Hypertrophic cranial pachymeningitis induced by long-term administration of nonsteroidal antiinflammatory drugs.

OBJECTIVE: To report a case of hypertrophic cranial pachymeningitis (HCP) associated with the long-term administration of nonsteroidal antiinflammatory drugs (NSAIDs). CASE SUMMARY: A 23-year-old man presented with recurrent headaches as the primary clinical manifestation. After the administration of the NSAIDs indomethacin and aceclofenac for 2 years, he developed signs of progressive cranial polyneuropathies (eg, II, III, V, VI, and VII palsy) and damage to the brainstem. Cranial contrast-enhanced magnetic resonance imaging (MRI) revealed curvilinear subdural enhancement and significant tentorium cerebelli and falx cerebri enhancements. Since antituberculosis treatment combined with corticosteroid therapy and analgesia with celecoxib for 40 days had not achieved satisfactory results, NSAIDs were discontinued and a single oral dose of a corticosteroid was given. No headaches were reported at a 6-month follow-up appointment. In addition, his cranial polyneuropathy improved significantly. Reexamination by contrast-enhanced MRI scan demonstrated that tentorial enhancement and thickening of the falx cerebri were markedly alleviated. DISCUSSION: No additional causes of HCP were found during systematic investigation in this patient. In addition to headache, cranial polyneuropathy and thickened cerebral dura mater appeared after administration of NSAIDs for 2 years. The symptoms that appeared during the NSAID therapy were remarkably alleviated 5 months after medication discontinuation. Adverse drug reaction (ADR) assessment revealed that long-term administration of NSAIDs may be associated with the occurrence and development of HCP. CONCLUSIONS: Long-term administration of NSAIDs is a probable cause of HCP. Clinicians should be aware of this ADR and avoid prescribing NSAIDs for an extended period.

miR­210 enhances mesenchymal stem cell­modulated neural precursor cell migration.

The migration of endogenous neural stem cells and neural precursor cells (NPCs) to sites of injury is essential for neuroregeneration following hypoxic­ischemic events. Bone marrow­derived mesenchymal stem cells (BMSCs) are a potential therapeutic source of cells following central nervous system damage; however, few studies have investigated the effects of BMSCs on cell migration. Thus, in the present study, the effects of BMSCs on NPC migration were investigated. In the present study, BMSCs and NPCs were isolated and cultured from mice. The effects of BMSCs on the migration of NPCs were analyzed using a Transwell cell migration assay. BMSCs were transfected with microRNA­210 (miR­210) mimics and inhibitors to examine the effects of the respective upregulation and downregulation of miR­210 in BMSCs on the migration of NPCs. Then, miR­210 expression in BMSCs were quantified and the expression levels of vascular endothelial growth factor­C (VEGF­C), brain derived neurotrophic factor (BDNF) and chemokine C­C motif ligand 3 (CCL3) in the supernatant under hypoxic conditions were investigated via reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and ELISA. Subsequently, the expression of VEGF­C, BDNF and CCL3 in BMSCs overexpressing miR­210 or BMSCs suppressing miR­210 was examined by RT­qPCR and western blot analyses. BMSCs promoted the migration of NPC, particularly when pre­cultured with BMSCs for 24 h and co­cultured with NPCs for 24 h; the miR­210 expression levels increased under hypoxic conditions. Additionally, the migration of NPCs was also increased when the BMSCs overexpressed miR­210 compared with the BMSCs transfected with a negative control miR and BMSCs with downregulated miR­210 levels. The expression levels of VEGF­C increased in the BMSCs that overexpressed miR­210 and were decreased in BMSCs transfected with a miR­210 inhibitor. The results of the present study indicated that BMSCs promote the migration of NPCs. Overexpression of miR­210 in BMSCs enhanced NPC migration and may be associated with increases in VEGF­C expression levels.

[The progress of research on the influencing factor of nuclear transfer].

Nuclear transfer (NT) is a new cloning technology developed in recent years. NT methods consist of electrofusion, NT mediated by polyethylene glycol (PEG) and microinjection. The success of somatic nuclear transfer depends on the source of donor nucleus, developmental stage of recipient cytoplasts, cell cycle synchrony of donor nucleus. Different methods of harvesting cells have effect on the efficiency of NT. The somatic nucleus will be reprogrammed after NT and will restore a totipotent state in order to undergo development.

