The effect of periaqueductal gray’s metabotropic glutamate receptor subtype 8 activation on locomotor function following spinal cord injury

1 Introduction

Spinal cord injury is one of the major causes of motor dysfunction. Spine diseases, road accidents, falls from height and knife injuries are the most common causes of spinal cord injury. In the long run, spinal cord injury will be associated with many complications such as pain, difficulty in urinating and movement problems. The partial recovery that will occur after injury depends on several factors, including patient age, cause of injury and location of injury in spinal cord [1]. In this study, we decided after spinal cord injury and complete limb paralysis below the lesion surface, evaluate the effect of glutamate metabotropic receptor (mGluR) type8 agonist (DCPG) in the periaqueductal gray (PAG) area on motor function improvement.

Glutamate is one of the most important excitatory neurotransmitters in the central nervous system which is involved in several pathological processes, including the transmission of pain [2]. Although glutamate is responsible for pain and its intensification, the action of this neurotransmitter is highly dependent on the type of its receptors and their locations. Group II and III of metabotropic glutamate receptors are pre-synaptic and have a suppressive role in glutamate transmissions due to their association with inhibitory G-Proteins [3]. It has also been observed that mGluRs in group III (mGluR 4, 6, 7 and 8) play an autoreceptor role in the glutaminergic ends, i.e. these receptors inhibit the release of more than the usual amounts of glutamate in pathologic conditions [3]. Interestingly, all of these inhibitory effects on glutamate release have been observed at the spinal cord level, and the behavior of these receptors in the brain, and especially in the PAG, is quite different. In these areas, mGluR 8 activity decreases GABA release and increases glutamate release, thereby activating the descending antinociceptive pathway to the spinal cord [4]. In addition, another function of glutamate through mGluRs is to enhance the recall of stem cells to the lesion site. This role of the mGluRs has been demonstrated in some of the mGluR8-family receptors (it is observed that the mGluR4 agonist can attenuates oxidative stress-induced death of neural stem cells) [5], [6].

Therefore, in this study we decided to investigate the effect of mGluR8 (fewer known species among the mGluR III) stimulation on the rate of axonal regeneration of the corticospinal tract and functional recovery and compare the results with the animals that their mGluR8 is knocked down by specific mGluR8 siRNA (GRM8) administration.

2 Materials and methods

3 Results

3.1 Behavioral study

3.1.1 BBB

Motor function was evaluated by the BBB score. Features such as fore/hind limb coordination, weight support and paw placement were considered according to the 21-point BBB scores. The score of normal (sham) animals were 21. The animals with spinal cord injury showed significantly lower BBB score when compared to sham animals [F (30, 180)=472.90; p<0.001)]. There was no significant difference between the DCPG and other groups up to the third week after injury (p<0.05). However, from the fourth to sixth weeks post injury, the motor recovery process started. The averages of BBB score in the DCPG group were 6.7±0.2 in the fourth, 8.3±0.2 in the fifth, and 11.5±0.3 in the sixth weeks post injury. These values in the SCI-control group were 4.0±0.2, 4.6±0.2 and 5.0±0.2, respectively (p<0.001) (Fig. 1).

Fig. 1:

BBB scores of experimental animals. The locomotor function of the animals is compared with the sham and other treated groups. Administration of DCPG could improve the animal’s motor function compared with injured animals. SCI-c: SCI-control. *** indicates a significant difference between all intervention groups and the sham group at the level of 0.001. ### indicates a difference between the DCPG-treated group and the SCI-control and siRNA group at the level of 0.001.

3.2 Histological study

3.2.1 Axonal regeneration on the corticospinal tract

Anterograde tracing of corticospinal tracts (CST) was done to investigate the relationship between motor function improvement and axonal regeneration. For this purpose, we injected Biotin Dextran Amine (BDA) into the motor cortex bilaterally (Fig. 2A). Our results on the evaluation of axonal regeneration in the corticospinal pathway showed that the number of axons in the SCI-control group was significantly lower than sham group (p<0.001) (Fig. 2B). However, administration of DCPG could increase the number of regenerated axons in the corticospinal tracts (p<0.01). As expected, administration of mGluR8 siRNA could not change the regeneration rate in the corticospinal tracts compared with the SCI-control group (Fig. 2).

Fig. 2:

Axonal Regeneration in the myelinated spinal cord. Anterograde axonal tracing with BDA was performed 6 weeks post-SCI. (A) Image of fluorescence microscope from longitudinal sections of the thoracic spinal cord. The longest regenerating axons extended several millimeters beyond the caudal end of the cavity. As shown in the picture, the rate of regeneration of axons in this pathway improved after treatment with DCPG compared with the SCI-control and siRNA groups. Scale bar=20 μm. (B) The average number of regenerating axons/sections was measured. The results show a greater number of regenerated axons in injured rats treated with DCPG (n=3 independent rats/condition; three to four sections/rat). Arrowheads indicate turning fibers. *** indicates a significant difference with the sham group at the level of p<0.001; ## indicates a significant difference with the DCPG group at the level of p<0.01.

