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Effects of Leukemia Inhibitory Factor on Galanin Expression and on Axonal Growth in Adult Dorsal Root Ganglion Neurons in Vitro
Experimental Neurology 169, 376 –385 (2001) doi:10.1006/exnr.2001.7667, available online at http://www.idealibrary.com on
Effects of Leukemia Inhibitory Factor on Galanin Expression and on Axonal Growth in Adult Dorsal Root Ganglion Neurons in Vitro ¨ ztu¨rk and D. A. Tonge G. O Neuroscience Research Centre, GKT School of Biomedical Sciences, King’s College London, Hodgkin Building, Guy’s Hospital, London SE1 9RT, United Kingdom Received November 11, 1999; accepted February 22, 2001
Synthesis of leukemia inhibitory factor (LIF) is increased in lesioned peripheral nerves and it is thought that this may cause increased expression of galanin (GAL) in axotomized dorsal root ganglia (DRG) neurons and also to promote axonal regeneration. We therefore compared effects of LIF and nerve growth factor (NGF) on galanin expression and axonal growth using cultured intact DRGs of adult mice. In control lumbar DRGs cultured for 3 days, only 16% of neurons were immunoreactive for GAL, but this was increased to 38% in preparations cultured with LIF. NGF by itself had no effect on GAL expression, but the proportion of GAL-positive neurons in cultures incubated with LIF and NGF together (22%) was less than that observed in DRGs cultured with LIF alone. Similar results were obtained using thoracic DRGs. In collagen gels, NGF caused marked increases in the numbers and lengths of outgrowing axons as observed in previous studies. In contrast, LIF did not stimulate axonal outgrowth but increased the proportions of axons which were immunoreactive for GAL. The results indicate that expression of LIF in lesioned nerves may affect expression of neuropeptides such GAL rather than stimulating axonal regeneration. ©
2001 Academic Press
Key Words: LIF; galanin; NGF; axon; regeneration; DRG.
INTRODUCTION
It is well known that axotomy of neurons in the peripheral nervous system (PNS) causes striking changes in expression of various classes of protein, reflecting a shift in metabolism from production of substances for synaptic transmission to synthesis of proteins required for regeneration (1). In dorsal root ganglia (DRGs) synthesis of some neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin is reduced while that of others such as galanin (GAL) neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and cholecystoki0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
nin (CCK) is increased, causing important effects on synaptic transmission within the spinal cord (9). Some of these changes may have protective functions (9) while others, such as increased expression of CCK which inhibits opioid action (47), may contribute to chronic pain. In DRGs of adult animals the proportion of neurons which express GAL is low, but increases dramatically after axotomy (9, 3, 20, 39). In the spinal cord, GAL may have both excitatory and inhibitory actions on nociceptive reflexes (4, 46, 14, 13). However, it is also expressed by developing trigeminal and DRG neurons suggesting that it may also be involved in axonal growth (48). Consistent with this hypothesis, recent studies have shown that axonal regeneration after peripheral nerve injuries is impaired in GAL knock-out mice (10). The mechanisms responsible for the changes in expression of GAL and other neuropeptides after axotomy are uncertain, but one possibility is that they result from reduced availability of trophic factors which are normally derived from innervated tissues. There is good evidence for this in the case of nerve growth factor (NGF) whose sequestration by chronic infusion of chimeric trk–IgG fusion molecules reduces CGRP levels in sensory neurons in vivo (21). Conversely, its expression and that of SP are increased by NGF in cultured sensory neurons (18). Chronic infusion of NGF restores levels of CGRP and SP in DRGs of lesioned peripheral nerves in vivo (44) and also reduces the proportions of neurons expressing NPY, GAL, VIP and CCK. However, the failure of NGF infusion to completely suppress the increased expression of these four neuropeptides after axotomy suggests that this may also be regulated by other factors. One factor which has recently been shown to induce GAL expression in DRG neurons in vivo (39) is leukemia inhibitory factor (LIF). This cytokine has important effects on the phenotype and survival of a number of different types of neuron, including embryonic and neonatal sensory and motor neurons (15, 36). Levels of LIF in peripheral nerves under normal conditions are
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EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
very low, but following axotomy its expression increases rapidly in lesioned nerves (5). Furthermore, LIF injected into peripheral nerves shows increased retrograde transport to sensory and motor neurons following peripheral injury (5), suggesting that this factor may have a role as a survival or growth factor in adult animals. LIF has been shown to increase expression of the neuropeptide VIP in superior sympathetic ganglia (SCGs) in culture (35) and conversely, transgenic LIF knock-out mice show lower levels of VIP, substance P and GAL expression after peripheral axotomy (28, 34). Subsequent studies have shown that LIF may also regulate GAL expression in DRGs since after axotomy, LIF-deficient transgenic mice had a substantially lower increase in GAL expression in DRGs compared to wild-type animals (3, 33). Recently, it was shown that intraneurally injected LIF causes up-regulation of GAL in corresponding DRGs (39). The targets of LIF in the DRGs are mostly those neurons that normally express TrkA and CGRP (40). Another factor that affects GAL expression in DRGs is NGF since its infusion partially antagonizes the increased GAL expression seen after axotomy (44). NGF also partially inhibits the increased galanin expression which occurs in SCGs following explantation or axotomy in vivo (31), which is consistent with the known interaction of NGF with LIF signaling (27). This raises the possibility that target-derived NGF normally inhibits GAL expression and that lack of this factor after axotomy may play a role in the production of this neuropeptide. Support for this hypothesis comes from another study, in which NGF antiserum injected into unoperated rats caused an increase in GAL mRNA and immunoreactivity in SCGs (31). Although several recent studies have shown that LIF affects expression of certain neuropeptides, relatively little is known about the effects of this factor on axonal regeneration. However, LIF promotes neurite extension and morphological maturation in cultured embryonic neuroblasts (22, 25). In mature DRG neurons, LIF promotes neurite extension in response to NGF (2). Furthermore, sprouting from postganglionic sympathetic efferents into DRGs after peripheral axotomy is induced by LIF (38). LIF has also been reported to enhance sciatic nerve regeneration in a silicone chamber model by increasing diameters of axons, numbers of myelinated fibers, and conduction velocity (37). The effects of growth factors and cytokines on neuronal phenotype and axonal growth have generally been studied using certain in vivo and in vitro experimental models although both types suffer some disadvantages. The in vivo models usually involve application of the factors to the cut end of a peripheral nerve or intrathecal infusion of these factors. However, the interpretation of results from such experiments may be complicated by the fact that the procedures may have indirect effects including inflammation. For example,
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although NGF infusions have effects on DRG neurons in vivo (21, 44) the widespread expression of receptors for neurotrophins on cells of the immune system, including mast cells (19) raises the possibility that factors released by these cells might also influence neuronal phenotype. Similar issues apply to the in vivo study of other growth factors. In view of these problems, in vitro studies offer certain advantages. Traditionally, dissociated neurons from sensory and autonomic ganglia of neonatal animals have been used for such studies but the enzymatic and mechanical procedures used to dissociate the neurons may cause changes in neuronal phenotype. Furthermore, the normal relationship between neurons, glial cells and extracellular matrix (ECM) is disrupted and this is likely to influence neuronal phenotype. For example, expression of neuropeptides by dissociated neurons in culture is usually very low unless growth factors such as NGF are added (18, 23). In contrast, neurons in vivo continue to express neuropeptides such as CGRP for several days after axotomy (44) although levels do eventually decline. An alternative method for investigating the regulation of neuropeptide expression was suggested by a recent study of regeneration of peripheral nerves of adult mice in vitro (43). In these experiments, peripheral nerves together with their attached DRGs were explanted into gels of Matrigel (the commercially available extracellular matrix extract of the Engelbreth– Holm–Swarm tumor cell line) without growth factors in serum-free medium. Axonal outgrowth from the tips of the cut nerves and dorsal roots began within 24 h and continued for at least a week. Conventional fluorescence microscopy showed that many axons had strong CGRP-like immunoreactivity (43), suggesting that such preparations could be used to study expression of neuropeptides and effects of growth factors. To determine the suitability of these preparations for such studies, we investigated the survival of neurons in the explanted DRG preparations and found that more than 90% of neurons remained viable over 3 days in culture. We therefore used these preparations to study effects of LIF and NGF on GAL expression and axonal regeneration in vitro. Our results suggest that these preparations could also be used to study the regulation of expression of other neuropeptides. METHODS