Brain regional synchronous activity predicts tauopathy in 3×TgAD mice.

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and by extensive neuronal loss associated with extracellular amyloid ß-peptide (Aß) plaques and intraneuronal tau pathology in temporal and parietal lobes. AD patients are at increased risk for epileptic seizures, and data from experimental models of AD suggest that aberrant neuronal network activity occurs early in the disease process before cognitive deficits and neuronal degeneration. The contributions of Aß and/or tau pathologies to dysregulation of neuronal network activity are unclear. Using a transgenic mouse model of AD (3×TgAD mice) in which there occurs differential age-dependent development of tau and Aß plaque pathologies, we applied analysis of resting state functional magnetic resonance imaging regional homogeneity, a measure of local synchronous activity, to discriminate the effects of Aß and tau on neuronal network activity throughout the brain. Compared to age-matched wild-type mice, 6- to 8-month-old 3×TgAD mice exhibited increased regional homogeneity in the hippocampus and parietal and temporal cortices, regions with tau pathology but not Aß pathology at this age. By 18-24 months of age, 3×TgAD mice exhibited extensive tau and Aß pathologies involving the hippocampus and multiple functionally related brain regions, with a spatial expansion of increased local synchronous activity to include those regions. Our findings demonstrate that age-related brain regional hypersynchronous activity is associated with early tau pathology in a mouse model, consistent with a role for early tau pathology in the neuronal circuit hyperexcitability that is believed to precede and contribute to neuronal degeneration in AD.

MicroRNA-210 Promotes Accumulation of Neural Precursor Cells Around Ischemic Foci After Cerebral Ischemia by Regulating the SOCS1-STAT3-VEGF-C Pathway.

BACKGROUND: Neural precursor cell (NPC) migration toward lesions is key for neurological functional recovery. The neovasculature plays an important role in guiding NPC migration. MicroRNA-210 (miR-210) promotes angiogenesis and neurogenesis in the subventricular zone and hippocampus after cerebral ischemia; however, whether miR-210 regulates NPC migration and the underlying mechanism is still unclear. This study investigated the role of miR-210 in NPC migration. METHODS AND RESULTS: Neovascularization and NPC accumulation was detected around ischemic foci in a mouse model of middle cerebral artery occlusion (MCAO) and reperfusion. Bone marrow-derived endothelial progenitor cells (EPCs) were found to participate in neovascularization. miR-210 was markedly upregulated after focal cerebral ischemia/reperfusion. Overexpressed miR-210 enhanced neovascularization and NPC accumulation around the ischemic lesion and vice versa, strongly suggesting that miR-210 might be involved in neovascularization and NPC accumulation after focal cerebral ischemia/reperfusion. In vitro experiments were conducted to explore the underlying mechanism. The transwell assay showed that EPCs facilitated NPC migration, which was further promoted by miR-210 overexpression in EPCs. In addition, miR-210 facilitated VEGF-C (vascular endothelial growth factor C) expression both in vitro and in vivo. Moreover, the luciferase reporter assay demonstrated that miR-210 directly targeted the 3' untranslated region of SOCS1 (suppressor of cytokine signaling 1), and miR-210 overexpression in HEK293 cells or EPCs decreased SOCS1 and increased STAT3 (signal transducer and activator of transcription 3) and VEGF-C expression. When EPCs were simultaneously transfected with miR-210 mimics and SOCS1, the expression of STAT3 and VEGF-C was reversed. CONCLUSIONS: miR-210 promoted neovascularization and NPC migration via the SOCS1-STAT3-VEGF-C pathway.

[Triplex-forming oligonucleotide inhibits the expression of tissue factor gene in endothelial cells induced by the blood flow shear stress in rats].

