Correlation between Carotid Blood Flow Velocity and Total Magnetic Resonance Imaging Burden of Cerebral Small Vessel Disease in Patients with Recent Small Subcortical Infarcts.

BACKGROUND: The common and internal carotid arteries are the upstream vessels of the small cerebral vessels. The relationship between hemodynamic changes in the significant cervical vessels and cerebral small vessel disease (CSVD) remains uncertain. This research sought to analyze the correlation between carotid blood flow velocity and the total magnetic resonance imaging (MRI) burden of CSVD in patients with recent small subcortical infarcts (RSSIs). METHODS: Data were gathered from individuals diagnosed with RSSIs admitted to Changzhou Second People's Hospital between January 2022 and June 2023. Brain MRI was performed on every patient to determine the overall MRI burden of CSVD, along with carotid duplex ultrasound to evaluate carotid blood flow velocity and pulsatility index (PI) of the common carotid (CCA) and internal carotid (ICA) arteries. The association between carotid blood flow velocity and the total MRI load of CSVD was examined using univariate and multivariate analyses. RESULTS: For our investigation, 272 individuals with RSSIs were screened. 82 individuals had a moderate to severe load of CSVD, while 190 participants showed a mild burden. Patients with moderate to severe burden of CSVD had lower end-diastolic velocity (EDV) and higher PI in CCA and ICA than those with mild load (P < 0.001). After adjusting for variables like age, hypertension, systolic blood pressure, and blood homocysteine levels, multivariate logistic regression analysis showed that EDV in CCA (OR, 0.894; P = 0.011), PI in CCA (OR, 5.869; P = 0.017), EDV in ICA (OR, 0.909; P = 0.008), and PI in ICA (OR, 5.324; P = 0.041) were independently related to moderate to severe CSVD burden. Spearman correlation analysis showed that EDV in CCA and ICA was negatively related to the total MRI load of CSVD in patients with RSSIs (P < 0.001). PI in CCA and ICA was positively associated with the whole MRI load of CSVD (P < 0.001). CONCLUSION: Low carotid blood flow velocity and high carotid pulsatility index are independently associated with moderate to severe burden of CSVD.

Blocking postsynaptic density-93 binding to C-X3-C motif chemokine ligand 1 promotes microglial phenotypic transformation during acute ischemic stroke.

We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357-395 of C X3 C motif chemokine ligand 1 (CX3CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3CL1 (comprising amino acids 357-395 of CX3CL1) disrupts the interaction between postsynaptic density-93 and CX3CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3CL1 (357-395aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3CL1. Furthermore, the a disintegrin and metalloprotease domain 17 (ADAM17) inhibitor GW280264x, which inhibits metalloprotease activity and prevents CX3CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3CL1 formation. Additionally, Tat-CX3CL1 (357-395aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31-34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3CL1 (357-395aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3CL1 (357-395aa) is a potential therapeutic agent for ischemic stroke.

FK506-binding protein 5 inhibits proliferation and stimulates apoptosis of glioma cells.

INTRODUCTION: FK506-binding protein 5 (FKBP5) is reported to act as a scaffolding protein for Akt to promote the dephosphorylation of AKT Ser473 and suppress pancreatic cancer growth. However, other studies have shown that FKBP5 promotes tumor growth and chemoresistance through regulating NF-κB signaling in other cancers. In this study, we attempted to investigate the role and mechanism of action of FKBP5 in the regulation of proliferation and apoptosis of glioma cells. MATERIAL AND METHODS: The glioma U251 cell line was used as the model. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by annexin-V staining. Protein expression was detected by Western blot analysis. RESULTS: FKBP5 overexpression inhibited the proliferation of U251 cells significantly (p < 0.05), and promoted the apoptosis of U251 cells significantly (p < 0.05). In addition, FKBP5 overexpression inhibited the phosphorylation of Akt at Ser743, decreased the level of Bcl-2, increased the level of Bax, and enhanced the cleavage of caspase-9 and caspase-3 (p < 0.05 compared to control). In contrast, FKBP5 knockdown enhanced the proliferation of U251 cells, increased the phosphorylation of Akt significantly (p < 0.05), increased the expression of Bcl-2 and decreased the expression of Bax, and decreased the cleavage of caspase-9 and caspase-3 significantly (p < 0.05). CONCLUSIONS: FKBP5 plays the role of a tumor suppressor in glioma by inhibiting the activation of Akt and stimulating the intrinsic mitochondrial apoptotic pathway, and could be used as a new target for gene therapy of glioma.

