In vivo characterization of endogenous proliferating cells in adult rat subcortical white matter

Proliferating cells in adult rat subcortical white matter were characterized in vivo using stereotactic injections of a replication-deficient retrovirus containing the construct for βgalactosidase (BAG); BAG was deposited into the cingulum at the level of the septal nuclei. Morphological profiles, generated using Xgal substrate to visualize labeled cells, revealed a population of simple, immature cells. The antigenic profile, generated immunohistochemically with cell-specific markers 2 or 30 days post injection (dpi), showed a population of cells that primarily expressed nestin or an oligodendrocyte-specific glutathione-S-transferase isoform, Yp (GST-Yp) at 2 dpi and nestin, GST-Yp or Rip at 30 dpi. Occasionally, labeled cells differentiated in vivo into myelinating oligodendrocytes 30 dpi. Labeled cells did not express the astrocyte markers GFAP, GST-Yb, or S100β at 2 or 30 dpi. Comparisons of cell distribution 2 and 30 dpi indicated the non-migratory nature of these cells. Cell distribution patterns and nearest neighbor analyses confirmed the emergence of clusters of labeled cells 30 dpi, which bromodeoxyuridine (BrdU) incorporation studies suggested arose from continued proliferation of some labeled cells. In vivo characterization of proliferating cells in the adult revealed a non-migratory, primarily undifferentiated population of cells. © 1996 Wiley-Liss, Inc.     

Oligodendroglial structures and distribution shown by carbonic anhydrase immunostaining in the spinal cords of developing normal and shiverer mice

The spinal cords of young and adult normal and dysmyelinating mutant (shiverer) mice were immunostained with anticarbonic anhydrase to investigate the distribution of oligodendroglial populations into the gray- and white-matter regions in the developing normal and mutant animals; the morphology of oligodendrocytes and their processes at the light microscopic level in gray matter and white matter; and the apparent gliosis in the gray matter, as well as the white matter, of the mutants. Immunocytochemistry and enzyme assays revealed consistent increases in carbonic anhydrase antigenicity and specific activity in controls and mutants between the ages of approximately 15 days and approximately 60 days. As shown previously in adult animals, oligodendroglia in larger than normal proportions were situated at the periphery of the "white-matter" columns, as compared to gray matter, in the shiverers, with, however, significant numbers of oligodendroglia were heterogeneous with respect to shapes, configuration of processes, and intensity of carbonic anhydrase immunostaining. In the shiverer "white matter" the oligodendrocytes were smaller than normal, and their shapes and arrangement were relatively irregular. In the normal gray matter short oligodendroglial processes appeared to be associated with neuronal perikarya, and those processes were more pronounced at approximately 90 days than at approximately 20 days of age. Background staining in normal gray matter suggested that oligodendroglial processes were, in addition, tightly wound around many axons. In shiverer gray matter the oligodendrocytes were smaller, and their processes appeared to be wrapped more loosely around smaller numbers of conspicuous axons and to be associated less frequently with neuronal perikarya. This finding suggests that the deficiency in the myelin basic protein in the mutant may affect interactions between oligodendrocytes and neurons in the gray matter as well as in the white matter. The astrocytic "marker," glial fibrillary acidic protein, was detected in gray and white matter of shiverers as young as 16 days, and the differences from carbonic anhydrase localization supported the conclusion that the processes enwrapping axons in the shiverer mouse CNS are derived from oligodendrocytes, not astrocytes.     

A reappraisal of ganglioside GD3 expression in the CNS

G_D3 ganglioside is a major glycolipid component of the developing central nervous system but diminishes considerably as the CNS matures. Despite consistent biochemical data, the cellular localization of G_D3 expression has been controversial. In this commentary we will review the cellular expression of G_D3 during CNS development and in neuropathological circumstances as determined by studies with the two most commonly used anti G_D3 monoclonal antibodies, R24 and LB1. G_D3 is not restricted to any one cell lineage, being expressed in development to varying degrees by immature neuroectodermal cells, oligodendrocyte progenitors, ameboid microglia, and subpopulations of developing neurons and astrocytes. In the adult CNS, G_D3 is expressed in low amounts by some neuronal subpopulations, on reactive and resting microglia, and by reactive astrocytes. In the appropriate contexts of development or neuropathology, anti-G_D3 antibodies are useful for cell type identification and for cell isolation, but caution should be exercised because of the lack of cellular specificity. © 1996 Wiley-Liss, Inc.     

