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Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro
Int. J. Devl Neuroscience 18 (2000) 735 – 741 www.elsevier.com/locate/ijdevneu
Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro N. Wilson a, E. Esfandiary b, K.S. Bedi a,* a
Department of Anatomical Sciences, Uni6ersity of Queensland, St Lucia, Qld. 4072, Australia b Department of Anatomy, Isfahan Uni6ersity of Medical Sciences, Isfahan, Iran Received 15 May 2000; received in revised form 14 August 2000; accepted 15 August 2000
Abstract Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mm-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. © 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Dorsal root ganglion cells; X-irradiation; CNS; Neurites; Tissue culture
1. Introduction Axonal regeneration following injury in the adult mammalian central nervous system (CNS) is typically abortive, with little or no functional recovery. This is in spite of the fact that CNS neurons are inherently capable of axonal regeneration if provided with a conducive environment [1,2,6,10]. A number of possible growth-inhibitory factors present within the microenvironment of the post-lesion CNS have been identified. Abbre6iations: CNS, central nervous system; DABCO, (1,4)-diazbicyclo-(2,2,2)-octane; GAP-43, growth-associated protein 43; GFAP, glial fibrillary acidic protein; MAG, myelin-associated glycoprotein; PBS, phosphate buffered saline; PND, post-natal day. * Corresponding author. Tel.: + 617-3365-3058; fax: + 617-33651299. E-mail address: [email protected] (K.S. Bedi).
These include oligodendrocyte associated molecules such as NI-35 and NI-250 [37], myelin associated glycoprotein [30], and molecules secreted by astrocytes such as tenascins [3] and sulphated proteoglycans [44,45]. Astrocytes may also inhibit axonal regeneration through some contact-mediated mechanism [28]. Several researchers have attempted to alter the postlesion CNS environment in order to enhance axonal regeneration. Much current research is investigating the efficacy of using various neurotrophic growth factors (e.g. brain-derived neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, neurotrophin 3/4, leucocyte inhibitory factor), either singly or in combination, in order to enhance axonal regeneration [11,29,31,36,51]. Another approach involves neonatal X-irradiation of the spinal cord. Studies have shown that this treatment reduces the population of oligoden-
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droglia [7,13,14] microglia [17,42,43] and astrocytes [13,18,19,22] in the irradiated area. The glia-depleted state induced by such X-irradiation may remain for several months [17]. This presents an interesting opportunity to examine CNS regeneration in an environment in which many of the glial cells reactive to injury are removed. For example, the thick astrocytic scar that normally forms in response to dorsal root injury is diminished in animals previously treated with X-irradiation [41]. Furthermore, injured dorsal root axons are able to regenerate into the X-irradiated spinal cord [42]. In addition, Schwann cells from the adjacent peripheral nerves have been shown to invade into the X-irradiated spinal cord [7,15], where they may possibly act to enhance CNS axonal regeneration [42]. Taken together, these observations suggest that the microenvironment following X-irradiation may not be as inhibitory to axonal regeneration as that provided by the normal spinal cord. To examine the mechanisms of axonal regeneration, we have previously used a cryoculture technique that allows many of the complexities of events occurring in vivo to be circumvented [5,39,40]. In this technique, dissociated neurons are plated onto cryosections of various tissues to determine their capacity for neurite extension in vitro. In the present study, we aimed to investigate whether or not cryosections of X-irradiated regions of spinal cord tissue are capable of supporting neurite extension in vitro, compared with sections from non-irradiated animals. We believe that a model system such as this could offer important advantages over in vivo experiments in the study of the molecular mechanisms involved in the enhancement of axonal regeneration by X-irradiation treatment.
2.2. Irradiation treatment Rat pups of 1, 3 or 5 post-natal days were anaesthetised by cooling their bodies on blocks of ice for a few minutes, and placed in a lead box. This box had a rectangular hole measuring 10×5 mm cut in one surface. The pups were gently lodged into the box and held in position by a small piece of foam, so that the lumbosacral region of the spinal cord was exposed through the hole. The exposed regions of the rat pups were irradiated under an Ultrays low voltage X-ray unit (Ultrays Pty. Ltd., Melbourne). The irradiation conditions were, 50 kV; 0.25 mm aluminium added filter; 1 cm cone; 30 mA; 50-s duration. The machine was calibrated so that these conditions exposed the pups to a total dose of about 40 Gray (Gy). Following irradiation, the rat pups were allowed to recover under the warmth of a 100 W light before being returned to their litter. Eight pups were ‘sham-irradiated’ by anaesthetising them on ice, placing them under the lead shield, but not applying any X-irradiation. These pups acted as controls.
2.3. Preparation of ‘fresh-tissue’ cryosections
2. Materials and methods
The animals were allowed to survive until they were at least 70 days of age before being killed. The lumbosacral region of the spinal cord from irradiationtreated and control rats was dissected out, mounted on a small cork disc, frozen in 2-methylbutane (Aldrich) cooled in liquid nitrogen, and sectioned in a cryostat (American Optical Corporation) to yield transverse sections (10 mm). All sections were picked up on sterile 13 mm diameter glass coverslips that had been previously coated with sterile poly-L-lysine (20 mg/ml; 70 000– 150 000 mol wt.; Sigma). These sections were then stored in 24-well plates (Costar) at − 20°C until required.
2.1. Animals
2.4. Preparation of ‘fixed-tissue’ sections
All animals used in these experiments were Wistar rats, purchased from the University of Queensland’s Central Animal Breeding House. A total of fourteen rats were used to provide cryosections for study. The lumbosacral region of the spinal cord was irradiated in two rats at each of 1, 3, and 5 post-natal days (PND) of age. Eight rat pups were used as controls. A further eighteen irradiated and six control rats were used to investigate the cellular response to the treatment. All animal experiments and protocols were under clearance from the animals ethics committee of the University of Queensland and were in accordance with the guidelines published by the Australian National Health and Medical Research Council.
