An animal study to examine the effects of the bilateral, epidural cortical stimulation on the progression of amyotrophic lateral sclerosis

We examined the effects of the unilateral cortical stimulation on the survival of neurons showing degenerative changes and compared those in delaying the progression of amyotrophic lateral sclerosis (ALS) between the unilateral cortical stimulation and the bilateral one in an animal experimental model using mice. We used 19 G93A transgenic mice and randomly divided into three groups: the control group (n = 6) (the implantation of electrodes in the bilateral motor cortex without electrical stimulation), the unilateral stimulation group (n = 7) (the implantation of electrodes in the unilateral motor cortex with a 24-hour cortical stimulation) and the bilateral stimulation group (n = 6) (the implantation of electrodes in the bilateral motor cortex with a 24-hour cortical stimulation). The mean survival period was significantly longer in the bilateral stimulation group as compared with the control group (124.33 ± 11.00 days vs. 109.50 ± 10.41 days) (P < 0.05). In addition, on postoperative weeks 11, 12, 13, 14 and 15, the mean Rota-rod score was significantly higher in the unilateral stimulation group as compared with the control group (P < 0.05). Furthermore, despite a lack of statistical significance, it was the lowest in the bilateral stimulation group on postoperative weeks 13, 14, 15 and 17. On postoperative weeks 11, 12, 13, 14 and 16, the mean score of paw-grip endurance was significantly higher in the unilateral stimulation group as compared with the control group (P < 0.05). Furthermore, despite a lack of statistical significance, it was the lowest in the bilateral stimulation group on postoperative weeks 13, 14, 15 and 17. In conclusion, our results indicate that the bilateral epidural cortical stimulation might have a treatment effect in a murine model of ALS. But it is the limitation that we examined a small number of experimental animals. Further studies are therefore warranted to establish our results and to identify the optimal parameters of the epidural cortical stimulation in a larger number of experimental animals.


Introduction
Amyotrophic lateral sclerosis (ALS) is one of the fatal motor neuron diseases, for which there are no established treatment modalities. It has been reported that patients with ALS have a mean survival period of 3-4 years after the onset of symptoms [1]. Its exact etiology remains uncertain. It has been proposed, however, that it may occur as a result of defects in the clearance of extracellular glutamate because of a marked decrease in the activity of glial-specific glutamate transporter-1 (GLT-1). With the increased concentrations of extracellular glutamate, mediating the activation of the N-methyl-Daspartate (NMDA) receptor subtype, patients with ALS are at risks of neuroexcitotoxic cell death in the motor cortex [2][3][4]. To date, glutamate-release inhibitors have been used to treat patients with ALS in a limited scope. But they are effective in prolonging the survival period to only several months [5,6].
Several pre-clinical and clinical studies have shown that the cortical stimulation is effective in delaying the progression of ALS [7][8][9][10][11]; its efficacy originates from alterations in neurotransmitters and neurotrophic factors released from the motor cortex. Still, however, little is known about the optimal parameters that are involved in the cortical stimulation.
It has been reported that low-frequency repetitive transcranial magnetic stimulation (rTMS) is effective in delaying the progression of glutamate-mediated neurotoxic disease by suppressing the hyperexcitability of the motor cortex in patients with ALS [8,11]. In addition, high-frequency rTMS is effective in stimulating the release of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), and thereby has neuroprotective effects in patients with ALS [7,9,10]. Our preliminary study showed that an unilateral, epidural anodal stimulation was effective in delaying the progression of disease and in preserving the brain functions in a murine model of ALS [12]. Thus, we have previously found that symptoms occurred eight days later as compared with normal controls, but there was no significant difference in the survival period between the experimental and control group.
Given the above background, we examined the effects of the unilateral cortical stimulation on the survival of neurons showing degenerative changes and compared those in delaying the progression of ALS between the unilateral cortical stimulation and the bilateral one in an animal experimental model using mice.

