Alpha-motoneurons of the injured cervical spinal cord of the adult rat can reinnervate the biceps brachii muscle by regenerating axons through peripheral nerve bridges: combined ultrastructural and retrograde axonal tracing study.

Following our previous studies related to brachial plexus injury and repair, the present experimentation was designed to examine the ultrastructural features of those motoneurons of the locally injured cervical spinal cord of adult rats that were seen to regenerate into peripheral nerve (PN) bridges and to reinnervate nearby skeletal muscles. Here, the peripheral connection of the PN bridge was made with the biceps brachii (BB) muscle. Three months postsurgery, the spinal motoneurons labelled by retrograde axonal transport of horseradish peroxidase (HRP), after its injection into the BB, were selected on thick sections, using light microscopy, for the presence of dark amorphous granules of the HRP reaction product. Serial ultrathin sections were then made from the selected material. For the 10 labelled neurons studied, we examined the synaptic boutons present on the membrane of the neuronal soma. For five of them, we could observe three of the six types of synaptic boutons described for the alpha-motoneurons of the cat (S-type with spherical vesicles, F-types with flattened vesicles, and C-type with subsynaptic cistern). The largest boutons (type C) are specific to alpha-motoneurons. In comparison to normal material, we noticed a decrease in the number of boutons and an increase in the number of glial processes. After a transient phase of trophic changes, the reinnervated BB muscles showed a return of their fibers to nearly normal diameters as well as evidence of fiber type grouping. Simultaneous staining with silver and cholinesterase also revealed the presence of new motor endplates frequently contacted by several motoneurons. The present study indicates that, after a local spinal injury, typical alpha-motoneurons can reinnervate a skeletal muscle by regenerating axons into the permissive microenvironment provided by a PN graft. These data offer prospects for clinical reconstruction of the brachial plexus after avulsion of one or several nerve roots.

Transplantation of autologous dorsal root ganglia into the peroneal nerve of adult rats: uni- and bidirectional axonal regrowth from the grafted DRG neurons.

Previous studies have demonstrated that transplanted dorsal root ganglion neurons (DRGNs) can survive and differentiate in a variety of orthotopic and heterotopic locations. In order to develop strategies aimed at restoring the sensory function following traumatic injury to the spinal cord and to its peripheral sensory connections, we have transplanted adult autologous dorsal root ganglia (DRGs) into the peroneal nerve of adult rats. Twelve female Sprague-Dawley rats were used. A segment of the peroneal nerve was isolated by double transection and ligature to prevent undesirable reinnervation. The left fifth cervical (C5) DRG was removed from its normal location and inserted into the midportion of the isolated nerve segment. One month after the grafting procedure, a morphological study included axonal retrograde labeling with True Blue (TB) and/or Diamidino Yellow (DY) applied on each cut end of the nerve segment, cell counting, and cell measurement after staining with cresyl violet. Compared to the C5 ganglion maintained in situ, the mean number of surviving DRGNs in the transplant was 1381, corresponding to a survival rate of 20%. Both singly (TB or DY) and doubly (TB + DY) stained DRGNs were encountered. The proportion of surviving neurons that appeared to be doubly labeled was 23%. These neurons were considered as having grown two opposite axonal projections, one into the "central" part of the nerve segment and a second one into its "peripheral" part. The present results give new insights and interesting prospects concerning the possibilities of reconstructing the sensory circuitry after central and/or peripheral injuries.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Estimation of the number and size of female adult rat C4, C5 and C6 dorsal root ganglia (DRG) neurons.

In previous studies primary sensory neurons of adult rats have been counted in lumbar dorsal root ganglia. However, different counting methods have given very different results and at the cervical level, recent data are scarce. In the present study, the number of neurons in C4, C5 and C6 adult rat ganglia was determined using two previously calibrated techniques. The stereological tool was preferred because it directly identifies neurons instead of nucleoli and is more efficient. The C4, C5 and C6 dorsal root ganglia were found to contain 7508+/-299, 6825+/-950 and 6858+/-923 neurons, respectively, and statistical analysis indicated that there was no significant difference between the three levels. There was, however, a great interindividual variation, which was also found at other levels of the spinal cord. The mean diameter of neurons in the C4, C5 and C6 dorsal root ganglia was determined and was 17.52, 20.16 and 20.68 microm, respectively. It is important to know more about the organization of the sensory systems in the normal rat. Once established, the number of neurons in these dorsal root ganglia could be compared with different pathological situations or experimental treatments such as developmental conditions, nerve section or ganglion transplantation.

