Neuroscience Letters 593 (2015) 13–18
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
A combination of keratan sulfate digestion and rehabilitation promotes anatomical plasticity after rat spinal cord injury Yoshimoto Ishikawa a,b , Shiro Imagama a , Tomohiro Ohgomori c , Naoki Ishiguro a , Kenji Kadomatsu b,∗ a
Department of Biochemistry, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan Department of Orthopedics, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan c Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan b
h i g h l i g h t s • • • • •
KS-digestion plus rehabilitation was evaluated in a spinal cord injury model. KS-digestion/rehabilitation tended to improve functional plasticity. KS-digestion and rehabilitation synergistically improved anatomical plasticity. This effect was comparable with that of CS-digestion/rehabilitation. KS-digestion/rehabilitation might widen the therapeutic window of neuronal injury.
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 11 February 2015 Accepted 9 March 2015 Available online 12 March 2015 Keywords: Spinal cord injury Keratan sulfate Rehabilitation Keratanase II Chondroitinase ABC Chondroitin sulfate
a b s t r a c t Functional recovery after neuronal injuries relies on neuronal network reconstruction which involves many repair processes, such as sealing of injured axon ends, axon regeneration/sprouting, and construction and refinement of synaptic connections. Chondroitin sulfate (CS) is a major inhibitor of axon regeneration/sprouting. It has been reported that the combination of task-specific rehabilitation and CSdigestion is much more effective than either treatment alone with regard to the promotion of functional and anatomical plasticity for dexterity in acute and chronic spinal cord injury models. We previously reported that keratan sulfate (KS) is another inhibitor and has a potency equal to CS. Here, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury. Keratanase II or chondroitinase ABC was locally administered at the lesion after spinal cord injury at C3/4. Task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus, was done for 3 weeks before injury, and then again at 1–6 weeks after injury. The combination of KS-digestion and rehabilitation yielded a better rate of pellet removal than either KS-digestion alone or rehabilitation alone, although these differences were not statistically significant. The combination of CS-digestion and rehabilitation showed similar results. Strikingly, both KS-digestion/rehabilitation and CS-digestion/rehabilitation showed significant increases in neurite growth in vivo as estimated by 5-hydroxytryptamine and GAP43 staining. Thus, KS-digestion and rehabilitation exerted a synergistic effect on anatomical plasticity, and this effect was comparable with that of CS-digestion/rehabilitation. KS-digestion might widen the therapeutic window of spinal cord injury if combined with rehabilitation. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Functional recovery after neuronal injuries relies on the reconstruction of neuronal networks. Maximization of these functional
∗ Corresponding author at: Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel.: +81 52 744 2059; fax: +81 52 744 2060. E-mail address: [email protected] (K. Kadomatsu). http://dx.doi.org/10.1016/j.neulet.2015.03.015 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
and anatomical plasticities is desirable as a therapy for various diseases, such as spinal cord injury (SCI) and stroke. However, it has been hard to achieve ideal reconstruction of neuronal networks, and no efficient therapeutics have been established for these diseases. On the other hand, it has become accepted that rehabilitation promotes functional recovery to some extent [1,2,6], probably by promoting the reconstruction and refinement of specific networks. Reconstruction of neuronal networks may involve several processes, i.e., sealing of injured axon ends, axon regeneration/
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Fig. 1. In vivo experimental design. (A). In schematic cross section of the spinal cord. The red area indicates the unilateral dorsolateral lesion at level C3/4, which includes the unilateral dorsal CST. (B). Flow of the experiments. (C). The single pellet reaching training required forearm training. (D). PKC-␥ staining was performed to assess the CST lesion. At the C6 transverse section (caudal to the lesion), PKC-␥ could not be detected on the lesion side. Scale bar, 200 m.
sprouting, and construction and refinement of synaptic connections. Therefore, a comprehensive understanding of these processes is required to establish therapies for neuronal injuries [9]. Axons of the adult mammalian central nervous system (CNS) do not regenerate or sprout after injuries due to the low intrinsic regeneration capacity and emerging inhibitory molecules. Chondroitin sulfate (CS) is a strong inhibitor for axon regeneration/sprouting [13]. Ablation of CS by its degrading enzyme chondroitinase ABC (C-ABC) promotes not only axon regeneration/sprouting but also functional recovery [3,12]. Furthermore, CS-digestion/task-specific rehabilitation improved dexterity recovery in both acute and chronic SCI [4,16]. CS belongs to a class of long sugar chains known as glycosaminoglycans, which are composed of repeating disaccharide units. Along with CS, keratan sulfate (KS), heparan sulfate, and hyaluronan belong to the glycosaminoglycans. We previously reported that KS acts as an inhibitor for axon regeneration/sprouting [7,8]. 5D4-reactive KS-deficient mice (GlcNAc6ST-1 knockout) showed enhanced axon regeneration/sprouting and better motor function recovery as compared with wild-type mice [8]. Local administration of keratanase II (K-II), a KS-specific degradative enzyme, can also ameliorate SCI [7]. Notably, the effect of KS-digestion is comparable to that of CS-digestion [7]. In this study, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury.
