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Research Article

Epidural oscillating field stimulation increases axonal regenerative capacity and myelination after spinal cord trauma

Bacova, Maria; Bimbova, Katarina; Kisucka, Alexandra; Lukacova, Nadezda; Galik, Jan,*

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doi: 10.4103/1673-5374.339497
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Abstract

Introduction

Spinal cord injury (SCI) results in permanent or partial motor, sensory, and autonomic impairment. There is a need to seek interventions during the acute phase of injury to reduce secondary injury (Ahuja et al., 2017), but no clinically relevant therapies are available to manage the neurological loss. Electrical stimulation (ES) has been shown to effectively reduce neuropathic pain and muscle atrophy (Huang et al., 2019; Thomaz et al., 2019) and promote regeneration of motor functions. Therefore, ES has been considered a promising treatment for SCI (Ievins and Moritz 2017; Darrow et al., 2019). Various types of stimulation treatment have been used with promising results: epidural ES (Wagner et al., 2018), functional ES (Marquez-Chin and Popovich, 2020), intraspinal ES (Saigal et al., 2004; Mondello et al., 2014), and transcutaneous ES (Hofstoetter et al., 2015). Although ES promotes the anatomical plasticity of the central nervous system after SCI (Hassannejad et al., 2019) and activates residual neuronal pathways (Smith and Knikou, 2016), the underlying mechanisms are not fully understood.

The main reasons for nonfunctional signal transduction after SCI are limited growth capacity of axons, failed axonal sprouting, and loss of myelinization of surviving and newly sprouting axons due to the formation of scar tissue around the site of injury, lack of trophic support and limited regenerative capacity of axons caused by demyelination, followed by axonal degeneration (Alizadeh et al., 2015). ES has been proven to be beneficial in these posttraumatic events (Carmel et al., 2010; Jack et al., 2020). Thus, enhancing the functional connectivity of spared circuitry using ES may be a viable means of promoting functional recovery after SCI.

Myelin sheaths formed by oligodendrocytes are responsible for the fast action potential transmission of the CNS. Following SCI, some axons and oligodendrocytes are initially lost via necrosis and mechanical injury (Almad et al., 2011). As injury progresses, a massive loss of oligodendrocytes occurs through apoptosis and autophagy that results in demyelination of the injured and spared axons (Casha et al., 2001; Plemel et al., 2014). Although spontaneous remyelination occurs naturally in the CNS after a traumatic event (Salgado-Ceballos et al., 1998), these remyelination attempts are often insufficient and limited due to post-traumatic environment changes (Xing et al., 2014; Hesp et al., 2015).

Oscillating field stimulation (OFS) reverses the polarity of the electric field every 15 minutes to stimulate the regeneration of both ascending and descending neural pathways, which makes it a promising strategy in various models of SCI (Borgens et al., 1999; Li et al., 2019). In our previous experiments, we demonstrated that epidural OFS had neuroprotective effects on spinal tissue during the 4-week study period (Bacova et al., 2019). In this study, we investigated the effects of epidural OFS applied immediately after spinal compression injury on myelin regeneration, axonal and oligodendrocyte survival, and functional recovery during a 8-week study period. We also observed several major behavioral changes in rats after epidural OFS.

Materials and Methods

Ethics statement

All experiments were approved by the Animal Use Committee at the Institute of Neurobiology, Slovak Academy of Sciences, as well as by the State Veterinary and Food Administration in Bratislava (approval No. 715/19-221/3, approval date March 25, 2019; approvial No. 4434/16-221/3, approval date May 3, 2021) and conducted in accordance with the EC Council Directive (2010/63/EU) regarding the use of animals in research. All efforts were made to minimize the number of rats and their suffering during the experiments.

Experimental animals

Thirty-six 3–4 month-old female Wistar rats, weighing 250–300 g, were included in this study. They were randomly assigned to four experimental groups: Intact control (no surgical intervention; n = 6), SCI (T9 compression only; n = 10), OFS + SCI (SCI followed by OFS; n = 10), and nOFS + SCI (SCI followed by implantation of nonfunctional OF stimulator; n = 10) groups. Animals were housed individually with food and water provided ad libidum. The study design is shown in Figure 1.

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Figure 1:
Experimental timeline.

Spinal cord compression and OF stimulator implantation

Animals underwent spinal cord compression at the ninth thoracic vertebral region (T9) using a custom-made compression device with a steel rod with an arc-shaped plastic impactor (2.5 × 2.0 mm). After laminectomy, a small impactor was positioned over the midpoint of the T9. SCI was induced with a 40 g force for 15 minutes causing permanent paraplegia of the lower limbs. Immediately after spinal injury, two Ir/Pt wire electrodes connected to a custom-made miniature stimulator with an oscillating electric field (OF; 50 µA) (Figure 2A) were implanted into the epidural space cranially and caudally to the lesion site (for more details see Bacova et al., 2019). The miniature OF stimulator reverses the polarity of the electric field every 15 minutes to initiate the simultaneous regeneration of both ascendent and descendent neuronal pathways. The OF stimulator is powered by a lithium battery (3V/90 mA) and supplies energy for at least 6 weeks of continuous stimulation. To prevent post-operative infection, rats received antibiotics (amoksiklav, 30 mg/kg intramuscularly; Sandoz Pharmaceuticals, Ljubljana, Slovenia) and the analgesic drug (novasul, 2 mL/kg intramuscularly; Richterpharma, Wels, Austria) for 3 days after surgery. Afterwards, rats were housed individually, with food and water provided ad libidum. The animal’s health status was monitored daily with manual bladder emptying twice a day until restoration of the urination reflex. After 8 weeks of survival, rats were perfused transcardially using 250 mL of saline followed by 250 mL of 4% paraformaldehyde. Then, rats were decapitated and spinal tissue (T7–T11) was collected for further analysis.

