Biomedical subjects

J Vanselow

Publications and source records attributed to J Vanselow.

31 records · Page 2Linked to original sources

PubMed1994

GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43-like expression pattern during development and regeneration.

Using transgenic mice, we have examined the expression pattern conferred by regions of genomic GAP-43 coupled to beta-galactosidase. We demonstrate that gene constructions that include the GAP-43 5'-flanking region along with sufficient sequences of the first intron drive beta-galactosidase (lacZ) expression to mimic in many regards the complex spatial and temporal pattern of endogenous GAP-43 expression. Transgene expression reaches peak levels during development, and persists at high levels in particular adult brain regions, such as the hippocampus and olfactory bulb. The inclusion of a stretch of the first intron in the construction is necessary to prevent expression outside of the nervous system, indicating that some of the cell specificity of GAP-43 expression is due to suppression of expression in inappropriate tissues. Injury caused by sciatic nerve crush causes reexpression of the transgene in adult sensory and motor neurons. This genomic region of GAP-43, therefore, includes elements responsive to neuronal growth signals that regulate both development and regeneration.

Aging↗

PubMed1992

Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro.

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.

Affinity Labels↗

PubMed1991

Regenerating axons from adult chick retinal ganglion cells recognize topographic cues from embryonic central targets.

We investigated whether regenerating mature axons recapitulate embryonic features essential to successful reconnectivity within the injured nervous system. Strips from embryonic and adult chick retinae were cultured, and outgrowing axons were examined morphometrically and immunohistochemically. In addition, the target-recognition properties of adult neurites were analyzed. Regenerating adult axons elongate on a poly-L-lysine/laminin substratum with a speed about one order of magnitude slower than that of embryonic axons. Morphologically, adult axonal tips differ dramatically from embryonic growth cones in that they possess only filopodial extensions whereas embryonic growth cones possess both lamellipodial and filopodial processes. Both embryonic and adult neurites express the growth-associated protein GAP-43. When cultured on alternating stripes of anterior and posterior embryonic tectal membranes, both adult and embryonic retinal axons distinguish between the two membrane preparations. Our results demonstrate that during axonal regeneration the mature neurons express embryonic properties that are involved in the recognition of tectal positional cues.

Animals↗

PubMed1991

Expression of a novel Müller glia specific antigen during development and after optic nerve lesion.

To generate monoclonal antibodies, immunogen fractions were purified from embryonic chick retinae by temperature-induced detergent-phase separation employing Triton X-114. Under reducing conditions, the monoclonal antibody (mAb) 2M6 identifies a protein doublet at 40 and 46 x 10(3) Mr, which appears to form disulfide-coupled multimers. The 2M6 antigen is regulated developmentally during retinal histogenesis and its expression correlates with Müller glial cell differentiation. Isolated glial endfeet and retinal glial cells in vitro were found to be 2M6-positive, identified with the aid of the general glia marker mAb R5. mAb 2M6 does not bind to any other glial cell type in the CNS as judged from immunohistochemical data. Cell-type specificity was further substantiated by employing retinal explant and single cell cultures on laminin in conjunction with two novel neuron-specific monoclonal antibodies. MAb 2M6 does not bind either to neurites or to neuronal cell bodies. Incubation of retinal cells in vitro with bromodeoxyuridine (BrdU) and subsequent immunodouble labelling with mAb 2M6 and anti-BrdU reveal that mitotic Müller cells can also express the 2M6 antigen. To investigate whether Müller cell differentiation depends on interactions with earlier differentiating ganglion cells, transections of early embryonic optic nerves in vivo were performed. This operation eliminates ganglion cells. Müller cell development and 2M6 antigen expression were not affected, suggesting a ganglion-cell-independent differentiation process. If, however, the optic nerve of juvenile chicken was crushed to induce a transient degeneration/regeneration process in the retina, a significant increase of 2M6 immunoreactivity became evident. These data are in line with the hypothesis that Müller glial cells, in contrast to other distinct glial cell types, might facilitate neural regeneration.

Animals↗

PubMed1990

Target dependence of chick retinal ganglion cells during embryogenesis: cell survival and dendritic development.

