Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain

Our previous studies have shown that intracerebral administration of endotoxin, lipopolysaccharide (LPS), induces selective white matter injury and hypomyelination in the neonatal rat brain and that the LPS-induced brain injury is associated with activation of microglia. To test the hypothesis that inhibition of microglial activation may protect against LPS-induced white matter injury, we examined roles of minocycline, a putative suppressor of microglial activation, on LPS-induced brain injury in the neonatal rat. A stereotactic intracerebral injection of LPS (1 mg/kg) was performed in postnatal day 5 Sprague-Dawley rats and control rats were injected with sterile saline. Minocycline (45 mg/kg) was administered intraperitoneally 12 h before and immediately after LPS injection and then every 24 h for 3 days. Inflammatory responses, activation of microglia and brain injury were examined 1 and 3 days after LPS injection. LPS injection resulted in brain injury in selective brain areas, including bilateral ventricular enlargement, cell death at the sub- and periventricular areas, loss of O4+ and O1+ oligodendrocyte (OL) immunoreactivity and hypomyelination, as indicated by decreased myelin basic protein immunostaining, in the neonatal rat brain. Minocycline administration significantly attenuated LPS-induced brain injury in these rat brains. The protective effect of minocycline was associated with suppressed microglial activation as indicated by the decreased number of activated microglial cells following LPS stimulation and with consequently decreased elevation of interleukin 1beta and tumor necrosis factor-alpha concentrations induced by LPS and a reduced number of inducible nitric oxide synthase expressing cells. Protection of minocycline was also linked with the reduction in LPS-induced oxidative stress, as indicated by 4-hydroxynonenal positive OLs. The overall results suggest that reduction in microglial activation may protect the neonatal brain from LPS-induced white matter injury and inhibition of microglial activation might be an effective approach for the therapeutic treatment of infection-induced white matter injury.     

Oxidative and nitrative injury in periventricular leukomalacia: a review

Periventricular leukomalacia (PVL) is the major substrate of cerebral palsy in survivors of prematurity. Its pathogenesis is complex and likely involves ischemia/reperfusion in the critically ill premature infant, with impaired regulation of cerebral blood flow, as well as inflammatory mechanisms associated with maternal and/or fetal infection. During the peak period of vulnerability for PVL, developing oligodendrocytes (OLs) predominate in the white matter. We hypothesize that free radical injury to the developing OLs underlies, in part, the pathogenesis of PVL and the hypomyelination seen in long-term survivors. In human PVL, free radical injury is supported by evidence of oxidative and nitrative stress with markers to lipid peroxidation and nitrotyrosine, respectively. Evidence in normal human cerebral white matter suggests an underlying vulnerability of the premature infant to free radical injury resulting from a developmental mismatch of antioxidant enzymes (AOE) and subsequent imbalance in oxidant metabolism. In vitro studies using rodent OLs suggest that maturational susceptibility to reactive oxygen species is dependent, not only on levels of individual AOE, but also on specific interactions between these enzymes. Rodent in vitro data further suggest 2 mechanisms of nitric oxide damage: one involving the direct effect of nitric oxide on OL mitochondrial integrity and function, and the other involving an activation of microglia and subsequent release of reactive nitrogen species. The latter mechanism, while important in rodent studies, remains to be determined in the pathogenesis of human PVL. These observations together expand our knowledge of the role that free radical injury plays in the pathogenesis of PVL, and may contribute to the eventual development of therapeutic strategies to alleviate the burden of oxidative and nitrative injury in the premature infant at risk for PVL.     

