This study was designed to investigate the mechanism of salivary dysfunction in an experimental periodontitis rat model and to examine the improvements in salivary secretion following treatment of the experimental periodontitis.
The aim of the present study was to clarify an involvement of growth-associated protein-43 (GAP-43) in the regeneration of primary afferent trigeminal ganglion (TG) neurons following inferior alveolar nerve transection (IANX). A larger number of GAP-43 immunoreactive (GAP-43 IR) TG neurons was observed in rats 3 d after IANX compared with sham rats. Growth-associated protein-43 IR TG neurons were also detected for 30 d after IANX, and the number of GAP-43 IR TG neurons was significantly higher in the IANX model until day 30. The relative number of large (>600 ?m2) GAP-43 IR TG neurons was significantly lower, whereas the relative number of small (<400 ?m2) GAP-43 IR TG neurons was significantly higher than that at day 0 until 30 d after IANX. To evaluate the functional recovery of damaged IAN, the jaw opening reflex (JOR), elicited by the electrical stimulation of the IAN, was measured before and after IANX. Jaw opening reflex occurrence was gradually increased and the relative threshold of electrical stimulation eliciting JOR was gradually decreased over the 30-d duration of the study. On day 30 after IANX, the JOR occurrence and relative JOR threshold were similar to those in sham rats. The present findings suggest that changes in the expression of GAP-43 in TG neurons after IANX are involved in regeneration and functional recovery of the transected IAN.
It is well known that oral inflammation causes tenderness in temporomandibular joints or masseter muscles. The exact mechanism of such an orofacial ectopic hyperalgesia remains unclear. Here, we investigated the functional significance of interaction of nerve growth factor (NGF) and transient receptor potential vanilloid 1 (TRPV1) in relation to heat hyperalgesia in the whisker pad skin caused by complete Freunds adjuvant (CFA) injection into the lower lip. CFA injection induced heat hyperalgesia of the ipsilateral whisker pad skin. Moreover, it leads to enhancement of spontaneous activity and heat responses in trigeminal ganglion (TG) neurons that was elicited by heat stimulation of the whisker pad skin. The heat hyperalgesia was dose-dependently reversed by intraperitoneal TRPV1 antagonist administration, also diminished by neutralizing anti-NGF antibody administration into the lower lip and intraganglionic administration of K252a, a tyrosine kinase receptor inhibitor. Nerve fibers in bundle of mandibular nerve and TG neurons that innervates the whisker pad skin and lower lip both expressed labeled NGF, which was administrated into the lower lip. Moreover, the NGF concentrations in ophthalmic-maxillary and mandibular divisions of the TG increased after CFA injection into the lower lip. The number of TRPV1-positive neurons that innervates the whisker pad skin and lower lip was increased after CFA injection into the lower lip, and this increase was annulled by anti-NGF administration. The present findings suggest that inflammation in the lower lip induces release of NGF that regulates TRPV1 expression in TG neurons. This TRPV1 overexpression may underlie ectopic heat hyperalgesia in the whisker pad skin.
Many phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) cells are expressed in the trigeminal spinal subnucleus caudalis (Vc), upper cervical spinal cord (C1-C2), nucleus tractus solitarii (NTS) and paratrigeminal nucleus (Pa5) after capsaicin injection into the whisker pad (WP), masseter muscle (MM), digastric muscle (DM) or sternohyoideus muscle (SM). The pERK-IR cells also showed NeuN immunoreactivity, indicating that ERK phosphorylation occurs in neurons. The pERK-IR cells were significantly reduced after intrathecal injection of MEK 1/2 inhibitor PD98059. The pERK-IR cells expressed bilaterally in the Vc and C1-C2 after capsaicin injection into the unilateral DM or SM, whereas unilaterally in the Vc and C1-C2 after unilateral WP or MM injection. After capsaicin injection into the WP or MM, the pERK-IR cell expression in the Vc was restricted rostrocaudally within a narrow area. However, the distribution of pERK-IR cells was more wide spread without a clear peak in the Vc and C1-C2 after capsaicin injection into the DM or SM. In the NTS, the unimodal pERK-IR cell expression peaked at 0-720?m rostral from the obex following capsaicin injection into WP, MM, DM or SM. In the ipsilateral Pa5, many pERK-IR cells were observed following capsaicin injection into the SM. The number of swallows elicited by distilled water administration was significantly smaller after capsaicin injection into the WP, MM or DM but not SM compared to that of vehicle-injected rats. Various noxious inputs due to the masticatory or swallowing-related muscle inflammation may be differentially involved in muscle pain and swallowing reflex activity.