Six control and eighteen X-irradiated rats were anaesthetised and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The lumbosacral regions of the spinal cord from three of the control and nine of the treated animals were processed for embedding in Technovit 7100 resin (Kulzer Histo-Technik). Transverse sections (2 mm) through this tissue were cut an LKB Historange 2218 microtome and stained with 1% toluidine blue. The lumbosacral spinal cords from the remaining three control and nine treated rats were immersed overnight in a solution of 30% sucrose in 0.1 M phosphate buffer (pH 7.4), placed in an appropriate mould, completely covered with Tissue-Tek (OCT; California) and frozen with liquid nitrogen. Transverse cryosec-
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tions (8 mm) through this OCT-embedded tissue were cut on a cryostat and picked up on gelatinised slides. These frozen sections were immunolabelled with antiglial fibrillary acidic protein (GFAP — see below).
2.5. Preparation of dissociated dorsal root ganglion cells About 20–25 dorsal root ganglia (DRG) from a normal adult rat were dissected out for each preparation. These were placed into a few millilitres of modified Bottenstein and Sato’s tissue culture medium [8] containing 2% fetal bovine serum (Sigma). The DRG neurons were dissociated using a procedure modified slightly from that described by Lindsay [27]. Briefly, the ganglia were washed several times in fresh medium, incubated for 3 h in 0.125% collagenase (Sigma), re-washed in several changes of fresh medium and triturated through the tip of a flame-polished pipette. The resultant cell suspension was layered onto a 2 ml cushion of 15% bovine serum albumin (Sigma; essentially fatty acid free) in a sterile 15 ml conical tube and centifuged at 2000 g for 10 min. The supernatant was removed, and the cell pellet resuspended in a small volume of fresh medium. The cell concentration was adjusted to a plating density of approximately 3000 per ml, and 0.5 ml of this suspension was plated onto each coverslip bearing the cryosections (prepared as described above), using 4-well plates (Nunc). It was ensured that cryosections from both control and irradiated tissues were layered with the same batch of dissociated cell suspensions. Cultures were incubated in a humidified atmosphere containing 5% CO2 at 37°C for up to 10 days. The cryosections and any growing DRGs were then fixed in 2% paraformaldehyde in preparation for immunolabelling. The experiments were repeated with cell suspensions prepared from 10 different rats.
2.6. Antibodies and immunolabelling The primary antibodies used in this study included, 1. monoclonal anti-GAP43 (growth-associated protein; Sigma) at a dilution of 1:100 to label growing DRG cells. It should be noted that anti-GAP43 has been found [5] to be a useful marker for regenerating neurites; 2. rabbit anti-GFAP (glial fibrillary acid protein; Sigma) at a dilution of 1:100. This antibody was used primarily to ease the visualisation of the spinal cord cryosections used in the culture studies. It is known [21] to be specific for astrocytes in rats of this age. The secondary antibodies used included, 1. goat anti-mouse IgG conjugated to Cy3 (Amersham; 1:1000 dilution);
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2. goat anti-rabbit IgG conjugated to FITC (Sigma; 1:100 dilution). The fixed cultures and cryosections were washed in three changes of phosphate buffered saline (PBS), postfixed in cold ( − 20°C) 100% methanol for 10 min, re-washed in three changes of PBS, incubated with the primary antibody for up to 2 h, re-washed in PBS, and treated with the secondary antibody for 1–2 h, washed in PBS and distilled water before mounting in 90% glycerol in PBS which had a small amount of 1,4-diazabicyclo-[2.2.2]-octane (DABCO; Sigma) added to it. The cultures and fixed cryosections were viewed under an Olympus BH-2 fluorescent microscope and digital images taken using a SPOT digital camera. The digital images were enhanced and labeled using Adobe Photoshop software (version 5.0; Adobe Systems Inc., 1998).
3. Results
3.1. Effects of the irradiation treatment The body weights of the previously irradiated rats were between 25 and 30% smaller than the age-matched controls by 28 days of age. These differences were statistically significant (PB 0.0001; analysis of variance). We also observed that by 14 days of age, there was hindlimb paralysis in those rats irradiated on PND 1. Symptoms were consistent with the dysfunction of the corticospinal tract. Two of the rats irradiated on PND 3 showed similar symptoms, but none of those treated on PND 5 developed any obvious signs of hindlimb paralysis. All affected rats had recovered full or partial movement of their hindlimbs by PND 28. There were no obvious macroscopic differences observed between the spinal cords of the irradiated rats irrespective of the age at which the irradiation treatment was carried out. However, transverse sections through the spinal cords from the irradiated animals were somewhat smaller than those from sham operated controls (see Fig. 1A and B). This phenomenon has been reported previously [13] and may simply be a reflection of the fact that the treated animals were smaller than the controls. Fig. 1A and B show transverse sections through the lumbosacral region of the spinal cord in 14-days-old control rats, and 14-days-old rats previously irradiated on PND 3, respectively. In control animals, there was a clear distinction between the grey and white matter regions with a number of cells present in the white matter of both the dorsal and ventral funiculi (Fig. 1A). By comparison, the irradiated spinal cord (Fig. 1B) appeared hypomyelinated, which made the boundary between the grey and white matter regions less distinct in most cases. In irradiated rats there were fewer darkly-stained cells in the funiculi indicating a probable
