Effects of peripheral inflammation on activation of ERK in the rostral ventromedial medulla

In the present study, the activation of extracellular signal-regulated kinase (ERK) in the rostral ventromedial medulla (RVM) following the injection of complete Freund's adjuvant (CFA) into the rat hindpaw was examined in order to clarify the mechanisms underlying the dynamic changes in the descending pain modulatory system after peripheral inflammation. Phospho-extracellular signal-regulated kinase-immunoreactive (p-ERK-IR) neurons were observed in the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (GiA). Inflammation induced the activation of ERK in the RVM, with a peak at 7 h after the injection of CFA into the hindpaw and a duration of 24 h. In the RVM, the number of p-ERK-IR neurons per section in rats killed at 7 h after CFA injection (14.2 +/- 1.7) was significantly higher than that in the control group (4.5 +/- 0.9) [P < 0.01]. At 7 h after CFA injection, about 60% of p-ERK-IR neurons in the RVM were serotonergic neurons. The percentage of RVM serotonergic neurons that are also p-ERK positive in the rats with inflammation (20.5% +/- 2.3%) was seven times higher than that in control rats (2.7% +/- 1.4%) [P < 0.01]. These findings suggest that inflammation-induced activation of ERK in the RVM may be involved in the plasticity in the descending pain modulatory system following inflammation.     

Perineural clonidine reduces p38 mitogen-activated protein kinase activation in sensory neurons

Perineural injection of clonidine at the site of nerve injury reduces hypersensitivity while simultaneously reducing leukocyte number and cytokine expression and hyperexcitability in sensory neurons. The activation of p38 mitogen-activated protein kinase in sensory neurons contributes to the development and maintenance of inflammatory and neuropathic pain. Here, we tested whether perineural clonidine affected activation of p38 mitogen-activated protein kinase following partial sciatic nerve ligation. Perineural clonidine significantly increased withdrawal threshold and concomitantly reduced phosphorylation of p38 mitogen-activated protein kinase in sensory neurons ipsilateral to injury. Clonidine's effects were blocked by the alpha2-adrenoceptor antagonist, BRL44408. These data suggest that activation of alpha2-adrenoceptors at the site of nerve injury, probably by immune modulation, reduces intracellular signaling in primary afferents that leads to hypersensitivity.     

Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro

Mammalian mitogen-activated protein kinases include the extracellular signal-regulated protein kinase, the c-Jun amino-terminal kinase, and the p38 subgroups. Sustained activation of Jun kinase and p38 have been shown to precede apoptosis of PC12 pheochromocytoma cells induced by withdrawal of trophic factors. To investigate the possible role of p38 in neuronal apoptosis, we tested the effect of two selective p38 inhibitors, the pyridinyl imidazole compounds SB203580 and SB202190, on different populations of chick embryonic neurons in vitro. Both substances promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion. When assayed in nerve growth factor-stimulated PC12 cells, SB203580 pretreatment inhibited the activation of both ribosomal S6 kinases-1 and -2 with the same IC₅₀ (approximately 30 μM) that inhibited apoptosis in primary neurons. Thus, p38 inhibitor-sensitive pathways may be involved in apoptosis of neurotrophic factor-deprived primary neurons, and in activation of ribosomal S6 kinases. J. Neurosci. Res. 52:483–490, 1998. © 1998 Wiley-Liss, Inc.     

