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Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.
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Background@#More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model.@*Methods@#Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used.@*Results@#Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately -6.75 (-7.94 to -6.31) D, while the control group remained hyperopic with only a shift of -0.50 (-0.75 to 0.25) D (Z=-3.38, P < 0.01). The AL increased by 0.74 (0.61–0.76) and 0.10 (0.05–0.21) mm in FDM and control groups, respectively (Z = -3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01).@*Conclusion@#Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.
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BACKGROUND@#More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model.@*METHODS@#Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used.@*RESULTS@#Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately -6.75 (-7.94 to -6.31) D, while the control group remained hyperopic with only a shift of -0.50 (-0.75 to 0.25) D (Z = -3.38, P < 0.01). The AL increased by 0.74 (0.61-0.76) and 0.10 (0.05-0.21) mm in FDM and control groups, respectively (Z = -3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01).@*CONCLUSION@#Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.
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<p><b>BACKGROUND</b>In response to the inflammatory reaction, circulating leukocytes aggregate and adhere to the endothelial cells and eventually pervade into tissues, resulting in cell damage. This study was to detect the inflammatory reactions in mouse focal cerebral ischemia and their distinct characteristics in the ischemic basal ganglia and surrounding cortex.</p><p><b>METHODS</b>Mice were subjected to permanent occlusion of the left middle cerebral artery (MCAO) by introducing a suture for 2 to 120 hours. The expression of intercellular adhesion molecule 1 (ICAM-1) and Mac-1 was determined immunohistochemically. The myeloperoxidase (MPO) activity of the ischemic regions was measured.</p><p><b>RESULTS</b>Four hours after MCAO, the number of ICAM-1 positive vessels in the ischemic basal ganglia increased (9.2 +/- 2.8 per mm(2)), peaked at 48 hours (29.6 +/- 4.8 per mm(2)), and decreased after 72 hours. In the ischemic cortex, the number increased rapidly 4 hours after MCAO (19.4 +/- 6.1 per mm(2)), peaked at 48 hours (44.4 +/- 16.8 per mm(2)), and declined after 72 hours. Mac-1 positive cells were seen in the ischemic basal ganglia (3.4 +/- 1.2 per mm(2)) 12 hours after MCAO, peaked after 48 hours (20.2 +/- 6.3 per mm(2)), and decreased after 72 hours. In the ischemic cortex, however, the number increased 4 hours after MCAO (4.3 +/- 1.7 per mm(2)), peaked after 48 hours (20.9 +/- 8.4 per mm(2)), and remained high at 120 hours. The MPO activity increased in the ischemic basal ganglia 12 hours after MCAO (0.111 +/- 0.023 U/g), peaked after 24 hours (0.194 +/- 0.059 U/g), and decreased after 72 hours. In the ischemic cortex, the MPO activity increased 12 hours after MCAO (0.110 +/- 0.032 U/g), peaked after 24 hours (0.210 +/- 0.067 U/g), and remained elevated at 120 hours.</p><p><b>CONCLUSIONS</b>The increased expression of ICAM-1 in the ischemic brain of mouse in the early phase of MCAO followed by the over-expression of Mac-1 and the increased MPO activity suggests that focal ischemia leads to early onset of inflammation. The inflammatory response is more persistent and intensive in the ischemic cortex than in the ischemic basal ganglia.</p>
Assuntos
Animais , Masculino , Camundongos , Gânglios da Base , Química Encefálica , Isquemia Encefálica , Metabolismo , Patologia , Córtex Cerebral , Circulação Cerebrovascular , Inflamação , Molécula 1 de Adesão Intercelular , Antígeno de Macrófago 1 , Artéria Cerebral Média , PeroxidaseRESUMO
<p><b>OBJECTIVE</b>To study the role of extracellular signal-regulated kinase (ERK) in cerebral ischemia and the mechanism of protective effects of U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) on ischemic brain.</p><p><b>METHODS</b>Mice underwent left middle cerebral artery occlusion (MCAO) by introducing a suture in the lumen. U0126 was injected intravenously through the internal jugular vein. The immuno-activity of phosphorylated ERK1/2 (pERK1/2), phosphorylated mitogen activated protein kinase kinase (pMEK), and phosphorylated Elk-1 (pElk-1) was assessed by Western blot analysis and immunohistochemistry. Interleukin (IL)-1beta mRNA level was measured by ribonuclease protection assay.</p><p><b>RESULTS</b>Phosphorylated ERK1/2 in 2 hours MCAO mice was down-regulated after intravenous injection of U0126. The inhibition was dose dependent and treatment time related. pMEK and pElk-1 were also reduced in a similar fashion after U0126 treatment. IL-1beta mRNA increased after 1 and 2 hours of MCAO. After injection of U0126, it was down-regulated during 1 to 4 hours after MCAO.</p><p><b>CONCLUSION</b>Intravenous administration of the MEK inhibitor U0126 inhibits pMEK, pERK1/2, and pElk-1 up-regulation induced by cerebral ischemia. The protective effect of U0126 against ischemic injury is probably resulted from the reduction of IL-1beta mRNA via the inhibition of ERK pathway.</p>