European Journal of Neuroscience, Vol. I , No. I @ 1989 European Neuroscience Association 0953-816x/89 $3.00

Survival and Axonal Elongation of Adult Rat Retinal Ganglion Cells In vitro effects of lesioned sciatic nerve and brain derived neurotrophic factor

Solon Thanos, Mathias Bahr, Yves-Alain Barde,' and Jens Vanselow Max-Planck-lnstitut fur Entwicklungsbiologie, Spemannstr. 35/1, D-7400 Tubingen, FRG 'Max-Planck-lnstitut fur Psychiatrie, Abteilung Neurochemie, D-8033 Martinsried/Munich, FRG

Key words: Axon regeneration, central neurons, fluorescent dyes, neurotrophic factors, immunohistochernistry.

Abstract

A peripheral nerve exudate, collected in situ from the proximal end of a severed rat sciatic nerve, can induce substantial axonal elongation from ganglion cells when tested on explanted adult rat retinae. The responsive cells are identified on the basis of their Thy 1.1 immunostaining properties. Similar outgrowth can be obtained from explants when the culture medium is supplemented with brain-derived neurotrophic factor (BDNF). In addition, both BDNF and the sciatic nerve exudate can prevent ganglion cell degeneration as shown by the retrograde transport of a fluorescent dye that had been applied to the superior colliculus prior to explantation. The results demonstrate that soluble components, released by lesioned peripheral nerves, can effect adult retinal ganglion cells in a way that is reminiscent of that which has been described in vivo using sciatic nerve grafts after sectioning of the optic nerve. The molecular nature of these components is discussed.

Introduction

I t is established that in the adult mammalian central nervous system (CNS), axons have a very limited capacity to regrow after lesion. In particular, as shown for adult retina, most ganglion cells die after their axons have been transected. A series of well-documented studies have shown, however, that significant numbers of adult retinal ganglion cells can be rescued and induced to re-elongate axons when a segment of peripheral nerve, such as the sciatic nerve, is presented as an alternative to the cut optic nerve (So and Aguayo, 1985; Politis and Spencer, 1986; Berry et al., 1986; Vidal-Sanz et al., 1987). These results are of importance as they unequivocally indicate that in vivo some potential for regeneration still exists in the adult.

In vitro studies can be of considerable help in trying to understand which cellular and molecular components provide a suitable, or, on the contrary, unfavourable environment (Schwab and Thoenen, 1985) for neuronal survival and axonal regeneration. A number of reports suggest that in peripheral nerve grafts it is the Schwann cells that are responsible for the synthesis of constituents which favour axonal growth. The best defined candi- dates are molecules which either create a favourable substrate for axon elongation, in particular laminin or laminin-related molecules (Tohyama and Ide, 1984) or promote neurite outgrowth such as nerve growth factor (NGF), so far the only well-defined neurotrophic factor whose synthesis by Schwann cells has been conclusively established (Richardson and Ebendal, 1982; Bandt-

Correspondence to: Solon Thanos, as above

Received 24 June 1988, accepted 25 August 1988

low et al., 1987; Heumann et al., 1987). However, it has been shown that the adult sciatic nerve can synthesize in addition to NGF, other molecules that have an action clearly distinct from NGF: not all neurotrophic activity found in normal, cultured, or sectioned sciatic nerve can be blocked by NGF antibodies, and neuronal survival and fibre outgrowth can be elicited from neurons not responsive to NGF (Richardson and Ebendal, 1982; Longo et al., 1983). In general, studies dealing with sciatic nerve exudates or extracts make use of embryonic and peripheral nervous system neurons that are notoriously easier to examine in culture than adult CNS neurons. Using a recently established In vitro model of adult rat retinal explants (Bahr et al., 1988; Johnson et al., 1988), the present study quantifies the effects of soluble molecules contained in sciatic nerve exudates, on ganglion cell survival and axonal re-elongation. The effects are compared with those obtained with BDNF, previously shown to support both survival and fibre outgrowth of most ganglion cells in dissociated embryonic rat retinae (Johnson et al., 1986).

