of injured spinal cord [28, 29]. Vascular endothelial growth factor (VEGF) constitutes a sub-family of growth factors that stimulate the growth of new blood vessels and it is an important signaling protein involved in the vasculogenesis for injured tissue [30, 31]. Many studies have revealed that the improved functional recovery after SCI may contribute to the high expression of BDNF and VEGF [7, 29, 32]. In our study, administration of HA4 to the damaged site of spinal cord induced the expression of BDNF and VEGF in the astrocytes in vivo. Meanwhile, HA4 could also stimulate BDNF and VEGF expression of astrocytes in vitro. Thus, the modulating astrocytes is an important treatment and could get astrocytes to perform its positive effect for the SCI. The cluster determinant 44 (CD44) and toll-like receptor 4 (TLR-4) are the two best-known receptor of HA. It has
123
Neurochem Res (2015) 40:98–108
107
Fig. 8 The Western blot analysis showed the expression of BDNF in astrocyte blocked by the antibody against TLR-4 or CD44. *P \ 0.05, as compared with control group, error bars represent SD
been reported that the profile of expression of CD44 and TLR-4 was modulated by high level of hyaluronidase and it provided the exquisite condition for the regulatory mechanisms [33]. CD44 stimulation with HA plays the role of cell adhesion, cell–substrate interaction, and inflammation [34]. Toll-like receptors are a class of proteins that play a key role in the innate immune system. the glycosaminoglycan (GAG) hyaluronan (HA) has also been identified as an inducer of TLR-4 activation, capable of causing the release of pro-inflammatory cytokines [35]. Low molecular weight of HA are able to interact with TLR-4 and CD44 and the previous studies showed that HA interact with TLR-4 and CD44 was involved in the process of inflammation [36, 37]. In our study, the HA4 stimulation of BDNF and VEGF effect on astrocytes was suppressed by blocking the TLR-4 and CD44. Either blocking TLR-4 or CD44 could partially decrease BDNF and VEGF expression. From the results of immunofluorescent double-labeling and western blot analysis, the neuroprotective mechanism of HA4 may be exerted by interacting with CD44 and TLR-4. The expression of CD44 and TLR-4 at the injuried spinal cord was upregulated under HA4 treatment. There are some limitations to our findings. In this study, we didn’t assess the effect of low dose HA4 treatment on
the SCI. The choice of 1,000 lg/ml was mainly designed based on the previous study [7]. In this study, we only tested the effect of HA4 on the interaction of astrocytes and modulating the neurotrophic factor of low dose was unclear. We aim to attract much attention on the hyaluronan therapeutic effect on the SCI patients. But this aspect of study is in its infancy and the exact mechanism of HA on the SCI proves inconclusive. Thus, the basic research is in need of further study and the further study of HA is in the process of further investigation. In conclusion, the present study has shown that HA4 could improve the functional recovery by upregulating BDNF and VEGF expression after SCI in the rats and may have therapeutic potential for SCI in the clinical.
Conflict of interest I confirm on behalf of all the authors that there is no conflict of interest issues related to this work.
References 1. Jackson AB, Dijkers M, Devivo MJ, Poczatek RB (2004) A demographic profile of new traumatic spinal cord injuries: change and stability over 30 years. Arch Phys Med Rehabil 85:1740–1748
123
108 2. Meyer K, Palmer J (1934) The polysaccharide of the vitreous humor. J Biol Chem 107:629–634 3. Atkins EDT, Phelps CF, Sheehan JK (1972) The conformation of the mucopolysaccharides. Biochem J 128:1255–1263 4. Almond A (2007) Hyaluronan. Cell Mol Life Sci: CMLS 64:1591–1596 5. Wakao N, Imagama S, Zhang H, Tauchi R, Muramoto A, Natori T, Takeshita S, Ishiguro N, Matsuyama Y, Kadomatsu K (2011) Hyaluronan oligosaccharides promote functional recovery after spinal cord injury in rats. Neurosci Lett 488:299–304 6. Torigoe K, Tanaka HF, Ohkochi H, Miyasaka M, Yamanokuchi H, Yoshidad K, Yoshida T (2011) Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: in vivo analysis by film model method. Brain Res 1385:87–92 7. Wang J, Rong W, Hu X, Liu X, Jiang L, Ma Y, Dang G, Liu Z, Wei F (2012) Hyaluronan tetrasaccharide in the cerebrospinal fluid is associated with self-repair of rats after chronic spinal cord compression. Neuroscience 210:467–480 8. Basso D, Beattie M, Bresnahan J (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1–21 9. Zhu Z, Zhang Q, Yu Z, Zhang L, Tian D, Zhu S, Bu B, Xie M, Wang W (2007) Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo. Glia 55:546–558 10. Tator CH, Fehlings MG (1991) Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 75:15–26 11. Wang J, Ma C, Rong W, Jing H, Hu X, Liu X, Jiang L, Wei F, Liu Z (2012) Bog bilberry anthocyanin extract improves motor functional recovery by multifaceted effects in spinal cord injury. Neurochem Res 37:2814–2825 12. Sharp K, Yee KM, Steward O (2012) A re-assessment of the effects of treatment with an epidermal growth factor receptor (EGFR) inhibitor on recovery of bladder and locomotor function following