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Combine effect of Chondroitinase ABC and low level laser (660 nm) on spinal cord injury model in adult male rats
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Combine effect of Chondroitinase ABC and low level laser (660 nm) on spinal cord injury model in adult male rats Atousa Janzadeha, Arash Sarveazadb, Mahmoud Yousefifarda, Sima Damenia, Fazel Sahraneshin Samanic, Kobra Mokhtariand, Farinaz Nasirinezhada,⁎ a
Physiology Research Center, Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran c Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran d Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Glycogen synthase kinase-3β (GSK3β) Low level laser Chondroitinase ABC (ChABC) Chondroitin sulfate proteoglycan (CSPG) Inflammation Spinal cord injury
After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660 nm low level laser (LLL) and intraspinal injection of 1 μg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3β (GSk 3β), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p < 0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3β, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser + enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.
1. Introduction Spinal cord injury (SCI) is one of the most debilitating clinical problems which can affect patients lifelong and quality of life, occurring in both genders equally (White and Black, 2016). SCI more frequently affects younger individuals and the burden of these injuries is considerably high (Yousefifard et al., 2016a). Despite the vast improvements in medical sciences, no definite treatment has been found for these patients and the ongoing research aiming to solve this issue has proposed various options including medical treatments, cell therapy and gene therapy (Hosseini et al., 2014; Hosseini et al., 2015; Hosseini et al., 2016; Mojarad et al., 2016; Nasirinezhad et al., 2016; Sarveazad et al., 2017; Yousefifard et al., 2016b; Yousefifard et al., 2016c). The lack of success observed in these treatments can be attributed to the pathophysiology of SCIs and their associated factors. Various factors limit the response to treatment in SCI patients which include decrease in growth factors (Lu et al., 2012), the stunted recovery of neural tissues (Finnerup and Baastrup, 2012), myelin-associated outgrowth inhibitors ⁎
(Simonen et al., 2003) and inhibiting factors associated with glial scars such as chondroitin sulfate proteoglycans (CSPGs) (Silver and Miller, 2004; Yuan and He, 2013). Studies have proposed that glial scar and chronic inflammation might be the main mechanisms affecting the response to treatment in SCI patients (Yuan and He, 2013; Muramoto et al., 2013). Glial scar, with CSPG as its main component, leads to activation of pathways that inhibit tissue recovery in central nervous system. CSPG is an extracellular matrix molecule, the expression of which starts one day after the injury and peaks 2 week later (Jones et al., 2003). This molecule activates the cascade responsible for activation of glycogen synthase kinase-3β (GSK3β), a key factor in inhibition of CNS tissue and axon recovery (Dill et al., 2008). Recent studies have shown that digestion of glycosaminoglycan present in the molecular structure of CSPG by Chondroitinase ABC (ChABC) can enhance axonal regeneration and lead to improvements in sensory and motor function after SCIs (Dyck et al., 2015; Shinozaki et al., 2016). Although the efficacy of ChABC is found to be moderate in recovery after SCIs, it might be improved by
Corresponding author at: Department of Physiology, School of Medicine, Iran University of Medical Sciences, Hemmat Highway, Tehran, Iran. E-mail address: [email protected] (F. Nasirinezhad).
http://dx.doi.org/10.1016/j.npep.2017.06.002 Received 14 February 2017; Received in revised form 29 May 2017; Accepted 4 June 2017 0143-4179/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Janzadeh, A., Neuropeptides (2017), http://dx.doi.org/10.1016/j.npep.2017.06.002
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2.3. Laser therapy
simultaneous administration of other medications (Alluin et al., 2014; Zhao et al., 2013; Zhao and Fawcett, 2013). Complicated inflammatory processes are responsible for healing of tissue injuries but seem to worsen the problem in a chronic setting in the central nervous system (Faden et al., 2016). More than 30 inflammatory factors including membrane binding proteins, receptors and regulators lead to neural tissue injury after trauma and controlling these factors can minimize these injuries (Pritchard et al., 2014). Recently the use of Laser, as a noninvasive anti-inflammatory has been increased clinically. It clinically applied in two local and systemic forms to treat rheumatoid arthritis, pain management, periodontal diseases and regenerative effects on axons (Bingol et al., 2005; Ekim et al., 2007; de Carvalho et al., 2012; Shirani et al., 2009; Masoumipoor et al., 2014). However, Byrnes et al. showed that the efficacy of laser therapy alone on neuronal regeneration is limited, and so suggested combining other therapies with laser (Byrnes et al., 2005). Accordingly, low level laser therapy (LLLT) might be useful in treating SCI patients as an antiinflammatory factor (Ojaghi et al., 2014). we have recently demonstrated that photobiomodulation with LLLT is effective in neuropathic pain and apoptosis reduction(Masoumipoor et al., 2014; Jameie et al., 2014; Janzadeh et al., 2016). Taking all of this into account, the present study aimed to answer this question: Does the simultaneous use of the anti-inflammatory effects of LLLT against inflammatory processes during SCI along with the effects of ChABC on neuro-inhibitory environments provide additional treatment response as compared with each of these methods alone? In this regard, the efficacy of simultaneous treatment with both LLL and ChABC was evaluated on mature male Wistar rats with SCIs.
A CW diode laser emitter (Heltschl, model ME-TL10000-SK) with a wavelength of 660 nm, power of 100 mW energy density of 0.5 J/cm2, power intensity of 0.819 W/cm2 and ~ 0.197 cm2 beam area was used. The laser therapy started 30 min after surgery and continued daily (once a day) for 14 days. Nine points around the surgical incision were transcutaneously irradiated between 10 and 12 a.m. as follows: three points on the center level of the injury site, three points caudally and three points rostral. Total irradiation time was 45 s (5 s for each point). Laser calibration was done using power meter (FieldMaxII-TO, Coherent Inc.). 2.4. ChABC injection Seven days after surgery, subsequent to anesthesia with mixture of ketamine (80 mg/kg) and xylasine (10 mg/kg) injury site was re-exposed and 0.1 U/μl, totally (10 μl) ChABC (sigma, c36667, Germany) diluted in 0.01% bovine serum albumin and buffer phosphate saline (PBS) was slowly injected in 2 min, intraspinally using a 30 μm glass micropipette connected to Hamilton syringe (1 mm in depth and 2 mm rostral to injury site) (Sarveazad et al., 2017; Sarveazad et al., 2014; Cheng et al., 2015). To prevent backflow, after the termination of the injection the needle was kept in injection site for 1 m. 2.5. Locomotor function assay The Basso-Beattie-Bresnahan (BBB) test was used to evaluate motor behavior before surgery and weekly until the end of the fourth week (Basso et al., 1995). All behavior evaluations were performed by two blinded observers. Rats were placed in an open-field area, and the behavior of the animals was observed for 4 min. The BBB score involves closely monitoring limb movement, weight-bearing capability, coordination, and gait. The scores range from 0 to 21. Data were quantified as the average of the two hind limbs.
2. Material and methods 2.1. Animals This study was performed on 44 male Wistar rats (150–170 g). Animals were purchased from Laboratory Animal Breeding Center of Iran University of Medical Sciences. Animal -care and handling were conducted with the approval of the local ethics committee. Rats used in this study had access to food and water freely and kept in a 12-hour light/dark cycle condition and were randomly divided into 6 groups (Table 1).
2.6. Cryosection preparation Four weeks after SCI, transcardial perfusion was done by 4% paraformaldehyde, and then cryo-protection was established with graded sucrose (10, 20, and 30%). Serial transverse sections were prepared (13 μm thickness) by cryostat (Histo-Line, Italy) from injury site. For all histological assays five sections (from injury site with 50 μm interval) from each rat were selected (three to four animals per group).
2.2. Surgery All the surgical procedures were performed under aseptic techniques. For spinal cord injury, the clip compression method was used (Yousefifard et al., 2016b; Azim and Butt, 2011). Briefly, after anesthesia with mixture of ketamine (80 mg/kg) and xylasine (10 mg/kg), laminectomy was performed at T13-L1 level following skin and muscles incision. The spinal cord was compressed by a micro-vascular clip (Fine surgical Tools, Germany) that induced 20 g/cm2 pressure for 90 s. After compression, the clip was removed, and the muscles and skin were separately sutured. Post-operative care included antibiotic prescription (gentamycin 0.8 mg/100 g, i.p) and bladder empting (twice daily until full recovery). Animals were followed for 4 weeks after surgery.
