Kazuyuki Miyamoto

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Regulation of oxidative stress by pituitary adenylate cyclase-activating polypeptide (PACAP) mediated by PACAP receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional peptide that has been shown to be neuroprotective following a diverse range of cell injuries. Although several mechanisms regulating this effect have been reported, no direct evidence has linked PACAP to the regulation of oxidative stress, despite the fact that oxidative stress is a factor in the injury progression that occurs in most models. In the present study, we investigated the plasma oxidative metabolite and anti-oxidation potential levels of PACAP-deficient mice, as well as those of wild-type animals treated with PACAP38. These were assayed by the determination of Reactive Oxidative Metabolites (d-ROMs) and the Biological Anti-oxidant Potential (BAP) using the Free Radical Electron Evaluator system. We also investigated the direct radical scavenging potency of PACAP38 and the functional role of its receptor in the regulation of oxidative stress by PACAP, by using vasoactive intestinal peptide (VIP) and the PACAP receptor antagonist, PACAP6–38. Although younger PACAP null mice displayed no significant effect, greater d-ROMs and lower BAP values were recorded in older animals than in their wild-type littermates. Intravenous injection of PACAP38 in wild-type mice decreased the plasma d-ROMs and BAP values in a dose-dependent manner. These effects were not reproduced using VIP and were abolished by co-treatment with PACAP38 and the PAC1R antagonist PACAP6-38. Taken together, these results suggest that PACAP plays an important role in the physiological regulation of oxidative stress.

Status of Systemic Oxidative Stress during Therapeutic Hypothermia in Patients with Post-Cardiac Arrest Syndrome

Therapeutic hypothermia (TH) is thought to be due to the downregulation of free radical production, although the details of this process remain unclear. Here, we investigate changes in oxidative stress and endogenous biological antioxidant potential during TH in patients with post-cardiac arrest syndrome (PCAS). Nineteen PCAS patients were enrolled in the study. Brain temperature was decreased to the target temperature of 33°C, and it was maintained for 24 h. Patients were rewarmed slowly (0.1°C/h, <1°C/day). The generation of reactive oxygen metabolites (ROMs) was evaluated in plasma samples by d-ROM test. Plasma antioxidant capacity was measured by the biological antioxidant potential (BAP) test. Levels of d-ROMs and BAP levels during the hypothermic stage (33°C) were suppressed significantly compared with pre-TH induction levels (P < 0.05), while both d-ROM and BAP levels increased with rewarming (33–36°C) and were correlated with brain temperature. Clinical monitoring of oxidative stress and antioxidant potential is useful for evaluating the redox state of patients undergoing TH after PCAS. Additional therapy to support the antioxidant potential in the rewarming stage following TH may reduce some of the observed side effects associated with the use of TH.

Therapeutic time window for edaravone treatment of traumatic brain injury in mice.

Traumatic brain injury (TBI) is a major cause of death and disability in young people. No effective therapy is available to ameliorate its damaging effects. Our aim was to investigate the optimal therapeutic time window of edaravone, a free radical scavenger which is currently used in Japan. We also determined the temporal profile of reactive oxygen species (ROS) production, oxidative stress, and neuronal death. Male C57Bl/6 mice were subjected to a controlled cortical impact (CCI). Edaravone (3.0 mg/kg), or vehicle, was administered intravenously at 0, 3, or 6 hours following CCI. The production of superoxide radicals (O2 ∙−) as a marker of ROS, of nitrotyrosine (NT) as an indicator of oxidative stress, and neuronal death were measured for 24 hours following CCI. Superoxide radical production was clearly evident 3 hours after CCI, with oxidative stress and neuronal cell death becoming apparent after 6 hours. Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress, particularly at the 3-hour time point. Moreover, the greatest decrease in O2 ∙− levels was observed when edaravone was administered 3 hours following CCI. These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI.

The anti-inflammatory property of human bone marrow-derived mesenchymal stem/stromal cells is preserved in late-passage cultures.

Human mesenchymal stem/stromal cells (hMSCs) have been reported to improve neural damage via anti-inflammation and multi-differentiation abilities. Here, we investigated immunosuppression effects of hMSCs by mixed-culturing with interferon-γ (IFNγ) stimulated BV-2 mouse microglial cells. We show that hMSCs decreased nitrite oxide (NO) production from BV-2 cells in cell density dependent manner. Aged hMSCs and peroxisome proliferator-activated receptor-γ (PPARγ) knockdown hMSCs decreased differentiation abilities but maintained NO suppressive function. We finally confirmed NO suppression activities of hMSCs in IFNγ-stimulated primary microglia/macrophages. It suggested that hMSCs significantly modified NO production in activated phagocytes and it might be preserved in late passage cultures.

Establishment and characterization of primary adult microglial culture in mice.

Microglial cells account for approximately 12-15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1β (IL-1β) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1β treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.

