Hideo Iwasaka

Mechanisms of analgesic action of pulsed radiofrequency on adjuvant-induced pain in the rat: Roles of descending adrenergic and serotonergic systems

Pulsed radiofrequency (PRF) has been reported to be effective in the treatment of several types of pain. The mechanism of action, however, is not well known. In a recent study, the antinociceptive effects of acute thermal pain were shown to be mediated via descending pain inhibitory pathways. In this study we observed an analgesic effect of PRF treatment in an adjuvant induced inflammatory pain model in rats. In this model, sciatic nerves were treated with PRF at 37° and 42°, which inhibited hyperalgesia in the inflammatory groups when compared to RF and sham treatment. This effect was attenuated after intrathecal administration of the alpha2‐adrenoceptor antagonist yohimbine, the selective 5‐HT3 serotonin receptor antagonist MDL72222, and the non‐selective serotonin receptor antagonist methysergide. All three drugs were found to significantly inhibit the analgesic effect of PRF. The results suggest that the analgesic action of PRF involves the enhancement of noradrenergic and serotonergic descending pain inhibitory pathways.

Effects of an angiotensin-converting enzyme inhibitor on the inflammatory response in in vivo and in vitro models.

Objective:Sepsis remains a major health threat in intensive care medicine. The renin-angiotensin system (ACE) affects inflammatory responses. In addition, angiotensin-converting enzyme inhibitors act to ameliorate lung injury. To investigate whether the widely used ACE inhibitor enalapril, used to treat hypertension, could inhibit secretion of cytokines and high-mobility group box 1 (HMGB1) protein, thus reducing lung damage in a rat model of lipopolysaccharide (LPS)-induced sepsis. Design:Randomized, prospective animal study. Setting:University medical center research laboratory. Subjects:Male Wistar rats. Interventions:LPS was administered intravenously to rats, with or without intraperitoneal pretreatment with enalapril. In addition, mouse macrophage RAW264.7 cells were stimulated with LPS, with and without simultaneous enalapril treatment. Measurements and Main Results:Histologic examination showed marked reduction of interstitial congestion, edema, inflammation, and hemorrhage in lung tissue harvested 12 hours after treatment with both agents compared with LPS administration alone. Plasma concentration of angiotensin II was strongly induced by LPS; this induction was inhibited by the enalapril pretreatment. Likewise, LPS-induced secretion of proinflammatory cytokines and HMGB1 protein was inhibited by enalapril. The presence of HMGB1 protein in the lung was examined directly by immunohistochemistry; the number of stained cells was significantly lower in LPS-treated animals that also received enalapril. In the in vitro studies, enalapril administration inhibited the phosphorylation of IkappaB. Conclusions:The ACE inhibitor enalapril blocked the LPS-induced inflammatory response and protected against the acute lung injury normally associated with endotoxemia in this rat sepsis model. Given these results, enalapril is a strong candidate as a therapeutic agent for sepsis.

Effects of hyperglycemia and insulin therapy on high mobility group box 1 in endotoxin-induced acute lung injury in a rat model.

Objective:Hyperglycemia and insulin resistance are commonly seen in septic patients and are associated with increased morbidity and mortality. High mobility group box 1 (HMGB1) protein has been shown to play a key role as a significant factor in sepsis pathogenesis. This study investigated the increase in lung damage because of hyperglycemia and HMGB1 increase in a lipopolysaccharide-induced septic rat model and the potential for insulin therapy to reduce this lung damage by decreasing the serum level of HMGB1. Design:Randomized, prospective animal study. Setting:University medical center research laboratory. Subjects:Male Wistar rats. Interventions:Septic hyperglycemia was induced by infusion of glucose immediately after administration of lipopolysaccharide in rats. Measurements and Main Results:Animals were monitored for blood glucose. Separate cohorts were killed at 12 and 24 hrs postlipopolysaccharide administration and analyzed for HMGB1 and lung damage. The effects of insulin treatment were also examined. Hyperglycemic septic animals had significantly higher blood glucose and enhanced lung damage. In addition, HMGB1 was increased in the serum of hyperglycemic rats. On the other hand, insulin treatment for hyperglycemia resulted in significantly lower blood glucose and decreased both the lung damage and the serum level of HMGB1. In an in vitro study, insulin treatment inhibited the activation of NF-kappaB. Conclusions:Hyperglycemia is associated with higher HMGB1 levels and lung damage in sepsis. Insulin therapy significantly reduced lung damage, suggesting that management of hyperglycemia with insulin might decrease HMGB1 levels in the serum and lung tissue. One of the mechanisms that could contribute to the inhibition of HMGB1 secretion might be related to the inhibition of NF-&kgr;B.