2.2. Surgical procedure
2. Material and methods
The animals were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After C4 laminectomy, we exposed the dura mater and induced a dorsolateral cut injury between C3 and C4 ipsilateral to the preferred paw (Fig. 1A). A cut was made with the tips of sharpened fine micro-blade inserted 2 mm in depth into the spinal cord. This injury included the descending dorsal CST and the ascending sensory dorsal columns. Immediately, after the dorsolateral cut injury, we performed C6 partial laminectomy and inserted a thin silicone tube with an osmotic mini-pump into the subarachnoid cavity, and set the tube tip at the C3 level under a surgical microscope. This tube was sufficiently soft and thin that we could minimize damage to the spinal cord. The osmotic mini-pumps (Model 2006; ALZET, Cupertino, CA; 200 l of solution, 0.5 l/h, 14 d delivery) were filled with K-II (0.05 U/200 l; Seikagaku), C-ABC (0.05 U/200 l; Seikagaku), or saline (as a vehicle control). The tube was sutured to the spinous process to anchor it in place, and the mini-pump was placed under the skin on the animal’s back. Afterward, the muscles and skin were closed in layers [7,14]. The dose of C-ABC or K-II was decided from a previous report [7]. One day following SCI, rats were visually inspected and evaluated in regard to the degree of paralysis. Rats with bilateral deficits or deficits of the shoulder were excluded from the study in all groups as they were inappropriate for further evaluation [5]. All animals were treated and cared for in accordance with the Nagoya University School of Medicine Guidelines pertaining to the treatment of experimental animals.
2.1. Animal and experimental groups
2.3. Single pellet reaching task
Adult female Sprague–Dawley rats weighing 200–230 g were used in this SCI study. All the animals (N = 58) received the SCI at C3/4, and K-II, C-ABC or saline with or without training in the single pellet reaching task. The animals were divided into six experimental groups: a K-II training group (N = 5); K-II no-training group (N = 5), C-ABC training group (N = 5), C-ABC no-training group (N = 5), saline training group (N = 5), and saline no-training group (N = 5). For histological analysis of the association of PNNs and KS, un-injured normal rats (N = 3) were used.
Before SCI, rats were trained to reach through a slot (1.5 cm wide) in an acrylic box (15 cm × 36 cm × 30 cm) to grasp food pellets of 45 mg each (Bio-serv, USA). The pellets were set in a small indentation on a tray and offered one at a time (the pellets were 2 cm away from the front wall at a height of 3 cm above the elevated grid floor) [11]. Success rates per session were calculated as the number of pellets successfully grasped and eaten out of 20 pellets offered. Starting on day 7 after SCI, training was performed for 20 min per day, 5 days per week for 5 weeks (Fig. 1B and C).
Y. Ishikawa et al. / Neuroscience Letters 593 (2015) 13–18
Un-trained rats were reintroduced to the training for 2 days before final testing. After baseline testing and final testing, the recovery rates to baseline were calculated, and then statistical analysis (oneway ANOVA) was performed. Values of P < 0.05 were defined as statistically significant. 2.4. Immunohistochemistry Three weeks after final testing, rats were killed with transcardial PBS perfusion followed by 4% paraformaldehyde. The spinal cords were dissected and post-fixed at 4 ◦ C overnight in 4% paraformaldehyde buffer, followed by 30% sucrose for 48 h. Tissue was frozen in OCT mounting medium and then cut into transverse 20 m sections with a cryostat and processed for immunohistochemistry. The sections were blocked in PBS containing 10% normal goat serum and 0.1% Triton X-100 at room temperature for immunohistochemistry. For visualization of CST damage, protein kinase C-␥ (1:500), a marker for the CST, was used with the sections at C2 (rostral to the lesion site) and C6 (caudal to the lesion site). For visualization of 5-HT- or Gap 43-positive fibers, sections were immunostained with primary antibody 5-HT (1:1000; Immunostar) and Gap43 (1:2000; Millipore), and then
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incubated with the Alexa Fluor 568 goat anti-rabbit IgG (1:2000; Invitrogen) for 1 h at room temperature. For histological analysis of the association between PNNs and KS, transverse sections from an uninjured rat were incubated with NeuN (1:500; Millipore), BCD4 anti-KS antibody (1:500; Seikagaku), lectin Wisteria fluribunda (WFA) (1:150; Sigma) and anti-aggrecan (1:300; Millipore) after blocking. After rinsing, the sections were incubated with the secondary antibody for 1 h at room temperature: Alexa Fluor 488–conjugated streptavidin (1:400; Invitrogen), alexa Fluor 568 goat anti-mouse IgG (1:2000, Invitrogen), and Alexa Fluor 568 goat anti-rabbit IgG (1:2000; Invitrogen). For evaluation of the effect of K-II or C-ABC, after blocking with normal goat serum and 0.1% Triton X-100, the sections were treated with C-ABC (0.5 U/ml) or K-II (0.5 U/ml) at 37 ◦ C for 1 h, and then incubated with the primary antibodies. For quantification of 5HT- and GAP43-positive fibers, we measured the axonal density at C2, a region close and rostral to the lesion. The total pixels of these positive fibers were calculated by ImageJ software, and statistical analysis (one-way ANOVA) was performed. Values of P < 0.05 were defined as statistically significant.
