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, 23 (10), 4117-26

Differential Activation of Extracellular Signal-Regulated Protein Kinase in Primary Afferent Neurons Regulates Brain-Derived Neurotrophic Factor Expression After Peripheral Inflammation and Nerve Injury

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Differential Activation of Extracellular Signal-Regulated Protein Kinase in Primary Afferent Neurons Regulates Brain-Derived Neurotrophic Factor Expression After Peripheral Inflammation and Nerve Injury

Koichi Obata et al. J Neurosci.

Abstract

To investigate the intracellular signal transduction pathways involved in regulating the gene expression of brain-derived neurotrophic factor (BDNF) in primary afferent neurons, we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons after peripheral inflammation and sciatic nerve transection. Peripheral inflammation induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons. The treatment of the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126 reversed the pain hypersensitivity and the increase in phosphorylated-ERK (p-ERK) and BDNF in DRG neurons induced by complete Freund's adjuvant. On the other hand, axotomy induced the activation of ERK mainly in medium-and large-sized DRG neurons and in satellite glial cells. U0126 suppressed the axotomy-induced autotomy behavior and reversed the increase in p-ERK and BDNF. The intrathecal application of nerve growth factor (NGF) induced an increase in the number of p-ERK-and BDNF-labeled cells, mainly small neurons, and the application of anti-NGF induced an increase in p-ERK and BDNF in some medium-to-large-diameter DRG neurons. The activation of MAPK in the primary afferents may occur in different populations of DRG neurons after peripheral inflammation and axotomy, respectively, through alterations in the target-derived NGF. These changes, including the changes in BDNF expression, might be involved in the pathophysiological changes in primary afferent neurons.

Figures

Figure 1.
Figure 1.
A, B, Photomicrographs showing the p-ERK-IR in the ipsilateral (A) and contralateral (B) L4/5 DRG 1 d after peripheral inflammation. There was an increase in the number of p-ERK-IR neurons in the ipsilateral DRG (large arrows). In contrast to DRG neurons, satellite cells show high basal levels of p-ERK-IR (small open arrows). Scale bar: (in B) A, B, 100 μm. C, Time course of the mean percentages of p-ERK-IR neurons relative to the total number of neurons in the L4/5 DRG. The mean percentages of p-ERK-IR neurons on the ipsilateral side significantly increased at 1 d after CFA injection compared with the naive control rats. *p < 0.05 compared with the naive control. D, Size-distribution histogram of p-ERK-IR neuron profiles in the ipsilateral DRG 1 d after the CFA. At least 300 neuron profiles with visible nuclei were counted in four to six randomly chosen sections from two animals (two to three sections per animal).E, Western blot analysis of phosphorylated ERK and the total amount of ERK in the ipsilateral L4/5 DRG after CFA injection. CFA induced an increase in the intensity of the band for p-ERK at 1 d. Bottom, Levels of total ERK, as loading controls. Fold. represents comparative levels over the naive control group after normalizing to the total ERK levels. The two rows of numbers correspond to measurements for ERK1 and ERK2. E, right, ERK1 corresponds to p44, and ERK2 corresponds to p42.
Figure 2.
Figure 2.
Effects of MEK1/2 inhibitor U0126 delivered intrathecally on CFA-induced inflammatory pain and BDNF expression in the DRG. A, The response frequencies of paw withdrawals to repeated mechanical stimuli applied to the pads of the hindpaws with a von Frey filament of 23.0 mN are expressed as a percentage of trials. B, The difference score (latency on the operated side − latency on the contralateral side) to the radiant heat stimuli was obtained from the same rats with the mechanical stimuli. Data from the vehicle group (open columns), the 0.05μg · μl −1 · hr −1 U0126 group (gray columns), and the 0.5μg · μl1 · hr −1 U0126 group (black columns) at 1 d after CFA injection are shown (means ± SEM) (n = 8 per group). *p < 0.05 compared with the preoperative value; #p < 0.05 compared with vehicle control. C, Western blot indicates that the CFA-induced ERK activation in the DRG at 3 d was inhibited by MEK inhibitor U0126 delivered by osmotic pump (0.5μg · μl −1 · hr −1). E, F, Photomicrographs showing the BDNF-IR in the L4/5 DRG in the vehicle group (E) and in the U0126 group (F) at 3 d after CFA injection. Scale bar: (in F) E, F, 100μm. D, Quantification of the percentage of BDNF-IR neurons 3 d after CFA injection. #p < 0.05 compared with vehicle control. G, Bright-field photomicrograph shows in situ hybridization signals for BDNF mRNA in the ipsilateral DRG in the vehicle group at 3 d after the CFA. Scale bar, 50 μm in G. H, I, Expression of BDNF mRNA in the ipsilateral DRG, using dark-field photomicrographs of ISHH, was observed in the vehicle group (H) and in the U0126 group (I) 3 d after the CFA. J, K, Immunohistochemistry of BDNF and p-ERK in DRG neurons after peripheral inflammation. BDNF-IR (J) and p-ERK-IR (K) in the ipsilateral L4/5 DRG 3 d after the CFA. Some BDNF-IR neurons in the DRG also coexpressed p-ERK (J, K, arrows). L, M, Immunohistochemistry of p-ERK and trkA in DRG neurons after peripheral inflammation. p-ERK-IR (L) and trkA-IR (M) in the ipsilateral L4/5 DRG 3 d after the CFA. Double labeling of neurons with p-ERK and trkA was observed (L, M, arrows). Scale bar: (in I), H–M, 100 μm.
Figure 3.
Figure 3.
A, B, Photomicrographs showing the p-ERK-IR in the ipsilateral (A) and contralateral (B) L4/5 DRG 7 d after sciatic nerve transection. Axotomy increased p-ERK expression in neurons and/or satellite cells in the ipsilateral DRG. The p-ERK-IR was present in both neurons and surrounding satellite cells (large arrows) or only in satellite cells (small open arrows); the inset shows that both the neuron and surrounding satellite cell expressed p-ERK-IR. Scale bar: (in B) A, B, 100 μm. C, The time course of the mean percentages of p-ERK-IR neurons relative to the total number of neurons in the L4/5 DRG. The mean percentages of p-ERK-IR neurons on the ipsilateral side significantly increased at 3, 7, and 14 d after axotomy compared with the naive control rats. *p < 0.05 compared with the naive control. D, Size-distribution histogram of p-ERK-IR neuron profiles in the ipsilateral DRG at 7 d after axotomy. At least 300 neuron profiles with visible nuclei were counted in four to six randomly chosen sections from two animals (two to three sections per animal). E, Western blot analysis of phosphorylated ERK and total amount of ERK in the ipsilateral L4/5 DRG after axotomy. Axotomy induced an increase in the intensity of the band for p-ERK at 7 and 14 d.
Figure 4.
Figure 4.
Immunohistochemical colocalization of p-ERK (A, C, E) and NF200 (B), GFAP (D), or ATF3 (F) in the ipsilateral L4/5 DRG 7 d after sciatic nerve transection. Double labeling of neurons with p-ERK and NF200 was detected (A, B, filled arrows). There was also an increase in p-ERK expression in satellite cells, particularly around large-diameter neurons that were positive for NF200 (A, B, open arrows). Double labeling of satellite cells with p-ERK and GFAP was detected (C, D, arrows). p-ERK was detected in a subpopulation of ATF3-labeled neurons, and p-ERK-IR was also present insatellite glial cells, surrounding large neurons that were labeled for ATF3-IR (E,F, arrows). Scale bar: (in F) A–F, 100 μm.
Figure 5.
Figure 5.
Effects of MEK1/2 inhibitor U0126 delivered intrathecally on axotomy-induced autotomy behavior and BDNF expression in the DRG at 7 d after surgery. A, The autotomy score used is described in the text and the average score achieved by the animals (means ± SEM) (n = 8 per group). #p < 0.05 compared with vehicle control. B, Western blotting indicates that the axotomy-induced ERK activation in the DRG at 7 d was inhibited by the MEK inhibitor U0126 delivered by osmotic pump (0.5 μg · μl1 · hr −1). Both immunohistochemistry (E, F) and in situ hybridization histochemistry (G–I) showed inhibition of the axotomy-induced increase in BDNF-labeled neurons in the DRG by U0126 at 7 d after surgery. A decrease in the expression of BDNF can be seen in the ipsilateral DRG in the U0126 group (F, I) compared with the vehicle control rats (E, G, H). C, Quantification of the percentage of BDNF-IR neurons at 7 d after axotomy. #p < 0.05 compared with vehicle control. D, Mean cross-sectional areas of BDNF-IR neuron profiles in the ipsilateral DRG both in the vehicle and U0126 groups 7 d after axotomy. At least 300 neuron profiles with visible nuclei were counted in four to six randomly chosen sections from two animals (two to three sections per animal). J, K, Immunohistochemical colocalization of BDNF (J) and p-ERK (K) in the ipsilateral L4/5 DRG 7 d after sciatic nerve transection. Some BDNF-IR neurons in the DRG also expressed p-ERK (J, K, arrows). L, M, Immunohistochemistry of p-ERK and trkA in DRG neurons after sciatic nerve transection. p-ERK-IR (L) and trkA-IR (M) in the ipsilateral L4/5 DRG 7 d after the nerve lesion are shown. Double labeling of neurons with p-ERK and trkA was observed (L, M, arrows). Scale bars: (in F) E, F, 100μm; (in G) G,50μm; (in I) H–M, 100 μm.
Figure 6.
Figure 6.
Effects of intrathecal injections of recombinant rat β-NGF or anti-NGF on the increase in the phosphorylation of ERK and BDNF expression in the DRG 3 d after surgery. Photomicrographs showing the p-ERK-IR (A–C), BDNF-IR (D–F), and BDNF mRNA-positive (G–I) neurons in the L4/5 DRG 3 d after surgery. The DRG neurons in the 10μg NGF group had clear increases in the number of both p-ERK-and BDNF-labeled neurons (B, E, H), compared with the saline group (A, D, G), 3 d after surgery. Most of these p-ERK-and BDNF-positive neurons were small-to-medium-diameter sensory neurons, as indicated by arrows in B, E, and H, respectively. An increase in the number of p-ERK-and BDNF-labeled neurons was detected in the 10 μg anti-NGF group (C, F, I) compared with the saline group (A, D, G) 3 d after surgery. The anti-NGF-induced increase in p-ERK and BDNF expression was seen mainly in medium-and large-sized neurons (C, F, and I, respectively). Scale bar: (in I) A–I, 50 μm. J, Quantification of the percentage of BDNF-IRneurons 3 dafter the injection. #p<0.05 compared with the saline group. K,L, Size-distribution histograms of BDNF-IR neuron profiles 3 d after the injection in the 10 μg NGF and 10μg anti-NGF groups are shown in K and L, respectively. Atleast 300 neuron profile swith visibl enuclei were counted in four to six randomly chosen sections from two animals(two to three sections per animal). M, Western blot analysis of phosphorylated ERK and total amount of ERK in the L4/5 DRG after the intrathecal injection of NGF and anti-NGF. NGF and anti-NGF induced an increase in the intensity of the band for p-ERK at 1 d. Bottom, Levels of total ERK, as loading controls. N, O, Immunohistochemical colocalization of BDNF (N) and trkA (O) in the L4/5 DRG in the anti-NGF 10μg group at 3 d. Some BDNF-IR neurons in the DRG also expressed trkA (N, O, arrows). Scale bar: (in O) N, O, 100μm.

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