Hiroki Imbe

Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly.

Daily restraint for 3 weeks was shown to atrophy dendrites of hippocampal pyramidal neurons in rats. Brain-derived neurotrophic factor (BDNF), which maintains neuronal survival and morphology, has been shown to decrease in response to acute stress. Plasma glucocorticoid (GC) and serotonergic projections from the raphe nuclei play major roles in reducing BDNF synthesis in the hippocampus. We investigated BDNF mRNA levels there, together with plasma GC levels, GC receptors in the hippocampus/hypothalamus and 5-HT synthesizing enzyme, tryptophan hydroxylase in the raphe nuclei, in animals chronically stressed for 1-3 weeks, using in situ hybridization and immunohistochemistry. In these animals, BDNF mRNA levels were significantly decreased in the hippocampus after 6 h of restraint, but the ability of restraint to reduce BDNF synthesis seemed less robust than that seen in acute stress models. HPA axis response to stress in these animals assessed by plasma GC levels was delayed and sustained, and the GC receptor in the paraventricular hypothalamic nucleus was increased at 1 week. Tryptophan hydroxylase immunoreactivity was increased in the median raphe nucleus at 2-3 weeks. Repetitive stress-induced reduction of BDNF may partly contribute to the neuronal atrophy/death and reduction of hippocampal volume observed both in animals and humans suffering chronic stress and/or depression.

Stress-induced hyperalgesia: animal models and putative mechanisms.

Stress has been shown to affect brain activity and promote long-term changes in multiple neural systems. A variety of environmental and/or stressful stimuli have been shown to produce analgesia, a phenomenon often referred to as stress-induced analgesia (SIA). However, acute and chronic stresses also produce hyperalgesia in various behavioral tests. There are now several animal models in which stress enhances nociceptive responses. The dysfunction of the hypothalamo-pituitary-adrenocortical axis (HPA axis) and multiple neurotransmitter systems in the central nervous system (CNS), including endogenous opioid, serotonergic and noradrenergic systems, has been reported. These stress-induced hyperalgesia models may contribute to a better understanding of chronic pain and provide a more rational basis for drug therapies in a variety of pain syndromes.

Persistent fos protein expression after orofacial deep or cutaneous tissue inflammation in rats: Implications for persistent orofacial pain

This study was designed to systematically examine the effects of persistent orofacial tissue injury on prolonged neuronal activation in the trigeminal nociceptive pathways by directly comparing the effects of orofacial deep vs. cutaneous tissue inflammation on brainstem Fos protein expression, a marker of neuronal activation. Complete Freunds adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. Rats were perfused 2 hours, 24 hours, 3 days, or 10 days following CFA injection. The TMJ and PO inflammation‐induced Fos expression paralleled the intensity and course of inflammation over the 10‐day observation period, suggesting that the increase in intensities and persistence of Fos protein expression may be associated with a maintained increase in peripheral input. Compared to PO CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a greater Fos expression. In TMJ‐ but not in PO‐inflamed rats, Fos‐like immunoreactivity (LI) spread from superficial to deep upper cervical dorsal horn as the inflammation persisted and there was a dominant ipsilateral Fos‐labeling in the paratrigeminal nucleus. Common to TMJ and PO inflammation, Fos‐LI was induced in the trigeminal subnuclei interpolaris and caudalis, C1–2 dorsal horn, and other medullary nuclei. Substantial bilateral Fos‐LI was found in the interpolaris‐caudalis trigeminal transition zone. Further analysis revealed that Fos‐LI in the ventral transition zone was equivalent bilaterally, whereas Fos‐LI in the dorsal transition zone was predominantly ipsilateral to the inflammation. The differential induction of Fos expression suggests that an increase in TMJ C‐fiber input after inflammation and robust central neuronal hyperexcitability contribute to persistent pain associated with temporomandibular disorders. J. Comp. Neurol. 412:276–291, 1999.

5-HT2A receptor subtype in the peripheral branch of sensory fibers is involved in the potentiation of inflammatory pain in rats

&NA; One of the major serotonin (5‐HT) receptor subtypes expressed in the rat dorsal root ganglion (DRG) neurons is the 5‐HT2A receptor. We have previously shown that 5‐HT2A receptors in the peripheral sensory terminals are responsible for 5‐HT‐induced pain and hyperalgesia. In the present study, we characterized neurons expressing 5‐HT2A receptors in the rat DRG neurons by means of in situ hybridization, immunohistochemistry, reverse transcription‐polymerase chain reaction (RT‐PCR) and behavioral tests. In situ hybridization on consecutive sections revealed that 5‐HT2A receptor mRNA is colocalized with calcitonin‐gene related peptide (CGRP) mRNA (100/104; 96.2%) but not with c‐Ret mRNA (1/115; 0.9%). Signals for 5‐HT2A receptor mRNA were found in 9.4±2.2% of normal DRG (L5) neurons, most of which were small to medium in size. Four days of complete Freunds adjuvant‐induced inflammation of the hindpaw doubled the incidence of 5‐HT2A receptor mRNA‐expressing neurons to 19.3±2.8%. The level of 5‐HT2A receptor mRNA in DRGs of normal and various pathological conditions was then determined by RT‐PCR. The level was up‐regulated by peripheral inflammation, but not by axotomy or chronic constriction of the peripheral nerve. Systemic administration of 5‐HT2A receptor antagonist (Sarpogrelate HCI) produced analgesic effects on thermal hyperalgesia caused by peripheral inflammation, but failed to attenuate thermal hyperalgesia in chronic constriction injury model. These findings suggest that 5‐HT2A receptors are mainly expressed in CGRP‐synthesizing small DRG neurons and may be involved in the potentiation of inflammatory pain in the periphery.