Experimental animals
For the current experimental study, we used 19 G93A transgenic male mice carrying the G93A human SOD1 mutation (Jackson Lab., Bar Harbor, ME, USA). Thus, SOD1-G93A mice were housed in a controlled animal husbandry unit at 21 ± 1°C with water ad libitum.
The experimental animals were randomly divided into three groups: (1) The control group (n = 6): The mice underwent implantation of electrodes in the bilateral motor cortex without electrical stimulation.
(2) The unilateral stimulation group (n = 7): The mice underwent implantation of electrodes in the unilateral motor cortex and daily received a 24-hour cortical stimulation.
(3) The bilateral stimulation group (n = 6): The mice underwent implantation of electrodes in the bilateral motor cortex and daily received a 24-hour cortical stimulation.
We conducted the current experimental study in compliance with guidelines for the Institutional Animal Care and Use Committee (IACUC) of Wonkwang University. This study was approved by the IACUC of Wonkwang University (approval number: # WMS-2009-001).

Experimental procedures
We performed operations for all the experimental animals at 70 ± 3 days after birth. The average body weight of mice at operation was 25.90 ± 2.25 g. Anesthesia was achieved by isoflurane (2% induction and 1.5% maintenance, in 80% N2O and 20% O2) administered via a face mask. The sufficient depth of anesthesia was checked by the absence of cardiovascular reflexes in response to tail pinch. To determine the frequency of the cortical stimulation for the movement of the forelimb and/or face, contralateral to the sites of electrode implantation, we stimulated the motor cortex of the experimental animals with a pulse of 1.0 ms in width and 3-5 seconds in duration and the minimal direct current. Then, we measured the threshold of movement twice a week.
At the onset of the symptoms, defined as the initial presentation of the abnormal gait or limb weakness, we implanted the electrodes and then initiated the cortical stimulation in the experimental animals. To do this, we implanted the circular electrode with a diameter of 2.0 mm on the exposed dura of the motor cortex, which is located anterior 1.5 mm and posterior 0.5 mm to the bregma and leaves a 1-mm margin from the midline. We also implanted the reference electrode with a rectangular shape of 2.0 × 4.0 mm in size on the subcutaneous layer of the posterior neck ( Figure 1). We fixed the electrodes in the skull with screws and a visible-lightactivated dental resin. In the experimental animals, we connected the stimulating electrode cable to the swivel conductor, thus attempting to prevent the kinking, and then to the programmable electrical stimulator (HSRG Neuro, Cybermedic, Iksan, Korea). From postoperative day 2 on, we delivered the electrical stimulation to the experimental animals at an amplitude of 50% of the movement threshold, a frequency of 50 Hz and a duration of 220 μs. In addition, we performed the anodal stimulation for 24 hours.
On postoperative weeks 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, we performed the Rota-rod test and paw-grip endurance test for the experimental animals. Then, we compared the effects on the motor functions between the three groups.

Histopathologic examinations
To examine the effects of the unilateral cortical stimulation on the survival of neurons, we additionally operated three mice and then performed the unilateral cortical stimulation for them at varying frequencies of 2, 50 and 70 Hz. Then, we extracted their brain and spinal cord at two weeks after the onset of symptoms and performed histopathologic examinations. To do this, we stained the motor cortex and spinal cord at the cervical, thoracic and lumbar levels using the hematoxylin-eosin (H-E) dye.

Statistical analysis
All data was expressed as mean ± SD (SD: standard deviation). We used the analysis of variance (ANOVA) to compare the variables between the three groups. A P-value of <0.05 was considered statistically significant.