Edaravone Increases Regional Cerebral Blood Flow After Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is a major cause of preventable death and serious morbidity, with subsequent low cerebral blood flow (CBF) considered to be associated with poor prognosis. In the present study, we demonstrated the effect of the free radical scavenger edaravone on regional CBF (rCBF) after TBI. Male mice (C57/BL6) were subjected to TBI using a controlled cortical impactor device. Immediately after TBI, the animals were intravenously administered 3.0 mg/kg of edaravone or a vehicle saline solution. Two-dimensional rCBF images were acquired before and 24 h post-TBI, and were quantified in the ipsilateral and contralateral hemispheres (n = 5 animals per group). CBF in the vehicle-treated animals decreased broadly over the ipsilateral hemisphere, with the region of low rCBF spreading from the frontal cortex to the occipital lobe. The zone of lowest rCBF matched that of the contusion area. The mean rCBF at 24 h for a defined elliptical region between the bregma and lambda was 73.7 ± 5.8 %. In comparison, the reduction of rCBF in edaravone-treated animals was significantly attenuated (93.4 ± 5.7 %, p < 0.05). The edaravone-treated animals also exhibited higher rCBF in the contralateral hemisphere compared with that seen in -vehicle-treated animals. It is suggested that edaravone reduces neuronal damage by scavenging reactive oxygen species (ROS) and by maintaining intact the autoregulation of the cerebral vasculature.

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.

Accumulation of autofluorescent storage material in brain is accelerated by ischemia in chloride channel 3 gene‐deficient mice

Autofluorescent storage material (ASM) is an aging pigment that accumulates during the normal course of senescence. Although the role of ASM has yet to be fully elucidated, ASM has been implicated in age‐related neurodegeneration. In this study, we determined the level of ASM in chloride channel 3 (ClC‐3) gene‐deficient (KO) mice both in response to aging and following mild global ischemia. To understand the mechanism of action of the ASM, mice subjected to ischemia were treated with the cyclooxygenase (COX) inhibitor indomethacin or with the noncompetitive glutamate receptor antagonist MK‐801. ClC‐3 KO mice displayed age‐related neurodegeneration of the neocortex as well as the hippocampus. The cortical layers in particular granular layers became thinner with aging. ASM accumulated in the brains of ClC‐3 KO mice was increased seven‐ to 50‐fold over that observed in the corresponding regions of their wild‐type littermates. Young wild‐type mice survived longer than age‐matched ClC‐3 KO mice after permanent global ischemia. However, in the case of older animals, the survival curves were similar. The ASM also increased four‐ to fivefold 10 days after mild global ischemia, an effect that was suppressed by treatment with indomethacin and MK‐801. These results suggest that temporary ischemia might trigger a process similar to aging in the brain, mimicking the effect of age‐related neurodegenerative diseases.

533: ORAL REHYDRATION SOLUTION INCREASES SGLT1 AND IMPROVES DEHYDRATION IN A MOUSE HEATSTROKE MODEL

www.ccmjournal.org Critical Care Medicine • Volume 46 • Number 1 (Supplement) Learning Objectives: Since proton pump inhibitors (PPI) can quickly establish optimal gastric pH conditions to support hemostasis, they are the mainstay for treating non-variceal upper gastrointestinal bleeding (NVUGIB). However, somatostatin analogs have been found to be more effective at maintaining a pH > 4 during the first 12 hours of infusion compared to placebo or PPI. Despite these findings, there is a paucity of data evaluating the impact on hospital outcomes when a somatostatin analog is added to a PPI in NVUGIB. The primary objective of this study was to elucidate the association of adjunctive octreotide with a PPI (O+PPI) versus PPI therapy alone with hospital length of stay (LOS). Methods: A retrospective cohort study of adults admitted our healthcare system between 2011 and 2016 with a NVUGIB treated with a PPI continuous infusion with or without an octreotide infusion for at least 12 hours was conducted. Exclusion criteria included varices, history of cirrhosis without endoscopy, or active gastrointestinal cancer. After a univariate analysis was conducted, a linear regression model with propensity score adjustment was conducted for hospital LOS. Results: A total of 180 patients were included (O+PPI n = 90; PPI n = 90). Some baseline differences between the two groups which were higher in the O+PPI group included alcohol abuse (49% vs 17%; p < 0.001) and liver disease (28% vs 4%; p < 0.001). In a univariate analysis, the median hospital and intensive care unit LOS for O+PPI versus PPI alone was 6.1 days [interquartile range or IQR: 3.3–9.4 days] vs 4.9 days [IQR: 3.1–8.5 days; p = 0.25] and 2.3 days [IQR: 1.3–4 days] vs 1.9 days [IQR: 1.2–3.1 days; p = 0.83]. After propensity score adjustment, no significant differences in hospital LOS were identified (p = 0.36). There were no differences in median duration of packed red blood cells (15.7 hours O+PPI; IQR: 0–46.9 hours vs 7.1 hours PPI; IQR: 0–44.9 hours; p = 0.47) or units required (3 units O+PPI vs 2 units PPI; p = 0.43), re-bleeding (9% O+PPI vs 12% PPI; p = 0.63), or mortality (6.7% O+PPI vs 5.6% PPI; p = 1.00). Conclusions: In patients presenting with NVUGIB, the addition of octreotide to PPI infusion did not impact hospital LOS, re-bleeding, or mortality compared to PPI alone. Based on our results, PPI monotherapy appears safe and effective in the initial treatment of NVUGIB.