PDGF-AA and bFGF mediate B104CM-induced proliferation of oligodendrocyte precursor cells

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of οligodendrocyte precursor cells (OPCs) in vitro, which indicates that certain factors contained within B104CM give instructional signals that direct the proliferation of OPCs. However, the OPC-proliferative factors present in B104CM have yet to be identified. Platelet-derived growth factor?AA (PDGF-AA), basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) have been reported to act as potent mitogens for OPC proliferation. This raises the possibility that B104CM induces proliferation of OPCs through secretion of PDGF?AA, bFGF and/or IGF-1. In the present study, we detected the expression and levels of PDGF-AA, bFGF and IGF-1 in B104 cells and B104CM, and observed the expression of their receptors in OPCs. The results indicated that these growth factors were expressed in B104 cells and B104CM. All 3 receptors, PDGFR, FGFR2 and IGF-1R, were also detected in OPCs. Furthermore, B104CM-stimulated OPC proliferation could be markedly decreased by both AG1295 (an inhibitor of PDGFR) and PD173074 (an inhibitor of FGFR). However, the inhibition of IGF-1R with AG1204 did not affect the proliferation of OPCs. Our study suggests that the PDGF-AA and bFGF in B104CM are 2 key factors that stimulate OPC proliferation.     

葛根素对脂多糖诱导N9小胶质细胞激活的抑制作用

目的:探索葛根素对脂多糖(LPS)诱导N9小胶质细胞激活的抑制作用,为其在神经退行性疾病的防治方面提供依据。方法:用MTT法检测脂多糖和葛根素对小胶质细胞的细胞毒性作用;用流式细胞术(FCM)检测葛根素对LPS诱导N9细胞内活性氧(ROS)产生的抑制作用;分别用Griess法和FCM检测细胞培养液和细胞内一氧化氮(NO)含量;用HE染色法观察LPS激活N9细胞,及葛根素恢复细胞静息状态下细胞的形态;以FCM检测细胞凋亡和细胞周期。结果:葛根素(100~200μmol/L)能使LPS激活的N9细胞,从阿米巴样的活化形状明显恢复至静息态的圆形;LPS激活的N9细胞能产生大量的NO到培养液中,葛根素(50~200μmol/L)能使NO的产生减少,其中,高浓度组(200μmol/L)与LPS模型组相比,NO从23.45±0.19μmol/L降至12.43±0.11μmol/L(P0.01)。胞内ROS检测表明:葛根素(200μmol/L)同样明显降低ROS的产生。葛根素对激活的N9细胞胞内ROS的产生具有抑制作用,高浓度组(200μmol/L)能使其恢复至对照组水平;此外,葛根素能显著抑制LPS诱导的细胞凋亡,并恢复LPS对N9细胞G0/G1期的阻滞作用。结论:葛根素具有抑制小胶质细胞激活的作用。     

The influence of DHEA pretreatment on prepulse inhibition and the HPA-axis stress response in rat offspring exposed prenatally to polyriboinosinic-polyribocytidylic-acid (PIC)

Periventricular leukomalacia (PVL) is the dominant form of brain injury in premature infants and no specific treatment is currently available. Neotrofin, a neurotrophin agonist, has been shown to provide neuroprotection in several in vivo and in vitro studies. The aim of this study was to investigate the neuroprotective effect of neotrofin treatment after endotoxin induced PVL in a rat model. Wistar rat pups were divided into four groups as: (1) control, (2) lipopolysaccharide (LPS)-administered group, (3) LPS-administered and prenatal maternal neotrofin-treated group and (4) LPS-administered and postnatal neotrofin-treated group. Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) was administered consecutively at the 18th and 19th embryonic days to establish endotoxin-induced PVL model. In the prenatal treatment group dams received an i.p. injection of neotrofin (60 mg/kg) following after the second LPS dose; and in the postnatal treatment group rat pups received i.p. injection of neotrofin (60 mg/kg) at birth. At P7, apoptosis and hypomyelination in periventricular white matter were evaluated by immunohistochemical assessments. The prenatal maternal neotrofin treatment significantly reduced the number of apoptotic cell death and greatly prevented LPS-stimulated loss of hypomyelinization. However, neotrofin treatment in the postnatal period was not as effective as intrauterine treatment. Given our results, neotrofin may be useful in reducing brain injury and possessing clinical relevance for the treatment of white matter injury in newborns.     