Baseline characteristics of the experimental animals
The mean disease duration was 9.83 ± 9.00 days, 14.86 ± 7.73 days and 24.00 ± 19.39 days in the corresponding order. But these differences reached no statistical significance (P > 0.05). In addition, the mean movement threshold was 2.50 ± 0.1 V in the control group, 1.83 ± 0.35 V in the unilateral stimulation group and 2.2 ± 0.2 V in the bilateral stimulation group. But these differences reached no statistical significance (P > 0.05).
As shown in Figure 2, the mean onset of the symptoms was 101.00 ± 8.76 days in the control group, 100.86 ± 4.45 days in the unilateral stimulation group and 102.33 ± 10.97 days in the bilateral stimulation group. But these differences reached no statistical significance (P > 0.05). In addition, the mean survival period was 109.50 ± 10.41 days, 114.71 ± 8.40 days and 124.33 ± 11.00 days in the corresponding order. These results indicate that it was significantly longer in the bilateral stimulation group as compared with the control group (P < 0.05).

Histopathologic findings
As shown in Figure 3, there was no significant difference in the number of neurons with degenerative changes between the stimulating and reference sites. This indicates that the frequency of stimulation with the number of neurons with degenerative changes.

Rota-rod scores
As shown in Figure 4, the mean Rota-rod score was 264.90 ± 20.68 s in the control group, 300.00 ± 0.00 s in the unilateral stimulation group and 274.67 ± 17.42 s in the bilateral stimulation group on postoperative week 11. In addition, on postoperative weeks 11, 12, 13, 14 and 15, it was significantly higher in the unilateral Figure 2 The survival period in each group. The survival period was significantly longer in the bilateral stimulation group as compared with the control group (P < 0.05). stimulation group as compared with the control group (P < 0.05). Furthermore, despite a lack of statistical significance, it was the lowest in the bilateral stimulation group on postoperative weeks 13, 14, 15 and 17.

Paw-grip endurance
As shown in Figure 5, on postoperative week 11, the mean score of paw-grip endurance was 72.85 ± 6.47 s in the control group, 90.00 ± 0.00 s in the unilateral stimulation group and 65.55 ± 7.63 s in the bilateral stimulation group. On postoperative weeks 11, 12, 13, 14 and 16, it was significantly higher in the unilateral stimulation group as compared with the control group (P < 0.05). Furthermore, despite a lack of statistical significance, it was the lowest in the bilateral stimulation group on postoperative weeks 13, 14, 15 and 17.

