Axon-myelin sheath relations of oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

Axon-oligodendrocyte relations of Rip-immunolabelled and dye-injected oligodendrocyte units are characterised in the adult rat anterior medullary velum (AMV). Each oligodendrocyte unit comprised the oligodendrocyte cell body, processes and the internodal myelin segments they support. Oligodendrocyte units corresponded to classically described type I/II or type III/IV unit phenotypes which respectively myelinated discrete populations of small and large diameter axons, delineated by a myelinated fire diameter of 2-4 microns (diameter of the axon plus its myelin sheath). Within units, mean fibre diameter was directly related to mean internodal length and inversely related to the number of myelin sheaths in the unit. The relationship between fibre diameter and internodal length was retained in units which myelinated axons of different diameters, indicating that axon diameter was an important determinant of the longitudinal dimensions of myelin sheaths. We also show that type III/IV units maintained a far greater volume of myelin than type I/II units. It was concluded that type I/II and III/IV oligodendrocytes represent two functionally and morphologically distinct phenotypes whose distribution densities were determined by the diameter and spatial dispersion of axons.     

Differential expression of the L- and S-isoforms of myelin associated glycoprotein (MAG) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum

We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the anterior medullary velum (AMV) of the adult rat (each unit comprises the cell body, processes and myelin sheaths). Others have indicated that myelin composition may also vary with respect to myelin basic protein (MBP) and proteolipid protein (PLP), and the small (S)- and large (L)-isoforms of myelin associated glycoprotein (MAG). In this study, we have determined the expression of myelin proteins in oligodendrocyte unit phenotypes I–IV, which myelinate fibres ranging in diameter from 0.3–12 m diameter in the AMV, by using double immunolabelling for Rip, which labels entire units, and MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), L-MAG and S-MAG. We show differences in the expression of L- and S-MAG in units which myelinate different diameter fibres: (1) type I/II units myelinating small diameter fibres had a L-MAG+/S-MAG–/CAII+ phenotype; (2) type II/III units myelinating different diameter fibres had a L-MAG+/S-MAG+/CAII+ phenotype; (3) type III/IV units myelinated large diameter fibres had a L-MAG+/S-MAG+/CAII– phenotype. All units, irrespective of fibre diameter, expressed Rip, MBP, PLP and MOG. The results indicate that type I–IV units may be variants of a single oligodendrocyte population and that phenotypic differences are determined by the diameter of fibres within the unit. The possible significance of metabolic and biochemical differences between oligodendrocytes myelinating small and large diameter axons are discussed with reference to the pathology of demyelination.     

Axon-glial relations during regeneration of axons in the adult rat anterior medullary velum

The anterior medullary velum (AMV) of adult Wistar rats was lesioned in the midsagittal plane, transecting all decussating axons including those of the central projection of the IVth nerve. At selected times up to 200 days after transection, the degenerative and regenerative responses of axons and glia were analyzed using transmission and scanning electron microscopy and immunohistochemistry. In particular, both the capacity of oligodendrocytes to remyelinate regenerated fibers and the stability of the CNS/PNS junctional zone of the IVth nerve rootlet were documented. Transected central AMV axons exhibited four patterns of fiber regeneration in which fibers grew: rostrocaudally in the reactive paralesion neuropil (Group 1); randomly within the AMV (Group 2); into the ipsilateral IVth nerve rootlet, after turning at the lesion edge and growing recurrently through the old degenerated contralateral central trochlear nerve trajectory (Group 3); and ectopically through paralesion tears in the ependyma onto the surface of the IVth ventricle (Group 4). Group 1–3 axons regenerated unperturbed through degenerating central myelin, reactive astrocytes, oligodendrocytes, microglia, and large accumulations of hematogenous macrophages. Only Group 3 axons survived long term in significant numbers, and all became myelinated by oligodendrocytes, ultimately establishing thin sheaths with relatively normal nodal gaps and intersegmental myelin sheath lenghts. Schwann cells at the CNS/PNS junction of the IVth nerve rootlet did not invade the CNS, but astrocyte processes grew across the junction into the PNS portion of the IVth nerve. The basal lamina of the junctional glia limitans remained stable throughout the experimental period.     