Fig. 2. Keratan sulfate was present in the PNNs. (A–C). Fluorescence images of WFA surrounding NeuN-positive neurons reveal the PNN structure. (D–F). Fluorescence images of BCD4 surrounding neurons reveal the KS in the PNN structure. (G–I). BCD4-positive KS co-localized with the PNN marker WFA. J-L. Magnified images of (G–I). Scale bar, 50 m.
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3. Results 3.1. SCI and rehabilitation protocol Rats received a C3/4 dorsolateral cut followed by C-ABC, K-II or saline as a control (Fig. 1A). The cut was made ipsilateral to the preferred paw. Rats either received or did not receive task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus (Fig. 1B and C). The rehabilitation training was started 3 weeks before injury, and was restarted 1 week after injury. The rehabilitation continued until 6 weeks after injury, when manual dexterity was estimated. Rats were finally killed 9 weeks after injury for histological analyses. Positivity for PKC-␥, a marker for the CST fibers, was observed in the CST on both sides at C2, which was rostral to the lesion, whereas PKC-␥ staining was negative on the side ipsilateral to the lesion at C6, which was caudal to the lesion (Fig. 1D). 3.2. KS was present in perineuronal nets Perineuronal nets (PNNs) are involved in various kinds of neural plasticity, and may also be important for plasticity after injury [4,16]. Since it is known that CS proteoglycans (CSPGs) are major components of PNNs, we wondered whether KS proteoglycans (KSPGs) would be detectable in PNNs. The lectin Wisteria floribunda agglutinin (WFA)-positive perineuronal nets surrounded a subset of neurons which expressed NeuN (Fig. 2A and C). The antibody BCD4 that specifically recognized low-sulfated KS also stained a subset of NeuN-positive neurons (Fig. 2D–F). Indeed, most of the WFA-positive neurons were BCD4-positive (Fig. 2G–L). It is believed that WFA recognizes 4-O-sulfation of CS. Consistent with this idea, C-ABC treatment completely abolished WFA staining, but did not affect KS expression as detected by BCD4 (data not shown). K-II treatment completely removed BCD4-reactive KS expression, but did not affect WFA staining (data not shown). Taken together, these results suggested that both CS and KS were involved in PNNs, and were completely digested by C-ABC and K-II, respectively. 3.3. Effects of KS digestion and rehabilitation on functional recovery after SCI We next estimated the effects of the combination of KS or CS digestion plus rehabilitation on functional recovery. Since it was previously reported that task-specific rehabilitation, but not general rehabilitation, enhanced the effect of C-ABC on recovery of dexterity [4], we employed a Whishaw apparatus to train rats in a single pellet removal task. As expected, the task-specific rehabilitation alone could improve recovery regardless of CSor KS-digestion (Fig. 3). The combination of rehabilitation and KS- or CS-digestion tended to achieve better recovery than that of rehabilitation and saline, but the difference was not significant (Fig. 3). These results were consistent with a previous report on CS-digestion/rehabilitation [4]. It is noteworthy that KSdigestion/rehabilitation showed almost the same outcome as the combination of CS-digestion/rehabilitation (Fig. 3). 3.4. Anatomical plasticity was promoted by KS-digestion/rehabilitation We next investigated anatomical plasticity. To this end, we quantified serotonergic neuron fibers (5HT-positive fibers) and regrowing neurites (GAP43-positive fibers). We found a striking increase in 5HT-positive fibers in KS-digestion/rehabilitation as compared with KS-digestion alone or saline/rehabilitation (Fig. 4). This effect was comparable with that of CS-digestion/rehabilitation (Fig. 4). We also found that GAP43-positive fibers exhibited a
Fig. 3. Recovery of dexterity. The trained animals showed significant improvement compared to the untrained baseline levels in each treatment group. However, compared with each trained rats with saline, C-ABC or K-2, there were no significant difference.
