Biol Cell (1990) 68, 221-225 221 © Elsevier, Paris

Original article

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Christian Dussartre, Jeanine Koenig*

CNRS LIRA 1136, Universit~ de Bordeaux II, Laboratoire de Neurobiologie Cellulaire, avenue des Facultds, 33405 Talence Cedex, France

(Received 22 June 1989; accepted 10 April 1990)

Summary - Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.

cryoculture / muscle sections / spinal cord cells / neuritic outgrowth / fibroblastic proliferalion

Introduction

Numerous in vitro studies [5] have demonst ra ted the ex- istence o f various factors that p romote cellular adhesion, neuronal survival and neurite extension. The mode o f ac- t ion o f these factors has mainly been studied using either tissue extracts o f target neurons, or culture media contain- ing factors synthesized and released by target or glial cells [2, 12, 14].

Carbonet to et al [1] have developed a system for analysis o f the effects o f various molecules on the growth o f nerve fibres culture on tissue substrata. More recently, Covaul t et al [4] have used this technique to study neurite outgrowth o f chick embryo ciliary ganglion neuron cultured on cryostat sections o f innervated and denervated rat mus- cle. Their results suggest that muscle fibres, part icularly when denervated, use cell surface (membrane and ex- tracellular matrix) molecules to mediate the growth o f neurons where they contact tissue substrata. These authors analyzed the effect o f these mediators on neurite outgrowth in the 48 h following the start o f culture. We have studied the longer- term effects on neurite ou tgrowth o f muscle tissue substrate in various physiological and pathological states : denervated or innervated s ternomastoid muscle o f adult rats, and soleus muscle f rom normal adult mice or f rom mice presenting the trembler mutat ion. This domi- nant mutat ion principally affects the Schwann cells, which are unable to fo rm and maintain a normal myelin sheath, but is also characterized by numerous other abnormalit ies including permanent multiplication o f glial cells in the m o t o r nerve [6], and a tendency o f axons to branch ex- tensively where they contact muscle fibres [13].

* Correspondence and reprints

Materials and Methods

Growth of rat embryo spinal cord cells on cryostat sections of denervated or innervated rat muscle

Three weeks before use, the right sternomastoid of young adult rats (Ofa Sprague Dawley) was denervated by section and ligature of the motor nerve at its insertion in the muscle. The left muscle served as control. Cryostat sections (10/~m) were collected on glass coverslips as described by Covault et al [4]. Spinal cord cells from 14-d-old rat embryos were plated on cryostat sections 005 cells/35-mm diameter plate, Falcon) as described by Koenig et al [8] and were cultured in MEM 199+ 10°70 HS: Eagle's minimal essential medium plus 10°70 199 medium containing glutamine (Gibco), penicillin (20 U/ml), streptomycin (10 mg/ml) and 10°/o horse serum (Boehringer). After 5 d of culture, the neurites were visualized by fluorescence after treatment with tetanus toxin and fluorescein-labelled antitoxin antibodies [16]. Total neurite length, that is the length of all neurites emanating from one perikaryon plus the lengths of their branches, was measured with a Biocom 200 image analyzer. Fifty neurons were analysed for each set of conditions. The initial results indicated that the differences in neuron behavior noted with sections of normal, innervated muscles were much more marked in muscle zones containing synapses than in synapse-free areas. We localized the neural regions of the muscle using the method of histochemicai labelling of cholinesterase activity of Koelle and Friedenwald [9], as modified by Couteaux and Taxi [3].

Rat embryo spinal cord cells and fibroblasts cultured in the presence of conditioned media based on cryostat sections o f soleus muscles from normal and trembler mice

Adult normal (B6D2 +) mice and heterozygote trembler mice (B6D2/Tr) were used. The neural (NZ) and synapse-free (AZ) zones were easily distinguished since the soleus, like the sterno- mastoid muscle, has a central neural zone.

Conditioned culture media To study molecules released by the cryostat sections, we placed about 100 sections (10tam) from each muscle substratum in each

222 C Dussartre, J Koenig

35-mm diameter culture plate. The sections in each plate were incubated in 2 ml of serum-free MEM 199 medium for 24 h, and the medium was then collected, filtered (MILLEX-8 tzm) and stored at -25°C before use.

Spinal cord cell cultures Various culture media were added (1 ml/plate) to cultures of spinal cord cells from 14-d-old rat embryos (105 cells/35-mm diameter plate) grown on a poly-L-lysine substrate (M,,.= 109000, 1 mg/100 ml distilled water) in MEM 199-10°/0 HS. After 5 d of culture, the total neurite length was measured after labelling with tetanus toxin (50 neurons for each set of con- ditions). Non-neuron.al cells were characterized by immuno- cytochemistry using antibodies against antigens specific to different cell types: anti-thy-l-I (New England Nuclear, Boston, SA) for rat fibroblasts, anti-6-17 for Schwann ceils [11], and anti- GFAP for astrocytes (glial fibrillary acid protein) followed by staining with rhodamine-or fluorescein-second antibody.

