Immunolocalization of p38 MAP kinase in mouse brain

p38 has been implicated to play a critical role in regulating apoptosis in PC12 and cerebellar granule cells, and is inactivated in cultured fetal neurons in response to insulin. Though p38 is activated in microglia after ischemia, the physiological functions of p38 in the brain are not well understood. As a first step to elucidate the physiological functions of p38 in the central nervous system, we raised a polyclonal antibody against p38 and performed immunohistochemical examination to demonstrate the localization of p38 in mouse brain. Strong p38 immunoreactivity was apparent in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, intense p38 immunoreactivity was often observed in myelin sheath-like structures but not in axons. This is the first demonstration of the localization of p38 in the central nervous system and provides an anatomical basis for understanding physiological roles of p38.     

Invited review: focal nerve injury: guidance factors during axonal regeneration

Regeneration of axons after nerve injury is not random but is often not precise, particularly after injuries that involve nerve transection. The factors that guide regenerating axons toward appropriate terminations are complex, but developments in neurobiology are beginning to indicate the mechanism that are involved. These include neurotrophic and neurite promoting factors, chemotactic influences from peripheral tissues, and the properties of the extracellular matrix. Understanding these mechanisms may indicate means of improving functional recovery after nerve injury.     

p38MAK基因诱导胶质瘤细胞凋亡机制的初步研究

目的 初步探讨p38MAPK基因诱导大鼠胶质瘤细胞C6发生凋亡的机制。方法 利用脂质体介导法将p38MAPK基因导入C6细胞中,用夹心法ELISA检测细胞培养液中sTNF-α水平的变化,用流式细胞仪检测膜TNF-α和膜TNFRI水平的变化。结果 转染pCMV5-p38MAPK后,细胞培养液中sTNF-α水平明显升高,膜TNF-α水平无明显变化,膜TNFRI表达升高。结论 p38MAPK可能通过上调sTNF-α和膜TNFRI水平而诱导C6细胞凋亡。     

Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury

Recent work regarding chronic central neuropathic pain (CNP) following spinal cord injury (SCI) suggests that activation of key signaling molecules such as members of the mitogen activated protein kinase (MAPK) family play a role in the expression of at-level mechanical allodynia. Previously, we have shown that the development of at-level CNP following moderate spinal cord injury is correlated with increased expression of the activated (and thus phosphorylated) forms of the MAPKs extracellular signal related kinase and p38 MAPK. The current study extends this work by directly examining the role of p38 MAPK in the maintenance of at-level CNP following spinal cord injury. Using a combination of behavioral, immunocytochemical, and electrophysiological measures we demonstrate that increased activation of p38 MAPK occurs in the spinal cord just rostral to the site of injury in rats that develop at-level mechanical allodynia after moderate SCI. Immunocytochemical analyses indicate that the increases in p38 MAPK activation occurred in astrocytes, microglia, and dorsal horn neurons in the spinal cord rostral to the site of injury. Inhibiting the enzymatic activity of p38 MAPK dose dependently reverses the behavioral expression of at-level mechanical allodynia and also decreases the hyperexcitability seen in thoracic dorsal horn neurons after moderate SCI. Taken together, these novel data are the first to demonstrate causality that increased activation of p38 MAPK in multiple cell types play an important role in the maintenance of at-level CNP following spinal cord injury.     

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。     

Roles of glial p75NTR in axonal regeneration

The neurotrophin receptor p75 (p75NTR) is expressed by both neurons and glia. Nerve injury triggers up-regulation of p75NTR in Schwann cells (SC) but not in central glia. In contrast to neuronal p75NTR, which mediates negative signals from myelin-associated proteins resulting in neurite collapse, glial p75NTR may play a positive role in nerve regeneration by forming neurotrophin chemoattractant gradients or by competitively antagonizing the NOGO/NgR/LINGO-1 signal through cell-cell contact or regulated intramembranous proteolysis (RIP) of p75NTR. This piece presents some recent evidence supporting this hypothesis.     

