Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats

doi:10.1016/j.neures.2016.07.005

Neuroscience Research

Volume 113, December 2016, Pages 37-47

https://doi.org/10.1016/j.neures.2016.07.005 Get rights and content

Highlights

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C-ABC and rehabilitation ameliorate severe and chronic spinal cord injury.
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C-ABC digests CSPG in chronic contusional spinal cord injury.
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C-ABS boosts functional recovery during rehabilitation.

Abstract

There are more than 50 times the number of chronic-phase spinal cord injury (SCI) patients than there are acute patients, and over half of all SCI patients are severely disabled. However, research focusing on chronic severe contusional SCI remains very rare. Here, we evaluated whether chondroitinase ABC (C-ABC), a degradative enzyme directed against chondroitin sulfate proteoglycans (CSPGs), and treadmill rehabilitation could exert synergistic therapeutic actions against chronic severe contusional SCI. First, we induced severe contusional SCI in adult rats, and administered C-ABC intrathecally at 6 weeks post-injury for a period of one week. Next, we performed treadmill rehabilitation from weeks 6 to 14 after SCI, for a total period of eight weeks. The initiation of treadmill rehabilitation triggered slight recovery between weeks 6 and 9, whereas C-ABC administration stimulated a third phase of recovery between weeks 12 and 14. Histologically, the C-ABC-treated rats showed an increase in the transverse residual tissue area and the extent of neuronal fiber regeneration at a site caudal to the lesion epicenter, and regrowth of putatively regenerating serotonergic fibers was significantly increased at the epicenter. We suggest that, when combined with intensive rehabilitation, C-ABC may play a beneficial role, even in severe and chronic SCI.

Graphical abstract

Section snippets

Significance statement

Approximately one-half of admitted spinal-cord-injury patients suffer from severe motor symptoms and are non-ambulatory in the chronic phase, but most studies of spinal cord injury focus on mild-to-moderate contusion injury in the acute-to-subacute phase. The present study utilized a combination of chondroitinase ABC and rehabilitation in severe and chronic contusional spinal cord injury in rats and revealed the effectiveness, and limits, of this combined regimen.

Animals

Adult female Sprague-Dawley rats (n = 61, weight = 200–220 g, CLEA Japan, Inc., Tokyo, Japan) were used in this study. The animals were housed doubly in standard plastic cages (26 × 42 cm) under the conditions of a 12-h light/dark cycle with ad libitum access to food and water. General activity, the urine condition, and the absence of symptoms of allodynia were checked twice daily after SCI. Hardwood sawdust bedding was replaced by soft paper bedding for several days. Antibiotics (ampicillin, Meiji

Behavioral performance after severe SCI

Immediately after contusion SCI, all animals showed complete paraplegia (BBB score = 0), which was followed by slight recovery until the chronic phase. The no-treatment control group reached a behavioral plateau (BBB score = 3.3 ± 0.12) and showed no increase in functional recovery at 5 weeks after SCI and thereafter (Fig. 2A). By contrast, the C-ABC and vehicle control with rehabilitation groups exhibited a second recovery phase after the initiation of treadmill training. Notably, the C-ABC group

Discussion

There are more than 50 times more chronic SCI patients than acute patients in the United States. More than half of all SCI patients are severely disabled and non-ambulatory (National Spinal Cord InjuryStatistical, 2014, Zorner et al., 2010); contusive trauma is the most common cause of SCI (Anderson et al., 2005). Nevertheless, studies of “chronic” and “severe” contusional SCI are rare (Du et al., 2015, Granger et al., 2012, Granger et al., 2013, Hall et al., 2010, Munoz-Quiles et al., 2009,

Role of authors

All authors had full access to all the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis.

Study concept and design: MS, KF.

Acquisition of data: MS, ST, KK, HF.

Analysis and interpretation of data: MS, KF, ST, KK, HF.

Drafting the manuscript: MS.

Critical revision of the manuscript for important intellectual content: MN, HO.

Statistical analysis: MS, ST.

Obtained funding: MN, HO.