significant increase in response to KS-digestion/rehabilitation as compared with KS-digestion alone or saline/rehabilitation (Fig. 4). This effect was also similar to that of CS-digestion/rehabilitation (Fig. 4). 4. Discussion Here, we demonstrated that KS-digestion, when combined with task-specific rehabilitation training, showed a synergistic effect on anatomical plasticity which was as strong as that of CS-digestion. Therefore, these findings confirmed the concept that KS and CS play closely related roles in the inhibition of anatomical plasticity after neuronal injury. Improved anatomical plasticity may reflect an improvement in functional plasticity. However, we could not observe a significant difference between KS-digestion/rehabilitation and KS-digestion alone, although the former tended to more effective. This was also the case with respect to CS-digestion/rehabilitation versus CS-digestion alone. These results may reflect a limitation of the dexterity assessment employed, i.e., single pellet removal with a Whishaw apparatus. Indeed, these data are consistent with a previous report by Garcia-Alias et al. [4]. It must be kept in mind that K-II and C-ABC are both originated from bacteria. The repeated use of these reagents may not be practical because of their immunogenicity. Therefore, alternative methods, i.e., small compounds inhibiting KS or CS production, and human enzymes that exert effects similar to K-II and C-ABC, may be taken into consideration for clinical application [15]. Furthermore, it has recently been reported that a membrane-permeable peptide mimetic of the wedge domain of PTP, a receptor for CS, binds to PTP, serving as its inhibitor, and successfully promotes functional recovery after SCI [10]. Thus, the targeting of CS- or KS-receptors is an alternative approach for ameliorating neuronal injury. In this context, it would also be an intriguing approach to use a sugar chain mimetic of a CS or KS functional stretch to promote anatomical plasticity.
Y. Ishikawa et al. / Neuroscience Letters 593 (2015) 13–18
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Fig. 4. Histological analysis of regrown 5HT fibers and regrown GAP43 fibers. (A). 5-HT-positive fibers were counted at C2 (rostral to the lesion site). The fibers were calculated as the sum of 5HT-positive pixels. Quantification of axonal sprouting of 5HT staining in grey matter was performed. (B). Low magnification image of a spinal cord section. (C). An enlargement of the boxed area from (B). (D). An enlargement of the boxed area from (C). (E). Saline control. (F). Quantification of neurite regrowth by GAP43 staining in grey matter is shown. Scale bar, 300 m.
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Acknowledgments This work was supported in part by Grants-in-Aid (No. 23110002 to K.K. and No. 22791370 to S.I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] D.L. Adkins, J.E. Hsu, T.A. Jones, Motor cortical stimulation promotes synaptic plasticity and behavioral improvements following sensorimotor cortex lesions, Exp. Neurol. 212 (2008) 14–28. [2] L.D. Beazley, J. Rodger, P. Chen, L.B. Tee, R.V. Stirling, A.L. Taylor, S.A. Dunlop, Training on a visual task improves the outcome of optic nerve regeneration, J. Neurotrauma 20 (2003) 1263–1270. [3] E.J. Bradbury, L.D. Moon, R.J. Popat, V.R. King, G.S. Bennett, P.N. Patel, J.W. Fawcett, S.B. McMahon, Chondroitinase ABC promotes functional recovery after spinal cord injury, Nature 416 (2002) 636–640. [4] G. Garcia-Alias, S. Barkhuysen, M. Buckle, J.W. Fawcett, Chondroitinase ABC treatment opens a window of opportunity for task-specific rehabilitation, Nat. Neurosci. 12 (2009) 1145–1151. [5] J. Girgis, D. Merrett, S. Kirkland, G.A. Metz, V. Verge, K. Fouad, Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery, Brain 130 (2007) 2993–3003. [6] D.A. Hovda, D.M. Fenney, Amphetamine with experience promotes recovery of locomotor function after unilateral frontal cortex injury in the cat, Brain Res. 298 (1984) 358–361. [7] S. Imagama, K. Sakamoto, R. Tauchi, R. Shinjo, T. Ohgomori, Z. Ito, H. Zhang, Y. Nishida, N. Asami, S. Takeshita, N. Sugiura, H. Watanabe, T. Yamashita, N. Ishiguro, Y. Matsuyama, K. Kadomatsu, Keratan sulfate restricts neural plasticity after spinal cord injury, J. Neurosci. 31 (2011) 17091–17102. [8] Z. Ito, K. Sakamoto, S. Imagama, Y. Matsuyama, H. Zhang, K. Hirano, K. Ando, T. Yamashita, N. Ishiguro, K. Kadomatsu, N-acetylglucosamine
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6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury, J. Neurosci. 30 (2010) 5937–5947. K. Kadomatsu, K. Sakamoto, Mechanisms of axon regeneration and its inhibition: roles of sulfated glycans, Arch. Biochem. Biophys. 558 (2014) 36–41. B.T. Lang, J.M. Cregg, M.A. DePaul, A.P. Tran, K. Xu, S.M. Dyck, K.M. Madalena, B.P. Brown, Y.L. Weng, S. Li, S. Karimi-Abdolrezaee, S.A. Busch, Y. Shen, J. Silver, Modulation of the proteoglycan receptor PTPsigma promotes recovery after spinal cord injury, Nature 518 (2015) 404–408. C.P. Montoya, L.J. Campbell-Hope, K.D. Pemberton, S.B. Dunnett, The staircase test: a measure of independent forelimb reaching and grasping abilities in rats, J. Neurosci. Methods 36 (1991) 219–228. L.D. Moon, R.A. Asher, K.E. Rhodes, J.W. Fawcett, Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC, Nat. Neurosci. 