Cellular proliferation of 3 T3 fibroblasts Cells from a homogeneous population of 3T3 fibroblasts were seeded (10 4 cells/35-mm diameter plate, Falcon) in MEM 199-5% HS medium (Boehringer) and were cultured for 1 or 2 d. The medium was then replaced by the same medium, but this time serum-free, in order to render most cells quiescent (G o phase of cell cycle). After 24 h, the medium was replaced by MEM 199-1 °7o HS and conditioned medium (1 ml/dish). At this serum concentration the 3T3 cells are likely to divide, in particular as a result of factors in the conditioned medium. Daily cell counts were performed using a Thoma cell with 4 dishes in each case after detachment of cells by treatment with trypsin (0,25°70 in calcium-free 0.02 M phosphate buffer).

Results

Study o f neurite outgrowth o f rat embryo spinal cord neu- rons cultured on cryostat sections o f denervated and in- nervated adult rat sternomastoid muscle

The average lengths of neurites of spinal cord neurons cultured on innervated and denervated sternomastoid muscle sections were 201.29-+90.03 /zm and 282.12-+ 109.99 tzm respectively. The large standard deviations doubtless reflect the extreme variability of the lengths of the neurites branches and render these figures non signifi- cant. However, histograms of the neurite length distribu- tion (fig 1),,revealed the differences between the 2 types of culture: each histogram presents a unimodal profile, with a shift between the 2 histograms, with a major peak at 100-200 ~m for the cultures on sections of innervated muscle and a peak at 300-400/zm for neurons cultured on sections of denervated muscle. This suggests that the neuritic branches tend to be longer when the neurons are cultured on sections of denervated muscle (fig 2).

Study of the effect on neuron growth and proliferation of fibroblasts o f conditioned media obtained from cryostat sections o f soleus muscle from normal or trembler mice

Spinal cord cells from 14-d-old rat embryos were cultured on sections of soleus muscle from normal and trembler mice. The total neurite length was measured after 5 d of culture. The preliminary results indicated that neuron adhesion to sections of normal or trembler soleus muscle was much weaker, proportionally to the surface area, than adhesion to sections of rat sternomastoid (data not shown). Adhesion accompanied by marked neurite outgrowth occurred directly on the glass coverslip near sections of

mouse soleus muscle, but was not seen in the vicinity of sections of rat sternomastoid muscle. On empty control coverslips adhesion was reduced and neurite outgrowth limited. Attachment of neurons and their growth near muscle sections seemed to depend on contact with a layer of non-neuronal cells: using immunocytochemistry, we have shown that non-neuronal cells in cultures of rat spinal cord comprise approximately 3°70 of fibroblasts, 1°7o of Schwann cells and 17070 of astrocytes.

The average length of neurites emanating from cultured spinal cord ceils was 455.94_+ 200.32 ~m, for the control without conditioned medium. The values were, in the presence of conditioned media, based on normal soleus muscle, 500.14-+ 181.20 gm (AZ), 473.93+ 192.87 tzm (NZ), and based on trembler soleus muscle 507.61_+ 186.57 ~m (ZA) and 523.52-+287.23/zm (NZ). The diff- erences between these values were not significant due to the size of the standard deviations, which reflect the cellular heterogeneity of the spinal cord. No particular tendency can be demonstrated from the histograms of neurite length distribution.

Proliferation o f 3 T3 cells Proliferation of 3T3 cells in the presence and absence of conditioned media suggested that the muscle sections released growth factors (fig 3). As of the first day of culture, the cellular density in conditioned media was approximately twice that in the control. This difference was accentuated with time since in the control the number of ceils tended to stabilize around day 3, whereas the number continued to increase until day 5 in the presence of conditioned media. It should be noted that in the presence of conditioned media some ceils reached con- fluence on day 3 and that cells undergoing mitosis in an

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Fig 1. Histogram of the distribution of the average total neurite length for each perikaryon of spinal cord neurons from 14-d-old rat embryos cultured for 5 d on cryostat sections of the neural zone of innervated or denervated 3 weeks prior (hatching) adult rat sternomastoid muscle.

In vitro neurite outgrowth 223

increasingly dense cellular layer are very easily detached from the support. Cell counts relate to cells attached to the bot tom of the plate and the differences between the conditioned media and the control are therefore underestimated. There is little or no significant differences between the various conditioned media, whether derived from the muscles of normal or trembler mice.