2.7. Cavity size and myelin area assay Luxol Fast Blue (LFB) staining was performed to assess the cavity size and the myelin area in studding animals (Ek et al., 2012). In LFB staining, myelin including phospholipids appears blue to green and neurons appear pink to violet. Cavity size for each slide was recorded by Image J software and eventually the mean cavity size for each group was measured. The cavity size was presented as percent of the total area of the section using the following formula:
Table 1 Experimental groups
Procedure
Control (n = 8) Spinal cord injury (SCI) (n = 8) Vehicle (n = 6) Laser (n = 8) Enzyme (n = 6) Laser + enzyme (n = 8)
Intact animals Animals with spinal Animals with spinal Animals with spinal Animals with spinal Animals with spinal
cord cord cord cord cord
2
injury injury injury injury injury
(no treatment) that received,10 μl buffer phosphate saline injection (intra-spinal) treated by low level laser (660 nm) that recived,10 μl of 100 U/ml of Chondroitinase ABC injection (intra spinal) treated with both low level laser and Chondroitinase ABC
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Fig. 1. Effect of low level laser therapy (n = 8), Chondroitinase ABC (n = 6; enzyme) and combination (n = 8) of laser + enzyme on locomotion recovery following spinal cord injury (SCI). After SCI induction, Basso, Beattie and Bresnahan (BBB) score significantly reduced in all experimental groups compared to control rats (n = 8). Administration of laser and enzyme alone and combination of laser + enzyme caused significant improvement in locomotor function in comparison to SCI animals (n = 8). Differences of single therapies with combination group were significant after two weeks. Data were expressed as means ± SEM. **, significant difference compared to SCI at level of p < 0.001; $$, significant difference compared to laser + enzyme group at level of p < 0.001, $, significant difference compared to laser + enzyme group at level of p < 0.05.
Percent of cavity size =
Cavity size (pixel) × 100 Total area of the section (pixel)
non-fat dry milk for 4 h at 37 °C. The membrane was washed 3 times with PBST and was incubated with the Rabbit polyclonal to Aquaporin 4 (abcam, ab46182, AB_955676, UK) and Rabbit Polyclonal antibodies GSK3 beta (biorbyt, orb89070, UK) primary antibodies overnight at 4 °C. The membrane was incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (biorbyt, orb216204, UK) for 2 h at 37 °C and was developed using chemiluminescence substrate kit and exposed against X-ray film in the darkroom. β-Actin (sc-130656) was used as an internal control. Densitometry analysis for proteins was achieved by using the Alpha EaseFC software.
2.8. Axonal position and density assay For axonal position around cavity assessment Bielschowsky method was used. In this staining method axons and neurofibrillary tangles are shown in black (Mavroudis et al., 2010). Stained slides were observed and captured by a light microscope equipped with a camera (Olympus, magnification ×20), and axonal position and density was quantified by image J software.
2.9.3. Statistical analysis All the data were analyzed by SPSS version 21.0 (SCR:002865) and were presented as the means ± SEM. The differences of motor function recovery between the studied groups were assessed by two-way repeated measures ANOVA. One-way ANOVA was used for assessment of differences in histological evaluation. For all analysis a Bonferroni post hoc test was applied to evaluate between group differences. Probability values (p) < 0.05 were considered to represent significant difference.
2.9. Hematoxylin and eosinophil (H & E) assay H & E staining was performed for assessment of fibroblast invasion into cavity of injury site (Llewellyn, 2009). After staining, digital images were captured (Olympus, magnification × 40) and invasion of fibroblast was quantified. 2.9.1. Immunohistochemistry Sections were washed three times with PBS, Triton X-100/10% and subsequently blocked with BSA 1% and normal goat serum for 1 h at room temperature. The primary Rabbit Polyclonal antibodies GSK3 beta (biorbyt, orb89070, UK) and Monoclonal Anti-Chondroitin Sulfate (sigma, c8035, Germany) were diluted in blocking solution and incubated overnight. Secondary antibodies were goat anti mouse(abcam,ab6785,UK) and goat anti rabbit (abcam,ab6717, UK) diluted in PBS/0.3% BSA incubated for 1 h, respectively. Images were capture from each section by Fluorescent microscope (magnification ×10). Where ChABC was present, the reactive area (Fluorescent) was identified and area was measured by Image J software based on the following formula (Sarveazad et al., 2017).
3. Result 3.1. Mortality Initially a total of 48 animals were included. During the induction of SCI 4 animals died in the enzyme (2 rats) and vehicle (2 rats) groups. Accordingly, data from 44 animals were included in the final analysis. 3.2. Behavioral assessment After SCI induction, motor function significantly was reduced in all groups compared to the control rats (df: 16, 136; F = 17.9; n =; p < 0.0001). During the 4 week follow up some degree of motor function recovery was observed but it did not reach the level of the control group (p < 0.001) (Fig. 1). In the first week following SCI a significant improvement was observed in the laser + enzyme group compared to SCI animals (p < 0.001). In the second, third, and fourth weeks the motor function recovery of animals in the laser, enzyme, and laser + enzyme groups significantly improved compared to the SCI group (p < 0.001). Two weeks after SCI induction, the laser + enzyme group showed a significant improvement in motor function recovery compared to the enzyme (p = 0.003) and laser (p < 0.001) groups. These differences were observed during the third (p = 0.04 for laser group; p = 0.04 for
immunoreactive area Percent of immunoreactive area = × 100 Total area of the section 2.9.2. Western blotting After carefully removing the dura and washing in PBS, spinal cord (T13-L1 level) was homogenized on ice in lysis buffer (Universal DNA/ RNA/Protein Purification kit, EURx). Protein concentration was assayed by Nano dropper (Thermo Science). The samples were loaded onto wells and electrophoresed on 12% SDS-polyacrylamide gel (SDSPAGE, BIORAD) for 1 h at a constant voltage (120 V). The proteins were transferred from the gel to a polyvinylidene difluoride membrane and were blocked with PBS containing 0.05% Tween-20 (PBST) and 5% 3
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Fig. 2. Luxol Fast Blue (LFB) staining was used for assessment of cavity size and myelin area (n = 3 per each group). Qualitative assessment showed that the majority of area observed in the samples of combination therapy group consisted of myelin, while less myelin area was observed in the SCI and laser-treated animals. Size of the cavity was significantly lower in combination group of laser + enzyme compared to the other groups. (A) Control; (B) Spinal Cord injury (SCI); (C) Vehicle; (D) Laser; (E) Chondroitinase ABC (enzyme); (F) laser + enzyme groups; (G) Quantitative analysis of cavity size. Data were expressed as the mean ± SEM. *, significant difference compared to SCI at level of p < 0.05; #, significant difference compared to laser group at level of p < 0.01; $, significant difference compared to Enzyme group at level of p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
animals (p < 0.001), Combination group has most axonal number (p < 0.001) (Fig. 3D–F).
enzyme group) and forth weeks (p = 0.032 for laser group; p = 0.045 for enzyme group) after SCI induction. 3.3. Assessment of myelin area and cavity size
3.5. Fibroblast invasion assessment
Qualitative assessment of spinal cord after LFB staining showed that the majority of the area observed in samples of the combination therapy group consisted of myelin, while less myelin area was found in the samples of SCI and laser-treated animals (Fig. 2A–F). Four weeks after SCI, a big cavity was seen in SCI (mean cavity size: 20.8 ± 3.6%) and vehicle (mean cavity size: 24.0 ± 4.2%) groups (df: 4, 31; F = 7.0; n = 3; p = 0.001). Cavity size in laser (mean cavity size: 15.1 ± 1.7%; p = 0.99 vs. SCI) and enzyme (mean cavity size: 21.0 ± 3.1%; p = 0.99 vs. SCI) groups were not significantly different compared to SCI animals. However, the size of the cavity was significantly smaller in the combination group of laser + enzyme (mean cavity size: 5.2 ± 2.0%) compared to SCI (p = 0.006), laser (p = 0.04), and enzyme (p = 0.003) groups (Fig. 2-G).