Fasudil hydrochloride protects neurons in rat hippocampal CA1 region through inhibiting GluR6-MLK3-JNKs signal pathway.

Fasudil hydrochloride (FH), a Rho kinase (ROCK) inhibitor, has been reported to prevent cerebral ischemia in vivo from increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain by what mechanism a ROCK inhibitor can directly protect neurons against ischemic damage. The present study was designed to evaluate whether FH decreased the increased phosphorylation of glutamate receptor 6 (GluR6) and its downstream in GluR6-MLK3-JNKs signal transduction pathway following global transient cerebral ischemia, as a result of protecting against neuronal apoptosis and death. Transient cerebral ischemia was induced by the Pulsinelli-Brierley four-vessel occlusion method. FH (15 mg/kg) was administered to rats by intraperitoneal injection 30 min before ischemia. The phosphorylation and protein expression of GluR6 at 6 h during reperfusion were detected using immunoprecipitation and immunoblotting analysis. The phosphorylation and protein expression of Mixed lineage kinase 3 (MLK3) at ischemia/reperfusion (I/R) 6 h and c-Jun N-terminal kinase (JNK) at I/R 3 d were detected using immunoblotting analysis, respectively. The same method was used to detect the expression of caspase-3 at I/R 6 h. Furthermore, we also use TUNEL staining and Cresyl violet staining to examine the survival neurons in rat hippocampal CA1 regions after 3 and 5 d reperfusion, respectively. Our study indicated that FH could inhibit the increased phosphorylation of GluR6 and MLK3 and the expression of caspase-3 at peaked 6 h of reperfusion and the phosphorylation of JNK (3 d) (p < 0.5). The results of TUNEL staining and Cresyl violet showed that the number of surviving pyramidal neurons in rats hippocampal CA1 subfield increased markedly in FH-treated rats compared with ischemic groups after 3 or 5 d of reperfusion following ischemia (p < 0.5). These results suggested that FH, as a ROCK inhibitor, may be partly responsible for its protective effects against such damage by taking part in GluR6-MLK3-JNKs signaling pathway which modulates ischemic damage. Taken together, this is the first study investigating Rho and ROCK as the upstream of GluR6 taking part in GluR6-MLK3-JNKs signal transduction pathway following cerebral ischemia.

Assembly of the FKBP51-PHLPP2-AKT signaling complex in cerebral ischemia/reperfusion injury in rats.

The imbalance of cell pro-death and pro-survival signaling pathways determines the neuronal fate during cerebral ischemia/reperfusion (I/R) injury. However, the biological mechanisms regulating the balance between activation of the pro-death or the pro-survival signaling pathways remain unclear. In this study, a rat model of I/R injury was established using four-vessel occlusion followed by different times of reperfusion. I/R injury did not affect the level of FK506 binding protein 51 (FKBP51), PH domain and leucine rich repeat protein phosphatases (PHLPP)-2, and AKT, but induced assembly of the FKBP51-PHLPP2-AKT signaling complex, as indicated by the enhancement of interactions among these compounds following reperfusion. Using an antisense oligonucleotide, PHLPP2 expression was effectively inhibited. Critically, the inhibition of PHLPP2 prohibited the interactions of FKBP51, PHLPP2 and AKT, reversed the decrease of p-AKT expression and increased the expression of p-JNKs and p-c-Jun elicited by I/R injury. In addition, PHLPP2 inhibition reversed I/R-injury-induced Caspase-3 cleavage and loss of pyramid neurons in the CA1 region of hippocampus. The results of the current study indicate that the assembly of the FKBP51-PHLPP2-AKT signaling complex plays a critical role in mediating cell death in I/R injury. The inhibition of PHLPP2 via antisense oligonucleotide treatment may be an effective method to prohibit the assembly of the FKBP51-PHLPP-AKT signaling complex, thus balancing the cell pro-survival and pro-death signaling pathways ultimately mitigating cell death in I/R injury.