Materials and Methods

Implantation of silicone tubes and collection of the nerve exudate Adult female Lewis rats weighing 180 to 220 g were anaesthetized by intraperitoneal injection of chloral hydrate (0.42 mg/kg body weight). The left or right sciatic nerve was mobilized from the

20 Axonal regeneration in vitro

NERVE INTERIOR (SCHWANN CELLS ,FIBROBLASTS

CRUSH PERINEURIUM MACROPHAGES, DEBRIS)

SCIATIC NERVE SILICON TUBE S4- MEDIUM LIGATURE DISTAL SCIATIC NERVE

FIG. I . Collection of sciatic nerve exudate. Incision of the perineurium permits contact of non-neuronal cells with the S4 medium during the time (2 weeks after surgery) of regrowth of crushed axons into this nerve segment. The S4 medium was supplemented with protease inhibitors to prevent proteolytic activities within the tube.

sciatic notch to the tibial-peroneal bifurcation and the epineural sheath was incised longitudinally, proximal to the bifurcation, and over a length of about 10 mm. The split proximal branches of the nerve were transected at the bifurcation level and forced in to the lumen of a silicone tube (length about 15 mm; i.d. 1.2 mm; 0.d. 2 mm) which was distally tied to prevent leakage of fluid (Fig. I). The silicone tube was filled with sterile S4 medium containing protease iahibitors (a-neuraminidase 50 pM, aprotinin 200 UI/ml, pepstatin 2 pM, leupeptin 50 pM).

The epineurium adjacent to the tube opening was affixed to the wall of the tube with a 0.6 nylon suture (Ethicon). The tube opening around the nerve was sealed with semi-viscous silicone paste (Bayer) and the tube wall was anchored to the thigh muscles. The nerve was then crushed proximal to the implanted tube by means of jeweller's forceps.

After 1-3 weeks, the rats were re-anaesthetized and the chambers removed. The fluid contents of the tubes were collected and centrifuged at 10,000 g for 10 min to remove cellular components and debris. The supernatants (10 to 15 pl) were diluted 10- to 100-fold in S4 medium, divided into aliquots, and used either immediately or frozen and stored at -70 "C. At these dilutions the fluid appeared to have its maximal neurotrophic activity. The activity could still be observed at dilutions 1 :2000.

In addition, the tibia1 and peroneal nerves distal to the site of transection were dissected up to their fine muscle branches, freed from their epineural sheath, placed in S4 medium and cut with microscissors into 20-30 small slices (0.5-1 mm length). These nerve segments were tested in co-cultures with retinal pieces.

Pretreatment of the retina and explantation procedures

Under chloral hydrate anaesthesia, the left optic nerve was exposed in its intraorbital segment and crushed by means of jeweller's for- ceps, in order to produce a conditioning lesion (Ford-Holevinski et al., 1986; Bahr et al., 1988). One week after the crush, the left retina was dissected under sterile conditions. The retina was flat- mounted on sterile filters (Sartorius) and chopped into eight wedge-like pieces centred in the optic disc. The retinal pieces were then explanted on petriperm dishes (Heraeus) coated with polylysine (MW 375,000 to 410,000 Da, Boehringer; 200 pg/ml overnight a t 37 "C) and laminin (BRL; 20 pg/ml, 1 h, 37 "C) with the ganglion cell layer towards the substratum. The retinal pieces were attached to the dish bottom by 20 pl drops of MatrigelcR) (Dunn). For examining the effects of the sciatic nerve exudate and BDNF on ganglion cell death, retinae from 12 rats were

retrogradely prelabelled with rhodamine isothiocyanate (RITC) from the contralateral superior colliculus (Thanos et al., 1987) one week prior to explantation.