Quantitative evaluation of fibroblast invasion by H & E staining showed that SCI was led to fibroblast invasion around the cavity (df: 5,42; F = 48.35, n = 3; p < 0.001). This phenomenon is more prominent in the laser group. Aggregation of the fibroblasts in the laser group was compact around the cavity and higher than other groups (p < 0.0001) (Fig. 4). Moreover, the presence of fibroblast around the cavity was less in enzyme and combination groups compared to SCI animals (p < 0.05, p < 0.01) (Fig. 4D & E).
3.6. Evaluation of chondroitin sulfate proteoglycans expression Spinal cord injury caused a significant CSPG expression compared to the control group (df: 5, 18; F = 35.33; n = 3; p < 0.001). Although administration of Chondroitinase ABC (p < 0.001) and low level laser (p = 0.003) decreased the CSPG immunoreactivity compared to the SCI group, it did not lower it to that of the control group (p < 0.001 for laser and enzyme groups). Also, laser + enzyme therapy decreased CSPG immunoreactivity (p < 0.001) in a way that the difference with the control group was not statistically significant (p = 0.28) (Fig. 5).
3.4. Assessment of nerve fibers and axons around cavity Bielschowsky's staining revealed that a few axons exist around the cavity in the SCI and vehicle groups compared to the normal rats (df: 5,54; F = 126.9,n = 3; p < 0.001). (Fig. 3A–C). In the enzyme, laser, and the combination group the amount of axons was higher than SCI 4
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Fig. 3. Effect of Chondroitinase ABC (enzyme group), low level laser (laser group), and combination therapy (laser + enzyme group) on demyelination after spinal cord injury. Axons appear black. Bielschowsky's silver staining showed that spinal cord injury led to a reduction in axon density around damaged areas. Enzyme, Laser, and laser + enzyme were found to have protective effects on the axons and fibers. (A) Control; (B) Spinal cord injury; (C) Vehicle, (D) enzyme, (E) Laser, (F) laser + enzyme. Magnification ×20. ***p < 0.001, *p < 0.05 vs control group, ###, p < 0.001, vs SCI group & vehicle. $$ $P < 0.001 vs laser, p < 0.001 £££ difference between Enzyme and laser + enzyme group (n = 3 per group).
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Fig. 4. Effect of SCI induction, Laser, Enzyme and combination of laser + enzyme on invasion and entrance of fibroblasts around the cavity. Presence of fibroblasts is higher in the laser group. Rush of fibroblasts are indicated by yellow arrows, respectively. (A) Control; (B) Spinal cord injury; (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + Enzyme. Magnification ×40. ***p < 0.001, vs control group, ##, p < 0.01, vs. # p < 0.05, vs. SCI group & vehicle, $$$ p < 0.001, $$ p < 0.01 vs laser group, £ £ difference between Enzyme and laser + enzyme group. (n = 3 per group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.7. Assessment of GSK3β in the spinal cord
4. Discussion
GSK3β expressions significantly increased in the SCI group compared to the control animals (df: 5. 13; F = 50.0; n = 3; p < 0.001). Laser therapy (p = 0.001), administration of Chondroitinase ABC (p = 0.002), and combination of laser + enzyme (p < 0.001) reduced the expression of GSK3β and brought it down to the level of control animals' (Fig. 6).
In the present study, we examined the effects of LLLT (660 nm) and ChABC administrated alone and in combination on SCI. Results showed that combination of LLLT and ChABC had more beneficial effects on motor function recovery, cavity size and GSK3β, CSPG, and AQp4 expression than using each separately. There are two major defensive barriers that halt recovery after SCI, including flux of inflammatory agents and production of CSPG that together lead to glial scar formation (Shechter et al., 2011). Although these two barriers seem to be separate from each other, but a direct correlation exist between secretions of inflammatory cytokines with expression of CSGP (Shechter et al., 2011). Inflammatory factors begin to attack immediately after SCI and cause cell death, demyelination, cavitation, invasion of microglia and eventually motor function impairment (Byrnes et al., 2005; Levine, 2015; Zhou et al., 2014). Inflammation after SCI leads to other changes as well. Inflammation is associated with an increase in GSK3β expression in the spinal cord (Cuzzocrea et al., 2006; Renault-Mihara et al., 2011). GSK3β is one of the factors inducing demyelination and plays an important role in
3.8. Aquaporin 4 (AQP4) expression Induction of SCI increased AQP4 expression compared to the control group (df: 5, 13; F = 23.89; n = 3; p < 0.001). Treatment with laser (p = 0.006) and laser + enzyme (p = 0.001) significantly decreased AQP4 expression after SCI. Administration of Chondroitinase ABC did not affect AQP4 expression (p = 0.43). There is a significant difference between laser + enzyme group and enzyme group in expression of AQP4 (p = 0.03) (Fig. 7).
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Fig. 5. Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on chondroitin sulfate proteoglycan (CSPG) immunoreactivity in the study groups. Treatments by laser and enzyme reduced CSPG expression. (A) Control; (B) spinal cord injury (SCI); (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + enzyme groups; (G) Quantitative assessment of CSPG expression. Data were expressed as mean ± SEM (n = 3 per group). ***p < 0.001, vs control group, **p < 0.01 vs control group, *p < 0.05, vs control group, ###, p < 0.001, vs. SCI group, ##, p < 0.01, vs. SCI group.
enzyme secretion starts in the first day of injury and reaches its maximum level one week later (Jones et al., 2003). Another beneficial effect of ChABC is its anti-inflammatory effect induced through increasing IL10 secretion (Didangelos et al., 2014). The results of the present study showed that the expression of CSPG decreased in the animals treated with LLLT and ChABC. This change might be due to the anti-inflammatory effect of laser, but ChABC probably has acted through both CSPG decomposition and anti-inflammatory effects (Lee et al., 2010). The results showed that GSK3β decreased in both treatments, leading to a decrease in apoptosis and subsequently improvement in motor function after SCI which is indicative of reduced demyelination and axon regeneration (Dill et al., 2008; Cuzzocrea et al., 2006). It seems that both LLLT and ChABC treatments acted through decreasing CSPG and GSK3β, by which a permeable environment was provided for the axons of mature neurons to grow and elongate (Tohda and Kuboyama, 2011; Yilmaz and Kaptanoglu, 2015). Accordingly, the size of the cavity decreased and motor function improvement was observed. Axons were also visualized around the cavity using bielschowsky staining. Simultaneous treatment with both LLLT and ChABC was found to be more effective in improving SCI induced complications. In the combined treatment group, the expression of CSPG and GSK3β returned to basic levels; the size of the cavity was smaller compared to laser and
Wallerian degeneration, during which the axons separate from their cell bodies (Dill et al., 2008; Nagai et al., 2016). It has also been shown to affect the release of CSGP which prevents axon regeneration after SCI through multiple mechanisms (Dill et al., 2008; Nagai et al., 2016; Wakatsuki et al., 2011). Proliferation of immune cells, such as macrophages has also been found to play a role in increasing CSGP expression which disrupts axonal connections, hinders axonal regeneration and leads to motor function defects (Nagai et al., 2016). All these mechanisms might have contributed to the cavity formation and BBB decrease in SCI animals. In the present study the mentioned defense barrier is attacked by two mechanisms. Laser is the first treatment strategy used to destroy this barrier. Based on the study carried out by Goncalves et al., LLLT decreases the inflammation in the spinal cord by controlling the invasion of inflammatory cells in the central nervous system and inhibiting axon demyelination (Goncalves et al., 2016). Since studies have shown that invasion of inflammatory cells occur shortly after SCI, laser therapy was initiated 30 min after the injury in this survey (Zhou et al., 2014). The second selected treatment was administration of ChABC enzyme which plays a direct role in destruction of the barrier through decomposition of CSPG and creates a permeable environment for axon regeneration (Bradbury et al., 2002; Lee et al., 2010). Enzyme injection was done one week after SCI since previous studies had shown that 7
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Fig. 6. Assessment of glycogen synthase kinase-3β (GSK3β) expression in experimental groups. IHC study showed that spinal cord injury (SCI) increased GSK3 beta expression mostly around the cavity. Pattern of GSK 3β expression after SCI is similar to that of the chondroitin sulfate expression (A–F). Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on GSK 3β expression, 4 week after SCI induction (G). GSK 3β protein expression was normalized to β-actin protein. Western blot assay showed an increase in GSK 3β expression after spinal cord injury (SCI). Treatment by laser and laser + enzyme decreased its expression (H). Data are expressed as the mean ± SEM. (A) Control; (B) spinal cord injury (SCI); (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + enzyme groups; (G) western blotting bonds; (H) quantitative assay of GSK3β. *p < 0.05 **p < 0.01, vs control group, # #p < 0.01, vs. SCI group & vehicle. (n = 3 per group). ***p < 0.001, vs control group, ###, p < 0.001, vs. SCI group, ##, p < 0.01, vs. SCI group.
