Distinct sites of origin of oligodendrocytes and somatic motoneurons in the chick spinal cord: oligodendrocytes arise from Nkx2.2-expressing progenitors by a Shh-dependent mechanism.

In the vertebrate spinal cord, oligodendrocytes arise from the ventral part of the neuroepithelium, a region also known to generate somatic motoneurons. The emergence of oligodendrocytes, like that of motoneurons, depends on an inductive signal mediated by Sonic hedgehog. We have defined the precise timing of oligodendrocyte progenitor specification in the cervico-brachial spinal cord of the chick embryo. We show that ventral neuroepithelial explants, isolated at various development stages, are unable to generate oligodendrocytes in culture until E5 but become able to do so in an autonomous way from E5.5. This indicates that the induction of oligodendrocyte precursors is a late event that occurs between E5 and E5.5, precisely at the time when the ventral neuroepithelium stops producing somatic motoneurons. Analysis of the spatial restriction of oligodendrocyte progenitors, evidenced by their expression of O4 or PDGFR(&agr;), indicate that they always lie within the most ventral Nkx2.2-expressing domain of the neuroepithelium, and not in the adjacent domain characterized by Pax6 expression from which somatic motoneurons emerge. We then confirm that Shh is necessary between E5 and E5.5 to specify oligodendrocyte precursors but is no longer required beyond this stage to maintain ongoing oligodendrocyte production. Furthermore, Shh is sufficient to induce oligodendrocyte formation from ventral neuroepithelial explants dissected at E5. Newly induced oligodendrocytes expressed Nkx2.2 but not Pax6, correlating with the in vivo observation. Altogether, our results show that, in the chick spinal cord, oligodendrocytes originate from Nkx2.2-expressing progenitors.

Early specification of oligodendrocytes in the chick embryonic brain.

Oligodendrocytes are the myelin-forming cells in the central nervous system of vertebrates. In the rodent embryo, these cells have been shown to emerge from restricted territories of the neuroepithelium. However, a comprehensive view of the development of oligodendroglial populations from their ventricular sources remains to be established. As a first step toward this aim, we have examined in vivo the spatiotemporal emergence of oligodendrocytes in the chick embryonic brain. We have detailed the patterns of expression of three early markers of the oligodendroglial lineage: the plp/dm-20 and PDGFRalpha transcripts and the O4-reactive antigen. During embryonic development, these molecules showed a similar segmental pattern of expression. However, plp/dm-20(+) cells were already observed, in the ventricular layer, at E2.5, i.e., 2 days before the appearance of O4(+) and PDGFRalpha(+) cells, suggesting that oligodendrocyte precursors arise nearly simultaneously with neurons. In the chick embryonic brain, the onset of expression of plp/dm-20 appears therefore to be the earliest event indicative of oligodendroglial specification and we propose, based on the expression of plp/dm-20 transcript, a ventricular map of the foci at which oligodendrocytes originate. In addition, we document the precocious segregation, from E5, of plp/dm-20(+) and PDGFRalpha(+) oligodendroglial cells in the subventricular and mantle layers of the brain.

Critical analysis of the supraspinatus outlet view: rationale for a standard scapular Y-view.

The supraspinatus outlet view has been standardized under fluoroscopic control to become reproducible and comparable; it is the normalized scapular Y-view. Four hundred four healthy shoulders from patients 20 to 80 years old and 63 shoulders with rotator cuff tears underwent x-ray evaluation and were compared. A qualitative study was carried out on the shape of the acromion in relation to Bigliani's types (I, II, III and on the presence or absence of a bony spur on the anterior part of the acromion. Quantitative measurements were determined; the subacromial peak and the spinoacromial angle were assessed for a statistical comparison among the various populations. The acromions of healthy shoulders varied with age (8% type III in patients younger than 60 years, 27% in those older than 60 years), although not significantly. The dominant side and sex of the subjects had no effect. With 29% type III acromions the shoulders with cuff tears differed from those of the healthy shoulders (14% type III). No close correlation was seen between type III acromions and cuff ruptures. The quantitative values of the acromions had no predictive value for cuff rupture. Acromion typology failed to confirm that a type III acromion was responsible for rotator cuff rupture. Spurs are found in increasing incidence with age and in the presence of cuff rupture; whether they are caused by subacromial impingement or by natural aging of the acromion is uncertain.