The aim of this study is to clarify the neural mechanisms underlying orofacial pain abnormalities after cervical spinal nerve injury. Nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) expression and astroglial cell activation in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal dorsal horn (C1-C2) neurons were analyzed in rats with upper cervical spinal nerve transection (CNX).
By means of retrograde transneuronal transport of rabies virus, ascending multisynaptic pathways from the trigeminal ganglion (TG) to the anterior cingulate cortex (ACC) were identified in the rat. After rabies injection into an electrophysiologically defined trigeminal projection region of the ACC, transsynaptic labeling of second-order neurons via the medial thalamus (including the parafascicular nucleus) was located in the spinal trigeminal nucleus pars caudalis. Third-order neuron labeling occurred in the TG. Most of these TG neurons were medium- or large-sized cells giving rise to myelinated A? or A? afferent fibers, respectively. By contrast, TG neurons labeled transsynaptically from the orofacial region of the primary somatosensory cortex contained many small cells associated with unmyelinated C afferent fibers. Furthermore, the TG neurons retrogradely labeled with fluorogold injected into the mental nerve were smaller in their sizes compared to those labeled with rabies. Our extracellular unit recordings revealed that a majority of ACC neurons responded to trigeminal nerve stimulation with latencies of shorter than 20ms. Thus, somatosensory information conveyed to the ACC by multisynaptic ascending pathways derived predominantly from myelinated primary afferents (i.e., the medial nociceptive system) and may be used to subserve affective-motivational aspects of pain. Lack of overlap with the lateral nociceptive system is notable and suggests that the medial and lateral nociceptive systems perform separate and non-overlapping functions.
Increased expression of the transient receptor potential vanilloid 1 (TRPV1) channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP)--induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN) transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG) neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect dynamic changes in TRPV1 expression, in various pathological conditions.
To clarify whether changes to the cellular properties of sensory neurons occur after a brief culture, we compared the electrophysiological and immunohistochemical properties of rat trigeminal ganglion neurons. We compared these neurons after acute dissociation and after a 1-day culture under serum-free and neurotrophin-free conditions. In whole-cell patch-clamp recordings, the 1-day cultured neurons required a lower current threshold to induce an action potential in both small- and medium-sized neurons. Furthermore, the input resistance was higher in the medium-sized neurons after a 1-day culture compared to the acutely dissociated medium-sized neurons. Immunofluorescent studies demonstrated that both the translocation of the activating transcription factor 3 (ATF3) into the nucleus and the expression of a low threshold Na(+) channel (Na(v)1.3) were upregulated after 1-day of culture. However, in the acutely dissociated neurons, ATF3 translocation occurred at low levels, and Na(v)1.3 was not expressed. These electrophysiological and immunohistochemical changes after 1-day of culture were very similar to the reported changes that occur after nerve injury. Our study demonstrated that injury-like characteristics appear to be manifested in the 1-day cultured sensory neurons, which do not occur in acutely dissociated neurons. Overall, our results are relevant and will help when interpreting the results of studies examining dissociated sensory neurons in pain research.