Culture media and additives

The serum-free medium (S4) was prepared from Modified Eagle's Medium and substituted with additives described by Needham et al. (l987), except for the endothelial cell growth factor. Vitamin C was added daily in concentrations of 1-2 pg/ml. Three to 4 retinal pieces were cultured in 3 ml S4 medium (pH 7.4) at 37.5 "C in an incubator with an atmosphere containing 5% CO,. Increase of the 0, content up to about 70% increased the speed of axonal regrowth and, to some extent, the number of regrowing axons.

Fluid collected from the silicone tubes was added to the culture medium at the time of explantation and then daily in concentrations corresponding to dilutions of 1 : 100. Alternatively, the cut slices from the distal part of the denervated sciatic nerve branches were added to the retinal cultures. For inactivation studies, the exudate was heated for 5 min at 90 "C and the sciatic nerves were frozen in solid CO, and rethawed prior to explantation (Smith and Stevenson, 1988). Nerve growth factor purchased from Boehringer was diluted in S4 medium and used a t concentrations of 5-20 ng/ml. Fibroblast growth factor (FGF, basic component, purified and kindly provided by D r W. Risau) was used in concentrations of 1-5 ngiml). Purified BDNF (Barde et al., 1982, as modified by Hofer and Barde, 1988) was diluted in adult rat serum, divided into 10 p1 aliquots amounts and pipetted to the retinal explants in final concentrations of 100 pg/ml medium.

Measurements of outgrowing jibres and ganglion cell survival

More than 200 pieces explanted from 30 retinae were scored for axon regrowth 1-2 days after explantation by means of an inverted Zeiss phase contrast photomicroscope. In experiments, where ganglion cell survival was assayed, RITC prelabelled retinal ganglion cells (RGC) were counted by means of the same microscope equipped for epifluorescence. The RITC labelling allowed the identification of ganglion cells as well as examination of their formations. After quantification, the retinal pieces were fixed in 4% phosphate-buffered paraformaldehyde, washed in phosphate-buffered saline (pH 7.2) and processed for Thy 1.1 (Barnstable and Drager, 1984) immunohistochemistry (SER- OTEC). Glial fibrillary acidic protein (GFAP) antiserum

Axonal regeneration in vitro 21

FIG. 2. Phase contrast (A) and fluorescence (B) micrographs illustrating the regrowth of T h y 1.1-labelled fibres from adult retinal tissue in vitro. The ON was crushed 1 week prior to dissection of the retina and axon growth was evaluated 2 days after explantation on polylysine/larninin substratum. Thy 1.1 immunohistochemistry was performed on fixed material. All regrowing fibres were stained with Thy 1.1. Scale bar: 50 pm.

22 Axonal regeneration in vitro

(SEROTEC) was used at dilutions of I :50 to label glial cells in the retinal explants.

Results

Axonal regrowth In the serum-free S4 medium, substituted daily with ascorbic acid (Needham et al., 1987), transected axons started to re-elongate within the first 24 h after explantation (Bahr et al., 1988). Typically, several fasciculated or single fibres grew in their original orientation and appeared a t the tip of the retinal piece that had been adjacent to the optic disc in vivo (Fig. 2). Presumably, the technique of cutting the retina in optic disc-centred triangular pieces permitted the maintenance of intraretinal axon stumps and their immediate re-elongation in the cultures (Bahr et al., 1988).

CHAMBER TlBlAL FGF NQF RAT BONF

(1 WEEK) NERVES NTA +PERONEAL SERUM

FIG. 3. The number of outgrowing axons was determined after 2 days in v i m . The fluid content removed after I week from the silicone tubes ( I ) gave rise to effective regrowth of axons. Heating of the fluid (2) prior to its addition to the medium resulted in the loss of its neurite-promoting activity. The distal part of the sciatic nerve (3) showed a similar activity which was abolished after freezing and thawing of the nerve (4). FGF (5) was used at 1-5 ng/ml and as NGF at 5-20 ng/ml. The low level of outgrowth on polylysine/laminin substratum (7) could be due to the promoting effects of laminin. BDNF (100 pg/ml) was found to promote neurite outgrowth (8) and was quantitatively comparable to sciatic nerve fluid and co-cultured sciatic nerve segments.

