Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2.

It has been reported that neuroinflammation occurs in the central nervous system (CNS) in patients with neuropathic pain, Alzheimer's disease and autism spectrum disorder. The 18-kDa translocator protein TSPO is used as an imaging target in positron emission tomography to detect neuroinflammation, and its expression is correlated with microglial activation. However, the mechanism underlying the transcriptional regulation of Tspo induced by inflammation is not clear. Here, we revealed that lipopolysaccharide (LPS) -induced Tspo expression was activated by the AP-1 complex in a mouse microglial cell line, BV-2. Knockdown of c-Fos and c-Jun, the components of AP-1, reduced LPS-induced Tspo expression. Furthermore, the enrichment of Sp1 in the proximal promoter region of Tspo was increased in the presence of LPS. In addition, the binding of histone deacetylase 1 (HDAC1) to the enhancer region, which contains the AP-1 site, was decreased by LPS treatment, but there were no significant differences in HDAC1 binding to the proximal promoter region with or without LPS. These results indicated that HDAC1 is involved not in the proximal promoter region but in the enhancer region. Our study revealed that inflammatory signals induce the recruitment of AP-1 to the enhancer region and Sp1 to the proximal promoter region of the Tspo gene and that Sp1 may regulate the basal expression of Tspo.

Phospholipase C-related inactive protein type-1 deficiency affects anesthetic electroencephalogram activity induced by propofol and etomidate in mice.

PURPOSE: The general anesthetics propofol and etomidate mainly exert their anesthetic actions via GABA A receptor (GABAA-R). The GABAA-R activity is influenced by phospholipase C-related inactive protein type-1 (PRIP-1), which is related to trafficking and subcellular localization of GABAA-R. PRIP-1 deficiency attenuates the behavioral reactions to propofol but not etomidate. However, the effect of these anesthetics and of PRIP-1 deficiency on brain activity of CNS are still unclear. In this study, we examined the effects of propofol and etomidate on the electroencephalogram (EEG). METHODS: The cortical EEG activity was recorded in wild-type (WT) and PRIP-1 knockout (PRIP-1 KO) mice. All recorded EEG data were offline analyzed, and the power spectral density and 95% spectral edge frequency of EEG signals were compared between genotypes before and after injections of anesthetics. RESULTS: PRIP-1 deficiency induced increases in EEG absolute powers, but did not markedly change the relative spectral powers during waking and sleep states in the absence of anesthesia. Propofol administration induced increases in low-frequency relative EEG activity and decreases in SEF95 values in WT but not in PRIP-1 KO mice. Following etomidate injection, low-frequency EEG power was increased in both genotype groups. At high frequency, the relative power in PRIP-1 KO mice was smaller than that in WT mice. CONCLUSIONS: The lack of PRIP-1 disrupted the EEG power distribution, but did not affect the depth of anesthesia after etomidate administration. Our analyses suggest that PRIP-1 is differentially involved in anesthetic EEG activity with the regulation of GABAA-R activity.

Anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice.

BACKGROUND: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. METHODS: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. RESULTS: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. CONCLUSIONS: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. SIGNIFICANCE: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

Effect of repeated oral administration of chondroitin sulfate on neuropathic pain induced by partial sciatic nerve ligation in mice.

We examined whether chondroitin sulfate (CS), a compound used to treat osteoarthritis and joint pain, is effective against partial sciatic nerve ligation (PSNL)-induced neuropathic pain. Repeated oral administration of CS (300 mg/kg, b.i.d. for 20 days) resulted in inhibition of tactile allodynia observed 21 days after PSNL. On day 21, phosphorylation of spinal p38 mitogen-activated protein kinase (MAPK) was attenuated by CS. CS also inhibited c-Fos upregulation in ipsilateral deep dorsal horn (laminae III-IV) neurons, which receive Aß-fiber afferent inputs. These findings suggest that CS attenuates PSNL-induced tactile allodynia by inhibiting spinal p38 MAPK phosphorylation and Aß-fiber activation.

Inhibitory effect of angiotensin (1-7) on angiotensin III-induced nociceptive behaviour in mice.