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reduction of the neuroglial cell population in comparison to control animals. Fig. 1C–F shows higher power micrographs of the dorsal funiculus region in control and irradiated animals. C and E are, respectively, from 14 and 30-day-old control rats, and D and F are from age-matched rats previously irradiated at 3 days of age. In control animals, many darkly-stained neuroglial cells could be seen in the dorsal funiculus at 14 days of age; by PND 30, however, these cells were not so obvious (Fig. 1C and E). In contrast, there was an obvious lack of neuroglia in the dorsal funiculus of the previously irradiated rats at PND 14, whilst at PND 30, the dorsal funiculus was hypercellular (Fig. 1D and F). At all ages, a number of small ‘holes’ were present in the sections from the irradiated spinal cords. Their appearance matched the description of the vascular and haemorrhagic effects of x-irradiation reported by previous investigators [4]. Fig. 1G and H show sections through the dorsal horn region of the spinal cord at PND 14, immunolabelled with anti-GFAP. Fig. 1G is from a control animal and Fig. 1H is from a rat previously irradiated on PND 3. The distribution of GFAP immunoreactivity in control rats was characterised by labelling of many thick radial fibres in the white matter and stellate astrocytes in the grey matter. In the irradiated spinal cords, qualitative observations seemed to indicate that the number and density of the GFAP-positive cells was less than that seen in controls. The stellate astrocytes within the grey matter appeared to be more affected than the radial glial cells found around the periphery of the spinal cords. Fig. 1. A and B show 2 mm-thick transverse sections through the lumbosacral region of the 14-days-old rat spinal cord, stained with toluidine blue. A is from a sham-irradiated control and B from a previously irradiated animal. The boundary regions between the grey (g) and white matter (w) regions were more distinct in the sham-irradiated controls than in the irradiated spinal cords. The dorsal and ventral funiculi appeared more cellular in the controls compared to the irradiated animals. *, Dorsal funiculus region; v, ventral horn of gray matter; d, dorsal horn of gray matter. Scale bar, 250 mm. C – F show higher power micrographs of the dorsal funiculus region (demarcated by a dotted line in each case) of the spinal cord. C and E are from 14 and 30-days-old control animals, respectively, and D and F are from age-matched, previously irradiated animals. The dorsal funiculi in irradiated spinal cords appeared to have fewer cells present at 14 days of age than in controls. By 30 days of age, the cellularity of the dorsal funiculus in the irradiated spinal cord had increased in comparison to control animals, probably by the invasion of Schwann cells (arrowheads in F). Scale bar, 50 mm. G and H show the dorsal horn region of the spinal cord in PND 14 animals, immunolabelled with anti-GFAP. G is from a control animal and 1H is from an animal previously irradiated on PND 3. The distribution of GFAP immunoreactivity was characterised by labeled stellate astrocytes in the grey matter (arrowheads) and coarse radial fibres in the white matter (small arrows). There appeared to be a reduction in the extent of immunoreactivity in the spinal cords from the previously irradiated animals compared with controls. This reduction seemed to be more pronounced for the stellate astrocytes than the radial glial cells. Scale bar, 100 mm.
3.2. Growth of DRG neurons CNS tissue substrates Fig. 2 A and B show sections of spinal cord, obtained from sham-irradiated animals. It was found that whilst the DRG cells grew extensive neurites on the adjacent regions of the poly-L-lysine coated coverslips, they were incapable of growing such neurites on the CNS substrates. Some neurites were observed to have grown towards the cryosections, but were incapable of growing onto them. Fig. 2C–F show DRG cells growing on sections of pre-irradiated spinal cord tissue. Neurites successfully regenerated on the substrate-free regions of the coverslips and in addition, many were also seen to extend onto regions of the irradiated cryosections (Fig. 2C and F). Other DRG cells clearly settled onto the cryosections themselves and were capable of extending neurites on to those sections (Fig. 2D, E and F). However, these neurites were not as extensively branched as those that grew on adjacent portions of the poly-L-lysine coated coverslips. There appeared to be no differences in the extent of neurite regeneration on spinal cord tissue irradiated on PND 1, 3 or 5 in our experiments.
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Fig. 2. In all cases, the DRG neurones (n) were plated onto the cryosections and allowed to grow in culture for 8–10 days before being fixed and double-labeled with anti-GFAP (for the substrate sections) and anti-GAP43 (for the regenerating neurites). The boundary between the coverslip (c) and the spinal cord cryosection (sc) is shown by a dotted line. A and B show cryosections of the spinal cord taken from a sham-irradiated adult control rat, with adjacent DRG neurons growing in tissue culture. Extensive neurite growth was seen on the poly-L-lysine-coated regions of the glass coverslips but no growth was observed on the tissue section itself. Some neurites could be seen to grow towards the section but on contact appeared to have turned away (arrow). Other neurites grew close to the edges of the sections but in all instances failed to actually grow onto them (arrowheads). C–F show examples of DRG neurones that are growing on and adjacent to cryosections of previously irradiated adult spinal cord tissue. C shows numerous neurites (arrowheads) that grew from the adjacent regions of the coverslip and onto the irradiated tissue sections (arrows). D–F show examples of DRG neurones that settled on the tissue sections themselves and then extended branching neurites (arrowheads). Scale bar, 50 mm.