Ganglion cell axons were identified, based on their positive staining with a monoclonal antibody (Fig. 2B) to the glycoprotein Thy 1.1; this antibody is known to specifically label retinal ganglion cells and their axons in the adult retina (Barnstable and Drager, 1984). When stained with antibodies to GFAP, all fibres stained with Thy 1.1 were G F A P negative.

Whereas the morphology, the degree of fasciculation and the growth rate of retinal axons were similar under the various culture conditions tested, the number of regrowing ganglion cell axons varied. In the presence of adult rat serum (diluted 1 : 50 to 1 : 1000) or N G F ( 5 to 20 ng/ml), the number of axons per explant was similar to control: 36f I I axons per explant. F G F (1-5 ng/ml)

which was reported to rescue RGC when applied to the optic nerve stump (Sievers et al., 1987), increased the number of fibres per explant: 78k23. Substitution of the culture medium with sciatic nerve exudate (210 & 51 axons) or co-cultivation of retinal pieces with distal sciatic nerve segments (186f35 axons) resulted in massive outgrowth of axons (Fig. 3). When extrapolated to the whole explanted retina, values of over 1,600 regrowing ganglion cell axons per retina can be calculated. These numbers are slightly lower than those calculated for the in vivo regenerating retina when sciatic nerve pieces were grafted to replace the optic nerve (Vidal-Sanz et al., 1987; Thanos and Vanselow, 1988). The activity of the sciatic nerve fluid was completely abolished by heating (Fig. 3, column 2) and that of the co-cultured sciatic segment by freezing of the nerve prior to co-cultivation (Fig. 3, column 4).

Administration of BDNF (100 ng/ml) to the S4 medium also resulted in massive re-elongation of RGC axons soon after explantation (Figs. 3 and 4). The length of Thy 1.1-positive axons was measured 2 days after explantation. The length and distribution of the total population of axons are shown in Figure 4B. The longest axons (> 500 pm) were observed when the medium was supplemented with BDNF. The axons seen in S4 medium alone or in the presence of various concentrations of rat serum were shorter and never exceeded 500 pm. Co-administration of BDNF and sciatic nerve exudate did not increase the number of regrowing axons. In this case, the number was almost identical (208f62, n = 28 explants) to that described for the sciatic nerve exudate alone.

Survival of adult ganglion cells in vitro In addition to promoting axonal regrowth, the sciatic nerve exudate and BDNF could also support RGC survival. To ap- proach this question, cell viability in explanted retinae was assessed using RITC labelling in situ. When RITC was injected into the superior colliculus and the contralateral retina was explanted one week later, highly fluorescent ganglion cells (Fig. 5A) were uniformly distributed across the entire retina. The density of labelled cells was approximately 2,000 mm2. This value corre- sponds to the normal ganglion cell density in the retina (Perry, 1979) and indicates that most, if not all RGC were retrogradely labelled (Thanos et al., 1987). An example of retina cultured for 6 days in the presence of the sciatic nerve fluid is shown in Figure 5B. Typically, labelling of the ganglion cells with the RITC was visible in about one-third of the neurons (Fig. 5B). Live cells can be distinguished by their elaborate dendritic trees and by the complete delineation of cell-body images. Numerous degenerating ganglion cells were also visible. They are spherical and display an atypical rhodamine fluorescence (Fig. 5B). During the cultivation in vitro, under control conditions (rat serum +O,), the density of identifiable ganglion cells decreased after 6 days to about 30% of the density measured a t the time of explantation (data not shown). The decrease was probably caused by cell degeneration and subsequent leakage of the dye into the environment. The presence of sciatic nerve exudate or BDNF significantly delayed the speed of ganglion cell degeneration in explants cultured for up to 6 days in culture (Fig. 6): with BDNF nearly 51% (Fig. 6), and with sciatic nerve fluid about 44% (Fig. 6) could be seen after 6 days in culture. At this stage, the corresponding control values ranged from 20 to 30% in the,presence of either rat serum or in the absence of any additive (Fig. 6).