Comparison of neurite outgrowth induced by intact and injured sciatic nerves: a confocal and functional analysis.

Mechanisms regulating axon growth in the peripheral nervous system have been studied by means of an in vitro bioassay, the tissue section culture, in which regenerating neurons are grown on substrata made up of tissue sections. Sections from intact and degenerated sciatic nerves proved to be different in their ability to support neurite outgrowth of embryonic chick sensory neurons from both qualitative and quantitative points of view. On denervated nerve sections, the total length of neurites elaborated per neuron was almost twice that found on intact nerve sections. In addition, confocal microscopy revealed a striking difference between intact and denervated nerve substrata: on denervated nerve sections, neurites grew inside the internal structures of endoneurial Schwann cell tubes, within the underlying tissue sections, whereas on intact nerve sections neurites extended along endoneurial basal laminae but never entered Schwann cell tubes. Perturbation experiments were used to analyze some of the molecular determinants that control neurite outgrowth in this system. Antibodies directed against the beta1-integrin subunit inhibited neurite extension on both normal and degenerated rat sciatic nerve tissue. Strikingly, however, differential inhibition was observed using antibodies directed against extracellular matrix molecules. Anti-laminin-2 (merosin) antibodies drastically reduced both the percentage of growing neurons and the total length of neurites on denervated nerve sections, but they did not modify these parameters on sections of normal nerve. Taken together, these results suggest that laminin-2/merosin promotes neurite outgrowth in peripheral nerve environments but only after Wallerian degeneration, which is when axons are allowed to extend within endoneurial tubes.

CNS axonal regeneration with peripheral nerve grafts cryopreserved by vitrification: cytological and functional aspects.

To test cool-warm protocols for storing peripheral nerves, 4-cm-long-nerve segments were removed from the hindleg of adult rats and cryopreserved using a vitrification solution (or cryoprotective mixture) containing a mixture of polyalcohols (2,3-butanediol, 1,2-propanediol, polyethylene glycol, and Belzer U.W. medium). Schwann cell viability and morphology were studied with regard to the effect of (i) cryoprotective mixture concentration (100, 50, and 30% diluted in human serum albumin at 4%), (ii) duration of exposure (10, 15, or 30 min in a single step) of nerves to the cryoprotective mixture, (iii) cooling rate (F1/F2, F3, and F4: 3, 12, and 231 degrees C/min, respectively), and (iv) type of replacement of cryoprotectant (T1, one step; or T2, perfusion) after warming. Nerves exposed 10 min to cryoprotective mixture 50% (2,3-butanediol, 1.926 mol.liter-1; 1,2-propanediol, 3.063 mol.liter-1; polyethylene glycol, 0.084 mol.liter-1; and Belzer U.W., 22.4 mosm-1) and cooled-warmed with the F2/F3/F4-T2 protocols contained live and correctly cryopreserved Schwann cells. The capacity of these cryopreserved nerve segments (n = 6) to be subsequently repopulated by regenerating axons from central neurons was compared to that of fresh nerves when used as peripheral nerve autografts implanted within the spinal cord at the level of the descending respiratory pathways. All cryopreserved nerve grafts were successfully reinnervated by regenerated central axons. Unitary spontaneous action potentials propagated along these axons were assessed by recording the discharge of tested nervous filaments (T) from the grafts in artificially ventilated and paralyzed animals. Out of 535 T, 32 (6 +/- 1.2%) presented spontaneous unitary activity with respiratory (R, n = 2) and nonrespiratory (NR, n = 30) pattern of discharge. The T mean number, the occurrence rate referenced to the total number of T (R/T, NR/T, and R + NR/T) and the mean number of spontaneous units (R, NR, R + NR) were compared to those of fresh spinal peripheral nerve grafts. Except for T, cryopreserved peripheral nerve grafts contained statistically significantly (P < 0.05) less spontaneous R and NR unitary activity, which represented, respectively, 6.2 +/- 6.2 and 26.8 +/- 5.7% of that found in the control group. These data indicate that nerves cryopreserved with the protocols described above contain viable Schwann cells which constitute a suitable support to induce regeneration of central fibers. The effectiveness of nerve cryopreservation by vitrification is discussed with regard to Schwann cell viability following cool-warm protocols and to subsequent reinnervation of the cryopreserved peripheral nerve grafts.

Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.

A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.

Induction of oligodendrocyte progenitors in the trunk neural tube by ventralizing signals: effects of notochord and floor plate grafts, and of sonic hedgehog.

Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. We have documented in chick embryos the effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation. Notochord or floor plate grafted at stage 10 in dorsal position induced the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiated from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induced oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage is related to the establishment of the dorso-ventral polarity of the neural tube.

Antibodies directed against the beta 1-integrin subunit and peptides containing the IKVAV sequence of laminin perturb neurite outgrowth of peripheral neurons on immature spinal cord substrata.

Neuron-substratum interactions regulating axon growth in the developing central nervous system of the rat have been studied by means of an in vitro bioassay: the tissue section culture. We have previously shown that purified chicken sensory or sympathetic neurons grown on natural substrata consisting of cryostat sections of neonatal rat spinal cord elaborate numerous long neurites [Sagot et al. (1991) Brain Res. 543, 25-35]. Perturbation experiments, in which neuron-substratum interactions are modified by antibodies and peptides, have allowed us to analyse some of the molecular determinants which control neurite outgrowth in this system. Antibodies directed against the beta 1-integrin subunit, one of the neuronal receptors for extracellular matrix molecules, reduced the percentage of growing neurons by about 30% and the length of neurites by about 50%. In contrast, antibodies directed against laminin-1 or fibronectin, two extracellular matrix proteins transiently expressed in various areas of the developing central nervous system, were unable to block neurite outgrowth. Paradoxically, a peptide containing the IKVAV sequence, which mimics an active sequence of the laminin alpha 1 chain responsible for neurite extension, also blocked neurite outgrowth on neonatal spinal cord substrata. These results indicate that integrin receptors containing the beta 1 subunit may play a role in regulating axon growth in the developing nervous system. Among the putative extracellular matrix ligands for these receptors, laminin and fibronectin do not appear as prominent candidates in the neonatal spinal cord. However, our data also suggest that the developing central nervous system may contain neurite outgrowth-promoting proteins carrying the IKVAV sequence, different from laminin-1.

Notochord and floor plate stimulate oligodendrocyte differentiation in cultures of the chick dorsal neural tube.

The regionalization of oligodendrocyte potentialities and the cellular interactions leading to the expression of the oligodendrocyte phenotype have been analyzed in the embryonic chick spinal cord. Dorsal and ventral regions of the spinal cord of 4-day-old embryos (E4) were cultivated separately. Oligodendrocyte differentiation was monitored at various times after explantation, using specific oligodendrocyte markers. After 2 weeks, several hundreds of differentiated oligodendrocytes were invariably observed in ventral cultures whereas significant numbers of oligodendrocytes failed to develop in dorsal spinal cord cultures. However, the E7 dorsal spinal cord was found to produce large numbers of oligodendrocytes, indicating that the ventral restriction of oligodendrocyte potentialities is transient. To test whether ventrally derived signals might influence oligodendrocyte differentiation, E4 dorsal spinal cord microexplants were cocultivated with notochord segments or with floor plate tissue. Numerous oligodendrocytes were found in dorsal explants associated with either tissue, notochord or floor plate, but not in dorsal explants cultivated alone, indicating that cells competent to be induced along the oligodendrocyte phenotype exist in the dorsal spinal cord. These results show that oligodendrocyte differentiation potentialities are initially restricted to the ventral spinal cord and suggest that ventrally derived signals from notochord and floor plate influence oligodendrocyte differentiation in the embryonic spinal cord.