4. Discussion Our study showed that DRG neurons are capable of regenerating neurites in vitro, on a substrate consisting of cryosections taken from irradiated, but not from non-irradiated, spinal cord tissue. This finding is consistent with the observation that lesioned dorsal root axons can successfully regenerate into the spinal cord of neonatally-irradiated rats, but not into non-irradiated spinal cord [42]. It seems that neonatal X-irradiation of the CNS tissue converts its environment from one that does not normally support axonal regeneration to one that does. The cryoculture technique may therefore be able to provide a convenient model system in which to
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investigate the irradiation-induced changes in the CNS that make it permissive for axonal regeneration. This model is particularly useful in that it allows some of the complexities of events occurring in vivo to be circumvented, whilst still providing the growing neurons with physiologically relevant substrata. It is also possible to present neurons with a potential to regenerate neurites (such as dissociated DRG cells) with uninjured CNS tissue as the substrate. Such a combination is difficult, if not impossible, to achieve in vivo. Studies of this type may therefore provide an insight into the mechanisms involved in the process of successful axonal regeneration Our observation that cryosections of normal adult rat CNS did not support neurite growth is consistent with the findings of previous investigators [9,20,33,34,39]. This observation suggests that, constitutively, normal CNS tissue does not support axonal regeneration. Any changes to the CNS environment following injury may make it even more difficult to achieve axonal regeneration than is normally the case. These changes may include the up-regulation, or release of inhibitory factors, down-regulation of growth-enhancing factors, or the formation of physical barriers to axonal regeneration (e.g. glial scar tissue). The non-permissive or inhibitory molecules within the CNS may include tenascins, sulphated proteoglycans, NI-35, NI250, and possibly hyaluronectin and myelin-associated glycoprotein [3,30,37,44,45]. The presence of some contact-mediated inhibitory molecules is indicated by our observation that neurites growing on the poly-L-lysine coated glass coverslips turned and grew away in a new direction on contact with the cryosections of normal spinal cord tissue. Our present results support the hypothesis that neonatal X-irradiation of the CNS alters the non-permissive nature of its environment, perhaps by altering the quantitative and qualitative properties of the glial population. This may in turn influence the distribution and amount of inhibitory molecules produced by glial cells. It should be noted, however, that the extent of DRG neurite growth on the irradiated cryosections was generally not as widespread, or robust as that seen on surrounding areas of the coverslips. This strongly suggests that some inhibitory influences are still present in the irradiated tissue. Immunohistochemistry showed that there were several GFAP-positive cells present within the irradiated CNS tissues. Whether these were newly-generated glial cells, or whether they were cells that survived the irradiation treatment is uncertain. Whatever the case, it seems possible that the remaining glial cells may continue to be partially inhibitory to axonal regeneration. Our observations are consistent with the finding that radial astroglia may be less sensitive to the effects of X-irradiation during early postnatal life than other types of glia [43].
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Our experiments on Nissl-stained sections confirmed that the number of neuroglia markedly decreased in the neonatally-irradiated regions of spinal cord in comparison to controls, at 7 (data not shown) and 14 days of age [4,23]. By 30 days of age, the number of cells in the dorsal funiculus regions had increased. This increase was thought to be due to Schwann cells invading the injured CNS region from the adjacent peripheral nerve tissue [4,7,16,22,23]. It is possible that the presence of these Schwann cells may be one of the factors that enhances axonal regeneration in these previously irradiated regions of the spinal cord. Schwann cells are known to be crucial in peripheral nerve axonal regeneration [32,46] due to the fact that they can secrete a host of neurotropic and neurotrophic factors [12]. Grafts of purified Schwann cells, or peripheral nerve segments containing Schwann cells, have been previously shown to enhance axonal regeneration in the CNS [26,35,38,47–50]. The question arises whether X-irradiation treatment could be used as a form of therapy following CNS injury in order to enhance axonal regeneration at the injured site. This possibility has been investigated [24,25]. In these studies, the delivery of 17 – 23 Gy of X-irradiation, 2–3 weeks post-injury, averted some of the pathological consequences which normally follow such injury. For example, the treatment modulated the formation of reactive astrocytes and reduced the degree of cavitation and neuronal apoptosis [25]. Furthermore, some of the severed axons regrew across the lesion site and formed new synaptic contacts [24]. In our study, neonatal rats between 1 and 5 days of age were X-irradiated. It remains uncertain whether similar neurite growth-enhancing properties could be adduced by such treatment in adult CNS tissues. In addition, the minimal level of exposure to X-irradiation required to achieve the desired growth-enhancing properties is unknown and awaits further research. It may also be possible to use X-irradiation treatment in combination with some other interventions to enhance neurite regeneration even further than that achieved by using just a single type of treatment. In conclusion, we have shown that cryosections of pre-irradiated spinal cord tissue support the regeneration of adult DRG neurones in vitro. This suggests that neonatal X-irradiation of CNS tissue results in a longterm alteration in its environment such that it supports, rather than inhibits, at least some axonal regeneration. This alteration may be due to depletion in the number of and/or a change in the characteristics of the glial cells and their associated inhibitory molecules. This in vitro technique may provide a convenient and unique way to investigate the constitutive inhibitory properties of CNS tissues. It may also provide a convenient biological assay to clarify the role of glial cells in the success, or otherwise, of neuronal regeneration in the CNS.