Developmental stage of oligodendrocytes determines their response to activated microglia in vitro

Background  

Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.     

Complement C3a induces axonal hypomyelination in the periventricular white matter through activation of WNT/β‐catenin signal pathway in septic neonatal rats experimentally induced by lipopolysaccharide

Neuroinflammation is thought to play a pivotal role in the pathogenesis of periventricular white matter (PWM) damage (PWMD) induced by neonatal sepsis. Because the complement cascade is implicated in inflammatory response, this study was carried out to determine whether C3a is involved in PWMD, and, if so, whether it would induce axonal hypomyelination. Furthermore, we explored if C3a would act through its C3a receptor (C3aR) and thence inhibit maturation of oligodendrocyte precursor cells (OPCs) via the WNT/β‐catenin signal pathway. Sprague Dawley (SD) rats aged 1 day were intraperitoneally injected with lipopolysaccharide (LPS) (1 mg/kg). C3a was upregulated in activated microglia and astrocytes in the PWM up to 7 days after LPS injection. Concomitantly, enhanced C3aR expression was observed in NG2⁺ oligodendrocytes (OLs). Myelin proteins including CNPase, PLP, MBP and MAG were significantly reduced in the PWM of 28‐day septic rats. The number of PLP⁺ and MBP⁺ cells was markedly decreased. By electron microscopy, myelin sheath thickness was thinner and the average g‐ratios were higher. This was coupled with an increase in number of NG2⁺ cells and decreased number of CC1⁺ cells. Olig1, Olig2 and SOX10 protein expression was significantly reduced in the PWM after LPS injection. Very strikingly, C3aRa administration for the first 7 days could reverse the above‐mentioned pathological alterations in the PWM of septic rats. When incubated with C3a, expression of MBP, CNPase, PLP, MAG, Olig1, Olig2, SOX10 and CC1 in primary cultured OPCs was significantly downregulated as opposed to increased NG2. Moreover, WNT/β‐catenin signaling pathway was found to be implicated in inhibition of OPCs maturation and differentiation induced by C3a in vitro. As a corollary, it is speculated that C3a in the PWM of septic rats is closely associated with the disorder of OPCs differentiation and maturation through WNT/β‐catenin signaling pathway, which would contribute ultimately to axonal hypomyelination.     

Oligodendroglial Cells Express and Secrete Reelin

Oligodendrocyte (OL) progenitor cells (OPCs) give rise to the myelinating cells of the central nervous system (CNS), the OL. To examine molecular changes involved in OPC differentiation, a microarray analysis was performed at several time points during OPC maturation. The results revealed significant expression levels of mRNA for reelin, one reelin receptor, very low density lipoprotein receptor (Vldlr), and the cytoplasmic adaptor molecule, disabled homolog 1 (Dab1). The expression of these proteins in oligodendroglial (ODG) cells was confirmed by immunocytochemistry and Western blot analysis. It was also discovered that both progenitors and mature OLs secrete reelin. Although there is no known effect of reelin on ODG cells, the data suggest that these cells may be a source of reelin in the CNS. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

