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, 21 (13), 4891-900

Brain-derived Neurotrophic Factor Increases in the Uninjured Dorsal Root Ganglion Neurons in Selective Spinal Nerve Ligation Model

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Brain-derived Neurotrophic Factor Increases in the Uninjured Dorsal Root Ganglion Neurons in Selective Spinal Nerve Ligation Model

T Fukuoka et al. J Neurosci.

Abstract

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two major members of the neurotrophin family. Using immunohistochemistry and in situ hybridization histochemistry, we examined the effect of L5 spinal nerve ligation (SPNL), a neuropathic pain model, on the expression of BDNF in the uninjured L4 dorsal root ganglion (DRG). After L5 SPNL, both immunoreactivity for BDNF and the hybridization intensity for BDNF mRNA increased mainly in the small- and medium-sized neurons. The percentage of BDNF mRNA-expressing neurons increased in the ipsilateral L4 DRG compared with the contralateral DRG from the third to 28th day after ligation. A significantly greater number of BDNF-immunoreactive neurons were observed in the ipsilateral L4 DRG than contralateral side 14 d after ligation. To test the contribution of BDNF to the thermal hyperalgesia produced in this model, we intrathecally injected anti-BDNF antibody at third day after ligation. This treatment clearly attenuated thermal hyperalgesia for a few hours. Almost all BDNF mRNA-expressing neurons coexpressed trkA, a high-affinity NGF receptor, mRNA. The percentage of BDNF mRNA-expressing cells of trkA cells significantly increased in the ipsilateral L4 DRG 14 d after ligation. Furthermore, we examined the contribution of NGF on this phenotypic change using ELISA, Northern blot analysis, and anti-NGF antibody. NGF content in the ipsilateral L4 DRG linearly increased and reached a statistical significant level 14 d after L5 SPNL. Moreover, at this time point, the increase in NGF mRNA was observed in the ipsilateral L5 DRG and sciatic nerve, but not in the ipsilateral L4 DRG or L4 spinal nerve. Local application of anti-NGF antibody to the L4 spinal nerve beside the L5 spinal nerve-ligation site prevented the development of thermal hyperalgesia for 5 d after ligation. Our data suggest that BDNF, which increased in the uninjured L4 DRG neurons, acts as a sensory neuromodulator in the dorsal horn and contributes to thermal hyperalgesia in this neuropathic pain model. The contribution of locally synthesized NGF to thermal hyperalgesia was also demonstrated. These dynamic alterations in the expression and content of BDNF and NGF in the uninjured DRG neurons might be involved in the pathomechanisms of neuropathic pain.

