The Primary Motor Cortex Stimulation Attenuates Cold Allodynia in a Chronic Peripheral Neuropathic Pain Condition in Rattus norvegicus

Background: The primary motor cortex (M1) stimulation (MCS) is a useful tool for attenuation of the peripheral neuropathic pain in patients with pharmacologically refractory pain. Furthermore, that neurological procedure may also cause antinociception in rodents with neuropathic pain. Cold allodynia is a frequent clinical finding in patients with neuropathic pain, then, we evaluated if an adapted model of neuropathy induced by chronic constriction injury (CCI) of the ischiadicus nervus (sciatic nerve) produces cold allodynia in an animal model of chronic pain. In addition, we also investigated the effect of the electrical stimulation of the M1 on chronic neuropathic pain condition in laboratory animals. Methods: Male Wistar rats were used. An adapted model of peripheral mononeuropathy induced by CCI was carried out by placing a single loose ligature around the right sciatic nerve. The acetone test was used to evaluate the cold allodynia in CCI or Sham (without ligature) rats. The MCS (M1) was performed at low-frequency (20 μA, 100 Hz) during *These authors equally contributed to this work. How to cite this paper: Medeiros, P., Negrini-Ferrari, S.E., Medeiros, A.C., Ferreira, L.L., da Silva, J.R.T., da Silva, J.A., Coimbra, N.C. and de Freitas, R.L. (2019) The Primary Motor Cortex Stimulation Attenuates Cold Allodynia in a Chronic Peripheral Neuropathic Pain Condition in Rattus norvegicus. World Journal of Neuroscience, 9, 138-152. https://doi.org/10.4236/wjns.2019.93009 Received: April 12, 2019 Accepted: July 29, 2019 Published: August 1, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
The most recent definition of pain is given by Williams and Craig [1] in 2016, who say that "pain is a distressing experience associated with actual or potential tissue damage with the sensory, emotional, cognitive and social component".
When such injury or dysfunction is related to the central nervous system activity, the pain is classified as neuropathic pain [2].
The incidence rate of chronic pain in the world is about 8% [3] [4]; however, the prevalence of neuropathic pain is estimated to be between 1% and 23% [4].
The Institute of Medicine reported that 100 million Americans suffer from chronic pain at the cost of $600 billion [5].
Recently, Carvalho and collaborators [6] reported high prevalence and severity of chronic pain and suggest that it is a public health problem in Brazil. In fact, there is a evidence that chronic pain affects more than two-thirds of the population of Brazil. Risk factors are being a woman, advanced age and low levels of household income. There is a need for improvement of health policies in Brazil for patients with chronic pain.
Pain can also be defined as acute when self-limited and caused by a specific disease or injury, or as chronic pain, considered a disease when it exceeds a normal cure time [7]. Neuropathic pain affects 5% of the Brazilian population [8] affecting the quality of life and reducing productivity [9]. Neuropathic pain is often severe, and it can afflict patients for their entire lifetime. Many studies have been performed to better understand the neurobiology of pain, considering its mechanisms, neural bases and comorbidities, as well as to find new forms of treatment. A novel target structure of these studies is the motor cortex (MC). Motor cortex stimulation (MCS) is a novel treatment modality used to modulate neuropathic pain by electrical stimulation of the brain [10].
That procedure was introduced by Tsubokawa et al. in 1991 [11] and has been used in clinical practice as an additional treatment of neuropathic pain [12] [13].
Since then, several studies have been done corroborating the pioneering results by Tsubokawa team [14] [15], although the neural bases of the analgesic mechanisms involved in that procedure have not been well established yet.
As a matter of fact, MCS is a neuromodulation therapy used for the treatment of neuropathic pain and it has shown good efficacy in the treatment of patients suffering from a pain disorder [14] [15]. Mixed results after MCS relieving pain in approximately 50% in patients with neuropathic pain were already demonstrated [13]. Based on that evidence, we evaluated the effect of MCS (M 1 ) in rats with chronic neuropathic pain investigating the modulation of cold allodynia threshold by M 1 -stimulation in rats with chronic constriction injury (CCI) of the ischiadicus nervus (sciatic nerve).
The chronic and neuropathic pain present a heightened experience of pain caused by a noxious stimulus (hyperalgesia) and a stimulus that was innocuous becomes painful (allodynia) [16]. The prevalence of allodynia in neuropathic pain is likewise difficult to assess. In a questionnaire study recruiting more than 1600 patients with painful diabetic neuropathy [17], 18% reported that light touching was painful, and 14% reported that cold or heat stimulus was occasionally painful. Only 47% with post-herpetic neuralgia had touch-evoked allodynia, although this is usually reported to be present in at least 70% of cases [18].
In another study involving 482 patients with different causes of neuropathic pain, 55% had brush-evoked allodynia, whereas pain evoked by contact with cold objects was reported by 31% of patients, with pressure-evoked pain reported by 52% of patients. Any pain evoked by brush, pressure, or cold stimuli was present in 52% of patients with painful diabetic polyneuropathy and reported by 92% of patients with post-herpetic neuralgia [19].
The perception of innocuous and noxious cold is mediated by unmyelinated (C) and thinly myelinated (Aδ) fibres. Differential blocks of A fibres in human volunteers have shown that the sensitivity to innocuous cold is mediated by Aδ fibres [20], although C fibres have also been shown to respond to innocuous cold [21] [22]. The existence of two types of neurons has been suggested: a low-threshold cool type, responding to activating temperatures close to 30˚C, and a high-threshold cold nociceptor neuron population, activated at temperatures less than 20˚C [23].
Thus, the study of pain can be performed using chemical models, such as those using acetone, in which it is possible to evaluate the cold allodynia in animals that were submitted to an experimental procedure for the induction of neuropathy, such as the CCI, that was described by Bennet and Xie [24], and In this sense, the study of the role played by MCS in the modulation of the cold allodynia becomes interesting because there are no reports in the literature using MCS procedure for cold allodynia/hipernociception control in CCI rats.
For this, in the present work, the effect of neurostimulation on the primary (M 1 ) motor cortex (MC) in animals with chronic and neuropathic pain were submitted to the acetone test 21 days after chronic constriction injury (CCI) of the ischiadicus nervus.