4 (2001) 465–466. D.M. Snow, V. Lemmon, D.A. Carrino, A.I. Caplan, J. Silver, Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro, Exp. Neurol. 109 (1990) 111–130. K. Takeuchi, N. Yoshioka, S. Higa Onaga, Y. Watanabe, S. Miyata, Y. Wada, C. Kudo, M. Okada, K. Ohko, K. Oda, T. Sato, M. Yokoyama, N. Matsushita, M. Nakamura, H. Okano, K. Sakimura, H. Kawano, H. Kitagawa, M. Igarashi, Chondroitin sulphate N-acetylgalactosaminyl-transferase-1 inhibits recovery from neural injury, Nat. Commun. 4 (2013) 2740. R. Tauchi, S. Imagama, T. Natori, T. Ohgomori, A. Muramoto, R. Shinjo, Y. Matsuyama, N. Ishiguro, K. Kadomatsu, The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury, J. Neuroinflammation 9 (2012) 53. D. Wang, R.M. Ichiyama, R. Zhao, M.R. Andrews, J.W. Fawcett, Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury, J. Neurosci. 31 (2011) 9332–9344.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
A combination of keratan sulfate digestion and rehabilitation promotes anatomical plasticity after rat spinal cord injury Yoshimoto Ishikawa a,b , Shiro Imagama a , Tomohiro Ohgomori c , Naoki Ishiguro a , Kenji Kadomatsu b,∗ a
Department of Biochemistry, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan Department of Orthopedics, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan c Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan b
h i g h l i g h t s • • • • •
KS-digestion plus rehabilitation was evaluated in a spinal cord injury model. KS-digestion/rehabilitation tended to improve functional plasticity. KS-digestion and rehabilitation synergistically improved anatomical plasticity. This effect was comparable with that of CS-digestion/rehabilitation. KS-digestion/rehabilitation might widen the therapeutic window of neuronal injury.
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 11 February 2015 Accepted 9 March 2015 Available online 12 March 2015 Keywords: Spinal cord injury Keratan sulfate Rehabilitation Keratanase II Chondroitinase ABC Chondroitin sulfate
a b s t r a c t Functional recovery after neuronal injuries relies on neuronal network reconstruction which involves many repair processes, such as sealing of injured axon ends, axon regeneration/sprouting, and construction and refinement of synaptic connections. Chondroitin sulfate (CS) is a major inhibitor of axon regeneration/sprouting. It has been reported that the combination of task-specific rehabilitation and CSdigestion is much more effective than either treatment alone with regard to the promotion of functional and anatomical plasticity for dexterity in acute and chronic spinal cord injury models. We previously reported that keratan sulfate (KS) is another inhibitor and has a potency equal to CS. Here, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury. Keratanase II or chondroitinase ABC was locally administered at the lesion after spinal cord injury at C3/4. Task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus, was done for 3 weeks before injury, and then again at 1–6 weeks after injury. The combination of KS-digestion and rehabilitation yielded a better rate of pellet removal than either KS-digestion alone or rehabilitation alone, although these differences were not statistically significant. The combination of CS-digestion and rehabilitation showed similar results. Strikingly, both KS-digestion/rehabilitation and CS-digestion/rehabilitation showed significant increases in neurite growth in vivo as estimated by 5-hydroxytryptamine and GAP43 staining. Thus, KS-digestion and rehabilitation exerted a synergistic effect on anatomical plasticity, and this effect was comparable with that of CS-digestion/rehabilitation. KS-digestion might widen the therapeutic window of spinal cord injury if combined with rehabilitation. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Functional recovery after neuronal injuries relies on the reconstruction of neuronal networks. Maximization of these functional
∗ Corresponding author at: Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel.: +81 52 744 2059; fax: +81 52 744 2060. E-mail address: [email protected] (K. Kadomatsu). http://dx.doi.org/10.1016/j.neulet.2015.03.015 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
and anatomical plasticities is desirable as a therapy for various diseases, such as spinal cord injury (SCI) and stroke. However, it has been hard to achieve ideal reconstruction of neuronal networks, and no efficient therapeutics have been established for these diseases. On the other hand, it has become accepted that rehabilitation promotes functional recovery to some extent [1,2,6], probably by promoting the reconstruction and refinement of specific networks. Reconstruction of neuronal networks may involve several processes, i.e., sealing of injured axon ends, axon regeneration/
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Y. Ishikawa et al. / Neuroscience Letters 593 (2015) 13–18
Fig. 1. In vivo experimental design. (A). In schematic cross section of the spinal cord. The red area indicates the unilateral dorsolateral lesion at level C3/4, which includes the unilateral dorsal CST. (B). Flow of the experiments. (C). The single pellet reaching training required forearm training. (D). PKC-␥ staining was performed to assess the CST lesion. At the C6 transverse section (caudal to the lesion), PKC-␥ could not be detected on the lesion side. Scale bar, 200 m.
sprouting, and construction and refinement of synaptic connections. Therefore, a comprehensive understanding of these processes is required to establish therapies for neuronal injuries [9]. Axons of the adult mammalian central nervous system (CNS) do not regenerate or sprout after injuries due to the low intrinsic regeneration capacity and emerging inhibitory molecules. Chondroitin sulfate (CS) is a strong inhibitor for axon regeneration/sprouting [13]. Ablation of CS by its degrading enzyme chondroitinase ABC (C-ABC) promotes not only axon regeneration/sprouting but also functional recovery [3,12]. Furthermore, CS-digestion/task-specific rehabilitation improved dexterity recovery in both acute and chronic SCI [4,16]. CS belongs to a class of long sugar chains known as glycosaminoglycans, which are composed of repeating disaccharide units. Along with CS, keratan sulfate (KS), heparan sulfate, and hyaluronan belong to the glycosaminoglycans. We previously reported that KS acts as an inhibitor for axon regeneration/sprouting [7,8]. 5D4-reactive KS-deficient mice (GlcNAc6ST-1 knockout) showed enhanced axon regeneration/sprouting and better motor function recovery as compared with wild-type mice [8]. Local administration of keratanase II (K-II), a KS-specific degradative enzyme, can also ameliorate SCI [7]. Notably, the effect of KS-digestion is comparable to that of CS-digestion [7]. In this study, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury.
2.2. Surgical procedure
2. Material and methods
The animals were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After C4 laminectomy, we exposed the dura mater and induced a dorsolateral cut injury between C3 and C4 ipsilateral to the preferred paw (Fig. 1A). A cut was made with the tips of sharpened fine micro-blade inserted 2 mm in depth into the spinal cord. This injury included the descending dorsal CST and the ascending sensory dorsal columns. Immediately, after the dorsolateral cut injury, we performed C6 partial laminectomy and inserted a thin silicone tube with an osmotic mini-pump into the subarachnoid cavity, and set the tube tip at the C3 level under a surgical microscope. This tube was sufficiently soft and thin that we could minimize damage to the spinal cord. The osmotic mini-pumps (Model 2006; ALZET, Cupertino, CA; 200 l of solution, 0.5 l/h, 14 d delivery) were filled with K-II (0.05 U/200 l; Seikagaku), C-ABC (0.05 U/200 l; Seikagaku), or saline (as a vehicle control). The tube was sutured to the spinous process to anchor it in place, and the mini-pump was placed under the skin on the animal’s back. Afterward, the muscles and skin were closed in layers [7,14]. The dose of C-ABC or K-II was decided from a previous report [7]. One day following SCI, rats were visually inspected and evaluated in regard to the degree of paralysis. Rats with bilateral deficits or deficits of the shoulder were excluded from the study in all groups as they were inappropriate for further evaluation [5]. All animals were treated and cared for in accordance with the Nagoya University School of Medicine Guidelines pertaining to the treatment of experimental animals.
2.1. Animal and experimental groups
2.3. Single pellet reaching task
Adult female Sprague–Dawley rats weighing 200–230 g were used in this SCI study. All the animals (N = 58) received the SCI at C3/4, and K-II, C-ABC or saline with or without training in the single pellet reaching task. The animals were divided into six experimental groups: a K-II training group (N = 5); K-II no-training group (N = 5), C-ABC training group (N = 5), C-ABC no-training group (N = 5), saline training group (N = 5), and saline no-training group (N = 5). For histological analysis of the association of PNNs and KS, un-injured normal rats (N = 3) were used.
