Combined treatment of high‐intensity interval training with neural stem cell generation on contusive model of spinal cord injury in rats

Spinal cord injury (SCI) leads to inflammation, axonal degeneration, and gliosis. A combined treatment of exercise and neural stem cells (NSC) has been proposed to improve neural repair. This study evaluated a combined treatment of high‐intensity interval training (HIIT) with NSC generation from adipose‐derived stem cells (ADSCs) on a contusive model of SCI in rats.


INTRODUCTION
Spinal cord injury (SCI) is a traumatic neurological condition that is characterized by the complete or incomplete loss of motor, sensory, and autonomic neural functions (Rouanet et al., 2017). SCI is caused by a primary damage mechanism that leads to secondary tissue loss through a cascade of cellular and molecular reactions (Gaudet & Fonken, 2018;Kim et al., 2017). These changes are distinct in two phases: the first is an acute phase that causes edema, hemorrhage, demyelination, inflammation, oxidation, apoptosis, and necrosis of both neurons and oligodendrocytes (Allison & Ditor, 2015;Jendelova, 2018;Kim et al., 2017). The second phase leads to cavity formation, microglial activity, and glial scar formation (astrogliosis).
Morphological changes associated with secondary damage are barriers to replacing the cavity with endogenous neural stem cells (NSCs) and axonal regeneration (Akhtar et al., 2008;Darvishi et al., 2014).
To improve axonal regeneration and replacement of cell loss following central nervous system (CNS) injury, stem cells, and environmental factors, such as neurotrophins, are critical (Gazdic et al., 2018;Hodgetts & Harvey, 2017;Perea & Araque, 2007). Although promising results have been obtained for the treatment of SCI, there are few to no options to improve functional recovery (Hodgetts & Harvey, 2017;Shahrezaie et al., 2017).
A variety of neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, and neurotrophin 3 (NT3) and 4 (NT4), were capable of inducing axon and dendritic extension (McTigue et al., 1998;Sainath & Gallo, 2015;Tanaka et al., 2000). There was also an effect on neuronal activity, survival, and remodeling. Of these, BDNF improved the regeneration of axonal damage and synaptogenesis. In addition, NT3 and BDNF restrained the formation of a glial scar after CNS injury (Xu et al., 1995;Yan & Wood, 2000), while a combination of NT3/BDNF and basic fibroblast growth factor (FGF2) mediated survival and axon regeneration following optic nerve injury (Blanco et al., 2000). Moreover, cell transplantation has been tested for the replacement of dead cells.
Therefore, transdifferentiation of adipose-derived stem cells (ADSC) was proposed as an appropriate source of neural lineage, since these can be easily obtained and can generate neurotrophic factors, extracellular matrix molecules that promote axonal growth (Mazini et al., 2019;Ohta et al., 2017). One of the limitations of using cell transplantation may be inadequate survival and integration of graft cells. Several studies have described a decrease in the number of graft cells after transplantation in the injury area and then modest functional recovery (Radhakrishnan et al., 2019). Some recent studies have proposed that functional recovery can be promoted in a combination with other therapeutic approaches such as physical exercise. Physical activity can have a positive effect on CNS activity through promotion of synaptic plasticity and survival neurons (Liu et al., 2019;Mattsson et al., 2008;Uysal et al., 2015). Exercise facilitates motor and sensory function as well, and improves the expression of genes and neurotrophic factors such as BDNF and NT3 in the injured spinal cord (Fritsch et al., 2010;Jung et al., 2016). In addition, there is an increased generation of Schwann cells, axonal growth, and suppressed muscle atrophy follow-ing CNS injury (Tashiro et al., 2016;Theisen et al., 2017). Schwann cell implantation into a contusion lesion resulted in unregulated expression of neurotrophic factors, myelination, and axon regeneration and promoted motor function (Flora et al., 2013;Golden et al., 2007;Tran et al., 2018). One exercise protocol is high-intensity interval training (HIIT), which is characterized by high-intensity exercise associated with short rest intervals. The result of this training strategy is aerobic-like effects.
This exercise protocol is used for different kinds of disorders such as cardiovascular failure, obesity, pulmonary disease, and type 2 diabetes (Engel et al., 2018;Francois & Little, 2015;Ito, 2019). However, the effect of a combined treatment of HIIT protocols with NSC generation from ADSC on the functional recovery of locomotion is not fully understood. Thus, the purpose of this study was to investigate whether HIIT with NSC transplantation would stimulate histological and functional recovery after SCI.