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Figure 2:
Principle of stimulation using oscillating electric field.It is known that the axons respond immediately to the voltage gradient and tend to orient themselves parallel to the long axis of such a gradient. Nerve fibers grow faster and preferentially towards the cathode (negative pole of applied electric field), and are distracted from the opposite pole. (A) Schematic picture of oscillating field stimulator before an implantation. (B) Subcutaneous placement of the miniature stimulator with epidural implantation of stimulating Ir/Pt stimulation wires. (C) Caudally placed cathode stimulates the regeneration and growth of motor nerve fibers. Cathode placed cranially to the lesion promotes the recovery of sensory fibers. Oscillating polarity of electric field every 15 minutes allows stimulation of the outgrowth of sensory and motor axons simultaneously.

Spinal cord tissue processing

Morphometric analysis of tissue integrity

Isolated frozen spinal cord segments for histological analysis were carefully cut into 25 µm-thick slices using Kryostat (Leica CM1850, Wetzlar, Germany) and placed on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). Morphometric analysis of the spinal cord tissue integrity was performed using standard Luxol fast blue/Cresyl violet (LFB/CV) staining. LFB is primarily used for staining of the myelinated axons in white matter, and CV stains the Nissl substance in neurons. Spinal cord slices were incubated for 10 minutes in 0.1 M phosphate-buffered saline (PBS), 120 minutes in 70% ethanol, and then overnight in 0.1% LFB solution. The next day, the spinal sections were rinsed with distilled water, decolored with 0.05% of lithium carbonate and 40% ethanol to highlight the white and gray matter of the spinal cord, and then incubated with 0.2% CV for 10 minutes. Afterwards, the slices were rinsed with distilled water and dehydrated with a graded series of ethanol (80%, 90% and 99.8%), cleared with xylene, and covered with coverslips. Stained slices were scanned using an automatic digital scanner Aperio AT2 (Leica Biosystems, Nussloch, Germany) with 20× magnification and analyzed with ImageJ software (Version 1.52, NIH, Bethesda, MD, USA; Schneider et al., 2012).

Immunohistochemical analyses

For immunohistochemical analysis, the following primary antibodies were used: anti-neurofilament (NF-l) light chains, to visualize and quantify neurofilaments, anti-growth-associated proteins (GAP-43), as newly sprouting axons; anti-APC (adenomatous polyposis coli) for oligodendrocytes; and anti-myelin basic protein (MBP), myelin.

Spinal cord sections for immunohistochemistry were washed in 0.1 M PBS with 0.3% Triton X-100 (PBS-T; Sigma-Aldrich, St. Louis, MO, USA) three times for 10 minutes each. After washing, the spinal slices were blocked for 30 minutes at room temperature in PBS with 5% normal goat/rabbit serum and 0.3% Triton-X100. To visualize oligodendrocytes, neurofilaments, and outgrowing axons, the following primary antibodies were used: APC (1:200, mouse; Milipore, Burlington, MA, USA, Cat# 0P80), NF-l (1:100, rabbit; Cell Signaling, Danvers, MA, USA, Cat# C28E10), GAP-43 (1:500, rabbit; Abcam, Cambridge, UK, Cat# ab129990). To visualize myelin (MBP) (1:200, rabbit; Sigma, Cat# M3821), the slices were pre-incubated with 99.8% ethanol for 15 minutes. After overnight incubation with primary antibodies at 4°C, the sections were washed in PBS with 0.3% Triton X-100 and incubated with secondary antibodies (FITC goat anti-mouse IgG, 1:200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA, Cat# 115-095-149; FITC goat anti-rabbit IgG, 1:200, Cat# 111-095-003, Rhodamine Red anti-rabbit IgG, 1:400, Cat# 111-295-14, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 hours at room temperature. Labeled spinal slices were washed again with PBS with 0.3% Triton X-100 and incubated with DAPI (1:10,000, Sigma, Cat# 102362760000001) to stain cell nuclei for 30 minutes. After incubation, the slices were finally washed with PBS-T, rinsed with distilled water, and placed on the slides. Selected regions (dorsal and lateral funiculi of the white matter) were captured by fluorescent microscope (Olympus BX51, Tokyo, Japan) with 40x magnification and then analyzed using ImageJ software (Version 1.52).