The relationship between developing oligodendrocyte units and maturing axons during myelinogenesis in the anterior medullary velum of neonatal rats

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between 0.2 and 0.4 m, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.     

Oligodendrocyte progenitor cells in the adult rat CNS express myelin oligodendrocyte glycoprotein (MOG)

While the effects of high dose X-irradiation on mitotically active progenitor cells and remyelination are well-documented, its effects on myelinating oligodendrocytes are less clear, due in part to divergent views on their mitotic capacity. To examine the effect of X-irradiation on oligodendrocytes, the spinal cord of rats was exposed to 40 Gy of X-irradiation and the number of oligodendrocytes and oligodendrocyte progenitors in the dorsal funiculi at T12 and L1 was determined by in situ hybridization using cRNA-probes for platelet derived growth factor alpha receptor (PDGFRalpha) (to identify oligodendrocyte progenitors), exon 3b of proteolipid protein (PLP) (to identify mature oligodendrocytes) and myelin oligodendrocyte glycoprotein (MOG). X-irradiation resulted in no change in the number of PLP positive cells and no loss of myelin internodes, but caused an almost complete loss of PDGFRalpha-expressing cells, and a reduction in the number of MOG positive cells to a number similar to that found using the PLP exon 3b probe. Importantly, the number of radiation-sensitive MOG-expressing cells was similar to the number of PDGFRalpha positive cells. To determine if the radiation-sensitive MOG positive cells were the same population as the radiation sensitive PDGFRalpha-expressing cells, MOG and PDGFRalpha-expressing cells were isolated from the adult CNS using antibody coated magnetic beads. Twelve to thirteen percent of MOG positive cells were PDGFRalpha positive and nearly all the PDGFRa isolated cells were MOG and galactocerebroside positive. Double immunofluorescence revealed colocalization of NG2 and MOG on cells in the normal adult rat spinal cord. These results show that in situ in the adult rat spinal cord white matter oligodendrocyte progenitors are MOG positive and indicates that expression of MOG cannot be regarded a marker that only identifies mature myelin-supporting oligodendrocytes in tissue.     

Electrical stimulation of the medullary pyramid promotes proliferation and differentiation of oligodendrocyte progenitor cells in the corticospinal tract of the adult rat

Endogenous tri-potential neural stem cells (eNSCs) exist in the adult spinal cord and differentiate primarily into oligodendrocytes (OLs) and astrocytes. Previous in vivo and in vitro studies have shown that during development proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) depend on activity in neighboring axons. However, this activity-dependent development of OPCs has not been examined in the adult CNS. In the present study, we stimulated unilateral corticospinal (CS) axons of the adult rat and investigated proliferation and differentiation of OPCs in dorsal corticospinal tract (dCST). eNSCs were labeled with the mitotic indicator 5-bromo-2′-deoxyuridine (BrdU). Phenotypes of proliferating cells were identified by double-immunolabeling of BrdU with a panel of antibodies to cell markers: NG2, Nkx2.2, APC, GFAP, and Glut-1. Electrical stimulation of CS axons increased BrdU labeled eNSCs and promoted the proliferation and differentiation of OPCs, but not astrocytes and endothelial cells. Our findings demonstrate the importance of neural activity in regulating OPC proliferation/differentiation in the mature CNS. Selective pathway electrical stimulation could be used to promote remyelination and recovery of function in CNS injury and disease.     

Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro

The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF -receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5–2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.     