Animal model
The rats were anesthetized (80 mg/kg ketamine and 10 mg/kg xylazine, intraperitoneally), and a laminectomy was performed at the T11 level to expose the T12-L1 spinal cord without the dura matter. A contusion injury was produced on the exposed dorsal surface of the spinal cord. The contusion was carried out by dropping a 10 g rod with a 2.5 mm diameter, from a height of 25 mm. After an injury, muscles and skin were sutured. The rats in the PC group and in the L/NSC and L/HIIT groups received laminectomy without injury.

High-intensity interval training protocol
One week after surgery, the rats in the exercise groups (L/HIIT, SCI/HIIT, and SCI/HIIT/NSC groups) were trained by swimming as described by Terada et al. (2004). The training session consisted of 14 bouts of 20/s swimming periods with 10/s resting time between each session. This method was performed three times a week on periodical days. Exercise compatibility was assessed at the end of 6 weeks, when all of the rats were investigated in an acute test of swimming while bearing a load of 14% of their body weight.

ADSC isolation
ADSCs were isolated according to the method explained by Darvishi et al. (2020). In brief, adipose tissue was obtained from the abdominal regions of female rats. The specimen was washed with phosphatebuffered saline (PBS) containing 100 U/mL penicillin and 100 μg streptomycin (Gibco  Moayeri et al., 2020). After 21 days, the induced adipocytes were stained using oil red stain. Osteogenic induction was performed in DMEM containing 10 nM dexamethasone, 50 mg/mL L-ascorbic acid, and 10 mM b-glycerophosphate for 3 weeks. Mineralization of the extracellular matrix was displayed using alizarin red. Chondrogenic induction was carried out using 6.25 lg/mL insulin, 6.25 lg/mL transferrin, 1.25 lg/mL bovine serum albumin, 50 lg/mL ascorbic acid, 10-7 M dexamethasone, and 10 ng/mL TGF-b3 (Sigma-Aldrich, Steinheim, Germany) for 3 weeks, and chondrogenesis was evaluated with 0.1% Safranin O Moayeri et al., 2020).

Cell labeling and transplantation
Transfection was carried out using the pEGFP-C1 plasmid reporter

Basso-Beattie-Bresnahan (BBB) locomotor scale
Functional recovery was evaluated using the Basso-Beattie-Bresnahan (BBB) locomotor scale. The test was carried out 2 days before surgery and again following surgery on days 1, 3, 7, 8, 9, and 14, then once a week for 12 weeks. The BBB test is a standard method to assess hindlimb locomotion in an open field (80 × 130 × 30 cm) with two blind investigators. The BBB test was assessed using the 21-locomotion scale (0 = flat paralysis and 21 = normal gait) with animal observation for 3 min (Darvishi et al., 2014).

Hoffman reflex
The Hoffman reflex (H-reflex) was performed as described earlier (Darvishi et al., 2014). Briefly, the ratio of the maximum H to maximum M reflexes (H/M ratio) was done with an electromyography (EMG) or NCV instrument (Cadwell, Series II, USA). The H/M ratio was recorded at preoperation and again at 1, 6, and 12 weeks postoperation. After anesthesia, the sciatic nerve on the left side was exposed 0.5 cm above the nerve bifurcation. To record reflexes, stimulator electrodes (cathode electrode located above the anode) were inserted adjacent to the sciatic nerve. The active, reference, and ground electrodes were located in the plantar muscles, the digital interosseous muscles, and the skin at the base of the tail, respectively. To record the first H-wave, the sciatic nerve was stimulated with 0.2 ms at 0.

Immunocytochemistry
For immunofluorescence, ADSCs and NSCs were seeded on gelatincoated coverslips and washed three times with PBS, and then fixed in 4% paraformaldehyde in PBS for 15 min and exposed to 0.1% bovine serum albumin solution containing triton x-100 (0.3%) for 30 min at room temperature. Next, cells were incubated overnight with primary antibodies that included CD90, CD49, CD105, CD45, Nestin, NF68, Sox2, Oct4, NeuroD, and NeuN (see Table 1

Immunohistochemistry
After washing, cross-tissue sections were exposed to triton x-100 (0.3%), and blocked in 10% goat serum for 45 min at room temperature. Tissue sections were then placed overnight in primary antibodies including anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:150, Millipore, Germany), anti-S100β cell monoclonal antibody (1:400, Cosmo Bio Co., Japan) and anti-neurofilament rabbit polyclonal antibody (1:400, Sigma, USA). The sections were incubated for 2 h with FITC secondary antibodies at 1:400 dilution. The sections were evalu-ated using a fluorescing microscope, and the intensity was calculated for each image (Image J software 1.43U). The coefficient of variation was then measured (20% for all groups).

Histology of spinal tissue
The animals were fully anesthetized using ketamine (80 mg/kg intraperitoneally; Alfasan Company, Woerden, The Netherlands).
Thereafter, rats were perfused with 4% paraformaldehyde and 1. xylene. The slides were mounted and observed using a light microscope (Olympus).