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peripheral nerve autografts in adult hamsters. Exp. Neurol., 1996, 137 (1), 94 – 104. Schnell, L., Schneider, R., Kolbeck, R., Barde, Y. A. and Schwab, M. E., Neurotrophin-3 enhances sprouting of corticospinal tract during development and after spinal cord lesion. Nature, 1994, 367, 170 – 173. Schwab, M. E., Kapfhammer, J. P. and Bandtlow, C. E., Inhibitors of neurite growth. Annu. Re6. Neurosci., 1993, 16, 565 – 595. Senoo, E., Tamaki, N., Fujimoto, E. and Ide, C., Effects of preslesioned peripheral nerve graft on nerve regeneration in the rat spinal cord. Neurosurgery, 1998, 42 (6), 1347 – 1356. Shewan, D., Berry, M., Bedi, K. and Cohen, J., Embryonic optic nerve tissue fails to support neurite outgrowth by central and peripheral nerves in vitro. Eur. J. Neurosci., 1993, 5 (7), 809– 817. Shewan, D. A., Bedi, K. S., Berry, M., Winter, J. and Cohen, J., Axon regeneration in vitro on physiologically relevant substrata. Neuroprotocols, 1994, 4, 142 – 145. Sims, T. J. and Gilmore, S. A., Glial response to dorsal root lesion in the irradiated spinal cord. Glia, 1992, 6, 96 – 107. Sims, T. J. and Gilmore, S. A., Regrowth of dorsal root axons into a radiation-induced glial-deficient environment in the spinal cord. Brain Res., 1994, 234, 113 – 126. Sims, T. J., Davies, D. L. and Gilmore, S. A., Glial development in primary cultures established from normal and X-irradiated neonatal spinal cord. Glia, 1994, 12, 319 – 328. Snow, D. M. and Letourneau, P. C., Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG). J. Neurobiol., 1992, 23 (3), 322 – 336. Snow, D. M., Watanabe, M., Letourneau, P. C. and Silver, J., A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth. De6elopment, 1991, 113 (4), 1473 – 1485. Son, Y. J. and Thompson, W. J., Schwann cell processes guide regeneration of peripheral axons. Neuron, 1995, 14 (1), 125–132. Tan, M. M., Harvey, A. R. and So, K. F., Regeneration of retinal axons in grafts of peripheral and central nervous tissue in the adult rat. Neurosci. Lett., 1990, 117 (1 – 2), 14 – 19. Vaudano, E., Campbell, G., Hunt, S. P. and Lieberman, A. R., Axonal injury and peripheral nerve grafting in the thalamus and cerebellum of the adult rat: upregulation of c-jun and correlation with regenerative potential. Eur. J. Neurosci., 1998, 10 (8), 2644 – 2656. Xu, X. M., Chen, A., Guenard, V., Kleitman, N. and Bunge, M. B., Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. J. Comp. Neurol., 1995, 351 (1), 145 – 160. Xu, X. M., Chen, A., Guenard, V., Kleitman, N. and Bunge, M. B., Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord. J. Neurocytol., 1997, 26 (1), 1 – 16. Ye, J. H. and Houle, J. D., Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons. Exp. Neurol., 1997, 143 (1), 70 – 81.
Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro N. Wilson a, E. Esfandiary b, K.S. Bedi a,* a
Department of Anatomical Sciences, Uni6ersity of Queensland, St Lucia, Qld. 4072, Australia b Department of Anatomy, Isfahan Uni6ersity of Medical Sciences, Isfahan, Iran Received 15 May 2000; received in revised form 14 August 2000; accepted 15 August 2000
Abstract Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mm-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. © 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Dorsal root ganglion cells; X-irradiation; CNS; Neurites; Tissue culture
1. Introduction Axonal regeneration following injury in the adult mammalian central nervous system (CNS) is typically abortive, with little or no functional recovery. This is in spite of the fact that CNS neurons are inherently capable of axonal regeneration if provided with a conducive environment [1,2,6,10]. A number of possible growth-inhibitory factors present within the microenvironment of the post-lesion CNS have been identified. Abbre6iations: CNS, central nervous system; DABCO, (1,4)-diazbicyclo-(2,2,2)-octane; GAP-43, growth-associated protein 43; GFAP, glial fibrillary acidic protein; MAG, myelin-associated glycoprotein; PBS, phosphate buffered saline; PND, post-natal day. * Corresponding author. Tel.: + 617-3365-3058; fax: + 617-33651299. E-mail address: [email protected] (K.S. Bedi).
These include oligodendrocyte associated molecules such as NI-35 and NI-250 [37], myelin associated glycoprotein [30], and molecules secreted by astrocytes such as tenascins [3] and sulphated proteoglycans [44,45]. Astrocytes may also inhibit axonal regeneration through some contact-mediated mechanism [28]. Several researchers have attempted to alter the postlesion CNS environment in order to enhance axonal regeneration. Much current research is investigating the efficacy of using various neurotrophic growth factors (e.g. brain-derived neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, neurotrophin 3/4, leucocyte inhibitory factor), either singly or in combination, in order to enhance axonal regeneration [11,29,31,36,51]. Another approach involves neonatal X-irradiation of the spinal cord. Studies have shown that this treatment reduces the population of oligoden-
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droglia [7,13,14] microglia [17,42,43] and astrocytes [13,18,19,22] in the irradiated area. The glia-depleted state induced by such X-irradiation may remain for several months [17]. This presents an interesting opportunity to examine CNS regeneration in an environment in which many of the glial cells reactive to injury are removed. For example, the thick astrocytic scar that normally forms in response to dorsal root injury is diminished in animals previously treated with X-irradiation [41]. Furthermore, injured dorsal root axons are able to regenerate into the X-irradiated spinal cord [42]. In addition, Schwann cells from the adjacent peripheral nerves have been shown to invade into the X-irradiated spinal cord [7,15], where they may possibly act to enhance CNS axonal regeneration [42]. Taken together, these observations suggest that the microenvironment following X-irradiation may not be as inhibitory to axonal regeneration as that provided by the normal spinal cord. To examine the mechanisms of axonal regeneration, we have previously used a cryoculture technique that allows many of the complexities of events occurring in vivo to be circumvented [5,39,40]. In this technique, dissociated neurons are plated onto cryosections of various tissues to determine their capacity for neurite extension in vitro. In the present study, we aimed to investigate whether or not cryosections of X-irradiated regions of spinal cord tissue are capable of supporting neurite extension in vitro, compared with sections from non-irradiated animals. We believe that a model system such as this could offer important advantages over in vivo experiments in the study of the molecular mechanisms involved in the enhancement of axonal regeneration by X-irradiation treatment.
2.2. Irradiation treatment Rat pups of 1, 3 or 5 post-natal days were anaesthetised by cooling their bodies on blocks of ice for a few minutes, and placed in a lead box. This box had a rectangular hole measuring 10×5 mm cut in one surface. The pups were gently lodged into the box and held in position by a small piece of foam, so that the lumbosacral region of the spinal cord was exposed through the hole. The exposed regions of the rat pups were irradiated under an Ultrays low voltage X-ray unit (Ultrays Pty. Ltd., Melbourne). The irradiation conditions were, 50 kV; 0.25 mm aluminium added filter; 1 cm cone; 30 mA; 50-s duration. The machine was calibrated so that these conditions exposed the pups to a total dose of about 40 Gray (Gy). Following irradiation, the rat pups were allowed to recover under the warmth of a 100 W light before being returned to their litter. Eight pups were ‘sham-irradiated’ by anaesthetising them on ice, placing them under the lead shield, but not applying any X-irradiation. These pups acted as controls.
