Early electrical field stimulation prevents the loss of spinal cord anterior horn motoneurons and muscle atrophy following spinal cord injury.

Our previous study revealed that early application of electrical field stimulation (EFS) with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury (SCI). The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T10. SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov's motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.

Introduction

Spinal cord injury (SCI) is damage to the spinal cord that, depending on the injury level and severity, can produce severe impairments of motor function at and below the level of the damage (McDonald and Sadowsky, 2002; Lynskey et al., 2008; David and Steward, 2010). Although spontaneous regeneration is rare in the adult spinal cord, the central nervous system is able to show significant functional recovery from incomplete SCI with appropriate treatment (Raineteau and Schwab, 2001; Han et al., 2015; Li et al., 2015).

It is believed that secondary injury processes including programmed cellular apoptosis are activated in the spinal cord after SCI (David and Kroner, 2011; Zhang et al., 2012). Cellular apoptosis aggravates irreversible tissue damage and function loss that worsens the primary spinal cord lesion (Borgens, 2012; Oudega, 2012; Rong et al., 2012). Neuronal apoptosis at the lesion site has been revealed (Liu et al., 1997). An extensive study confirmed the spatial and temporal profiles of the loss of neurons (Kieran and Greensmith, 2004). A main trigger for apoptosis seems to be injury-induced Ca2+ influx that exceeds the homeostatic capacity of cells and ultimately triggers cell apoptosis in the tissue surrounding the injury (Oyinbo, 2011; Rong et al., 2012). Many agents that impede Ca2+ influx-induced injury have shown protective function after nerve injuries (Liverman et al., 2005; Yan et al., 2010).

Furthermore, Ca2+ influx is also the main cause of injury potential, which refers to a direct current voltage between the normal and damaged tissue (Goodman et al., 1985; Zuberi et al., 2008). Our recent investigations elaborated a positive correlation between the damage potential and injury severity. The injury potential increased quickly within 30 minutes after the damage and then decreased gradually to 0 mV several hours after injury (Zhang et al., 2013). Electrical field stimulation (EFS) treatment with the anode at the lesion and the cathode distal to the lesion offset the Ca2+ influx and accordingly delayed and reduced the formation of injury potential. The results revealed that EFS could efficiently exert neuroprotective effects against apoptosis and promote axon regeneration in the dorsal corticospinal tract of the spinal cord after SCI (Zhang et al., 2015a).

Secondary damage continues for weeks following SCI and some neurons, including anterior horn motoneurons, may be eventually damaged (Liu and Xu, 2010; Pastor et al., 2014; Liu et al., 2016) and the ventral roots may also be involved (Moschilla et al., 2001; Karalija et al., 2012). Some studies have reported that the muscles supplied by spinal cord motoneurons and the motor nerves through the ventral roots at and distal to the injury level undergo denervation and atrophy (Gordon and Mao, 1994; Byers et al., 2012). The recovery process after injury depends not only on the motoneurons in the spinal cord but also their target musculature. Therefore, the objective of this study was to further evaluate the neuroprotective efficacy of early EFS on functional recovery. We also evaluated the effect of EFS on protecting spinal anterior horn motoneurons and lower limb musculature after severe SCI.

Materials and Methods

Animals

Fifty-four female Sprague-Dawley rats aged 8 weeks and weighing 200–220 g were bought from Beijing HFK Bio-Technology (Beijing, China; animal produce license No. SCXK (Jing) 2014-0004). The study protocol was approved by the Animal Welfare Committee of Beijing Key Laboratory of Bioelectromagnetism. The experimental procedures followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animal (NIH Publication No. 85-23, revised 1985).

The 54 rats were equally randomized into three groups. EFS group rats received EFS immediately after SCI; T8, T10, and T12 spinal cord segments were exposed. One electric field stimulator contains one anode and two cathodes, and each rat received stimulation from two electrical field stimulators. Two anodes were sutured at both sides of the T10 paravertebral muscle, and four cathodes were sutured at both sides of T8 and T12 paravertebral muscle (). A severe impact injury at T10 (10 g, 50 mm) was induced by a trained technician using the weight-drop method (Zhang et al., 2015b). The electric field was applied (injury potential compensation) immediately after the measurement of injury potentials. The caudal and rostral injury potential was adjusted to 0 ± 0.5 mV. EFS lasted for 30 minutes. All electrodes were removed following the stimulation. The rats of the SCI group were subjected to the same severe SCI procedures but without electric field stimulator suture and electric field stimulation. The sham group rats were subjected to the same procedure of laminectomy without weight-drop injury and electric field stimulation.