Pre-Irradiation of Blood by Gallium Aluminum Arsenide (830 nm) Low-Level Laser Enhances Peripheral Endogenous Opioid Analgesia in Rats

BACKGROUND:Low-level laser therapy (LLLT) has been reported to relieve pain, free of side effects. However, the mechanisms underlying LLLT are not well understood. Recent studies have also demonstrated that opioid-containing immune cells migrate to inflamed sites and release β-endorphins to inhibit pain as a mode of peripheral endogenous opioid analgesia. We investigated whether pre-irradiation of blood by LLLT enhances peripheral endogenous opioid analgesia. METHODS:The effect of LLLT pretreatment of blood on peripheral endogenous opioid analgesia was evaluated in a rat model of inflammation. Additionally, the effect of LLLT on opioid production was also investigated in vitro in rat blood cells. The expression of the β-endorphin precursors, proopiomelanocortin and corticotrophin releasing factor, were investigated by reverse transcription polymerase chain reaction. RESULTS:LLLT pretreatment produced an analgesic effect in inflamed peripheral tissue, which was transiently antagonized by naloxone. Correspondingly, β-endorphin precursor mRNA expression increased with LLLT, both in vivo and in vitro. CONCLUSION:These findings suggest that that LLLT pretreatment of blood induces analgesia in rats by enhancing peripheral endogenous opioid production, in addition to previously reported mechanisms.

Landiolol, an ultrashort-acting beta1-adrenoceptor antagonist, has protective effects in an LPS-induced systemic inflammation model.

Previous studies suggest that the blockade of &bgr;-adrenoceptors augments the release of inflammatory regulators in response to proinflammatory stimuli. High-mobility group box 1 (HMGB-1) is a key mediator in the development of sepsis. We investigated whether landiolol, a short-acting selective &bgr;1-adrenoceptor-blocking agent, can attenuate acute lung injury and cardiac dysfunction in a rat model of endotoxin-induced sepsis. We administered LPS i.v. to rats, with or without simultaneous treatment with landiolol (0.1 mg/kg per min). After the induction of sepsis by LPS treatment, we measured cytokine and HMGB-1 levels in the serum and lung tissue. In addition, we performed histopathology, determined wet-to-dry weight ratios, and measured cardiac function and cell signaling in the lung. Cotreatment with landiolol was associated with significantly less severe disease, as assessed by lung histopathology and cardiac function metrics. Serum and lung HMGB-1 levels were lower over time among landiolol-treated animals. Furthermore, nuclear factor-&kgr;B activity was inhibited by the administration of landiolol. Cotreatment with the selective &bgr;1-adrenoceptor-blocking agent landiolol protects against acute lung injury and cardiac dysfunction in a rat model of LPS-induced systemic inflammation. Treatment was associated with a significant reduction in serum levels of the inflammation mediator HMGB-1 and histological lung damage.

C-reactive protein induces high-mobility group box-1 protein release through activation of p38MAPK in macrophage RAW264.7 cells

BACKGROUND C-reactive protein (CRP) is widely used as a sensitive biomarker for inflammation. Increasing evidence suggests that CRP plays a role in inflammation. High-mobility group box-1 (HMGB1), a primarily nuclear protein, is passively released into the extracellular milieu by necrotic or damaged cells and is actively secreted by monocytes/macrophages. Extracellular HMGB1 as a potent inflammatory mediator has stimulated immense curiosity in the field of inflammation research. However, the molecular dialogue implicated between CRP and HMGB1 in delayed inflammatory processes remains to be explored. METHODS AND RESULTS The levels of HMGB1 in culture supernatants were determined by Western blot analysis and enzyme-linked immunosorbent assay in macrophage RAW264.7 cells. Purified CRP induced the release of HMGB1 in a dose- and time-dependent fashion. Immunofluorescence analysis revealed nuclear translocation of HMGB1 in response to CRP. The binding of CRP to the Fc gamma receptor in RAW264.7 cells was confirmed by fluorescence-activated cell sorter analysis. Pretreatment of cells with IgG-Fc fragment, but not IgG-Fab fragment, efficiently blocked this binding. CRP triggered the activation of p38MAPK and ERK1/2, but not Jun N-terminal kinase. Moreover, both p38MAPK inhibitor SB203580 and small interfering RNA significantly suppressed the release of HMGB1, but not the MEK1/2 inhibitor U-0126. CONCLUSION We demonstrated for the first time that CRP, a prominent risk marker for inflammation including atherosclerosis, could induce the active release of HMGB1 by RAW264.7 cells through Fc gamma receptor/p38MAPK signaling pathways, thus implying that CRP plays a crucial role in the induction, amplification, and prolongation of inflammatory processes, including atherosclerotic lesions.