thoracic spinal cord injury in rats. Exp Neurol 233:649–659 13. Chen L, Wang Z, Ghosh-Roy A, Hubert T, Yan D, O’Rourke S, Bowerman B, Wu Z, Jin Y, Chisholm AD (2011) Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71:1043–1057 14. Jager C, Lendvai D, Seeger G, Bruckner G, Matthews RT, Arendt T, Alpar A, Morawski M (2013) Perineuronal and perisynaptic extracellular matrix in the human spinal cord. Neuroscience 238:168–184 15. Jeong SR, Kwon MJ, Lee HG, Joe EH, Lee JH, Kim SS, Suh-Kim H, Kim BG (2012) Hepatocyte growth factor reduces astrocytic scar formation and promotes axonal growth beyond glial scars after spinal cord injury. Exp Neurol 233:312–322 16. Huang X, Kim JM, Kong TH, Park SR, Ha Y, Kim MH, Park H, Yoon SH, Park HC, Park JO, Min BH, Choi BH (2009) GM-CSF inhibits glial scar formation and shows long-term protective effect after spinal cord injury. J Neurol Sci 277:87–97 17. Bradbury EJ, Carter LM (2011) Manipulating the glial scar: chondroitinase ABC as a therapy for spinal cord injury. Brain Res Bull 84:306–316 18. Hunanyan AS, Garcia-Alias G, Alessi V, Levine JM, Fawcett JW, Mendell LM, Arvanian VL (2010) Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord. J Neurosci Off J Soc Neurosci 30:7761–7769 19. Herrmann JE, Shah RR, Chan AF, Zheng B (2010) EphA4 deficient mice maintain astroglial-fibrotic scar formation after spinal cord injury. Exp Neurol 223:582–598 20. Sherman LS, Struve JN, Rangwala R, Wallingford NM, Tuohy TM, Kuntz CT (2002) Hyaluronate-based extracellular matrix: keeping glia in their place. Glia 38:93–102
123
Neurochem Res (2015) 40:98–108 21. Cheng G, Swaidani S, Sharma M, Lauer ME, Hascall VC, Aronica MA (2011) Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma. Matrix Biol J Int Soc Matrix Biol 30:126–134 22. Struve J, Maher PC, Li YQ, Kinney S, Fehlings MG, Kuntz CT, Sherman LS (2005) Disruption of the hyaluronan-based extracellular matrix in spinal cord promotes astrocyte proliferation. Glia 52:16–24 23. Seidlits SK, Khaing ZZ, Petersen RR, Nickels JD, Vanscoy JE, Shear JB, Schmidt CE (2010) The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation. Biomaterials 31:3930–3940 24. Austin JW, Kang CE, Baumann MD, DiDiodato L, Satkunendrarajah K, Wilson JR, Stanisz GJ, Shoichet MS, Fehlings MG (2012) The effects of intrathecal injection of a hyaluronan-based hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis. Biomaterials 33:4555–4564 25. Wei YT, He Y, Xu CL, Wang Y, Liu BF, Wang XM, Sun XD, Cui FZ, Xu QY (2010) Hyaluronic acid hydrogel modified with nogo-66 receptor antibody and poly-L-lysine to promote axon regrowth after spinal cord injury. J Biomed Mater Res B Appl Biomater 95:110–117 26. Lin CM, Lin JW, Chen YC, Shen HH, Wei L, Yeh YS, Chiang YH, Shih R, Chiu PL, Hung KS, Yang LY, Chiu WT (2009) Hyaluronic acid inhibits the glial scar formation after brain damage with tissue loss in rats. Surg Neurol 72(Suppl 2):S50–S54 27. Yamada T, Sawada R, Tsuchiya T (2008) The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes. Biomaterials 29:3503–3513 28. Tolwani RJ, Cosgaya JM, Varma S, Kuo LE, Shooter EM (2004) BDNF overexpression produces a long-term increase in myelin formation in the peripheral nervous system. J Neurosci Res 77:662–669 29. Han X, Yang N, Xu Y, Zhu J, Chen Z, Liu Z, Dang G, Song C (2011) Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487:255–259 30. Chen CH, Huang SY, Chen NF, Feng CW, Hung HC, Sung CS, Jean YH, Wen ZH, Chen WF (2013) Intrathecal granulocyte colony-stimulating factor modulate glial cell line-derived neurotrophic factor and vascular endothelial growth factor A expression in glial cells after experimental spinal cord ischemia. Neuroscience 242:39–52 31. Yang PW, Hsieh MS, Huang YC, Hsieh CY, Chiang TH, Lee JM (2014) Genetic variants of EGF and VEGF predict prognosis of patients with advanced esophageal squamous cell carcinoma. PLoS One 9:e100326 32. Rong W, Wang J, Liu X, Jiang L, Wei F, Hu X, Han X, Liu Z (2012) Naringin treatment improves functional recovery by increasing BDNF and VEGF expression, inhibiting neuronal apoptosis after spinal cord injury. Neurochem Res 37:1615–1623 33. Stern R (2004) Hyaluronan catabolism: a new metabolic pathway. Eur J Cell Biol 83:317–325 34. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–1313 35. Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Gallo RL (2004) Hyaluronan fragments stimulate endothelial recognition of injury though TLR4. J Biol Chem Biol Interact 279:17079–17084 36. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes. Biochem Pharmacol 80:480–490 37. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes. Biochimie 92:204–215
ORIGINAL PAPER
Hyaluronan Tetrasaccharide Exerts Neuroprotective Effect and Promotes Functional Recovery After Acute Spinal Cord Injury in Rats Jun Wang • Xiaofang Wang • Jie Wei Manyi Wang
•
