Chen-Yu Chiang

Astroglia in Medullary Dorsal Horn (Trigeminal Spinal Subnucleus Caudalis) Are Involved in Trigeminal Neuropathic Pain Mechanisms

The aim of this study was to investigate whether astroglia in the medullary dorsal horn (trigeminal spinal subnucleus caudalis; Vc) may be involved in orofacial neuropathic pain following trigeminal nerve injury. The effects of intrathecal administration of the astroglial aconitase inhibitor sodium fluoroacetate (FA) were tested on Vc astroglial hyperactivity [as revealed by glial fibrillary acid protein (GFAP) labeling], nocifensive behavior, Vc extracellular signal-regulated kinase phosphorylation (pERK), and Vc neuronal activity in inferior alveolar nerve-transected (IANX) rats. Compared with sham-control rats, a significant increase occurred in GFAP-positive cells in ipsilateral Vc at postoperative day 7 in IANX rats, which was prevented following FA administration. FA significantly increased the reduced head withdrawal latency to high-intensity heat stimulation of the maxillary whisker pad skin in IANX rats, although it did not significantly affect the reduced escape threshold to low-intensity mechanical stimulation of the whisker skin in IANX rats. FA also significantly reduced the increased number of pERK-like immunoreactive cells in Vc and the enhanced Vc nociceptive neuronal responses following high-intensity skin stimulation that were documented in IANX rats, and glutamine administration restored the enhanced responses. These various findings provide the first documentation that astroglia is involved in the enhanced nociceptive responses of functionally identified Vc nociceptive neurons and in the associated orofacial hyperalgesia following trigeminal nerve injury.

Involvement of glia in central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn)

Central sensitization is a crucial mechanism underlying the increased excitability of nociceptive pathways following peripheral tissue injury and inflammation. We have previously demonstrated that the small-fiber excitant and inflammatory irritant mustard oil (MO) applied to the tooth pulp produces glutamatergic- and purinergic-dependent central sensitization in brainstem nociceptive neurons of trigeminal subnucleus caudalis (Vc). Recent studies have implicated both astrocytes and microglia in spinal nociceptive mechanisms, showing, for example, that inhibition of spinal astroglial metabolism or spinal microglial p38MAPK activation can attenuate hyperalgesia in inflammatory pain models but have not tested effects of glial inhibitors on central sensitization in functionally identified spinal nociceptive neurons. The aim of the present study was to determine whether glial cells are involved in the MO-induced central sensitization in Vc nociceptive neurons, by examining the effects of intrathecally applied SB203580 (SB), an inhibitor of p38MAPK, and fluoroacetate (FA), an inhibitor of the astroglial metabolic enzyme aconitase. During continuous superfusion of phosphate-buffered saline over Vc, MO application to the pulp-induced central sensitization in Vc nociceptive neurons reflected in significant increases in cutaneous mechanoreceptive field (RF) size and responses to noxious mechanical stimuli and a decrease in mechanical activation threshold. The i.t. application of SB or FA markedly attenuated the MO-induced increases in pinch RF size and responses to noxious stimuli and the decrease in activation threshold. Neither SB nor FA application significantly affected the baseline (i.e., pre-MO application) RF and response properties. These results suggest that glial metabolic processes are important in the development of Vc central sensitization.

Astroglial Glutamate–Glutamine Shuttle Is Involved in Central Sensitization of Nociceptive Neurons in Rat Medullary Dorsal Horn

Growing evidence suggests that astroglia are involved in pain states, but no studies have tested their possible involvement in modulating the activity of nociceptive neurons per se. This study has demonstrated that the central sensitization induced in functionally identified nociceptive neurons in trigeminal subnucleus caudalis (the medullary dorsal horn) by application of an inflammatory irritant to the rats tooth pulp can be significantly attenuated by continuous intrathecal superfusion of methionine sulfoximine (MSO; 0.1 mm), an inhibitor of the astroglial enzyme glutamine synthetase that is involved in the glutamate–glutamine shuttle. Simultaneous superfusion of MSO and glutamine (0.25 mm) restored the irritant-induced central sensitization. In control experiments, superfusion of either MSO or glutamine alone, or vehicle, did not produce any significant changes in neuronal properties. These findings suggest that the astroglial glutamate–glutamine shuttle is essential for the initiation of inflammation-induced central sensitization but that inhibition of astroglial function may not affect normal nociceptive processing.

Role of Glia in Orofacial Pain

Several acute and chronic pain conditions in the face or mouth are very common, and some are unique to the orofacial region. However, the etiology and pathogenesis of most orofacial chronic pain conditions are unresolved, and they are difficult to diagnose and manage. This article provides a brief overview of the neural mechanisms underlying orofacial pain and then highlights recent findings indicating that nonneural cells, specifically satellite cells in the sensory ganglia and astroglia and microglia cells in the central nervous system, are important players in both acute and chronic inflammatory and neuropathic orofacial pain conditions and may offer new targets for management of these conditions.