The effects of acute and chronic restraint stress on activation of ERK in the rostral ventromedial medulla and locus coeruleus

&NA; Extracellular signal‐regulated kinase (ERK) is a key molecule in numerous cellular and physiological processes in the CNS. Exposure to stressors causes substantial effects on the perception and response to pain. The rostral ventromedial medulla (RVM) and the locus coeruleus (LC) play crucial roles in descending pain modulation system. In the present study, the activation of ERK in the RVM and the LC in rats following acute and chronic restraint stress was examined in order to characterize the mechanisms underlying stress induced analgesic and hyperalgesic responses. Rats were stressed by restraint 6 h daily for 3 weeks. The acute and chronic restraint stresses produced analgesic and hyperalgesic reactions, respectively, to thermal stimuli applied to the tail. The phospho‐ERK‐immunoreactive (p‐ERK‐IR) neurons were observed in the nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis pars alpha (GiA) and LC. In the RVM, the number of p‐ERK‐IR neurons per section in the 3‐week restraint rats (14.3±1.2) was significantly higher than that in the control rats (8.9±0.7) [P<0.01]. About 75% of p‐ERK‐IR neurons in the RVM in the 3‐week restraint rats were serotonergic neurons. Protein levels of tryptophan hydroxylase were significantly increased in the RVM region in the 3‐week restraint rats. On the other hand, the chronic restraint stress significantly decreased p‐ERK‐IR in the LC [P<0.05]. These findings suggest that chronic restraint stress‐induced activation of ERK in the RVM and the suppression in the LC may be involved in the modulation of the pain threshold by chronic stress.

Orofacial Deep and Cutaneous Tissue Inflammation and Trigeminal Neuronal Activation

A rat model has been developed to characterize the responses of brainstem trigeminal neurons to orofacial deep and cutaneous tissue inflammation and hyperalgesia. Complete Freund’s adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. The TMJ and PO inflammation resulted in orofacial behavioral hyperalgesia and allodynia that peaked within 4–24 h and persisted for at least 2 weeks. Compared to cutaneous CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a selective upregulation of preprodynorphin mRNA in the trigeminal spinal complex, an enhanced medullary dorsal horn hyperexcitability, and a greater trigeminal Fos protein expression, a marker of neuronal activation. The Fos-LI induced by TMJ inflammation persisted longer, was more intense, particularly in the superficial laminae, and more widespread rostrocaudally. Thus, the inflammatory irritant produces a stronger effect in deep than in cutaneous orofacial tissue. As there is heavy innervation of the TMJ by unmyelinated nerve endings, a strong nociceptive primary afferent barrage is expected following inflammation. An increase in TMJ C-fiber input after inflammation and strong central neuronal activation may initiate central hyperexcitability and contribute to persistent pain associated with temporomandibular disorders. Since deep inputs may be more effective in inducing central neuronal excitation than cutaneous inputs, greater sensory disturbances may occur in pain conditions involving deep tissues than in those involving cutaneous tissues.

Masseteric inflammation-induced Fos protein expression in the trigeminal interpolaris/caudalis transition zone: contribution of somatosensory–vagal–adrenal integration

The effects of vagotomy and adrenalectomy on the expression of Fos protein in brainstem neurons following the inflammation of masseter muscle were examined in order to differentiate the Fos activation related to nociceptive processing in contrast to that due to somatoautonomic processing. The inflammation was induced by a unilateral injection of complete Freunds adjuvant (CFA) into the masseter muscle under methohexital anesthesia after a small skin-cut (S-cut). After the CFA injection, Fos positive neurons were identified in bilateral spinal trigeminal nucleus (VSP), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM) and inferior medial olivary nucleus (IOM). At the level of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition zone, there was a selective induction of Fos-like immunoreactivity (LI) in the VSP and NTS, when compared to control rats (anesthesia with or without S-cut). A major portion of the Fos-LI in the VSP at the level of the caudal Vc was apparently activated by S-cut. Bilateral adrenalectomy or a unilateral vagotomy resulted in a selective reduction of inflammation-induced Fos-LI in the VSP at the Vi/Vc transition zone (P<0.05) and NTS (P<0.05), but had less effect on Fos-LI in the caudal Vc. These results suggest that the inflammation of the masseter muscle, an injury of orofacial deep tissue, results in a widespread change in neuronal activity in the VSP and NTS that depends in part on the integrity of the adrenal cortex and vagus. Thus, in addition to somatotopically organized nociceptive responses, orofacial deep tissue injury also is coupled to somatovisceral and somatoautonomic processing that contribute to central neural activation.