Received: 25 June 2014 / Revised: 11 September 2014 / Accepted: 30 October 2014 / Published online: 6 November 2014 Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to explore the therapeutic efficiency of hyaluronan tetrasaccharide (HA4) treatment after spinal cord injury (SCI) in rats and to investigate the underlying mechanism. Locomotor functional and electrophysiological evaluations revealed that the behavioral function of rats in the HA4-treated group was significantly improved compared with the vehicletreated group. The expression of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), cluster determinant (CD44) and Toll-like receptor-4 (TLR-4) was obviously upregulated in the HA4treated group than that in the sham and vehicle-treated group. Furthermore, HA4 could induce BDNF and VEGF expression in the astrocytes in vitro. In addition, the high expression of BDNF and VEGF could be inhibited by blocking CD44 and TLR-4. These findings indicate that HA4 could be useful as a promising therapeutic agent for SCI and might exert the effect by interaction with the CD44 and TLR-4. Keywords Spinal cord injury Hyaluronan tetrasaccharide Astrocytes Neurotrophic factor
J. Wang J. Wei M. Wang (&) Traumatology Department,Beijing Jishuitan Hospital, No. 31 East Street of Xin Jie Kou Beijing, Beijing 100035, People’s Republic of China e-mail: [email protected] J. Wang Medical Center Tsinghua University, No. 30 Shuangqing Rd, Haidian District, Beijing 100084, People’s Republic of China X. Wang Department of Pharmacy, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, People’s Republic of China
123
Introduction Spinal cord injuries (SCI) are highly disabling and deadly injuries. With the modern traffic development, the incidence and prevalence of spinal injuries have been increasing, with the incidence rate of traumatic SCI estimated at 15–40 cases per million worldwide, although injury prevention initiatives have tried to reduce the occurrence of SCIs [1]. The adult central nervous system (CNS) has limited regenerative capacity and is unable to achieve full functional recovery after traumatic injury. Thus, the patients with complete SCI always have poor outcomes and the problem causes a huge economic burden to society and family. Hyaluronan (HA) was first described by Meyer and Palmer and composed of repeating units of disaccharides [-Dglucuronic-acid-b1, 3-N-acetyl-D-glucosamine b1, 4-]n [2]. It has been reported that different molecular weight HA has distinct biological function [2–4]. In the literature, Many studies have showed that low molecular weight (LMW)-HA has the neuroprotective effect on the nervous system. The previous study has demonstrated that NMDA-induced neuronal cell death was partially blocked by hyaluronan tetrasaccharide (HA4) in vitro, and HA4 promotes motor function recovery after SCI in vivo [5]. It has been reported that, at an optimal dose, HA4 promotes neural regeneration and axonal outgrowth [6]. In our previous study, it has been reported that HA4 in the cerebrospinal fluid was associated with self-repair of rats after chronic spinal cord compression. Furthermore, HA4 could induce the neurotrophic factor expression in the astrocytes in vitro and these phenomenons illustrated that LMW-HA4 could exert positive influence on the SCI [7]. Thus, we performed this experiment to assess the neuroprotective effect of HA4 on the injured spinal cord in the rats.
Neurochem Res (2015) 40:98–108
Materials and Methods A total of 36 Sprague–Dawley rats, aged 12 weeks (250–300 g), were used for the experiment. Anesthesia was induced with pentobarbital sodium (50 mg/kg). All protocols were approved by the animal care committee of the Peking University Health Science Center. We have minimized the number of animals used and their suffering. A laminectomy was carried out on the T8 vertebra. SCI was induced using the weight-drop A moderate SCI (force, 40 g/cm) was induced by dropping a weight of 10 g from a height of 4 cm onto an impounder (diameter, 0.2 cm) gently placed on the spinal cord. Sham animals received the same surgical procedure but sustained no impact injury. Muscle and skin were sutured in layers, and an antibacterial Baytril (3 mg/kg) was applied subcutaneously each morning for 3 days. After the operation, animals were maintained in the same postoperative condition of lying on a heated bed. At 24 h after SCI, a short behavior test was conducted. Only the rats with a Basso, Beattie, and Bresnahan (BBB) locomotor score of \1 were included in the study. The SCI animals were randomly divided into three groups (n = 12): sham group; vehicle-treated group; HA4treated group. HA4 or vehicle was administered to the injured site by the ALZET mini-osmotic pump with a catheter at 1 day after SCI for 4 weeks. Saline was applied to the vehicle-treated group. Neurobehavioral Evaluations Locomotor activity was evaluated in the rats (n = 12, per group) before surgery and at days 1, 3 postoperation, and then weekly until the rats were killed, using the BBB scoring system that measures locomotor ability during 5 min [8]. Two independent examiners who were blinded to experiment observed the hind limbs movements and scored the locomotor function according to the Basso, Beattie and Bresnahan (BBB) scale, wherein 0 indicates no motor activity and 21 indicates a normal performance. The score of each animal was obtained by averaging the values from both examiners. Electrophysiological Evaluations Animals (n = 12, per group) were anesthetized with pentobarbital sodium and placed in a prone position. At 10 weeks after the operation, electrophysiological tests were performed on both hind limbs using the Cadwell Cascade system (Cadwell Laboratories Inc., Kennewick, WA, USA). To assess the motor function after injury, motor evoked potentials (MEPs) were elicited by transcranial stimulation with two needle electrodes placed over the skull. The anode was placed over the sensorimotor