脂多糖诱导小胶质细胞依赖的早产鼠脑白质损伤机制研究

目的研究中枢神经系统小胶质细胞（MG）正常发育尤其是少突胶质细胞前体细胞（OPCs）最易受损阶段的发育,探讨宫内感染早产鼠MG依赖的OPCs损伤机制。方法①观察正常C57B/L鼠不同胎龄（孕10、15d）和生后（0、5、10d）MG和OPCs在脑白质的发育分布情况,明确两者在发育和分布上的关联。②建立脂多糖（LPS）宫内感染新生鼠模型（宫内分别接种LPS5、10和20μg·mL-1为感染A-C组）,以PBS溶液接种为对照组。以Tomato lectin作为静息状态MG标志,CD68作为活化MG的特殊抗体,O4＋作为OPCs抗体,抗体浮片法进行免疫组化染色并计数分析。③Western blot法检测各组脑室周围白质组织Toll样受体-4（TLR-4）蛋白表达。④采用ELISA法检测各组MG活化后IL-2、TNF-α和SOD水平变化。结果①MG在孕10d胎鼠Tomato lectin表达低下,孕15d胎鼠表达显著增高,MG主要分布在脑室周围白质区域,灰质皮质几乎不表达。出生后,脑室周围白质区域MG的表达有所下降,灰质皮质的表达逐渐增高。②感染A-C组CD68＋细胞数量均显著增加,与对照组差异有统计学意义（P〈0.01）,但感染C组与B组CD68＋细胞数量差异无统计学意义（P〉0.05）。与对照组比较,感染A-C组均可见O4＋细胞数量显著性下降（P〈0.01）,其中以感染C组下降最为明显。③对照组未检测到TLR-4蛋白表达,感染A-C组均可见LPS剂量依赖的TLR-4蛋白表达增加,与对照组差异有统计学意义（P〈0.05）。④随接种LPS剂量增大,IL-2和TNF-α水平较对照组呈显著增加趋势,SOD水平较对照组呈显著降低趋势。结论新生鼠发育依赖的MG在脑白质受损区域过度表达,表明活化MG起到本底激活效应,是早产儿脑白质损伤的物质基础。     

Paeonol Suppresses Neuroinflammatory Responses in LPS-Activated Microglia Cells

In this work, we assessed the anti-inflammatory effects of paeonol (PAE) in LPS-activated N9 microglia cells, as well as its underlying molecular mechanisms. PAE had no adverse effect on the viability of murine microglia N9 cell line within a broad range (0.12～75 μM). When N9 cell line was activated by LPS, PAE (0.6, 3, 15 μM) significantly suppressed the release of proinflammatory products, such as nitric oxide (NO), interleukin-1β (IL-1β), and prostaglandin E2 (PGE2), demonstrated by the ELISA assay. Moreover, the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly reduced in PAE-treated N9 microglia cells. We also examined some proteins involved in immune signaling pathways and found that PAE treatment significantly decreased the expression of TLR4, MyD88, IRAK4, TNFR-associated factor 6 (TRAF6), p-IkB-α, and NF-kB p65, as well as the mitogen-activated protein kinase (MAPK) pathway molecules p-P38, p-JNK, and p-ERK, indicating that PAE might act on these signaling pathways to inhibit inflammatory responses. Overall, we found that PAE had anti-inflammatory effect on LPS-activated N9 microglia cells, possibly via inhibiting the TLR4 signaling pathway, and it could be a potential drug therapy for inflammation-associated neurodegenerative diseases.     

Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT–PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-α, IL-1β, and iNOS and release of TNF-α and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-α, IL-1β and iNOS and production of TNF-α and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.     

Myelin abnormalities without oligodendrocyte loss in periventricular leukomalacia

The cellular basis of myelin deficits detected by neuroimaging in long-term survivors of periventricular leukomalacia (PVL) is poorly understood. We tested the hypothesis that oligodendrocyte lineage (OL) cell density is reduced in PVL, thereby contributing to subsequent myelin deficits. Using computer-based methods, we determined OL cell density in sections from 18 PVL and 18 age-adjusted control cases, immunostained with the OL-lineage marker Olig2. Myelination was assessed with myelin basic protein (MBP) immunostaining. We found no significant difference between PVL and control cases in Olig2 cell density in the periventricular or intragyral white matter. We did find, however, a significant increase in Olig2 cell density at the necrotic foci, compared with distant areas. Although no significant difference was found in the degree of MBP immunostaining, we observed qualitative abnormalities of MBP immunostaining in both the diffuse and necrotic components of PVL. Abnormal MBP immunostaining in PVL despite preserved Olig2 cell density may be secondary to arrested OL maturation, damage to OL processes, and/or impaired axonal-OL signaling. OL migration toward the "core" of injury may occur to replenish OL cell number. This study provides new insight into the cellular basis of the myelin deficits observed in survivors of PVL.     

Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia

The activation of microglia plays an important role in a variety of brain disorders by the excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂) and proinflammatory cytokines. We investigated here whether pinoresinol isolated from the fruits of Forsythia koreana Nakai inhibits the inflammatory responses in LPS-activated microglia. Pinoresinol inhibited the production of NO, PGE₂, TNF-α, IL-1β and IL-6 in LPS-activated primary microglia. Also, pinoresinol attenuated mRNA and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cytokines in LPS-activation. However, most of these inhibitory effects of pinoresinol have been mediated by extracellular-signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) phosphorylation and the NF-κB dependent. The results suggest that pinoresinol attenuates inflammatory responses of microglia and could be potentially useful in modulation of inflammatory status in brain disorders.     

Sesamin inhibits lipopolysaccharide-induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-kappaB

Sesame seed oil increases the survival after cecal ligation and puncture in mice and the increased IL-10 levels with non-lethal lipopolysaccharides (LPS) challenge. We showed that sesamin and sesamolin, major lignans of sesame oil, regulated LPS-induced nitric oxide production in the murine microglia and BV-2 cell line. In this study, we studied the effect of sesamin on cytokine production by LPS stimulation. The result showed that sesamin significantly inhibited LPS-stimulated IL-6 mRNA and protein, and to a lesser degree TNF-alpha, in BV-2 microglia. Sesamin and sesamolin also reduced LPS-activated p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activations. Furthermore, SB203580, a specific inhibitor of p38 MAP kinase, specifically inhibited LPS-induced IL-6 production. These results suggest that sesamin inhibited LPS-induced IL-6 production by suppression of p38 MAPK signal pathway and NF-kappaB activation.     

Central role of microglia in neonatal excitotoxic lesions of the murine periventricular white matter

Periventricular leukomalacia (PVL) is the main cause of neurologic handicap in pre-term infants. The understanding of cellular and molecular mechanisms leading to white matter damage is critical for development of innovative therapeutic strategies for PVL.The pathogenesis of PVL remains unclear but possibly involves glutamate excitotoxicity as an important molecular pathway. We previously described a neonatal mouse model of excitotoxic white matter lesion mimicking human PVL. In the present study, we used this experimental tool to investigate the cellular populations and the glutamate receptor subtypes involved in excitotoxic white matter lesions. Combined immunohistochemical, electron microscopic, and cell death detection data revealed that microglial activation and astrocytic death were the primary responses of white matter to excitotoxic insult. In vitro experiments suggested that microglia activated by ibotenate released soluble factors that kill astrocytes. The use of selective agonists and antagonists of glutamate receptors revealed that N-methyl-D-aspartate (NMDA) receptor activation was essential and sufficient to produce cystic white matter lesions. NMDA receptor immunohistochemistry labeled microglial cells in the neonatal periventricular white matter. The developing white matter displayed a window of sensitivity to excitotoxic damage that was paralleled by the transient presence of NMDA receptor-expressing white matter cells. Assuming that similar pathophysiologic mechanisms are present in human pre- term infants, microglia and NMDA receptors could represent key targets for treatment of PVL.     

Plexin A3 is involved in semaphorin 3F-mediated oligodendrocyte precursor cell migration

Class 3 semaphorins are expressed in the neurodevelopmental or damage repair phase of the central nervous system (CNS). They play an important role in guiding axon growth and directing cell migration, including the migration of oligodendrocyte precursor cells (OPCs). As co-receptors for semaphorin 3F(sema3F), the expression and role of neuropilin-2 (NRP2) and plexin A3 in OPC migration are unclear. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrated that primary OPCs and immature oligodendrocytes from neonatal rats express NRP2 and plexin A3. After transfection with NRP2 siRNA and plexin A3 siRNA, the number of migrating OPCs attracted to sema3F remarkably decreased. These results suggest that plexin A3 is expressed in OPCs and immature oligodendrocytes and is involved in OPC migration.     