Respiratory mechanics and arterial blood gases during and after laparoscopic cholecystectomy

PurposeThe purpose of this study was to assess the effects of increased intra-abdominal pressure due to CO2 insufflation on the mechanical characteristics of the respiratory system and arterial blood gases during and after laparoscopic cholecystectomy.MethodsRespiratory mechanics and arterial blood gases were examined in 12 patients undergoing laparoscopic cholecystectomy with CO2 insufflation. Respiratory mechanics were continuously monitored with in-line spirometry. In the recovery room, PaCO2 was measured in this group at 30 min and compared with PaCO2s in 23 patients who had undergone open cholecystectomy retrospectively, to evaluate the effects of insufflation on CO2 elimination.ResultsMinute ventilation was decreased by about 500 ml·min−1 during abdominal insufflation. Dynamic lung compliance decreased from 49.6 ± 4.7 to 30.7 ±2.3 (mean ± SEM) ml·cmH2O−1 with abdominal insufflation (P < 0.005), and returned to 45.1 ±3.1 after the release of pneumoperitoneum. Peak inspiratory pressure increased from 15.9 ± 0.9 to 18.9 ± 1.0 cmH2O with abdominal insufflation (P < 0.05). Arterial blood gas determinations indicated a decrease in arterial pH, with CO2 retention during insufflation and in the recovery room (P < 0.05). PaCO2 of the laparoscopic patients was higher than that of the open patients in the recovery room.ConclusionThe results indicate that respiratory acidosis was caused during CO2 insufflation for laparoscopic cholecystectomy, that was due to (1) decreased compliance, (2) increased CO2 load and (3) insufficient ventilation. Accumulated CO2 during laparoscopic cholecystectomy increased PaCO2 level in the recovery room.RésuméObjectifEvaluer les effets de l’augmentation de pression intraabdominale provoquée par l’insufflation de CO2 sur les caractéristiques du système respiratoire et des gaz du sang artériel pendant et après la cholécystectomie laparoscopique.MéthodeLa mécanique respiratoire et les gaz du sang artériels ont été étudiés chez 123 patients soumis à une cholé-cystectomie laparoscopique avec insufflation de CO2. La mécanique respiratore a été monitorée en continu par spirométrie. A la salle de réveil, la PaCO2 a été mésurée à la 30e min de l’admission et comparée rétrospectivement à la PaCO2 de 23 patients qui avaient subi une cholécystectomie ouverte, dans le but d’évaluer les effets de l’insufflation sur l’élimination du CO2.RésultatsLa ventilation minute a diminué d’environ 500 ml·min−1 pendant l’insufflation abdominale. La compliance dynamique pulmonaire diminuait de 49,6 ± 4,7 à 30,7 ± 2,3 (moyenne ± SEM) ml·cmH2O−1 avec l’insufflation (P < 0,005) et revenait à 45,1 ± 3,1 après le relâchement du pneumopéritoine. L’analyse des gaz artériels a révélé une diminution du pH artériel avec rétention de CO2 pendant l’insufflation et à la salle de réveil (P < 0,005). La PaCO2 des patients opérés sous laparoscopie était plus élevée que celle des patients opérés par chirurgie ouverte.ConclusionCes résultats indiquent que l’insufflation de CO2 pour la cholécystectomie laparoscopique provoque de l’acidose respiratoire causée 1) par la baisse de la compliance, 2) l’augmentation du volume de CO2 et 3) l’insuffisance ventilatoire. L’accumulation du CO2 pendant la cholécystectomie laparoscopique augmente la PaCO2 à la salle de réveil.