Western blot analysis

Spinal cord segments (T7–T11) were homogenized and the protein concentration was quantified with Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific). The samples (20 µg per lane) were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12%, 90 minutes/100 V) and then transferred into a polyvinylidene difluoride membrane (Bio-Rad Lab, Hercules, CA, USA). Afterwards, the membranes were blocked in 5% skimmed milk (Blotting-Grade Blocker, Bio-Rad Lab) for 90 minutes at room temperature and then incubated overnight at 4°C with primary antibodies diluted in 2.5% milk/Tris-buffered saline + Tween 20 (TBS-T). For western blot analysis, the following primary antibodies were used: anti-rabbit NF-l (1:1000; Cell Signaling, Danvers, MA, USA), anti-rabbit GAP-43 (1:500; Merck, Millipore, Burlington, MA, USA) and anti-mouse MBP (1:500; Abcam). The next day, the membrane was washed in TBS-T for four times for 5 minutes each and incubated with secondary antibodies (mouse anti-rabbit IgG HRP, 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc2357; goat anti-mouse IgG HRP, 1:50000, Merck Millipore, Darmstadt, Germany, Cat# AP124P) diluted in 2.5% milk/TBS-T for 90 minutes. The blotted protein bands were visualized by enhanced chemiluminescent solution (SuperSignal™ West Pico, Thermo Fisher Scientific) and exposed to Fusion XT scanner (Vilber, Collégien, France). After exposure, the membranes were stripped (Restore™ Plus Western Slot Stripping Buffer; Thermo Fisher Scientific) and incubated with monoclonal antibody against β-actin HRP (1:20,000, Sigma, Cat# A3854) as a loading control. Band optical density was evaluated using Quantity One 4.6 software (Bio-Rad Lab) and the obtained results were determined as a ratio of protein to the level of β-actin.

Behavioral assessment

The recovery of neurological functions after experimental SCI was analyzed using multiple behavioral tests. Rat sensory and motor functions were evaluated using the hot plate test, open field test and Basso, Baettie, Breshnahan (BBB) locomotor rating scale during the survival period. Each rat in the experiment was assessed and evaluated individually. All behavioral tests were performed by the same investigator blinded to study parameters to minimize rat stress level and achieve objective results.

Exploration and anxiety tests

The spontaneous activity of rats was assessed using the modified open field test (Martinez et al., 2009), which started 4 weeks post-SCI and was conducted once weekly until the end of study period. The rats were tested in the open field, which was a 60 cm × 45 cm transparent plexiglass box with a non-slippery floor. During a 5-minute interval, basic motor activity, exploration, anxiety, and depressive behavior were observed and evaluated. All monitored parameters were obtained by the descriptive method of direct observation and using the automated system for data processing and analysis (EthoVision XT, Noldus, Wageningen, The Netherlands). The rats were acclimated to the testing room for 20 minutes before testing. The test consisted of placing a single rat in the center of the arena during a 5-minute experiment. Locomotion was automatically registered using a video-tracking system and reported as the distance traveled (in centimeters). The duration of exploratory behavior and time spent grooming or freezing were manually scored from video recordings. The arena was cleaned with 70% ethanol between each trial.

Sensory function test

To assess sensory functions and nociception, the hot plate test (Giglio et al., 2006) was carried out in all experimental groups at 4 weeks post-SCI, and conducted once weekly until the end of survival. Rats were placed on an enclosed hot plate (52 ± 0.5°C) until the nociceptive symptoms started to occur. Once the rat was placed on the hot plate, the response latency to lick a hindpaw or jump out of the enclosure was measured as a primary sign of nociception. To prevent tissue damage, the rats were immediately removed after the pain manifestation, or if no response occurred within 40 seconds.

Neurological assessment

The restoration of hind limb motor functions was analyzed using the BBB locomotor scale once every 3 days over the entire study period. The BBB locomotor scale was a 0–21 rating scale that classifies and evaluates hind limb movement, torso stability, gait coordination, paw placement, and tail positioning (Basso et al., 1995).

Cell and tissue analysis

Spinal tissue integrity and the extent of spinal cord injury were analyzed using ImageJ software 1.52 (NIH). After calibrating the scale, the RGB image was converted to 8-bit and then into black-white (BW) format using the Huang threshold (Fedorova and Pavel, 2019) which separates the colored part of the spinal cord and the background. Adjusted images were then used to assess total area of preserved spinal tissue and white matter area. For quantitative analysis of the spinal tissue, selected spinal cord sections were captured by fluorescent microscope (Olympus BX51) with 40x magnification. The same exposure time was used for all compared microphotographs. Fluorescently stained oligodendrocytes and neurofilaments were quantitatively analyzed using Image J software in predetermined regions of white matter – dorsal and lateral spinal cord funiculi (Figure 3). The size of analyzed area was the same in all evaluated images (1.1 mm × 1 mm). Selected images were converted into 8 bit BW format, and the number of fluorescent cells were analyzed using a particle analysis module. For densitometric analysis (oligodendrocytes and neurofilaments), the density of fluorescent cells was measured as a mean fluorescence intensity relative to the total sample area. From each spinal segment, five spinal sections were selected for analysis.

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Figure 3:
Illustration of spinal cord white matter areas analyzed by immunofluorescent and densitometric analyses.