Statistical Analysis
For all experiments, the data were analyzed using SPSS 16 (www.spss. com  of ADSCs were immunoreactive to these markers (Figure 1d-h).

Rat NSC generation and characterization
The morphology of the floating neurosphere is shown in Figure 2a.

GFP labeling and transplantation
Before transplanting cells in vivo, they were labeled with GFP. Figure 3 shows transfection of NSCs using the pEGFP-C1 plasmid by lipofec-tamine™ 2000, which shows green fluorescence. The efficiency of nucleofection using the pEGFP-C1 plasmid was 55.2 ± 1.94%.
As shown in Figure

BBB locomotor scale
The results of the BBB scores are shown in Figure 5 across all eight groups over 12 weeks. The BBB score significantly decreased after SCI, and this condition remained during the experimental study. However, the HIIT, NSC, and combined treatment of HIIT/NSC groups showed increased BBB scores in SCI rats after 12 weeks (p < .05).
Functional recovery of lower limbs was confirmed by cell therapy and exercise (HIIT). In addition, among three experimental groups (SCI/HIIT, SCI/NSC, and SCI/HIIT/NSC), the highest BBB score was found in the SCI/HIIT/NSC group, which was significantly different from all other experimental groups. The BBB scores for the SCI/HIIT, SCI/NSC, and SCI/HIIT/NSC groups were 12.71 ± 0.24, 13.57 ± 0.14, and 15.28 ± 0.45 at 12 weeks after SCI, respectively.

Hoffman reflex (H-reflex) analysis
The electromyography was done using the H-reflex as a parameter for evaluating the improvement of the treated animals by an EMG/NCV instrument (Cadwell, Series II). The H/M ratio: H max and M max waves were recorded in order to calculate the H max /H max (M max ), which was done preoperatively, and at 1, 6, and 12 week(s) postoperatively. The F I G U R E 5 Effect of cell therapy and exercise on locomotor function using BBB score. The data were obtained at days 1, 3, 7, 8, 9 and at weeks 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 lowest H/M ratio was 0.579 ± 0.016 in the SCI/HIIT/NSC group, which was significantly different from that seen in the SCI+ HIIT group, and was also significantly lower compared to other times in the same group after injury (Figure 6), but it was not significantly different from the SCI + NSC group.

Gliosis and axon regeneration after SCI
Results also showed the effect of exercise and NSC transplantation on the astrogliosis following SCI by immunostaining for GFAP, a marker for astrocytes. The expression of GFAP-positive cells declined in both the SCI/HIIT and SCI/NSC groups compared to the untreated group.
The significantly lowest level was noticed in the SCI/HIIT/NSC groups (p < .05) (Figure 7i).

Cavitation
At the epicenter of the SCI group, the cavity is large compared with the SCI/HIIT, SCI/NSC, SCI/HIIT/NSC, and sham-operation groups.
Morphometric evaluation shows that the lowest volume density of cavitation was in the SCI/HIIT/NSC group, which was significantly

