Expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 messenger RNAs in spinal ganglia and sciatic nerve: regulation after peripheral nerve lesion

doi:10.1016/S0306-4522(96)00355-7

Neuroscience

Volume 76, Issue 1, 5 December 1996, Pages 123-135

https://doi.org/10.1016/S0306-4522(96)00355-7 Get rights and content

Abstract

In order to determine functional roles of basic fibroblast growth factor (FGF-2) in the peripheral nervous system we have analysed the expression of FGF-2 and FGF receptor 1 (FGFR1) in spinal ganglia and the sciatic nerve under normal conditions and after nerve crush using RNAse protection assay and in situ hybridization. In intact spinal ganglia, both FGF-2 and FGFR1 messenger RNAs are expressed, albeit at different levels. In situ hybridization identifies satellite cells as the source of FGF-2 and sensory neurons as the source of FGFR1 suggesting a paracrine mode of action of FGF-2 on sensory neurons. One day after crush lesion FGF-2 is significantly up-regulated in sensory ganglia L4–L6. Highest levels are found at day 7; control levels are approached after 28 days. FGFR1 messenger RNA, which is strongly expressed in intact spinal ganglia, displays no significant change after lesion. In the intact sciatic nerve, FGFR1 messenger RNA is detected at higher levels than FGF-2 messenger RNA. After injury, both transcripts display a time-dependent up-regulation in both the proximal and distal nerve stump. Schwann cells, as a putative source of the sciatic nerve-derived FGF-2, express both FGF-2 and FGFR1 messenger RNAs in vitro. The FGFR1 transcript level is increased in the presence of forskolin. FGF-2 does not affect expression of FGFR1 messenger RNA but stimulates its own expression.

These results show that during peripheral nerve regeneration FGF-2 is up-regulated in both the crushed nerve and the respective spinal ganglia suggesting a possible physiological function of FGF-2 during the regeneration process.

Section snippets

Animal surgery

Experiments were performed on adult female Hanover–Wistar rats (Charles River Wiga, Sulzfeld, Germany) weighing approximately 180 g. For surgery, animals were anaesthetized intraperitoneally using sodium pentobarbital (50 mg/kg). The left sciatic nerve was exposed and crushed with a fine forceps at the mid-thigh level. After survival intervals, from one to 28 days (d), animals were killed and L4–L6 DRG ipsilateral and contralateral to the lesion were collected. Sciatic nerve segments extending 8

Expression of FGF-2 and FGF receptor 1 in intact spinal ganglia and sciatic nerve

FGF-2 and FGFR1 transcripts were constitutively expressed in spinal ganglia and sciatic nerve.

In spinal ganglia FGF-2 mRNA was localized to satellite cells as shown by in situ hybridization. Almost all of the satellite cells were labelled (Figs 1, 2). FGFR1 displayed a neuronal localization in DRG, most of the neurons showed the signal albeit at different intensities (Fig. 1Fig. 2). Using ribonuclease protection assay expression of both transcripts FGF-2 and FGFR1 was also found in intact DRG (

Discussion

In the present study we demonstrate for the first time, that FGF-2 and FGFR1 mRNAs are constitutively expressed in spinal ganglia and sciatic nerve of the adult rat. Furthermore, we show that after crush lesion of the sciatic nerve expression of FGF-2 is increased in both the proximal and distal stump and in the DRG whereas the level of FGFR1 mRNA is exclusively increased in the lesioned sciatic nerve. This different spatial regulation of FGF-2 and FGFR1 argues for a diverse, region-specific

Conclusion

The presence and up-regulation after injury of FGF-2 and FGFR1 mRNAs in sensory ganglia and in the peripheral nerve suggests that FGF-2 is involved in regeneration phenomena of the peripheral nervous system. In DRG FGF-2 could mediate survival promoting activity or other, non-trophic functions. At the lesion side FGF-2 could help to control the onset and rate of myelination in regenerating nerves.

Acknowledgements

We thank Dr A. Baird for kindly providing the FGF-2 cDNA and Dr J. Milbrandt for the FGFR-1 cDNA. We also thank C. Zeschnigk, H. Arnold, and C. Micucci for excellent technical assistance and help in preparing the figures and Dr S. Wewetzer for reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft (SFB 505/TPA2).