The survival of retinal ganglion cells and the dendritic development were investigated a) in normal chick embryos, b) in embryos whose primordial optic lobes and adjacent areas were removed (target reduced embryos), and c) in embryos whose optic nerves were transected (target deprived embryos) in order to study the influences of central targets on developing ganglion cells. The ganglion cells were stained postmortem with the carbocyanine dye DiI. Cell body and dendritic field diameters were measured in whole-mounted retinae before and after the period of cell death at embryonic day 10 (E10) and E16. The cell densities within the ganglion cell layer were counted in cresyl violet/thionine stained retinae. The central retinal projection in target reduced embryos was studied with the anterogradely transported fluorescent marker rhodamine-B-isothiocyanate (RITC). In normal embryos, the earliest dendritic processes were observed at E6 in the central retina, whereas at E10 elaborate dendritic branching was found across the retina. Different morphological types of ganglion cells could be identified at E16. In both, target reduced embryos and target deprived embryos, the initial dendritic growth and pattern of ramification were indistinguishable from those of normal embryos up to E10. Cell body diameters, dendritic tree diameters, and cell densities were not significantly different. At the end of the naturally occurring cell death period (E16), the ganglion cell density was strongly reduced in both experimental groups compared to controls. In particular, when the optic nerve was transected, it resulted in the almost complete degeneration of ganglion cells. In target reduced embryos, a small population (about 5% of the normal number) of ganglion cells survived. The proportion of large cells was increased within the total population compared to normal retinae. Displaced ganglion cells were not affected by partial target removal but strongly affected by transection of the optic nerve. Anterograde labelling from the retina revealed that in target reduced embryos the remaining ganglion cells innervated non-tectal primary visual nuclei. The present results suggest the following: a) Before the onset of the cell death period, the growth and ramification of ganglion cell dendrites occur independently of central visual targets. b) In target reduced embryos, a small population of ganglion cells survives, namely, those cells that project to remaining central areas. Complete disconnection from central targets by transecting the optic nerve leads to the degeneration of almost all ganglion cells. c) The surviving ganglion cell population consists mainly of large ganglion cells.

Animals↗

PubMed1989

Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides.

The ability of lesioned adult retina ganglion cells (RGC) to survive and regrow axons in vitro was investigated in retina organ cultures under chemically defined conditions. Factors which are known to either affect the RGC survival like the basic fibroblast growth factor (FGF) or influence neurite outgrowth like gangliosides were assayed by recording the course of prelabeled RGC degeneration in vitro and the number and length of regrowing RGC axons from explanted retinal pieces. Administration of basic FGF significantly slowed down the decrease in the number of RITC-prelabeled RGC in the cultured retinae. In addition, in the presence of gangliosides (GM1, GD1a, GD1b GT1b), the numbers of regrown RGC axons (Thy 1-immunostained) increased dramatically as compared to controls. The data indicate that adult neurons with an intrinsic ability to regenerate axons can respond to substances with neurotrophic or neurite-promoting activities in tissue cultures.

Animals↗

PubMed1989

Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development.

Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection.

Animals↗

PubMed1989

Adult retinal ganglion cells retain the ability to regenerate their axons up to several weeks after axotomy.

The present work was to elucidate whether the ability of adult central neurons to regrow their lesioned axons is retained for long periods of time. Using the rat retina as an experimental paradigm, the optic nerve was lesioned by crush in situ. Up to 6 weeks after the trauma, the optic nerve (ON) was again exposed and transected close to the first lesion and autologous sciatic nerve segments were anastomosed at the ocular ON stump. Alternatively, the retina corresponding to the lesioned ON was dissected for in vitro cultivation 1-6 weeks after the crush-axatomy was applied. Both experimental strategies revealed a regrowth of the lesioned retinal ganglion cell (RGC) axons. When peripheral nerve (PN) segments were grafted without previous ON crush, axon stumps started to reelongate and to penetrate the grafted piece of nerve 6 days later. In contrast, when the PN graft was apposed to the ON stump 1-6 weeks after crush, it was penetrated by regrowing axons within 24 hr. Maximal numbers of regenerating axons were observed if transplantation occurred within the first week after the crush. The numbers of axons decreased progressively if transplantation was performed later than 1 week postcrush and approximated zero values at the 6th week. When the noncrushed retina was explanted and cultured in vitro in a chemically defined, serum-free medium, there was almost no extending fiber. In contrast, explantation of the retina for which the ON had been precrushed in situ resulted in massive regrowth of RGC axons. The numbers of regenerating axons and their temporal changes paralleled those described for the transplantation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

[The effect of central and peripheral neuroglia on the regeneration of the optic nerve].

The post-traumatic response of adult ganglion cell axons to the presence of regeneration-permissive environments was examined in the optic nerve of the adult rat. As a first goal, we elucidated the reasons why the optic nerve was not able to regenerate. We were able to show that ganglion cell axons growing in vitro could not penetrate an impediment consisting of purified central nervous system myelin. The results demonstrated that the intrinsic ability of central neurons to regrow their axons is suppressed by non-permissive central myelin. After total intraorbital transection of the optic nerve and either simultaneous anastomosis of an autologous transplant from the peroneus communis nerve, or anastomosis delayed for up to 6 weeks several thousand ganglion cell axons reelongated into the transplant and reached lengths of about 30 mm within 5 to 6 weeks. Regeneration-stimulating substances could be accumulated within silicone tubes implanted at the site of the lesion in the N. peroneus communis. Their neurotrophic activity was tested in vitro in the explanted adult retina. The results indicate that the regeneration-permissive peripheral neuroglia responds to lesions with the production and supply of neuritogenic substances. Such substances are also active in the explanted adult retina where they support central axonal reelongation.