DISCUSSION
SCI leads to neuronal apoptosis, hemorrhage, and inflammation, cavity formation, loss of trophic factors and glial scarring, resulting in altered neuronal connections and functional disabilities (Gaudet & Fonken, 2018;Kim et al., 2017). Gliosis prevents the growth and regeneration of nerve fibers (Yiu & He, 2006). Stem cell transplantation is a technology used to decrease the inflammatory response, inhibit neural loss, and promote neuronal and axonal regeneration (Zhou et al., 2019).
Nevertheless, immunological rejection and poor survival of grafted NSCs are major obstacles to the success of this therapeutic measure (Mothe et al., 2013;Parr et al., 2008). There is little information that explains the cause of transplanted cell death and the effects of environmental factors. Several studies have shown that the death of transplanted NSCs can be induced by reactive nitrogen species or reactive oxygen species (ROS) (Hwang et al., 2014). Exercise can reduce ROS-related damages through mechanisms involving the antioxidant system, trophic factor expression, and modulation of signaling pathways (Asimakos et al., 2018;Ristow et al., 2009;Simioni et al., 2018). Wang et al. (2006) reported that HIIT enhanced tumor necrosis factor alpha, the transcription factor for BDNF synthesis, and the CREB pathway.
The present study found that a combination of NSCs derived from ADSC and HIIT promoted histological and behavioral recovery in a contusive model of SCI. Therefore, simultaneous cellular and exercise therapy could be a promising method for functional improvement. In recent years, several studies have been conducted on the production of neurospheres from MSC. In 2014, Abbaszadeh and Darabi showed that BMSCs differentiate into the neurosphere and then NSCs using EGF, bFGF, and B27 factors (Abbaszadeh et al., 2014;Mukai et al., 2016).
Also, Graf (2011) showed that neurons derived from fibroblasts could be reverted into fibroblasts or even to primitive stem cell populations.
For this reason, to stabilize transplanted cells, Monni et al. (2011) andJoo et al. (2012) proposed a neurosphere culture medium. In a previous study, we showed that ADSCs are capable of differentiating into the neurosphere and expressing nestin and stemness markers (Sox2, Oct4, and Nanog). Moreover, NSCs derived from the neurosphere are immunoreactive to nestin (Yang et al., 2015). On the other hand, cellnestin positive is known as a lineage-reprogramming factor with a high differentiation ability and a low risk of tumorigenesis (Bernal & Arranz, 2018;Zhou & Melton, 2008). We carried out an indirect protocol for transdifferentiation as similar as possible to the path of natural differentiation. In the present study, NSCs expressed the embryonic stem cell markers including SOX2, OCT4, and nestin. In addition, no tumorigenesis was detected 12 weeks after transplantation of these NSCs derived from ADSC. Our results showed that GFP-positive NSCs are able to integrate into the spinal cord, and we confirmed survival and migration. Moreover, transplantation of NSCs led to significantly lower numbers of GFAP immunoreactive cells as well as significantly higher S100β immunodensity of the spinal cord. Riemann et al. (2018) also discussed neural precursor cell transplantation where a significant reduction in astrogliosis and post-traumatic apoptosis was seen. We observed promotion of functional recovery in NSC-transplanted animals compare to the untreated group as well as a trend toward a decline in cavity size. Abbaszadeh (2014) reported that neurospherederived oligodendrocyte-like cells decreased cavity formation at the epicenter of a transplantation area and improved functional recovery in a contusive model of SCI (Abbaszadeh et al., 2014). In this study, we found that neurosphere-derived NSC transplantation decreased cavity formation and GFAP expression as well as increased S100β followed by axon regeneration compared to the untreated group and this finding is consistent with previous reports (Darvishi et al., 2014;Li & Lepski, 2013). The results of EMG and BBB tests were consistent; our findings showed that the H-reflex was enhanced after the SCI (Lee et al., 2007).
We observed that the H/M ratio increased after SCI and decreased with the implantation of NSCs which is consistent with other studies (Darvishi et al., 2014;Lee et al., 2007). The effects of different types of exercise on functional improvement and expression of neurotrophic factors have been investigated in several models of SCI (Côté et al., 2011;Gómez-Pinilla et al., 2002). In one study (Heng, 2009), treadmill training increased locomotor function following rat SCI (Heng & de Leon, 2009). In the current study, HIIT decreased the H/M ratio and improved the BBB test 5 weeks after contusion of spinal cord. HIIT promoted the expression of BDNF in the spinal cord and brain (Afzalpour et al., 2015). Moreover, this finding indicates that this neurotrophin, through a tyrosine kinase b receptor (TrkB), increases neurogenesis, axonal regeneration, and synaptogenesis (Tyler & Pozzo-Miller, 2001;Vaynman et al., 2004). However, BDNF expression decreased in SCI, accompanied with augmentation of axonal sprouting and promotion of this neurotrophin. Ying et al. (2008) reported that the mRNA levels of BDNF and neurotrophin-3 (NT-3) were decreased by SCI, and exercise training increased the mRNA levels. Accordingly, it appears that HIIT exercise improves motor recovery after SCI (Leech & Hornby, 2017). These findings are in agreement with the data obtained in this study. Our results showed that SCI led to cavity formation, and HIIT decreases the size of this cavity as well as promoting axonal regeneration and enhancing Schwann cells. Schwann cell proliferation is correlated with both axonal regeneration and migration. These cells increase nerve regeneration by secreting neurotrophic factors. Previous studies have found that implantation of Schwann cells enhanced and guided axonal growth following SCI (Fortun et al., 2009;Imaizumi et al., 2000). In the current study, we observed that HIIT promoted S100β expression while decreasing GFAP expression, which is in agreement with other studies. Neurosphere-derived NSC transplantation leads to improved motor recovery and axonal growth in the injured spinal cord through the induction of neurotrophic factors and induction of Schwann cell proliferation through exercise, along with reducing gliosis. The simultaneous effects of cell therapy (NSC) and exercise (HIIT) on the survival of neurons and regrowth of axons were greater than either treatment alone.

AUTHOR CONTRIBUTIONS
The Authors confirm contribution to the paper as follows: study con-

CONFLICT OF INTEREST STATEMENT
Authors declare that they have no conflict of interest.