Administrative, technical, and material support: AI, SS, HF.

Study

Acknowledgments

We thank Drs. T. Ikegami, N. Nagoshi, O. Tsuji, M. Mukaino, F. Renault-Mihara, T. Harada, and K. Yasutake for their technical assistance and scientific discussions and the members of the Okano laboratory for encouragement and generous support.

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The chondroitinases (Chase) are bacterial lyases that specifically digest chondroitin sulfate and/or dermatan sulfate glycosaminoglycans via a β-elimination reaction and generate unsaturated disaccharides. In recent decades, these enzymes have attracted the attention of many researchers due to their potential applications in various aspects of medicine from the treatment of spinal cord injury to use as an analytical tool. In spite of this diverse spectrum, the application of Chase is faced with several limitations and challenges such as thermal instability and lack of a suitable delivery system. In the current review, we address potential therapeutic applications of Chase with emphasis on the challenges ahead. Then, we summarize the latest achievements to overcome the problems by considering the studies carried out in the field of enzyme engineering, drug delivery, and combination-based therapy.
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Citation Excerpt :
Although the present study did not provide the clear evidence of regenerating axons passing through the fibrous lesion center, it is possible that descending spinal projections such as the corticospinal tract regenerate beyond the lesion site to project to the lumbo-sacral spinal motor area within 3 weeks after SCI. A study in which treadmill training was combined with the administration of an enzyme that digests CSPGs, however, demonstrated that corticospinal tracts did not regenerate beyond the lesion area (Shinozaki, et al. 2016). On the other hand, another study demonstrated that treadmill training changes the plasticity of lumbar spinal neurons, particularly activating interneurons by increasing the peripheral sensory projections (Tillakaratne, et al. 2010).
In the acute phase of spinal cord injury, the initial injury triggers secondary damage due to neuroinflammation, leading to the formation of cavities and glial scars that impair nerve regeneration. Following injuries to the central nervous system, early mobilization promotes the recovery of physical function. Therefore, in the present study, we investigated the effects of early mobilization on motor function recovery and neuroinflammation in rats. Early mobilization of rats with complete spinal cord transection resulted in good recovery of hindlimb motor function after 3 weeks. At 1 week after spinal cord injury, the early-mobilized rats expressed fewer inflammatory M1 microglia/macrophages and more anti-inflammatory M2 microglia. In addition, significantly more matrix metalloproteinase 2 (MMP2)-positive cells were observed at the lesion site 1 week after injury in the early-mobilized rats. Multiple labeling studies suggested that many MMP2-positive cells were M2 microglia. MMP9-positive cells that highly co-expressed GFAP were also observed more frequently in the early-mobilized rats. The density of growth-associated protein-positive structures in the lesion center was significantly higher in the early-mobilized rats at 3 weeks after spinal cord injury. The present results suggest that early mobilization after spinal cord injury reduced the production of M1 microglia/macrophages while increasing the production of M2 microglia at the lesion site. Early mobilization might also activate the expression of MMP2 in M2 microglia and MMP9 in astrocytes. These cellular dynamics might suppress neuroinflammation at the lesion site, thereby inhibiting the progression of tissue destruction and promoting nerve regeneration to recover motor function.
Treadmill training based on the overload principle promotes locomotor recovery in a mouse model of chronic spinal cord injury
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Although there have been very few studies on the effect of rehabilitation for chronic SCI, Tashiro et al. failed to show a significant functional improvement with voluntary bipedal treadmill training in a severely spinal cord–contused mouse model (Tashiro et al., 2016). Shinozaki et al. also failed to show significant recovery with quadrupedal treadmill training in a very severely spinal cord–contused rat model (Shinozaki et al., 2016). In contrast, our method resulted in improvements in gait function and quality.
Rehabilitative treatment, including treadmill training, is considered an important strategy for restoring motor function after spinal cord injury (SCI). However, many unexplained problems persist regarding the appropriate rehabilitative method and the mechanism underlying the beneficial effects of rehabilitation. Moreover, only a few preclinical studies have been performed on rehabilitative interventions for chronic SCI, although most patients have chronic injuries. In fact, several preclinical studies reported that rehabilitative training was less effective when applied during the chronic phase than when applied sooner. While numerous studies have examined the effects of treadmill training during the subacute phase, the training conditions vary considerably among preclinical reports. Therefore, establishing a standard training protocol is essential for achieving beneficial rehabilitation effects at the chronic stage. Since the difficulty of applying an appropriate training load hinders training at constant speeds, it is important to adjust the training intensity in accordance with the exercise tolerance of an individual animal to provide further functional recovery benefits. Here, we created a novel quadrupedal treadmill training protocol based on the overload principle for mice with incomplete thoracic SCI. We subjected SCI model mice to rehabilitative training according to the protocol for two consecutive weeks starting at 42 days after injury. We examined the treadmill speeds at which the mice were able to run based on the severity of paresis and investigated the impact of the protocol on functional recovery. Assessment of running speed changes during the treadmill training period revealed faster treadmill speeds for mice with mild paresis than for those with severe paresis. The training parameters, including the speed and distance traveled, were positively correlated with the changes in motor function. These results suggest that the most suitable running speed during treadmill training differs according to the level of motor dysfunction and that running longer distances has a positive impact on motor functional recovery. Based on this established protocol, we compared functional and histological results between the chronic SCI groups with and without rehabilitation. The gait analyses showed significantly better functional improvement in the rehabilitation group than in the nonrehabilitation group. Histological analyses revealed that the BDNF- and VGLUT1-positive areas of lumbar enlargement were significantly increased in the rehabilitation group. These findings implied that rehabilitation promoted not only motor performance but also motor control, including forelimb-hindlimb coordination, even in chronic SCI, resulting in functional improvement by treadmill training alone. Therefore, rehabilitative training based on the overload principle appears to be one of the appropriate treatment options for incomplete thoracic SCI, and evidence of its efficacy exists in actual clinical settings.
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^☆: Funding: This work was supported by the Research Center Network for the Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST) and Japan Agency for Medical Research and Development (A-MED) to M.N. and H.O.

