Eon-Jeong Shim

Involvement of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse formalin pain model.

In the present study, we investigated the role of phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMK-II) and phosphorylated extracellular signal-regulated protein kinase (pERK) in nociceptive processing at the spinal and supraspinal levels in the formalin subcutaneous induced mouse pain model. In the immunoblot assay, subcutaneous (s.c.) injection with formalin increased the pERK and pCaMK-IIalpha level in the spinal cord, and an immunohistochemical study showed that the increase of pERK and pCaMK-IIalpha immunoreactivity mainly occurred in the laminae I and II areas of the spinal dorsal horn. At the supraspinal level, although pERK was not changed in the hippocampus induced by formalin s.c. injection, pCaMK-IIalpha was increased in the hippocampus and hypothalamus by s.c. formalin injection, and an increase of pCaMK-IIalpha immunoreactivity mainly occurred in the pyramidal cells and the stratum lucidum/radiatum layer of the CA3 region of hippocampus and paraventricular nucleus of the hypothalamus. Moreover, pERK immunoreactivity in the hypothalamic paraventricular nucleus was also increased. The second phase of nociceptive behavior induced by formalin administered either i.t. or intracerebroventricularly (i.c.v.) was attenuated by PD98059 (ERK inhibitor) as well as KN-93(a CaMK-II inhibitor). On the other hand, the first phase of nociceptive behavior induced by formalin s.c. injection was not affected by i.t. KN-93. Our results suggest that pERK and pCaMK-II located at both the spinal cord and supraspinal levels are an important regulator during the nociceptive processes induced by formalin administered s.c. respectively.

The effect of single or repeated restraint stress on several signal molecules in paraventricular nucleus, arcuate nucleus and locus coeruleus.

The effect of single or repeated restraint stress on several signal molecules in the hypothalamus was studied in ICR mice. Single restraint stress was induced for 30, 60, and 120 min. A repeated restraint stress was induced for 2 h daily during four consecutive days, and then induced in the same time course on the fifth day. In the immunoblot assay, we observed that the signal molecules c-Fos, phosphorylated extracellular cell-regulated protein kinase (pERK), phosphorylated calcium/calmodulin dependent protein kinase II (pCaMKII) and phosphorylated cyclic-AMP response element binding protein (pCREB) in the hypothalamus were increased by single restraint, and the increased c-Fos and pERK levels were attenuated by repeated restraint stress. However, pCaMKII and pCREB levels were increased by both single and repeated restraint stress. We also observed in the immunohistochemistry study that immunoreactivities (IR) of these signal molecules were changed in paraventricular (PVN) and arcuate nuclei (ArcN) of the hypothalamus in accordance with immunoblot results. Furthermore, in confocal immunofluorescence, the pCaMKII and pCREB up-regulated by repeated restraint stress were co-localized within many neurons of PVN and ArcN. In addition, we found that c-Fos and pCaMKII IR in locus coeruleus (LC) were increased by single restraint, and were attenuated by repeated restraint stress. However, the pERK and pCREB IR were increased by both single and repeated restraint stress. The confocal study revealed that pERK and pCREB up-regulated by repeated restraint stress were co-localized within many neurons of LC. Our results suggest that single and repeated restraint stress differentially triggers the induction and phosphorylation of several signal molecules in the PVN, ArcN, and LC. In addition, single and repeated stress stimuli elicited the brain-region specific changes of signal molecules examined. Furthermore, the upstream signal molecule activating CREB may be also brain-region specific, especially in repeated stress stimuli.

Increase of phosphorylation of calcium/calmodulin-dependent protein kinase-II in several brain regions by substance p administered intrathecally in mice

In the present study, we investigated the role of phosphorylated calcium/calmodulin-dependent protein kinase-II (pCaMK-II) in nociceptive processing at the spinal and supraspinal levels in the substance P (SP)-induced mouse pain model. In the immunoblot assay, intrathecal (i.t.) injection with SP increased the pCaMK-II level in the spinal cord, and an immunohistochemical study showed that the increase of pCaMK-II immunoreactivity mainly occurred in the laminae I and II areas of the spinal dorsal horn. At the supraspinal level, pCaMK-II was increased in the hippocampus and hypothalamus by i.t. SP injection, and an increase of pCaMK-II immunoreactivity mainly occurred in the pyramidal cells and the stratum lucidum/radiatum layer of the CA3 region of hippocampus and paraventricular nucleus of the hypothalamus. Moreover, pCaMK-II immunoreactivity in the locus coelureus of the brain stem was also increased. The nociceptive behavior induced by SP administered either i.t. or intracerebroventricularly (i.c.v.) was attenuated by KN-93 (a CaMK-II inhibitor). Our results suggest that pCaMK-II located at both spinal cord and supraspinal levels is an important regulator during the nociceptive processes induced by SP administered i.t.

Effect of Aspirin and Acetaminophen on Proinflammatory Cytokine-Induced Pain Behavior in Mice

Aspirin (ASA) is a widely used oral analgesic which acts as an inhibitor of cyclooxygenase. Acetaminophen (ACET) is also an effective analgesic and may selectively inhibit brain prostaglandin synthetase. Various proinflammatory cytokines injected into the central nervous system show pain behavior. In the present study, the effects of orally administered ASA and ACET on pain behaviors induced by various proinflammatory cytokines were examined. At a dose of 100 mg/kg, ASA or ACET did not affect the pain behavior induced by TNF-α (100 pg), IL-1β (100 pg) or IFN-γ (100 pg) administered intrathecally. However, at doses of 200 and 300 mg/kg, ASA or ACET significantly and dose-dependently attenuated pain behavior induced by TNF-α, IL-1β or IFN-γ administered intrathecally. Our results suggest that orally administered ASA and ACET produce antinociception by inhibiting the nociceptive action of TNF-α, IL-1β or IFN-γ administered intrathecally.