[Oligodendrocyte lineage]. / Le lignage oligodendrocytaire.

The mechanisms leading to cell diversification in the Vertebrate central nervous system are still poorly understood. We have analyzed neural differentiation potentialities of the embryonic chick optic nerve. In the adult, the optic nerve is made up of astrocytes and oligodendrocytes ensheathing retinal axons, but it is entirely devoid of neuronal cell bodies. Using explant cultures and specific cell type markers, we demonstrate that in fact the embryonic optic nerve contains cells endowed with neuronal potentialities but is initially devoid of a potential for oligodendrogenesis. Studies by other groups in rodents suggest that oligodendrocyte precursors may be initially restricted to the ventral region of the developing spinal cord. Taken together, these results indicate that early in development, oligodendrocyte precursors are not distributed homogeneously in the neuroepithelium. Preliminary results in our laboratory show that the specification of the oligodendrocyte lineage in the chick spinal cord may depend on ventral signals from the notochord.

Long-lasting accumulation of hemopexin in permanently transected peripheral nerves and its down-regulation during regeneration.

We have previously demonstrated that hemopexin is present in the intact sciatic nerve and is overproduced in the distal stump after nerve transection (Swerts et al.: J Biol Chem 267:10596-10600, 1992). To get further insight into the function of this hemoprotein in nervous tissue, we have documented long-term changes in hemopexin levels in permanently degenerated (transected) and regenerating (crush-lesioned) sciatic nerves of adult rats, using immunochemical techniques. As early as a couple of days after nerve transection, the amount of hemopexin was raised in the distal stump and at the end of the proximal stump. Similarly, after a crush lesion hemopexin was rapidly increased at the injury site and in the distal part of the nerve. Subsequently, in transected nerves the level of hemopexin rose steadily and remained elevated, representing, three months after injury, over 20 times the amount found in intact contralateral nerves. In contrast, in crush-lesioned nerves, hemopexin level declined progressively in a proximodistal direction and returned to basal values 2 months after injury, together with axonal regeneration. This long-term increase in hemopexin in permanently degenerated nerves and its progressive return to normal levels during nerve regeneration suggests that hemopexin content could be regulated negatively, directly or indirectly, by growing axons. In turn, these results support the idea that hemopexin could be involved in the process of Wallerian degeneration and/or in nerve repair.

Lineage analysis of early neural plate cells: cells with purely neuronal fate coexist with bipotential neuroglial progenitors.

To investigate the lineage relationships of neurons and astroglial cells early in central nervous system development, we have analyzed the progeny of neural plate cells in an amphibian embryo (Pleurodeles waltl). A fluorescent tracer, lysinated rhodamine-dextran, was iontophoretically injected into individual precursor cells in various areas of the early neural plate. The phenotypes of clonally related cells were identified in the hindbrain and spinal cord by morphological and immunohistochemical criteria 12 days later, at larval stages. We found that the large majority of clones (83%) contained both neurons and astroglial cells, whereas the remainder (17%) were homogeneous and were only composed of neurons. We never observed purely astroglial clones. These results clearly demonstrate the predominance of bipotential progenitors in the neural plate. Interestingly, the progenitors with a restricted neuronal fate were always located along the intermediate axes of the neural plate, while mixed progenitors were found in all areas examined. The analysis of migratory paths has shown that sister cells first migrated together along radial pathways without dispersion along the rostrocaudal axis. From larval stages, some neurons migrated away from the original clonal cohort along dorsoventral and ventrodorsal tangential routes, but only after they had reached the border between the intermediate and marginal zones.

Cells from the early chick optic nerve generate neurons but not oligodendrocytes in vitro.