Statistical analysis

Power Analysis was performed to verify that the number of animals in groups was sufficient (GPower 3.1 software, University of Kiel, Kiel Germany) based on our statistical tests (one-way analysis of variance). The actual power of the analyzed tests was more than 80%. The experimental data were analyzed using GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA, USA). All data are expressed as means ± standard deviation. One-way analysis of variance followed by the post hoc Tukey’ Honest Significant Difference (HSD) test was used to determine statistical significance between all experimental groups. Correlation analyses were performed with Pearson correlation test and linear regression analysis. Correlation analyses were performed with Pearson correlation test. A level of P < 0.05 was considered statistically significant.

Results

Changes in spinal tissue integrity and myelin preservation after epidural OFS

It is well known that after spinal cord trauma, there is massive tissue damage of spinal parenchyma, leading to great white and gray matter loss. To examine spinal tissue preservation after SCI and OFS, LFB/CV-stained spinal sections were analyzed. Quantitative histological evaluation revealed remarkable degradation of white and gray matter in all experimental groups. There was obvious visible difference in spinal tissue integrity between whole spinal cord slices (Figure 4A) and white matter area (Figure 4B) in rats from the OFS + SCI groups. Spinal tissue loss mainly occured within the epicenter of injury in all experimental groups. Histopathological analysis, however, showed that the percentage of spared tissue at the epicenter of injury (T9) was signifiantly (P < 0.05) increased in the OFS + SCI group (54.46 ± 8.60%) than in the nOFS + SCI (42.92 ± 8.69%) and SCI (37.92 ± 2.85%) groups. To determine the extent of myelin loss after SCI and OFS, we also analyzed white matter integrity in selected spinal segments (Figure 4C). Epidural OFS strongly promoted myelin preservation, with the most evident changes evaluated in the cranial (T8) spinal segment (78.75 ± 6.5%), compared to the non-stimulated (nOFS + SCI; 59.45 ± 11.6%) and SCI (53.27 ± 11.7%) groups. There was significant difference in myelin preservation in the area caudal to the lesion site (T10 and T11) in the OFS + SCI group than in the intact control groups (P < 0.05). Severe tissue loss and damage after spinal compression injury mainly occurred in the dorsal and lateral white matter areas. These regions in the spinal cord consist of nerve fibers (corticospinal tracts) that primarily regulate motor functions and movement control. Therefore, we will focus on these areas using fluorescent and densitometric analyses in future researches.

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Figure 4:
Spinal tissue integrity assessment after spinal cord compression and OFS.(A) Histological staining of the spinal injury epicenter in all experimental groups (scale bar: 600 µm). (B) Spinal cord tissue integrity at the site of injury and in adjacent craniocaudal regions of the spinal cord. (C) Preservation of white matter in all experimental groups. Data are presented as the mean ± SD (n = 5) and were analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, ***P < 0.001. nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Effect of epidural stimulation on oligodendrocyte survival and regeneration after spinal trauma

To address the assumption whether OFS applied early modulates the process of remyelination by oligodendrocyte regeneration after trauma was to determine the number and density of oligodendrocytes (APC+ cells) in the most SCI-affected regions. We noticed that the number of oligodendrocytes in both the dorsal and lateral regions was considerably higher in the OFS + SCI group compared to the nOFS + SCI and SCI groups (Figure 5A). The greatest increase in the number of APC+ cells in the dorsal funiculi was observed in the segments adjacent to the injury site (Figure 5B), compared to the SCI and nOFS + SCI groups (T8 segment: OFS + SCI group, 70.6 ± 2.9; nOFS + SCI group, 52 ± 2.5; SCI group, 50.6 ± 3.9; T10 segment: OFS + SCI group, 72.4 ± 5.3; nOFS + SCI group, 49.1 ± 2.9; SCI group, 45.9 ± 2.1). Similarly, the most signifiant change in the number of oligodendrocytes in the lateral region was observed in T10 spinal segment (OFS + SCI group, 81.4 ± 4.1; nOFS + SCI group, 55.2 ± 3.6; SCI group, 43.6 ± 2.4, P < 0.01). As shown in Figure 5C, the number and density of APC+ cells were increased in the OFS + SCI group than in the nOFS + SCI and SCI groups. The mean fluorescence intensity of APC+ cells in both the dorsal and lateral white matter areas was signifiantly higher in the OFS + SCI group than Intact control groups (P < 0.01). The biggest differences in oligodendrocyte density in the dorsal region were assessed in the T10 and T11 spinal segments. In the lateral funiculi, major differences between all experimental groups were observed in the spinal segments closest to the injury site at T8 (OFS + SCI group, 15.4 ± 0.7%; nOFS + SCI group, 11.5 ± 0.6%; SCI group, 10.6 ± 0.6%) and T11 (OFS + SCI group, 14.7 ± 0.4%; nOFS + SCI group, 10.7 ± 0.5%; SCI group, 10.1 ± 0.6%).