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Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats☆

Highlights

Abstract

Graphical abstract

Section snippets

Significance statement

Animals

Behavioral performance after severe SCI

Discussion

Role of authors

Acknowledgments

Neuron

Trends Neurosci.

J. Neurosci. Methods

Brain Res.

J. Pain

Neurosci. Lett.

Neurosci. Res.

Behav. Brain Res.

Exp. Neurol.

Exp. Neurol.

Exp. Neurol.

Exp. Neurol.

Stem Cell Reports

Mol. Brain

Neurosci. Lett.

Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury

J. Clin. Invest.

Functional regeneration of respiratory pathways after spinal cord injury

Nature

Recommended guidelines for studies of human subjects with spinal cord injury

Spinal Cord

A sensitive and reliable locomotor rating scale for open field testing in rats

J. Neurotrauma

Chondroitinase ABC promotes functional recovery after spinal cord injury

Nature

Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo

J. Comp. Neurol.

Proteoglycan-specific molecular switch for RPTPsigma clustering and neuronal extension

Science

Overcoming neurite-inhibitory chondroitin sulfate proteoglycans in the astrocyte matrix

Glia

Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord

J. Neurosci.

Pten deletion promotes regrowth of corticospinal tract axons 1 year after spinal cord injury

J. Neurosci.

Brain extracellular matrix affects AMPA receptor lateral mobility and short-term synaptic plasticity

Nat. Neurosci.

Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery

Brain

Perineuronal nets protect fear memories from erasure

Science

Autologous olfactory mucosal cell transplants in clinical spinal cord injury: a randomized double-blinded trial in a canine translational model

Brain

Use of an implanted sacral nerve stimulator to restore urine voiding in chronically paraplegic dogs

J. Vet. Intern. Med.

A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord

J. Neurosci.

Chondroitinase ABC combined with neural stem/progenitor cell transplantation enhances graft cell migration and outgrowth of growth-associated protein-43-positive fibers after rat spinal cord injury

Eur. J. Neurosci.