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The transmission of signals to the cell body from injured axons induces significant alterations in primary sensory neurons located in the ganglion tissue, the site of the perikaryon of the affected nerve fibers. Disruption of the continuity between the proximal and distal ends leads to substantial adaptability in ganglion cells and induces macrophage-like activity in the satellite cells. Research findings have demonstrated the plasticity of satellite cells following injury. Satellite cells work together with sensory neurons to extend the interconnected surface area in order to permit effective communication. The dynamic cellular environment within the ganglion undergoes several alterations that ultimately lead to differentiation, transformation, or cell death. In addition to necrotic and apoptotic cell morphology, phenomena such as histomorphometric alterations, including the development of autophagic vacuoles, chromatolysis, cytosolic degeneration, and other changes, are frequently observed in cells following injury. The use of electron microscopic and stereological techniques for assessing ganglia and nerve fibers is considered a gold standard in terms of investigating neuropathic pain models, regenerative therapies, some treatment methods, and quantifying the outcomes of pharmacological and bioengineering interventions. Stereological techniques provide observer-independent and reliable results, which are particularly useful in the quantitative assessment of three-dimensional structures from two-dimensional images. Employing the fractionator and disector techniques within stereological methodologies yields unbiased data when assessing parameters such as number. The fundamental concept underlying these methodologies involves ensuring that each part of the structure under evaluation has an equal opportunity of being sampled. This review describes the stereological and histomorphometric evaluation of dorsal root ganglion neurons and satellite cells following nerve injury models.
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However, Cuevas et al. (1995) demonstrated that treatment with FGF-1 increases the survival of axotomized neonatal rat facial motoneurons from 18% to 80%. FGF2 plays a role in peripheral nerve regeneration: FGF-2 promotes the early regenerative response after peripheral nerve transection and surgical repair by stimulating Schwann cell proliferation, which, in turn, facilitates axon regrowth from the proximal to the distal stump as well as modulating both neurite elongation and axon branching (Grothe et al., 1997; Jungnickel et al., 2006). FGF2 also stimulates axon regrowth in vivo and contributes to the enlargement of axon calibre during regeneration (Aebischer et al., 1989; Laquerriere et al., 1994; Piehl et al., 1998).
It is well-known that, after nerve transection and surgical repair, misdirected regrowth of regenerating motor axons may occur in three ways. The first way is that the axons enter into endoneurial tubes that they did not previously occupy, regenerate through incorrect fascicles and reinnervate muscles that they did not formerly supply. Consequently the activation of these muscles results in inappropriate movements. The second way is that, in contrast with the precise target-directed pathfinding by elongating motor nerves during embryonic development, several axons rather than a single axon grow out from each transected nerve fiber. The third way of misdirection occurs by the intramuscular terminal branching (sprouting) of each regenerating axon to culminate in some polyinnervation of neuromuscular junctions, i.e. reinnervation of junctions by more than a single axon. Presently, “fascicular” or “topographic specificity” cannot be achieved and hence target-directed nerve regeneration is, as yet, unattainable. Nonetheless, motor and sensory reinnervation of appropriate endoneurial tubes does occur and can be promoted by brief nerve electrical stimulation.
This review considers the expression of neurotrophic factors in the neuromuscular system and how this expression can promote functional recovery, with emphasis on the whisking of vibrissae on the rat face in relationship to the expression of the factors. Evidence is reviewed for a role of neurotrophic factors as short-range diffusible sprouting stimuli in promoting complete functional recovery of vibrissal whisking in blind Sprague Dawley (SD)/RCS rats but not in SD rats with normal vision, after facial nerve transection and surgical repair. Briefly, a complicated time course of growth factor expression in the nerves and denervated muscles include (1) an early increase in FGF2 and IGF2, (2) reduced NGF between 2 and 14 days after nerve transection and surgical repair, (3) a late rise in BDNF and (4) reduced IGF1 protein in the denervated muscles at 28 days. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of nerve injury-associated neurotrophic factors and cytokines at the neuromuscular junctions of denervated muscles. In particular, the increase of FGF2 and concomittant decrease of NGF during the first week after facial nerve-nerve anastomosis in SD/RCS blind rats may prevent intramuscular axon sprouting and, in turn, reduce poly-innervation of the neuromuscular junction.
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After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates.
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After transection and repair, the animals that received treatment with NGF showed recovery in both motor and sensory nerve functions [48,20]. During peripheral nerve regeneration, FGF-2 is up-regulated in both the crushed nerve and the respective spinal ganglia, suggesting a possible physiological function of FGF-2 during the regeneration process [11]. Treatment with acupuncture has been seen to promote effects on the expression of GDNF and FGF-2 in the left sixth lumbar DRG following removal of adjacent dorsal root ganglia [49].