of inflammatory cytokines occurs, that are associated with cell death, demyelination, cavity formation, paralysis and even death (Li et al., 2014; Yang and Piao, 2003). In this stage AQP4 helps in reducing the edema by excretion of excessive water (Kimura et al., 2010). However, increased expression of AQP4 in the chronic stage, which is the target in this study, is associated with different results. In the chronic stage AQP4 is expressed as mislocalized and abnormal shaped in the glial barrier which is not capable of excreting excessive water (Vajda et al., 2002). Nesic et al. showed that the increased AQP4 in the chronic stage might
ChABC groups alone, more neuron aggregation was observed around the cavity and the motor function outcome was better compared to other groups. Despite the improvement observed in LLLT and ChABC treated groups, it seems that the application of these two treatments alone might have some limitations. Previous studies have shown that immediately after SCI, local edema (cytotoxic and vasogenic) occurs due to the injuries of the blood brain barrier which contribute to exacerbation of SCIs. Following edema, an increase in microglia and invasion 8
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Fig. 7. Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on aquaporin4 protein (AQP4) expression 4 week after SCI induction. AQP4 protein expression was normalized to β-actin protein. Western blot assay showed an increase in AQP4 expression after spinal cord injury (SCI). Treatment by laser and laser + enzyme decreased its expression. Data are expressed as the mean ± SEM. *p < 0.05 **p < 0.01, vs control group, # #p < 0.01, vs. SCI group & vehicle, $ p < 0.05, vs enzyme group. (n = 3 per group).
Conflict of interest
be indicative of continued water retention and cytotoxic edema that hinders recovery after SCI (Nesic et al., 2006). The results of the present study showed that expression of AQP4 in animals treated with LLLT decreased compared to the SCI group, but administration of ChABC was not able to decrease AQP4 expression. Signs and symptoms of cytotoxic edema can be a limitation for treatment with ChABC alone. Results obtained from H & E staining revealed invasion of fibroblasts around the lesion in animals treated with LLLT, while the presence of fibroblasts was significantly lower in other groups even the SCI and vehicle groups. The impermeable glial barrier which includes CSPG, not only inhibits growth and invasion of axons in the lesion, but also prohibits invasion of fibroblasts (Soderblom et al., 2013). In this regard, one hypothesis states that some factors might be able to break the glial barrier and make it permeable for axonal regeneration, but fibroblast invasion creates fibrotic scar which is hard and impermeable (Soderblom et al., 2013; Cregg et al., 2014; Liesi and Kauppila, 2002). This phenomenon can limit the effects of laser and ChABC treatments since previous studies have shown that in cases where fibroblasts were aggregated, no improvements were observed (Soderblom et al., 2013). Nevertheless, in the present study aggregation of fibroblasts was decreased in the combined treatment group with LLLT and ChABC to the control level. In these cases, laser might have provided a suitable environment for ChABC to take effect by controlling inflammation and edema. Probably LLLT has reduced inflammation and ChABC has helped preserve the integrity of the tissue by decomposing CSPG and reducing inflammation. The observed reduction of CSPG and GSk3β to that of the control levels, the improvement in the motor function and the decreased size of the cavity are confirmed for the proposed hypothesis. Understanding detail mechanism will need to study more in the future.
The authors have declared no conflict of interest. Acknowledgment This study was supported by Iran University of Medical Sciences (IUMS) (25049), Faculty of Medicine and Physiology Research Center. The authors would like to thank the Heltschl Company for giving laser as a present. References Alluin, O., Delivet-Mongrain, H., Gauthier, M.K., Fehlings, M.G., Rossignol, S., KarimiAbdolrezaee, S., 2014. Examination of the combined effects of chondroitinase ABC, growth factors and locomotor training following compressive spinal cord injury on neuroanatomical plasticity and kinematics. PLoS One 9 (10), e111072. Azim, K., Butt, A.M., 2011. GSK3beta negatively regulates oligodendrocyte differentiation and myelination in vivo. Glia 59 (4), 540–553. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12 (1), 1–21. Bingol, U., Altan, L., Yurtkuran, M., 2005. Low-power laser treatment for shoulder pain. Photomed. Laser Surg. 23 (5), 459–464. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., et al., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416 (6881), 636–640. Byrnes, K.R., Waynant, R.W., Ilev, I.K., Wu, X., Barna, L., Smith, K., et al., 2005. Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury. Lasers Surg. Med. 36 (3), 171–185. de Carvalho, Pde T., Leal-Junior, E.C., Alves, A.C., Rambo, C.S., Sampaio, L.M., Oliveira, C.S., et al., 2012. Effect of low-level laser therapy on pain, quality of life and sleep in patients with fibromyalgia: Study protocol for a double-blinded randomized controlled trial. Trials 13, 221. Cheng, C.H., Lin, C.T., Lee, M.J., Tsai, M.J., Huang, W.H., Huang, M.C., et al., 2015. Local delivery of high-dose chondroitinase ABC in the sub-acute stage promotes axonal outgrowth and functional recovery after complete spinal cord transection. PLoS One 10 (9), e0138705. Cregg, J.M., DePaul, M.A., Filous, A.R., Lang, B.T., Tran, A., Silver, J., 2014. Functional regeneration beyond the glial scar. Exp. Neurol. 253, 197–207. Cuzzocrea, S., Genovese, T., Mazzon, E., Crisafulli, C., Di Paola, R., Muia, C., et al., 2006. Glycogen synthase kinase-3 beta inhibition reduces secondary damage in experimental spinal cord trauma. J. Pharmacol. Exp. Ther. 318 (1), 79–89. Didangelos, A., Iberl, M., Vinsland, E., Bartus, K., Bradbury, E.J., 2014. Regulation of IL10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury. J. Neurosci. 34 (49), 16424–16432. Dill, J., Wang, H., Zhou, F., Li, S., 2008. Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J. Neurosci. 28 (36), 8914–8928. Dyck, S.M., Alizadeh, A., Santhosh, K.T., Proulx, E.H., Wu, C.L., Karimi-Abdolrezaee, S., 2015. Chondroitin sulfate proteoglycans negatively modulate spinal cord neural precursor cells by signaling through LAR and RPTPσ and modulation of the rho/ ROCK pathway. Stem Cells 33 (8), 2550–2563. Ek, C.J., Habgood, M.D., Dennis, R., Dziegielewska, K.M., Mallard, C., Wheaton, B., et al., 2012. Pathological changes in the white matter after spinal contusion injury in the rat. PLoS One 7 (8), e43484. Ekim, A., Armagan, O., Tascioglu, F., Oner, C., Colak, M., 2007. Effect of low level laser therapy in rheumatoid arthritis patients with carpal tunnel syndrome. Swiss Med. Wkly. 137 (23–24), 347–352.
5. Conclusion The results of this survey showed that although the application of LLLT and ChABC alone can reduce the cavity size and GSk3β and CSPG expression and lead to improvements in motor recovery, but they are associated with complications that limit their use. Administration of ChABC cannot reduce AQP4 which is an indicator for cytotoxic edema, an inhibitory factor for improvement after and SCI. Application of LLLT was also associated with aggregation of fibroblasts around the lesion which is indicative of the fibrotic scar formation. However, in the combined treatment group, these limitations were not present. Expression of GSk3β and CSPG returned to baseline; the size of the cavity decreased and presence of axons around the lesion and the motor function recovery was improved. Therefore, combined treatment with LLLT and ChABC was found to be more effective than application of these treatments alone. 9
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Combine effect of Chondroitinase ABC and low level laser (660 nm) on spinal cord injury model in adult male rats Atousa Janzadeha, Arash Sarveazadb, Mahmoud Yousefifarda, Sima Damenia, Fazel Sahraneshin Samanic, Kobra Mokhtariand, Farinaz Nasirinezhada,⁎ a
Physiology Research Center, Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran c Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran d Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Glycogen synthase kinase-3β (GSK3β) Low level laser Chondroitinase ABC (ChABC) Chondroitin sulfate proteoglycan (CSPG) Inflammation Spinal cord injury
After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660 nm low level laser (LLL) and intraspinal injection of 1 μg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3β (GSk 3β), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p < 0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3β, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser + enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.