Figure 1

Stimulation setup.

(A) Schematic diagram of the applied electric field stimulator. (B) Electrode suture method. The cathodes were sutured at the paravertebral muscle of T8 and T12, whereas the anodes were sutured at T10.

Electrical field stimulation and injury potential measurement system

The electrical field stimulation system and the injury potential measurement system have been introduced comprehensively in our previous study (Zhang et al., 2015b); here we described them briefly.

Two 9-volt-batteries supplied energy for the electric field stimulator (Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing, China). The central terminal of the potentiometer in the voltage regulation unit provided an electric potential between ± 0.43 V. The amplification unit included a noninverting amplifier, which could be adjusted from 1 to 21, and a voltage follower. The electrode system was composed of one spiral reference electrode and two spiral stimulating electrodes. A resistor was set up to limit the current flow in the tissues, which was between the output of the voltage follower and the stimulating electrode. Platinum-iridium (90:10) wires were used for each spiral electrode ().

Figure 2

Schematic of the applied electrical field stimulator.

The electric field stimulator was powered by two 9 V batteries. A voltage regulation unit comprising R0, R1, and R2 provided a voltage range between ± 0.43 V. The amplification unit included a noninverting amplifier and a voltage follower. Two stimulating electrodes and one reference electrode comprised the electrode system. GND: Ground; R: resistance; Vcc: volt current condenser.

The injury potentials were measured by two glass electrodes. The upper glass tube of the glass electrode contained a calomel electrode and was filled with 3 M KCl solution. The lower glass tube was plugged by porous ceramic and filled with normal saline. The tip of one glass electrode was gently placed at T10 and the tip of another glass electrode was lightly put on the surface of T8 or T12. The rostral injury potential (the voltage between T8 and T10) and the caudal injury potential (the voltage between T10 and T12) were measured and recorded.

Luxol fast blue staining

At 8 weeks after the operation, the rats were executed by pentobarbital overdose (70 mg/kg; Abbott, North Chicago, IL, USA) and then transcardially perfused sequentially with 37°C phosphate buffered saline and cold 4% paraformaldehyde. The spinal cord segments (n = 5) compassing the injury center were removed and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Serial spinal cord sagittal sections (20 μm thick, 500 μm intervals) were stained with luxol fast blue (ZSGB-Bio, Beijing, China). Transverse sections of spinal cord showing the largest proportion of cavity were taken to represent the injury center. The cavity area and perimeter of injured spinal cord were measured with Image Pro Plus 5.0 software (Cybernetics, Rockville, MD, USA).

Immunofluorescence assessment

Eight weeks after the injury, spinal cord frozen sections (5 μm, n = 5/group) 2 mm caudal from the epicenter were prepared for immunofluorescence staining. Motoneurons in the anterior horn were marked by a rabbit anti-choline acetyltransferase (ChAT) antibody (1:500; Millipore, Bedford, MA, USA) and a Red-X-conjugated goat anti-rabbit antibody (1:100; ZSGB-Bio, Beijing, China). Following immunostaining, all sections were coverslipped with 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech, Birmingham, AL, USA) to stain the nuclei.

ChAT/DAPI co-labeled cells were counted in an area of the bottom right and bottom left quarter of the spinal cord gray matter. The quarter areas were defined by vertical and horizontal crossed lines, which were drawn through the central canal (Ling et al., 2013). Photos from the anterior horn were taken by a fluorescence microscope (Nikon E600, Tokyo, Japan). ChAT/DAPI co-labeled cells were counted in three randomized fields (450 µm × 450 µm) of 10 sections per group (30 optical fields per group). The motoneuron quantity in each group was expressed as the mean cell number per mm2.

Transmission electron microscopy

Spinal cords (n = 3/group) 2 mm caudal from the center were fixed in 2.5% glutaraldehyde and 2% osmic acid (Klamar, Shanghai, China) 8 weeks after the injury. The spinal cords were embedded in epoxy resin and ultrathin sections mounted on copper grids were observed using transmission electron microscopy (Hitachi-7650, Tokyo, Japan).

Five grids in different parts of the gray matter in the anterior horn were prepared in each group. The morphological alterations of the nucleus and chromatin condensation were assessed in approximately 20 motoneurons from rats of each group.