2.3. Preparation of ‘fresh-tissue’ cryosections
2. Materials and methods
The animals were allowed to survive until they were at least 70 days of age before being killed. The lumbosacral region of the spinal cord from irradiationtreated and control rats was dissected out, mounted on a small cork disc, frozen in 2-methylbutane (Aldrich) cooled in liquid nitrogen, and sectioned in a cryostat (American Optical Corporation) to yield transverse sections (10 mm). All sections were picked up on sterile 13 mm diameter glass coverslips that had been previously coated with sterile poly-L-lysine (20 mg/ml; 70 000– 150 000 mol wt.; Sigma). These sections were then stored in 24-well plates (Costar) at − 20°C until required.
2.1. Animals
2.4. Preparation of ‘fixed-tissue’ sections
All animals used in these experiments were Wistar rats, purchased from the University of Queensland’s Central Animal Breeding House. A total of fourteen rats were used to provide cryosections for study. The lumbosacral region of the spinal cord was irradiated in two rats at each of 1, 3, and 5 post-natal days (PND) of age. Eight rat pups were used as controls. A further eighteen irradiated and six control rats were used to investigate the cellular response to the treatment. All animal experiments and protocols were under clearance from the animals ethics committee of the University of Queensland and were in accordance with the guidelines published by the Australian National Health and Medical Research Council.
Six control and eighteen X-irradiated rats were anaesthetised and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The lumbosacral regions of the spinal cord from three of the control and nine of the treated animals were processed for embedding in Technovit 7100 resin (Kulzer Histo-Technik). Transverse sections (2 mm) through this tissue were cut an LKB Historange 2218 microtome and stained with 1% toluidine blue. The lumbosacral spinal cords from the remaining three control and nine treated rats were immersed overnight in a solution of 30% sucrose in 0.1 M phosphate buffer (pH 7.4), placed in an appropriate mould, completely covered with Tissue-Tek (OCT; California) and frozen with liquid nitrogen. Transverse cryosec-
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tions (8 mm) through this OCT-embedded tissue were cut on a cryostat and picked up on gelatinised slides. These frozen sections were immunolabelled with antiglial fibrillary acidic protein (GFAP — see below).
2.5. Preparation of dissociated dorsal root ganglion cells About 20–25 dorsal root ganglia (DRG) from a normal adult rat were dissected out for each preparation. These were placed into a few millilitres of modified Bottenstein and Sato’s tissue culture medium [8] containing 2% fetal bovine serum (Sigma). The DRG neurons were dissociated using a procedure modified slightly from that described by Lindsay [27]. Briefly, the ganglia were washed several times in fresh medium, incubated for 3 h in 0.125% collagenase (Sigma), re-washed in several changes of fresh medium and triturated through the tip of a flame-polished pipette. The resultant cell suspension was layered onto a 2 ml cushion of 15% bovine serum albumin (Sigma; essentially fatty acid free) in a sterile 15 ml conical tube and centifuged at 2000 g for 10 min. The supernatant was removed, and the cell pellet resuspended in a small volume of fresh medium. The cell concentration was adjusted to a plating density of approximately 3000 per ml, and 0.5 ml of this suspension was plated onto each coverslip bearing the cryosections (prepared as described above), using 4-well plates (Nunc). It was ensured that cryosections from both control and irradiated tissues were layered with the same batch of dissociated cell suspensions. Cultures were incubated in a humidified atmosphere containing 5% CO2 at 37°C for up to 10 days. The cryosections and any growing DRGs were then fixed in 2% paraformaldehyde in preparation for immunolabelling. The experiments were repeated with cell suspensions prepared from 10 different rats.
2.6. Antibodies and immunolabelling The primary antibodies used in this study included, 1. monoclonal anti-GAP43 (growth-associated protein; Sigma) at a dilution of 1:100 to label growing DRG cells. It should be noted that anti-GAP43 has been found [5] to be a useful marker for regenerating neurites; 2. rabbit anti-GFAP (glial fibrillary acid protein; Sigma) at a dilution of 1:100. This antibody was used primarily to ease the visualisation of the spinal cord cryosections used in the culture studies. It is known [21] to be specific for astrocytes in rats of this age. The secondary antibodies used included, 1. goat anti-mouse IgG conjugated to Cy3 (Amersham; 1:1000 dilution);
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2. goat anti-rabbit IgG conjugated to FITC (Sigma; 1:100 dilution). The fixed cultures and cryosections were washed in three changes of phosphate buffered saline (PBS), postfixed in cold ( − 20°C) 100% methanol for 10 min, re-washed in three changes of PBS, incubated with the primary antibody for up to 2 h, re-washed in PBS, and treated with the secondary antibody for 1–2 h, washed in PBS and distilled water before mounting in 90% glycerol in PBS which had a small amount of 1,4-diazabicyclo-[2.2.2]-octane (DABCO; Sigma) added to it. The cultures and fixed cryosections were viewed under an Olympus BH-2 fluorescent microscope and digital images taken using a SPOT digital camera. The digital images were enhanced and labeled using Adobe Photoshop software (version 5.0; Adobe Systems Inc., 1998).