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside G_D3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP⁺) and oligodendroglia (CNP⁺) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1⁺ and OX-42⁺ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of G_D3⁺ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype G_D3⁺ ED1⁺ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of G_D3⁺ cells exhibited cell membrane G_D3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. G_D3⁺ cells were present within the lesion sites from 6 DPI until 10–12. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP⁺ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP⁺Po^? myelin) with only small pockets of peripheral myelin (MBP⁺Po⁺ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley-Liss, Inc.     

Unmasking the responses of the stem cells and progenitors in the subventricular zone after neonatal and pediatric brain injuries

There is great interest in the regenerative potential of the neural stem cells and progenitors that populate the subventricular zone (SVZ). However, a comprehensive understanding of SVZ cell responses to brain in-juries has been hindered by the lack of sensitive approaches to study the cellular composition of this niche. Here we review progress being made in deciphering the cells of the SVZ gleaned from the use of a recently designed lfow cytometry panel that allows SVZ cells to be parsed into multiple subsets of progenitors as well as putative stem cells. We review how this approach has begun to unmask both the heterogeneity of SVZ cells as well as the dynamic shifts in cell populations with neonatal and pediatric brain injuries. We also discuss how lfow cytometric analyses also have begun to reveal how speciifc cytokines, such as Leuke-mia inhibitory factor are coordinating SVZ responses to injury.     

Morphometric analysis of oligodendrocytes in the adult mouse frontal cortex

Oligodendrocytes (OLs), the myelinating cells of the central nervous system, have specialized morphologies that subserve their function. Numerous qualitative studies suggest that OLs in different brain regions can differ in their morphological characteristics, including number of branches and internodes, internode length, etc. However, progress in identifying and characterizing the diverse types of OLs and their distribution in the brain has been made difficult by several technical constraints. Here we report a new strategy to analyze OL morphology with a high degree of quantitative power and throughput. We used confocal microscopy and three-dimensional cell tracing software to study OLs in the frontal cortex of mice expressing enhanced green fluorescent protein (eGFP) under the control of the proteolipid protein (Plp) gene promoter. Three-dimensional reconstructions were then used to analyze and quantify cell morphology, including total process length, total process surface area, total internode length, number of primary processes, number of branch points, and number of internodes. In addition, these reconstructions were subjected to Sholl analysis, which allows for the quantitative measure of OL arbor complexity. By using this approach, we identified and characterized a previously undescribed population of small OLs with a compact but complex morphology that includes numerous branching processes and a large number of short internodes. Our data suggest that other populations of OLs remain to be identified and characterized and that the tools we have developed could help in the process of characterizing them.     

Neural proliferation in the dorsal root ganglia of the adult rat following capsaicin‐induced neuronal death

Glial proliferation is a major component of the nervous system's response to injury. In addition to glial proliferation, injury may induce neuronal proliferation in areas of the adult nervous system not considered neurogenic. We have previously reported increased neural proliferation within adult nodose ganglia following capsaicin‐induced neuronal death. However, proliferation within the dorsal root ganglia (DRG) remains to be characterized. We hypothesized that capsaicin‐induced neuronal death would increase proliferation of satellite glial cells (SGCs) within the DRG. To test this hypothesis, 6‐week‐old Sprague‐Dawley rats received a neurotoxic dose of capsaicin, and proliferation was quantified and characterized at multiple time points thereafter. Proliferation of satellite glial cells expressing the progenitor cell marker nestin was increased at 1 and 3 days following capsaicin administration as shown by BrdU incorporation. In addition to SGCs was a large population of proliferating resident macrophages, as shown by retrovirally mediated expression of GFP. SGC proliferation at these early time points was followed by recovery of neuronal numbers after a loss of 40% of the neuronal population in the DRG. This recovery in neuronal number correlated with recovery of function as shown by paw withdrawal from a noxious heat source. Further understanding of the role that glial proliferation plays in the recovery of neuronal numbers and function may lead to the development of therapeutic treatments for neurodegenerative conditions. J. Comp. Neurol. 522:3295–3307, 2014. © 2014 Wiley Periodicals, Inc.     