Neurotrophic factors and peripheral nerves are known to be good substrates for bridging CNS trauma. The involvement of fibroblast growth factor-2 (FGF-2) activation in the dorsal root ganglion (DRG) was examined following spinal cord injury in the rat. We evaluated whether FGF-2 increases the ability of a sciatic nerve graft to enhance neuronal plasticity, in a gap promoted by complete transection of the spinal cord. The rats were subjected to a 4 mm-long gap at low thoracic level and were repaired with saline (Saline or control group, n = 10), or fragment of the sciatic nerve (Nerve group, n = 10), or fragment of the sciatic nerve to which FGF-2 (Nerve + FGF-2 group, n = 10) had been added immediately after lesion. The effects of the FGF-2 and fragment of the sciatic nerve grafts on neuronal plasticity were investigated using choline acetyl transferase (ChAT)-immunoreactivity of neurons in the dorsal root ganglion after 8 weeks. Preservation of the area and diameter of neuronal cell bodies in dorsal root ganglion (DRG) was seen in animals treated with the sciatic nerve, an effect enhanced by the addition of FGF-2. Thus, the addition of exogenous FGF-2 to a sciatic nerve fragment grafted in a gap of the rat spinal cord submitted to complete transection was able to improve neuroprotection in the DRG. The results emphasized that the manipulation of the microenvironment in the wound might amplify the regenerative capacity of peripheral neurons.
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Heparan sulfate proteoglycans (HSPGs) have important functions in development of the central nervous system; however, their functions in nerve injury are not yet fully understood. We previously reported the expression of syndecan-1, a type of HSPG, in cranial motor neurons after nerve injury, suggesting the importance of syndecan-1 in the pathology of motor nerve injury. In this study, we examined the expression of syndecan-1, a type of HSPG, in primary sensory neurons after nerve injury in mice. Sciatic nerve axotomy strongly induced the expression of syndecan-1 in a subpopulation of injured dorsal root ganglion (DRG) neurons, which were small in size and had CGRP- or isolectin B4-positive fibers. Syndecan-1 was also distributed in the dorsal horn of the spinal cord ipsilateral to the axotomy, and located on the membrane of axons in lamina II of the dorsal horn. Not only sciatic nerve axotomy, infraorbital nerve axotomy also induced the expression of syndecan-1 in trigeminal ganglion neurons. Moreover, syndecan-1 knockdown in cultured DRG neurons induced a shorter neurite extension. These results suggest that syndecan-1 expression in injured primary sensory neurons may have functional roles in nerve regeneration and synaptic plasticity, resulting in the development of neuropathic pain.
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Injury to a peripheral nerve induces a complex cellular and molecular response required for successful axon regeneration. Proliferating Schwann cells organize into chains of cells bridging the lesion site, which is invaded by macrophages. Approximately half of the injured neuron population sends out axons that enter the glial guidance channels in response to secreted neurotrophic factors and neuropoietic cytokines. These lesion-associated polypeptides create an environment that is highly supportive for axon regrowth, particularly after acute injury, and ensure that the vast majority of regenerating axons are directed toward the distal nerve stump. Unfortunately, most neurotrophic factors and neuropoietic cytokines are also strong stimulators of axonal sprouting. Although some of the axonal branches will withdraw at later stages, the sprouting effect contributes to the misdirection of reinnervation that results in the lack of functional recovery observed in many patients with peripheral nerve injuries. Here, we critically review the role of neuronal growth factors and cytokines during axon regeneration in the peripheral nervous system. Their differential effects on axon elongation and sprouting were elucidated in various studies on intraneuronal signaling mechanisms following nerve lesion. The present data define a goal for future therapeutic strategies, namely, to selectively stimulate a Ras/Raf/ERK-mediated axon elongation program over an intrinsic PI3K-dependent axonal sprouting program in lesioned motor and sensory neurons. Instead of modulating growth factor or cytokine levels at the lesion site, targeting specific intraneuronal molecules, such as the negative feedback inhibitors of ERK signaling, has been shown to promote long-distance regeneration while avoiding sprouting of regenerating axons until they have reached their target areas.

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Expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 messenger RNAs in spinal ganglia and sciatic nerve: regulation after peripheral nerve lesion

Abstract

Section snippets

Animal surgery

Expression of FGF-2 and FGF receptor 1 in intact spinal ganglia and sciatic nerve

Discussion

Conclusion

Acknowledgements

Curr. Opin. Neurobiol.

Biochem. biophys. Res. Commun.

Analyt. Biochem.

Expl Cell Res.

Curr. Opin. Neurobiol.

Analyt. Biochem.

Cell

Neuron

Mech. Devl.

Biochim. biophys. Acta

Cell

Molec. Brain Res.

Cell

Neurosci. Lett.

Curr. Biol.

Curr. Opin. Neurobiol.

J. Immunol. Meth.

Tissue distribution, developmental expression and response to injury of rat CDF/LIF and its receptor

Soc. Neurosci. Abstr.

Interleukin-6 production by Schwann cells and induction in sciatic nerve injury

J. Neurochem.

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells

J. Cell Biol.

FGF-2: apical ectodermal ridge growth signal for chick limb development

Science