Neuroprotection of GST, an extract of traditional Chinese herb, against ischemic brain injury induced by transient brain ischemia and reperfusion in rat hippocampus.

In this study, we investigated the effect of GST, an extract of Chinese traditional herb, on transient brain ischemia/reperfusion-induced neuronal cell death. Immunoblotting was used to detect the phosphorylation of MLK, JNK and c-jun. Transient (15 minutes) brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. GST was administrated to the SD rats 20 minutes before ischemia or 1 hour after ischemia. Our data showed that the pretreatment of GST could inhibit phosphorylation of MLK, JNK and c-jun. Moreover, GST showed potent neuroprotective effects on ischemic brain damage in vivo and administration of it 1 hour after ischemia also achieved the protective effects. These results indicate that GST has a prominent neuroprotection action against brain ischemic damage and provides a promising therapeutic approach for ischemic brain injury.

[Frequency of ABO type IgM antibody in 276 infants younger than six months].

The aim of study was to explore the frequency of ABO type IgM antibody in infants younger than six months. 309 hospitalized infants younger than six months were selected at first and their EDTA K(3) anticoagulant blood samples were taken. All the infants were divided into five groups: neonates within 1 week as group I; neonates aged 8 to 14 days as group II; neonates aged 15 days to 1 month as group III; infants aged two to 3 months as group IV and infants aged 4 to 6 months as group V. The monocolonal anti-A, anti-B serums, A cells, B cells and O cells were utilized to carried out the blood typing with tube test. The results indicated that from 309 samples tested 33 AB type sample were excluded. Out of the remains of 276 samples, 29 of 46 samples in group I were positive and with the ABO type consistent rate 63% (29/46); 41 of 64 samples in group II were positive and with the ABO type consistent rate 64% (41/64); 47 of 74 samples in group III were positive and with the ABO type consistent rate 63% (47/74); 28 of 45 samples in group IV were positive and with the ABO type consistent rate 62% (28/45); 40 of 47 samples in group V were positive and with the ABO type consistent rate 85%. It is concluded that the ABO type IgM antibody appear in most infants younger than six months and these IgM antibodies may be regarded as the important evidence for ABO typing in infants.

Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia.

Our previous study indicates that global ischemia facilitates the assembly of the GluR6.PSD-95.MLK3 signaling module, which in turn activated MLK3, leading to exacerbated ischemic neuron death. In addition, JIP1, functioning as a scaffold protein, could couple MLK3-MKK7-JNK to form a specific signaling module and facilitate the activation of the JNK signal pathway. However, the organization, regulation, and function between the two signaling modules and the effects they have on MLK3 activation remain incompletely understood. Here, we show that JIP1 maintains MLK3 in an inactive and monomeric state; once activated, MLK3 binds to PSD-95 and then dimerizes and autophosphorylates. In addition, a GluR6 C-terminus-containing peptide (Tat-GluR6-9c) and antisense oligonucleotides (AS-ODNs) against PSD-95 inhibit the integration of PSD-95 and MLK3 and the dimerization of MLK3, facilitate the interaction of JIP1 and MLK3, and, consequently, perform neuroprotection on neuron death. However, AS-ODNs against JIP1 play a negative role compared to that mentioned above. The findings show that the crosstalk occurs between PSD-95 and the JIP1-mediated signaling module, which may be involved in brain ischemic injury and contribute to the regulation of MLK3 activation. Thus, specific blockade of PSD-95-MLK3 coupling may reduce the extent of ischemia-reperfusion-induced neuronal cell death.

Blockade of the translocation and activation of mitogen-activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia-reperfusion.