Human glial-restricted progenitors survive, proliferate, and preserve electrophysiological function in rats with focal inflammatory spinal cord demyelination

Transplantation of glial progenitor cells results in transplant-derived myelination and improved function in rodents with genetic dysmyelination or chemical demyelination. However, glial cell transplantation in adult CNS inflammatory demyelinating models has not been well studied. Here we transplanted human glial-restricted progenitor (hGRP) cells into the spinal cord of adult rats with inflammatory demyelination, and monitored cell fate in chemically immunosuppressed animals. We found that hGRPs migrate extensively, expand within inflammatory spinal cord lesions, do not form tumors, and adopt a mature glial phenotype, albeit at a low rate. Human GRP-transplanted rats, but not controls, exhibited preserved electrophysiological conduction across the spinal cord, though no differences in behavioral improvement were noted between the two groups. Although these hGRPs myelinated extensively after implantation into neonatal shiverer mouse brain, only marginal remyelination was observed in the inflammatory spinal cord demyelination model. The low rate of transplant-derived myelination in adult rat spinal cord may reflect host age, species, transplant environment/location, and/or immune suppression regime differences. We conclude that hGRPs have the capacity to myelinate dysmyelinated neonatal rodent brain and preserve conduction in the inflammatory demyelinated adult rodent spinal cord. The latter benefit is likely dependent on trophic support and suggests further exploration of potential of glial progenitors in animal models of chronic inflammatory demyelination.     

Insulin-like growth factor actions during development of neural stem cells and progenitors in the central nervous system

Insulin-like growth factor-I (IGF-I) plays a key role in normal development. Recent studies show that IGF-I exerts a wide variety actions in the central nervous system during development as well as in adulthood. This report reviews recent developments on IGF-I actions and its mechanisms in the central nervous system, with a focus on its actions during the development of neural stem cells and progenitors. Available data strongly indicate that IGF-I shortens the length of the cell cycle in neuron progenitors during embryonic life and has an influence on the growth of all neural cell types. The phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase pathways seem to be the predominant mediators of IGF-I-stimulated neural cell proliferation and survival. IGF-I actions, however, likely depend on cell type, developmental stage, and microenvironmental milieu.     

Proliferation and differentiation of O4+ oligodendrocytes in postnatal rat cerebellum: analysis in unfixed tissue slices using anti-glycolipid antibodies.

We report the study of the in vivo morphology, differentiation, and proliferation of oligodendrocytes (OLs) and their progenitors identified by the antiglycolipid antibodies O4, R-mAb, and O1 in postnatal rat cerebellum, using a novel immunocytochemical staining protocol which allows the analysis of the expression of OL-specific glycolipids in live, unfixed brain slices. An analysis of the individual cells identified in double label immunocytochemistry indicated that the order of antigen expression in OLs during in vivo development is, first, antigens recognized by O4, second, antigens recognized R-mAb, and third, antigens recognized by O1. This order of antigen expression is correlated with increasing morphological complexity and is a pattern mimicked in many culture systems. In vivo O4 identified 3 distinct stages of the OL lineage: (1) morphologically simple proligodendrocyte antigen+ (POA+) R-mAb- blast cells localized at the leading edge of myelinogenesis; (2) morphologically more complex R-mAb+O1- cells; and (3) actively myelinating O1+ [i.e., galactocerebroside+ (GalC)] OLs residing within the white matter. Only the POA+R-mAb- cells incorporated BrdU in animals that were prelabeled 3 hr before immunocytochemistry. We have demonstrated in vivo the subdivision of pre-GalC+ OL progenitors into shorter, biologically noteworthy, stages of maturation. A spatial comparison of the cell populations identified by O4, R-mAb, and O1 demonstrated a progressive wave of OL maturation from the base of the cerebellum toward the folia. The data are consistent with the hypothesis that multiprocessed O4+GalC- progenitors are the most mature stage of the OL lineage with significant proliferative capacity and the first postmigratory stage in normal development.     