Periaqueductal gray matter and nucleus raphe magnus involvement in anterior pretectal nucleus-induced inhibition of jaw-opening reflex in rats

In previous studies we have shown that electrical stimulation of the cortex or anterior pretectal nucleus (APT) inhibits the jaw-opening reflex (JOR). In the present study we investigated whether these effects are mediated by a relay in the periaqueductal gray matter (PAG) or rostroventromedial medulla (RVM). Experiments were performed on chloralose-urethane anesthetized rats. The JOR which was elicited by electrical stimulation of the mandibular incisor tooth was monitored by recording the evoked digastric muscle activity. Conditioning stimulation (20 ms train of 0.2 ms pulses at 400 Hz) was delivered to the facial area of the sensorimotor cortex, APT, PAG or nucleus raphe magnus (NRM) 50 ms prior to the test stimulus to the tooth that evoked the JOR. In addition, the effects of microinjections of glutamate into APT, PAG and NRM on the tooth-evoked JOR were also evaluated. The inhibition of the JOR by electrical and glutamate conditioning stimulation was found to be most potent for activation of the NRM and least potent for the APT. Local anesthetic (2% lidocaine, 0.3-0.6 microliters) block of the PAG could partially, significantly (P less than 0.05) and reversibly reduce both the APT and cortical-induced depression of the JOR. Lidocaine block of the ventromedial pons reversibly reduced the PAG, APT and cortical-induced inhibition of the JOR (P less than 0.05). Lidocaine block of the lateral RVM had powerfully (P less than 0.01) and reversibly reduced the PAG-induced inhibition, but had only a small effect (P less than 0.05) on the APT-induced inhibition and no significant effect on the cortical-induced inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of trigeminal subnucleus caudalis (medullary dorsal horn) in craniofacial nociceptive reflex activity

We have previously shown that an increase in electromyographic (EMG) activity of digastric (DIG) and masseter (MASS) muscles can be reflexly evoked by injection into the rats temporomandibular joint (TMJ) region of the small-fibre excitant and inflammatory irritant mustard oil (MO). Since the trigeminal (V) subnucleus caudalis (Vc, i.e. medullary dorsal horn) has traditionally been viewed as an essential brainstem relay site of nociceptive information from craniofacial tissues, an EMG study was carried out in 45 anaesthetized rats to determine if Vc is involved in the MO-evoked increases in jaw muscle EMG activity. The effects of histologically confirmed surgical or chemical lesions of Vc on this evoked EMG activity were tested in different groups of rats. MO injection into the left TMJ region of intact rats evoked bilateral increases in EMG activity of DIG and MASS which could be significantly reduced by surgical transection of the left caudal brainstem at the obex level; MO injection into the right TMJ region in these same rats still readily evoked increases in EMG activity. A sagittal section medial to Vc or transection at the level of the second cervical spinal segment did not produce any significant reduction in the reflexly evoked EMG activity. Neurones in Vc, as opposed to fibres of passage, appear to be important for the MO-evoked EMG activity, since injection into Vc of the neurotoxic chemical ibotenic acid significantly reduced the mustard oil-evoked EMG activity. The Vc also appears to play a role in the activation of contralateral V motoneurons, as evidenced by the activation of the contralateral DIG and MASS muscles by the injection of MO into the left TMJ region of intact rats and by the reduction of this evoked EMG activity in the contralateral DIG and MASS of rats with a surgical transection or ibotenic acid lesion of the left Vc. These findings suggest that Vc may be a critical element in the neural pathways underlying the reflex responses evoked bilaterally in DIG and MASS muscles by noxious stimulation of the TMJ region.

Responses of neurons in the rat thalamic nucleus submedius to cutaneous, muscle and visceral nociceptive stimuli

&NA; The findings of recent studies have suggested that nucleus submedius (Sm) may be an important thalamic relay for nociceptive information. The aim of the present electrophysiological study was to examine in greater detail the activity and response properties of neurons in the rat Sm in order to further evaluate this hypothesis. Single unit extracellular recordings from neurons histologically verified to be in Sm were obtained in urethane/chloralose‐anesthetized rats. Noxious but not innocuous mechanical stimulation elicited responses in 75% of the 204 neurons studied. Most (85%) of these neurons were excited, 10% were inhibited and a few neurons (5%) were excited by stimulation at some sites on the body and inhibited from other sites. The receptive fields were usually very large and bilateral. No marked differences were observed in the incidence, response type, or spontaneous activity of neurons located in dorsal, ventral, rostral or caudal parts of Sm. Most of these neurons (99 of 108, 92%) also responded to noxious heating and had a mean threshold of 47°C. The majority of the neurons (19 of 21, 90%) also responded to subcutaneous, intramuscular or intraperitoneal injections of noxious chemicals (formalin or hypertonic saline). The responses elicited by pinching skin or squeezing muscle were frequently facilitated by the subcutaneous or intramuscular injections of formalin. Single electrical stimuli delivered to the cutaneous receptive field rarely produced responses. However, short trains (15–25 msec trains of 200 Hz, 3 msec pulses at 5–10 mA) delivered repetitively elicited responses in 90% (n = 73) of the neurons. These responses appearing after repetitive stimulation frequently resembled the ‘wind‐up’ pattern observed in spinal cord dorsal horn. The conduction velocities of the primary afferents which elicited the Sm neuronal responses as estimated from the latency differences of responses elicited by stimulation at two points along the tail, were indicative of recruitment of A&dgr; and C fibers. These findings provide further support for the proposed role of Sm in thalamic nociceptive mechanisms.