Sodium phenylacetate (NaPa) induces modifications of the proliferation, the adhesion and the cell cycle of tumoral epithelial breast cells.

Sodium phenylacetate (NaPa), a physiological product of phenylalanine metabolism, present in micromolar concentrations in human plasma, has been shown to induce in vivo and in vitro cytostatic antiproliferative effects at millimolar concentrations. Cadherin molecules are powerful invasion suppressor molecules and the reduction of E-cadherin expression plays an important role in the invasion and metastasis of human breast cancer. In this study, we demonstrated, on one hand, that NaPa stimulated aggregation by increasing the expression of E-cadherin at the surface of breast cancer MCF-7ras cells transformed by Ha-ras oncogene and inhibited its expression in MCF-7 cells. We demonstrated that NaPa increased the formation of MCF-7ras cell aggregates and did not alter the formation of MCF-7 cell aggregates. By Northern blot, we demonstrated that the E-cadherin expression was not regulated at the transcriptional level. On the other hand, we analyzed the cell cycle of these 2 cell lines after NaPa treatment and showed that NaPa induced arrest at the G1/S phase in both MCF-7 and MCF-7ras cells. bFGF increased the growth of MCF-7 cells, but inhibited MCF-7ras cell proliferation. NaPa treatment suppressed the stimulation of MCF-7 cell proliferation and increased MCF-7ras cell growth inhibition. We have demonstrated a new target of NaPa action in blocking the cell cycle of tumor cells in G0/G1. We suggest that the anti-proliferative effect of NaPa associated to the restoration of the cadherin function in human mammary carcinoma cells indicates that NaPa could be a novel therapeutic agent in breast cancer.

Rat embryo fibroblasts transformed by c-Jun display highly metastatic and angiogenic activities in vivo and deregulate gene expression of both angiogenic and antiangiogenic factors.

The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors.

Sodium phenylacetate modulates the synthesis of autocrine and paracrine growth factors secreted by breast cancer cell lines.

Sodium Phenylacetate (NaPq) has been shown to suppress tumor growth and promote differentiation in experimental models. Thus, we have previously shown an inhibition of MCF-7ras cell proliferation by NaPa both in vitro and in vivo on xenographed tumors. In order to study the action of NaPa on the synthesis of paracrine or autocrine growth factors, conditioned media were prepared from breast pretumoral HBL100 cells, tumoral MCF-7 and MCF-7ras cells in the presence of NaPa. Growth factor activities of these media were tested on Balb c/3T3 fibroblasts and on the above breast tumor cells. Conditioned media from the 3 cell types contained different mitogenic activities when tested on the same cell lines. NaPa treatment for 24 hours inhibited differentially and dose-dependently the mitogenic activity of conditioned media. Inhibitions of HBL100 and MCF-7 cell proliferation by MCF-7ras medium conditioned with 20 mM NaPa reached 75% and 48% respectively. In contrast, NaPa treated MCF-7 conditioned medium decreased HBL100 and MCF-7ras proliferation by 49% and 72%, respectively, at the same NaPa concentration. The efficiency of NaPa inhibition reached an optimum as soon as one day after treatment. Among growth factors secreted by MCF-7 and MCF-7ras, TGF beta synthesis is inhibited and stimulated in MCF-7 and MCF-7ras cells respectively after NaPa treatment. We showed that NaPa modifies the synthesis of growth factors secreted by MCF-7 and MCF-7ras tumor cells leading to cell proliferation inhibitions. The synthesis of these previously identified factors was more involved in MCF-7 cells than fibroblast cell proliferation. In vitro and in vivo NaPa inhibition of MCF-7ras cells which secreted higher levels of these growth factors could be explained by this mechanism of action.

[Post-traumatic reconnection of the cervical spinal cord with skeletal striated muscles. Study in adult rats and marmosets]. / Reconnexion post-traumatique de la moelle épinière cervicale avec la musculature striée squelettique. Etude chez le rat et le marmouset adultes.

