Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology

doi:10.1016/S0306-4522(98)00405-9

Neuroscience

Volume 89, Issue 4, April 1999, Pages 1123-1134

https://doi.org/10.1016/S0306-4522(98)00405-9 Get rights and content

Abstract

After transection of the sciatic nerve there is a dramatic increase in both galanin/galanin message-associated peptide-like immunoreactivities and preprogalanin messenger RNA levels in rat and mouse lumbar 4 and 5 dorsal root ganglion neurons. There is strong evidence that after nerve injury leukemia inhibitor factor is a key molecule in the control of peptide expression both in sympathetic neurons and in dorsal root ganglion neurons, although the cells of origin of endogenous leukemia inhibitory factor remain to be established. We have therefore studied the effect of leukemia inhibitory factor on galanin expression in 72 h cultured dorsal root ganglion neurons from normal mice, leukemia inhibitory factor-deficient and heterozygous mice with immunohistochemistry and in situ hybridization. In cultures of leukemia inhibitory factor-deficient (−/−) mice only 13% of the dorsal root ganglion neurons expressed galanin message-associated peptide and in cultures from heterozygous (+/−) and wild-type (+/+) mice the corresponding figures were, respectively, 24 and 40%. After addition of leukemia inhibitory factor (10 or 50 ng/ml) to the culture medium, the number of neurons expressing galanin message-associated peptide was increased (up to 41%) in cultures from (−/−) animals after the high concentration and reached similar values in cultures from heterozygous animals incubated with the low concentration. These findings were supported by parallel analysis of prepro-galanin messenger RNA levels, where similar transcript levels and effects in the various cultures were observed in the non-radioactive in situ hybridization experiments.

These results support the hypothesis that leukemia inhibitory factor is an important regulator of galanin/galanin message-associated peptide expression following axotomy, and may therefore be involved in the defence mechanisms against neuropathic pain at the level of dorsal root ganglion neurons.

Section snippets

Animals

LIF-deficient (−/−), heterozygous (+/−) and wild-type (+/+) mice were bred (see [11]), and genetic typing was performed on four-week-old animals. Approximately 5–10 mm of the mouse tail was cut off and placed in an Eppendorf tube containing 700 μl lysis buffer (50 mM Tris, pH 8; 100 mM EDTA; 100 mM NaCl; 1% sodium dodecyl sulphate, 350 μg proteinase K) and incubated overnight with shaking at 55°C. DNA was extracted with 500 μl isopropanol. The precipitate was transferred to Eppendorf tubes containing

Determination of genotype

As shown in Fig. 1, the genotype of four-week-old mice was determined by the presence only of the wild-type LIF gene in wild-type mice (+/+), indicated by an amplified band at 651 bp after electrophoresis on the agarose gel. The LIF knockout mice (−/−) had only the reporter gene, indicated by a band at 822 bp. Heterozygous (+/−) animals carry both the wild-gene and one copy of the reporter gene, and had both bands amplified (651 bp and 822 bp). Control reaction without DNA showed an absence of both

Regulation of galanin/galanin message-associated peptide expression is leukemia inhibitory factor dependent

Galanin, VIP and some other peptides are strongly up-regulated in DRG neurons after peripheral axotomy (see [20] and [75] and Introduction). In most studies rats have been analysed, but an equally strong up-regulation has been found in mice in vivo,[6] suggesting that similar mechanisms operate in both species. VIP,[38] and GMAP and galanin[30] levels also increase dramatically, when rat DRG neurons are dispersed in culture. These findings suggest that DRG neurons in culture are in an

Conclusions

In conclusion, considering all aspects of the four approaches used here, it is clear that the fastest and perhaps most reliable approach to detect galanin/GMAP-synthesizing neurons is immunohistochemistry. In this particular case it also appears to be as sensitive as in situ hybridization expression, which is often not the case, for example, when studying various peptides in brain sections. The non-radioactive approaches are fast and simple, whereby the most reproducible results were obtained

Acknowledgements

This work was supported by Marianne and Marcus Wallenbergs Stiftelse, the Swedish MRC (04X-2887) and the European Commission (BMH4-CT95-0172). We thank Professor Tamás Bártfai and Dr Katarina Bedecs, Stockholm University, Stockholm, Sweden, for generous supply of GMAP peptide and GMAP antiserum, and Dr Elvar Theodorsson, University of Linköping, Linköping, Sweden, for galanin antiserum. We thank Professor Philippe Brulet, Pasteur Institute, Paris, France, for the generous gift of the