Inhibition of p38 mitogen-activated protein kinase interferes with cell shape changes and gene expression associated with Schwann cell myelination

In the present study we demonstrate that p38, a member of the mitogen-activated protein kinase (MAPK) family, is essential for ascorbate- and laminin-induced myelination in Schwann cell-dorsal root ganglion neuron cocultures. The inhibitory effect of the specific p38 blockers, PD 169316 and SB 203580, on ascorbate-induced myelination was exerted during the early stages (1-2 days) of ascorbate treatment. Inhibition of p38 was further shown to prevent the alignment of Schwann cells along axons in laminin-treated cocultures. The addition of laminin to Schwann cell-dorsal root ganglion neuron cocultures stimulated phosphorylation of p38, thereby demonstrating a link between laminin-induced myelination and p38 activation. Similarly, the small heat shock protein, Hsp27, which is phosphorylated by MAPKAPK2, a downstream substrate of p38, was phosphorylated in response to the addition of laminin to the cocultures. The p38 inhibitors did not affect the proliferation or survival of Schwann cells in the cocultures as assessed by BrdU incorporation and total cell counts. However, p38 inhibition interfered with an early stage in myelination, thereby preventing ascorbate-induced increases in the levels of mRNAs encoding MBP, MAG, and P(0) and reducing laminin deposition. These results indicate that activation of p38 by a signaling pathway(s) involving laminin and appropriate integrin receptor(s) is required for the alignment of Schwann cells with axons that precedes myelination.     

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.     

转染热休克蛋白70对p38MAPK信号通路的影响

目的：探讨热休克蛋白（HSP70）在人胶质瘤细胞BT-325p38MAPK信号通路中的作用。方法：用脂质体介导法将hsp70基因导入人胶质瘤细胞BT-325中，倒置显微镜观察转染细胞的形态学及粘附性变化，紫外线照射30min后，采用免疫组化和Western-blot方法测定转染前后HSP70的表达水平及照射前后p38MAPK表达情况。结果：免疫组化和Western-blot证实hsp70基因成功转染入BT-325中，转染细胞受到紫外线照射后p38MAPK表达减弱。结论：体外转染hsp70基因可抑制紫外线照射后BT-325细胞p38MAPK的表达。     

In vitro pre-degenerated nerve autografts support CNS axonal regeneration

In the present study we compared, in adult rats, the axonal regeneration of central respiratory neurons within autologous fresh (f-; grafted immediately after removal) and pre-degenerated (pd-; grafted after being stored during 3 days in saline at + 8°C) peripheral nerve grafts (PNGs) implanted within the C2 cervical spinal cord. The proximal end of the left peroneal nerve was implanted in the site of projection of medullary respiratory neurons (ventro-lateral quadrant) and the distal part of each nerve graft was left unconnected (blind-ended graft). PNGs were examined 2 to 4 months after grafting. Central neurons regenerating axons within the PNGs were studied by recording spontaneous unit activity from small strands teased from the grafts. In control f-PNGs (n = 9), 248 filaments had spontaneous activities, 58 of these were respiratory-related, i.e. had discharge patterns identical to those of normal respiratory (inspiratory and expiratory) neurons. The presence of regenerated nerve fibers with spontaneous unitary impulse traffic (n = 216) was found in all pd-PNGs (n = 5). Thirty-four had respiratory patterns identical to those found within f-PNGs and corresponded to efferent activity. No statistically significant differences in axonal regrowth were found between f- and pd-PNGs. In conclusion, f- and pd-PNGs were equally capable of promoting axonal regeneration of central neurons. The neural components (Schwann cells and others) required for axonal regeneration of adult central neurons are still effective following 3 days of in vitro peripheral nerve degeneration without special storage conditions (oxygenation, medium inducing ATP synthesis). These results have clinical implications for nerve graft surgery when time is required for typing the tissues of both donor and recipient (post-mortem allografts) or transportation of graft material.     

Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord

Previous studies have shown that activation of p38 mitogen-activating kinase (MAPK) in spinal microglia participates in the generation of inflammatory and neuropathic pain in various rodent models. However, these studies focused on male mice to avoid confounding effects of the estrous cycle of females. Recent studies have shown that some spinal pro-inflammatory signaling such as Toll-like receptor 4-mediated signaling contributes to pain hypersensitivity only in male mice. In this study we investigated the distinct role of spinal p38 in inflammatory and neuropathic pain using a highly selective p38 inhibitor skepinone. Intrathecal injection of skepinone prevented formalin induced inflammatory pain in male but not female mice. Furthermore, intrathecal skepinone reduced chronic constriction injury (CCI) induced neuropathic pain (mechanical allodynia) in male mice on CCI-day 7 but not CCI-day 21. This male-dependent inhibition of neuropathic pain also occurred in rats following intrathecal skepinone. Nerve injury induced spinal p38 activation (phosphorylation) in CX3CR1-GFP⁺ microglia on CCI-day 7, and this activation was more prominent in male mice. In contrast, CCI induced comparable microgliosis and expression of the microglial markers CX3CR1 and IBA-1 in both sexes. Notably, intraperitoneal or local perineural administration of skepinone inhibited CCI-induced mechanical allodynia in both sexes of mice. Finally, skepinone only reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices of males 7 days post CCI. Therefore, the sex-specific p38 activation and signaling is confined to the spinal cord in inflammatory and neuropathic pain conditions.     

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.     

p38MAPK对耐苯妥英钠和卡马西平癫痫模型大鼠电生理和行为的影响

目的 观察p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)信号通路抑制剂SB202190对耐苯妥英钠和卡马西平大鼠电生理指标和行为学的影响.方法 建立慢性杏仁核点燃癫痫模型,筛选出耐药大鼠,通过侧脑室注射SB202190,对照组注射生理盐水.观察其对各组大鼠后放电阈值(after discharge threshold,ADT)和行为学的影响.结果 相对于对照组,耐药组大鼠在给予SB202190后,ADT明显高于对照组(P＜0.05);Racine行为分级明显下降(P＜0.05).结论 阻断p38MAPK信号通路可以协助AEDs改善耐药大鼠的电生理活动,降低点燃后发作分级,提示p38MAPK信号通路参与了难治性癫痫的耐药.     

电针夹脊穴通过p38信号通路提高实验大鼠痛阈机制的研究

目的观察电针夹脊穴对佐剂性关节炎大鼠背根神经节内磷酸化p38MAPK表达的影响,从信号转导的角度研究电针镇痛的机制。方法以完全福氏佐剂（CFA）佐剂性关节炎大鼠为疼痛模型,采用免疫组化技术,分别检测空白对照组、模型组、及电针治疗组大鼠DRG内磷酸化p38丝裂原活化蛋白激酶表达特征。结果与空白对照组相比,造模后24h、48h及7d后,模型组大鼠痛阈均有明显下降,血浆内β-Ep含量略有上升,同时DRG内p38MAPK磷酸化明显增多,尤以48h后增加明显;电针治疗组大鼠在电针夹脊穴治疗后,24h、48h及7d后后痛阈检测发现其痛阈回升,血浆内β-Ep含量明显上升,同时DRG内p38MAPK磷酸化明显增多。结论p38MAPK可能为电针镇痛中一个重要的信号转导分子,其磷酸化在疼痛及电针镇痛过程中其重要作用。     

Anisomycin activates p38 MAP kinase to induce LTD in mouse primary visual cortex

Anisomycin is both a well-established protein synthesis inhibitor and a potent activator of the p38/JNK MAPK pathway. It has been used to block the late phase of long-term potentiation (LTP) and long-term depression (LTD) in hippocampus. In this study, we have found that anisomycin produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in acute brain slices of mouse primary visual cortex. This anisomycin-mediated fEPSP depression occludes NMDA receptor-dependent LTD induced by low-frequency stimulation (LFS). In contrast, two other protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission or on LTD. Moreover, the decline of the fEPSP caused by anisomycin can be rescued by the application of the p38 inhibitor SB203580 but not by the JNK inhibitor SP600125. These results indicate that activation of p38 MAPK by anisomycin induces LTD and subsequently occludes electrically induced LTD. Also, the occlusion of LFS-LTD by anisomycin suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of a membrane permeant peptide derived from the carboxyl tail of GluR2 subunit of AMPA receptor, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-induced LTD, significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, our results indicate that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating p38 MAPK-mediated endocytosis of APMA receptors in mouse primary visual cortex.     

Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model

Background Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen‐activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E₂ and KCl‐induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine. Methods Rabbits were injected with (i) saline, (ii) LPS, (iii) SB203580, a specific p38 MAPK inhibitor or (iv) SB203580 + LPS. Duodenal contractility was studied in an organ bath. SB203580 was also tested in vitro. The protein expression of p‐p38 and total p38 was measured by Western blot and p‐p38 was localized by inmunohistochemistry. The formation of products of oxidative damage to proteins (carbonyls) and lipids (MDA+4‐HDA) was quantified in intestine and plasma. Key Results ACh, PGE₂ and KCl‐induced contractions decreased with LPS. LPS increased phospho‐p38 expression and the levels of carbonyls and MDA+4‐HDA. SB203580 blocked the effect of LPS on the ACh, PGE₂ and KCl‐induced contractions in vivo and in vitro and the levels of carbonyls and MDA+4‐HDA. P‐p38 was detected in neurons of the myenteric plexus and smooth muscle cells of duodenum. Conclusions & Inferences Lipopolysaccharide decreases the duodenal contractility in rabbits and increases the production of free radicals. p38 MAPK is a mediator of these effects.     

p38MAPK activation and DUSP10 expression in meningiomas

The mitogen activated protein kinase (MAPK) p38MAPK has been implicated in regulation of cell proliferation and apoptosis. However, expression, activation and regulation has not been studied in meningiomas, to our knowledge. p38MAPK is regulated, in part, by dual specificity phosphatases (DUSP) that inactivate signaling by dephosphorylation. DUSP10 is also a likely participant in regulating meningioma proliferation. Five fetal and an adult human leptomeninges and 37 meningioma cultures (MC) were evaluated for DUSP10 as well as phosphorylation of its substrates p38MAPK and p44/42MAPK by western blot and DUSP10 expression by polymerase chain reaction. Platelet derived growth factor-BB (PDGF-BB), transforming growth factor B1 (TGFB1) and cerebrospinal fluid effects on DUSP10 and signaling were also studied in vitro. DUSP10 and phospho-p38MAPK and phospho-p44/42MAPK were detected in all six leptomeninges. DUSP10 was detected in 13 of 17 World Health Organization grade I, 11 of 14 grade II and four of six grade III meningiomas. Phospho-p38MAPK was detected in nine of 17 grade I, two of six grade II, and four of six grade III meningiomas. In the majority of meningiomas DUSP10 expression correlated inversely with phosphorylation of p38MAPK. PDGF-BB increased DUSP10 in MC2 and MC4 and weakly in MC3. TGFB1 increased phosphorylation of p38MAPK and caspase 3 activation. Thus p38MAPK and DUSP10 likely participate in the pathogenesis of meningiomas.     

Inhibitors of p38 MAP kinase increase the survival of transplanted dopamine neurons

Fetal cell transplantation therapies are being developed for the treatment of a number of neurodegenerative disorders including Parkinson's disease [10-12,21,22,24,36,43]. Massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. We have explored a novel protein kinase pathway for its role in apoptosis of dopamine neurons. We have discovered that inhibitors of p38 MAP kinase (the pyridinyl imidazole compounds: PD169316, SB203580, and SB202190) improve survival of rat dopamine neurons in vitro and after transplantation into hemiparkinsonian rats. In embryonic rat ventral mesencephalic cultures, serum withdrawal led to 80% loss of dopamine neurons due to increased apoptosis. Incubation of the cultures with p38 MAP kinase inhibitors at the time of serum withdrawal prevented dopaminergic cell death by inhibiting apoptosis. In the hemiparkinsonian rat, preincubation of ventral mesencephalic tissue with PD169316 prior to transplantation accelerated behavioral recovery and doubled the survival of transplanted dopamine neurons. We conclude that inhibitors of stress-activated protein kinases improve the outcome of cell transplantation by preventing apoptosis of neurons after grafting.