We have previously demonstrated that the intrathecal (i.t.) administration of angiotensin (Ang) II into mice produces a nociceptive behaviour consisting of scratching, biting and licking accompanied by the phosphorylation of p38 MAPK in the spinal cord, which was mediated through AT1 receptors. Both the p38 MAPK phosphorylation and subsequent nociceptive behaviour were attenuated by the i.t. co-administration of Ang (1-7), an N-terminal fragment of Ang II, that acted via Mas receptors. On the other hand, a C-terminal fragment of Ang II, namely Ang III, was also shown to induce a nociceptive behaviour by acting upon AT1 receptors on spinal astrocytes and neurons, and was found to be more potent than Ang II. However, the inhibitory effect of Ang (1-7) on the Ang III-induced nociceptive behaviour remains unclear. Thus, here we examined whether Ang (1-7) can attenuate the Ang III-induced nociceptive behaviour and activation of spinal p38 MAPK. The i.t. administration of Ang (1-7) (1-100fmol) dose-dependently attenuated the Ang III (1pmol)-induced nociceptive behaviour in mice. Moreover, the inhibitory effect of Ang (1-7) at a dose of 100fmol was prevented by A779 (30fmol), a Mas receptor antagonist. Western blot analysis showed that the phosphorylation of p38 MAPK induced by the i.t. administration of Ang III (1pmol) was also attenuated by Ang (1-7) (100fmol), and this inhibition was prevented by A779 (30fmol). Furthermore, we showed that in the lumbar superficial dorsal horn, Mas receptors are expressed in neurons and microglia but absent from astrocytes. Together, these results suggest that the i.t. administration of Ang (1-7) attenuates the nociceptive behaviour and accompanying p38 MAPK phosphorylation induced by Ang III, and that this effect is likely mediated through Mas receptors on spinal neurons.

Chondroitin sulfate attenuates formalin-induced persistent tactile allodynia.

We examined the effect of chondroitin sulfate (CS), a compound used in the treatment of osteoarthritis and joint pain, on the formalin-induced tactile allodynia in mice. A repeated oral administration of CS (300 mg/kg, b.i.d.) significantly ameliorated the formalin-induced tactile allodynia from day 10 after formalin injection. On day 14, the phosphorylation of spinal p38 MAPK and subsequent increase in c-Fos-immunoreactive dorsal lumbar neurons were attenuated by the repeated administration of CS. These findings suggest that CS attenuates formalin-induced tactile allodynia through the inhibition of p38 MAPK phosphorylation and subsequent up-regulation of c-Fos expression in the dorsal lumbar spinal cord.

Involvement of Spinal Angiotensin II System in Streptozotocin-Induced Diabetic Neuropathic Pain in Mice.

Renin-angiotensin system (RAS) activity increases under hyperglycemic states, and is thought to be involved in diabetic complications. We previously demonstrated that angiotensin (Ang) II, a main bioactive component of the RAS, might act as a neurotransmitter and/or neuromodulator in the transmission of nociceptive information in the spinal cord. Here, we examined whether the spinal Ang II system is responsible for diabetic neuropathic pain induced by streptozotocin (STZ). Tactile allodynia was observed concurrently with an increase in blood glucose levels the day after mice received STZ (200 mg/kg, i.v.) injections. Tactile allodynia on day 14 was dose-dependently inhibited by intrathecal administration of losartan, an Ang II type 1 (AT1) receptor antagonist, but not by PD123319, an AT2 receptor antagonist. In the lumbar dorsal spinal cord, the expression of Ang II, Ang converting enzyme (ACE), and phospho-p38 mitogen-activated protein kinase (MAPK) were all significantly increased on day 14 after STZ injection compared with vehicle-treated controls, whereas no differences were observed among AT1 receptors or angiotensinogen levels. Moreover, the increase in phospho-p38 MAPK was significantly inhibited by intrathecal administration of losartan. These results indicate that the expression of spinal ACE increased in STZ-induced diabetic mice, which in turn led to an increase in Ang II levels and tactile allodynia. This increase in spinal Ang II was accompanied by the phosphorylation of p38 MAPK, which was shown to be mediated by AT1 receptors.

Modified sympathetic nerve regulation in AKAP5-null mice.