p38MAPK在蛛网膜下腔出血后脑血管痉挛中的作用

目的探讨促分裂原活化蛋白激酶p38(p38MAPK)在蛛网膜下腔出血(SAH)后脑血管痉挛(CVS) 中的作用。方法采用枕大池二次注血的方法建立SAH模型。用酶联免疫吸附测定法(ELISA)、免疫组织化学、测量基底动脉横截面积的方法分别检测兔脑脊液肿瘤坏死因子-α(TNF—α)浓度变化及平滑肌细胞p38MAPK的表达与CVS程度变化的关系。结果 TNF—α浓度在注血后第3天达高峰,持续到第5天时,与注血前有明显差异(P< 0．01);平滑肌细胞p38MAPK表达增强;基底动脉横截面积则显著小于对照组(P<0．01)。应用p38MAPK特异性抑制剂SB203580干预后,TNF—α浓度显著降低到注血前水平,平滑肌细胞p38MAPK表达也明显减弱,基底动脉横截面积与对照组相比无明显差异(P>0．05)。结论 SAH后继发性的CVS可能是激活的p38MAPK通过对细胞因子增量调节机制作用的结果。     

Gp120-mediated cytotoxicity of human brain microvascular endothelial cells is dependent on p38 mitogen-activated protein kinase activation

Breakdown of the blood-brain barrier has been shown to contribute to neurological disorders that are prevalent in human immunodeficiency virus type 1 (HIV-1)-infected individuals, but the mechanisms involved in HIV-1-associated blood-brain barrier dysfunction remain incompletely understood. Using human brain microvascular endothelial cells (HBMECs) that constitute the blood-brain barrier, the authors determined the cytotoxic effects of gp120 on HBMECs. The authors showed that gp120 induced cytotoxicity of HBMECs derived from children, which required cotreatment with interferon (IFN)-gamma. IFN-gamma treatment exhibited up-regulation of the chemokine receptors CCR3 and CCR5 in children's HBMECs. In contrast, HBMECs isolated from adults were not responsive to gp120-mediated cytotoxicity. Peptides of gp120 representing binding regions for CD4 and chemokine receptors as well as CD4 antibody inhibited gp120-mediated cytotoxicity of HBMECs. RANTES, as expected, inhibited M-tropic gp120-mediated HBMEC cytotoxicity, whereas stromal cell-derived factor (SDF)-1alpha failed to inhibit T-tropic gp120-mediated cytotoxicity. Of interest, gp120 peptides representing non-CD4/non-chemokine receptor binding regions inhibited gp120-mediated HBMEC cytotoxicity. In addition, the authors showed that gp120-mediated HBMEC cytotoxicity involved p38 mitogen-activated protein kinase pathway. Taken together, these findings showed that gp120, in the presence of IFN-gamma, can cause dysfunction of the blood-brain barrier endothelium via MAPK pathways involving several gp120-HBMEC interactions.     

Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.     

p38MAPK信号通路在小胶质细胞激活介导多巴胺能神经元变性中的作用研究

目的:研究p38丝裂原激活蛋白激酶(p38MAPK)在脂多糖(LPS)诱导小胶质细胞激活介导多巴胺(DA)能神经元变性中的作用。方法:脑立体定位注射LPS于大鼠脑黑质,Western blot印记法检测不同时间点(0、0.5h、1h、6h、12h)黑质p38磷酸化水平。酪氨酸羟化酶(Tyrosine hydroxylase,TH)免疫组织化学染色观察蛋白激酶(MAPK)特异性抑制剂SB203580预处理后LPS对DA能神经元变性的影响。结果:黑质注射LPS后,Western blot结果显示p38MAPK总体蛋白水平在各组均存在表达,无显著性差异(P>0.05),而其磷酸化p-p38MAPK却发生了明显变化。正常对照组和PBS注射侧几乎无p-p38的表达,LPS注射后30min,p-p38即有少量的表达;1h表达量增加;6h表达量达高峰;12h后表达量逐渐下降。与PBS对照侧相比,LPS注入黑质导致TH阳性细胞数下降至38%;SB203580预处理可以显著增加TH+细胞数达63%(P<0.05)。结论:p38MAPK信号通路参与了LPS诱导小胶质细胞激活介导DA能神经元变性,可通过阻断信号通路来减轻LPS诱导DA能神经元变性,为PD治疗提供新的思路。     

Albumin causes increased myosin light chain kinase expression in astrocytes via p38 mitogen-activated protein kinase

Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton, leading to disruption of vascular barrier integrity in multiple organs, including the blood-brain barrier (BBB), after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia, and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase of MLCK in astrocytes and the signaling pathways involved in this increase. After midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MLCK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK, or rho kinase, also prevented this increase. These results are further evidence of a role of MLCK in the mechanisms of BBB compromise following TBI and identify astrocytes as a cell type, in addition to endothelium in the BBB, that expresses MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB.     