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The neuropeptide galanin (GAL) has recognized physiological actions in the nervous system and other tissues, but there is no documented evidence of GAL influencing normal or pathological bone metabolism. GAL expression, however, is upregulated in central and peripheral nerves following axotomy and is known to influence neural regeneration. Thus, severance of skeletal-associated nerves during fracture could similarly increase local GAL concentrations and thereby influence fracture healing. The initial aim of this study was therefore to identify the presence of GAL in normal bone and/or fracture callus by assessing the concentration and cellular localization of GAL in intact and/or fractured rat rib, using radioimmunoassay and immunohistochemistry, respectively. Groups of Sprague–Dawley rats (13 weeks old) had their left sixth ribs surgically fractured or underwent sham surgery and then calluses and nonfractured rib samples were analyzed at 1 and 2 weeks postsurgery (n = 5–6 per group). Low (basal) concentrations of GAL were detected in control ribs, whereas at 1 and 2 weeks postfracture, callus samples contained markedly increased levels of peptide (∼32- and 18-fold increase, respectively, relative to controls; P < 0.01), revealing a strong upregulation during bone healing. Plasma GAL concentrations were also increased at 2 weeks postfracture (P < 0.005). In normal (nonfractured) rib, minimal levels of GAL-like immunoreactivity (LI) were present in cortical bone, periosteum, endosteum, and surrounding skeletal muscle. In costal cartilage plates, intense GAL-LI was present in all chondrocytes of the hypertrophic zone and in a population of chondrocytes in the reserve zone. GAL-LI was not present, however, in chondrocytes in the proliferative zone of costal cartilage or skeletal muscle fibers. In fracture callus, levels of GAL-LI were moderate to intense in osteoprogenitor cells and osteoblasts, in some chondrocytes, and in cartilaginous, osseous, and periosteal matrices. Subsequent studies revealed the presence of galanin receptor-1-like immunoreactivity (GALR1-LI) in most cell types shown to contain GAL-LI, although the distribution of GALR1-LI was more extensive in reserve zone chondrocytes than that of GAL-LI; and GALR1-LI also appeared in late proliferative zone chondrocytes of costal cartilage. In summary, GAL concentrations were significantly increased in fracture callus and plasma of rats that underwent rib fracture. In addition, GAL- and GALR1-LI was also detected in specific cells and structures within costal cartilage, bone, and fracture callus. These results strongly implicate GAL in aspects of cartilage growth plate physiology and fracture repair, possibly acting in an autocrine/paracrine fashion via GALR1.
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Expression levels of mRNA are commonly measured as a ratio of test to reference gene. The assumption is that reference genes such as β-actin or cyclophilin are unaffected by treatment and act as steady-state controls. TaqMan™ real-time RT-PCR was used to test these assumptions in a rat model of cerebral ischaemia (tMCAO). Following measurement of 24 genes, we show that reference genes in this animal model fail the criteria for steady-state controls. Neuronal loss, glial proliferation and an influx of leukocytes into the lesioned brain result in major disturbance to cell populations. The mRNA for reference genes, as for test genes, reflects these changes. Specific mRNA levels vary according to the choice of reference gene to which they are normalised. In the process of resolving reference gene issues, mRNA increases were discovered for leukaemia inhibitory factor, nestin and galanin in rat brain hemispheres affected by ischaemia. Results are reported for a further 21 genes and mathematical and statistical methods are described that allow in this study fraction-fold changes in mRNA to be detected.
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¹: Present address: Laboratoire de Biologie Cellulaire, Université Bordeaux II, Bâtiment 3B, 3ème étage; 146, rue Léo Saignat, 33076 Bordeaux Cedex, France.

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Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology

Abstract

Section snippets

Animals

Determination of genotype

Regulation of galanin/galanin message-associated peptide expression is leukemia inhibitory factor dependent

Conclusions

Acknowledgements

Neuron

Brain Res.

Expl Neurol.

Neurosci. Lett.

Trends Neurosci.

Neurosci. Lett.

Brain Res.

Brain Res.

Brain Res.

Neuroscience

Neuroscience

Neuroscience

Pain

Molec. Brain Res.

Molec. Brain Res.

Neuron

Devl. Biol.

Analyt. Biochem.

Brain Res.

Molec. cell. Neurosci.

Neuroscience

Neuroscience

Neurosci. Lett.

Brain Res.

Neuropeptides

Neuroscience

Expression in brain of messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide

Science

Regulation of HILDA/LIF gene expression in activated human monocytic cells

J. Immunol.

mRNA encoding IL-1β, IL-1 receptor and IL-1 receptor antagonist are induced following sympathetic axotomy

Soc. Neurosci. Abstr.

Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia

Proc. natn. Acad. Sci. U.S.A.

LIF is an autocrine factor for sympathetic neurons

Molec. cell. Neurosci.

Influence of leukemia inhibitory factor on galanin/GMAP and neuropeptide Y expression in mouse primary sensory neurons after axotomy

Expl Brain Res.

NGF and LIF both regulate galanin gene expression in primary DRG cultures

NeuroReport

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Histochemistry

Differential regulation of calcitonin gene-related peptide (CGRP) in regenerating rat facial nucleus and dorsal root ganglion

Eur. J. Neurosci.

Leukaemia inhibitory factor is necessary for maintenance of haemotapoietic stem cells and thymocyte stimulation

Nature

Increased levels of GMAP, VIP and nitric oxide synthase, and their mRNAs, in lumbar dorsal root ganglia of the rat following systemic resiniferatoxin treatment

NeuroReport