AIM: To study the effect of antiparallel phosphorothioate triplex-forming oligonucleotide (apsTFO) matching with the shear stress response element (SSRE) of tissue factor (TF) gene promoter region on the expression of TF in endothelial cells (ECs) of rat common carotid artery stenosis. METHODS: The model of common carotid artery middle segment stenosis was established by silica gel pipe loop ligation in SD rats. The mRNA expression and protein synthesis of TF, early growth response-1 (Egr-1) and specificity protein 1 (Sp1) were measured by in situ hybridization (ISH) and immunohistochemistry (IHC) technique. GT21-apsTFO, GT20-apsTFO, GT20-psTFO and FITC-labeled apsTFO, matching with the SSRE of TF gene promoter region, were designed, and intravenously injected into rats at 0.5 h before operation. TFO was detected 4 h after the operation, and the mRNA expression and protein synthesis of TF, Egr-1 and Sp1 were detected 6 h after the operation. RESULTS: There were much fluorescence in vascular tissue, especially in the nuclear of ECs 4.5 h after the injection of apsTFO. The mRNA expression and protein synthesis of TF reduced by 22% - 23% with injection of GT20-apsTFO 6.5 h after stenosis (P < 0.01) and by 10% - 11% with GT21-apsTFO at the same time (P < 0.05). The inhibition by GT20-apsTFO was stronger than that of the GT21-apsTFO (P < 0.05). The expression of TF was not inhibited by the GT20-psTFO (P > 0.05). The mRNA expression and protein synthesis of Egr-1 and Sp1 did not change in the rat treated with GT20-apsTFO, GT20-psTFO and GT21-apsTFO (P > 0.05). CONCLUSION: apsTFO could mero-inhibit the expression of TF gene but could not change the expression of Egr-1 and Sp1 protein.

[The study of the effect of the administration of mannitol on flash visual evoked potentials].

OBJECTIVE: To study the relationship between the change of N2 wave in flash visual evoked potentials (fVEP) and the intracranial pressure after the administration of mannitol. METHODS: Fifty-two patients with elevated intracranial pressure were chosen and are divided into two groups: the former (n = 32) were treated with mannitol, the latter (n = 20) were treated with mannitol and glycerol and sodium chloride injection. The latency and amplitude of N2 wave of fVEP was measured by NIP-200 noninvasive intracranial pressure apparatus before and after the 1st, 4th, 10th administration of mannitol. RESULTS: The latency of N2 wave began to reduce at 30 min after the injection of mannitol and was lowest at 2 h. At 4 h after the injection of mannitol, the latency of N2 wave increased but was still shorter than that before the injection of mannitol. The amplitude of N2 wave did not change significantly. The reduction of the latency of N2 wave after the administration of mannitol decreased with the multiple mannitol injection. The latency of N2 wave did not change significantly after the administration of mannitol when combined with glycerol and sodium chloride injection. CONCLUSION: The latency of N2 wave of fVEP changes after the administration of mannitol, which suggests that the change of the latency of N2 wave of fVEP can reflect the change of intracranial pressure.

Specific locations within the white matter and cortex are involved in the cognitive impairments associated with periventricular white matter lesions (PWMLs).

The aim of this study was to test the hypothesis that both white matter disruption and the corresponding cortical dysfunction are involved in the cognitive impairments associated with periventricular white matter lesions (PWMLs). Twenty-two PWMLs subjects were divided into cognitively impaired (PWMLs-CI) and normal (PWMLs-CN) groups. Twelve subjects with normal magnetic resonance imaging (MRI) and cognition were recruited as controls. After cognitive evaluation, diffusion tension image (DTI) and resting-state functional MRI (rfMRI) scans, the fractional anisotropy (FA) values of DTI and the fractional amplitude of low-frequency fluctuation (fALFF) values of rfMRI were measured. Finally, correlations between the cognitive scores and MRI values were analyzed in PWMLs subjects. Our results demonstrated that compared with the other groups, the PWMLs-CI group demonstrated significantly decreased scores in Trail-Making Test (TMT), Symbol Digit Modalities Test (SDMT) and Logical Memory Test (LMT). Compared with the PWMLs-CN group, the PWMLs-CI group displayed decreased FA values in the right splenium of the corpus callosum and right posterior cingulum bundle; lower fALFF values in the left frontal middle gyrus, left precentral gyrus, right angular gyrus and right precuneus; and higher fALFF values in the right mid cingulum cortex, right hippocampus amygdala, right cerebellar hemisphere and left vermis. Meanwhile, the cognitive assessment scores were significantly correlated with the FA or fALFF values in some of the above-mentioned white matter or cortical regions. Conclusively, our results indicate that specific regions of WMLs and cortical dysfunction are involved in the cognitive impairments associated with PWMLs.

Fractalkine promotes chemotaxis of bone marrow-derived mesenchymal stem cells towards ischemic brain lesions through Jak2 signaling and cytoskeletal reorganization.