Expression of the myelin and oligodendrocyte progenitor marker sulfatide in neurons and astrocytes of adult rat brain

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti-sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I-positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered.     

Increased neural progenitors in individuals with cerebral small vessel disease

A. Ekonomou, M. Johnson, R. H. Perry, E. K. Perry, R. N. Kalaria, S. L. Minger and C. G. Ballard (2012) Neuropathology and Applied Neurobiology 38, 344–353 Increased neural progenitors in individuals with cerebral small vessel disease Aims: Recent work has highlighted a significant increase of neural stem/progenitor cells after stroke in humans. In this study, we examined neurogenesis in small vessel disease, a key concurrent pathology in Alzheimer's disease. Methods: We assayed autopsy tissue from 13 vascular dementia patients with small vessel disease and 12 age‐matched subjects without cerebrovascular pathology, undertaking immunohistochemistry in the affected brain area and the subventricular zone with a well‐characterized battery of antibodies to detect neural stem cells/progenitors and immature neurones, as well as choline acetyltransferase immunoreactivity. Results: We showed significant increases ranging from 33% to 92% (P < 0.05) in neural progenitor cells around the areas of microvascular pathology and in the subventricular zone in patients with small vessel disease compared to individuals without cerebrovascular changes, even in patients with severe cerebrovascular disease, as defined by neuropathological assessment. Some of the progenitor cells give rise to immature neurones in the affected areas. These alterations were associated with vascular changes, but were unrelated to the cholinergic deficit observed in the cortex and subventricular zone in these patients, in contrast to other dementias examined such as dementia with Lewy bodies. Conclusions: This study provides evidence for neurogenesis in small vessel disease and may have important implications for the development of new therapies for neurodegenerative diseases.     

Platelet-derived growth factor delays oligodendrocyte differentiation and axonal myelination in vivo in the anterior medullary velum of the developing rat

The AA dimeric form of platelet-derived growth factor (PDGF-AA) is implicated in the differentiation of cells of the oligodendrocyte lineage, which express PDGF receptors of the alpha subunit type (PDGF-αR). In the present study, we show that a single injection of PDGF-AA into the cerebrospinal fluid of neonatal rats delays oligodendrocyte differentiation and interrupts the progress of myelination in the anterior medullary velum (AMV), a white matter tract roofing the IVth ventricle of the brain. PDGF-AA or saline was injected intrathecally in postnatal day (P) 7 rats, and the AMV was subsequently removed and immunolabelled with the oligodendrocyte-specific antibody Rip, at P9, P12, and P21, corresponding to postinjection days (PID) 2, 5, and 14. At P9 (PID2), myelination was retarded in PDGF-AA-treated rats as opposed to saline-treated controls but progressed rapidly after P12 (PID5). Quantification supported the qualitative observations that PDGF-AA mediated an acute decrease in the number of Rip+ oligodendrocytes at P9–12, which largely recovered by P21, suggesting that PDGF-AA may have delayed recruitment of myelinating oligodendrocytes. However, the definitive number of Rip+ oligodendrocytes in the AMV was not increased, suggesting that its action as a promoter of early oligodendrocyte survival may not ultimately affect the definitive number of myelinating oliogdendrocytes in vivo. We discuss the possibilities that excess PDGF-AA may have acted on early oligodendrocytes (precursors or preoligodendrocytes) to either (1) delay their differentiation by maintaining them in the cell cycle or (2) accelerate their differentiation, which may result in premature cell death in the absence of synchronised survival signals. This study supports a role for PDGF-AA in the timing of oligodendrocyte differentiation in vivo, as has been shown in vitro. J. Neurosci. Res. 48:588–596, 1997. © 1997 Wiley-Liss Inc.     