Wet weight and cross-sectional area of vastus lateralis muscles

The target musculature (n = 5/group) of the motoneurons were further examined to evaluate regressive muscle changes post-injury. The bilateral vastus lateralis muscles were removed 4 and 8 weeks post-injury and their wet weights were measured.

The cross-sectional area of hematoxylin and eosin (ZSGB-Bio, Beijing, China) stained sections (10 μm) of vastus lateralis muscles were examined and approximately 150 muscle fibers per rat were evaluated.

Behavioral assessment

Assessment of functional recovery was performed independently by two investigators before injury and once per week until 8 weeks after SCI (n = 13/group).

The inclined plane test was used to evaluate the ability of the rats to maintain postural stability (Rivlin and Tator, 1977; Pal et al., 2010). The rats stood on an inclined plane, which was tilted slowly, and the maximum angle that the rats could hold their position for 5 seconds was considered as the final incline.

The modified Tarlov’s motor grading scale was used to evaluate motor function (Behrmann et al., 1992; Liu et al., 2011): grade 5, rats can walk normally; grade 4, rats can walk with uncoordinated hind limbs; grade 3, rats can stand but cannot walk; grade 2, rats cannot stand but can slightly perform voluntary hind limb activity; grade 1, rats have no voluntary hind limb activity. Three measurements were taken for each rat, and the average angle and scores were calculated.

Statistical analysis

All data were expressed as the mean ± SD, and analyzed using SPSS 15.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance followed by Tukey’s post hoc test was used to compare the indexes among groups. Repeated-measures analysis of variance followed by Bonferroni’s post hoc test, unless otherwise stated, was used to compare the indexes of groups over time. A P value of < 0.05 was considered statistically significant.

Results

Injury potential compensation by EFS

The rostral and caudal injury potentials of the SCI group rats increased rapidly with the onset of injury, and then decreased gradually until approximately 30 minutes post-injury. EFS group rats were given simulation (injury potential compensation) immediately following SCI. The injury potential of the EFS group was 0.5 mV because of injury potential compensation. The compensation voltages were 2.7 ± 0.1 V, and the amount of current was 400 ± 10 μA during EFS ().

Figure 3

Injury potential compensation by EFS

(A, B) Rostral and caudal injury potentials of the SCI and EFS groups. The rostral and caudal injury potentials of the SCI group rats increased rapidly with the onset of injury and then decreased gradually approximately 30 minutes post-injury. At the end of the stimulation, the stitched electrodes were removed; injury potential restarted, then gradually decreased 60 minutes post-injury. EFS: Electrical field stimulation; SCI: spinal cord injury; min: minute.

At the end of stimulation, the stitched electrodes were removed; injury potential restarted and then gradually decreased to 60 minutes post-injury. The rostral and caudal injury potentials of the SCI group were 4.21 ± 1.77 mV and 5.86 ± 1.86 mV, respectively, 60 minutes post-injury ().

Characteristic cavitation was produced by damage in EFS and SCI groups at the injury center. The degree of tissue disruption was high in the SCI group. Significant tissue preservation and decreased cavity expansion were seen in the EFS group (). The cavity area (F0.05(2,12) = 3.88, F = 5.03, P < 0.05) and perimeter (F0.05(2,12) = 3.88, F = 6.18, P < 0.05) were larger in the SCI group than in the EFS group (Figure , ).

Figure 4

Spinal cord tissue preservation at the injury center 8 weeks post-injury.

(A) Luxol fast blue staining of 20 μm sagittal sections from three groups of rats. The dotted lines in the images indicate the border of the lesion area. Scale bars: 100 mm. (B) Quantitative data on the cavity area of injured spinal cord. (C) Quantitative data on the cavity perimeter of injured spinal cord; *P < 0.05, vs. SCI group (mean ± SD, n = 5, one-way analysis of variance followed by Tukey’s post hoc test). EFS: Electrical field stimulation; SCI: spinal cord injury.

Immunofluorescence assessment of spinal anterior horn motoneurons

To evaluate the protective effect of EFS on anterior horn motoneurons, spinal cord sections 2 mm caudal from the center were immunostained for ChAT, a marker of motoneurons. The number of ChAT/DAPI-positive cells was quantified in the spinal anterior horn (). The statistical results showed that the EFS group rats preserved spinal anterior horn motoneurons better than the rats in the SCI group 8 weeks post-injury (F0.05(2,12) = 3.88, F = 4.05, P < 0.05) ().