1. Introduction Spinal cord injury (SCI) is one of the most debilitating clinical problems which can affect patients lifelong and quality of life, occurring in both genders equally (White and Black, 2016). SCI more frequently affects younger individuals and the burden of these injuries is considerably high (Yousefifard et al., 2016a). Despite the vast improvements in medical sciences, no definite treatment has been found for these patients and the ongoing research aiming to solve this issue has proposed various options including medical treatments, cell therapy and gene therapy (Hosseini et al., 2014; Hosseini et al., 2015; Hosseini et al., 2016; Mojarad et al., 2016; Nasirinezhad et al., 2016; Sarveazad et al., 2017; Yousefifard et al., 2016b; Yousefifard et al., 2016c). The lack of success observed in these treatments can be attributed to the pathophysiology of SCIs and their associated factors. Various factors limit the response to treatment in SCI patients which include decrease in growth factors (Lu et al., 2012), the stunted recovery of neural tissues (Finnerup and Baastrup, 2012), myelin-associated outgrowth inhibitors ⁎
(Simonen et al., 2003) and inhibiting factors associated with glial scars such as chondroitin sulfate proteoglycans (CSPGs) (Silver and Miller, 2004; Yuan and He, 2013). Studies have proposed that glial scar and chronic inflammation might be the main mechanisms affecting the response to treatment in SCI patients (Yuan and He, 2013; Muramoto et al., 2013). Glial scar, with CSPG as its main component, leads to activation of pathways that inhibit tissue recovery in central nervous system. CSPG is an extracellular matrix molecule, the expression of which starts one day after the injury and peaks 2 week later (Jones et al., 2003). This molecule activates the cascade responsible for activation of glycogen synthase kinase-3β (GSK3β), a key factor in inhibition of CNS tissue and axon recovery (Dill et al., 2008). Recent studies have shown that digestion of glycosaminoglycan present in the molecular structure of CSPG by Chondroitinase ABC (ChABC) can enhance axonal regeneration and lead to improvements in sensory and motor function after SCIs (Dyck et al., 2015; Shinozaki et al., 2016). Although the efficacy of ChABC is found to be moderate in recovery after SCIs, it might be improved by
Corresponding author at: Department of Physiology, School of Medicine, Iran University of Medical Sciences, Hemmat Highway, Tehran, Iran. E-mail address: [email protected] (F. Nasirinezhad).
http://dx.doi.org/10.1016/j.npep.2017.06.002 Received 14 February 2017; Received in revised form 29 May 2017; Accepted 4 June 2017 0143-4179/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Janzadeh, A., Neuropeptides (2017), http://dx.doi.org/10.1016/j.npep.2017.06.002
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2.3. Laser therapy
simultaneous administration of other medications (Alluin et al., 2014; Zhao et al., 2013; Zhao and Fawcett, 2013). Complicated inflammatory processes are responsible for healing of tissue injuries but seem to worsen the problem in a chronic setting in the central nervous system (Faden et al., 2016). More than 30 inflammatory factors including membrane binding proteins, receptors and regulators lead to neural tissue injury after trauma and controlling these factors can minimize these injuries (Pritchard et al., 2014). Recently the use of Laser, as a noninvasive anti-inflammatory has been increased clinically. It clinically applied in two local and systemic forms to treat rheumatoid arthritis, pain management, periodontal diseases and regenerative effects on axons (Bingol et al., 2005; Ekim et al., 2007; de Carvalho et al., 2012; Shirani et al., 2009; Masoumipoor et al., 2014). However, Byrnes et al. showed that the efficacy of laser therapy alone on neuronal regeneration is limited, and so suggested combining other therapies with laser (Byrnes et al., 2005). Accordingly, low level laser therapy (LLLT) might be useful in treating SCI patients as an antiinflammatory factor (Ojaghi et al., 2014). we have recently demonstrated that photobiomodulation with LLLT is effective in neuropathic pain and apoptosis reduction(Masoumipoor et al., 2014; Jameie et al., 2014; Janzadeh et al., 2016). Taking all of this into account, the present study aimed to answer this question: Does the simultaneous use of the anti-inflammatory effects of LLLT against inflammatory processes during SCI along with the effects of ChABC on neuro-inhibitory environments provide additional treatment response as compared with each of these methods alone? In this regard, the efficacy of simultaneous treatment with both LLL and ChABC was evaluated on mature male Wistar rats with SCIs.
A CW diode laser emitter (Heltschl, model ME-TL10000-SK) with a wavelength of 660 nm, power of 100 mW energy density of 0.5 J/cm2, power intensity of 0.819 W/cm2 and ~ 0.197 cm2 beam area was used. The laser therapy started 30 min after surgery and continued daily (once a day) for 14 days. Nine points around the surgical incision were transcutaneously irradiated between 10 and 12 a.m. as follows: three points on the center level of the injury site, three points caudally and three points rostral. Total irradiation time was 45 s (5 s for each point). Laser calibration was done using power meter (FieldMaxII-TO, Coherent Inc.). 2.4. ChABC injection Seven days after surgery, subsequent to anesthesia with mixture of ketamine (80 mg/kg) and xylasine (10 mg/kg) injury site was re-exposed and 0.1 U/μl, totally (10 μl) ChABC (sigma, c36667, Germany) diluted in 0.01% bovine serum albumin and buffer phosphate saline (PBS) was slowly injected in 2 min, intraspinally using a 30 μm glass micropipette connected to Hamilton syringe (1 mm in depth and 2 mm rostral to injury site) (Sarveazad et al., 2017; Sarveazad et al., 2014; Cheng et al., 2015). To prevent backflow, after the termination of the injection the needle was kept in injection site for 1 m. 2.5. Locomotor function assay The Basso-Beattie-Bresnahan (BBB) test was used to evaluate motor behavior before surgery and weekly until the end of the fourth week (Basso et al., 1995). All behavior evaluations were performed by two blinded observers. Rats were placed in an open-field area, and the behavior of the animals was observed for 4 min. The BBB score involves closely monitoring limb movement, weight-bearing capability, coordination, and gait. The scores range from 0 to 21. Data were quantified as the average of the two hind limbs.
2. Material and methods 2.1. Animals This study was performed on 44 male Wistar rats (150–170 g). Animals were purchased from Laboratory Animal Breeding Center of Iran University of Medical Sciences. Animal -care and handling were conducted with the approval of the local ethics committee. Rats used in this study had access to food and water freely and kept in a 12-hour light/dark cycle condition and were randomly divided into 6 groups (Table 1).
2.6. Cryosection preparation Four weeks after SCI, transcardial perfusion was done by 4% paraformaldehyde, and then cryo-protection was established with graded sucrose (10, 20, and 30%). Serial transverse sections were prepared (13 μm thickness) by cryostat (Histo-Line, Italy) from injury site. For all histological assays five sections (from injury site with 50 μm interval) from each rat were selected (three to four animals per group).
2.2. Surgery All the surgical procedures were performed under aseptic techniques. For spinal cord injury, the clip compression method was used (Yousefifard et al., 2016b; Azim and Butt, 2011). Briefly, after anesthesia with mixture of ketamine (80 mg/kg) and xylasine (10 mg/kg), laminectomy was performed at T13-L1 level following skin and muscles incision. The spinal cord was compressed by a micro-vascular clip (Fine surgical Tools, Germany) that induced 20 g/cm2 pressure for 90 s. After compression, the clip was removed, and the muscles and skin were separately sutured. Post-operative care included antibiotic prescription (gentamycin 0.8 mg/100 g, i.p) and bladder empting (twice daily until full recovery). Animals were followed for 4 weeks after surgery.