Before SCI, rats were trained to reach through a slot (1.5 cm wide) in an acrylic box (15 cm × 36 cm × 30 cm) to grasp food pellets of 45 mg each (Bio-serv, USA). The pellets were set in a small indentation on a tray and offered one at a time (the pellets were 2 cm away from the front wall at a height of 3 cm above the elevated grid floor) [11]. Success rates per session were calculated as the number of pellets successfully grasped and eaten out of 20 pellets offered. Starting on day 7 after SCI, training was performed for 20 min per day, 5 days per week for 5 weeks (Fig. 1B and C).
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Un-trained rats were reintroduced to the training for 2 days before final testing. After baseline testing and final testing, the recovery rates to baseline were calculated, and then statistical analysis (oneway ANOVA) was performed. Values of P < 0.05 were defined as statistically significant. 2.4. Immunohistochemistry Three weeks after final testing, rats were killed with transcardial PBS perfusion followed by 4% paraformaldehyde. The spinal cords were dissected and post-fixed at 4 ◦ C overnight in 4% paraformaldehyde buffer, followed by 30% sucrose for 48 h. Tissue was frozen in OCT mounting medium and then cut into transverse 20 m sections with a cryostat and processed for immunohistochemistry. The sections were blocked in PBS containing 10% normal goat serum and 0.1% Triton X-100 at room temperature for immunohistochemistry. For visualization of CST damage, protein kinase C-␥ (1:500), a marker for the CST, was used with the sections at C2 (rostral to the lesion site) and C6 (caudal to the lesion site). For visualization of 5-HT- or Gap 43-positive fibers, sections were immunostained with primary antibody 5-HT (1:1000; Immunostar) and Gap43 (1:2000; Millipore), and then
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incubated with the Alexa Fluor 568 goat anti-rabbit IgG (1:2000; Invitrogen) for 1 h at room temperature. For histological analysis of the association between PNNs and KS, transverse sections from an uninjured rat were incubated with NeuN (1:500; Millipore), BCD4 anti-KS antibody (1:500; Seikagaku), lectin Wisteria fluribunda (WFA) (1:150; Sigma) and anti-aggrecan (1:300; Millipore) after blocking. After rinsing, the sections were incubated with the secondary antibody for 1 h at room temperature: Alexa Fluor 488–conjugated streptavidin (1:400; Invitrogen), alexa Fluor 568 goat anti-mouse IgG (1:2000, Invitrogen), and Alexa Fluor 568 goat anti-rabbit IgG (1:2000; Invitrogen). For evaluation of the effect of K-II or C-ABC, after blocking with normal goat serum and 0.1% Triton X-100, the sections were treated with C-ABC (0.5 U/ml) or K-II (0.5 U/ml) at 37 ◦ C for 1 h, and then incubated with the primary antibodies. For quantification of 5HT- and GAP43-positive fibers, we measured the axonal density at C2, a region close and rostral to the lesion. The total pixels of these positive fibers were calculated by ImageJ software, and statistical analysis (one-way ANOVA) was performed. Values of P < 0.05 were defined as statistically significant.
Fig. 2. Keratan sulfate was present in the PNNs. (A–C). Fluorescence images of WFA surrounding NeuN-positive neurons reveal the PNN structure. (D–F). Fluorescence images of BCD4 surrounding neurons reveal the KS in the PNN structure. (G–I). BCD4-positive KS co-localized with the PNN marker WFA. J-L. Magnified images of (G–I). Scale bar, 50 m.