Adult female mice aged 6 –12 weeks (BK1 TO strain; bred at King’s College) were used in all experiments. Prior to removal of DRGs, animals were anesthetized by ether and an intraperitoneal injection of 0.25 ml of a mixture of 3% Hypnorm (Jansen) and 33% diazepam (Phoenix Pharmaceuticals) as described (7) and killed by cervical transection. In experiments to determine neuronal survival, thoracic DRGs (T10 –T12) from both
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sides of two mice were removed rapidly and the ganglia from one side fixed immediately in 4% paraformaldehyde in phosphate buffer, pH 7.0 (PB). The remaining DRGs were incubated free-floating in 0.5 ml of RPMI 1640 medium containing 100 units of penicillin, 100 g streptomycin, and 250 ng amphotericin B per milliliter (all from Sigma) at 36°C in a sealed box containing 95% O 2/5% CO 2 in a humidified incubator for 3 days prior to fixation. To determine the total number of neurons, the fixed DRGs were split into four pieces with a razor blade under the microscope. Then they were transferred onto coverslips and incubated overnight with the monoclonal antibody A60 (Chemicon) diluted 1/100 that recognizes a protein (NeuN) in the nuclei of most types of neurons in adult rodents (21, 41). The washes and application of secondary antibody [fluorescein (FITC)conjugated goat anti-mouse (Sigma)]. were as for preparations in collagen gels as described below. Preparations were mounted in Citifluor (Agar Scientific) and scanned using a Leica scanning confocal microscope. Optical sections were obtained at 7-m intervals and the images digitally stored. Commercial image editing software (Adobe Photoshop) was used to create cumulative templates from series of sections by marking labeled nuclei on each one. Double counting was avoided by marking on sections, only those nuclei which did not appear marked on the latest form of the template. The numbers of marks on the final cumulative templates were counted using an image analysis program (NIH Image). In a second experiment to determine the proportions of viable neurons, T10 –T12 DRGs were removed from six mice. Some of the DRGs were immediately fixed to use as controls while the remaining preparations were cultured free-floating for 3 days. After 2.5 days incubation, a solution of 75 M propidium iodide (PI) and 0.5 mg/ml 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide; thiozolyl blue (MTT) in RPMI was added to the cultures which were then returned to incubator for 12 h. The next day all the preparations were fixed by immersion in 4% paraformaldehyde in PB. MTT is a water-soluble tetrazolium salt that is converted to insoluble purple formazan crystals in the cells with active mitochondria (8) and in our experiments demonstrated live neurons very successfully. PI is a fluorescent stain for nucleic acids which can not cross intact cell membranes but stains the cytoplasm of dead/dying cells bright pink/red as nuclear material is dispersed into the cytoplasm as a result of nuclear disintegration at a very early stage of cell death (45). In order to determine the reliability of PI labeling and its staining pattern, some preparations were incubated with MTT and PI together overnight in a refrigerator for 12 h where the unbuffered culture medium became alkaline and would therefore be expected to increase the proportion of dead cells. In these preparations,
about 30% of neurons were diffusely stained with PI (and therefore presumably dead), while the rest of the cell profiles were laden with formazan crystals but lacked PI labeling of the cytoplasm. After the sections had been mounted in Citifluor (which is an aqueous mounting medium), PI gradually diffused across the tissues and eventually labeled all cell nuclei. However, the dead cells could be clearly distinguished by the intense PI labeling of both the nucleus and the cytoplasm, providing a convenient method for live/dead assessment of cells in these experiments since although the MTT method identified live neurons, the accumulation of formazan crystals often made it difficult to determine their numbers accurately. This finding indicated that dead/dying cells could be distinguished by PI labeling of the nucleus and cytoplasm and that it was therefore not necessary to use MTT to mark the live cells. To study regulation of GAL expression, experiments were carried out using thoracic and also L4 DRGs since a substantial number of fibers from this DRG run through the sciatic nerve (6), so that it was possible to compare effects of axotomy in vivo with those of LIF and NGF in vitro. In these experiments, thoracic and L4 DRGs were removed from mice and the spinal roots and peripheral nerves cut off. The DRGs were then cultured free-floating for 2 days in RPMI medium containing 30 ng/ml LIF (Chemicon) or 50 ng/ml 2.5S NGF (Alamone Labs or Promega) or in some cases both factors. Control preparations were cultured for 2 days without growth factors, after which all preparations were fixed in 4% paraformaldehyde in PB. In some experiments, mice were anesthetized with Hypnorm and diazepam as described above and the intercostal nerves (T11 and T12) or sciatic nerve on one side cut 2 days prior to removal of the corresponding thoracic or L4 DRGs, which were immediately fixed in paraformaldehyde. After fixation L4 DRG preparations were covered with OCT compound (BDH), frozen by immersing into liquid nitrogen, and kept at ⫺20°C (or ⫺70°C for longer periods of storage) until cutting. Sections of 10 m thickness were cut using a cryostat and mounted on coverslips which had been treated with aminopropyltriethoxysilane (Sigma) as described (29). In experiments to determine effects of LIF and NGF on axonal growth and phenotype, thoracic DRGs with short lengths of intercostal nerves attached (about 3 mm) were quickly removed from the mice by dissection and placed on 13-mm glass coverslips in multiwells in shallow gels of type 1 collagen (26). The preparations were covered with 0.5 ml of RPMI medium and subsequently maintained at 36°C in a sealed box containing 95% O 2/5% CO 2 in a humidified incubator 2 days, prior to fixation with 4% paraformaldehyde in PB. LIF and 2.5S NGF separately and in combination (30 and 50 ng/ml, respectively) were added to some of the preparations at the start of the incubation period.
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EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
TABLE 1
Histological Methods Frozen sections were blocked with 3% bovine serum albumin (BSA) ⫹ 0.1% Triton X-100 in phosphatebuffered saline (PBS) for 1 h. The primary antibody to GAL, raised in rabbit (Affinity) at a dilution of 1/100, was added to the sections for 2 h, followed by 3 ⫻ 10-min washes in PBS. The preparations were then incubated with FITC-conjugated goat anti-rabbit (Jackson) for an additional 2 h with three subsequent washes in PBS, before mounting in Citifluor. In order to label the explant cultures, the fixed preparations were first washed with several changes of PBS and then blocked with 3% BSA and 0.1% Triton X-100 in PBS for 1 h. Primary antibodies to GAL and a mouse monoclonal antibody (SDL.3D10) to the panaxonal marker III-tubulin (Sigma) were diluted 1/50 in PBS containing 3% BSA and left on the fixed preparations overnight. The next day preparations were given five washes with PBS, each wash lasting about 1 h, and secondary antibodies (diluted 1/100) to rabbit and mouse IgGs, conjugated with FITC and tetramethylrhodamine isothiocyanate (Jackson and Sigma, respectively), were added overnight. During the third day they were washed again in the same way and mounted in Citifluor. The preparations which had been labeled using the antibodies (sections and whole mounts) were viewed using an epifluorescence microscope equipped with phase contrast illumination and images captured directly off the microscope using a Hamamatsu C5810 digital camera system Stereography To determine the lengths of outgrowing axons, preparations which had already been fixed and stained using fluorescent antibodies were incubated overnight with biotinylated goat anti-mouse (1/100; Sigma). After a further series of washes the following day, the preparations were incubated overnight with peroxidaseconjugated avidin (1/100; Sigma). The following day, after a final series of washes with Tris buffer (pH 7.6), preparations were incubated in Tris buffer containing 0.6 mg diaminobenzidine and 15 l of 1% CoCl 2 per milliliter for 10 min prior to addition of 10 g/ml of 3% H 2O 2 to visualize peroxidase activity. The stained preparations were mounted in glycergel (Dako) and viewed under dark-ground illumination using a microscope with an image projection tube. Camera lucida drawings were made of the trajectories of the longest axons growing out of the cut ends of the peripheral nerves (usually 10 –20 per preparation) and their lengths were determined using the NIH Image software. To determine the thickness of axons growing in collagen, images were captured directly off the microscope using a Hamamatsu C5810 digital camera and analyzed using Scion Image PC.