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Figure 5:
Analysis of changes in the number of oligodendrocytes after spinal trauma and epidural OFS treatment.(A) Imunohistological representative microphotographs of spinal cord dorsal funiculi within the T11 spinal segment 8 weeks post-spinal compression injury (scale bar: 500 µm). Immunoreactivity of marker for oligodendrocytes (APC, red) and neurofilaments (NF-l, green) was increased in OFS + SCI group compared with the nOFS + SCI and SCI groups (scale bar in magnified images: 100 µm). (B) Quantitative analysis of oligodendrocytes (APC+ cells) in dorsal and lateral funiculi. (C) Results of oligodendrocyte densitometry in dorsal and lateral white matter areas. Data are presented as the mean ± SD (n = 5). Results were statistically analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, **P < 0.01. nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Regenerative capacity of axons after spinal trauma and OFS treatment

Damage to the spinal cord caused severe disruption of nerve fiber integrity, mainly at the epicenter of injury, which is one of the main issues for restoring spinal cord functions after SCI. To evaluate the effect of OFS applied early on survival of spared axons after spinal trauma, we quantified neurofilaments (NF-l+), the major components of nerve cell cytoskeleton (Figure 6). Our results showed that epidural OFS had a neuroprotective effect on axonal preservation in the most affected areas of white matter (Figure 6B). Quantitative analysis of NF-l+ filaments in the dorsal funiculus revealed an increased number of NF-1+ filaments in OFS + SCI group compared with the nOFS + SCI and SCI groups. The most evident increase in the number of NF-1+ filaments was observed cranially to the lesion site in the T8 segment (OFS + SCI, 1462.8 ± 199; nOFS + SCI, 830.2 ± 183; SCI, 1241.1 ± 138, P < 0.01). Similar results were observed in the lateral funiculus where the most prominent (P < 0.01) changes in the number of NF+ fibers were seen in the dorsal T11 spinal segment (OFS group, 1887.2 ± 201; nOFS + SCI group, 978.1 ± 253; SCI group, 1348.7 ± 186).

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Figure 6:
Regenerative capacity of damage axons after spinal trauma and OFS treatment.(A) Fluorescent microphotographs of longitudinal spinal cord sections at the site of injury after 8 weeks of survival (scale bar: 1000 µm). The fluorescent intensity of spared axons (neurofilaments [NF-l]; green) and newly sprouted axons (growth-associated proteins [GAP-43]; red) after spinal compression and OFS (scale bar: 100 µm). (B) Quantitative analysis of spared axons in the dorsal and lateral funiculus (transverse spinal cord sections). (C) Densitometric analysis of axons in the longitudinal sections of the spinal cord lesion. Epidural OFS greatly supported axonal sprouting in the spinal cord lesion after 8 weeks of animal survival. Data are presented as the mean ± SD (n = 5). Results were statistically analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, **P < 0.01. nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

To investigate the regenerative capacity of the axons at the lesion site, we performed a densitometric analysis of the spared (NF-l+) fibers and newly sprouted axons (GAP-43+) in the lateral funiculi of the longitudinal spinal sections (Figure 6C). The immunoreactivity of the NF-l+ axons at the site of injury was slightly elevated (7.3 ± 0.4%) in the OFS + SCI group, compared with the nOFS + SCI group (5.9 ± 0.2%) and SCI group (6.2 ± 0.6%). Interestingly, there was signifiant change in the number of newly sprouted GAP43+ axons at the lesion site between the experimental groups (all P < 0.05). The density of GAP43+ axons was significantly higher in the OFS + SCI group (3 ± 0.5%) than in the nOFS + SCI group (1.7 ± 0.6%) and SCI (1.4 ± 0.4%) group (all P < 0.05).

Changes in NF-l and GAP-43 protein levels after spinal trauma followed by epidural OFS treatment

To determine whether OFS affected axonal regeneration at the protein level, we performed western blot analyses of NF-l and GAP-43 proteins. As shown in Figure 7, at 8 weeks after spinal compression, there continued to exist a considerable decline in NF-l and GAP43 protein levels in all experimental groups compared to the intact congrol group. NF-1 protein level elevated in all studied spinal segments in the OFS + SCI group compared to the nOFS + SCI and SCI groups. Statistically significant differences were observed in only the segments (T8, T10) adjacent to the epicenter of injury (P < 0.05; Figure 7A). There was a more significant difference in GAP-43 protein level in the outgrowing nerve fibers between groups. Spinal segment T8 was the most affected spinal cord region by OFS. A significant increase in GAP-43 protein level was observed in the OFS + SCI group compared to the nOFS + SCI and SCI groups. A neuroregenerative effect of OFS treatment was also observed in the caudal spinal segments, in which GAP-43 protein level was significantly elevated (T10: P < 0.05; T11: P < 0.01) in the OFS + SCI group compared to the nOFS + SCI and SCI groups (Figure 7B).