Although recent evidence suggests that central glial hyperactivation is involved in cancer-induced persistent pain, the time course of this hyperactivation and the glial contribution to pain hypersensitivity remain unclear. The present study investigated the time-dependent spatial changes of microglial and astrocytic hyperactivation in the trigeminocervical complex, which consists of the medullary (MDH) and upper cervical (UCDH) dorsal horns, and pain-related behaviors in a rat facial cancer model in which Walker 256B-cells are inoculated into the vibrissal pad. In this model, the tumors grew within the vibrissal pad, from which sensory nerve fibers project into the MDH, but did not expand into the infraorbital region, from which fibers project into the UCDH. Nevertheless, mechanical allodynia and thermal hyperalgesia were observed not only in the vibrissal pad but also in the infraorbital region. Western blotting and immunofluorescence studies indicated that microglia were widely activated in the trigeminocervical complex on day 4 and gradually inactivated by day 11. In contrast, astrocytes were only activated in the MDH on day 4; the hyperactivation later expanded into the UCDH. Daily administration of the glial hyperactivation inhibitor propentofylline beginning on day 4 suppressed the glial hyperactivation on later days. Propentofylline treatment largely prevented allodynia/hyperalgesia in the infraorbital region beginning on day 5, although established allodynia/hyperalgesia in the vibrissal pad was less sensitive to the treatment. These results suggest that central glial hyperactivation, transient microglial hyperactivation and persistent astrocytic hyperactivation, contributes to the development of pain hypersensitivity but not to the maintenance of pain in this model.
Chronic pain often develops in the orofacial region after inferior alveolar nerve (IAN) injury. In animal models IAN injury often causes severe neuropathic pain-like behavior in the IAN-innervated region as well as the adjacent region that includes the whisker pad skin. However, the basis for the spreading of pain to adjacent facial areas after IAN injury is still unknown. In this study we determined if the transient receptor potential vanilloid 1 (TRPV1) was associated with altered nocifensive behavior evoked by stimulation of the whisker pad skin following IAN transection. Grooming behavior after capsaicin injection into the whisker pad region was significantly increased after IAN transection and the increase in the behavior was reversed by systemic administration of a TRPV1 antagonist. The number of phosphorylated extracellular signal-regulated kinase immunoreactive (IR) neurons in trigeminal spinal subnucleus caudalis and upper cervical spinal cord following capsaicin injection into the whisker pad region was significantly greater in IAN-transected rats than sham-operated rats. The number of TRPV1-IR trigeminal ganglion (TG) neurons innervating the whisker pad skin was also increased significantly after IAN transection. The present findings suggest that an increase in TRPV1 expression in TG neurons innervating the whisker pad skin after IAN transection may underlie the spreading of pain to the adjacent whisker pad skin.
The aims of this study were to investigate the involvement of dopamine (DA) in drinking behaviour related to body fluid balance. All experiments were performed in rats. Water intake induced by intracerebroventricular injection of angiotensin II (ANGII) was suppressed by co-injection of DA in a dose-dependent manner. RT-PCR revealed the presence of mRNAs for all known DA receptors, D1-D5, in the subfornical organ (SFO), a brain region that plays a key role in regulating drinking behaviour. Extracellular recordings and whole-cell patch-clamp recordings from SFO neurons showed that DA or the D4 selective agonist PD168077 inhibited spontaneous electrical activity. The D4 antagonist L745870 blocked DA-induced inhibition of spontaneous electrical activity in SFO neurons. Under conditions of synaptic blockade, the inhibitory effects of DA and PD168077 still remained, but the D2/D3 agonist quinpirole and the D1/D5 agonist SKF38393 had almost no effect on electrical activity. While DA induced excitation in a small number of neurons, these excitatory responses almost disappeared following synaptic blockade. All neurons with firing rates that were suppressed by DA were excited by ANGII. In voltage clamp mode, we found that DA and quinpirole, but not SKF38393, suppressed GABAergic miniature inhibitory post-synaptic currents. These results suggest that DA inhibits neuronal activity in ANGII-sensitive SFO neurons primarily through the postsynaptic D4 receptor subtype. This may be a cause of the suppression of ANGII-induced water intake by DA. In addition, the inhibitory DA responses in SFO neurons may be modulated by presynaptic suppression of GABAergic inhibitory inputs through D2/D3 receptor subtypes.
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