Axonal regeneration in vitro 23

80 LL LL 0 a m r 3 2

w

60

40

20

0 0 0 0 0 0 0 0 0 0 0 I v)

v)

v) , = , " 8 8 A A

0 0 0 0 0

0 1 1 0 1 I A A 0 0 " 0 0 % ? z % s s

control + 02 % F g - ( u BDNF + 0 2

I ratserum + 02

FIBRE L E N G T H [pm] FIG. 4. (A) Phase contrast photomicrograph of a retinal explant supplemented with 100 pg/ml BDNF and evaluated 2 days after explantation. Massive regrowth of axons can be observed. They emerge from the explant localized to the left and outside of the field view. Scale bar: 50 pm. (B) Length distribution of fibres extending from explants (2 days in virro). The length distribution was similar in all cases, although some fibres seemed to exceed the length of 500 pm in the presence of BDNF. Mean numbers of fibres were 168k43 in BDNF (18 explants), 4 6 k 2 9 (18 explants) in rat serum and 50k31 (16 explants) in serum-free S4 medium. The length distribution of fibres growing in the presence of the sciatic nerve exudate was almost identical with that drawn for BDNF.

24 Axonal regeneration in vitro

t

FIG. 5 . Fluorescence photomicrographs showing RITC-labelled ganglion cells on retinal whole mounts during the time of explantation (A) and 6 days later in culture (B). This retina was cultured in the presence of fluid from the sciatic nerve. About 30% of the RGC have survived this period of cultivation in vitro. Some degenerating RGC are indicated with small white arrows. Scale bar: 50 wm.

Discussion

The present study demonstrates that axonal re-elongation from adult retinal ganglion cells can be observed in vitro, and that this re-elongation can be markedly stimulated by addition of sciatic nerve exudate or BDNF to the culture medium. Furthermore, both can rescue retinal ganglion cells from degeneration.

The release of neuronal survival and neurite-promoting activity from the axotomized sciatic nerve is in agreement with previous observations demonstrating that peripheral nerves contain and/or release such activity acting on neurons. For the most part, these observations were made on embryonic peripheral nervous system

. days after explantation

Od 2d 4d 6d

FIG. 6. Fate of RGC prelabelled with RITC from the superior colliculus and explanted 7 days later. The numbers of labelled RGC (mean 1850+ 200 cells at the time of explantation; 6 retinae) were converted to percentages. Both the sciatic nerve exudate (x) and BDNF (A) promoted the survival of about 25% more RITC labelled cells compared to controls. Explantation and culture in S4 medium ( 0 ) or S4 medium+rat serum (m) resulted in a decrease of RGC to values ranging after 6 days between 20 and 30% of the initial RITC-labelled population of cells. The means and SEM for each time point of measurement were obtained from 6-8 explants and were significantly different between controls and experimental groups (p < 0.001).

neurons (see, for examples, Richardson and Ebendal, 1982; Longo et al., 1983; Assouline et al., 1987). So far, there are only two defined soluble molecules with survival and fibre outgrowth promoting activities known to be present in peripheral nerves, N G F and ciliary neuronotrophic factor (CNTF; Barbin et al., 1984). Small amounts of N G F can be found in the adult unlesioned sympathetic and sensory axons as the result of the retrograde axonal transport of N G F from the peripheral sites of synthesis to the neuronal cell bodies (Richardson and Ebendal, 1982; Korsching and Thoenen, 1983; Heumann et al., 1984; Korsching and Thoenen, 1985). In addition, in lesioned or explanted adult peripheral nerves, N G F is also known to be synthesized in substantial amounts by the nerve sheath, in particular by Schwann cells (Richardson and Ebendal, 1982; Rush, 1984; Heumann et al., 1987; Bandtlow et al., 1987). Thus, N G F is most probably contained in the sciatic nerve exudate used in this study. However, there is general agreement that in mammals (as opposed to fish and amphibia, for example), N G F does not play any role in terms of retinal ganglion cell survival and axon outgrowth, and in accordance with this, the data reported here also show that the addition of N G F to the retinal explants produces no measurable effects. CNTF, the other molecule with neuronal survival activity, is known to act on most embryonic peripheral neurons, and could explain many of the non-NGF effects seen in other studies on such neurons using sciatic nerve extracts, co-culture, or exudate (Richardson and Ebendal, 1982; Longo et al., 1983). However, it is unlikely to be exerting an effect on these explanted adult