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Figure 7:
Assessment of neurofilament and newly formed axon protein levels after SCI and OFS treatment.(A, B) Graphs depict the protein levels of neurofilaments (NF-l, A) and newly sprouted axons (growth-associated proteins [GAP-43], B) relative to the β-actin level, compared to the intact control group. Data are presented as the mean ± SD (n = 5). The results were statistically analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, **P < 0.01, ***P < 0.001. Con: Intact control; nOFS: implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Differences in myelin immunoreactivity and protein levels after spinal cord compression and OFS treatment

MBP is the most abundant protein component in the myelin membrane in the CNS. By interacting with lipids in the myelin membrane, MBPs maintain the correct structure of myelin and are therefore considered important for the myelination process (Deber and Reynolds, 1991). To evaluate the changes after epidural OFS treatment, we analyzed the immunoreactivity of MBP and NF-l co-localization in each experimental group. MBP fluorescent staining showed moderate fluorescent signals in all groups, with a slight loss of signal in the dorsal and lateral white matter areas. Co-localization with markers specific for NF-1 showed visible differences between the stimulated and unstimulated animals. Figure 8A shows reduced MBP/NF-l+ signals in the SCI and nOFS + SCI groups, compared to the OFS + SCI group.

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Figure 8:
Immunoreactivity and protein level of myelin basic protein in spinal tissue 8 weeks after spinal compression injury and OFS treatment.(A) Fluorescent illustrations of co-localization of myelin basic protein (MBP; green) and neurofilament (NF-l; red) within the T11 spinal segment in all experimental groups (right, scale bar, 100 µm; left, scale bar, 1000 µm). (B) Western blot analysis revealed major changes in MBP protein level in treated (OFS + SCI) and untreated (SCI and nOFS + SCI) groups of animals compared to the intact control animals. (C) Immunoblotting picture of MBP. Data presented as means ± standard deviation (n = 5). The results were statistically analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, ***P < 0.001. Con: Intact control; nOFS: implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

To confirm the results, we performed protein analysis of MBP. Western blot results revealed major differences in MBP level (Figure 8C) between experimental groups at 8 weeks after surgery. MBP protein expression was markedly decreased in the nOFS + SCI and SCI groups than in the intact control group in all studied segments (Figure 8B). Interestingly, an elevated level of MBP protein relative to that in the intact control group was observed only in the OFS + SCI group at the site of injury and in craniocaudal segments. While MBP protein levels were decreased in the unstimulated groups, the OFS + SCI group showed significantly higher values at the caudal and cranial segments (P < 0.001, P < 0.05).

Functional recovery of spinal cord after early epidural OFS

Motor function restoration

After T9 spinal compression, rats were completely paraplegic with a neurological score of 0. BBB scores increase in each group over time (Figure 9A). In the first 3 weeks after surgery, no statistically significant differences in BBB score were observed between OFS + SCI and nOFS + SCI and SCI groups. As shown in Figure 9B, the first prominent motor improvement (P < 0.05) in BBB score was observed in the OFS + SCI group 4 weeks after SCI, compared with the nOFS + SCI and SCI groups. An increasing trend was observed in the OFS + SCI group until the end of study period. At 8 weeks post-surgery (Figure 9C), rats subjected to implantation of OF stimulators were able to move extensively in all three joints with an independent posture and slight infrequent coordination of hind limb movement (BBB score, 12.1 ± 1.1). Rats in the SCI and nOFS + SCI groups were partially able to stand alone with frequent “sweeping” movements of the hind limbs (nOFS + SCI group, 9.7 ± 0.6; SCI group, 9.8 ± 1.3).

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Figure 9:
Neurological evaluation of hind limb locomotor activity after spinal injury and OFS using rat BBB scale.(A) Rats with OFS showed better recovery of hind limb functions during the 8-week study period. (B) Four weeks post-SCI, significant differences in functional recovery started to appear. (C) At the end of experiments (8 weeks post-SCI), the differences between the experimental groups were even more pronounced. Data are presented as the mean ± SD (n = 10). The results were statistically analyzed using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, **P < 0.01. BBB: Basso, Baettie, Breshnahan locomotor rating scale; nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Behavior associated with stress or depression

Behavioral analysis data showed significant changes (P < 0.01) in locomotor activity between the experimental groups during the 8-week study period (Figure 10A and E). Rats not subjected to OFS treatment showed lower spontaneous locomotor activity and velocity compared to rats subjected to an active OFS. Considering that reduced locomotor functions induced by SCI could interfere with exploratory behavior, which is the rodent’s fundamental type of behavior, we evaluated exploration along with locomotor activity. An increased exploratory activity was observed in the OFS + SCI group than in the nOFS + SCI and SCI groups (both P < 0.05; Figure 10D). On the contrary, significantly more manifestations of depressive behavior (freezing frequency, P < 0.01; freezing duration, P < 0.05) were observed in the nOFS + SCI and SCI groups compared to the nOFS + SCI group (Figure 10C). Grooming and self-cleaning licking, as a sign of comfort behavior, was also evaluated. Rats subected to active OFS spent profoundly more time in grooming than did rats not subjected to stimulation (P < 0.01; Figure 10B). There were no significant differences in the frequencies of urination and defection during the OFS between groups.