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.

Neuroprotection of Tat-GluR6-9c against neuronal death induced by kainate in rat hippocampus via nuclear and non-nuclear pathways.

Previous studies have suggested that glutamate receptor 6 (GluR6) subunit- and JNK-deficient mice can resist kainate-induced epileptic seizure and neuronal toxicity (Yang, D. D., Kuan, C.-Y., Whitmarsh, A. J., Rinocn, M., Zheng, T. S., Davis, R. J., Rakic, P., and Flavell, R. A. (1997) Nature 389, 865-870; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). In this study, we show that kainate can enhance the assembly of the GluR6-PSD95-MLK3 module and facilitate the phosphorylation of JNK in rat hippocampal CA1 and CA3/dentate gyrus (DG) subfields. More important, a peptide containing the Tat protein transduction sequence (Tat-GluR6-9c) perturbed the assembly of the GluR6-PSD95-MLK3 signaling module and suppressed the activation of MLK3, MKK7, and JNK. As a result, the inhibition of JNK activation by Tat-GluR6-9c diminished the phosphorylation of the transcription factor c-Jun and down-regulated Fas ligand expression in hippocampal CA1 and CA3/DG regions. The inhibition of JNK activation by Tat-Glur6-9c attenuated Bax translocation, the release of cytochrome c, and the activation of caspase-3 in CA1 and CA3/DG subfields. Furthermore, kainate-induced neuronal loss in hippocampal CA1 and CA3 subregions was prevented by intracerebroventricular injection of Tat-Glur6 - 9c. Taken together, our findings strongly suggest that the GluR6-PSD95-MLK3 signaling module mediates activation of the nuclear and non-nuclear pathways of JNK, which is involved in brain injury induced by kainate. Tat-GluR6-9c, the peptide we constructed, gives new insight into seizure therapy.

Neuroprotective effects of GluR6 antisense oligodeoxynucleotides on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region.

To investigate whether the kainate (KA) receptors subunit GluR6 is involved in the neuronal cell death induced by cerebral ischemia followed by reperfusion, the antisense oligodeoxynucleotides (ODNs) of GluR6 were used to suppress the expression of GluR6 by intracerebroventricular infusion once per day for 3 days before ischemia. Transient brain ischemia was induced by four-vessel occlusion in Sprague-Dawley rats. The effects of GluR6 antisense ODNs on the phosphorylation of MLK3 and JNK and the interactions of MLK3 and PSD-95 with GluR6 were examined by immunoprecipitation and immunoblotting. Our results show that GluR6 antisense ODNs can knock down the expression of GluR6 and suppress the assembly of the GluR6.PSD-95.MLK3 signaling module and, therefore, inhibit JNK activation and phosphoralation of c-jun. On the other hand, the GluR6 antisense ODNs also show a protective role against neuronal cell death induced by cerebral ischemia/reperfusion. Administration of GluR6 antisense ODNs once per day for 3 days before cerebral ischemia significantly decreased neuronal degeneration. In conclusion, our results demonstrate that kainate receptor subunit GluR6 plays an important role in neuronal death induced by cerebral ischemia followed by reperfusion.

N-Acetylcysteine inhibit the translocation of mixed lineage kinase-3 from cytosol to plasma membrane during transient brain ischemia in rat hippocampus.

Mixed lineage kinase-3 (MLK3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase (MAPK) pathways. In this study, we investigated the translocation of MLK3 during transient cerebral ischemia in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. Our data show that MLK3 can translocate from cytosolic fraction to the membrane fraction during ischemia and the increased MLK3 in the membrane fraction bind to postsynaptic density protein 95 (PSD-95). The antioxidant N-acetylcysteine (NAC) could inhibit the translocation of MLK3 from cytosolic fraction to the membrane fraction and decrease the interactions of MLK3 and PSD-95 in the membrane fraction. Consequently, these results indicate that reactive oxygen species (ROS) was closely associated with MLK3 translocation induced by transient global ischemia in rat hippocampus.