Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.
Animals ; Animals, Newborn ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; genetics ; Cells, Cultured ; Cuprizone ; toxicity ; Demyelinating Diseases ; chemically induced ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Regulation, Developmental ; genetics ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Myelin Proteolipid Protein ; metabolism ; Myelin Sheath ; drug effects ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector. The recombinant vector p5TMP was transformed into E. coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning. Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1:16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.
Animals ; Antibody Formation ; Autoantibodies ; Brain ; immunology ; metabolism ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Myelin Basic Protein ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology

OBJECTIVETo investigate the protective effects of allopurinol (ALLO) on white matter damage in premature rats.

METHODSAn animal model for white matter damage was established by bilateral carotid artery occulation (BCAO). Eighty-four newborn SD rats (1 day old) were used in this study and were divided randomly into three groups [sham surgery (Sham); BCAO group (BCAO); allopurinol-treated group (ALLO)]. Pathological changes were studied 7 days and 14 days after BCAO, respectively. Myelin basic protein (MBP) was detected by immunohistochemistry 7 days and 14 days after BCAO, respectively. MBP-mRNA expression was determined 7 days and 14 days after BCAO respectively by reverse transcription-polymerase chain reaction (RT-PCR) with fluorescent quantitative method.

RESULTSIn BCAO group, mild or severe rarefaction was found in 10 cases in the corpus callosum area, especially at the cingulum. Pathological changes of white matter were found in 4 cases in internal capsule. Subcortex white matter rarefaction was found in 8 cases. The extent of white matter rarefaction in ALLO group was reduced significantly. Enlargement of bilateral ventricles was found in 6 of 8 cases in BCAO group. The average ventricle size in ALLO group (2.44 +/- 0.71)% was reduced significantly as compared with that in BCAO group (3.27 +/- 0.73)% (P < 0.05). Strong MBP positive staining was found in sub-cortex, corpus callosum, hippocampus gyrus, and internal capsule of P14 sham surgery group. In BCAO group the MBP staining extent was reduced. The extent of MBP staining of ALLO group was between the other two groups. The optical density (OD) of MBP positive staining in BCAO group (6.60 +/- 0.68) was found higher than that in sham surgery group (9.40 +/- 0.53), the difference was statistically significant (P < 0.05). Compared with BCAO group, OD value in ALLO group (7.10 +/- 0.18) increased significantly (P < 0.05). RT-PCR data showed that MBP-mRNA copies (log10) in P7 and P14 rats of both BCAO and ALLO groups were lower than that in sham surgery group (P < 0.01); However, MBP-mRNA copies in ALLO group were higher than that in BCAO group (P < 0.05).

CONCLUSIONSBCAO could be used in newborn rats (1 day old) to establish a premature white matter damage (WMD) animal model. Allopurinol may have a potential protective effect on premature SD rat with ischemic WMD.

Allopurinol ; pharmacology ; Animals ; Animals, Newborn ; Brain ; drug effects ; Brain Diseases ; metabolism ; pathology ; prevention & control ; Disease Models, Animal ; Immunohistochemistry ; Myelin Basic Protein ; genetics ; metabolism ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of caffeine citrate on myelin basic protein (MBP) expression in the cerebral white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the related mechanism.

METHODSForty-eight seven-day-old Sprague-Dawley neonatal rats were randomly assigned to 3 groups: sham operation (n=16), HIBD (n=16) and HIBD+caffeine citrate (n=16). The rats in the HIBD and HIBD+caffeine citrate groups were subjected to left common carotid artery ligation, and then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen for 2 hours to induce HIBD. The rats in the sham operation group were only subjected to a sham operation, without the left common carotid artery ligation or hypoxia exposure. Caffeine citrate (20 mg/kg) was injected intraperitoneally before hypoxia ischemia (HI) and immediately, 24 hours, 48 hours and 72 hours after HI. The other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time points. On postnatal day 12, the expression of MBP in the left subcortical white matter was detected by immunohistochemistry, and the levels of adenosine A1 receptor mRNA and A2a receptor mRNA in the left brain were detected by real-time PCR.

RESULTSThe expression of MBP in the left subcortical white matter in the HIBD group was lower than in the sham operation group (P<0.05). The MBP expression in the HIBD+caffeine citrate group was significantly higher than in the HIBD group, but was still lower than the sham operation group (P<0.05). Real-time PCR showed that the adenosine A1 receptor mRNA expression was significantly higher in the HIBD group than in the sham operation group, and it was significantly lower in the HIBD+caffeine citrate group than in the HIBD group (P<0.05).

CONCLUSIONSCaffeine citrate can improve brain white matter damage following HIBD in neonatal rats and the protection mechanism might be related with the down-regulation of adenosine A1 receptor expression.

Animals ; Animals, Newborn ; Caffeine ; pharmacology ; Citrates ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Male ; Myelin Basic Protein ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Adenosine A1 ; genetics ; Receptor, Adenosine A2A ; genetics ; White Matter ; chemistry

OBJECTIVETo investigate the protective effect of pSVPoMcat (myelin basic protein microgene) modifying Schwann cell on injured spinal neurons.

METHODSA model of rat spinal cord injured by hemisection was used. One hundred and twenty healthy SD rats of both sexes weighing 250-300 g were divided into three groups: Group A (n=40, treated with implantation of pSVPoMcat modifying Schwann cell), Group B (n= 40, treated with implantation of Schwann cell only) and Group C (n=400, treated with sham operation as the control). One week after operation the rat functional recovery was observed dynamically by using combined behavioral score (CBS) and cortical somatasensory evoked potentials, the spinal cord sections were stained by Nissl, acid phosphatase enzyme histochemistry and cell apoptosis was examined by methye green, terminal deoxynucleotidyl and the dUTP Nick end labeling technique. Quantitative analysis was done by computer image analysis system.

RESULTSIn Group A the injured neurons recovered well morphologically. The imaging analysis showed a result of Group A

CONCLUSIONSpSVPoMcat modifying Schwann cell implantation has protective effect on injured spinal neurons and promotes recovery of injured spinal cord function in rats.

Acid Phosphatase ; metabolism ; Animals ; Apoptosis ; Cell Transplantation ; Disease Models, Animal ; Evoked Potentials, Somatosensory ; Female ; Gene Transfer Techniques ; Male ; Methyl Green ; Myelin Basic Protein ; genetics ; Nerve Regeneration ; Rats ; Rosaniline Dyes ; Schwann Cells ; metabolism ; transplantation ; Spinal Cord Injuries ; physiopathology ; surgery

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism