Ingrid Liebmann

Increased expression of TRPV1 receptor in dorsal root ganglia by acid insult of the rat gastric mucosa

It is still unknown which receptors of peripheral sensory pathways encode and integrate an acid‐induced nociceptive event in the gastric mucosa. The transient receptor potential vanilloid receptor 1 (TRPV1) and the acid‐sensing ion channel 3 (ASIC3) are two nociception‐related receptors. Here we investigated (i) to what extent these receptors are distributed in stomach‐innervating neurons of dorsal root and nodose ganglia, using immunohistochemistry and retrograde tracing, and (ii) whether their expression is altered in response to a noxious acid challenge of the stomach. We also explored the presence of TRPV1 in the gastric enteric nervous system because of its possible expression by intrinsic sensory neurons. Most stomach‐innervating neurons in nodose ganglia were immunoreactive for TRPV1 (80%) and ASIC3 (75%), these results being similar in the dorsal root ganglia (71 and 82%). RT‐PCR and Western blotting were performed up to 6 h after oral application of 0.5 m HCl to conscious rats. TRPV1 protein was increased in dorsal root but not in nodose ganglia whereas TRPV1 and ASIC3 mRNAs remained unchanged. TRPV1 mRNA was detected in longitudinal muscle–myenteric plexus preparations of control stomachs and was not altered by the acid challenge. Combined vagotomy and ganglionectomy abolished expression of TRPV1, indicating that it may derive from an extrinsic source. In summary, noxious acid challenge of the stomach increased TRPV1 protein in spinal but not vagal or intrinsic sensory afferents. The TRPV1 receptor may be a key molecule in the transduction of acid‐induced nociception of the gastric mucosa and a mediator of visceral hypersensitivity.

Stimulation by nerve growth factor of neuropeptide synthesis in the adult rat in vivo: bilateral response to unilateral intraplantar injections

Unilateral intraplantar injections (1/day for 3 days) of 4 mu g nerve growth factor (NGF) into the rat hindpaw increased the expression of prepro-tachykinin (PPT)- and prepro-calcitonin gene-related peptide (ppCGRP)-mRNA in bilateral L5 dorsal root ganglia (DRGs). This was accompanied by an increase of CGRP-like immunoreactivity in the ipsi- and contralateral sciatic nerve but by no detectable change of CGRP-IR in other afferents. NGF injections into the skin of one ear or into the plantar side of one forepaw increased CGRP-IR in the respective afferents (trigeminal ganglion, or nerves arising from the brachial plexus, respectively), but had no effect on sciatic CGRP-IR. This suggests that the NGF-induced symmetrical increase of CGRP synthesis in L5 DRGs was not caused by systemic action of NGF, which, therefore, may be a useful tool to further investigate mechanisms which are responsible for contralateral effects of unilateral inflammation.

Nociceptive transmitter release in the dorsal spinal cord by capsaicin-sensitive fibers after noxious gastric stimulation

Little is known about transmitters that encode noxious gastric stimuli in the spinal cord. The release of glutamate, substance P, and CGRP from the spinal cord was therefore investigated in response to acid injury of the gastric mucosa. Dorsal halves of the caudal thoracic spinal cord (T7-T13) were removed 6 h after oral application of 0.5 M HCl or saline, transferred to a superfusion chamber, and the basal and capsaicin-stimulated (3.3 microM) transmitter release was determined. After acid injury, basal glutamate release increased 134% as compared to saline-treated animals. Capsaicin-stimulated release of CGRP and SP was 48% and 58% lower in acid- than in saline-treated animals, indicating that capsaicin-sensitive fibers in the dorsal spinal cord were already partially depleted by acid treatment. Capsaicin denervation reduced basal glutamate release by 33% after acid injury as compared to non-denervated acid-treated animals. Gastric origin and capsaicin sensitivity of glutamatergic, CGRP- and SP-containing primary afferents in thoracic dorsal root ganglia were then determined by retrograde tracing with True Blue and immunohistochemical labeling with the vanilloid receptor TRPV1. About 65% of True Blue-labeled cells were glutamatergic and more than 73% of this population expressed the TRPV1 receptor. Nearly all True Blue/CGRP (85%)- and True Blue/SP-positive cells (97%) coexpressed TRPV1. We conclude that noxious gastric stimulation with acid induces release of glutamate, SP, and CGRP from capsaicin-sensitive sensory afferents in the dorsal horn of the spinal cord where they may play an important role in gastric nociception and hyperalgesia.

Differential Regulation of 3-Beta-Hydroxysteroid Dehydrogenase and Vanilloid Receptor TRPV1 mRNA in Sensory Neurons by Capsaicin and NGF

It was the aim of the present study to investigate by RT-PCR the regulation of the mRNA of the neurosteroid-synthesizing enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) and of the vanilloid receptor TRPV1 in dorsal root ganglia (DRGs) of rats during the process of capsaicin denervation of primary sensory neurons and the following regeneration. The expression of 3β-HSD in DRG was increased 3 days after the capsaicin treatment, and it remained at that level during a 22 day observation period. The expression of TRPV1, a specific marker of capsaicin-sensitive small sensory neurons connected to C- and Aδ-fibers, was markedly reduced 3 days after the capsaicin treatment. It slowly recovered during the 22 days observation period reaching almost control levels on day 22. When the capsaicin-treated rats received 5 intraplantar injections of nerve growth factor (NGF), the prototypical neurotrophin for capsaicin-sensitive neurons, on day 1, 2, 3, 5 and 6, both the 3β-HSD and the TRPV1 mRNA had returned to control levels at the time point 8 days after capsaicin. The present results demonstrate that both 3β-HSD and TRPV1 are markers for neurodegeneration and neuroregeneration in capsaicin-sensitive primary afferent neurons, and that NGF is an effective tool to induce recovery after peripheral nerve injury.

Extracellular signal-regulated kinase-1 and -2 are activated by gastric luminal injury in dorsal root ganglion neurons via N-methyl-D-aspartate receptors

Mitogen activated protein kinases such as phosphorylated extracellular signal-regulated kinase-1 and -2 (pERK 1/2) have been recently demonstrated to play an important role in somatic nociception and hyperalgesia. In the present study we examined whether pERK 1/2 is involved in the response of sensory neurons to a noxious visceral stimulation, in particular, of the gastric mucosa. After induction of gastric injury by oral administration of 0.5M HCl pERK 1/2 expression was determined by Western blotting of caudal thoracic dorsal root ganglia and by immunohistochemistry in stomach-innervating dorsal root ganglion neurons which were retrogradely labeled with True Blue. The content of pERK 1/2 remained unchanged in dorsal root ganglia until 2 h post-HCl, however, was found elevated 4 (approximately 80%) and 6 h (approximately 100%) after HCl administration. True Blue-labeled pERK 1/2-immunoreactive neurons were likewise increased 6 h post-HCl (204%) and were mainly of small size (20-40 microm) and negative for neurofilament 200 (approximately 76%). The majority of these cells also expressed the nociceptive transient receptor potential vanilloid receptor 1 (approximately 70%). The gastric mucosa was simultaneously examined for lesion formation showing highest percentage of damage 6 h post-HCl. Application of a N-methyl-D-aspartate receptor antagonist (MK-801; 100 microg/kg s.c.) significantly reduced HCl-induced pERK 1/2 expression and mucosal lesions 6 h post-HCl. Activation of the extracellular signal-regulated kinase-1 and -2 signaling cascade indicates that visceral primary afferents may sensitize after gastric noxious stimulation involving N-methyl-D-aspartate receptors. The extracellular signal-regulated kinase-1 and -2 pathway therefore may not only be of importance for somatic but also for visceral nociception.

A fluorescence-immunohistochemical study on phosphorylation of ERK1/2, p38, and STAT3 in rat dorsal root ganglia following noxious stimulation of hind paw sensory neurons.

A fluorescence-immunohistochemical investigation was performed in lumbar dorsal root ganglia (DRGs) neurons of the rat with regard to ERK1/2-, p38- and STAT3-phosphorylation in response to nociceptor activation in the rat. The stimuli applied were perineural capsaicin treatment of the sciatic nerve, mustard oil application to the hind paw and heat or cold stimulation of the hind paw. The time points of investigations were 15 min/30 min after perineural capsaicin, 30 min/2 h/4 h for mustard oil, 10 min/4 h for cold and 30 min/2 h/8 h for the heat stimulus. All four stimuli lead to a time-dependent, significant 2-3 fold increase in the number of small and medium size DRG cells displaying cytoplasmic staining for p-ERK1/2, but to no activation of satellite cells. Phosphorylated p38 immunoreactivity was increased in the cytoplasma of DRG cells at 2 h after the mustard oil treatment of the hind paw and 30 min after the perineural capsaicin application to the sciatic nerve axons, but not following heat or cold stimuli to the hind paws. Phospho-STAT3 staining was characteristically observed as nuclear and cytoplasmic staining. It was found increased after the perineural capsaicin application to the sciatic nerve axons, however, no marked increase was found with the other 3 noxious stimuli. The present results show that sensory neurons respond with a selective long-lasting increase in p-ERK1/2 in small and medium-size DRG cells, when their axons or axon terminals are stimulated by capsaicin, mustard oil, noxious heat or noxious cold.

ERK and STAT3 phosphorylation in sensory neurons during capsaicin-induced impairment and nerve growth factor treatment.

Distinct signal transduction pathways have been shown to regulate injury responses and regeneration in peripheral nerves. In the present investigation, the time courses of the induction of phospho-MAPK/ERK1/2 and of phospho-STAT3 were investigated in the dorsal root ganglia (DRG) and in the sciatic nerve of rats following a systemic capsaicin treatment without or with concomitant intraplantar NGF injections. Western blots were probed with polyclonal antibodies that specifically detect phosphorylated ERK 1/2 and STAT3. Phosphorylation of ERK clearly peaked in the sciatic nerve and in the lumbar DRGs at 6 and 10 h after the capsaicin treatment. In the following 8 days phospho-ERK decreased to very low levels and was found recovered to basal values at the time point 16 days. An additional intraplantar nerve growth factor (NGF) injection at time points 20, 44 and 92 h after the capsaicin treatment, and collection of tissues 4 h later, markedly increased the level of phospho-ERK in the sciatic nerve as well as in the DRG, as compared to the samples taken from rats at the same time points with a capsaicin treatment only. Posphorylated STAT3, which was almost non-detectable in the control sciatic nerve, clearly peaked at 6 h after the capsaicin treatment and decreased again during the following days to almost undetectable levels. The intraplantar NGF injections slightly stimulated phosho-STAT3 in the sciatic nerve. A basal level of phosphorylated STAT3 was present in DRGs of control animals, it remained at a high level up to 6 h after the capsaicin treatment, then markedly decreased and recovered on day 8 and day 16. NGF increased STAT3 phosphorylation in DRG on day 1 and day 2 above the level observed in samples taken from rats at the same time points with a capsaicin treatment only. The present study demonstrates that a capsaicin impairment of small diameter primary sensory neurons followed by an NGF treatment evokes a characteristic pattern of ERK and STAT3 activation indicative of neuronal degeneration and regeneration.

Phosphorylation of ERK1/2 in dorsal root ganglia following sequential mustard oil and thermal stimulation of the rat hind paw.

The effect of a sequential stimulation by mustard oil and noxious heat or cold on the phosphorylation of ERK1/2 in sensory afferents was investigated. The stimuli were applied to the rat hind paw and phospho-ERK1/2 (p-ERK1/2) was measured by fluorescence-immunohistochemistry in the lumbar dorsal root ganglia (DRGs) neurons. All stimuli lead to a significant increase in the number of small size DRG cells displaying cytoplasmic staining for p-ERK1/2. The combination of mustard oil with cold significantly increased the number of cells with cytoplasmic staining above the level obtained with cold stimulus alone, however this was not observed with the combination of mustard oil and heat. Nuclear staining was weak and was found increased by mustard oil combined with cold stimulation. Mustard oil is known to activate TRPA1 and TRPV1 channels, heat TRPV1, and cold TRPA1 and TRPM8. The present findings shed new light on the DRG cell populations reacting with cytoplasmic and nuclear staining for p-ERK1/2 following sequential irritant chemical and thermal stimuli to the skin.

Dorsal root ganglion neurons respond with prolonged extracellular signal-regulated protein kinase phosphorylation following noxious heat and cold stimulation

In the present study the time course of extracellular signal-regulated protein kinase phosphorylation (pERK1/2 appearance) in lumbar sensory dorsal root ganglia (DRG) was determined following a 5-min noxious heat or a noxious cold stimulus to the hind paw of the rat. The thermal stimuli were chosen to activate transient receptor potential (TRP) channels, but not to induce tissue damage. A quantitative analysis of phospho-ERK1/2 was performed by protein extraction and Western blot analysis. Western blot analysis showed that following the heat stimulus, phosphorylation of ERK1/2 increased 2-3-fold between 10 and 30min in the DRG on the ipsilateral side. High levels were maintained from 30min up to 16h. Following the cold stimulus to the paw, pERK1/2 immediately increased 2-fold within 2min in the DRG on the ipsilateral side, it declined within 2h and reached a second peak at 4h. In the DRGs on the contralateral side of the paws heat or cold immersion the pERK1/2 remained low at all time points investigated. Fluorescence immunohistochemistry of the DRG following the thermal stimuli revealed an increased cytoplasmic staining for pERK1/2 in neurons. The present results show that following a 5-min nociceptive thermal stimulus sensory neurons respond with a characteristic long-lasting phosphorylation of ERK1/2.

The Pain Pathway in the Rat following Noxious Thermal Stimulation: Effect of Morphine on pERK1/2 and TRPV1 at the Dorsal Horn Level, and on Hyperalgesia

The aim of the present study was to investigate the phosphorylation of ERK1/2 in the lumbar dorsal horn of the rat by fluorescence immunohistochemistry following a noxious thermal stimulation of the hind paw. The protein level of TRPV1 in the dorsal spinal cord and the development of a heat hyperalgesia after the acute noxious thermal stimulation were also measured. The protein content of TRPV1 was determined by Western blot and heat hyperalgesia by the plantar test. At 2 and 10 min after the thermal stimulation a 4-fold increase in pERK1/2 immunoreactivity was observed in cells of lamina I/II of the L3-L5 dorsal horn. A pretreatment with the opioid analgesic morphine markedly attenuated ERK1/2 phosphorylation. The protein content of TRPV1 in the lumbar dorsal spinal cord was not significantly altered at 1 and 4 h after the thermal hind paw stimulation and by the morphine pretreatment. Heat hyperalgesia in the plantar test was observed at 8 h, but not at 24 h after the noxious stimulation. This temporary hyperalgesia was prevented by the morphine pretreatment. The present findings indicate that ERK1/2 activation in dorsal horn nociceptive neurons may be linked to the development of hyperalgesia, and that opioid analgesics are effective agents to prevent sensitization in the pain pathway at spinal level.