Numbers of Neurons in Matched Pairs of Freshly Fixed (Control) and 3-Day Cultured Thoracic DRGs from Two Mice DRGs
Control
Cultured
T10 T10 T11 T11 T12 T12
1832 1901 2115 2039 2642 2416
1748 (0.95%) 1872 (0.98%) 2047 (0.97%) 1989 (0.98%) 2518 (0.95%) 2340 (0.97%)
Note. The ratios of numbers of neurons in the cultured DRGs compared with the numbers in the fresh DRGs are given in parentheses.
Statistics Results are expressed as means ⫾ SEM. The differences between means were evaluated by Student’s t test or 2 test where appropriate and considered significant at P ⬍ 0.05. RESULTS
Neuronal Survival in Explant DRG Cultures The antibody to NeuN clearly labeled neuronal nuclei as described in other studies (45). The numbers of identifiable neurons in optical sections of freshly dissected and cultured thoracic DRGs are summarized in Table 1. These results show that the numbers of neurons after 3 days in culture were 95–98% (mean 97 ⫹ 0.49%; n ⫽ 6) of the numbers in freshly fixed ganglia indicating neuronal loss of only 2–5%. The differences between the numbers of neurons in cultured and fresh preparations were not statistically significant on a 2 test. However, it is possible that some of these neurons could be dying and occasionally some distorted nuclear profiles were observed which may be an indication of ongoing disintegration. To determine the proportion of viable cells, the preparations were therefore incubated with PI. The percentage of dead neurons identified on 20 frozen sections of 12 DRGs by this method was 6.2 ⫹ 0.7% (n ⫽ 1203) indicating that some of the neurons counted in the optical sections were probably dead or dying. However, even combining the percentages of identified dead cells with the estimated percentages of lost cells, the results suggest that more than 90% of neurons remain viable after 3 days in culture. Expression of Galanin in Dorsal Root Ganglia In sections of normal DRGs, a few neurons were labeled by the antibody to GAL (Fig. 1A), but the proportion was very low as has been found in other studies (9). Two days after cutting the thoracic or sciatic nerves, increased numbers of GAL-positive neurons were observed in the DRGs (Fig. 1B) and their propor-
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FIG. 1. GAL expression in sections of DRGs: normal (A), 2 days axotomized (B), 2 days cultured without growth factors (C), and 2 days cultured with LIF (D). Arrows indicate GAL-immunoreactive neurons.
tions were significantly higher (P ⬍ 0.001) than in normal DRGs. In DRGs cultured without growth factors, many of the neurons were clearly labeled (Fig. 1C) and their proportions were similar to that in DRGs after axotomy in vivo. However, in the preparations cultured with LIF, the numbers of GAL-positive neurons were clearly greater (Fig. 1D) and their proportions were significantly increased (P ⬍ 0.001) compared to preparations cultured without LIF. NGF by itself did not show any obvious effect on GAL expression in culture, since the proportions of GAL-positive neurons were similar to that of preparations cultured without growth factors. However, the proportions of GAL-positive neurons in DRGs cultured with NGF ⫹ LIF were lower than in preparations cultured with LIF alone (P ⬍ 0.001) (Table 2).
Table 3. In control preparations after 2 days in culture, a few axons had grown into the gels spontaneously (Fig. 2A) and similar axonal growth was also observed in preparations cultured with LIF (Fig. 2B). However, preparations cultured with NGF showed a marked enhancement in the numbers and lengths of outgrowing axons (Fig. 2C) as observed in previous studies (16, 42), but these were thinner than in controls and those in preparations cultured with LIF. Axonal outgrowth in preparations cultured with NGF and LIF together was similar (Fig. 2D) but interestingly, the axons were thicker than with NGF alone. There were no obvious differences in the lengths and numbers of axons between NGF and NGF ⫹ LIF-treated preparations.
Effects of LIF and NGF on Axonal Growth in Collagen Gels
In control preparations after 2 days in culture, the proportion of axons which were immunoreactive for GAL (Fig. 3A) was 13 ⫾ 3% (n ⫽ 64; six preparations). In the presence of LIF the proportion of labeled axons (Fig. 3B) had increased significantly (P ⬍ 0.05) to 33 ⫾
The mean lengths and diameters of axons growing from thoracic DRGs in collagen gels are summarized in
GAL Expression in Outgrowing Axons
TABLE 2 The Mean Percentages (⫾SEM) of GAL-Positive Neurons in Thoracic and Lumbar DRGs after 2 Days in Culture or Axotomy in Vivo
Thoracic DRGs Lumbar DRGs
Normal
Axotomized
Control culture
LIF
NGF
LIF ⫹ NGF
2.8 ⫾ 0.8 n ⫽ 428 (6) 3.0 ⫾ 0.9 n ⫽ 475 (4)
18.5 ⫾ 1.4 n ⫽ 350 (4) 13.1 ⫾ 1.0 n ⫽ 560 (8)
19.2 ⫾ 1.3 n ⫽ 415 (6) 16 ⫾ 1.0 n ⫽ 541 (4)
35.8 ⫾ 1.2 n ⫽ 465 (6) 38 ⫾ 2.0 n ⫽ 612 (4)
18.7 ⫾ 0.1 n ⫽ 448 (6) 16 ⫾ 1.0 n ⫽ 539 (4)
24.4 ⫾ 0.1 n ⫽ 451 (6) 22 ⫾ 2.0 n ⫽ 547 (4)
Note. The numbers of preparations are given in parenthesis. n, number of neurons counted.
EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
TABLE 3
DISCUSSION
Mean Lengths of Axons (in m ⫾ SEM) after 2 Days in Culture with LIF or NGF Controls
LIF
381
NGF
LIF ⫹ NGF
Axon 334 ⫾ 20 282 ⫾ 21 693 ⫾ 18* 660 ⫾ 68* lengths n ⫽ 85 (6) n ⫽ 85 (6) n ⫽ 127 (6) n ⫽ 83 (6) Axon 2.0 ⫾ 0.06 1.8 ⫾ 0.05 1.6 ⫾ 0.03* 1.8 ⫾ 0.04* diameters n ⫽ 78 (6) n ⫽ 96 (6) n ⫽ 247 (6) n ⫽ 224 (6) Note. The number of preparations is given in parentheses. n, total number of axons. (* P ⬍ 0.01 compared to control preparations.)
6% (n ⫽ 69; six preparations). Preparations cultured with NGF showed marked increases in numbers of outgrowing axons as described and of these 24 ⫾ 3% (n ⫽ 196; six preparations) were GAL positive (Fig. 3C). However, when LIF and NGF were added together, this increased the proportion of GAL-immunoreactive axons (Fig. 3D) to 36 ⫾ 4% (n ⫽ 153; six preparations), which was significantly higher than in the presence of NGF alone (P ⬍ 0.05) or in control cultures (P ⬍ 0.001). In view of previous suggestions that GAL might be involved in axonal regeneration (10, 47), we examined the explant cultures carefully to see if the GAL-positive axons were longer than unlabeled axons but saw no evidence for this. This result suggests that GAL expression may not directly affect axonal regeneration under the experimental conditions used in this study.
