PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells

We have been studying the differing characteristics of oligodendrocyte- type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O- 2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.     

A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.     

Estimation problems associated with stochastic modeling of proliferation and differentiation of O-2A progenitor cells in vitro

Our previous research effort has resulted in a stochastic model that provides an excellent fit to our experimental data on proliferation and differentiation of oligodendrocyte type-2 astrocyte progenitor cells at the clonal level. However, methods for estimation of model parameters and their statistical properties still remain far away from complete exploration. The main technical difficulty is that no explicit analytic expression for the joint distribution of the number of progenitor cells and oligodendrocytes, and consequently for the corresponding likelihood function, is available. In the present paper, we overcome this difficulty by using computer-intensive simulation techniques for estimation of the likelihood function. Since the output of our simulation model is essentially random, stochastic optimization methods are necessary to maximize the estimated likelihood function. We use the Kiefer-Wolfowitz procedure for this purpose. Given sufficient computing resources, the proposed estimation techniques significantly extend the spectrum of problems that become approachable. In particular, these techniques can be applied to more complex branching models of multi-type cell systems with dependent evolutions of different types of cells.     

Histochemical alterations in the adult rat brain after X-ray irradiation: effects of O-(beta-hydroxyethyl)-rutosides

X-ray-induced tissue damage in the brain of adult rats was investigated in the presence or absence of O-(beta-hydroxyethyl)-rutosides (OHR; active agents of Venoruton, Zyma-Blaes, München, FRG). Histochemical methods were used for the detection of glycogen (periodic acid-Schiff), acid mucopolysaccharides (Hale) and acid phosphatases (Gomori) by light microscopy. The tissue alterations were reduced after drug application in the dose range between 5 and 7.5 Gy (300 kV, 12 mA). Exposure of the animals to higher irradiation doses (10, 20 Gy) led to an inversion of the drug effect, now exerting pronounced tissue injury. For a possible explanation we discuss the inhibitory influence of rutosides (e.g. OHR) on the glycolytic pathway. Hence, a vital energy source of brain tissue could be impaired by the drug after reaching a threshold of 10 Gy.     

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

Neuroglia of the adult rat optic nerve in the course of wallerian degeneration

The neurological reactions in Wallerian degeneration have been studied by electron microscopy in the optic nerve of adult albino rats from 7 to 120 days after unilateral enucleation. Reactive astrocytes contained abundant dense bodies, numerous microtubules and hyperplastic glial filaments. These astrocytes also assisted phagocytosis of degenerated myelin sheaths and in glial scar formation. Oligodendrocytes disconnected their cytoplasmic extensions, which were phagocytosed by microglial cells and astrocytes, by increased production of lysosomes. Microglial cells consisted of crinkled, long, rough endoplasmic reticula, several highly-active Golgi complexes, laminar inclusions and globoid lipid droplets. Microglia engulfed and lysed the disintegrated axons and myelin sheaths.     

Ion channel expression by white matter glia: the O-2A glial progenitor cell.

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.     

Enzyme histochemical changes in retinal ganglion cells and the optic nerve after goldfish optic nerve lesion. A regenerative and hypertrophic phenomena study

After sectioning of the goldfish optic nerve a number of enzyme histochemical changes are observed in the hypertrophied retinal ganglion cells and in the optic nerve. Between one and eighteen days postoperatively an increase in the amount of acid phosphatase reaction product is noted. The enhanced activity decreased to normal first in the optic nerve, followed by the optic tract and tectum. Four days postoperatively higher levels of activity were noted in the hypertrophic retinal ganglion cells for the enzymes NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase. The same enzymes also showed an activity increase in the lesioned optic nerve after four to ten days postoperatively, beginning at the cut and gradually spreading towards the optic tectum. Between fifteen and eighteen days the activity dropped to normal in the hypertrophic retinal ganglion cells, while in the lesioned nerve raised levels of reaction products could be seen till days thirty-five and/or forty-five. It was concluded that the degeneration of the optic pathway is marked by the increase of acid phosphatase activity, whereas the process of regeneration is characterized by an increase of NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase activities. The possible functional implications of these enzymes in the regenerative phenomena are discussed.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

Upregulation of Gem relates to retinal ganglion cells apoptosis after optic nerve crush in adult rats