Figure 5

EFS effects on spinal anterior horn motoneurons 8 weeks post-injury.

(A) Representative images taken from the spinal anterior horn 2 mm caudal from the center in the three groups of rats. Scale bars: 100 μm. (B) Quantification of ChAT/DAPI-positive cells; *P < 0.05, vs. SCI group (mean ± SD, n = 5, one-way analysis of variance followed by Tukey’s post hoc test). ChAT: Choline acetyltransferase, a marker of motoneurons; EFS: electric field stimulation; SCI: spinal cord injury.

Ultrastructure in the spinal anterior horn after SCI

Transmission electron microscopy revealed that the shape of neuronal nuclei was normal and the mitochondria contained sparse cristae in the sham group (). At 8 weeks after injury, in the SCI group spinal anterior horn motoneurons had morphological changes, such as cell shrinkage and chromatin condensation (). In the EFS group, the distributions of organelles were more normal and the shapes of motoneurons were better preserved (). The proportion of cells with morphological alterations in the EFS group was significantly lower than that in the SCI group (F0.05(2,6) = 5.14, F = 7.82, P < 0.05) ().

Figure 6

EFS effects on ultrastructure of spinal cord anterior horn neurons 2 mm caudal from the injury center 8 weeks post-injury.

(A) In the sham group, the shape of neuronal nuclei was almost normal. (B) In the SCI group, some anterior horn neurons had morphological alterations (cell shrinkage and chromatin condensation). (C) In the EFS group, the extent of chromatin condensation and the shape of mitochondria were much better than in the SCI group. Scale bars: 2 μm. (D) The proportion of the cells with morphological alterations in the EFS group was significantly lower than that in the SCI group; *P < 0.05, vs. SCI group (mean ± SD, n = 3, one-way analysis of variance followed by Tukey’s post hoc test). EFS: Electrical field stimulation; SCI: spinal cord injury.

Wet weight and cross-sectional area changes of vastus lateralis muscle fibers

The degree of vastus lateralis muscle loss was estimated among the three groups of rats ().The SCI group showed significantly lower wet weights compared with the EFS group at 4 (F0.05(2,12) = 3.88, F = 6.37, P < 0.05) and 8 (F0.05(2,12) = 3.88, F = 5.47, P < 0.05) weeks after the injury.

Figure 7

EFS effects on wet weight and cross-sectional area of vastus lateralis muscle fibers 4 and 8 weeks after SCI.

(A) Light micrographs of transversely sectioned vastus lateralis muscles 4 and 8 weeks after the SCI. Scale bars: 100 μm. (B) Wet weight of vastus lateralis muscles was significantly lower in the SCI group than in the EFS group. (C) The mean cross sectional area of vastus lateralis muscle fibers was significantly smaller in the SCI group than in the EFS group; *P < 0.05, **P < 0.01, vs. SCI group (mean ± SD, n = 5, one-way analysis of variance followed by Tukey’s post hoc test). EFS: Electrical field stimulation; SCI: spinal cord injury.

Hematoxylin-eosin stained micrographs of transversely sectioned vastus lateralis muscle further showed that SCI caused vastus lateralis muscle fiber atrophy in both injury groups after SCI (). Cross-sectional areas in the injured groups initially became smaller. The mean area of the SCI group was significantly smaller than the EFS group at 4 (F0.05(2, 12) = 3.88, F = 6.37, P < 0.05) and 8 (F0.01(2, 12) = 6.93, F = 11.25, P < 0.01) weeks ().

Locomotor function testing after SCI

The mean angles of all groups of rats in the inclined-plane test were approximately 75.00° preoperatively. The mean inclined-plane angles decreased and were then followed by a slow decline in both SCI and EFS groups after SCI. Four weeks after injury, the angles recorded in the SCI and EFS groups were 50.21 ± 4.27° and 58.24 ± 4.35°, respectively. The angles were significantly larger in the EFS group than in the SCI group and this advantage persisted until the end of the study (F0.05(2,21) = 3.47, F = 5.26, P < 0.05) (). The angles of the sham rats were approximately 75.00°.

Figure 8

EFS effect on behavioral outcome of rats from three groups.