2.7. Cavity size and myelin area assay Luxol Fast Blue (LFB) staining was performed to assess the cavity size and the myelin area in studding animals (Ek et al., 2012). In LFB staining, myelin including phospholipids appears blue to green and neurons appear pink to violet. Cavity size for each slide was recorded by Image J software and eventually the mean cavity size for each group was measured. The cavity size was presented as percent of the total area of the section using the following formula:
Table 1 Experimental groups
Procedure
Control (n = 8) Spinal cord injury (SCI) (n = 8) Vehicle (n = 6) Laser (n = 8) Enzyme (n = 6) Laser + enzyme (n = 8)
Intact animals Animals with spinal Animals with spinal Animals with spinal Animals with spinal Animals with spinal
cord cord cord cord cord
2
injury injury injury injury injury
(no treatment) that received,10 μl buffer phosphate saline injection (intra-spinal) treated by low level laser (660 nm) that recived,10 μl of 100 U/ml of Chondroitinase ABC injection (intra spinal) treated with both low level laser and Chondroitinase ABC
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Fig. 1. Effect of low level laser therapy (n = 8), Chondroitinase ABC (n = 6; enzyme) and combination (n = 8) of laser + enzyme on locomotion recovery following spinal cord injury (SCI). After SCI induction, Basso, Beattie and Bresnahan (BBB) score significantly reduced in all experimental groups compared to control rats (n = 8). Administration of laser and enzyme alone and combination of laser + enzyme caused significant improvement in locomotor function in comparison to SCI animals (n = 8). Differences of single therapies with combination group were significant after two weeks. Data were expressed as means ± SEM. **, significant difference compared to SCI at level of p < 0.001; $$, significant difference compared to laser + enzyme group at level of p < 0.001, $, significant difference compared to laser + enzyme group at level of p < 0.05.
Percent of cavity size =
Cavity size (pixel) × 100 Total area of the section (pixel)
non-fat dry milk for 4 h at 37 °C. The membrane was washed 3 times with PBST and was incubated with the Rabbit polyclonal to Aquaporin 4 (abcam, ab46182, AB_955676, UK) and Rabbit Polyclonal antibodies GSK3 beta (biorbyt, orb89070, UK) primary antibodies overnight at 4 °C. The membrane was incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (biorbyt, orb216204, UK) for 2 h at 37 °C and was developed using chemiluminescence substrate kit and exposed against X-ray film in the darkroom. β-Actin (sc-130656) was used as an internal control. Densitometry analysis for proteins was achieved by using the Alpha EaseFC software.
2.8. Axonal position and density assay For axonal position around cavity assessment Bielschowsky method was used. In this staining method axons and neurofibrillary tangles are shown in black (Mavroudis et al., 2010). Stained slides were observed and captured by a light microscope equipped with a camera (Olympus, magnification ×20), and axonal position and density was quantified by image J software.
2.9.3. Statistical analysis All the data were analyzed by SPSS version 21.0 (SCR:002865) and were presented as the means ± SEM. The differences of motor function recovery between the studied groups were assessed by two-way repeated measures ANOVA. One-way ANOVA was used for assessment of differences in histological evaluation. For all analysis a Bonferroni post hoc test was applied to evaluate between group differences. Probability values (p) < 0.05 were considered to represent significant difference.
2.9. Hematoxylin and eosinophil (H & E) assay H & E staining was performed for assessment of fibroblast invasion into cavity of injury site (Llewellyn, 2009). After staining, digital images were captured (Olympus, magnification × 40) and invasion of fibroblast was quantified. 2.9.1. Immunohistochemistry Sections were washed three times with PBS, Triton X-100/10% and subsequently blocked with BSA 1% and normal goat serum for 1 h at room temperature. The primary Rabbit Polyclonal antibodies GSK3 beta (biorbyt, orb89070, UK) and Monoclonal Anti-Chondroitin Sulfate (sigma, c8035, Germany) were diluted in blocking solution and incubated overnight. Secondary antibodies were goat anti mouse(abcam,ab6785,UK) and goat anti rabbit (abcam,ab6717, UK) diluted in PBS/0.3% BSA incubated for 1 h, respectively. Images were capture from each section by Fluorescent microscope (magnification ×10). Where ChABC was present, the reactive area (Fluorescent) was identified and area was measured by Image J software based on the following formula (Sarveazad et al., 2017).
3. Result 3.1. Mortality Initially a total of 48 animals were included. During the induction of SCI 4 animals died in the enzyme (2 rats) and vehicle (2 rats) groups. Accordingly, data from 44 animals were included in the final analysis. 3.2. Behavioral assessment After SCI induction, motor function significantly was reduced in all groups compared to the control rats (df: 16, 136; F = 17.9; n =; p < 0.0001). During the 4 week follow up some degree of motor function recovery was observed but it did not reach the level of the control group (p < 0.001) (Fig. 1). In the first week following SCI a significant improvement was observed in the laser + enzyme group compared to SCI animals (p < 0.001). In the second, third, and fourth weeks the motor function recovery of animals in the laser, enzyme, and laser + enzyme groups significantly improved compared to the SCI group (p < 0.001). Two weeks after SCI induction, the laser + enzyme group showed a significant improvement in motor function recovery compared to the enzyme (p = 0.003) and laser (p < 0.001) groups. These differences were observed during the third (p = 0.04 for laser group; p = 0.04 for
immunoreactive area Percent of immunoreactive area = × 100 Total area of the section 2.9.2. Western blotting After carefully removing the dura and washing in PBS, spinal cord (T13-L1 level) was homogenized on ice in lysis buffer (Universal DNA/ RNA/Protein Purification kit, EURx). Protein concentration was assayed by Nano dropper (Thermo Science). The samples were loaded onto wells and electrophoresed on 12% SDS-polyacrylamide gel (SDSPAGE, BIORAD) for 1 h at a constant voltage (120 V). The proteins were transferred from the gel to a polyvinylidene difluoride membrane and were blocked with PBS containing 0.05% Tween-20 (PBST) and 5% 3
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Fig. 2. Luxol Fast Blue (LFB) staining was used for assessment of cavity size and myelin area (n = 3 per each group). Qualitative assessment showed that the majority of area observed in the samples of combination therapy group consisted of myelin, while less myelin area was observed in the SCI and laser-treated animals. Size of the cavity was significantly lower in combination group of laser + enzyme compared to the other groups. (A) Control; (B) Spinal Cord injury (SCI); (C) Vehicle; (D) Laser; (E) Chondroitinase ABC (enzyme); (F) laser + enzyme groups; (G) Quantitative analysis of cavity size. Data were expressed as the mean ± SEM. *, significant difference compared to SCI at level of p < 0.05; #, significant difference compared to laser group at level of p < 0.01; $, significant difference compared to Enzyme group at level of p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
animals (p < 0.001), Combination group has most axonal number (p < 0.001) (Fig. 3D–F).
enzyme group) and forth weeks (p = 0.032 for laser group; p = 0.045 for enzyme group) after SCI induction. 3.3. Assessment of myelin area and cavity size
3.5. Fibroblast invasion assessment
Qualitative assessment of spinal cord after LFB staining showed that the majority of the area observed in samples of the combination therapy group consisted of myelin, while less myelin area was found in the samples of SCI and laser-treated animals (Fig. 2A–F). Four weeks after SCI, a big cavity was seen in SCI (mean cavity size: 20.8 ± 3.6%) and vehicle (mean cavity size: 24.0 ± 4.2%) groups (df: 4, 31; F = 7.0; n = 3; p = 0.001). Cavity size in laser (mean cavity size: 15.1 ± 1.7%; p = 0.99 vs. SCI) and enzyme (mean cavity size: 21.0 ± 3.1%; p = 0.99 vs. SCI) groups were not significantly different compared to SCI animals. However, the size of the cavity was significantly smaller in the combination group of laser + enzyme (mean cavity size: 5.2 ± 2.0%) compared to SCI (p = 0.006), laser (p = 0.04), and enzyme (p = 0.003) groups (Fig. 2-G).