Changes in cell culture temperature alter release of inflammatory mediators in murine macrophagic RAW264.7 cells

Abstract.Objectives:To examine whether moderate changes in cell culture temperature influence the production of various cytokines and associated mediators of inflammation.Methods:We performed lipopolysaccharide (LPS) stimulation of the murine macrophagic RAW264.7 cell line under hyperthermic (40 °C), normothermic (37 °C) and hypothermic (34 °C) conditions. We then measured the levels of heat shock protein 70 (HSP70), heat shock factor protein (HSF) and nuclear factor–kB (NF-kB) dimers (p50 and p65) in the cells, and the levels of high mobility group box 1 (HMGB1) and the cytokines tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in the culture supernatants.Results:Levels of HMGB1, IL-1β, IL-6, and TNF-α, as well as NF-kB dimers (p50 and p65), were all reduced following LPS stimulation at 40 °C and 34 °C compared with those at 37 °C. Levels of HSP70 and HSF increased at 40 °C and 34 °C.Conclusions:The application of moderate hyperthermia and hypothermia after LPS-induced cell activation attenuated the inflammatory response and reduced the likelihood of cell damage. These findings suggest that moderate temperature changes modulate the inflammatory response and could be a useful therapy against sepsis.

Adenosine diphosphate receptor antagonist clopidogrel sulfate attenuates LPS-induced systemic inflammation in a rat model.

Septic shock is characterized by systemic inflammation and can lead to hemorrhage and necrosis in multiple organs. Septic shock is one of the leading causes of death. Studies have reported that septic shock is strongly associated with coagulation abnormality. The adenosine diphosphate (ADP) receptor antagonist, clopidogrel sulfate (CS), inhibits platelet function. Thus, we hypothesized that CS could inhibit LPS-induced systemic inflammation in a rat model. Male Wistar rats weighing 250 to 300 g received an LPS injection, followed 6 h later by filtration leukocytapheresis or mock treatment for 30 min under sevoflurane anesthesia. Five days before LPS injection, rats were given an oral dose of water or CS (10 mg/kg body weight). Levels of proinflammatory markers were determined in serum and tissue samples, and high-mobility group box 1 (HMGB1) expression was evaluated in lung and liver tissues. Compared with LPS-treated rats, induction of cytokines (IL-6 and TNF-&agr;) was reduced in rats pretreated with CS. In addition, histological changes observed in lung and liver tissue samples of LPS-treated rats were attenuated in CS-pretreated rats. Clopidogrel sulfate pretreatment also reduced LPS-induced HMGB1 expression in lung and liver tissues. Collectively, our findings demonstrate that CS pretreatment may have value as a new therapeutic tool against systemic inflammation.

In vivo and in vitro effects of the anticoagulant, thrombomodulin, on the inflammatory response in rodent models.

Sepsis remains a major health threat in intensive care medicine. The physiological functions of the coagulation cascade extend beyond blood coagulation and play a pivotal role in inflammation. We investigated whether the use of recombinant thrombomodulin (rTM), which has activity comparable with antithrombin, tissue factor pathway inhibitor, and activated protein C, could inhibit secretion of cytokines and high-mobility group box 1 (HMGB1) protein, thus reducing lung damage in a rat model of LPS-induced systemic inflammation. Rats treated with an intravenous injection of either rTM or saline were injected concurrently with intravenous LPS. In addition, mouse macrophage RAW264.7 cells were stimulated with LPS, with or without simultaneous rTM treatment. Histological examination revealed marked reductions of interstitial congestion, edema, inflammation, and hemorrhage in lung tissue harvested 12 h after treatment with both agents compared with LPS administration alone. LPS-induced secretion of proinflammatory cytokines and HMGB1 protein was inhibited by treatment with rTM. The presence of HMGB1 protein in the lung was examined by immunohistochemistry; the number of HMGB1-positive cells was significantly lower in LPS-treated animals that also received rTM. In the in vitro studies, rTM administration inhibited the activation of nuclear factor-kappa B by inhibiting I kappa B phosphorylation. The anticoagulant rTM blocked the LPS-induced inflammatory response and protected against acute lung injury normally associated with endotoxemia in this rat sepsis model. Given these results, rTM is a strong candidate as a therapeutic agent for various systemic inflammatory diseases.