99
cortex and the cathode was placed on the hard palate. Single electrical pulses (30 V intensity, 100 ls duration, and train of four pulses) were delivered. MEPs were recorded from the tibialis anterior muscle and the reference electrode inserted into the footpad. The signals were amplified and filtered (amplifier gain of 10; bandpass frequency, 10–3,000 Hz). To ensure reproducibility, five consecutive responses were recorded, with a time interval of 30 s between stimuli. The recording with the average amplitude and latency was used for analysis. All signals of MEP were obtained on a computer by measuring the peak latency and peak-to-peak amplitude of the evoked potentials. Tissue Processing for Electron Microscopy Tissue (n = 3, per group) was post-fixed with osmium tetroxide and potassium ferrocyanide. Following five 10-min sodium cacodylate washes and five quick washes with 0.1 M acetate buffer, the tissue was stained with 1 % uranylacetate overnight at 4 °C. Five 10-min washes with acetate buffer were followed by progressive dehydration in 50, 70, 80, 90, 95, and 100 % ethanol, each with a 10-min duration. Three additional 10-min washes with 100 % ethanol were followed by two short-duration washes with propylene oxide and rotation overnight with 1:1 propylene oxide and epon. The tissue was then embedded in inverted polyethylene beam capsules with the tips removed and allowed to cure for 48 h at 60 °C under a vacuum of 20 mm Hg. Immunofluorescent Double-Labeling For tissue staining, 8 lm of spinal cord frozen sections (n = 4, per group) were permeabilized with 1 % Triton X-100 and 10 % normal goat serum at room temperature for 15 min. A monoclonal mouse anti-GFAP antibody (1:250, Santa Cruz Biotechnology) with either a polyclonal rabbit anti-BDNF antibody (1:500; Abcam) or a rabbit vascular endothelial growth factor (VEGF) antibody (1:500; Abcam) were applied to the sections at 4 °C overnight. Next, sections were incubated with an FITCconjugated goat anti-rabbit IgG antibody (1:200; SigmaAldrich) and a Texas Red-conjugated goat anti-mouse antibody (1:200; Sigma-Aldrich). Following immunostaining, all of the sections were coverslipped with Mowiol containing DAPI to counterstain the nuclei. Images were taken using a Leica DM3000. Western Blot Analysis For Western blotting, 1.5-cm-long spinal cord samples centered at the injured site from the sham, vehicle-treated,
123
100
and HA4-treated groups (n = 5, per group) were washed twice in ice-cold PBS. The samples were individually homogenized in 5 mM Tris–HCI (4 mM EDTA, pH 7.4, containing 1 lM pepstatin, 100 lM leupeptin, 100 lM phenylmethyl sulfonylfluoride, and 10 lg/ml aprotinin) and cleared by centrifugation at 14,000g for 10 min at 4 °C. Protein concentrations were determined by the Lowry method. Approximately 100 lg of protein were run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5 % skim milk in TBS containing 0.05 % Tween 20 and were incubated with the following primary antibodies: a polyclonal rabbit anti-VEGF antibody (1:1000; Abcam); and a rabbit anti-BDNF antibody (1:100; Abcam). The optical density (OD) of the signals was quantified and expressed as the ratio of OD of the tested proteins to that of b-actin. Astrocytic Primary Culture and Treatment For astrocytic cultures, the cortex was homogenized by mechanical fragmentation, and the cell suspension passed sequentially through steel screens of 230-, 104-, and 73.3lm pore size. Cells were subsequently collected by centrifugation at 700 g for 10 min and resuspended in DMEM supplemented with 10 % fetal calf serum. Finally, 2.0 9 105 cells/cm2 were plated on tin plates and maintained at 37 °C in a humidified atmosphere of 5 % CO2. The culture medium was replaced at day 7, and the confluent cultures were used at day 10. The detailed information was followed by the previous study [9]. The experimental cells were incubated in a medium containing 1,000 lg/ml of HA4 for 24 h. The control cells were cultured without HA4. A separate set of plates were also first treated with the specific antibody against either TLR-4 or CD44 alone or with both antibodies. Immunofluorescent double-labeling is performed for the cells with the procedure described previously using the following antibodies: a polyclonal mouse anti-GFAP antibody (1:250, Santa Cruz Biotechnology); a polyclonal rabbit anti-VEGF antibody (1:500; Abcam); and a rabbit antiBDNF antibody (1:500; Abcam). The control group, group treated with 1,000 lg/ml of HA4 and groups treated with the specific antibody against either TLR-4 or CD44 alone or with both antibodies were analyzed by the western blot analysis. Statistical Analysis Comparisons of data with more than two variables were analyzed by two-way repeated measure-ANOVAs with post hoc Tukey’s analysis. All data are plotted as the mean ± standard error.