Kv1.1 expression in microglia regulates production and release of proinflammatory cytokines,endothelins and nitric oxide

Potassium channels play an important role in microglial activation but their involvement in main functions of microglia including secretion of proinflammatory cytokines has remained uncertain. This study has revealed the specific expression of Kv1.1 in microglia both in vivo and in vitro. Kv1.1 immunoreactivity was localized in the amoeboid microglia in the rat brain between postnatal (P) day 1 (P1) and day 10 (P10); it was, however, progressively reduced with age and was hardly detected at P14 and P21 in ramified microglia, a derivative cell of amoeboid microglia. Following hypoxic exposure, Kv1.1 expression in amoeboid microglia was enhanced or induced in ramified microglia in more mature brain at P21 when compared with their matching controls. RT-PCR and Western blot analysis confirmed Kv1.1 mRNA and protein expression in murine BV-2 cells which was up-regulated by hypoxia or lipopolysaccharide (LPS) treatment; it was reduced significantly by dexamethasone. Neutralization with Kv1.1 antibody suppressed the expression and release of tumor necrosis factor-α, interleukin-1β, endothelins and nitric oxide (NO) in LPS-activated BV-2 cells. It is concluded that Kv1.1, constitutively expressed by microglia, is elicited by hypoxia and LPS and this may be linked to production of proinflammatory cytokines, endothelins and NO.     

Releasing factors from mature neurons modulate microglial survival via purinergic receptor activation

Activated microglia release many types of substances to neurons. However, little is known concerning how information from neurons is received by microglia prior to the induction of these substances. Here, we examined whether neurons modulate microglial function. Treatment with conditioned medium of mature cerebellar granule neurons (CGCs) and cortical neurons significantly induced the death of lipopolysaccharide (LPS)-stimulated microglia. On the other hand, treatment with conditioned medium of mature superior ganglion neurons induced microglial cell death in neither the presence nor absence of LPS. Conditioned medium of mature CGCs induced nuclear condensation. In contrast, treatment with heat-treated conditioned medium or low-calcium ion medium prevented the death of LPS-stimulated microglia. Pretreatment with P2X7 agonist enhanced microglial cell death in neither the presence nor absence of LPS. These findings suggest that unknown pyrolytic releasing factors of brain-derived mature neurons influence microglial survival.     

Lipopolysaccharide-activated microglia lower P-glycoprotein function in brain microvascular endothelial cells

P-glycoprotein, an efflux transporter that is highly expressed at the blood–brain barrier (BBB), is involved in the traffic of several compounds across the BBB. BBB disruption under pathological conditions is observed in parallel with microglial activation. Previous studies of the interaction between rat brain endothelial cells (RBECs) and microglia have shown that lipopolysaccharide (LPS) activated microglia increase the permeability of RBECs through a mechanism involving NADPH oxidase. In this study, to investigate whether LPS-activated microglia are linked to P-gp dysfunction at the BBB, we examined the effect of LPS on P-gp function in a coculture system with RBECs and rat microglia. When LPS at a concentration showing no effect on the RBEC monolayer was added for 6 h to the abluminal side of the RBEC monolayer and RBEC/microglia cocultures, cellular accumulation of the P-gp substrate rhodamine 123, in RBECs, was increased by LPS in the RBEC/microglia coculture. This increased accumulation of rhodamine 123 in RBECs was blocked by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor. P-gp expression on RBECs was not influenced by treatment with LPS in either RBEC monolayers or RBEC/microglia cocultures. These findings suggest that activated microglia induce P-gp dysfunction at the BBB through an NADPH oxidase-dependent pathway.