The present study revealed that glutamate is increased significantly in trigeminal neurons of all sizes and their enveloping SGCs, but predominantly in small neurons and their SGCs, in a model of neuropathic pain associated with peripheral nerve injury. This suggests that each populations of satellite glial cells (SGCs) and their surrounding trigeminal neurons of different type are involved in the glutamate signaling associated with neuropathic pain at a different level.
Ever since the elegant studies by the Ottersen's group with postembedding immunogold showed that the density of gold particles coding for glutamate is linearly correlated with the concentration of glutamate in the brain12,13, we, among others, have routinely used immunogold labeling as a reliable method for a semiquantitative estimation of level of glutamate as a signal substance in the neural tissue14,15. In the present study, we employed this method to evaluate changes in the levels of glutamate in trigeminal neurons and their enveloping SGCs in a model of neuropathic pain following peripheral nerve injury.
That the levels of glutamate in trigeminal neurons of all sizes were higher in rats with CCI-ION than in sham-operated rats is consistent with the results of previous light microscopic immunohistochemical studies in the DRG and TG neurons following peripheral nerve injury and inflammation16,17. Since there is a positive correlation between the size of DRG neurons and the conduction velocity of their fibers18,19, it is now widely accepted that the small, medium-sized, and large neurons in the DRG are roughly equivalent to neurons with C, Aδ, and Aβ fibers, respectively. Our results, then, support the idea that the release of glutamate from sensory fibers of all types is increased following CCI-ION16,20,21,22, potentially leading to an increase in the excitability of their postsynaptic neurons in the superficial laminae (by C and Aδ fibers) as well as in the deep laminae (by Aβ fibers) of the spinal and medullary dorsal horn23,24, which may further contribute to the development of pathologic pain.
The increase in the levels of glutamate following CCI-ION was the highest in small neurons (1.9 and 3.0 times higher than in medium-sized and in large neurons, respectively). This supports the notion that peripheral nerve injury leads to increased synaptic transmission primarily by central C fibers. Also, the increased synaptic transmission at the central synapses of C fibers in the superficial lamina of the medullary dorsal horn may cause more robust sensitization of their (nociceptive) postsynaptic neurons, compared to the A fiber synapses and their (non-nociceptive) postsynaptic neurons in the deep laminae of Vc. On the other hand, the synaptic facilitation of A fiber synapses in the deep laminae may also contribute to activation of nociceptive neurons in the superficial laminae via secondary activation of silent synapses or polysynaptic pathways associated with A fibers25,26,27.
It has been shown previously that following peripheral nerve injury and inflammation, SGCs are activated by the neurons they are enveloping1,28. In turn, the activated SGCs release signaling molecules, such as ATP and the cytokines TNFa and IL-1b, causing enhancement of the excitability of the neurons they are enveloping, and adjacent neurons via SGC-neuron and SGC-SGC communication, and thus contributing to the development of pathologic pain1,7.
We observed significant increase of glutamate level in trigeminal SGCs that envelop neurons of all sizes following peripheral nerve injury, suggesting involvement of SGCs that envelop primary sensory neurons of all types, including nociceptive and mechanosensitive neurons, in the SGC-neuron and SGC-SGC communication following nerve injury. It also raises the possibility, albeit remote, that a mechanism underlying the mechanical allodynia following nerve injury exists in the TG (and DRG) besides that in the brain stem (and the spinal cord).
In rats with nerve injury, the glutamate concentration was higher in SGCs of small neurons than in SGCs of medium or large neurons, suggesting a predominant involvement small (C fiber) neurons and their enveloping SGCs in the neuron-SGC and SGC-SGC communication that contribute to neuronal sensitization and pathologic pain. This complements previous observations of increased expression of substance P (SP), CGRP and nNOS primarily in the small DRG neurons following nerve injury29,30,31, which can then activate the respective receptors in the SGCs that envelop them32,33,34. Expression NMDA glutamate receptors are also reported in the small nociceptive neurons and their enveloping SGCs in the DRG 35,36,37.
When viewed in the light of these results, our findings suggest that glutamate released from the SGCs following nerve injury can intensely activate the nociceptive neurons they envelop, and also neighboring neurons via activation of the SGCs that envelop them. This can contribute to the spread of sensitization to multiple nociceptive and non-nociceptive primary sensory neurons, and ultimately to the development of pathologic pain 1,4,7.