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Figure 10:
The effect of epidural OFS on spontaneous locomotor activity and stress/comfort-related behavior in rats after spinal compression injury.(A) Locomotor activity (distance, velocity) in animals after spinal trauma and followed OFS were evaluated during 5-minute testing interval. (B, C) Stress/comfort-related behavior, frequency and time spent in freezing and self-grooming were evaluated. (D) Exploratory behavior analysis of animals during the open field test. (E) Locomotor activity changes in experimental animals. (B–E) The data were obtained at the end of experiment (8 weeks post-SCI). Data are presented as the mean ± SD (n = 5). The results were statistically evaluated using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; *P < 0.05, **P < 0.01, ***P < 0.01; nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Sensory function

During the hot plate test, a radical prolongation of response latency (time when the rat started to show nociceptive signals as forepaw/hindpaw withdrawal or licking) was observed in rats with SCI. At 4 weeks post-SCI, the mean response latency was 7.9 ± 0.2 seconds in the intact control group and it was signifiantly prolonged in all experimental groups (17.9 ± 2.1 seconds). In following weeks, we observed a gradual shortening of the response latency in all tested rats, with more pronounced differences in the OFS + SCI group (Figure 11B). Our results showed that rats subjected to OFS had profoundly shorter latency 8 weeks post-SCI (10.9 ± 0.6 seconds) compared to rats subjected to non-functional stimulation (14.6 ± 0.8 seconds), and rats only subjected to SCI (14.4 ± 0.7 seconds). At 8 weeks post-SCI, a strong reduction in hot plate response latency was observed in rats with high BBB score, as confirmed by correlation analysis (Figure 12A). Similarly, hind limb regeneration as evaluated by neurological BBB score was strongly correlated with the percentage of spared spinal cord tissue after injury (Figure 12B).

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Figure 11:
Analysis of sensory function recovery after spinal trauma and OFS treatment using the hot plate test.(A) In the hot plate test, the rat was placed on a metal surface maintained at a constant temperature (52 ± 0.5°C) and the response latency. The time taken to observe a nociceptive behavior was recorded by the investigator. (B) Weekly hot plate testing of sensory function recovery during 8-week study period showed significant changes in response latency between the treated and untreated groups. Data presented as means ± standard deviation (n = 5). The results were statistically evaluated using one-way analysis of variance followed by the post hoc Tukey’s honest significant difference test; **P < 0.01; #P < 0.01, vs. other three group; ††P < 0.01, †††P < 0.001, vs. nOFS + SCI and SCI groups. nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.
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Figure 12:
Correlation analysis of response latency in hot plate test and spared tissue analysis versus BBB neurological function scores (Pearson’s correlation test).Significant negative correlation was found between neurological function evaluation versus hot plate response latency (A), while BBB score was significantly positively correlated with histological analysis of tissue integrity (B) at 8 weeks post-SCI and OFS. Scatterplots of individual values (n = 5) with regression line correlation coefficient (r) and coefficients of determination (R 2) calculated by linear regression analysis. BBB: Basso, Baettie, Breshnahan locomotor rating scale; nOFS: Implanted electrical stimulator without stimulation; OFS: oscillating field stimulation; SCI: spinal compression injury.

Discussion

Application of OFS is known to facilitate the bidirectional regeneration of axons after SCI (Borgens et al., 1999; Hamid and Hajek 2008; Walters, 2010; Zhang et al., 2014, 2015). Results of our previous study clearly indicated the beneficial properties of short-term epidural OFS via the implantation of the miniature, originally designed OF stimulator (Bacova et al., 2019). The main objective of the present study was to investigate the further impact of constant epidural OFS on functional recovery after T9 spinal compression during long-term survival.

One of the most evident changes after SCI is the massive loss of spinal cord tissue (Smith and Jeffrey, 2006). In this study, early epidural stimulation using an oscillating electric field was shown to be protective on spinal cord tissue and myelin preservation. LFB-positive staining of the myelin showed that epidural OFS markedly increased the myelin area in the segments adjacent to the lesion site. Previous studies have proposed that ES can promote spinal tissue integrity and contribute to remyelination after SCI via improving differentiation of oligodendrocyte precursor cells (Zhang et al., 2014; Jing et al., 2015). Oligodendrocytes, as specialized glial cells, are known to play a key role in myelin formation, integrity, and maintenance (Simons and Nave, 2015). To investigate the association between myelin recovery and oligodendrocyte regeneration after OFS, a quantitative and densitometric analysis of immunolabeled oligodendrocytes was performed. Our results confirmed that epidural OFS not only reduces oligodendrocyte loss, but also promotes their density directly in areas of greatest tissue damage (T8, T10 spinal segments). Mature oligodendrocytes are also the main cells that express myelin protein, such as MBP (Bernardo et al., 2013). Therefore, we used a western blot analysis of MBP to verify the impact of epidural OFS on the promotion of remyelination after SCI. We found that OFS strongly increased MBP level in cranial and caudal spinal segments of the lesion. A similar beneficial outcome was reported in a study of epidural stimulation after SCI, in which the stimulation upregulated MBP and mRNA levels and reduced oligodendrocyte loss by promoting their differentiation and inhibiting apoptosis (Li et al., 2020). Our findings in this study indicate that long-term epidural OFS may contribute to the recovery of myelination after T9 spinal injury by inhibiting oligodendrocyte loss and promoting tissue and myelin regeneration.