The Majority of Thoracic DRG Neurons Survived 3 Days in Explant Cultures Most studies of neuronal survival and loss in DRGs have been based on cell counts from histological sections and have used various mathematical correction factors to avoid double counting (30). The DRGs used in this study were small enough to count the total numbers of neurons directly using a confocal microscope. Using this technique, it was established that after 3 days in culture, preparations had approximately the same numbers of neurons as fresh DRGs although dead and living cells may not be differentiated. However, incubation with PI showed that only about 5% of neurons were dead or dying, confirming that the majority of the DRG neurons remained viable during the 3 days in culture. The ability of adult DRG neurons to survive in culture in defined medium without growth factors is consistent with previous studies, in which up to 80% neuronal survival rate in dissociated cell cultures has been found (17). The higher rates of neuronal survival observed in the present study may be due to the avoidance of the mechanical and enzymatic treatments used in preparation of dissociated cell cultures. In addition, the neurons retain their normal contact with ECM and satellite cells which may also provide trophic support.
FIG. 2. Axonal outgrowth from the cut end of peripheral nerves in collagen gels after 2 days in culture. Control (A) and preparations cultured with LIF (B) show sparse axonal growth. The lengths and numbers of outgrowing axons in preparations cultured with NGF (C) or NGF and LIF (D) are markedly increased. Arrows indicate some of the outgrowing axons, although owing to the three-dimensional nature of the gels not all axons are in focus.
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FIG. 3. Outgrowing axons in collagen gels after 2 days in culture labeled with antibodies to III-tubulin (A and C) and GAL (B and D). In a preparation cultured with NGF profuse axonal outgrowth is visualized with the antibody to III-tubulin (A) but only a few of these are immunoreactive for GAL (B). In preparations cultured with NGF and LIF, the proportion of axons which are immunoreactive for GAL is clearly increased (C and D). Straight arrows indicate axons which are GAL positive while some of the axons which are labeled only by the antibody to III-tubulin are indicated by curved arrows. Note that owing to the three-dimensional nature of the gels, many axons are out of focus. Bar, 100 m.
Galanin Expression It is well established that DRG neurons axotomized in vivo show increased GAL expression but the reasons for this are uncertain. This could be due to lack of target-derived factors such as NGF or lesion-induced factors such as LIF. In vivo experiments have provided results which suggest that both mechanisms may be involved since treatment with antiserum to NGF causes increased GAL expression in DRGs (31) and conversely, NGF infusion partially antagonizes the increased GAL expression seen after axotomy (44). LIF injection into the sciatic nerve of normal animals causes increased GAL expression in L4 and L5 DRGs (39) and the up-regulation of GAL expression usually seen following axotomy is reduced in LIF-deficient mice (33). In the present study, the number of GALexpressing neurons increased in axotomized DRGs in vivo and also in cultured control preparations. The proportions of GAL-positive neurons were similar in these two groups. However, addition of LIF to cultures caused a marked increase in the proportion of GALpositive neurons confirming in vivo observations (39) that this factor regulates GAL expression. Up-regulation of GAL in explant cultures without addition of any factors may be due to spontaneous production of LIF which occurs in cultured DRGs within 24 h (33). Consistent with this hypothesis, GAL up-regulation in cultures of dissociated DRG neurons of LIF-deficient mice (12) is less than in normal mice, but can be increased
by addition of LIF. Interestingly, in this study, addition of LIF did not increase the proportion of GALpositive DRG neurons in cultures from normal mice, which was much higher (⬎40%) than the cultures of control DRGs in our experiments (16%). It is possible that the lower incidence of spontaneous GAL expression observed in the cultured intact DRGs used in our study may provide a more favorable model for detection of changes induced by LIF than in cultures of dissociated neurons, with their high spontaneous levels of GAL expression. Addition of NGF by itself had no effect on GAL expression in the cell bodies of cultured DRG neurons. This indicates that the spontaneous expression of GAL is not due to lack of NGF. However in preparations cultured with both LIF and NGF, the proportion of GAL-positive neurons was lower than in preparations cultured with LIF alone. It could be argued that the lower proportion of GAL-positive neurons in DRGs cultured with LIF and NGF might be due to preferential survival of GAL-negative neurons. However, since neuronal loss in the thoracic DRGs was estimated to be less than 10% in these preparations it is unlikely that selective survival of GAL-negative neurons could explain this result, although this possibility is not excluded in the case of the lumbar DRGs. One explanation for the reduction in numbers of GAL-positive neurons in DRGs cultured with LIF and NGF compared to LIF alone is that NGF may prevent
EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
the expression of GAL in a proportion of neurons responding to LIF. Interestingly, LIF is retrogradely transported by mainly small-diameter DRG neurons, of which a subpopulation express trkA, the high-affinity receptor for NGF and would therefore be expected to respond to this factor (40). The results suggest that NGF and LIF may therefore have reciprocal effects on GAL expression in a subpopulation of neurons expressing receptors for both factors. The failure of NGF to suppress spontaneous GAL expression in culture might be explained by the presence of LIF-sensitive DRG neurons that are TrkA-negative and in fact it has been demonstrated that many DRG neurons which retrogradely transport LIF in vivo do not express trkA (40). LIF Has No Effect on the Extension of Outgrowing Axons but Increases Axonal Diameter In our experiments, LIF appeared to have no effect on the lengths and numbers of outgrowing axons in the collagen gels, in contrast to NGF which caused vigorous axonal outgrowth as previously reported (11, 42, 16). However, axons growing in the presence of NGF were thinner than in control preparations. This might be expected as the majority of NGF sensitive neurons are of small size and have unmyelinated axons. Interestingly, in preparations cultured with NGF and LIF together, axonal thickness was similar to that in control preparations, suggesting that in this situation, LIF could increase the diameter of the regenerating axons. LIF has been shown to enhance sciatic nerve regeneration in a silicone chamber model by increasing diameters of axons, numbers of myelinated fibers, and conduction velocity (37), although the mechanisms involved are uncertain. Although LIF did not affect the number and lengths of outgrowing axons, it increased the proportion which were GAL-positive when added to cultures (from 13 to 33%), which may be expected from the increase in proportion of GAL-positive neurons in the DRGs. In preparations cultured with NGF, the proportion of GAL-positive axons (24%) was higher than in control preparations (13%), although NGF did not increase the proportion of GAL-positive neurons in the cultured DRGs. This paradox may be explained by the fact that NGF increases the number of outgrowing trkA axons, many of which would be expected to be GAL positive (39). In preparations cultured with NGF, LIF also caused a further increase in the proportion of GALpositive axons but the magnitude of the increase (from 24 to 36%) appears to be less than that observed in the preparations cultured without NGF. This difference might be explained by failure of a proportion of the trkA-expressing axons to respond to LIF or by NGF antagonizing the effects of LIF. The latter explanation would be consistent with our observation that NGF
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appears to reduce the proportion of GAL-positive neurons in DRGs cultured with LIF. Interestingly in SCGs, it was recently demonstrated that LIF could not induce GAL without neutralization of NGF (32). The present study shows that this is not the case with DRGs since even in the presence of exogenous NGF, LIF was still able to significantly increase GAL expression. ACKNOWLEDGMENTS The digital camera used in this study was purchased out of funds provided by alumni, staff, and other members of the KCLA through the King’s College London Annual Fund. We thank Dr. S. Thompson for reading an earlier version of the manuscript.