We have recently described neuronal potentialities in neuroepithelial cells of the embryonic chicken optic nerve (Giess et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 1643-1647). To further investigate the developmental repertoire of optic nerve cells, oligodendroglial development was studied in cultures of optic nerve explanted at various developmental stages. Oligodendrocyte differentiation was analyzed using antibodies directed against galactocerebrosides (Gal-C) and against sulfatides. Optic nerves removed at embryonic days 5 and 6 (E5-E6) never gave rise in culture to differentiated oligodendrocytes, even after 3 weeks in vitro. In contrast, in cultures of optic nerves removed from E7 or older embryos, cells expressing both oligodendrocyte markers were rapidly and invariably observed. Absence of oligodendrocytes before E7 was not due to culture conditions being inadequate to support the differentiation of early precursors along this pathway, since neuroepithelial cells from E2 and E4 trunk neural tube cultivated in the same conditions expressed Gal-C after respectively 16 and 10 days. These results demonstrate that the optic nerve territory is initially devoid of oligodendrocyte potentialities. Whether oligodendrocyte precursors that, around E7, populate the optic nerve are induced by a specific developmental signal occurring at this stage or migrate from outside the optic nerve remains to be determined.

Hemopexin is synthesized in peripheral nerves but not in central nervous system and accumulates after axotomy.

In adult mammals, injured axons regrow over long distances in peripheral nerves but fail to do so in the central nervous system. Analysis of molecular components of tissue environments that allow axonal regrowth revealed a dramatic increase in the level of hemopexin, a heme-transporting protein, in long-term axotomized peripheral nerve. In contrast, hemopexin did not accumulate in lesioned optic nerve. Sciatic nerve and skeletal muscle, but not brain, were shown to be sites of synthesis of hemopexin. Thus, hemopexin expression, which can no longer be considered to be liver-specific, correlates with tissular permissivity for axonal regeneration.

[2 roentgen projections for the subacromial space before and following acromioplasty. Results of a study series of 40 patients]. / Zwei Röntgenzielaufnahmen für den Subakromialraum vor und nach Akromioplastik. Ergebnisse einer Untersuchungsserie von 40 Patienten.

It is suggested that two radiological projections of the scapula, an AP view and a lateral (tunnel) view, be obtained in order to estimate the form and size of the acromion before and after acromioplasty. In order to evaluate the standardized films precisely, a film must have been taken for comparison before the surgery. As the method described is easily reproducible, it can be used to evaluate the effect of an acromioplasty irrespective of whether the procedure was an open or an arthroscopic one. The radiological results can be correlated with the functional results at the beginning of rehabilitation and at the end of this period. More sophisticated studies might require a more elaborate X-ray technique than the one proposed in this article.

Changes in permissivity for neuronal attachment and neurite outgrowth of spinal cord grey and white matters during development: a study with the 'cryoculture' bioassay.

We have used the recently developed cryoculture bioassay (Carbonetto et al., J. Neurosci., 7 (1987) 610-620) to document changes during development of CNS tissular ability to support nerve fiber growth. Neuronal attachment and neurite outgrowth of purified neurons cultured on tissue sections of rat spinal cord at various stages of development were quantified. Nerve fiber growth permissivity increased during embryonic stages, reaching as postnatal days 2-4 (P2-P4) a maximum value, higher than that found on adult PNS tissue sections. This permissivity diminished rapidly thereafter, indicating that early postnatally, the nerve fiber growth supporting ability of the CNS environment shifts abruptly from an increasingly permissive mode to an increasingly non-permissive status. Furthermore, after P4, neurite outgrowth permissivity diminished in parallel on white and grey matters, whereas neuronal attachment declined much more drastically on white matter than on grey matter. This indicates that the progressive loss of spinal cord ability to support nerve fiber growth is attributable to both grey and white matters. In several instances it also appeared that neuronal adhesion was not necessarily followed by a comparable level of nerve fiber growth, suggesting that these two processes could be regulated by different factors.

Astroglial differentiation from neuroepithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis.