Differential Modulatory Effects of Cholera Toxin and Pertussis Toxin on Pain Behavior Induced by TNF-a, lnterleukin-1β and Interferon- Injected Intrathecally

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in pro-inflammatory cytokine induced pain behaviors. Intrathecal injection of tumor necrosis factor-a (TNF-a; 100 pg), inter-leukin-1β (IL-1β; 100 pg) and interferon-γ (INF-γ; 100 pg) showed pain behavior. Intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 mg) attenuated pain behavior induced by TNF-α and INF-γ administered intrathecally. But intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 μg) did not attenuate pain behavior induced by IL-1β. On the other hand, intrathecal pretreatment with PTX further increased the pain behavior induced by TNF-a and IL-1 β administered intrathecally, especially at the dose of 0.5 μg. But intrathecal pretreatment with PTX did not affect pain behavior induced by INF-γ.Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play important roles in modulating pain behavior induced by pro-inflammatory cytokines administered spinally. Furthermore, TNF-α, IL-1 β and INF-γ administered spinally appear to produce pain behavior by different mechanisms.

Antinociceptive Effect of Nicotine in Various Pain Models in the Mouse

The antinociceptive effect of nicotine administered intracereboventricularly (i.c.v.) or intrathecally (i.t) in several pain models was examined in the present study. We found that i.t. treatment with nicotine (from 5 to 20 g) dose-dependently blocked pain behavior revealed during the second phase, but not during the first phase in the formalin test. In addition, i.c.v. treatment with nicotine (from 0.1 to 10 μg) dose-dependently attenuated pain behavior revealed during both the first and second phases. In addition to the formalin test, nicotine administered i.c.v. or i.t. attenuated acetic acid-induced writhing response. Furthermore, i.c.v. or i.t. administration of nicotine did not cause licking, scratching and biting responses induced by substance P, glutamate, TNF-α (100 pg), IL-1β (100 pg) and INF-γ (100 pg) injectied i.t. The antinociception induced by supraspinally-administered nicotine appears to be more effective than that resulting from spinally administered nicotine. Our results suggest that nicotine administration induces antinociception by acting on the central nervous system and has differing antinociceptive profiles according to the various pain models.

The differential effects of emotional or physical stress on pain behaviors or on c-Fos immunoreactivity in paraventricular nucleus or arcuate nucleus

Although many studies which explore on the interaction between stress and antinociception have been conducted, most of them do not divide stress into emotional stress (ES) and physical stress (PS). In the present study, we investigated the differential effects of ES or PS on pain behaviors or on c-Fos immunoreactivity (IR) in the paraventricular nucleus (PVN) or arcuate nucleus (ArcN) using electrical footshock-witness model. In addition, alteration of pain behaviors or c-Fos IR following stress repetition was examined. The electrical foot shock was applied to PS animal group in one chamber, whereas the witness animal group in another chamber without any electrical foot shock was regarded as an ES. In each group, either single (10 stimuli /10 min/1 day) or repeated stress for 5 consecutive days was applied. Our results suggest that ES and PS appear to play differential roles in the regulation of nociception produced by various types of pain stimuli (formalin, substance P, glutamate or pro-inflammatory cytokines) and on c-Fos IR in the PVN or ArcN. Moreover, such antinociceptive effect or c-Fos IR appears to be modified following stress repetition.

The intracerebroventricular kainic acid-induced damage affects animal nociceptive behavior.

In the present study, we examined nociceptive behaviors on various pain models after the pretreatment of kainic acid intracerebroventricularly. We found that intracerebroventricular administration of kainic acid shows significant neuronal damage on the hippocampal CA3 region in the brain slices stained with cresyl violet. Compared to the control group, intracerebroventricular pretreatment of kainic acid significantly attenuated nocifensive behaviors induced by intraplantar formalin (only in the 2nd phase), intrathecal glutamate, TNF-alpha or IL-1beta. However, nocifensive behaviors induced by intraperitoneal acetic acid (writhing test), intrathecal substance P or IFN-gamma were not affected by the pretreatment of kainic acid. These results suggest that (1) KA-induced alterations of nocifensive behaviors are related to the neuronal death of the hippocampal formation, especially CA3 pyramidal neurons and (2) nocifensive behaviors induced by formalin, acetic acid, SP, glutamate, and pro-inflammatory cytokines were modulated in a different manner.

Role of γ-aminobutyric acid B (GABAB) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of γ-aminobutyric acid B (GABAB) receptors in hippocampal cell death induced by KA (0.1 µg) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABAB receptors antagonist, 20 µg) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca2+ /calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABAB receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Role of nicotinic acetylcholine receptors in the regulation of kainic acid-induced hippocampal cell death in mice

Kainic acid (KA) is a well-known excitatory, neurotoxic substance. In mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the CA3 pyramidal neurons. In the present study, the possible role of nicotinic acetylcholine receptors (nAchRs) in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. Methyllycaconitine (MC; nAchRs antagonist, 20 microg) attenuated KA-induced CA3 pyramidal cell death. KA increased immunoreactivities (IRs) of phorylated extracellular signal-regulated kinase (p-ERK; at 30 min), p-CaMK II (at 30 min), c-Fos (at 2 h), c-Jun (at 2 h), glial fibrillary acidic protein (GFAP at 1 day), and the complement receptor type 3 (OX-42; at 1 day) in hippocampal area. MC attenuated selectively KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. Our results suggest that p-CaMK II may play as an important regulator responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Reactive astrocytes, which was meant by GFAP IR, and activated microglia, which was meant by OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA-induced excitotoxicity. Furthermore, it is implicated that niconitic receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.