3. Results
3.1. Effects of the irradiation treatment The body weights of the previously irradiated rats were between 25 and 30% smaller than the age-matched controls by 28 days of age. These differences were statistically significant (PB 0.0001; analysis of variance). We also observed that by 14 days of age, there was hindlimb paralysis in those rats irradiated on PND 1. Symptoms were consistent with the dysfunction of the corticospinal tract. Two of the rats irradiated on PND 3 showed similar symptoms, but none of those treated on PND 5 developed any obvious signs of hindlimb paralysis. All affected rats had recovered full or partial movement of their hindlimbs by PND 28. There were no obvious macroscopic differences observed between the spinal cords of the irradiated rats irrespective of the age at which the irradiation treatment was carried out. However, transverse sections through the spinal cords from the irradiated animals were somewhat smaller than those from sham operated controls (see Fig. 1A and B). This phenomenon has been reported previously [13] and may simply be a reflection of the fact that the treated animals were smaller than the controls. Fig. 1A and B show transverse sections through the lumbosacral region of the spinal cord in 14-days-old control rats, and 14-days-old rats previously irradiated on PND 3, respectively. In control animals, there was a clear distinction between the grey and white matter regions with a number of cells present in the white matter of both the dorsal and ventral funiculi (Fig. 1A). By comparison, the irradiated spinal cord (Fig. 1B) appeared hypomyelinated, which made the boundary between the grey and white matter regions less distinct in most cases. In irradiated rats there were fewer darkly-stained cells in the funiculi indicating a probable
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reduction of the neuroglial cell population in comparison to control animals. Fig. 1C–F shows higher power micrographs of the dorsal funiculus region in control and irradiated animals. C and E are, respectively, from 14 and 30-day-old control rats, and D and F are from age-matched rats previously irradiated at 3 days of age. In control animals, many darkly-stained neuroglial cells could be seen in the dorsal funiculus at 14 days of age; by PND 30, however, these cells were not so obvious (Fig. 1C and E). In contrast, there was an obvious lack of neuroglia in the dorsal funiculus of the previously irradiated rats at PND 14, whilst at PND 30, the dorsal funiculus was hypercellular (Fig. 1D and F). At all ages, a number of small ‘holes’ were present in the sections from the irradiated spinal cords. Their appearance matched the description of the vascular and haemorrhagic effects of x-irradiation reported by previous investigators [4]. Fig. 1G and H show sections through the dorsal horn region of the spinal cord at PND 14, immunolabelled with anti-GFAP. Fig. 1G is from a control animal and Fig. 1H is from a rat previously irradiated on PND 3. The distribution of GFAP immunoreactivity in control rats was characterised by labelling of many thick radial fibres in the white matter and stellate astrocytes in the grey matter. In the irradiated spinal cords, qualitative observations seemed to indicate that the number and density of the GFAP-positive cells was less than that seen in controls. The stellate astrocytes within the grey matter appeared to be more affected than the radial glial cells found around the periphery of the spinal cords. Fig. 1. A and B show 2 mm-thick transverse sections through the lumbosacral region of the 14-days-old rat spinal cord, stained with toluidine blue. A is from a sham-irradiated control and B from a previously irradiated animal. The boundary regions between the grey (g) and white matter (w) regions were more distinct in the sham-irradiated controls than in the irradiated spinal cords. The dorsal and ventral funiculi appeared more cellular in the controls compared to the irradiated animals. *, Dorsal funiculus region; v, ventral horn of gray matter; d, dorsal horn of gray matter. Scale bar, 250 mm. C – F show higher power micrographs of the dorsal funiculus region (demarcated by a dotted line in each case) of the spinal cord. C and E are from 14 and 30-days-old control animals, respectively, and D and F are from age-matched, previously irradiated animals. The dorsal funiculi in irradiated spinal cords appeared to have fewer cells present at 14 days of age than in controls. By 30 days of age, the cellularity of the dorsal funiculus in the irradiated spinal cord had increased in comparison to control animals, probably by the invasion of Schwann cells (arrowheads in F). Scale bar, 50 mm. G and H show the dorsal horn region of the spinal cord in PND 14 animals, immunolabelled with anti-GFAP. G is from a control animal and 1H is from an animal previously irradiated on PND 3. The distribution of GFAP immunoreactivity was characterised by labeled stellate astrocytes in the grey matter (arrowheads) and coarse radial fibres in the white matter (small arrows). There appeared to be a reduction in the extent of immunoreactivity in the spinal cords from the previously irradiated animals compared with controls. This reduction seemed to be more pronounced for the stellate astrocytes than the radial glial cells. Scale bar, 100 mm.
3.2. Growth of DRG neurons CNS tissue substrates Fig. 2 A and B show sections of spinal cord, obtained from sham-irradiated animals. It was found that whilst the DRG cells grew extensive neurites on the adjacent regions of the poly-L-lysine coated coverslips, they were incapable of growing such neurites on the CNS substrates. Some neurites were observed to have grown towards the cryosections, but were incapable of growing onto them. Fig. 2C–F show DRG cells growing on sections of pre-irradiated spinal cord tissue. Neurites successfully regenerated on the substrate-free regions of the coverslips and in addition, many were also seen to extend onto regions of the irradiated cryosections (Fig. 2C and F). Other DRG cells clearly settled onto the cryosections themselves and were capable of extending neurites on to those sections (Fig. 2D, E and F). However, these neurites were not as extensively branched as those that grew on adjacent portions of the poly-L-lysine coated coverslips. There appeared to be no differences in the extent of neurite regeneration on spinal cord tissue irradiated on PND 1, 3 or 5 in our experiments.
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Fig. 2. In all cases, the DRG neurones (n) were plated onto the cryosections and allowed to grow in culture for 8–10 days before being fixed and double-labeled with anti-GFAP (for the substrate sections) and anti-GAP43 (for the regenerating neurites). The boundary between the coverslip (c) and the spinal cord cryosection (sc) is shown by a dotted line. A and B show cryosections of the spinal cord taken from a sham-irradiated adult control rat, with adjacent DRG neurons growing in tissue culture. Extensive neurite growth was seen on the poly-L-lysine-coated regions of the glass coverslips but no growth was observed on the tissue section itself. Some neurites could be seen to grow towards the section but on contact appeared to have turned away (arrow). Other neurites grew close to the edges of the sections but in all instances failed to actually grow onto them (arrowheads). C–F show examples of DRG neurones that are growing on and adjacent to cryosections of previously irradiated adult spinal cord tissue. C shows numerous neurites (arrowheads) that grew from the adjacent regions of the coverslip and onto the irradiated tissue sections (arrows). D–F show examples of DRG neurones that settled on the tissue sections themselves and then extended branching neurites (arrowheads). Scale bar, 50 mm.