Animals↗

PubMed1988

In vitro regeneration of adult rat ganglion cell axons from retinal explants.

The potential for regeneration of adult rat ganglion cell (RGC) axons was investigated in vitro. Explants from RITC (rhodamine-B-isothiocyanate) retrogradely labeled and in vivo axotomized retinae were placed on dishes coated with various substrates. The retinal pieces were cultivated in a serum-free medium and incubated under 50 to 80% O2-enriched and 5% CO2-containing atmosphere. Under these conditions, massive outgrowth of fibers was observed as early as 24 h after explantation and over a period of time extending up to 7 days in culture. By various criteria, two main types of processes could be characterized: (1) Short, thick processes emerged from either migrated flat cells or from cells inside the retinal explant, and (2) long and thin processes emerged from cells in the ganglion cell layer (GCL). By immunohistochemistry, the short processes and the cells from which they had emerged, appeared to be glial acidic fibrillary protein (GFAP)-positive Thy 1 and L 1-negative, suggesting their glial nature. The second type of long, thin processes appeared to be GFAP-negative, L1- and Thy 1-positive, indicating that they were neuronal, probably RGC processes. Proof that long fibers emerged from RGCs was provided by retrograde labeling of RGCs with RITC prior to explanation. Numerous RITC-filled RGCs survived in vitro. Regrowing axons retransported part of the accumulated fluorescent dye in the orthograde direction and thus unequivocally permitted their identification as RGC axons. The fact that adult RGC axons can reelongate in vitro might provide a useful bioassay for investigations on the factors that either support or inhibit regrowth of axons from CNS neurons.

Animals↗

PubMed1987

A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.

Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985, 1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987). We report here that two of these dyes, diI (1,1',dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) and diO (3,3'-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells. This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13 1/2) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.

Animals↗

PubMed1983

Surface electrical stimulation in the treatment of idiopathic scoliosis: preliminary results in 30 patients.

From 1978 to 1981, 30 patients have been treated for idiopathic scoliosis with surface electrical stimulation using the E.S.O. (Electro Spinal Orthosis) Single Channel designed by Medtronics. The criteria for selection were: patients who were skeletally immature; single thoracic, thoracolumbar or lumbar curvatures between 30 degrees and 40 degrees; or the same curve pattern greater than 20 degrees with 5 degrees of documented progression in one year; patient and family reliability. Patients with previous treatment were excluded from the study. No one was more than 15 years of age. Curve amplitude was between 25 degrees and 35 degrees in 72% of the patients. Curve pattern was single thoracic in 28 patients, thoracolumbar in one, and lumbar in one. Five patients were excluded from the evaluation of the results of stimulation of the correction of the curve. Of the 25 patients remaining, one was improved, 14 were stable, two had mild acceptable progression (less than 10 degrees with no need for further treatment), and eight had an unacceptable progression greater than 10 degrees requiring some form of alternative treatment. The authors conclude that significant improvement in the curvatures under treatment was extremely unlikely, that progression may have been stopped in some curves, that the curvatures under 30 degrees had the best results, and that curvatures that do not respond to surface electrical stimulation are not likely to respond to a Milwaukee brace treatment.

Adolescent↗

PubMedDate not supplied

Effects of the mycotoxins alpha- and beta-zearalenol on regulation of progesterone synthesis in cultured granulosa cells from porcine ovaries.

Mycotoxins as contaminants of animal food can impair fertility and can cause abnormal fetal development in farm animals. Therefore, the present study has investigated whether derivatives of the mycotoxin zearalenone, alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL), influence progesterone synthesis via cytochrome p450 side chain cleavage enzyme (p450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) in cultured porcine granulosa cells. Both enzymes are essential for the conversion of cholesterol to progesterone. No differences in basal progesterone levels and numbers of viable cell were observed between untreated granulosa cells and those treated with alpha- or beta-ZOL (15 and 30 microM). FSH (0.01 microg/ml) or forskolin (10 microM) enhanced the basal progesterone secretion in the absence of mycotoxins. The addition of alpha- or beta-ZOL (7.5, 15 and 30 microM) to cultures stimulated with FSH (0.01 microg) or forskolin (10 microM) reduced progesterone synthesis and the levels of p450scc and 3beta-HSD transcripts in a dose-dependent manner (P<0.05). The enzymatic activity of 3beta-HSD and the abundance of p450scc protein were also reduced by these mycotoxins. In conclusion, effects of mycotoxins on FSH receptor-dependent and receptor-independent pathways indicate that adenylate cyclase activity and/or regulatory pathways further downstream are targets of mycotoxin actions. The apparent dose-dependent reduction of p450scc and 3beta-HSD transcripts implies an effect of alpha- and beta-ZOL on transcriptional regulation of these enzymes.

3-Hydroxysteroid Dehydrogenases↗