血小板衍生性生长因子对体外培养人肾小球系膜细胞生长的影响

目的观察血小板衍生性生长因子（ＰＤＧＦ）对体外培养的人肾小球系膜细胞（ＭｓＣ）生长、基质合成和分泌以及ｃ┐ｍｙｃ癌基因表达的影响。方法体外培养的ＭｓＣ培养液中掺入５┐溴脱氧尿嘧啶（ＢｒｄＵ）,采用ＢｒｄＵ单克隆抗体免疫组化法检测ＭｓＣ的增殖情况;应用［３Ｈ］脯氨酸掺入酶消化法测定ＭｓＣ细胞内、外胶原蛋白总量;采用Ｎｏｒｔｈｅｒｎ印迹法检测纤维连接蛋白（ＦＮ）、Ⅳ型胶原、核癌基因ｃ┐ｍｙｃｍＲＮＡ表达结果。结果（１）ＢｒｄＵ掺入法的阳性细胞标记指数在对照组为１９．５％,ＰＤＧＦ组为３４．５％（Ｐ＜０．０１）;（２）ＭｓＣ经ＰＤＧＦ作用后,细胞内、外胶原蛋白量分别为２．６９±０．６０％和３．８７±０．６５％,较正常对照组１．２５±０．５０％和１．６１±０．５１％明显增加（Ｐ＜０．０１）;（３））Ｎｏｒｔｈｅｒｎ印迹法显示ＰＤＧＦ组细胞的ＦＮ、Ⅳ型胶原及ｃ┐ｍｙｃｍＲＮＡ的表达明显高于正常组。结论ＰＤＧＦ不仅可刺激肾小球ＭｓＣ的增殖和胶原蛋白合成,而且可从基因转录水平增加ＦＮ、Ⅳ型胶原及ｃ┐ｍｙｃ癌基因的表达。由此推断ＰＤＧＦ在肾小球疾病的发生、发展中可能起着重要作用     

Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I_Na) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7–21 rats express low density I_Na (9.5–15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC₅₀ value of 39.3 nM. The I_Na expression correlated with a 25 mV more depolarized resting membrane potential (RMP) as compared with non-I_Na-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 μM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I_Na-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I_Na expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 μM TTX. Analysis of the I_Na activation/inactivation curves yields an I_Na “window current” at −40±20 mV, implying a persistent I_Na component being active around the NG2 glia RMP of −45 mV. According to the constant-field equation analysis, this active I_Na component leads to a pNa/pK ratio of 0.14 at RMP which is threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I_Na component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.     

新生大鼠大脑皮质O-2A祖细胞的体外诱导分化

目的　在获取高纯度O 2A祖细胞的基础上 ,探讨O 2A祖细胞在体外分化的形态学特点。方法　采用“两次恒温摇床振荡法”和差速贴壁法 ,结合使用神经营养因子 (bFGF、PDGF AA) ,进行O 2A祖细胞体外纯化和扩增培养 ,并观察O 2A祖细胞在有血清和无血清培养条件下的分化情况 ,用免疫细胞化学鉴定分化成熟的 2型星型胶质细胞和少突胶质细胞。结果　培养的O 2A祖细胞能形成克隆球 ,具有增殖能力 ,经免疫细胞化学鉴定细胞纯度达 90 %以上。在含血清的培养基中能定向分化为 2型星形胶质细胞 ,其胞体较大 ,从胞体上伸出细长突起 ,由突起上再发出更为细小的分支 ,GFAP和A2B5抗体标记均为阳性 ;在无血清的化学条件培养基中能定向分化为少突胶质细胞 ,其胞体较小 ,突起短而细 ,相互交织呈“蜘蛛网”样 ,CNPase抗体标记阳性。结论　 (1)培养的O 2A祖细胞保持定向干细胞的特性 ,具有增殖和双向分化的潜能 ;(2 )O 2A祖细胞定向分化为 2型星形胶质细胞过程中经历较长的增殖期 ,而分化为少突胶质细胞过程中则增殖期较短 ;(3)分化的 2型星形胶质细胞和少突胶质细胞在形态学和抗原表达上存在差异。     