GTP-binding protein Gem, a member protein of the Ras superfamily, can regulate actin cytoskeleton reorganization mediated by Rho-associated coiled-coil-containing protein kinase (ROCK). One attractive activity of the ROCK is playing a potential role in physiological and pathological process in retinal ganglion cells (RGCs) apoptosis. However, the function of Gem in retina is still with limited understanding. To investigate whether Gem is involved in optic nerve injury, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis indicated that Gem was significantly increased in the retina at the 3rd day after ONC. Meanwhile, double-immunofluorescent staining showed that Gem expression was mainly up-regulated in ganglion cell layer and co-localized with NeuN (a marker of RGCs). Additionally, the co-localizations of Gem/active-caspase-3 and Gem/TUNEL-positive cells were detected in RGCs. Furthermore, the expression of active-caspase-3 and TUNEL-positive cells was parallel with that of Gem. Finally, expression pattern of ROCK family (only ROCK2 but not ROCK1) was increased in the differentiated process, which was collected with the expression of GEM and active-caspase-3. Based on the present results, it is suggested that Gem might play a crucial role in RGCs apoptosis after ONC, which might be involved in ROCK pathway.     

Radiation sensitization by oxygen of in vitro mammalian cells: is .O-2 involved?

Oxygen is a potent sensitizer of cells exposed to ionizing radiation, and, although the exact chemical mechanisms are not fully understood, some evidence suggests that this sensitization may involve the formation of superoxide anion radicals (.O-2) [F. Lavelle, A. M. Michelson, and L. Dimitrijevic, Biochem. Biophys. Res. Commun. 55, 350-357 (1973); A. Petkau and W. S. Chelack, Int. J. Radiat. Biol. 26, 421-426 (1974); L. W. Oberley, A. L. Lindgren, S. A. Baker, and R. H. Stevens, Radiat. Res. 68, 320-328 (1976)] To test this hypothesis, we compared the sensitivity of Chinese hamster V79 cells irradiated in O2/N2 and O2/N2O gas mixtures with and without the addition of other radical scavenging agents. In these tests, although oxygen was present, be blocked the radiation-induced reactions of O2 which produce .O-2. We found that the total amount of biological damage depends simply on the concentration of O2 that is present; the overall sensitivity is not reduced when .O-2 cannot be formed. Thus radiation sensitization by O2--at least of this cell line--does not require the formation of superoxide anion radicals.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Ultrastructural study of the neuroglial and macrophagic reaction in Wallerian degeneration of the adult rat optic nerve.

The Wallerian degeneration of the optic nerve of adult rat has been studied after destroying the retina. Animals were sacrificed between 4 days and 1 year after the lesion. Different cell types of the optic nerve have been studied ultrastructurally. Our results demonstrate the existence of a population of macrophages, probably of microglial origin, responsible for scavenging degenerated myelin. Astrocytes suffer a process of proliferation and hypertrophy, and are massively stuffed by gliofilaments, leading to a glial scar. These cells apparently do not participate in phagocytic phenomena, while some cytoplasmic inclusions (e.g. lipid droplets) suggest some implication in the local metabolization of some tissue degradation products. Oligodendrocytes do not undergo ultrastructural changes, showing a rather quiescent appearance.     

The complex relationship between cell division and the control of differentiation in oligodendrocyte-type-2 astrocyte progenitor cells isolated from perinatal and adult rat optic nerves

By studying the response of a well-defined progenitor cell to two well-defined mitogens, we have been able to provide a dramatic example of the complex relationship which can exist between the control of cell division and the control of differentiation.In previous studies we have described the development of the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell, a glial progenitor cell isolated from the rat optic nerve. Although originally described as a bipotential cell, we have recently identified a new differentiation pathway in this lineage. We have found that O-2A^perinatal progenitors, with properties appropriate for early development, give rise to O-2A^adult progenitors, which have stem cell-like properties more appropriate to the physiological needs of adult animals. Our studies thus indicate that the population of O-2A^perinatal progenitors is tripotential, and also suggests a possible developmental origin for self-renewing stem cells. Moreover, the properties of O-2A^adult progenitor cells may provide a cellular biological basis for understanding the failure of remyelination in multiple sclerosis.The division of both O-2A^perinatal and O-2A^adult progenitors is stimulated by type-1 astrocytes (which are themselves derived from a separate glial lineage) but this cell-cell interaction promotes different programs of differentiation in the two progenitor populations. The effects of type-1 astrocytes on perinatal and adult progenitors appears to be mediated by platelet-derived growth factor (PDGF), and this mitogen will also induce different programs of differentiation in the two progenitor populations. Moreover, the patterns of differentiation promoted by PDGF are different from those promoted by fibroblast growth factor (FGF), demonstrating that the modulation of division can be distinguished from the modulation of differentiation.     

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.