Animals
Male Wistar rats were housed four per cage with free access to food and water under controlled illumination ( search on laboratory animals [25]. Experiments were conducted during the light phase of the cycle, and animals were randomly tested across that time-window.
Each animal was used only in a single independent experimental group, and all efforts were made to minimise discomfort for the animals.

Model of Neuropathic Pain: Chronic Constriction Injury (CCI) of the Ischiadicus Nervus
The sciatic nerve was exposed through dissection of the gluteus maximus muscle and biceps femoris, and a single ligature of 4 -0 catgut thread was placed around the nerve, in contrast to the procedure described by Bennett & Xie (1988). Finally, the skin incision was sutured with 5 -0 mononylon thread. Sham surgery was performed by exposing the sciatic nerve as described above, without nerve ligation. The animals were then transferred to their home cages and left to recover.
In our study, the rats were examined daily after the surgery and about weekly until 21 days. During this inspection, each animal was placed upon a table and carefully observed the condition of the affected hind paw (Figure 1).

Cold Allodynia-Acetone Test
The acetone test, conducted in an independent group of rodents, was used to measure cold allodynia. Each animal was placed in a transparent acrylic cubicle

Stereotaxic Surgery
An electrode was implanted in the primary (M 1 ) motor cortex of rats. Fourteen days after either the Sham procedure or the CCI surgery for chronic neuropathy induction, the animals were anaesthetised with 92 mg/kg ketamine (Ketamine) and 9.2 mg/kg xylazine (Dopaser) and fixed in a stereotaxic frame (David Kopf, USA). The upper incisor bar was set at 3.3 mm below the interaural line such that the skull was horizontal between bregma and lambda. Subsequently, the electrode was fixed to the skull with acrylic resin and two stainless steel screws above the M 1 (anteroposterior = −1.1 mm; mediolateral = +1.5; and dorsoventral = −1.5 mm from the skull) in accordance with Paxinos and Watson's rat brain in stereotaxic coordinates atlas [26].

Primary (M1) Motor Cortex Stimulation (MCS)
Three weeks after the CCI or Sham surgeries and one week after stereotaxic surgery, it was performed the baseline 2 of acetone test in the animals. After that procedure, the animals were placed in a circular arena (60 cm in diameter and 50 cm high with the floor divided into 12 sections) with the experimental compartment illuminated with a 40-W fluorescent lamp (350 lx at the arena floor level). The animals were allowed a 3 min period of habituation. Afterwards, the electrode implanted in the midbrain was connected to a stimulus generator (STG3008-FA, Multichannel Systems, Germany) which allowed to apply current pulses (cathode pulse width 100 μs, pulse interval 100 μs and anode pulse width 100 μs, repeated during 15 s). The M 1 stimulation was made during 15 s at 20 μA, immediately followed by acetone test for 30 min in the right paw. Each animal was used only once and received only one MCS. The temporal window of the experimental procedure was shown in Figure 2.

Histological Analysis
After testing, the rats were anaesthetised as previously described and perfused

Statistical Analysis
The data are expressed as the mean ± standard error of the mean (S.E.M.). Data regarding the cold allodynia threshold were subjected to a two-way repeated measures analysis of variance (split-plot ANOVA), followed, when appropriate, by Tukey's post hoc test. The procedure (electrical stimulation of M 1 ) was considered the independent factor, and the time was considered the dependent factor. Repeated measures ANOVA uses the same conceptual framework as classical ANOVA followed by Tukey's post hoc test [27] [28].