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EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs suppression in adult sensory neurons. Neuroscience 15: 2081– 2096. 45. Wilde, G. J., L. E. Sundstrom, and F. Iannotti. 1994. Propidium iodide in vivo: An early marker of neuronal damage in rat hippocampus. Neurosci. Lett. 180: 223–226. 46. Wiesenfeld-Hallin, Z., X. J. Xu, M. J. Villar, and T. Ho¨kfelt. 1989. The effect of intrathecal galanin on the flexor reflex in rat: Increased depression after sciatic nerve section. Neurosci. Lett. 105: 149 –154.
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Effects of Leukemia Inhibitory Factor on Galanin Expression and on Axonal Growth in Adult Dorsal Root Ganglion Neurons in Vitro ¨ ztu¨rk and D. A. Tonge G. O Neuroscience Research Centre, GKT School of Biomedical Sciences, King’s College London, Hodgkin Building, Guy’s Hospital, London SE1 9RT, United Kingdom Received November 11, 1999; accepted February 22, 2001
Synthesis of leukemia inhibitory factor (LIF) is increased in lesioned peripheral nerves and it is thought that this may cause increased expression of galanin (GAL) in axotomized dorsal root ganglia (DRG) neurons and also to promote axonal regeneration. We therefore compared effects of LIF and nerve growth factor (NGF) on galanin expression and axonal growth using cultured intact DRGs of adult mice. In control lumbar DRGs cultured for 3 days, only 16% of neurons were immunoreactive for GAL, but this was increased to 38% in preparations cultured with LIF. NGF by itself had no effect on GAL expression, but the proportion of GAL-positive neurons in cultures incubated with LIF and NGF together (22%) was less than that observed in DRGs cultured with LIF alone. Similar results were obtained using thoracic DRGs. In collagen gels, NGF caused marked increases in the numbers and lengths of outgrowing axons as observed in previous studies. In contrast, LIF did not stimulate axonal outgrowth but increased the proportions of axons which were immunoreactive for GAL. The results indicate that expression of LIF in lesioned nerves may affect expression of neuropeptides such GAL rather than stimulating axonal regeneration. ©
2001 Academic Press
Key Words: LIF; galanin; NGF; axon; regeneration; DRG.
INTRODUCTION
It is well known that axotomy of neurons in the peripheral nervous system (PNS) causes striking changes in expression of various classes of protein, reflecting a shift in metabolism from production of substances for synaptic transmission to synthesis of proteins required for regeneration (1). In dorsal root ganglia (DRGs) synthesis of some neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin is reduced while that of others such as galanin (GAL) neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and cholecystoki0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
nin (CCK) is increased, causing important effects on synaptic transmission within the spinal cord (9). Some of these changes may have protective functions (9) while others, such as increased expression of CCK which inhibits opioid action (47), may contribute to chronic pain. In DRGs of adult animals the proportion of neurons which express GAL is low, but increases dramatically after axotomy (9, 3, 20, 39). In the spinal cord, GAL may have both excitatory and inhibitory actions on nociceptive reflexes (4, 46, 14, 13). However, it is also expressed by developing trigeminal and DRG neurons suggesting that it may also be involved in axonal growth (48). Consistent with this hypothesis, recent studies have shown that axonal regeneration after peripheral nerve injuries is impaired in GAL knock-out mice (10). The mechanisms responsible for the changes in expression of GAL and other neuropeptides after axotomy are uncertain, but one possibility is that they result from reduced availability of trophic factors which are normally derived from innervated tissues. There is good evidence for this in the case of nerve growth factor (NGF) whose sequestration by chronic infusion of chimeric trk–IgG fusion molecules reduces CGRP levels in sensory neurons in vivo (21). Conversely, its expression and that of SP are increased by NGF in cultured sensory neurons (18). Chronic infusion of NGF restores levels of CGRP and SP in DRGs of lesioned peripheral nerves in vivo (44) and also reduces the proportions of neurons expressing NPY, GAL, VIP and CCK. However, the failure of NGF infusion to completely suppress the increased expression of these four neuropeptides after axotomy suggests that this may also be regulated by other factors. One factor which has recently been shown to induce GAL expression in DRG neurons in vivo (39) is leukemia inhibitory factor (LIF). This cytokine has important effects on the phenotype and survival of a number of different types of neuron, including embryonic and neonatal sensory and motor neurons (15, 36). Levels of LIF in peripheral nerves under normal conditions are
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very low, but following axotomy its expression increases rapidly in lesioned nerves (5). Furthermore, LIF injected into peripheral nerves shows increased retrograde transport to sensory and motor neurons following peripheral injury (5), suggesting that this factor may have a role as a survival or growth factor in adult animals. LIF has been shown to increase expression of the neuropeptide VIP in superior sympathetic ganglia (SCGs) in culture (35) and conversely, transgenic LIF knock-out mice show lower levels of VIP, substance P and GAL expression after peripheral axotomy (28, 34). Subsequent studies have shown that LIF may also regulate GAL expression in DRGs since after axotomy, LIF-deficient transgenic mice had a substantially lower increase in GAL expression in DRGs compared to wild-type animals (3, 33). Recently, it was shown that intraneurally injected LIF causes up-regulation of GAL in corresponding DRGs (39). The targets of LIF in the DRGs are mostly those neurons that normally express TrkA and CGRP (40). Another factor that affects GAL expression in DRGs is NGF since its infusion partially antagonizes the increased GAL expression seen after axotomy (44). NGF also partially inhibits the increased galanin expression which occurs in SCGs following explantation or axotomy in vivo (31), which is consistent with the known interaction of NGF with LIF signaling (27). This raises the possibility that target-derived NGF normally inhibits GAL expression and that lack of this factor after axotomy may play a role in the production of this neuropeptide. Support for this hypothesis comes from another study, in which NGF antiserum injected into unoperated rats caused an increase in GAL mRNA and immunoreactivity in SCGs (31). Although several recent studies have shown that LIF affects expression of certain neuropeptides, relatively little is known about the effects of this factor on axonal regeneration. However, LIF promotes neurite extension and morphological maturation in cultured embryonic neuroblasts (22, 25). In mature DRG neurons, LIF promotes neurite extension in response to NGF (2). Furthermore, sprouting from postganglionic sympathetic efferents into DRGs after peripheral axotomy is induced by LIF (38). LIF has also been reported to enhance sciatic nerve regeneration in a silicone chamber model by increasing diameters of axons, numbers of myelinated fibers, and conduction velocity (37). The effects of growth factors and cytokines on neuronal phenotype and axonal growth have generally been studied using certain in vivo and in vitro experimental models although both types suffer some disadvantages. The in vivo models usually involve application of the factors to the cut end of a peripheral nerve or intrathecal infusion of these factors. However, the interpretation of results from such experiments may be complicated by the fact that the procedures may have indirect effects including inflammation. For example,
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although NGF infusions have effects on DRG neurons in vivo (21, 44) the widespread expression of receptors for neurotrophins on cells of the immune system, including mast cells (19) raises the possibility that factors released by these cells might also influence neuronal phenotype. Similar issues apply to the in vivo study of other growth factors. In view of these problems, in vitro studies offer certain advantages. Traditionally, dissociated neurons from sensory and autonomic ganglia of neonatal animals have been used for such studies but the enzymatic and mechanical procedures used to dissociate the neurons may cause changes in neuronal phenotype. Furthermore, the normal relationship between neurons, glial cells and extracellular matrix (ECM) is disrupted and this is likely to influence neuronal phenotype. For example, expression of neuropeptides by dissociated neurons in culture is usually very low unless growth factors such as NGF are added (18, 23). In contrast, neurons in vivo continue to express neuropeptides such as CGRP for several days after axotomy (44) although levels do eventually decline. An alternative method for investigating the regulation of neuropeptide expression was suggested by a recent study of regeneration of peripheral nerves of adult mice in vitro (43). In these experiments, peripheral nerves together with their attached DRGs were explanted into gels of Matrigel (the commercially available extracellular matrix extract of the Engelbreth– Holm–Swarm tumor cell line) without growth factors in serum-free medium. Axonal outgrowth from the tips of the cut nerves and dorsal roots began within 24 h and continued for at least a week. Conventional fluorescence microscopy showed that many axons had strong CGRP-like immunoreactivity (43), suggesting that such preparations could be used to study expression of neuropeptides and effects of growth factors. To determine the suitability of these preparations for such studies, we investigated the survival of neurons in the explanted DRG preparations and found that more than 90% of neurons remained viable over 3 days in culture. We therefore used these preparations to study effects of LIF and NGF on GAL expression and axonal regeneration in vitro. Our results suggest that these preparations could also be used to study the regulation of expression of other neuropeptides. METHODS