123
Neurochem Res (2015) 40:98–108
Results Hyaluronan Tetrasaccharide Promotes Hindlimb Locomotion Recovery After SCI To assess if HA4 promoted the hindlimb locomotion recovery, we performed the BBB scores test. All animals had an initial BBB score of 21, and the injury resulted in a score of 0, with complete hindlimb paralysis. In the following weeks, 1000 lg/ml HA4-treated group (from 5 weeks post-SCI) showed more significant improvements compared with the vehicle-treated group (P \ 0.05), and sustained throughout the survival period. At 10 weeks after SCI, the average BBB scores for the HA4-treated rats was 16.51 ± 2.12. In comparison, although the vehicle-treated rats also regained some motor functional recovery due to the self-repair mechanism, their BBB score was significantly lower (11.13 ± 2.05) compared with HA4-treated rats (P \ 0.05; Fig. 1a). Electrophysiological changes were evaluated based on the amplitude and latency of MEPs. The representative MEP wave forms of sham, vehicle-treated, HA4-treated rats were showed in Fig. 1b. Interestingly, HA4-treated rats showed significantly higher amplitudes and shorter latencies than vehicle-treated at 10 weeks (P \ 0.05). Furthermore, there was no significant difference between the sham and HA4-treated rats (Fig. 1c). Hyaluronan Tetrasaccharide Induces BDNF and VEGF Expression in Astrocytes In Vivo After SCI To determine whether BDNF and VEGF were upregulated under HA4 treatment in astrocytes, we performed an immunofluorescent double-labeling and Western blot analysis at the epicenter of the injured sites. The results showed that the expression of BDNF and VEGF in the astrocytes in the HA4-treated group was significantly higher than that in the vehicle-treated group (P \ 0.05) (Fig. 2). Hyaluronan Tetrasaccharide Induces CD44 and TLR-4 Expression at the Injured Spinal Cord To investigate the expression of CD44 and TLR-4 under HA4 treatment in the astrocytes, we performed an immunofluorescent double-labeling analysis at the epicenter of the injured sites. We found that HA4 treatment could stimulate the CD44 and TLR-4 expression in the astrocytes. The expression of CD44 and TLR-4 in the HA4treated and vehicle-treated group was obviously increased compared with that in the sham group (P \ 0.05). Furthermore, There was significant difference between the HA4-treated and vehicle-treated group (P \ 0.05; Fig. 3).
Neurochem Res (2015) 40:98–108
101
Fig. 1 a HA4 improves behavioral recovery after SCI. We performed the BBB score test before and after SCI. The rats in the HA4-treated group showed significantly higher scores than that in the vehicletreated group from 5 weeks and sustained until sacrifice. b The representative waveforms of the MEPs in the sham, vehicle-treated
and HA4-treated groups. c Plots show the mean latency and amplitude of the MEPs. HA4-treated rats showed significantly higher amplitudes and shorter latencies than vehicle-treated at 10 weeks. *P \ 0.05, as compared with vehicle group; error bars represent SD
Hyaluronan Tetrasaccharide Promotes Axons Survival After SCI
the morphological change of astrocytes in vitro. The result showed that HA4 promoted dramatical stellation of astrocytes and it transformed the cell body of astrocytes into a thin cell body like a stella (Fig. 5).
To determine the condition of axons under HA4 treatment after SCI, we performed an electron micrograph analysis at the epicenter of the injured sites. The white matter from the sham tissue contained many axons encircled by compact myelin laminations. After SCI, ruptured axons were observed, with expelling their axoplasmic contents into the extracellular space. The conditions of the axons in the HA4-treated group were better than those in vehicle-treated group. Furthermore, the density of axons in the HA4-treated group was higher than that in the vehicle-treated group (Fig. 4).
Hyaluronan Tetrasaccharide Induces BDNF and VEGF Expression in Astrocytes In Vitro To observe whether HA4 could stimulate the BDNF and VEGF expression in astrocytes in vitro, we performed an immunofluorescent double-labeling analysis. We found that 1,000 lg/ml of HA4 obviously stimulated the BDNF and VEGF expression in astrocytes in vitro (Fig. 6).
Hyaluronan Tetrasaccharide Stimulates the Morphological Change of Astrocytes In Vitro
The BDNF and VEGF Expression Induced by Hyaluronan Tetrasaccharide was Blocked by the Antibody Against TLR-4 or CD44 In Vitro
To assess the effect of HA4 on the condition of astrocytes, we performed the immunofluorescent labeling to observe
To investigate whether the BDNF and VEGF expression induced by HA4 was blocked by the antibody
123
102 Fig. 2 a The upregulation of BDNF and VEGF expression in the immunofluorescent doublelabeling experiment. Scale bar 1,000 and 100 lm. b Western blot analysis also showed that BDNF and VEGF expression was increased under the HA4 treatment after SCI. #P \ 0.05, as compared with vehicle group, error bars represent SD
123
Neurochem Res (2015) 40:98–108
Neurochem Res (2015) 40:98–108
103
Fig. 3 The induction effect of HA4 treatment on the expression of CD44 and TLR-4 in vivo. The expression of CD44 and TLR-4 in the HA4-treated group was significantly increased compared with that in
the vehicle-treated group. *P \ 0.05, as compared with sham group; # P \ 0.05, as compared with vehicle group, error bars represent SD. Scale bar 100 lm
against TLR-4 or CD44, we performed the immunofluorescent double-labeling and western blot analysis of cultured astrocytes. In the immunofluorescent double-labeling analysis, the astrocyte didn’t expressed the BDNF without HA4 treatment (Fig. 7A1, A2, A). The BDNF expression in the astrocytes was upregulated by either blocking TLR-4 or CD44 (Fig. 7B1, B2, B and C1, C2, C). When both of TLR-4 and CD44 were blocked, the BDNF didn’t expressed in the astrocytes (Fig. 7D1, D2, D). Meanwhile, in the Western blot analysis, the results were similar to that in the immunofluorescent double-labeling analysis. Overall, the result showed that HA4 could stimulate the BDNF expression in astrocytes by interaction with either TLR-4 or CD44 (Fig. 8).