Experimental Design
Primary motor cortex stimulation (MCS) and the effect of that procedure on cold allodynia and chronic neuropathic pain induced by CCI. 1) The animals had their basal nociceptive thresholds (baseline 1) measured through the acetone test; on the same day, each animal was anaesthetised and subjected to either a CCI or sham procedure; 2) Fourteen days after the CCI and sham procedures, the animals were anaesthetised and subjected to stereotaxic surgery for implantation of an electrode in the M 1 ; 3) Seven days after the stereotaxic surgery and 21 days after the CCI or sham procedure, the nociceptive threshold of cold allodynia was measured by the acetone test (baseline 2) for each rat; World Journal of Neuroscience 4) Immediately after baseline 2, the rats were connected to the neurostimulator (DBS machine) for MCS (electric current intensity: 20 µA; duration: 15 s) in the open field apparatus, and after 1 min, the rats were subjected to cold allodynia threshold recordings at 0, 15, and 30 min after MCS; 5) Twenty-four hours after the experiment, each rat was anaesthetised and perfused for the histochemical procedure.

Results
The primary motor cortex (M 1 ) electrical stimulation (MCS) attenuates the neuropathic pain (NP) Histologically confirmed electrode tips situated in the primary (M 1 ) motor cortex in CCI or Sham rats are shown in Figure 3.
To considering cold allodynia recorded 21 days after CCI induction (Figure 4).
There was a significant decreased in cold allodynia in the neuropathic pain animals after the motor cortex stimulation at 20 µA for 15 seconds compared with the neuropathic animals that did not receive stimulation for 30 min after deep brain stimulation (Tukey's post hock test; P < 0.05), as shown in Figure 4.

Discussion
In the present study, it was demonstrated that the electrical MCS on the M 1 decreased cold allodynia assessment by acetone test in Wistar rats. The nociceptive measured evaluating cold allodynia was made 21 days after CCI in animals with chronic neuropathic pain. These results suggest an antinociceptive effect caused by motor cerebral cortex electric stimulation (see graphical abstract in Figure 5).
According to Calcutt et al., 1997 [29], the neural mechanism of the thermal stimulation is not completely known, since the studies performed until now were focused only on a cerebral cortex stimulation. Interestingly, we showed here that the deep brain stimulation of the primary motor cortex reduced the cold thermal stimulus-related hyperalgesia. Considering the relevance of glutamatergic projections from the cerebral cortex [27] [30], these telencephalic fugal projections have a putative involvement in the neurochemical mechanisms of motor cortex stimulation-induced antinociception. Indeed, there are reports highlighting the relevance of N-methyl-D-aspartic acid receptor in the transcranial magnetic stimulation-induced analgesia [31].
Considering the neuropathic pain mechanisms studied in the present experimental model of chronic pain, peripheral lesion of spinal nerves leads the individual to an altered perception of innocuous stimuli and noxious stimuli, and therefore, pain is often felt in the limb at the touch, as demonstrated by the inability to detect a fine probe applied to the skin [32], and the information resulting from the somatosensory cortex is altered. We might consider that the motor cortex stimulation causes a reorganisation of the sensory somatic information in the cerebral somatosensory cortex through a direct cortico-cortical functional brain connectivity, or through an indirect cortico-neostriatum-pallido-thalamocortical projection [33] Cortico-cortical neurons in superficial layers of motor cortex work with motor and sensory signals and might mediate sensorimotor integration [34].
Interestingly, there is evidence that tactile stimuli can be modulated by thalamus-efferent connections to reach both motor and somatosensory neocortical areas [35] [36]. The influence exerted by neocortical somatosensory areas on the motor cortex function is already known, and sensorimotor connexions are critical for efficient movements control, considering that the somatosensory connectivity has been shown to play relevant effects on the execution of motor plans [37]. Impairments of the connectivity between the motor and the somatosensory cortexes cause dexterity disfunctions [38]. Finally, reciprocal connections between the somatomotor cortical areas were already demonstrated in rodents [39]. Differences in synaptic function suggest specialisations within the sensorimotor connexions that may allow updating of sensory-motor inputs integration within the neocortex in response to changes in the sensory periphery [40].
Therefore, the acetone test procedure in animals with neuropathic pain was planned to evaluate sensitivity of low temperature-related ascending spino-thalamic pathways, and allows us to verify that there were decreases in the nociceptive threshold in these animals with chronic neurogenic mononeuropathy. These results corroborate studies performed in human beings, and considering that in the medical clinics, it is reported that the sensitivity for cold stimulus in patients with chronic neuropathic pain also showed increased [17] [18] [19]. It is known that the sensory dysfunction detected in patients with chronic neuropathic pain involves both mechanical and thermal dysfunctions and, as shown in the present study, rodents with neuropathic pain present markedly cold allodynia. Some studies using fRMI approaches before and after the injury demonstrated that the mechanical stimulation of the skin produced increased pain in the area of secondary hyperalgesia leading to greater activation in areas of the brain associated with pain signalling [41] [42].
In conclusion, the electrical stimulation of the primary motor cortical area caused analgesia in laboratory animals with neuropathic pain induced by CCI. Although additional studies are required to understand the relationship between MCS and neuropathic pain control, we might consider that the MCS acts not only as a cortico-cortical neuromodulator, but also as a putative descending modulator of pain pathways.
Further investigations of the neuroanatomical and neurochemical mechanisms involved in the anti-hypernociceptive phenomenon studied in the present work may contribute to the improvement of the clinical treatment of pharmacologically refractory chronic pain and neuropathy.