Quantitative evaluation of fibroblast invasion by H & E staining showed that SCI was led to fibroblast invasion around the cavity (df: 5,42; F = 48.35, n = 3; p < 0.001). This phenomenon is more prominent in the laser group. Aggregation of the fibroblasts in the laser group was compact around the cavity and higher than other groups (p < 0.0001) (Fig. 4). Moreover, the presence of fibroblast around the cavity was less in enzyme and combination groups compared to SCI animals (p < 0.05, p < 0.01) (Fig. 4D & E).
3.6. Evaluation of chondroitin sulfate proteoglycans expression Spinal cord injury caused a significant CSPG expression compared to the control group (df: 5, 18; F = 35.33; n = 3; p < 0.001). Although administration of Chondroitinase ABC (p < 0.001) and low level laser (p = 0.003) decreased the CSPG immunoreactivity compared to the SCI group, it did not lower it to that of the control group (p < 0.001 for laser and enzyme groups). Also, laser + enzyme therapy decreased CSPG immunoreactivity (p < 0.001) in a way that the difference with the control group was not statistically significant (p = 0.28) (Fig. 5).
3.4. Assessment of nerve fibers and axons around cavity Bielschowsky's staining revealed that a few axons exist around the cavity in the SCI and vehicle groups compared to the normal rats (df: 5,54; F = 126.9,n = 3; p < 0.001). (Fig. 3A–C). In the enzyme, laser, and the combination group the amount of axons was higher than SCI 4
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Fig. 3. Effect of Chondroitinase ABC (enzyme group), low level laser (laser group), and combination therapy (laser + enzyme group) on demyelination after spinal cord injury. Axons appear black. Bielschowsky's silver staining showed that spinal cord injury led to a reduction in axon density around damaged areas. Enzyme, Laser, and laser + enzyme were found to have protective effects on the axons and fibers. (A) Control; (B) Spinal cord injury; (C) Vehicle, (D) enzyme, (E) Laser, (F) laser + enzyme. Magnification ×20. ***p < 0.001, *p < 0.05 vs control group, ###, p < 0.001, vs SCI group & vehicle. $$ $P < 0.001 vs laser, p < 0.001 £££ difference between Enzyme and laser + enzyme group (n = 3 per group).
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Fig. 4. Effect of SCI induction, Laser, Enzyme and combination of laser + enzyme on invasion and entrance of fibroblasts around the cavity. Presence of fibroblasts is higher in the laser group. Rush of fibroblasts are indicated by yellow arrows, respectively. (A) Control; (B) Spinal cord injury; (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + Enzyme. Magnification ×40. ***p < 0.001, vs control group, ##, p < 0.01, vs. # p < 0.05, vs. SCI group & vehicle, $$$ p < 0.001, $$ p < 0.01 vs laser group, £ £ difference between Enzyme and laser + enzyme group. (n = 3 per group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.7. Assessment of GSK3β in the spinal cord
4. Discussion
GSK3β expressions significantly increased in the SCI group compared to the control animals (df: 5. 13; F = 50.0; n = 3; p < 0.001). Laser therapy (p = 0.001), administration of Chondroitinase ABC (p = 0.002), and combination of laser + enzyme (p < 0.001) reduced the expression of GSK3β and brought it down to the level of control animals' (Fig. 6).
In the present study, we examined the effects of LLLT (660 nm) and ChABC administrated alone and in combination on SCI. Results showed that combination of LLLT and ChABC had more beneficial effects on motor function recovery, cavity size and GSK3β, CSPG, and AQp4 expression than using each separately. There are two major defensive barriers that halt recovery after SCI, including flux of inflammatory agents and production of CSPG that together lead to glial scar formation (Shechter et al., 2011). Although these two barriers seem to be separate from each other, but a direct correlation exist between secretions of inflammatory cytokines with expression of CSGP (Shechter et al., 2011). Inflammatory factors begin to attack immediately after SCI and cause cell death, demyelination, cavitation, invasion of microglia and eventually motor function impairment (Byrnes et al., 2005; Levine, 2015; Zhou et al., 2014). Inflammation after SCI leads to other changes as well. Inflammation is associated with an increase in GSK3β expression in the spinal cord (Cuzzocrea et al., 2006; Renault-Mihara et al., 2011). GSK3β is one of the factors inducing demyelination and plays an important role in
3.8. Aquaporin 4 (AQP4) expression Induction of SCI increased AQP4 expression compared to the control group (df: 5, 13; F = 23.89; n = 3; p < 0.001). Treatment with laser (p = 0.006) and laser + enzyme (p = 0.001) significantly decreased AQP4 expression after SCI. Administration of Chondroitinase ABC did not affect AQP4 expression (p = 0.43). There is a significant difference between laser + enzyme group and enzyme group in expression of AQP4 (p = 0.03) (Fig. 7).
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Fig. 5. Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on chondroitin sulfate proteoglycan (CSPG) immunoreactivity in the study groups. Treatments by laser and enzyme reduced CSPG expression. (A) Control; (B) spinal cord injury (SCI); (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + enzyme groups; (G) Quantitative assessment of CSPG expression. Data were expressed as mean ± SEM (n = 3 per group). ***p < 0.001, vs control group, **p < 0.01 vs control group, *p < 0.05, vs control group, ###, p < 0.001, vs. SCI group, ##, p < 0.01, vs. SCI group.
enzyme secretion starts in the first day of injury and reaches its maximum level one week later (Jones et al., 2003). Another beneficial effect of ChABC is its anti-inflammatory effect induced through increasing IL10 secretion (Didangelos et al., 2014). The results of the present study showed that the expression of CSPG decreased in the animals treated with LLLT and ChABC. This change might be due to the anti-inflammatory effect of laser, but ChABC probably has acted through both CSPG decomposition and anti-inflammatory effects (Lee et al., 2010). The results showed that GSK3β decreased in both treatments, leading to a decrease in apoptosis and subsequently improvement in motor function after SCI which is indicative of reduced demyelination and axon regeneration (Dill et al., 2008; Cuzzocrea et al., 2006). It seems that both LLLT and ChABC treatments acted through decreasing CSPG and GSK3β, by which a permeable environment was provided for the axons of mature neurons to grow and elongate (Tohda and Kuboyama, 2011; Yilmaz and Kaptanoglu, 2015). Accordingly, the size of the cavity decreased and motor function improvement was observed. Axons were also visualized around the cavity using bielschowsky staining. Simultaneous treatment with both LLLT and ChABC was found to be more effective in improving SCI induced complications. In the combined treatment group, the expression of CSPG and GSK3β returned to basic levels; the size of the cavity was smaller compared to laser and
Wallerian degeneration, during which the axons separate from their cell bodies (Dill et al., 2008; Nagai et al., 2016). It has also been shown to affect the release of CSGP which prevents axon regeneration after SCI through multiple mechanisms (Dill et al., 2008; Nagai et al., 2016; Wakatsuki et al., 2011). Proliferation of immune cells, such as macrophages has also been found to play a role in increasing CSGP expression which disrupts axonal connections, hinders axonal regeneration and leads to motor function defects (Nagai et al., 2016). All these mechanisms might have contributed to the cavity formation and BBB decrease in SCI animals. In the present study the mentioned defense barrier is attacked by two mechanisms. Laser is the first treatment strategy used to destroy this barrier. Based on the study carried out by Goncalves et al., LLLT decreases the inflammation in the spinal cord by controlling the invasion of inflammatory cells in the central nervous system and inhibiting axon demyelination (Goncalves et al., 2016). Since studies have shown that invasion of inflammatory cells occur shortly after SCI, laser therapy was initiated 30 min after the injury in this survey (Zhou et al., 2014). The second selected treatment was administration of ChABC enzyme which plays a direct role in destruction of the barrier through decomposition of CSPG and creates a permeable environment for axon regeneration (Bradbury et al., 2002; Lee et al., 2010). Enzyme injection was done one week after SCI since previous studies had shown that 7
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Fig. 6. Assessment of glycogen synthase kinase-3β (GSK3β) expression in experimental groups. IHC study showed that spinal cord injury (SCI) increased GSK3 beta expression mostly around the cavity. Pattern of GSK 3β expression after SCI is similar to that of the chondroitin sulfate expression (A–F). Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on GSK 3β expression, 4 week after SCI induction (G). GSK 3β protein expression was normalized to β-actin protein. Western blot assay showed an increase in GSK 3β expression after spinal cord injury (SCI). Treatment by laser and laser + enzyme decreased its expression (H). Data are expressed as the mean ± SEM. (A) Control; (B) spinal cord injury (SCI); (C) Vehicle; (D) Laser; (E) Enzyme; (F) laser + enzyme groups; (G) western blotting bonds; (H) quantitative assay of GSK3β. *p < 0.05 **p < 0.01, vs control group, # #p < 0.01, vs. SCI group & vehicle. (n = 3 per group). ***p < 0.001, vs control group, ###, p < 0.001, vs. SCI group, ##, p < 0.01, vs. SCI group.
