In this study, we initially investigated cellular changes in trigeminal neurons and glia in response to GSE supplemented diets. We chose to study the effect of MegaNatural-BP GSE, a highly purified extract from Vitis vinifera seeds containing >90% polyphenols as gallic acid equivalents . Inclusion of this product in the diet of mice was reported to significantly attenuate cognitive deterioration and Aβ oligomerization. Importantly, the concentration of GSE shown to cause changes in Aβ oligomerization was identical to the amount used in our study and shown to suppress inflammatory responses caused by prolonged stimulation of trigeminal nerves. GSE supplemented diets significantly increased the basal expression of MKP-1 in both neurons and glia within the trigeminal ganglia, as well as in neurons and glia in the TNC. MKP-1 is reported to dephosphorylate, and therefore, inactivate several MAP kinases including p38, JNK, and ERK, in a concentration dependent manner . Thus, increased expression of MKP-1 in trigeminal ganglia and the TNC would function to control MAP kinase activity in response to trigeminal nerve activation. In support of this notion, we found that CFA stimulated levels of active p38 in trigeminal ganglia and TNC were significantly repressed in animals receiving GSE when compared to control animals. Importantly, MAP kinases such as p38, are implicated in peripheral and central sensitization, and blocking MAP kinases with specific inhibitors is reported to suppress nociceptive responses and sensitization [15, 37, 38]. To our knowledge, this is the first study to provide evidence that GSE can lead to sustained, elevated levels of MKP-1, an enzyme that plays a fundamental role in regulating the activity of MAP kinases and thus, controlling development of peripheral and central sensitization.
Our finding that natural products such as GSE increases MKP-1 expression is in agreement with our previous result that demonstrated animals fed an enriched diet containing 1% or 10% cocoa exhibited elevated levels of MKP-1 . Interestingly, the anti-inflammatory effects of dexamethasone have been shown to be meditated, at least to some degree, by inducing a sustained expression of MKP-1 . Furthermore, the anti-nociceptive effects of opioids are reported to involve increased expression of MKP-1 . Thus, it appears that daily consumption of GSE or cocoa may function to block inflammatory and nociceptive responses in the trigeminal ganglion by a similar cellular mechanism as has been reported for glucocorticoids and opioids.
Another interesting finding from our study was that dietary GSE decreases basal expression of the MAP kinase-responsive gene CGRP, while increasing expression of GLAST in the TNC. CGRP is a multifunctional neuropeptide known to promote inflammatory and nociceptive responses and is implicated in TMJ pathology . In response to trigeminal neuron activation, peripheral CGRP release promotes an inflammatory response within the joint capsule, while central release of CGRP mediates enhanced nociception and central sensitization. CGRP is thought to contribute to nociception and central sensitization by its stimulatory effects on second order neurons and glia within the TNC [42, 43]. For example, CGRP release causes upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor proteins , prolongs activity of substance P by preventing its degradation , and increases levels of the purinegic receptor P2X3
. In addition, CGRP via activation of protein kinase A (PKA) is proposed to modulate activity of (N-methyl-D-aspartic acid) NMDA receptors thereby regulating glutamate excitation of second order nociceptive neurons . Thus, decreased levels of CGRP are likely to reduce the stimulatory effects of glutamate, substance P, and ATP on second order neurons that promote nociception [25, 47]. Significantly, we also found that GSE caused increased expression of the glutamate transporter GLAST that functions to regulate the amount of extracellular glutamate. Sustained GLAST expression in astrocytes leads to reduced glutamate levels, thus decreasing the excitability state of neurons and minimizing spontaneous activation . To our knowledge, this is the first example of using natural products as dietary supplements to increase GLAST expression. Taken together, our findings provide evidence that GSE functions to regulate levels of proteins known to play important roles in regulating spinal neuronal and glial excitability by decreasing CGRP expression and increasing GLAST expression.
In our study, we observed increased expression of P-p38 in trigeminal ganglia neurons, and neurons and microglia in the TNC in response to CFA injection. Increased levels of P-p38 are thought to contribute to the development of peripheral and central sensitization [10, 16], a process characteristic of TMJ pathology, by increasing the synthesis and release of proinflammatory cytokines [49, 50]. Chronic stimulation by CFA is a well-established model for TMJ pathology and when injected into the joint capsule results in a long-lasting inflammatory event and prolonged activation of nociceptive neurons [9, 51, 52]. Importantly, we found that the stimulatory effects of CFA on P-p38 expression in trigeminal ganglia and TNC were significantly repressed in animals receiving GSE. We propose that the basal increase in MKP-1 mediated by dietary GSE is responsible, at least in part, for the repressed expression of P-p38 in trigeminal ganglia and TNC neurons and glia in response to CFA since MKP-1 is a primary regulator of p38 activity . Taken together, GSE repression of P-p38 is likely to prevent development of a persistent pain state in response to CFA, which is used as a model of TMJ pathology.
We found that bilateral injection of CFA caused enhanced activity of astrocytes and microglia and that pretreatment with GSE was sufficient to repress the stimulatory effect of CFA on these glial cells. Our results with respect to CFA are in agreement with previous studies that reported the inflammatory response to CFA injection increased activation of astrocytes and microglia, as demonstrated by increases in the expression of GFAP and OX-42, respectively [5, 9, 53]. While once thought to play mainly a supportive role for neurons, it is now known that astrocytes and microglia modulate the activation state of neurons and contribute to hypersensitivity and the chronification of pain states . Under normal conditions, both microglia and astrocytes are relatively quiescent. However, in response to noxious/inflammatory stimuli these cells release proinflammatory cytokines leading to activation of nearby neurons and other glia [55, 56]. Interestingly, GSE is able to repress the stimulatory effects of CFA in both astrocytes and microglia as demonstrated by reduced levels of GFAP, a structural protein and a marker of astrocyte activation, and OX-42, a marker of activated microglia [57, 58]. Enhanced cellular activity of both astrocytes and microglia in the trigeminal system have been implicated in the development of persistent pain states . Thus, it appears that GSE functions to modulate cellular responses to inflammatory stimuli by inhibiting glial cell activation, which in turn, would decrease the excitability state of nociceptive neurons in the TNC.