Discussion This study demonstrates that treatment with HA4 could improve the functional recovery by upregulating BDNF and VEGF expression after SCI in the rats. Spinal cord injuries (SCIs) can be divided into two different groups: traumatic spinal cord injuries (TSCI) and non-traumatic spinal cord injuries (NTSCI). Traumatic SCI evolves through three phases: the acute, mostly due to the direct mechanical damage, the secondary, and the chronic stages [10]. Mounting evidence indicates that glial scars constitute a major component of the post-injury environment that discourages spontaneous axonal regeneration [11–16]. Astrocytes expressing glial fibrillary acidic protein (GFAP) become hypertrophic and highly proliferative
123
104
Neurochem Res (2015) 40:98–108
Fig. 4 The condition of axons under HA4 treatment after SCI by the electron micrograph analysis. The density of axons in the HA4-treated group was higher than that in the vehicletreated group. Scale bar 10 lm. *P \ 0.05, as compared with sham group; #P \ 0.05, as compared with vehicle group, error bars represent SD
Fig. 5 The condition of astrocytes under HA4 treatment. HA4 promoted the morphological change of astrocytes in vitro
to form eventually a dense network of glial scars around the cavity [17–19]. Thus, modulating the condition of astrocytes is an important direction of the therapy.
123
It has been reported that different molecular weight HA has distinct biological effect. High molecular weight hyaluronic acid (HA) is an essential organizational and structural
Neurochem Res (2015) 40:98–108
105
Fig. 6 The effect of HA4 on the BDNF and VEGF expression in astrocytes in vitro. The intensity of BDNF and VEGF expression in astrocytes under 1,000 lg/ml of HA4 treatment was significantly higher than that under 0 lg/ml of HA4 treatment
component in the ECM of native tissues [20, 21]. High molecular weight hyaluronan (HMW-HA) surrounds neuronal cell bodies in the nervous system and maintains astrocytes in a state of quiescence. Furthermore, the degradation of HA following SCI relieves the astrocyte growth inhibition, resulting in increased astrocyte proliferation [22]. Seidlits et al. [23] reported that hyaluronic acid hydrogels could induce the neural progenitor cell differentiation. Austin et al. [24] revealed that intrathecal injection of
hyaluronic acid hydrogels reduced the extent of fibrosis and inflammation in the subarachnoid space, promoted neurobehavioral recovery, enhanced axonal conduction and reduced the extent of post-traumatic parenchymal fibrous scar formation. Wei et al. [25] reported that hyaluronic acid hydrogel modified with nogo-66 receptor antibody and polyL-lysine promoted axon regrowth after SCI. The previous studies illustrated that HMW-HA had the positive and negative effects on the functional recovery in the acute SCI.
123
106
Neurochem Res (2015) 40:98–108
Fig. 7 The immunofluorescent double-labeling analysis showed the expression of BDNF in astrocyte blocked by the antibody against TLR-4 or CD44 receptors. (A1, A2, A) the astrocytes without HA4 treatment. (B1, B2, B and C1, C2, C) Under HA4 treatment, the
astrocytes treated by the specific antibody against either TLR-4 or CD44 alone. (D1, D2, D) Under HA4 treatment, the astrocytes treated by both of specific antibodies against TLR-4 and CD44. Scale bar 10 lm
In the literature, many studies have revealed that low molecular weight (LMW)-HA could exert a profound neuroprotective effect on the injured nervous system [6, 26, 27]. It has been reported that hyaluronan tetrasaccharide (HA4) could induce astrocytes to express neurotrophic factor [7, 27]. In the previous study, we found that HA4 could induce astrocytes to express more BDNF and VEGF in vitro and was involved in the self-repair process of chronic spinal cord compression injury [7]. In this study, the results showed that HA4 could improve the motor function and induce the neurotrophic factor expression in the astrocytes. It illustrated that HA4 has the neuroprotective effect on the injured spinal cord. In our study, we determined the effects of hyaluronan tetrasaccharide treatment on the expression of BDNF and VEGF. Brain-derived neurotrophic factor (BDNF) is an important neurotrophic factor. It exerts an positive
influence on the survival of neurons and enhances the remyelination of injured spinal cord [28, 29]. Vascular endothelial growth factor (VEGF) constitutes a sub-family of growth factors that stimulate the growth of new blood vessels and it is an important signaling protein involved in the vasculogenesis for injured tissue [30, 31]. Many studies have revealed that the improved functional recovery after SCI may contribute to the high expression of BDNF and VEGF [7, 29, 32]. In our study, administration of HA4 to the damaged site of spinal cord induced the expression of BDNF and VEGF in the astrocytes in vivo. Meanwhile, HA4 could also stimulate BDNF and VEGF expression of astrocytes in vitro. Thus, the modulating astrocytes is an important treatment and could get astrocytes to perform its positive effect for the SCI. The cluster determinant 44 (CD44) and toll-like receptor 4 (TLR-4) are the two best-known receptor of HA. It has