In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG. After a post-operative delay ranging from 2 to 22 months, the animals were checked electrophysiologically for functional muscular reconnection and processed for a morphological study including retrograde axonal tracing (HRP, Fast Blue, True Blue), histochemistry (AChE, ATPase), immunocytochemistry (ChAT) and EM. It was thus demonstrated that host motoneurons of the cervical enlargement could extend axons all the way through the PNG bridge as: a) in anaesthetized animals, contraction of the reconnected muscle could be obtained by electrical stimulation of the grafted nerve; b) the retrograde axonal tracing studies indicated that a great number of host cervical neurons extended axons into the PNG bridge up to the muscle; c) many of them were assumed to be motoneurons (double labelling with True Blue and an antibody against ChAT); and even alpha-motoneurons (type C axosomatic synapses in HRP labelled neurons seen in EM in the rat); d) numerous ectopic endplates were seen around the intramuscular tip of the PNG. In larger (cavitation) spinal lesions (rat), foetal motoneurons contained in E14 spinal cord transplants could similarly grow axons through PNG bridges up to the reconnected muscle. Taking all these data into account, it can be concluded that neural transplants are interesting tools for evaluating both the plasticity and the repair capacities of the mammalian spinal cord and of its muscular connections.

Morphology and immunohistochemistry of the nerve endings on the chromaffin cells of adrenal medulla grafted into mouse brain.

Mouse adrenal medulla was transplanted to mouse brain for morphological and morphometric examination of the nerve endings abutting on the surface of the grafted adrenal chromaffin cells. To determine the types of these endings, they were treated with antibodies specific for phenylethanolamine N-methyltransferase (PNMT), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). Three types of vesicles were found in nerve fibers and endings: the first contained small clear synaptic vesicles 30-50 nm in diameter, the second was mixed with large granules with moderately electron-dense cores 80-100 nm in diameter, and the third exhibited small electron-dense cored vesicles 50 nm in diameter. The two first types occurred in nerve endings of normal and grafted medulla, but the third was only seen in the grafts. Grafted chromaffin cells carried two morphologically distinct types of synapse: small with a diameter of 1-2 microns, and large, as in normal adrenal medulla. The first type predominated after transplantation. In normal medulla, the number of synapses calculated per grafted chromaffin cells was about 4.5 for cells containing epinephrine (E) and 5.8 for those containing norepinephrine (NE), and in grafted medulla, 4 per cells. After grafting, nerve endings were labeled to ChAT, AChE and neuron-specific enolase (NSE), but only a few nerve fibers were immunoreactive to PNMT. The presence of NSE in nerve endings on the grafted cells, a marker of the glycolytic activity in neurons, suggests the formation of de novo functional synaptic connections.

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats.

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1-30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.

[Effects of NGF on chromaffin adrenaline-containing cells of adrenal medulla of rabbits transplanted into brains of mice]. / Effets du NGF sur les cellules chromaffines à adrénaline de la médullo-surrénale de lapin greffées dans le cerveau de la souris.

The graft of chromaffin adrenaline-containing (A) cells of rabbit adrenal medulla implanted to mouse brain and treated with NGF contains more survived cells 1 month after grafting than adrenal medulla alone. The cells developed either an intermediate (e.g. chromaffin cell and neuron) or a neuron-like phenotypes accompanied with a decrease in an immunoreactivity for PNMT (phenyletanolamine-N-methyltransferase). A gap junctions and attached plaques were found between grafted cells. The grafts received a synaptic input. The NGF influence on the fate of chromaffin A-containing cells is discussed.

Co-transplantation of fetal or adult dorsal root ganglia and of autologous peripheral nerve segments to the adult rat spinal cord: extensive reinnervation of the grafted nerves by the transplanted DRG cells.

Intraspinal transplantation of embryonic neurons and of autologous peripheral nerve segments is an essential tool for studying plasticity and repair in the adult mammalian spinal cord. Unlike adult central nervous system neurons, adult dorsal root ganglion (DRG) cells can be cultured in vitro and are assumed to survive transplantation. In the present work, we have co-transplanted adult (and also fetal, for comparison) DRG and peripheral nerve autografts to the cervical spinal cord of the adult rat. Similar results were obtained from both series: fetal as well as adult DRG cells did survive transplantation and nearly half of them grew lengthy axons into the grafted nerves. A few of them were seen to express a calcitonin gene-related peptide. Possibilities of central afferentation as well as of peripheral connectivity of these transplanted neurons is under study.