Discussion
The exact pathogenesis of ALS remains obscure. According to several in vivo and clinical studies, the hyperexcitability of the motor cortex is involved in its early onset [13,14]. It arises from the increased excitability of Figure 3 The histopathologic findings after stimulation on Brain (a), C-spine (b), T-spine (c) and L-spine (d). The stimulating electrode was implanted on the motor cortex. We stimulated the brain, C-spine, T-spine and L-spine at a frequency of 2 Hz and 50% of the movement threshold of 1 V (als-1), a frequency of 50 Hz and 50% of the movement threshold of 0.7 V (als-2) and a frequency of 70 Hz and 50% of the movement threshold of 0.6 V (als-3) for continuously 24 hours. There were no significant differences in the number of neurons showing degenerative changes between the stimulating and reference sites. the corticomotor neurons and the decreased inhibition of the cortical inhibitory interneurons due to the brain dysfunction. Based on the decreased uptake of glutamate in astrocytes and increased levels of glutamate in the cerebrospinal fluid, it can be inferred that the neuronal toxicity might be mediated by excessive glutamate. That is, due to the presence of excess glutamate, cortical cells might be vulnerable to the cell death. This leads to the decreased threshold of the motor cortex in the early stage of ALS. But there is an increase in it with the progression of the disease; it may be increased to such an extent that it cannot elicit motor-evoked potentials. It can therefore be inferred that patients with late-stage ALS would have functional deficits due to the decreased motor function [15].
Recent non-clinical and clinical studies have shown that both non-invasive and invasive cortical stimulation can modulate the excitability of glutamatergic circuit in the motor cortex [16][17][18][19][20]. This has led to the application of such treatment modalities as TMS, transcranial direct current stimulation (tDCS) and direct (epidural) cortical stimulation (DCS) to the recovery of the motor functions in patients with stroke [21][22][23][24]. Moreover, the direct brain stimulation has also been applied to the treatment of patients with Parkinson's disease, depression or epilepsy [25][26][27]. Because these types of cortical stimulation may cause alterations in the neurotransmitters such as glutamate, they are used to treat several neurological disorders such as stroke, depression, central pain syndrome, ALS and Parkinson's disease.
In the current study, we evaluated the effects of epidural cortical stimulation in a murine model of ALS. We found that the bilateral epidural cortical stimulation delayed the progression of ALS. Our results also showed, however, that scores of Rota-rod and paw-grip tests were significantly higher in the unilateral stimulation group as compared with the control group. Many previous studies have demonstrated that both the invasive and noninvasive cortical stimulation are effective in delaying the progression of ALS. But we failed to clarify the actual mechanisms by which the cortical stimulation delays it. We assume that the epidural stimulation of the motor cortex may modulate the excitability of the motor cortex and thereby protect the motor neurons from damages by altering neurotransmitters, such as BDNF, and the glutamatergic or GABAnergic transmission.
The survival rate was the highest in the bilateral stimulation group. As compared with the unilateral stimulation group, however, scores on the behavioral test were significantly lower in the bilateral one. It can therefore be concluded that bilateral cortical stimulation more strongly affected inter-hemispheric inhibition, thus producing a decrease in overall motor functions. In addition, it is possible that bilateral placement of electrodes and wiring could cause the unexpected result of lesser functional improvement despite the prolonged survival period. It is also important to recognize that a prolonged survival period in mice might not be interpreted in the same way in humans based on a genomic response in murine models [28,29].
In the current study, we analyzed the positive effects of unilateral cortical stimulation based on histopathological findings. But there were no significant differences in the number of neurons showing degenerative changes between the stimulating and reference sites in the motor cortex and the spinal cord at the cervical, thoracic and lumbar levels. These results indicate that the unilateral cortical stimulation affects both sides of the central nervous system.
In the current study, we stimulated the motor cortex at a frequency of 50 Hz. In addition, we also found that the epidural cortical stimulation was also effective in making a recovery of the stroke [23,30]. Furthermore, it has also been reported that it was effective in increasing the density of dendrites in the layer V and eliciting polysynaptic-evoked potentials to a greater extent when its frequency was 50 or 100 Hz in animal experimental models using rats or primates [31][32][33][34]. Taken together, we assume that there is a possible relationship between the progression of ALS and the anatomical and physiological changes in the motor cortex in a murine model of ALS.
In the current study, we stimulated our experimental animals for 24 hours a day. It is impossible to continuously perform the TMS for long periods of time. In a clinical setting, however, it is possible to perform the TMS using the implantable electrical stimulator with implanted epidural electrode in patients with ALS. It would be an invasive procedure to implant the electrode and Figure 5 Scores of paw-grip test. There was a significant difference in the score of paw-grip test between the control group and the unilateral stimulation group on postoperative weeks 11, 12, 13, 14 and 16 (*P < 0.05).
stimulator. Nevertheless, it is advantageous in localizing the sites of stimulation both exactly and continuously.
We first attempted to examine the effects of epidural cortical stimulation in a murine model of ALS. As compared with other several non-invasive methods, such as the concentration of the stimulation, the minimization of the stimulation intensity and the concurrent multifocal stimulation, epidural cortical stimulation has beneficial neuromodulatory effects on the brain. But further studies are warranted to develop advanced treatment modalities for patients with brain diseases.

Conclusions
In conclusion, our results indicate that the bilateral epidural cortical stimulation might have a treatment effect in a murine model of ALS. But it is the limitation that we examined a small number of experimental animals. Further studies are therefore warranted to establish our results and to identify the optimal parameters of the epidural cortical stimulation in a larger number of experimental animals.