Discussion

Neurite outgrowth o f rat embryo spinal cord neurons cultured on sections o f innervated or denervated rat muscle

Our results suggest that one or several factors existing in the neural zone of denervated muscles promote neurite outgrowth. It is possible, however, that the different ef- fects of innervated and denervated muscles on neurite length are due to promotion of the adhesion and survival of one type of neuron compared with another . Nonetheless, the shift in distribution of neurite lengths ap- parent from the histograms suggests that the denervated substratum affects the neurite outgrowth of most neurons. Moreover, these data are in agreement with those of Covault el a! [4]. However, in our study we used a homospecific muscle-nerve cell system in which neurite

outgrowth was determined in the long-term and not as the neurites emerged. The mechanisms involved and the pro- moting factors may prove to be different. Covault et al have shown that chicken ciliary ganglion neurons extend their neurites along the surface (extracellular matrix and sarcolemma) of sectioned muscle cells. In contrast, we have no strong evidence for orientation of neurite outgrowth by tissular tropism. This could be because, in our spinal cord cell cultures, heterogeneous cellular clusters, composed of neurons and fibroblasts or glial cells, interact strongly with one another or isolated neurons to form dense net- works of interconnected neurites. This apparent tropism could mask that exerted by the surface of muscle cells. However, another possibility is that long-term neurite outgrowth is only partly affected by such tropism.

Comparative immunohistological studies of cryostat sec- tions of innervated and denervated adult rat skeletal muscle [15] have revealed differences in the distribution and ex- pression of molecules involved in adhesion or comprising the basal lamina (J1, Ncam, fibronectin, heparan sulphate proteoglycan). These molecules accumulate after dener- vation in the interstitial spaces near the neuromotor junc- tions. They may be responsible for the changes in neurite outgrowth seen in cultures grown on cryostat sections, depending on the substrata. It would be interesting to use antibodies against these molecules to define their effects on neurite outgrowth on cryostat sections.

Fig 2. Rat embryo spinal cord cells cultured on cryostat sections of the neural zone of innervated (photo 1) or denervated (photo 2) adult rat sternomastoid muscles. The neurons were visualized by staining with tetanus toxin and then with fluorescein anti-toxin antibodies (GAR, polyclonal, KPL). G: x500.

224 C Dussartre, J Koenig

Effect o f conditioned media on neuronal development and fibroblast proliferation

The conditioned media produced from normal and trembler mouse soleus muscle did not seem to affect neurite outgrowth. Nonetheless, it is possible that quan- tification of a more refined nature or the use of a more homogeneous cell population would establish whether a factor released by normal or trembler muscles acts on neurite outgrowth.

Our results do, however, suggest that a mitotic factor in neural and aneural zones of skeletal muscles of normal and trembler adult mice acts on fibroblasts. It would be interesting to repeat the experiment with central and peripheral glial cells, but this would required very pure cell populations. It is probable that the sections of soleus muscle release into the medium one or several factors able to influence neuron growth, at least indirect- ly. They may activate the proliferation of non-neuronal cells, which in turn may promote adhesion, survival or perhaps neurite outgrowth of neurons not contacting cryostat sections.

It has recently been shown that fibroblasts interact with muscle cells to form the extracellular matrix [10] and that fibroblasts present in the synaptic interstitial space are able to respond to denervation by synthesizing molecules nor- mally concentrated at the neuromuscular synapses [7]. Changes in the fibroblast proliferation rate may have im- portant repercussions on muscle fibre growth. The regenerating capacity of muscle cells differs in myopathies, Duchenne's disease and its animal model, the mdx muta-

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Fig 3. Test of proliferation of 3T3 cells in the presence of condi- tioned media prepared from sections of normal or trembler mouse soleus muscle. The measurements were performed daily using a Thoma cell: 4 plates for each set of experimental condi- tions and two measurements per plate. The mean values and stan- dard deviations are indicated. Control: - - ; normal solear, NZ: • , AZ: ---*---; trembler solear, NZ: ..-*..., AZ: * .

tion in mice. In Duchenne's disease it is inadequate, and in the mdx mutation compensatory. Hyperproliferation of fibroblasts occurs in parallel in Duchenne's disease, but not in the mdx mutation [17]. The ability of a muscle to regulate the proliferation of fibroblasts could be a very important feature of its development. The determination and characterization of the factor we have found should improve understanding of the interactions between skeletal muscle cells and other neighbouring cells.

The preparation of conditioned medium from tissue sec- tions has certain advantages compared with more conven- tional techniques: it is a less drastic and gentler method for the study of tissular factors than the use of crude ex- tracts, in which cellular components may have been modified.

Acknowledgments

This work was supported by the french Muscular Dystrophy Association (AFM).

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