Axonal regeneration after SCI is significantly limited. The insufficient activation of regenerative processes in neuronal cells is one of the key factors contributing to the lack of axonal post-traumatic regeneration. Moreover, growing axons need to be oriented correctly to re-establish functional reconnections across the lesion site. Several experimental studies have reported that ES applied early after SCI might be crucial for the promotion of axonal overgrowth (Haan and Song 2014; Zhang et al., 2015). Our hypothesis is that OFS-induced sparing of spinal tissue should correlate with enhanced axonal regenerative capacity. For that reason, we performed quantitative and densitometric analyses of spared NF-l+ and GAP-43+ nerve fibers, which corresponded to newly outgrowing fibers with the ability to rebuild neural connections after CNS damage. Our data showed more pronounced regeneration, increased number of NF-l+ fibers and density of new GAP-43+ fibers in selected regions in the animals with active OFS. Our findings were also confirmed by western blot analysis, which indicated a profoundly increased protein level of NF-l and GAP-43 in rats with active OFS. We assume that the early application of weak OF current to the damaged spinal cord could be a trigger for the initiation of the regenerative processes, since the pro-regenerative and major inflammatory processes are initiated within the first week after SCI (Bimbova et al., 2018).

In the context of these findings, behavioral tests confirmed a progressive improvement of the motor function in rats with active OFS. We noticed the most obvious changes 7 weeks after the traumatic event, which indicates the gradual effect of the initial OFS to restore motor function. Tian et al. (2016) reported functional improvement after OFS (40 µA) within 4 weeks after SCI; however, the effect of stimulation remarkably decreased after 10 weeks. The BBB test performed in this study showed slow and gradual improvement in motor functions, which became statistically significant in rats subjected to active OFS at 4 weeks and continued further for up to 8 weeks.

The early neurological improvement might be due to the modulation of the inflammatory response caused by the reduction of reactive astrocytes involved in glial scar formation, as a response to OFS application (Bacova et al., 2019). This beneficial outcome correlates with the spared white matter area, which was approximately 20% higher in the stimulated group of animals. Besides locomotory impairment, the inflammatory response after SCI often affects the development of neuropsychiatric disorders, including anxiety and depression (do Espirito Santo et al., 2019). To exclude the negative impact of OFS applied early after injury on spontaneous activity, mental health and well-being of rats, we performed open field testing. In the OFS + SCI group, we noted significantly increased motility, exploration, and self-cleaning (comforting type of behavior – grooming), indicating that the behavior of rats in the OFS + SCI group was not adversely affected by OFS. On the contrary, rats not subjected to stimulation lacked exploration and comforting behaviors, which may also be associated with mood impairments, such as depression or anxiety (Burnett et al., 2006), especially post-SCI (do Espirito Santo et al., 2019). Another important factor affecting functional recovery after SCI is the regeneration of sensory functions. In a recent study, we used the hot plate test to examine the integrity of supraspinal pathways in animals with or without active OFS. The hot plate test is often used to detect sensory system disorders, such as hyper/hyposensibility, allodynia, or loss of sensory functions caused by SCI (Fischer and Peduzzi 2007; Sedy et al., 2008; Jalan et al., 2017). Our results showed that the recovery of sensory functions did not differ between the untreated animals and the animals that underwent OFS until 7 weeks post-SCI. This finding correlates with the results of the neurological assessment, in which the most visible changes between groups were also observed 7 weeks after SCI. Despite the fact that we noticed differences between the groups within the first weeks of testing, a statistical significance was demonstrated up to 7 weeks after SCI. We expect that, for optimal assessment of sensory and motor function recovery, long-term experiments in the future are necessary.

In our recent study, we used histological, immunohistochemical, molecular and behavioral methods to investigate the potential of weak, long-term OFS on tissue regeneration and functional recovery after severe spinal cord damage. Our results confirm that immediate, epidural OFS represents a suitable therapeutic strategy that can contribute to the activation of regenerative processes in the acute phase of SCI, with a long-term effect on the chronic stage of disease. Despite many beneficial properties of OFS, further research is needed due to the limited number of studies focused on OFS after SCI. Their outcomes are very difficult to compare because of wide divergence of models and methods of stimulation. OFS should not be considered as a sole treatment for SCI, it should potentiate recovery as a part of combined therapy together with pharmacological and non-pharmacological interventions. Clearly, the identification of the molecular mechanisms that promote spinal tissue regeneration in response to OFS, as well as the possibility to combine several therapeutic strategies to achieve maximal functional recovery, are areas for our further study.

Author contributions:Study conception and experimental design: MB, JG; surgical procedure and stimulator implantation: MB; animal monitoring, post-surgical care, behavioral testing: MB, KB, AK; tissue harvesting, material preparation, immunohistochemical and western blot analyses, data collection and evaluation: MB, BB; manuscript editing and correction: NL, JG. All authors approved the final version of this manuscript.

Conflicts of interest:None declared.

C-Editor: Zhao M; S-Editor: Li CH; L-Editor: Song LP; T-Editor: Jia Y

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Keywords:

axonal regenerative capacity; behavioral assessment; epidural stimulation; motor recovery; myelin regeneration; oscillating field stimulation; spinal cord injury

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