4. Discussion Our study showed that DRG neurons are capable of regenerating neurites in vitro, on a substrate consisting of cryosections taken from irradiated, but not from non-irradiated, spinal cord tissue. This finding is consistent with the observation that lesioned dorsal root axons can successfully regenerate into the spinal cord of neonatally-irradiated rats, but not into non-irradiated spinal cord [42]. It seems that neonatal X-irradiation of the CNS tissue converts its environment from one that does not normally support axonal regeneration to one that does. The cryoculture technique may therefore be able to provide a convenient model system in which to
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investigate the irradiation-induced changes in the CNS that make it permissive for axonal regeneration. This model is particularly useful in that it allows some of the complexities of events occurring in vivo to be circumvented, whilst still providing the growing neurons with physiologically relevant substrata. It is also possible to present neurons with a potential to regenerate neurites (such as dissociated DRG cells) with uninjured CNS tissue as the substrate. Such a combination is difficult, if not impossible, to achieve in vivo. Studies of this type may therefore provide an insight into the mechanisms involved in the process of successful axonal regeneration Our observation that cryosections of normal adult rat CNS did not support neurite growth is consistent with the findings of previous investigators [9,20,33,34,39]. This observation suggests that, constitutively, normal CNS tissue does not support axonal regeneration. Any changes to the CNS environment following injury may make it even more difficult to achieve axonal regeneration than is normally the case. These changes may include the up-regulation, or release of inhibitory factors, down-regulation of growth-enhancing factors, or the formation of physical barriers to axonal regeneration (e.g. glial scar tissue). The non-permissive or inhibitory molecules within the CNS may include tenascins, sulphated proteoglycans, NI-35, NI250, and possibly hyaluronectin and myelin-associated glycoprotein [3,30,37,44,45]. The presence of some contact-mediated inhibitory molecules is indicated by our observation that neurites growing on the poly-L-lysine coated glass coverslips turned and grew away in a new direction on contact with the cryosections of normal spinal cord tissue. Our present results support the hypothesis that neonatal X-irradiation of the CNS alters the non-permissive nature of its environment, perhaps by altering the quantitative and qualitative properties of the glial population. This may in turn influence the distribution and amount of inhibitory molecules produced by glial cells. It should be noted, however, that the extent of DRG neurite growth on the irradiated cryosections was generally not as widespread, or robust as that seen on surrounding areas of the coverslips. This strongly suggests that some inhibitory influences are still present in the irradiated tissue. Immunohistochemistry showed that there were several GFAP-positive cells present within the irradiated CNS tissues. Whether these were newly-generated glial cells, or whether they were cells that survived the irradiation treatment is uncertain. Whatever the case, it seems possible that the remaining glial cells may continue to be partially inhibitory to axonal regeneration. Our observations are consistent with the finding that radial astroglia may be less sensitive to the effects of X-irradiation during early postnatal life than other types of glia [43].
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Our experiments on Nissl-stained sections confirmed that the number of neuroglia markedly decreased in the neonatally-irradiated regions of spinal cord in comparison to controls, at 7 (data not shown) and 14 days of age [4,23]. By 30 days of age, the number of cells in the dorsal funiculus regions had increased. This increase was thought to be due to Schwann cells invading the injured CNS region from the adjacent peripheral nerve tissue [4,7,16,22,23]. It is possible that the presence of these Schwann cells may be one of the factors that enhances axonal regeneration in these previously irradiated regions of the spinal cord. Schwann cells are known to be crucial in peripheral nerve axonal regeneration [32,46] due to the fact that they can secrete a host of neurotropic and neurotrophic factors [12]. Grafts of purified Schwann cells, or peripheral nerve segments containing Schwann cells, have been previously shown to enhance axonal regeneration in the CNS [26,35,38,47–50]. The question arises whether X-irradiation treatment could be used as a form of therapy following CNS injury in order to enhance axonal regeneration at the injured site. This possibility has been investigated [24,25]. In these studies, the delivery of 17 – 23 Gy of X-irradiation, 2–3 weeks post-injury, averted some of the pathological consequences which normally follow such injury. For example, the treatment modulated the formation of reactive astrocytes and reduced the degree of cavitation and neuronal apoptosis [25]. Furthermore, some of the severed axons regrew across the lesion site and formed new synaptic contacts [24]. In our study, neonatal rats between 1 and 5 days of age were X-irradiated. It remains uncertain whether similar neurite growth-enhancing properties could be adduced by such treatment in adult CNS tissues. In addition, the minimal level of exposure to X-irradiation required to achieve the desired growth-enhancing properties is unknown and awaits further research. It may also be possible to use X-irradiation treatment in combination with some other interventions to enhance neurite regeneration even further than that achieved by using just a single type of treatment. In conclusion, we have shown that cryosections of pre-irradiated spinal cord tissue support the regeneration of adult DRG neurones in vitro. This suggests that neonatal X-irradiation of CNS tissue results in a longterm alteration in its environment such that it supports, rather than inhibits, at least some axonal regeneration. This alteration may be due to depletion in the number of and/or a change in the characteristics of the glial cells and their associated inhibitory molecules. This in vitro technique may provide a convenient and unique way to investigate the constitutive inhibitory properties of CNS tissues. It may also provide a convenient biological assay to clarify the role of glial cells in the success, or otherwise, of neuronal regeneration in the CNS.
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