Somatodendritic morphology of on- and off-cells in the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) contains two classes of physiologically defined neurons, on-cells and off-cells, that are implicated in nociceptive modulation. In a continuing effort to detail the neural circuitry that underlies the activity of these two distinct neuronal types, the somatodendritic morphology of on- and off-cells was studied in the cat, rat, and ferret. In lightly anesthetized animals, on-cells increased and off-cells decreased their discharge rate during a withdrawal reflex evoked by noxious stimuli. Following their physiological characterization by using intracellular recording, on- and off-cells were injected with either horseradish peroxidase or biocytin and their somatodendritic arborizations were examined. Labeled on- and off-cells included fusiform and stellate cells of all sizes as well as large multipolar neurons. Although the somatic shape of both on- and off-cells in RVM was heterogeneous, off-cells tended to be fusiform neurons whose long axis was oriented mediolaterally. The dendritic domains of both on- and off-cells extended bilaterally past the lateral edge of the trapezoid body or pyramid and ventrally to, and sometimes including, the trapezoid body or pyramid. In contrast to their extensive mediolateral spread, the dendritic domains of both cell types were limited to the ventral half of the reticular formation and were compressed along the rostrocaudal axis. The dendritic arbor of individual on- and off-cells extended well beyond the cytoarchitectonic boundaries of any single nuclear region, within the domain delineated as the RVM. The spatial domains of the dendritic arbors of on- and off-cells are further evidence that the on- and off-cells throughout the RVM constitute an integrated unit in the modulation of nociceptive transmission.     

Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance

Compelling evidence has suggested that spinal glial cells were activated by chronic morphine treatment and involved in the development of morphine tolerance. However, the mechanisms of glial activation were still largely unknown in morphine tolerance. In present study, we investigated the role of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal cord in the development of chronic morphine antinociceptive tolerance. We found that intrathecal administration of morphine (15 microg) daily for 7 consecutive days significantly induced an increase in number of phospho-p38 (p-p38) immunoreactive cells in the spinal cord compared with chronic saline or acute morphine treated rats. Double immunofluorescence staining revealed that p-p38 immunoreactivity was exclusively restricted in the activated spinal microglia, not in astrocytes or neurons. Repeated intrathecal administration of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (10 microg or 2 microg), a specific p38 inhibitor, 30 min before each morphine injection for 7 consecutive days significantly attenuated tolerance to morphine analgesia assessed by tail flick test. However, a single intrathecal administration of SB203580 (10 microg) did not antagonize the established tolerance to morphine analgesia. Taken together, these findings suggested that p38 MAPK activation in the spinal microglia was involved in the development of morphine antinociceptive tolerance. Inhibition of p38 MAPK by SB203580 in the spinal cord attenuated but not reversed the tolerance to morphine analgesia. The present study provides the first evidence that p38 activation in spinal microglia played an important role in the development of tolerance to morphine analgesia.     

Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling

Extracellular hypertonicity can induce the phosphorylation of mitogen-activated protein kinases (MAPKs). Of these, both extracellular signal-regulated kinases (ERKs) and the stress-activated kinase p38 have been implicated in neuronal cell survival. Resuscitation with hypertonic saline decreases secondary brain injury after trauma, as well as neuronal damage, after ischemia. Since hypertonicity has been shown to support somatic cell survival, we investigated if hypertonicity can also prevent neuronal cell death via MAPK signaling. Death of postnatal rat corticospinal motoneurons (CSMNs) was induced by serum deprivation, and survival in both isotonic and hypertonic media was assessed after 20 h. Addition of NaCl (4–250 mM) to isotonic medium significantly and dose dependently protected CSMN in enriched cultures, increasing cell survival by up to 70% over that in isotonic medium. This response was not restricted to NaCl; addition of KCl, choline chloride, and sucrose had similar effects on cell survival. In addition, hypertonicity supported the survival of pure CSMN populations, albeit with lower potency. In cortical cell suspensions, hypertonic NaCl (20–100 mM) increased basal phosphorylation of p38 and ERK. The activation of both MAPKs, which was induced by 40 mM NaCl, was transient. Cultivation of CSMNs in media containing the specific p38 inhibitor SB203580 abolished the protective effect of hypertonic NaCl, indicating a central role for p38. We therefore conclude that hypertonicity can prevent neuronal cell death via MAPK signaling.     

Spinal p38 mitogen-activated protein kinase mediates allodynia induced by first-degree burn in the rat

Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been implicated in the development and maintenance of pain states. In this study, we tested whether p38 MAPK is involved in the response to first-degree burn of the hind paw. This injury induces central sensitization leading to tactile allodynia and is mediated by activation of Ca(2+) permeable AMPA/kainate receptors through PKC and PKA. We demonstrate that p38 MAPK is rapidly and robustly activated in the superficial spinal dorsal horn after mild thermal injury to the hind paw. Activated p38 MAPK was localized primarily to microglia and to a lesser extent in oligodendrocytes and lamina II neurons. Astrocytes were not involved in the p38 MAPK response. Intrathecal pretreatment of pharmacological inhibitors of p38 MAPK (SB203580, SD-282) dose-dependently blocked development of tactile allodynia, a characteristic of the first-degree burn model. The effects of the inhibitors on tactile allodynia were lost when they were administered after injury. These studies identify p38 MAPK as a major mediator of tactile allodynia, most likely activated downstream of AMPA/kainate receptors.     

p38 mitogen-activated protein kinase inhibitor reduces neurocan production in cultured spinal cord astrocytes

Chondroitin sulfate proteoglycans are formed in scar tissue after a spinal cord injury and inhibit axon regrowth. The production of neurocan, one of these chondroitin sulfate proteoglycans, in cultured spinal cord astrocytes increased after the addition of epidermal growth factor (EGF) in a dose-dependent manner (2-200 ng/ml). In astrocytes stimulated by 20 ng/ml of EGF, neurocan production was inhibited after the addition of the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580: 3-10 μM) in a dose-dependent manner. These results suggest that the activation of p38 MAPK is one of the mechanisms of neurocan production in EGF-stimulated astrocytes. The p38 MAPK inhibitor may reduce neurocan production and accelerate axonal regrowth after a spinal cord injury.     

Evidence that p38 mitogen-activated protein kinase contributes to neonatal hypoxic-ischemic brain injury

We tested the response of stress-activated mitogen-activated protein kinases (MAPKs) - p38 MAPK and c-JUN NH2-terminal kinase (JNK) - following hypoxia-ischemia (H-I) induced by unilateral carotid artery ligation and hypoxia (8% O2 and 92% N2) for 2.5 h in postnatal-day-7 rats. Phosphorylation of p38 MAPK increased in the hippocampus and cortex immediately following H-I and returned to a basal level 6 h later. In contrast to p38 MAPK, phosphorylation of JNK decreased in the hippocampus and cortex immediately following H-I. Intracerebroventricular administration of two different p38 MAPK inhibitors prior to H-I significantly protected the neonatal brain from H-I injury. Interestingly, p38 MAPK inhibitors did not attenuate caspase-3 activation 24 h after H-I. Thus, these data suggest that p38 MAPKs contribute to the rapid, early component of brain injury following neonatal H-I.     