Genetic analyses have revealed an important association between A-kinase anchoring proteins (AKAPs) and the intracellular calcium modulating system. AKAP5, also known as AKAP79/150, is an anchoring protein between PKA and voltage-dependent calcium channels, ryanodine receptor-2, phospholamban and other molecules. The aim of the present study was to elucidate the physiological importance of AKAP5 in the creation of cardiac rhythm using AKAP5-null mice. ECG analysis showed a normal sinus rhythm and a decreased responsiveness to isoproterenol in AKAP5-null mice compared with wild-type mice. Analysis of heart rate variability revealed that the R-R interval was unstable in AKAP5-null mutants and that the low-frequency components had decreased, indicating that the tonus of the sympathetic nervous system was affected. Furthermore, the atrium of the AKAP5-null mice showed a decreased positive inotropic response to isoproterenol, indicating the involvement of AKAP5 in a PKA-dependent pathway. Thus, our present study revealed that AKAP5 plays a significant role in the regulation of sympathetic nerve activities.

Involvement of p38 MAPK activation mediated through AT1 receptors on spinal astrocytes and neurons in angiotensin II- and III-induced nociceptive behavior in mice.

We have previously demonstrated the possibility that angiotensin (Ang) II and its N-terminal metabolite Ang (1-7) act as neurotransmitters and/or neuromodulators in the spinal transmission of nociceptive information. Ang III, which is a C-terminal metabolite of Ang II, can also act on AT1 receptors, but its role in spinal nociceptive transmission remains unclear. Therefore, we examined the role of Ang III on the spinal nociceptive system in comparison with that of Ang II. Intrathecal (i.t.) administration of Ang III into mice produced a nociceptive behavior, which was dose-dependently inhibited by the co-administration of the AT1 receptor antagonist losartan and the p38 MAPK inhibitor SB203580, but not by the AT2 receptor antagonist PD123319, MEK1/2 inhibitor U0126 and JNK inhibitor SP600125. In addition, Ang III increased the phosphorylation of p38 MAPK in the dorsal lumbar spinal cord, which was inhibited by losartan. These effects were similar to those of observed with Ang II. The nociceptive behavior produced by Ang II or III was also attenuated by the administration of the astrocytic inhibitor L-α-aminoadipic acid, but not by the microglial inhibitor minocycline. Double immunohistochemical staining showed that spinal AT1 receptors were expressed on neurons and astrocytes, and that i.t. administration of either Ang II or III phosphorylated p38 MAPK in both spinal astrocytes and neurons. These results indicate that Ang III produces nociceptive behavior similar to Ang II, and suggest that the phosphorylation of p38 MAPK mediated through AT1 receptors on spinal astrocytes and neurons contributes to Ang II- and III-induced nociceptive behavior.

Liver hydrolysate attenuates the sickness behavior induced by concanavalin A in mice.

Liver hydrolysate (LH) is used as a pharmaceutical agent in Japan, to enhance liver function. However, the effects of LH on sickness behavior are unknown. This study investigated the effect of LH on sickness behaviors, such as concanavalin A (ConA)-induced reduction of locomotor activity. ConA treatment significantly decreased locomotor activity. The striatal tyrosine hydroxylase (TH) levels were also significantly decreased following ConA treatment. The decreased locomotor activity and TH levels were significantly reversed by LH treatment. LH may prove beneficial for preventing sickness behavior following ConA treatment, at least in part, by activating TH in the striatum.

The intrathecal administration of losartan, an AT1 receptor antagonist, produces an antinociceptive effect through the inhibiton of p38 MAPK phosphorylation in the mouse formalin test.

We have recently reported that an intrathecal (i.t.) administration of angiotensin II (Ang II) into mice induces a nociceptive behavior accompanied by the activation of p38 MAPK signaling via AT1 receptors (Nemoto et al., 2013, Mol. Pain 9, 38). These results suggested that Ang II participates in the facilitation of nociceptive transmission in the spinal cord. In the present study, we used formalin test to examine the effect of i.t.-administered losartan, an AT1 receptor antagonist, and determine whether Ang II acts as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information. When administered i.t. 5 min before the injection of a 2% formalin solution into the plantar surface of the hindpaw, losartan (30-100 nmol) produced a dose-dependent and significant antinociceptive effect during both the first and second phases of the test. In the superficial dorsal horn of the spinal cord (laminae I and II), the fluorescence intensities for Ang II and phospho-p38 MAPK were both significantly increased on the ipsilateral side 3 min after the injection of formalin compared to saline-treated controls. Moreover, the increase of phospho-p38 MAPK fluorescence intensity was significantly inhibited by the i.t. administration of losartan (54.8 nmol) 5 min prior to formalin. These results indicate that losartan produces an antinociceptive effect through the inhibition of p38 MAPK phosphorylation in the mouse formalin test and that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.