The fractalkine (FKN)-CX3CR1 (FKN receptor) axis reportedly plays an important role in the progression of many neural pathologies. However, its role in the recruitment of bone marrow-derived progenitor cells for neurogenesis remains elusive. The chemokine-based mechanism underlying the migration of bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a double-chamber transmigration model with recombinant FKN and endogenous FKN extract, and the results confirmed the involvement of FKN in migration. This chemotactic response was CX3CR1-dependent and FKN-sensitive. Western blotting, immunoprecipitation and transmigration assays revealed that the Janus kinase (Jak)2-signal transducer and activator of transcription (Stat)5α-extracellular signal-related kinase (ERK)1/2 pathway was activated by FKN. Confocal laser scanning microscopy was used to demonstrate cytoskeletal reorganization caused by remodeling of the surface receptor integrin α5ß1, intracellular phosphorylation of Fak and Pax, and upregulation of intercellular adhesion molecule-1 during BMSC migration. Moreover, significant inhibition of signaling and migration was detected after treatment of cells with Jak2-interfering RNA or the antagonist AG490. In addition, the results of a fluorescence immunohistochemical analysis of an in vivo chemotactic model, developed via transplantation of BMSCs into transient middle cerebral artery-occluded rats, were consistent with the in vitro results. These findings suggest that FKN activates Jak2-Stat5α-ERK1/2 signaling through CX3CR1, thereby triggering integrin-dependent machinery reorganization to allow chemotactic migration of BMSCs towards an ischemic cerebral lesion.

Adenosine A2A receptor deficiency up-regulates cystatin F expression in white matter lesions induced by chronic cerebral hypoperfusion.

In previous studies, we have shown that the inactivation of the adenosine A2A receptor exacerbates chronic cerebral hypoperfusion-induced white matter lesions (WMLs) by enhancing neuroinflammatory responses. However, the molecular mechanism underlying the effect of the adenosine A2A receptor remains unknown. Recent studies have demonstrated that cystatin F, a potent endogenous cysteine protease inhibitor, is selectively expressed in immune cells in association with inflammatory demyelination in central nervous system diseases. To understand the expression of cystatin F and its potential role in the effect of A2A receptor on WMLs induced through chronic cerebral hypoperfusion, we investigated cystatin F expression in the WMLs of A2A receptor gene knockout mice, the littermate wild-type mice and wild-type mice treated daily with the A2A receptor agonist CGS21680 or both CGS21680 and A2A receptor antagonist SCH58261 after chronic cerebral hypoperfusion. The results of quantitative-PCR and western blot analysis revealed that cystatin F mRNA and protein expression were significantly up-regulated in the WMLs after chronic cerebral hypoperfusion. In addition, cystatin F expression in the corpus callosum was significantly increased in A2A receptor gene knockout mice and markedly decreased in mice treated with CGS21680 on both the mRNA and protein levels. Additionally, SCH58261 counteracted the attenuation of cystatin F expression produced by CGS21680 after chronic cerebral hypoperfusion. Moreover, double immunofluorescence staining revealed that cystatin F was co-localized with the activated microglia marker CD11b. In conclusion, the cystatin F expression in the activated microglia is closely associated with the effect of the A2A receptors, which may be related to the neuroinflammatory responses occurring during the pathological process.

Phenotypic characteristics of hybrid cells generated by transferring neuronal nuclei into bone marrow stromal cell cytoplasts.

Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally.

Fractalkine and CX3CR1 are involved in the migration of intravenously grafted human bone marrow stromal cells toward ischemic brain lesion in rats.

Recent research has shown that transplanted bone marrow stromal cells (MSCs) migrate to the injured regions and exert their therapeutic effects in cases of intracranial trauma, stroke, inflammation and degenerative disease. The specific mechanisms involved in their migration to lesions are still to be fully elucidated. In the present study, a rat model of transient middle cerebral artery occlusion (MCAO) was established. At 24 h after reperfusion, human bone marrow stromal cells (hMSCs) were transplanted by intravenous injection to explore the effects of fractalkine/CX3CR1 on the migration of transplanted MSCs to lesions. In vitro study using real-time PCR and western blot revealed that CX3CR1, the only known receptor of fractalkine, was expressed in cultured hMSCs. The expression of fractalkine in the ischemic brain was significantly increased. The directional migration of transplanted hMSCs to the damaged region was observed through detection of green fluorescence protein (GFP). The results indicated the cells were mainly distributed in the ischemic boundary zone with high fractalkine expression. In a further study, lentivirus-mediated RNA interference of CX3CR1 expression was employed. The results of these experiments indicated that CX3CR1 knock-down dramatically decreased the migration of hMSCs to the ischemic brain. The present study suggests that fractalkine and its specific receptor CX3CR1 are involved in the directional migration of transplanted MSCs to the ischemic damaged brain region.