Initial development of astroglial phenotype has been studied in vitro in an amphibian embryo (Pleurodeles waltI), to document the differentiation potentialities acquired by neural precursor cells isolated at the early neurula stage. In particular, we sought to determine whether interactions between neuroepithelial cells and the inducing tissue, the chordamesoderm, are required beyond this stage to specify precursor cells along glial lineages. Glial cell differentiation was documented by examining the appearance of glial fibrillary acidic protein (GFAp), a specific marker of astroglial lineages. Cells expressing GFAp-immunoreactivity differentiated rapidly, after 48 hours of culture, from cultivated neural plate cells, irrespective of the presence or absence of the inducing tissue. The widespread expression of Pleurodeles GFAp protein in neural plate cultures, in which CNS precursor cells develop alone in a simple saline medium, showed that prolonged contact with chordamesodermal cells was not necessary for the emergence of the astroglial phenotype. In addition, the initial development of astroglial phenotype has been defined in vivo. The first detectable GFAp-immunoreactivity was visualized in the neural tube of stage-24 embryos, a stage corresponding to 2-3 days in culture, defining radial glial cell end-feet. Thus, dissociation and culture of neural precursor cells did not appear to modify the onset of astroglial differentiation. At stage 32, GFAp-immunoreactivity was observed over the entire length of radial glial fibers and was also evidenced in mitotic cells located in the ventricular zone, suggesting that radial glial cells were not all post-mitotic.

Neuronal potentialities of cells in the optic nerve of the chicken embryo are revealed in culture.

Neuronal potentialities in neuroepithelial cells of the chicken embryonic optic nerve were studied in culture by using neurofilament antibodies as neuronal markers. Embryonic day-4 and -5 (E4 and E5) optic stalks were explanted in vitro. Within the first few days of culture, numerous morphologically identifiable neurons extending long neurites developed. These neurons and their processes were specifically labeled with neurofilament antibodies. Similar results were obtained by explanting only the medial portion of E7 optic stalks away from possibly contaminating cerebral or retinal tissue. To determine whether neuronal potentialities persisted at later embryonic stages, cultures of dissociated optic stalks were established at E11, E15, and E18. Neurons labeled with the various neurofilament antibodies appeared in all cultures of E11 and E15 optic stalks. However, typical neurons could not be recognized in cultures of E18 optic nerves. These results indicate that cells with neuronal potentialities are present in the embryonic optic nerve from early stages of development and persist until at least E15. Since the adult optic nerve is devoid of nerve cell bodies, our observations are consistent with the hypothesis that axons of retinal ganglion cells, which course through the optic stalk, repress neuronal potentialities within a subpopulation of precursor cells during normal development.

Nerve fiber growth in culture on tissue substrata from central and peripheral nervous systems.

In adult mammals, injured neurons regenerate extensively within the PNS but poorly, if at all, within the CNS. We have studied the effect of substrata consisting of tissue sections from various nervous systems on nerve fiber growth in culture and correlated our results with the growth potential of these tissues in vivo. Ganglionic explants from embryonic chicks (9-12 d) fail to extend nerve fibers onto sections of adult rat optic nerve or spinal cord (CNS) but do so on sciatic nerve (PNS). Dissociated DRG neurons behave similarly whether in serum-containing or defined medium. Tissue substrata from nervous systems that support regeneration in vivo--i.e., goldfish optic nerve, embryonic rat spinal cord, degenerating sciatic nerve--also support fiber growth in culture. Within the same culture, neurons will grow onto sciatic nerve rather than neighboring optic nerve sections, suggesting that the responsible agent(s) is not soluble. In addition, neurons adhere more extensively to sciatic nerve substrata than to optic nerve. The occurrence of 3 molecules known to be involved in neuron-substratum adhesion and nerve fiber growth in vitro has been documented immunocytochemically in the tissue sections. One of these, laminin, is demonstrable in all tissues tested that supported nerve fiber growth. Immunoreactivities for fibronectin and heparan sulfate proteoglycan are found in only some of these tissues. None of these 3 molecules can be demonstrated in neural cells of normal adult rat CNS tissue. Our data suggest that these molecules may be important effectors of nerve regeneration in neural tissues.