Blockade of peripheral 5HT3 receptor attenuates the formalin-induced nocifensive behavior in persistent temporomandibular joint inflammation of rat

The role of peripheral 5HT3 receptors in the orofacial nocifensive behavior induced by the injection of formalin into masseter muscle was evaluated. The behavioral activities evoked by the formalin injection exhibited a biphasic response in the rats with or without temporomandibular joint (TMJ) inflammation (CFA group or non-CFA group). The orofacial nocifensive behavioral activity was enhanced after TMJ inflammation. Systemic administration of tropisetron, 5HT3 receptor antagonist, reduced the nocifensive behavioral activities in the late phase of orofacial formalin test in CFA group, but not in non-CFA group. Local administration of tropisetron into the masseter muscle in CFA group, but not in non-CFA group also attenuated the behavioral activities in the late phase. Unexpectedly, low dose of local tropisetron reduced the nocifensive behavioral activities in the early phase of orofacial formalin test in CFA group. These data suggest that induction of TMJ inflammation causes the elevation of the orofacial nocifensive behavioral activities evoked by formalin injection into masseter muscle, and that peripheral 5HT3 receptors may play a critical role in nociception and the transmission of orofacial pain.

Chronic restraint stress decreases glial fibrillary acidic protein and glutamate transporter in the periaqueductal gray matter.

Stress affects brain activity and promotes long-term changes in multiple neural systems. Exposure to stressors causes substantial effects on the perception and response to pain. In several animal models, chronic stress produces lasting hyperalgesia. Postmortem studies of stress-related psychiatric disorders have demonstrated a decrease in the number of astrocytes and the level of glial fibrillary acidic protein (GFAP), a marker for astrocyte, in the cerebral cortex. Since astrocytes play vital roles in maintaining neuroplasticity via synapse maintenance and secretion of neurotrophins, impairment of astrocytes is thought to be involved in the neuropathology. In the present study we examined GFAP and excitatory amino acid transporter 2 (EAAT2) protein levels in the periaqueductal gray matter (PAG) after subacute and chronic restraint stresses to clarify changes in descending pain modulatory system in the rat with stress-induced hyperalgesia. Chronic restraint stress (6h/day for 3 weeks), but not subacute restraint stress (6h/day for 3 days), caused a marked mechanical hypersensitivity and aggressive behavior. The chronic restraint stress induced a significant decrease of GFAP protein level in the PAG (32.0 ± 8.9% vs. control group, p<0.05). In immunohistochemical analysis the remarkable decrease of GFAP was observed in the ventrolateral PAG. The EAAT2 protein level in the 3 weeks stress group (79.6 ± 6.8%) was significantly lower compared to that in the control group (100.0 ± 6.1%, p<0.05). In contrast there was no significant difference in the GFAP and EAAT2 protein levels between the control and 3 days stress groups These findings suggest a dysfunction of the PAG that plays pivotal roles in the organization of strategies for coping with stressors and in pain modulation after chronic restraint stress.

Selective upregulation of the flip-flop splice variants of AMPA receptor subunits in the rat spinal cord after hindpaw inflammation.

Glutamate receptors are involved in spinal nociceptive transmission and the development of persistent inflammatory hyperalgesia. It is unclear, however, whether there are changes in glutamate receptor gene expression associated with tissue injury. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to examine the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor gene expression in the rat spinal cord by inflammation. Inflammation was introduced into the hindpaw by intraplantar injection of 0.2 ml of complete Freunds adjuvant (CFA). At 2 h-14 d after inflammation, total RNAs from L4,5 spinal cord were used for RT-PCR with primers targeted at eight flip-flop splice variants of the AMPA receptor subunits. It was found that the GluR1-flop mRNA was up-regulated at 2 h-5 h (P<0.05), down-regulated at 3 d (P=0.05), and returned to control levels at 7 d following inflammation. The GluR2-flip and GluR3-flop mRNAs were up-regulated at 5 h-1 d (P<0.05) and returned to control levels at 3 d after inflammation. The GluR1-flip mRNA was not detected in the samples and the mRNAs for other splice variants did not exhibit significant changes. Immunocytochemical analysis of GluR1 and GluR2 subunits indicate that the protein translation products of these subunits were also increased in the spinal cord. These results demonstrate an increased expression of AMPA receptor subunits that correlates with the acute phase of CFA-induced inflammation and hyperalgesia. Selective changes in the expression of the flip-flop splice variants of the AMPA receptor suggest a reorganization of the composition of the AMPA receptor complex and its involvement in the development of inflammatory hyperalgesia.