of inflammatory cytokines occurs, that are associated with cell death, demyelination, cavity formation, paralysis and even death (Li et al., 2014; Yang and Piao, 2003). In this stage AQP4 helps in reducing the edema by excretion of excessive water (Kimura et al., 2010). However, increased expression of AQP4 in the chronic stage, which is the target in this study, is associated with different results. In the chronic stage AQP4 is expressed as mislocalized and abnormal shaped in the glial barrier which is not capable of excreting excessive water (Vajda et al., 2002). Nesic et al. showed that the increased AQP4 in the chronic stage might
ChABC groups alone, more neuron aggregation was observed around the cavity and the motor function outcome was better compared to other groups. Despite the improvement observed in LLLT and ChABC treated groups, it seems that the application of these two treatments alone might have some limitations. Previous studies have shown that immediately after SCI, local edema (cytotoxic and vasogenic) occurs due to the injuries of the blood brain barrier which contribute to exacerbation of SCIs. Following edema, an increase in microglia and invasion 8
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Fig. 7. Effect of Chondroitinase ABC (enzyme), low level laser (laser) and combination therapy (laser + enzyme) on aquaporin4 protein (AQP4) expression 4 week after SCI induction. AQP4 protein expression was normalized to β-actin protein. Western blot assay showed an increase in AQP4 expression after spinal cord injury (SCI). Treatment by laser and laser + enzyme decreased its expression. Data are expressed as the mean ± SEM. *p < 0.05 **p < 0.01, vs control group, # #p < 0.01, vs. SCI group & vehicle, $ p < 0.05, vs enzyme group. (n = 3 per group).
Conflict of interest
be indicative of continued water retention and cytotoxic edema that hinders recovery after SCI (Nesic et al., 2006). The results of the present study showed that expression of AQP4 in animals treated with LLLT decreased compared to the SCI group, but administration of ChABC was not able to decrease AQP4 expression. Signs and symptoms of cytotoxic edema can be a limitation for treatment with ChABC alone. Results obtained from H & E staining revealed invasion of fibroblasts around the lesion in animals treated with LLLT, while the presence of fibroblasts was significantly lower in other groups even the SCI and vehicle groups. The impermeable glial barrier which includes CSPG, not only inhibits growth and invasion of axons in the lesion, but also prohibits invasion of fibroblasts (Soderblom et al., 2013). In this regard, one hypothesis states that some factors might be able to break the glial barrier and make it permeable for axonal regeneration, but fibroblast invasion creates fibrotic scar which is hard and impermeable (Soderblom et al., 2013; Cregg et al., 2014; Liesi and Kauppila, 2002). This phenomenon can limit the effects of laser and ChABC treatments since previous studies have shown that in cases where fibroblasts were aggregated, no improvements were observed (Soderblom et al., 2013). Nevertheless, in the present study aggregation of fibroblasts was decreased in the combined treatment group with LLLT and ChABC to the control level. In these cases, laser might have provided a suitable environment for ChABC to take effect by controlling inflammation and edema. Probably LLLT has reduced inflammation and ChABC has helped preserve the integrity of the tissue by decomposing CSPG and reducing inflammation. The observed reduction of CSPG and GSk3β to that of the control levels, the improvement in the motor function and the decreased size of the cavity are confirmed for the proposed hypothesis. Understanding detail mechanism will need to study more in the future.
The authors have declared no conflict of interest. Acknowledgment This study was supported by Iran University of Medical Sciences (IUMS) (25049), Faculty of Medicine and Physiology Research Center. The authors would like to thank the Heltschl Company for giving laser as a present. References Alluin, O., Delivet-Mongrain, H., Gauthier, M.K., Fehlings, M.G., Rossignol, S., KarimiAbdolrezaee, S., 2014. Examination of the combined effects of chondroitinase ABC, growth factors and locomotor training following compressive spinal cord injury on neuroanatomical plasticity and kinematics. PLoS One 9 (10), e111072. Azim, K., Butt, A.M., 2011. GSK3beta negatively regulates oligodendrocyte differentiation and myelination in vivo. Glia 59 (4), 540–553. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12 (1), 1–21. Bingol, U., Altan, L., Yurtkuran, M., 2005. Low-power laser treatment for shoulder pain. Photomed. Laser Surg. 23 (5), 459–464. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., et al., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416 (6881), 636–640. Byrnes, K.R., Waynant, R.W., Ilev, I.K., Wu, X., Barna, L., Smith, K., et al., 2005. Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury. Lasers Surg. Med. 36 (3), 171–185. de Carvalho, Pde T., Leal-Junior, E.C., Alves, A.C., Rambo, C.S., Sampaio, L.M., Oliveira, C.S., et al., 2012. Effect of low-level laser therapy on pain, quality of life and sleep in patients with fibromyalgia: Study protocol for a double-blinded randomized controlled trial. Trials 13, 221. Cheng, C.H., Lin, C.T., Lee, M.J., Tsai, M.J., Huang, W.H., Huang, M.C., et al., 2015. Local delivery of high-dose chondroitinase ABC in the sub-acute stage promotes axonal outgrowth and functional recovery after complete spinal cord transection. PLoS One 10 (9), e0138705. Cregg, J.M., DePaul, M.A., Filous, A.R., Lang, B.T., Tran, A., Silver, J., 2014. Functional regeneration beyond the glial scar. Exp. Neurol. 253, 197–207. Cuzzocrea, S., Genovese, T., Mazzon, E., Crisafulli, C., Di Paola, R., Muia, C., et al., 2006. Glycogen synthase kinase-3 beta inhibition reduces secondary damage in experimental spinal cord trauma. J. Pharmacol. Exp. Ther. 318 (1), 79–89. Didangelos, A., Iberl, M., Vinsland, E., Bartus, K., Bradbury, E.J., 2014. Regulation of IL10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury. J. Neurosci. 34 (49), 16424–16432. Dill, J., Wang, H., Zhou, F., Li, S., 2008. Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J. Neurosci. 28 (36), 8914–8928. Dyck, S.M., Alizadeh, A., Santhosh, K.T., Proulx, E.H., Wu, C.L., Karimi-Abdolrezaee, S., 2015. Chondroitin sulfate proteoglycans negatively modulate spinal cord neural precursor cells by signaling through LAR and RPTPσ and modulation of the rho/ ROCK pathway. Stem Cells 33 (8), 2550–2563. Ek, C.J., Habgood, M.D., Dennis, R., Dziegielewska, K.M., Mallard, C., Wheaton, B., et al., 2012. Pathological changes in the white matter after spinal contusion injury in the rat. PLoS One 7 (8), e43484. Ekim, A., Armagan, O., Tascioglu, F., Oner, C., Colak, M., 2007. Effect of low level laser therapy in rheumatoid arthritis patients with carpal tunnel syndrome. Swiss Med. Wkly. 137 (23–24), 347–352.
5. Conclusion The results of this survey showed that although the application of LLLT and ChABC alone can reduce the cavity size and GSk3β and CSPG expression and lead to improvements in motor recovery, but they are associated with complications that limit their use. Administration of ChABC cannot reduce AQP4 which is an indicator for cytotoxic edema, an inhibitory factor for improvement after and SCI. Application of LLLT was also associated with aggregation of fibroblasts around the lesion which is indicative of the fibrotic scar formation. However, in the combined treatment group, these limitations were not present. Expression of GSk3β and CSPG returned to baseline; the size of the cavity decreased and presence of axons around the lesion and the motor function recovery was improved. Therefore, combined treatment with LLLT and ChABC was found to be more effective than application of these treatments alone. 9
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