123
Neurochem Res (2015) 40:98–108
107
Fig. 8 The Western blot analysis showed the expression of BDNF in astrocyte blocked by the antibody against TLR-4 or CD44. *P \ 0.05, as compared with control group, error bars represent SD
been reported that the profile of expression of CD44 and TLR-4 was modulated by high level of hyaluronidase and it provided the exquisite condition for the regulatory mechanisms [33]. CD44 stimulation with HA plays the role of cell adhesion, cell–substrate interaction, and inflammation [34]. Toll-like receptors are a class of proteins that play a key role in the innate immune system. the glycosaminoglycan (GAG) hyaluronan (HA) has also been identified as an inducer of TLR-4 activation, capable of causing the release of pro-inflammatory cytokines [35]. Low molecular weight of HA are able to interact with TLR-4 and CD44 and the previous studies showed that HA interact with TLR-4 and CD44 was involved in the process of inflammation [36, 37]. In our study, the HA4 stimulation of BDNF and VEGF effect on astrocytes was suppressed by blocking the TLR-4 and CD44. Either blocking TLR-4 or CD44 could partially decrease BDNF and VEGF expression. From the results of immunofluorescent double-labeling and western blot analysis, the neuroprotective mechanism of HA4 may be exerted by interacting with CD44 and TLR-4. The expression of CD44 and TLR-4 at the injuried spinal cord was upregulated under HA4 treatment. There are some limitations to our findings. In this study, we didn’t assess the effect of low dose HA4 treatment on
the SCI. The choice of 1,000 lg/ml was mainly designed based on the previous study [7]. In this study, we only tested the effect of HA4 on the interaction of astrocytes and modulating the neurotrophic factor of low dose was unclear. We aim to attract much attention on the hyaluronan therapeutic effect on the SCI patients. But this aspect of study is in its infancy and the exact mechanism of HA on the SCI proves inconclusive. Thus, the basic research is in need of further study and the further study of HA is in the process of further investigation. In conclusion, the present study has shown that HA4 could improve the functional recovery by upregulating BDNF and VEGF expression after SCI in the rats and may have therapeutic potential for SCI in the clinical.
Conflict of interest I confirm on behalf of all the authors that there is no conflict of interest issues related to this work.
References 1. Jackson AB, Dijkers M, Devivo MJ, Poczatek RB (2004) A demographic profile of new traumatic spinal cord injuries: change and stability over 30 years. Arch Phys Med Rehabil 85:1740–1748
123
108 2. Meyer K, Palmer J (1934) The polysaccharide of the vitreous humor. J Biol Chem 107:629–634 3. Atkins EDT, Phelps CF, Sheehan JK (1972) The conformation of the mucopolysaccharides. Biochem J 128:1255–1263 4. Almond A (2007) Hyaluronan. Cell Mol Life Sci: CMLS 64:1591–1596 5. Wakao N, Imagama S, Zhang H, Tauchi R, Muramoto A, Natori T, Takeshita S, Ishiguro N, Matsuyama Y, Kadomatsu K (2011) Hyaluronan oligosaccharides promote functional recovery after spinal cord injury in rats. Neurosci Lett 488:299–304 6. Torigoe K, Tanaka HF, Ohkochi H, Miyasaka M, Yamanokuchi H, Yoshidad K, Yoshida T (2011) Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: in vivo analysis by film model method. Brain Res 1385:87–92 7. Wang J, Rong W, Hu X, Liu X, Jiang L, Ma Y, Dang G, Liu Z, Wei F (2012) Hyaluronan tetrasaccharide in the cerebrospinal fluid is associated with self-repair of rats after chronic spinal cord compression. Neuroscience 210:467–480 8. Basso D, Beattie M, Bresnahan J (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1–21 9. Zhu Z, Zhang Q, Yu Z, Zhang L, Tian D, Zhu S, Bu B, Xie M, Wang W (2007) Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo. Glia 55:546–558 10. Tator CH, Fehlings MG (1991) Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 75:15–26 11. Wang J, Ma C, Rong W, Jing H, Hu X, Liu X, Jiang L, Wei F, Liu Z (2012) Bog bilberry anthocyanin extract improves motor functional recovery by multifaceted effects in spinal cord injury. Neurochem Res 37:2814–2825 12. Sharp K, Yee KM, Steward O (2012) A re-assessment of the effects of treatment with an epidermal growth factor