Axonal regeneration in vitro 25

RGC, as CNTF has been shown not to support the survival of dissociated, embryonic rat retinal ganglion cells (Johnson et al., 1986), and, more generally, has not been shown so far to support the survival and fibre outgrowth of CNS neurons.

Two other candidates that could explain the effects observed with the sciatic nerve exudate were tested in the present study: basic FGF and BDNF. Although there is, to our knowledge, no clear demonstration that FGF is present or synthesized in normal peripheral nerves, it has been shown that macrophages, known to invade lesioned peripheral nerves (Abercrombie and Johnson, 1946), do contain basic FGF (Baird et al., 1985). It is therefore likely to be present in our sciatic exudate. Unlike NGF, FGF was found to be active (Fig. 3). Whether or not the effects of FGF are direct or indirect is unclear at the moment: FGF has been shown to act on a number of non-neuronal cells, and it could be that the actual target cells in the explants used in this study are the non-neuronal cells (see Gospodarowicz et al., 1987, for review). This possibility could explain the apparent discrepancy that although no effects of FGF (acidic or basic) could be seen using dissociated embryonic rat ganglion cells (Johnson et al., 1986), FGF has been shown to be active and to rescue adult rat ganglion cells in vivo when applied at the cut end of the optic nerve (Sievers et al., 1987). Though the effects of FGF on fibre outgrowth were less prominent than the results seen with the sciatic nerve exudate, the effects of BDNF, both in terms of fibre length and ganglion cell survival, were qualitatively and quantitatively identical. Con- cerning fibre elongation from adult neurons, it is also worth noting that BDNF has already been shown to increase the rate of fibre outgrowth from dissociated adult spinal sensory neurons (Lindsay, 1958). Whereas it is not unexpected to see an effect of BDNF in this system (BDNF is known to support the survival and fibre outgrowth of most embryonic rat ganglion cells in dissociated retinae, Johnson et al., 1986), the parallelism seen between the effects of the sciatic nerve exudate and those of BDNF is intriguing. Unfortunately, the lack of antibodies blocking the biological activity of BDNF do not allow us to directly test the straightforward hypothesis that the activity displayed by the sciatic nerve exudate is actually due to BDNF. Also, nothing is known about the tissue distribution of BDNF, apart from the fact that it can be isolated from adult CNS tissue. Circumstantial evidence in support of this hypothesis might be the observation that there is no additive effect of BDNF and nerve exudate on axonal regrowth and RGC survival.

In future studies, when adequate tools become available, such as cDNA probes, it will be important to test whether or not a lesioned segment of peripheral nerve, in particular the Schwann cells, can be shown to be a site of BDNF’s synthesis. The synthesis of neurotrophic molecules of this kind (already firmly established for NGF) could help to explain, at least in part, why segments of peripheral nerve, in addition to overcoming the problems resulting from the negative influences exerted by some CNS glial cells (Schwab and Thoenen, 1985; Schwab and Caroni, 1988), can be used to stimulate axon re-elongation from such a variety of CNS neurons (Aguayo, 1985).

Acknowledgements The authors are grateful to Dr W. Risau for generously providing the FGF. Drs F. Bonhoeffer and T. Alsopp contributed with critical comments on the manuscript.

Abbreviations BDNF CNS CNTF FGF GFAP NGF RGC RITC

brain derived neurotrophic factor central nervous system ciliary neurotrophic factor fibroblast growth factor glial fibrillary acidic protein Nerve growth factor retinal ganglion cells Rhodamine-B-isothiocyanate

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