Co-transplantation of embryonic neural tissue and autologous peripheral nerve segments to severe spinal cord injury of the adult rat. Guided axogenesis from transplanted neurons.

The present study is the first of a series of experiments designed to investigate the possibilities of reconstructing the severely injured spinal cord by means of transplantation techniques. Special attention has been given here to the capability of transplanted embryonic neurons to extend axons into autologous peripheral nerve grafts (PNGs). A cavity, made unilaterally in the cervical enlargement of the spinal cord of adult rats, was filled with solid pieces of different embryonic tissues: spinal cord (SC), cortex (CT) or dorsal root ganglia (DRG). In more than half of the transplanted animals, one end of a PNG was inserted into the center of the transplants, while the other, extraspinal end, was crushed and tied to peripheral tissues. After a postgrafting period ranging from 1 to 6 months, we found that the 3 types of transplants in general had survived and become integrated with the host spinal cord, although their overall organization remained atypical. Surviving graft neurons had developed processes, some of which had become myelinated. The ability of the grafted neurons to extend axons into the PNG differed strikingly from one type of graft to another, being apparently non-existent for cortical grafts, moderate for spinal cord grafts and quite extensive for dorsal root ganglia transplants. Interestingly, these differences reflected what was observed for the corresponding, fully differentiated qeurons in adult animals, when their cut axons were put in contact with non-neuronal components of peripheral nerves.

Invasion of the peripheral nervous systems of adult mice by the CVS strain of rabies virus and its avirulent derivative AvO1.

The penetration of the CVS strain of rabies virus and its avirulent derivative AvO1 into peripheral neurons was investigated after intramuscular inoculation into the forelimbs of adult mice. It was found that CVS directly penetrates both the sensitive and motor routes with equal efficiency, without prior multiplication in muscle cells. Infected neurons became detectable 18 h after infection. The second cycle of infection occurred within 2 days, and at day 3 there was a massive invasion of the spinal cord and sensory ganglia. In sensory ganglia, where it was possible to identify cell outlines, it was evident that the infection did not proceed directly from cell body to cell body. The avirulent strain AvO1 penetrated motor and sensory neurons with the same efficiency as CVS. Restriction of viral propagation was observed from the second and third cycles onwards. No further development of the infection could be seen after day 3, and by that time the lysis of primarily infected neurons seemed to occur.

Identification of spinal motoneurones in the weakly electric fish, Eigenmannia virescens.

The motoneurones of the spinal cord in the weakly electric fish, Eigenmannia virescens, were investigated by light microscopical observations. For identification of motoneurones HRP injections were made: (1) in the electric organ, (2) in different parts of the trunk muscle, and (3) in the muscle of the anal fin. When the spinal cord was considered in transverse section: (1) labelled cells (EMN), 20-25 micron, were observed in the first case in the dorsal grey between the central canal and dorsal border of the spinal cord; (2) in the second case, labelled cells (MN1), 35-40 micron, were found in the central grey ventro-laterally, in close vicinity of the ependymal canal; (3) in the last case, labelled cells (MN2), 18-20 micron, were seen in the ventro-lateral part of the anterior horn. When the spinal cord was considered in longitudinal sections, EMNs and MN1 constitute a continuous column along the spinal axis, whereas MN2 show a metametric distribution. The 3 groups of motor cells can be identified in the spinal cord for the whole length of the anal fin, whereas the MN1 and MN2 disappear at the level of the caudal peduncle. The 3 cell types bear dendritic processes; the EMN thin ones and the MN1 and 2 rather large ones. The results indicate that distinct cell groups innervate the electric organ (EMN), the large muscles of the trunk (MN1) and those of the anal fin (MN2).

Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to R-band-positive heterochromatin, which has a high G + C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R- and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.

Development of immunologically identified brain cells in culture: quantitative aspects.

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures.

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.

Identification of left-handed Z-DNA by indirect immunomethods in metaphasic chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia).

Antisera and antibodies against Z-DNA were used on metaphasic fixed chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia). By indirect immunofluorescence and indirect immunoperoxidase labelling a heavy staining was detected in a fraction of the R-band positive heterochromatic segments, which are presumed to be rich in G-C base pairs. A weak and non homogeneous staining was also observed on euchromatic segments. The presence of a left-handed Z-DNA in mammalian chromosomes is discussed.