4 Discussion

In our study, the effect of DCPG, as a selective agonist of mGluR8, on motor dysfunction following spinal cord injury was investigated. Results obtained from this research unraveled that the activation of PAG’s mGluR8 following spinal cord injury could restore motor functions through axonal regeneration in the CST and increase motor skills through BBB testing.

Despite the increasing prevalence of spinal cord injury, unfortunately, no definitive cure for multiple complications has been found yet. The most common drugs used to reduce the complications of spinal cord injury are as follows:

1. Non-steroidal anti-inflammatory drugs (also known as NSAIDs), such as aspirin, ibuprofen, Motrin, Advil and naproxen, are the most commonly used drugs to treat musculoskeletal pain. Side effects of these medications may include stomach upset or stomach bleeding problems.

2. Anticonvulsants such as gabapentin or neurontin and Pregabalin or Lyrica are used to treat neuropathic pain. Their main side effects include dizziness, drowsiness and body swelling.

3. Addicts, including morphine, codeine, hydrocodone, and oxycodone, are used to treat a variety of neuropathic and skeletal muscles pains. One of the most important side effects of these drugs is constipation and sleep apnea. They can also become habitual and addictive, and symptoms can return as they stop abruptly. Antidepressants, muscle relaxants and anti-spasm, topical anesthesia drugs are other common treatments [10], [11], [12], [13]. The combination of two or more of these drugs is nowadays used in combination therapy to allow more cellular pathways to be involved and to achieve better results in less time and at lower doses [14], [15].

Another treatment approach in recent years is the treatment of spinal cord lesions with stem cells. By using stem cell transplantation at the site of the lesion, scientists have now reached plausible results in improving the quality of life of people with spinal cord injury [16].

According to our behavioral findings, DCPG could significantly improve the motor performance of animals as compared to the SCI-control and siRNA groups. In the BBB motor test, it was found that indicators such as weight support, tail movement control, movement of hind limb’s joints and some degree of coordination of the front and hind limbs could be improved. As demonstrated in Faden’s study and the effect of glutamate on motor recovery after spinal cord injury, glutamate via NMDA receptors causes motor loss and worsens the complication of spinal cord injury. However, they found that a single injection of NMDA’s antagonist could improve motor functions [17]. There is extensive evidence that the increased release of glutamate under conditions of damage to the central nervous system contributes to damage and thereby to permanent impairments arising from such traumas [18]. Most of the evidence suggests that the administration of glutamate agonists exacerbates post-injury disorders, and that administration of glutamate antagonists can result in motor and sensory improvement [19], [20], [21], [22]. Therefore, it would not be inadvisable that an increase in mGluR8 activity due to its modulatory effect on glutamate release (at the spinal cord surface) in injury conditions could improve animal motor function in the BBB test. It doesn’t exist any study right now that investigate the direct effect of mGluR8 on motor function following spinal cord injury. In 2012, Johnson and et al. evaluated the effect of the single dose of DCPG on forelimb coordination in Parkinson disease. They showed that DCPG could improve neuronal regeneration in substantia nigra [23]. On the other hand, Guo-Ying Xu in 2005 proved that increase in glutamate following spinal cord injury and at the level of spinal cord through NMDA receptors will lead to exacerbation of pathological conditions especially functional impairment [18]. In fact, scores of animals in the BBB test following intrathecal injection of glutamate were significantly reduced compared to control animals that had only spinal cord injury. In addition, Faden, in a study in 1988 and 1990, also proved that glutamate antagonist injection (as well as NMDA and AMPA receptors antagonists) could significantly improve post-spinal cord injury’s complications including motor function [17], [24].

Histological examination of the animal’s spinal cord after injection of an axonal tracer (BDA) also showed axonal regeneration in the corticospinal tract. This regeneration was quite evident in the DCPG-treated group compared with the siRNA group. One reason for this is that increased mGluR8 activity after DCPG administration, due to coupling with inhibitory G-proteins can reduce glutamate release at the spinal cord level and thus prevent the deleterious effects of glutamate [19]. Wang and his colleagues in 2011 showed that the injection of mGluR4 aagonist (VU0155041) after peripheral nerve ligation (L5) and progression of neuropathic pain, could significantly increase the animal pain threshold [25]. Due to the many similarities between this receptor (mGluR4) and mGluR8, most probably, the proven role of mGluR4 in suppressing of oxidative stress and axonal regeneration can be generalized to mGluR8 [25], [26]. Molecular and more detailed examination of these documents will be the goals of our future studies. In our study, following the injection of tracer to determine the rate of corticospinal axonal regeneration, it was found that DCPG can significantly improve axonal repair in the cortico-spinal tract of adult rats compared with the siRNA and SCI-control groups.

5 Conclusion

In summary, the present study has shown that mGluR8 agonists (DCPG) improve functional recovery following spinal cord injury, as well as axonal regeneration rate in CST. These observations are consistent with the hypothesis that glutamate contributing to secondary tissue damage, and the use of mGluR type 8 agonists that modulate glutamate release can improve conditions after the injury.