Adult female mice aged 6 –12 weeks (BK1 TO strain; bred at King’s College) were used in all experiments. Prior to removal of DRGs, animals were anesthetized by ether and an intraperitoneal injection of 0.25 ml of a mixture of 3% Hypnorm (Jansen) and 33% diazepam (Phoenix Pharmaceuticals) as described (7) and killed by cervical transection. In experiments to determine neuronal survival, thoracic DRGs (T10 –T12) from both
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sides of two mice were removed rapidly and the ganglia from one side fixed immediately in 4% paraformaldehyde in phosphate buffer, pH 7.0 (PB). The remaining DRGs were incubated free-floating in 0.5 ml of RPMI 1640 medium containing 100 units of penicillin, 100 g streptomycin, and 250 ng amphotericin B per milliliter (all from Sigma) at 36°C in a sealed box containing 95% O 2/5% CO 2 in a humidified incubator for 3 days prior to fixation. To determine the total number of neurons, the fixed DRGs were split into four pieces with a razor blade under the microscope. Then they were transferred onto coverslips and incubated overnight with the monoclonal antibody A60 (Chemicon) diluted 1/100 that recognizes a protein (NeuN) in the nuclei of most types of neurons in adult rodents (21, 41). The washes and application of secondary antibody [fluorescein (FITC)conjugated goat anti-mouse (Sigma)]. were as for preparations in collagen gels as described below. Preparations were mounted in Citifluor (Agar Scientific) and scanned using a Leica scanning confocal microscope. Optical sections were obtained at 7-m intervals and the images digitally stored. Commercial image editing software (Adobe Photoshop) was used to create cumulative templates from series of sections by marking labeled nuclei on each one. Double counting was avoided by marking on sections, only those nuclei which did not appear marked on the latest form of the template. The numbers of marks on the final cumulative templates were counted using an image analysis program (NIH Image). In a second experiment to determine the proportions of viable neurons, T10 –T12 DRGs were removed from six mice. Some of the DRGs were immediately fixed to use as controls while the remaining preparations were cultured free-floating for 3 days. After 2.5 days incubation, a solution of 75 M propidium iodide (PI) and 0.5 mg/ml 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide; thiozolyl blue (MTT) in RPMI was added to the cultures which were then returned to incubator for 12 h. The next day all the preparations were fixed by immersion in 4% paraformaldehyde in PB. MTT is a water-soluble tetrazolium salt that is converted to insoluble purple formazan crystals in the cells with active mitochondria (8) and in our experiments demonstrated live neurons very successfully. PI is a fluorescent stain for nucleic acids which can not cross intact cell membranes but stains the cytoplasm of dead/dying cells bright pink/red as nuclear material is dispersed into the cytoplasm as a result of nuclear disintegration at a very early stage of cell death (45). In order to determine the reliability of PI labeling and its staining pattern, some preparations were incubated with MTT and PI together overnight in a refrigerator for 12 h where the unbuffered culture medium became alkaline and would therefore be expected to increase the proportion of dead cells. In these preparations,
about 30% of neurons were diffusely stained with PI (and therefore presumably dead), while the rest of the cell profiles were laden with formazan crystals but lacked PI labeling of the cytoplasm. After the sections had been mounted in Citifluor (which is an aqueous mounting medium), PI gradually diffused across the tissues and eventually labeled all cell nuclei. However, the dead cells could be clearly distinguished by the intense PI labeling of both the nucleus and the cytoplasm, providing a convenient method for live/dead assessment of cells in these experiments since although the MTT method identified live neurons, the accumulation of formazan crystals often made it difficult to determine their numbers accurately. This finding indicated that dead/dying cells could be distinguished by PI labeling of the nucleus and cytoplasm and that it was therefore not necessary to use MTT to mark the live cells. To study regulation of GAL expression, experiments were carried out using thoracic and also L4 DRGs since a substantial number of fibers from this DRG run through the sciatic nerve (6), so that it was possible to compare effects of axotomy in vivo with those of LIF and NGF in vitro. In these experiments, thoracic and L4 DRGs were removed from mice and the spinal roots and peripheral nerves cut off. The DRGs were then cultured free-floating for 2 days in RPMI medium containing 30 ng/ml LIF (Chemicon) or 50 ng/ml 2.5S NGF (Alamone Labs or Promega) or in some cases both factors. Control preparations were cultured for 2 days without growth factors, after which all preparations were fixed in 4% paraformaldehyde in PB. In some experiments, mice were anesthetized with Hypnorm and diazepam as described above and the intercostal nerves (T11 and T12) or sciatic nerve on one side cut 2 days prior to removal of the corresponding thoracic or L4 DRGs, which were immediately fixed in paraformaldehyde. After fixation L4 DRG preparations were covered with OCT compound (BDH), frozen by immersing into liquid nitrogen, and kept at ⫺20°C (or ⫺70°C for longer periods of storage) until cutting. Sections of 10 m thickness were cut using a cryostat and mounted on coverslips which had been treated with aminopropyltriethoxysilane (Sigma) as described (29). In experiments to determine effects of LIF and NGF on axonal growth and phenotype, thoracic DRGs with short lengths of intercostal nerves attached (about 3 mm) were quickly removed from the mice by dissection and placed on 13-mm glass coverslips in multiwells in shallow gels of type 1 collagen (26). The preparations were covered with 0.5 ml of RPMI medium and subsequently maintained at 36°C in a sealed box containing 95% O 2/5% CO 2 in a humidified incubator 2 days, prior to fixation with 4% paraformaldehyde in PB. LIF and 2.5S NGF separately and in combination (30 and 50 ng/ml, respectively) were added to some of the preparations at the start of the incubation period.
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TABLE 1
Histological Methods Frozen sections were blocked with 3% bovine serum albumin (BSA) ⫹ 0.1% Triton X-100 in phosphatebuffered saline (PBS) for 1 h. The primary antibody to GAL, raised in rabbit (Affinity) at a dilution of 1/100, was added to the sections for 2 h, followed by 3 ⫻ 10-min washes in PBS. The preparations were then incubated with FITC-conjugated goat anti-rabbit (Jackson) for an additional 2 h with three subsequent washes in PBS, before mounting in Citifluor. In order to label the explant cultures, the fixed preparations were first washed with several changes of PBS and then blocked with 3% BSA and 0.1% Triton X-100 in PBS for 1 h. Primary antibodies to GAL and a mouse monoclonal antibody (SDL.3D10) to the panaxonal marker III-tubulin (Sigma) were diluted 1/50 in PBS containing 3% BSA and left on the fixed preparations overnight. The next day preparations were given five washes with PBS, each wash lasting about 1 h, and secondary antibodies (diluted 1/100) to rabbit and mouse IgGs, conjugated with FITC and tetramethylrhodamine isothiocyanate (Jackson and Sigma, respectively), were added overnight. During the third day they were washed again in the same way and mounted in Citifluor. The preparations which had been labeled using the antibodies (sections and whole mounts) were viewed using an epifluorescence microscope equipped with phase contrast illumination and images captured directly off the microscope using a Hamamatsu C5810 digital camera system Stereography To determine the lengths of outgrowing axons, preparations which had already been fixed and stained using fluorescent antibodies were incubated overnight with biotinylated goat anti-mouse (1/100; Sigma). After a further series of washes the following day, the preparations were incubated overnight with peroxidaseconjugated avidin (1/100; Sigma). The following day, after a final series of washes with Tris buffer (pH 7.6), preparations were incubated in Tris buffer containing 0.6 mg diaminobenzidine and 15 l of 1% CoCl 2 per milliliter for 10 min prior to addition of 10 g/ml of 3% H 2O 2 to visualize peroxidase activity. The stained preparations were mounted in glycergel (Dako) and viewed under dark-ground illumination using a microscope with an image projection tube. Camera lucida drawings were made of the trajectories of the longest axons growing out of the cut ends of the peripheral nerves (usually 10 –20 per preparation) and their lengths were determined using the NIH Image software. To determine the thickness of axons growing in collagen, images were captured directly off the microscope using a Hamamatsu C5810 digital camera and analyzed using Scion Image PC.