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3. Results 3.1. SCI and rehabilitation protocol Rats received a C3/4 dorsolateral cut followed by C-ABC, K-II or saline as a control (Fig. 1A). The cut was made ipsilateral to the preferred paw. Rats either received or did not receive task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus (Fig. 1B and C). The rehabilitation training was started 3 weeks before injury, and was restarted 1 week after injury. The rehabilitation continued until 6 weeks after injury, when manual dexterity was estimated. Rats were finally killed 9 weeks after injury for histological analyses. Positivity for PKC-␥, a marker for the CST fibers, was observed in the CST on both sides at C2, which was rostral to the lesion, whereas PKC-␥ staining was negative on the side ipsilateral to the lesion at C6, which was caudal to the lesion (Fig. 1D). 3.2. KS was present in perineuronal nets Perineuronal nets (PNNs) are involved in various kinds of neural plasticity, and may also be important for plasticity after injury [4,16]. Since it is known that CS proteoglycans (CSPGs) are major components of PNNs, we wondered whether KS proteoglycans (KSPGs) would be detectable in PNNs. The lectin Wisteria floribunda agglutinin (WFA)-positive perineuronal nets surrounded a subset of neurons which expressed NeuN (Fig. 2A and C). The antibody BCD4 that specifically recognized low-sulfated KS also stained a subset of NeuN-positive neurons (Fig. 2D–F). Indeed, most of the WFA-positive neurons were BCD4-positive (Fig. 2G–L). It is believed that WFA recognizes 4-O-sulfation of CS. Consistent with this idea, C-ABC treatment completely abolished WFA staining, but did not affect KS expression as detected by BCD4 (data not shown). K-II treatment completely removed BCD4-reactive KS expression, but did not affect WFA staining (data not shown). Taken together, these results suggested that both CS and KS were involved in PNNs, and were completely digested by C-ABC and K-II, respectively. 3.3. Effects of KS digestion and rehabilitation on functional recovery after SCI We next estimated the effects of the combination of KS or CS digestion plus rehabilitation on functional recovery. Since it was previously reported that task-specific rehabilitation, but not general rehabilitation, enhanced the effect of C-ABC on recovery of dexterity [4], we employed a Whishaw apparatus to train rats in a single pellet removal task. As expected, the task-specific rehabilitation alone could improve recovery regardless of CSor KS-digestion (Fig. 3). The combination of rehabilitation and KS- or CS-digestion tended to achieve better recovery than that of rehabilitation and saline, but the difference was not significant (Fig. 3). These results were consistent with a previous report on CS-digestion/rehabilitation [4]. It is noteworthy that KSdigestion/rehabilitation showed almost the same outcome as the combination of CS-digestion/rehabilitation (Fig. 3). 3.4. Anatomical plasticity was promoted by KS-digestion/rehabilitation We next investigated anatomical plasticity. To this end, we quantified serotonergic neuron fibers (5HT-positive fibers) and regrowing neurites (GAP43-positive fibers). We found a striking increase in 5HT-positive fibers in KS-digestion/rehabilitation as compared with KS-digestion alone or saline/rehabilitation (Fig. 4). This effect was comparable with that of CS-digestion/rehabilitation (Fig. 4). We also found that GAP43-positive fibers exhibited a
Fig. 3. Recovery of dexterity. The trained animals showed significant improvement compared to the untrained baseline levels in each treatment group. However, compared with each trained rats with saline, C-ABC or K-2, there were no significant difference.
significant increase in response to KS-digestion/rehabilitation as compared with KS-digestion alone or saline/rehabilitation (Fig. 4). This effect was also similar to that of CS-digestion/rehabilitation (Fig. 4). 4. Discussion Here, we demonstrated that KS-digestion, when combined with task-specific rehabilitation training, showed a synergistic effect on anatomical plasticity which was as strong as that of CS-digestion. Therefore, these findings confirmed the concept that KS and CS play closely related roles in the inhibition of anatomical plasticity after neuronal injury. Improved anatomical plasticity may reflect an improvement in functional plasticity. However, we could not observe a significant difference between KS-digestion/rehabilitation and KS-digestion alone, although the former tended to more effective. This was also the case with respect to CS-digestion/rehabilitation versus CS-digestion alone. These results may reflect a limitation of the dexterity assessment employed, i.e., single pellet removal with a Whishaw apparatus. Indeed, these data are consistent with a previous report by Garcia-Alias et al. [4]. It must be kept in mind that K-II and C-ABC are both originated from bacteria. The repeated use of these reagents may not be practical because of their immunogenicity. Therefore, alternative methods, i.e., small compounds inhibiting KS or CS production, and human enzymes that exert effects similar to K-II and C-ABC, may be taken into consideration for clinical application [15]. Furthermore, it has recently been reported that a membrane-permeable peptide mimetic of the wedge domain of PTP, a receptor for CS, binds to PTP, serving as its inhibitor, and successfully promotes functional recovery after SCI [10]. Thus, the targeting of CS- or KS-receptors is an alternative approach for ameliorating neuronal injury. In this context, it would also be an intriguing approach to use a sugar chain mimetic of a CS or KS functional stretch to promote anatomical plasticity.
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Fig. 4. Histological analysis of regrown 5HT fibers and regrown GAP43 fibers. (A). 5-HT-positive fibers were counted at C2 (rostral to the lesion site). The fibers were calculated as the sum of 5HT-positive pixels. Quantification of axonal sprouting of 5HT staining in grey matter was performed. (B). Low magnification image of a spinal cord section. (C). An enlargement of the boxed area from (B). (D). An enlargement of the boxed area from (C). (E). Saline control. (F). Quantification of neurite regrowth by GAP43 staining in grey matter is shown. Scale bar, 300 m.
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