A combination of insulin-like growth factor-I and platelet-derived growth factor enhances myelination but diminishes axonal regeneration into Schwann cell grafts in the adult rat spinal cord

Insulin-like growth factor-I (IGF-I) promotes axonal regeneration in the peripheral nervous system and this effect is enhanced by platelet-derived growth factor (PDGF). We decided, therefore, to study the effects of these factors on axonal regeneration in the adult rat spinal cord. Semipermeable polymer tubes, closed at the distal end, containing Matrigel mixed with cultured rat Schwann cells and IGF-I/PDGF, were placed at the proximal stump of the spinal cord after removal of the thoracic T9-11 segments. Control animals received implants of only Matrigel and Schwann cells or only Matrigel and IGF-I/PDGF. Four weeks after implantation, electron microscopic analysis showed that the addition of IGF-I/PDGF resulted in an increase in the myelinated:unmyelinated fiber ratio from 1:7 to 1:3 at 3 mm in the Schwann cell graft, and that myelin sheath thickness was increased 2-fold. The reduced number of unmyelinated axons was striking in electron micrographs. These results suggested that IGF-I/PDGF enhanced myelin formation of regenerated axons in Schwann cell implants, but there was a 36% decrease in the total number of myelinated axons at the 3 mm level of the graft. This finding and the altered myelinated:unmyelinated fiber ratio revealed that the overall fiber regeneration into Schwann cell implants was diminished up to 63% by IGF-I/PDGF. Histological evaluation revealed that there were more larger cavities in tissue at the proximal spinal cord-graft interface in animals receiving a Schwann cell implant with IGF-I/PDGF. Such cavitation might have contributed to the reduction in axonal ingrowth. In sum, the results indicate that whereas the combination of IGF-I and PDGF enhances myelination of regenerating spinal cord axons entering implants of Matrigel and Schwann cells after midthoracic transection, the overall regeneration of axons into such Schwann cell grafts is diminished. GLIA 19:247–258, 1997. © 1997 Wiley-Liss, Inc.     

The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS

There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFRalpha) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.     

Transplantation of fetal spinal cord tissue into the chronically injured adult rat spinal cord

Transplants of fetal central nervous system (CNS) tissue into the acutely injured rat spinal cord have been demonstrated to differentiate and partially integrate with the adjacent host neuropil. In the present study, we examined the potential for applying a transplantation approach to chronic spinal cord lesions. In particular, we were interested in learning whether host-graft fusion would be adversely affected by an advanced histopathology characterized in part by glial scar formation. Hemisection cavities were prepared at lumbar levels of the adult rat spinal cord 2-7 weeks prior to the transplantation of spinal cord tissue obtained from 14-day rat fetuses. Graft survival, differentiation, and integration with the host spinal cord were subsequently evaluated by light microscopic techniques at post-transplantation intervals of 1-6 months. Immunocytochemistry was also employed to examine the extent of astrocytic scar formation at the host-graft interface and serotoninergic innervation of the grafts. In some other cases, anterograde and retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase was used to determine whether axonal projections were formed between the host spinal cords and grafts. By 2 weeks after injury the initial lesion cavities were surrounded by a continuous astrocytic scar which remained intact for at least 7 weeks after injury in nongrafted control animals. In other animals, transplantation into these advanced lesions resulted in well-differentiated grafts with a 90% long-term survival rate. Although dense gliosis was still present along the lesion surfaces of the recipient spinal cord, foci of confluent host-graft neuropil were observed where interruptions in the scar had occurred. Donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. Thus, some regions of confluent graft and host neuropil could be routinely identified, despite the presence of a dense glial scar along the walls of the chronic lesion site at the time of transplantation. Anterograde and retrograde tract-tracing results suggested that some axonal projections into these grafts had originated from host neurons located immediately adjacent to the donor-recipient interface. In addition, immunocytochemistry revealed some host serotoninergic axons (presumably of supraspinal origin) traversing nongliotic interfaces. The results of this study raise the possibility that grafted fetal CNS tissue has a capacity for stimulating partial regression of an established glial scar.(ABSTRACT TRUNCATED AT 400 WORDS)