Numbers of Neurons in Matched Pairs of Freshly Fixed (Control) and 3-Day Cultured Thoracic DRGs from Two Mice DRGs
Control
Cultured
T10 T10 T11 T11 T12 T12
1832 1901 2115 2039 2642 2416
1748 (0.95%) 1872 (0.98%) 2047 (0.97%) 1989 (0.98%) 2518 (0.95%) 2340 (0.97%)
Note. The ratios of numbers of neurons in the cultured DRGs compared with the numbers in the fresh DRGs are given in parentheses.
Statistics Results are expressed as means ⫾ SEM. The differences between means were evaluated by Student’s t test or 2 test where appropriate and considered significant at P ⬍ 0.05. RESULTS
Neuronal Survival in Explant DRG Cultures The antibody to NeuN clearly labeled neuronal nuclei as described in other studies (45). The numbers of identifiable neurons in optical sections of freshly dissected and cultured thoracic DRGs are summarized in Table 1. These results show that the numbers of neurons after 3 days in culture were 95–98% (mean 97 ⫹ 0.49%; n ⫽ 6) of the numbers in freshly fixed ganglia indicating neuronal loss of only 2–5%. The differences between the numbers of neurons in cultured and fresh preparations were not statistically significant on a 2 test. However, it is possible that some of these neurons could be dying and occasionally some distorted nuclear profiles were observed which may be an indication of ongoing disintegration. To determine the proportion of viable cells, the preparations were therefore incubated with PI. The percentage of dead neurons identified on 20 frozen sections of 12 DRGs by this method was 6.2 ⫹ 0.7% (n ⫽ 1203) indicating that some of the neurons counted in the optical sections were probably dead or dying. However, even combining the percentages of identified dead cells with the estimated percentages of lost cells, the results suggest that more than 90% of neurons remain viable after 3 days in culture. Expression of Galanin in Dorsal Root Ganglia In sections of normal DRGs, a few neurons were labeled by the antibody to GAL (Fig. 1A), but the proportion was very low as has been found in other studies (9). Two days after cutting the thoracic or sciatic nerves, increased numbers of GAL-positive neurons were observed in the DRGs (Fig. 1B) and their propor-
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FIG. 1. GAL expression in sections of DRGs: normal (A), 2 days axotomized (B), 2 days cultured without growth factors (C), and 2 days cultured with LIF (D). Arrows indicate GAL-immunoreactive neurons.
tions were significantly higher (P ⬍ 0.001) than in normal DRGs. In DRGs cultured without growth factors, many of the neurons were clearly labeled (Fig. 1C) and their proportions were similar to that in DRGs after axotomy in vivo. However, in the preparations cultured with LIF, the numbers of GAL-positive neurons were clearly greater (Fig. 1D) and their proportions were significantly increased (P ⬍ 0.001) compared to preparations cultured without LIF. NGF by itself did not show any obvious effect on GAL expression in culture, since the proportions of GAL-positive neurons were similar to that of preparations cultured without growth factors. However, the proportions of GAL-positive neurons in DRGs cultured with NGF ⫹ LIF were lower than in preparations cultured with LIF alone (P ⬍ 0.001) (Table 2).
Table 3. In control preparations after 2 days in culture, a few axons had grown into the gels spontaneously (Fig. 2A) and similar axonal growth was also observed in preparations cultured with LIF (Fig. 2B). However, preparations cultured with NGF showed a marked enhancement in the numbers and lengths of outgrowing axons (Fig. 2C) as observed in previous studies (16, 42), but these were thinner than in controls and those in preparations cultured with LIF. Axonal outgrowth in preparations cultured with NGF and LIF together was similar (Fig. 2D) but interestingly, the axons were thicker than with NGF alone. There were no obvious differences in the lengths and numbers of axons between NGF and NGF ⫹ LIF-treated preparations.
Effects of LIF and NGF on Axonal Growth in Collagen Gels
In control preparations after 2 days in culture, the proportion of axons which were immunoreactive for GAL (Fig. 3A) was 13 ⫾ 3% (n ⫽ 64; six preparations). In the presence of LIF the proportion of labeled axons (Fig. 3B) had increased significantly (P ⬍ 0.05) to 33 ⫾
The mean lengths and diameters of axons growing from thoracic DRGs in collagen gels are summarized in
GAL Expression in Outgrowing Axons
TABLE 2 The Mean Percentages (⫾SEM) of GAL-Positive Neurons in Thoracic and Lumbar DRGs after 2 Days in Culture or Axotomy in Vivo
Thoracic DRGs Lumbar DRGs
Normal
Axotomized
Control culture
LIF
NGF
LIF ⫹ NGF
2.8 ⫾ 0.8 n ⫽ 428 (6) 3.0 ⫾ 0.9 n ⫽ 475 (4)
18.5 ⫾ 1.4 n ⫽ 350 (4) 13.1 ⫾ 1.0 n ⫽ 560 (8)
19.2 ⫾ 1.3 n ⫽ 415 (6) 16 ⫾ 1.0 n ⫽ 541 (4)
35.8 ⫾ 1.2 n ⫽ 465 (6) 38 ⫾ 2.0 n ⫽ 612 (4)
18.7 ⫾ 0.1 n ⫽ 448 (6) 16 ⫾ 1.0 n ⫽ 539 (4)
24.4 ⫾ 0.1 n ⫽ 451 (6) 22 ⫾ 2.0 n ⫽ 547 (4)
Note. The numbers of preparations are given in parenthesis. n, number of neurons counted.
EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
TABLE 3
DISCUSSION
Mean Lengths of Axons (in m ⫾ SEM) after 2 Days in Culture with LIF or NGF Controls
LIF
381
NGF
LIF ⫹ NGF
Axon 334 ⫾ 20 282 ⫾ 21 693 ⫾ 18* 660 ⫾ 68* lengths n ⫽ 85 (6) n ⫽ 85 (6) n ⫽ 127 (6) n ⫽ 83 (6) Axon 2.0 ⫾ 0.06 1.8 ⫾ 0.05 1.6 ⫾ 0.03* 1.8 ⫾ 0.04* diameters n ⫽ 78 (6) n ⫽ 96 (6) n ⫽ 247 (6) n ⫽ 224 (6) Note. The number of preparations is given in parentheses. n, total number of axons. (* P ⬍ 0.01 compared to control preparations.)
6% (n ⫽ 69; six preparations). Preparations cultured with NGF showed marked increases in numbers of outgrowing axons as described and of these 24 ⫾ 3% (n ⫽ 196; six preparations) were GAL positive (Fig. 3C). However, when LIF and NGF were added together, this increased the proportion of GAL-immunoreactive axons (Fig. 3D) to 36 ⫾ 4% (n ⫽ 153; six preparations), which was significantly higher than in the presence of NGF alone (P ⬍ 0.05) or in control cultures (P ⬍ 0.001). In view of previous suggestions that GAL might be involved in axonal regeneration (10, 47), we examined the explant cultures carefully to see if the GAL-positive axons were longer than unlabeled axons but saw no evidence for this. This result suggests that GAL expression may not directly affect axonal regeneration under the experimental conditions used in this study.