Jaw electromyographic activity induced by the application of algesic chemicals to the rat tooth pulp

The aim of this study was to determine if the application of mustard oil (MO), a small-fiber excitant and inflammatory irritant, or other algesic chemicals (capsaicin, CAP, and bradykinin, BK) to the rat maxillary molar tooth pulp induces electromyographic (EMG) activity of the masseter and digastric muscles, and also to determine if endogenous opioid mechanisms may be involved in any documented EMG changes. Application of MO to the tooth pulp induced a significant increase in EMG activity of the ipsilateral masseter up to 30 min. The application of mineral oil to the pulp or MO application to the pulp-extirpated tooth did not induce any significant EMG increases. The application of CAP or BK to the pulp in contrast had much weaker effects on EMG activity of the jaw muscles. CAP produced a small but prolonged increase in masseter EMG activity, and BK induced a short-lasting increase in digastric EMG activity. The systemic administration of the opiate antagonist naloxone significantly reactivated (i.e. rekindled) the EMG response evoked by MO application to the pulp. Naloxone did not produce any such significant rekindling effect on EMG activity following CAP, BK or mineral oil application to the pulp or following MO application to the pulp-extirpated tooth. The MO, BK and especially CAP groups showed histological evidence of vasodilatation and polymorphonuclear leukocyte infiltration in the pulp tissue and a significant increase in plasma extravasation of Evans Blue dye, whereas mineral oil did not induce these changes. These findings suggest that pulp afferent inputs to the central nervous system evoked by BK. CAP and especially MO may induce enhanced jaw muscle activity. In addition, the naloxone data suggest that an opioid suppresive mechanism may be induced by the pulpal afferent inputs evoked by MO, and may serve to limit the jaw muscle activity elicited by these inputs.

Modulation of astroglial glutamine synthetase activity affects nociceptive behaviour and central sensitization of medullary dorsal horn nociceptive neurons in a rat model of chronic pulpitis.

Previous studies indicate that the astroglial glutamate–glutamine shuttle may be involved in acute pulpal inflammatory pain by influencing central sensitization induced in nociceptive neurons in the trigeminal subnucleus caudalis [the medullary dorsal horn (MDH)] by application of an inflammatory irritant to the rat tooth pulp. The aim of this study was to test if intrathecal application to the rat medulla of the astroglial glutamine synthetase inhibitor methionine sulfoximine (MSO) can influence the central sensitization of MDH nociceptive neurons and the animal’s associated behaviour that are manifested in a model of chronic pulpitis pain induced by exposure of a mandibular molar pulp. This model was found to be associated with nocifensive behaviour and enhanced reflex activity evoked by mechanical stimulation of the rat’s facial skin and with immunocytochemical evidence of astroglial activation in the MDH. These features were apparent for up to 28 days post‐operatively. During this post‐operative period, the nocifensive behaviour and enhanced reflex activity were significantly attenuated by intrathecal application of MSO (5 μL, 10 mM) but not by vehicle application. In electrophysiological recordings of nociceptive neuronal activity in the MDH, central sensitization was also evident in pulp‐exposed rats but not in intact rats and could be significantly attenuated by MSO application but not by vehicle application. These behavioural and neuronal findings suggest that the astroglial glutamate–glutamine shuttle is responsible for the maintenance of inflammation‐induced nocifensive behavioural changes and the accompanying central sensitization in MDH nociceptive neurons in this chronic pulpitis pain model.

Inhibitory effect of stimulation of the anterior pretectal nucleus on the jaw-opening reflex

The anterior pretectal nucleus (APT) has been recently implicated in sensorimotor integration and has been shown to have suppressive influences on tail flick behaviour and on nociceptive responses of spinal dorsal horn neurones in rats. The present study tested the effect of stimulation of the APT on the rats digastric jaw-opening reflex elicited by orofacial stimuli. Either ipsilateral or contralateral electrical stimulation at histologically confirmed sites within and immediately subjacent to the APT produced a suppression of the reflex that had an onset of 20-30 ms, peaked around 50 ms and lasted for 200-300 ms; in some cases, a brief period of reflex facilitation preceded the onset of inhibition and the inhibition was sometimes followed by a facilitatory period. No prolonged period of suppression induced by electrical stimulation was noted in these anaesthetized rats. The injection of monosodium glutamate at comparable sites within and subjacent to APT induced reflex suppression that lasted several minutes. These findings represent the first documentation of APT-induced modulation in the trigeminal sensorimotor system, but support recent evidence suggesting the involvement of APT in sensorimotor integration and modulation.