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla

Within the rostral ventromedial medulla (RVM), there are two classes of putative pain modulation neurons: ON cells and OFF cells, which respectively burst or pause prior to withdrawal reflexes elicited by noxious stimulation. Alpha-adrenergic agonists injected into the RVM produce changes in the latency of spinal nocifensive reflexes and, when iontophoretically applied, alter the firing of RVM ON but not OFF cells. To provide further information about the contribution of norepinephrine to RVM neuron function, we analyzed the distribution of tyrosine hydroxylase immunoreactive (TH-ir) appositions upon RVM ON and OFF cells. In the lightly anesthetized rat, seven ON and five OFF cells were identified by changes in their discharge rate in relation to nociceptive withdrawal reflexes and were labeled by intracellular injection of neurobiotin. Sections containing labeled cells were visualized by using avidin conjugated to a Texas Red fluorophore. Tissue with labeled cells was subsequently processed for TH-ir by using a Bodipy fluorophore conjugated secondary antibody. The distribution of the Bodipy-labeled fibers and terminals upon the Texas Red-labeled neurons was mapped using a confocal laser-scanning microscope. All the labeled neurons exhibited close TH-ir appositions. Appositions were of two types: swellings and fibers. Although the numbers and density of appositions varied among the cells, there were no consistent differences that correlated with physiological properties. Thus the overall density of appositions for ON cells (29.0 ± 22.2 × 10⁴ μm²) did not differ significantly from that for OFF cells (25.4 ± 22.2 × 10⁴ μm²). Tyrosine hydroxylase immunoreactive (TH-ir) appositions upon ON and OFF cells varied with their location along the dorso-ventral axis with more ventral neurons having a greater density of TH-ir swelling-type appositions. In a separate study, TH-ir and dopamine-β-hydroxylase-like immunoreactivity (DBH-ir) were mapped in the same sections by using confocal microscopy. Nearly 97% of the TH-ir profiles co-localized with DBH-ir. These observations provide evidence that both ON and OFF cells in the RVM are targeted by noradrenergic inputs. J. Comp. Neurol. 377:381–391, 1997. © 1997 Wiley-Liss, Inc.     

Trigeminal transition zone/rostral ventromedial medulla connections and facilitation of orofacial hyperalgesia after masseter inflammation in rats

Recent studies have implicated a role for the trigeminal interpolaris/caudalis (Vi/Vc) transition zone in response to orofacial injury. Using combined neuronal tracing and Fos protein immunocytochemistry, we investigated functional connections between the Vi/Vc transition zone and rostral ventromedial medulla (RVM), a key structure in descending pain modulation. Rats were injected with a retrograde tracer, FluoroGold, into the RVM 7 days before injection of an inflammatory agent, complete Freund's adjuvant, into the masseter muscle and perfused at 2 hours postinflammation. A population of neurons in the ventral Vi/Vc overlapping with caudal ventrolateral medulla, and lamina V of the trigeminal subnucleus caudalis (Vc), exhibited FluoroGold/Fos double staining, suggesting the activation of the trigeminal-RVM pathway after inflammation. No double-labeled neurons were found in the dorsal Vi/Vc and laminae I-IV of Vc. Injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin, into the RVM resulted in labeling profiles overlapped with the region that showed FluoroGold/Fos double labeling, suggesting reciprocal connections between RVM and Vi/Vc. Lesions of Vc with a soma-selective neurotoxin, ibotenic acid, significantly reduced inflammation-induced Fos expression as well as the number of FluoroGold/Fos double-labeled neurons in the ventral Vi/Vc (P<0.05). Compared with control rats, lesions of the RVM (n=6) or Vi/Vc (n=6) with ibotenic acid led to the elimination or attenuation of masseter hyperalgesia/allodynia developed after masseter inflammation (P<0.05-0.01). The present study demonstrates reciprocal connections between the ventral Vi/Vc transition zone and RVM. The Vi/Vc-RVM pathway is activated after orofacial deep tissue injury and plays a critical role in facilitating orofacial hyperalgesia.     

Inhibitors of p38 mitogen-activated protein kinase enhance proliferation of mouse neural stem cells

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.