The Majority of Thoracic DRG Neurons Survived 3 Days in Explant Cultures Most studies of neuronal survival and loss in DRGs have been based on cell counts from histological sections and have used various mathematical correction factors to avoid double counting (30). The DRGs used in this study were small enough to count the total numbers of neurons directly using a confocal microscope. Using this technique, it was established that after 3 days in culture, preparations had approximately the same numbers of neurons as fresh DRGs although dead and living cells may not be differentiated. However, incubation with PI showed that only about 5% of neurons were dead or dying, confirming that the majority of the DRG neurons remained viable during the 3 days in culture. The ability of adult DRG neurons to survive in culture in defined medium without growth factors is consistent with previous studies, in which up to 80% neuronal survival rate in dissociated cell cultures has been found (17). The higher rates of neuronal survival observed in the present study may be due to the avoidance of the mechanical and enzymatic treatments used in preparation of dissociated cell cultures. In addition, the neurons retain their normal contact with ECM and satellite cells which may also provide trophic support.
FIG. 2. Axonal outgrowth from the cut end of peripheral nerves in collagen gels after 2 days in culture. Control (A) and preparations cultured with LIF (B) show sparse axonal growth. The lengths and numbers of outgrowing axons in preparations cultured with NGF (C) or NGF and LIF (D) are markedly increased. Arrows indicate some of the outgrowing axons, although owing to the three-dimensional nature of the gels not all axons are in focus.
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FIG. 3. Outgrowing axons in collagen gels after 2 days in culture labeled with antibodies to III-tubulin (A and C) and GAL (B and D). In a preparation cultured with NGF profuse axonal outgrowth is visualized with the antibody to III-tubulin (A) but only a few of these are immunoreactive for GAL (B). In preparations cultured with NGF and LIF, the proportion of axons which are immunoreactive for GAL is clearly increased (C and D). Straight arrows indicate axons which are GAL positive while some of the axons which are labeled only by the antibody to III-tubulin are indicated by curved arrows. Note that owing to the three-dimensional nature of the gels, many axons are out of focus. Bar, 100 m.
Galanin Expression It is well established that DRG neurons axotomized in vivo show increased GAL expression but the reasons for this are uncertain. This could be due to lack of target-derived factors such as NGF or lesion-induced factors such as LIF. In vivo experiments have provided results which suggest that both mechanisms may be involved since treatment with antiserum to NGF causes increased GAL expression in DRGs (31) and conversely, NGF infusion partially antagonizes the increased GAL expression seen after axotomy (44). LIF injection into the sciatic nerve of normal animals causes increased GAL expression in L4 and L5 DRGs (39) and the up-regulation of GAL expression usually seen following axotomy is reduced in LIF-deficient mice (33). In the present study, the number of GALexpressing neurons increased in axotomized DRGs in vivo and also in cultured control preparations. The proportions of GAL-positive neurons were similar in these two groups. However, addition of LIF to cultures caused a marked increase in the proportion of GALpositive neurons confirming in vivo observations (39) that this factor regulates GAL expression. Up-regulation of GAL in explant cultures without addition of any factors may be due to spontaneous production of LIF which occurs in cultured DRGs within 24 h (33). Consistent with this hypothesis, GAL up-regulation in cultures of dissociated DRG neurons of LIF-deficient mice (12) is less than in normal mice, but can be increased
by addition of LIF. Interestingly, in this study, addition of LIF did not increase the proportion of GALpositive DRG neurons in cultures from normal mice, which was much higher (⬎40%) than the cultures of control DRGs in our experiments (16%). It is possible that the lower incidence of spontaneous GAL expression observed in the cultured intact DRGs used in our study may provide a more favorable model for detection of changes induced by LIF than in cultures of dissociated neurons, with their high spontaneous levels of GAL expression. Addition of NGF by itself had no effect on GAL expression in the cell bodies of cultured DRG neurons. This indicates that the spontaneous expression of GAL is not due to lack of NGF. However in preparations cultured with both LIF and NGF, the proportion of GAL-positive neurons was lower than in preparations cultured with LIF alone. It could be argued that the lower proportion of GAL-positive neurons in DRGs cultured with LIF and NGF might be due to preferential survival of GAL-negative neurons. However, since neuronal loss in the thoracic DRGs was estimated to be less than 10% in these preparations it is unlikely that selective survival of GAL-negative neurons could explain this result, although this possibility is not excluded in the case of the lumbar DRGs. One explanation for the reduction in numbers of GAL-positive neurons in DRGs cultured with LIF and NGF compared to LIF alone is that NGF may prevent
EFFECTS OF LIF ON GALANIN EXPRESSION IN DRGs
the expression of GAL in a proportion of neurons responding to LIF. Interestingly, LIF is retrogradely transported by mainly small-diameter DRG neurons, of which a subpopulation express trkA, the high-affinity receptor for NGF and would therefore be expected to respond to this factor (40). The results suggest that NGF and LIF may therefore have reciprocal effects on GAL expression in a subpopulation of neurons expressing receptors for both factors. The failure of NGF to suppress spontaneous GAL expression in culture might be explained by the presence of LIF-sensitive DRG neurons that are TrkA-negative and in fact it has been demonstrated that many DRG neurons which retrogradely transport LIF in vivo do not express trkA (40). LIF Has No Effect on the Extension of Outgrowing Axons but Increases Axonal Diameter In our experiments, LIF appeared to have no effect on the lengths and numbers of outgrowing axons in the collagen gels, in contrast to NGF which caused vigorous axonal outgrowth as previously reported (11, 42, 16). However, axons growing in the presence of NGF were thinner than in control preparations. This might be expected as the majority of NGF sensitive neurons are of small size and have unmyelinated axons. Interestingly, in preparations cultured with NGF and LIF together, axonal thickness was similar to that in control preparations, suggesting that in this situation, LIF could increase the diameter of the regenerating axons. LIF has been shown to enhance sciatic nerve regeneration in a silicone chamber model by increasing diameters of axons, numbers of myelinated fibers, and conduction velocity (37), although the mechanisms involved are uncertain. Although LIF did not affect the number and lengths of outgrowing axons, it increased the proportion which were GAL-positive when added to cultures (from 13 to 33%), which may be expected from the increase in proportion of GAL-positive neurons in the DRGs. In preparations cultured with NGF, the proportion of GAL-positive axons (24%) was higher than in control preparations (13%), although NGF did not increase the proportion of GAL-positive neurons in the cultured DRGs. This paradox may be explained by the fact that NGF increases the number of outgrowing trkA axons, many of which would be expected to be GAL positive (39). In preparations cultured with NGF, LIF also caused a further increase in the proportion of GALpositive axons but the magnitude of the increase (from 24 to 36%) appears to be less than that observed in the preparations cultured without NGF. This difference might be explained by failure of a proportion of the trkA-expressing axons to respond to LIF or by NGF antagonizing the effects of LIF. The latter explanation would be consistent with our observation that NGF
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appears to reduce the proportion of GAL-positive neurons in DRGs cultured with LIF. Interestingly in SCGs, it was recently demonstrated that LIF could not induce GAL without neutralization of NGF (32). The present study shows that this is not the case with DRGs since even in the presence of exogenous NGF, LIF was still able to significantly increase GAL expression. ACKNOWLEDGMENTS The digital camera used in this study was purchased out of funds provided by alumni, staff, and other members of the KCLA through the King’s College London Annual Fund. We thank Dr. S. Thompson for reading an earlier version of the manuscript.
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