receptor (EGFR) inhibitor on recovery of bladder and locomotor function following thoracic spinal cord injury in rats. Exp Neurol 233:649–659 13. Chen L, Wang Z, Ghosh-Roy A, Hubert T, Yan D, O’Rourke S, Bowerman B, Wu Z, Jin Y, Chisholm AD (2011) Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71:1043–1057 14. Jager C, Lendvai D, Seeger G, Bruckner G, Matthews RT, Arendt T, Alpar A, Morawski M (2013) Perineuronal and perisynaptic extracellular matrix in the human spinal cord. Neuroscience 238:168–184 15. Jeong SR, Kwon MJ, Lee HG, Joe EH, Lee JH, Kim SS, Suh-Kim H, Kim BG (2012) Hepatocyte growth factor reduces astrocytic scar formation and promotes axonal growth beyond glial scars after spinal cord injury. Exp Neurol 233:312–322 16. Huang X, Kim JM, Kong TH, Park SR, Ha Y, Kim MH, Park H, Yoon SH, Park HC, Park JO, Min BH, Choi BH (2009) GM-CSF inhibits glial scar formation and shows long-term protective effect after spinal cord injury. J Neurol Sci 277:87–97 17. Bradbury EJ, Carter LM (2011) Manipulating the glial scar: chondroitinase ABC as a therapy for spinal cord injury. Brain Res Bull 84:306–316 18. Hunanyan AS, Garcia-Alias G, Alessi V, Levine JM, Fawcett JW, Mendell LM, Arvanian VL (2010) Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord. J Neurosci Off J Soc Neurosci 30:7761–7769 19. Herrmann JE, Shah RR, Chan AF, Zheng B (2010) EphA4 deficient mice maintain astroglial-fibrotic scar formation after spinal cord injury. Exp Neurol 223:582–598 20. Sherman LS, Struve JN, Rangwala R, Wallingford NM, Tuohy TM, Kuntz CT (2002) Hyaluronate-based extracellular matrix: keeping glia in their place. Glia 38:93–102
123
Neurochem Res (2015) 40:98–108 21. Cheng G, Swaidani S, Sharma M, Lauer ME, Hascall VC, Aronica MA (2011) Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma. Matrix Biol J Int Soc Matrix Biol 30:126–134 22. Struve J, Maher PC, Li YQ, Kinney S, Fehlings MG, Kuntz CT, Sherman LS (2005) Disruption of the hyaluronan-based extracellular matrix in spinal cord promotes astrocyte proliferation. Glia 52:16–24 23. Seidlits SK, Khaing ZZ, Petersen RR, Nickels JD, Vanscoy JE, Shear JB, Schmidt CE (2010) The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation. Biomaterials 31:3930–3940 24. Austin JW, Kang CE, Baumann MD, DiDiodato L, Satkunendrarajah K, Wilson JR, Stanisz GJ, Shoichet MS, Fehlings MG (2012) The effects of intrathecal injection of a hyaluronan-based hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis. Biomaterials 33:4555–4564 25. Wei YT, He Y, Xu CL, Wang Y, Liu BF, Wang XM, Sun XD, Cui FZ, Xu QY (2010) Hyaluronic acid hydrogel modified with nogo-66 receptor antibody and poly-L-lysine to promote axon regrowth after spinal cord injury. J Biomed Mater Res B Appl Biomater 95:110–117 26. Lin CM, Lin JW, Chen YC, Shen HH, Wei L, Yeh YS, Chiang YH, Shih R, Chiu PL, Hung KS, Yang LY, Chiu WT (2009) Hyaluronic acid inhibits the glial scar formation after brain damage with tissue loss in rats. Surg Neurol 72(Suppl 2):S50–S54 27. Yamada T, Sawada R, Tsuchiya T (2008) The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes. Biomaterials 29:3503–3513 28. Tolwani RJ, Cosgaya JM, Varma S, Kuo LE, Shooter EM (2004) BDNF overexpression produces a long-term increase in myelin formation in the peripheral nervous system. J Neurosci Res 77:662–669 29. Han X, Yang N, Xu Y, Zhu J, Chen Z, Liu Z, Dang G, Song C (2011) Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487:255–259 30. Chen CH, Huang SY, Chen NF, Feng CW, Hung HC, Sung CS, Jean YH, Wen ZH, Chen WF (2013) Intrathecal granulocyte colony-stimulating factor modulate glial cell line-derived neurotrophic factor and vascular endothelial growth factor A expression in glial cells after experimental spinal cord ischemia. Neuroscience 242:39–52 31. Yang PW, Hsieh MS, Huang YC, Hsieh CY, Chiang TH, Lee JM (2014) Genetic variants of EGF and VEGF predict prognosis of patients with advanced esophageal squamous cell carcinoma. PLoS One 9:e100326 32. Rong W, Wang J, Liu X, Jiang L, Wei F, Hu X, Han X, Liu Z (2012) Naringin treatment improves functional recovery by increasing BDNF and VEGF expression, inhibiting neuronal apoptosis after spinal cord injury. Neurochem Res 37:1615–1623 33. Stern R (2004) Hyaluronan catabolism: a new metabolic pathway. Eur J Cell Biol 83:317–325 34. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–1313 35. Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Gallo RL (2004) Hyaluronan fragments stimulate endothelial recognition of injury though TLR4. J Biol Chem Biol Interact 279:17079–17084 36. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes. Biochem Pharmacol 80:480–490 37. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes. Biochimie 92:204–215