(A) The inclined-plane test after SCI. (B) Modified Tarlov’s grading scale after SCI. Functional recovery was assessed before injury and once per week until 8 weeks after SCI. Significant functional improvement was observed in the EFS group; *P < 0.05, vs. SCI group (mean ± SD, n = 13, repeated-measures analysis of variance followed by Bonferroni’s post hoc test). EFS: Electric field stimulation; SCI: spinal cord injury; Pre: pre-injury.

All the rats were graded according to the modified Tarlov’s grading scale standard (). The motor scores were 5.0 ± 0.0 in all groups before the injury. After injury, the injury ranking dropped and then gradually elevated. At 3, 4, 5 and 7 weeks, grading scores were significantly better in the EFS group than in the SCI group (F0.05(2,21) = 3.47, F= 4.48, P < 0.05). At 8 weeks, the grades of the SCI and EFS groups reached the maximal ranking, but the difference between them was not statistically significant.

Discussion

Electrical fields (10 mV/mm) could adjust Ca2+ migration into injured lamprey spinal axons, and the appropriate polarity of the electric field nearly canceled out the migration of Ca2+ at the injury end (Strautman et al., 1990). Accordingly, we conducted applied EFS (270 mV/mm) with the anode at the lesion site and the cathode distal to the lesion in injured spinal cord. The larger electrical field used in the present study may help offset the Ca2+ flux at injured regions. In our previous paper, we demonstrated neuroprotective effects of EFS and satisfactory results were obtained in the dorsal corticospinal tract of spinal cord post-injury (Zhang et al., 2015a).

SCI has been characterized by morphological hallmarks such as the loss of spinal cord tissue, including motoneurons (Sharp et al., 2010; Bains et al., 2012). In this study, luxol fast blue staining was performed 8 weeks after injury and the degree of white and gray tissue preservation was high in the EFS group. The presence of ChAT reliably reveals motoneurons (Xu et al., 2011; Sareen et al., 2012), so motoneurons were stained for ChAT and then quantified. The results confirmed that EFS significantly preserved the number of spinal cord anterior horn motoneurons 8 weeks post injury. Furthermore, results from transmission electron microscopy showed that EFS maintained the configuration of spinal cord anterior horn motoneurons 8 weeks after injury. The proportion of cells with morphological alterations was significantly lower in the EFS group than in the SCI group. These results indicate that EFS played an important role in neuroprotection of anterior horn motoneurons after SCI.

Muscle atrophy shown by weight and fiber size is typical of weight-bearing muscles that are innervated by spinal cord motoneurons below the lesion level after SCI (Peckham et al., 1976; Giangregorio and McCartney, 2006; Panisset et al., 2016). As expected, in the present study the injured rats exhibited vastus lateralis muscle atrophy, as shown by wet weight and cross-sectional area measurement at 4 and 8 weeks after SCI. However, the regressive changes of vastus lateralis muscle in the EFS group were prevented by EFS treatment. The results indicated better wet weight and morphology of vastus lateralis muscle in the EFS group than in the SCI group. This effect may be due to sparing of motoneuron function with EFS treatment after SCI (Morimoto, 2012; Jain, 2016).

Large motor deficits persist for a long time after SCI, and poor functional recovery of SCI groups has been shown by behavioral assessment (Rengachary et al., 2011; Kolb, 2013). Lower behavior assessment scores indicate a poorer ability to maintain postural stability and motor function (Marigold et al., 2004; Liu et al., 2011). After EFS intervention, behavioral recovery was improved and the magnitude of locomotor recovery was strongly paralleled by the extent of spared motoneurons (Mondello et al., 2015). This improvement was actually an increase of coordinated activity among the various muscles of the hind limbs, which could be attributed locally to sparing of enough spinal circuitry to maintain motor vitality and to prevent target muscle atrophy (Martin et al., 2012; Roy et al., 2012; Slawinska et al., 2014).

One limitation of the present study was the lack of a sham EFS group. To ensure that the neuroprotective effect was not the result of the EFS setup but indeed was the result of the EFS, a sham EFS group with exactly the same EFS setup should be conducted in our future experiments.

In summary, EFS with the anode at the lesion and the cathode distal to the lesion efficiently protected anterior horn motoneurons against apoptosis and vastus lateralis muscles from atrophy. The differences in the spinal cord and vastus lateralis muscle analyses correlated with greater functional improvement of EFS group rats at the endpoint. This kind of physical therapy will be valuable for acute SCI treatment.