Adenosine A2A receptor deficiency exacerbates white matter lesions and cognitive deficits induced by chronic cerebral hypoperfusion in mice.

Adenosine A2A receptor inactivation consistently protects against acute ischemic brain injury; however, the role of the A2A receptor in chronic cerebral ischemia is unknown. To elucidate that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in A2A receptor knock-out mice and their wild-type littermates in this study. White matter lesions were observed after stenosis of common carotid arteries in both A2A receptor knock-out mice and wild-type mice. The demyelination-related damage and proliferation of astrocytes and microglia in white matter was observed more seriously in A2A receptor knock-out mice compared with that in wild-type mice. Working memory was also more seriously impaired in A2A receptor knock-out mice relative to wild-type mice. The mRNA expression and protein level of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) increased more remarkably in the corpus callosum in the A2A receptor knock-out mice. In conclusion, inactivation of the A2A receptor exacerbates the white matter lesions and cognitive deficits induced by chronic cerebral hypoperfusion, and this effect may be associated with increased expression of the proinflammatory cytokines in the white matter.

[Effect of low serum culture on the synchrony of cell cycle of mesenchymal stem cells].

AIM: To investigate a suitable method of inducing cell cycle synchronization at G0/G1 stage of mesenchymal stem cells (MSCs). METHODS: MSCs were cultured and identified with CD44, CD90, CD71 and CD11b by flow cytometer. Cell cycle and apotosis under normal and low serum culture were detected by flow cytometer. RESULTS: MSCs were positive for CD44, CD90 and CD71 and negative for CD11b. The cells at G0/G1 cell stage decreased, while the cells at S and G2 cell stage increased in 1 day of 50 mL/L fetal bovine serum (FBS) and 1 to 3 days of 5 mL/L FBS. However, prolonged culture in serum-starvation medium induced cell cycle arrest at G0/G1 stage. The ratio of apoptotic cells did not increase in 50 mL/L FBS. In 5 mL/L FBS, the ratio of apoptotic cells increased in 3 days and then decreased in 4 to 5 days. The proportion of cells at G0/G1 phase was significantly increased from 75.9% to 89.4% while the ratio of apoptotic cells was only 0.162% in 5 days of 5 mL/L FBS. CONCLUSION: It is a good method to induce the synchrony of cell cycle of MSCs at G0/G1 stage in five days of 5 mL/L FBS.

[Approach to the relationship between the changes of the content of free zinc in hippocampus and ischemic neuronal damage].

AIM: To make approach to the relationship between the changes of free zinc and ischemic neuronal damage in hippocampus after forebrain ischemia/reperfusion. METHODS: The models of forebrain ischemia/reperfusion were established in rats. The contents of free Zn2+ were measured by TSQ fluorescence method. The Zn2+ chelator (CaEDTA) was injected into lateral ventricles in order to evaluate the effect of free Zn2+ on ischemic neuronal damage. RESULTS: (1) Zn2+ fluorescence in the hilus of dentate gyrus, CA3 region and the stratum radiatum and stratum oriens of CA1 decreased slightly at forty-eight hours after reperfusion. From seventy-two hours to ninety-six hour after reperfusion, the decreased fluorescence gradually returned to the normal level, but some fluorescence dots were found in pyramidal neurons of CA1 and the hilus of dentate gyrus. Seven days after reperfusion, all the changes of the fluorescence almost recovered. (2) The cell membrane-impermeable Zn2+ chelator CaEDTA could reduce the intracellular concentration of free Zn2+ and reduced neuronal damage after forebrain ischemia/reperfusion. CONCLUSION: (1) The synaptic vesicle Zn2+ released and then translocated into postsynaptic neurons after forebrain ischemia/reperfusion and played a role in ischemic neuronal damage. (2) The cell membrane-impermeable chelator CaEDTA could provide neuroprotection.