Cerebral endothelial derived vascular endothelial growth factor promotes the migration but not the proliferation of oligodendrocyte precursor cells in vitro

In gray matter, cerebral endothelium is known to provide trophic support for neighboring cells such as neurons. However, signaling from cerebral endothelium to white matter cells remains to be elucidated. Here, we show that vascular endothelial growth factor (VEGF-A) secreted from cerebral endothelial cells promotes the migration but not the proliferation of oligodendrocyte precursor cells (OPCs). Cultured OPCs were obtained from newborn rat cortex, and treatment with conditioned culture media of cerebral endothelial cells increased the OPC proliferation and migration. Importantly, co-treatment with anti-neutralizing antibody for Flk-1 (VEGF-receptor2) inhibited OPC movement but did not affect OPC propagation. Western blot and flow cytometry analyses confirmed that our cultured cerebral endothelial cells produced VEGF-A and our cultured OPCs expressed Flk-1. Taken together, our current data suggest that cerebral endothelium is supportive for oligodendrocyte lineage cells and VEGF-A may participate in the endothelium–OPC cell–cell signaling. This phenomenon may be important for white matter homeostasis.     

NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system

The mammalian adult central nervous system (CNS) is known to respond rapidly to demyelinating insults by regenerating oligodendrocytes for remyelination from a dividing precursor population. A widespread population of cells exists within the adult CNS that is thought to belong to the oligodendrocyte lineage, but which do not express proteins characteristic of mature myelinating oligodendrocytes, such as myelin basic protein (MBP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP). Instead, these cells have phenotypic characteristics of a more immature stage of the oligodendrocyte lineage. They express the NG2 chondroitin sulphate proteoglycan, in addition to O4 and the platelet-derived growth factor alpha-receptor, all widely accepted as markers for oligodendrocyte progenitor cells (OPCs) throughout development. However, NG2+ cells residing in the adult CNS do not resemble embryonic or neonatal NG2+ cells in terms of their morphology or proliferation characteristics, but instead represent a unique type of glial cell that has the ability to react rapidly to CNS damage. In this review, we present the evidence that adult NG2+ cells are part of the oligodendrocyte lineage and are capable of giving rise to new oligodendrocytes under both normal and demyelinating conditions. We also review the literature that these cells may have multiple functional roles within the adult CNS, notwithstanding their primary role as OPCs.     

A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus.

The adult brain contains a large population of oligodendrocyte precursor cells that can be identified using antibodies against the NG2 chondroitin sulfate proteoglycan. The functions of this newly recognized class of glial cells in the normal or pathological brain are not well understood. To begin to elucidate these functions, we have examined the morphology and distribution of oligodendrocyte precursor cells in the hippocampus and neocortex of normal and kainate-lesioned rats by anti-NG2 immunocytochemistry using light and electron microscopy. Large numbers of oligodendrocyte precursor cells were present in all layers of the neocortex and hippocampus. These cells differed in their morphology from astrocytes, oligodendrocytes and microglia. The processes of these cells often surrounded unlabeled areas of clear cytoplasm. At the electron microscopic level, some of the profiles that were enclosed by oligodendrocyte precursor cell processes contained synaptic vesicles. Other enclosed profiles were dendrites or dendritic spines. NG2-immunopositive processes were also observed to interpose between axon terminals containing round vesicles and dendrites with thick postsynaptic densities. After kainate injection, the NG2-positive oligodendrocyte precursor cells in the hippocampus displayed reactive changes characterized by swollen cell bodies, an increased number of small, filopodial-like processes, and higher levels of immunodetectable NG2. Both viable and degenerating oligodendrocyte precursor cells were observed with electron microscopy. These observations emphasize the dynamic nature of the oligodendrocyte precursor cell and suggest that, in addition to participating in the glial reactions to excitotoxic damage, oligodendrocyte precursor cells may regulate the stability, structure and function of synapses in the normal central nervous system.