Figures

Fig. 1.
Fig. 1.
Northern blot analysis of oligonucleotide probes designed for the present study. The probes for BDNF and trkA were hybridized onto the total RNA obtained from naive L4 DRG, and the probe for βNGF was hybridized onto the total RNA obtained from the sciatic nerve 14 d after L5 SPNL. Each probe was hybridized at the position or positions expected from the previous studies (see Materials and Methods). The top and bottom arrows indicate the positions of 28S and 18S rRNAs, respectively.
Fig. 2.
Fig. 2.
Time course of neuropathic pain behaviors.A, The response frequencies of paw withdrawals to repeated mechanical stimuli applied to the pads of the hindpaws with a von Frey filament of 72.2 mN are expressed as a percentage (mean ± SEM) of trials. Data from the ipsilateral (closed symbols) and contralateral (open symbols) side of 20 rats that received L5 SPNL (circles) and five sham-operated rats (triangles) are shown. There was a significant group effect between the ipsilateral and contralateral side of the L5 SPNL group (p < 0.05, two-way repeated measures ANOVA). Number signs indicate significant differences from the contralateral side (p < 0.05, paired t test). Asterisks indicate significant differences from the preoperative value (Pre) (p < 0.05, one-way ANOVA followed by Fisher's PLSD). B is the difference score (latency on the operated side − latency on the contralateral side) to the radiant heat stimuli obtained from the same rats with the mechanical stimuli. Data obtained from 20 rats that received L5 SPNL (closed circles) and five sham-operated rats (closed triangles) are expressed as seconds (mean ± SEM). There was a significant change with time in the L5 SPNL group (p < 0.05, one-way ANOVA).Asterisks indicate significant difference from the preoperative value (Pre) (p< 0.05 by Fisher's PLSD).
Fig. 3.
Fig. 3.
Effect of L5 spinal nerve ligation on BDNF protein and mRNA expression in L4 DRG neurons. A andB are dark-field photomicrographs showing in situ hybridization products for BDNF mRNA 14 d after surgery. In the ipsilateral L4 DRG (A), several neurons were intensely labeled (arrows) compared with the contralateral L4 DRG (B). Scale bars, 300 μm. C and D are scatterplot diagrams of BDNF mRNA expression in the ipsilateral (C, ipsi.) and contralateral (D, contra.) L4 DRG from one representative rat. Individual cell profiles are plotted according to the cross-sectional area (in square micrometers; along thex-axis) and S/N ratio (along the y-axis). The dashed lines indicate the borderline between the negatively and positively labeled neurons (S/N ratio = 2). A subpopulation of small (<600 μm2)- and medium (600–1200 μm2)-sized neurons showed an increase in S/N ratio on the ipsilateral side compared with the contralateral side. E is a bar graph showing means ± SEM (%) of BDNF mRNA-expressing neurons in the L4 DRG at various times after L5 spinal nerve ligation (n = 4 at each time point).Filled bars and open bars represent the values on the ipsilateral and contralateral sides, respectively. #p < 0.05; ##p < 0.01 by paired t test. F and G are photomicrographs of BDNF immunohistochemistry 14 d after surgery. On the ipsilateral side (F), the immunoreactive intensity of some small-sized neurons markedly increased compared with the contralateral side (G). Scale bars, 200 μm.
Fig. 4.
Fig. 4.
Effects of intrathecal injection (arrow) of anti-BDNF antibody (20 μg, closed circles) or normal sheep IgG (20 μg, open circles) on paw withdrawal response to noxious heat of the rats that received L5 SPNL. Difference scores were calculated by subtracting contralateral withdrawal latencies from ipsilateral withdrawal latencies and expressed as mean ± SEM (n = 8 for each treatment). Thermal hyperalgesia was abolished for at least 2.5 hr by anti-BDNF antibody injection, whereas normal sheep IgG injection had no significant effect on the behavior. *p < 0.05 versus before L5 SPNL (pre ope); #p < 0.05 versus before injection (pre inj.) by one-way ANOVA followed by Fisher's PLSD.
Fig. 5.
Fig. 5.
Coexpression of BDNF mRNA with trkA mRNA in the ipsilateral L4 DRG 14 d after L5 spinal nerve ligation. A pair of serial sections processed for in situ hybridization for BDNF mRNA (A) and trkA mRNA (B). Asterisks indicate the neurons that coexpress both mRNAs. Scale bars, 100 μm.
Fig. 6.
Fig. 6.
Change in NGF content in the L4 DRG, L4 SpN and ScN after L5 spinal nerve ligation (closed circles) and after sham operation (open circles). NGF content was measured by ELISA, and the values of the ipsilateral side are presented as mean ± SEM (%) of the contralateral side (n = 4 independent experiments). *p < 0.05 versus naive control (N) by one-way ANOVA followed by Fisher's PLSD.
Fig. 7.
Fig. 7.
Expression of NGF mRNA in the L4 and L5 DRG, L4 spinal nerve, and sciatic nerve 14 d after L5 spinal nerve ligation. Northern blot analysis (A) revealed clear upregulation of NGF mRNA in the ipsilateral (I.) L5 DRG and sciatic nerve compared with the contralateral side (C.). The NGF mRNA expression in the L4 DRG and L4 spinal nerve was just above the detectable level, and there was no difference between the ipsilateral and contralateral sides.B is a bar graph of relative expression of NGF mRNA obtained from four pooled samples. Relative expression is defined as the expression on the ipsilateral side relative to the mean ± SEM (%) of the contralateral side. The difference from 100% was tested byt test. *p < 0.05. In situ hybridization was performed to rule out the possibility that a very small number of cells expressed NGF mRNA in the ipsilateral L4 DRG (C). There was no accumulation of silver grains on any specific cells in the DRG. Scale bar, 300 μm.
Fig. 8.
Fig. 8.
The effect of local application of anti-NGF antibody on thermal hyperalgesia in the L5 spinal nerve ligation (L5 SPNL) model. A small piece of gelatin sponge saturated with sheep anti-NGF antibody (100 μg, filled circles,n = 5–9) or normal sheep IgG (100 μg,open circles, n = 5) was placed on the surface of the L4 spinal nerve when the L5 SPNL was performed. The anti-NGF antibody prevented the development of thermal hyperalgesia for up to 6 d after surgery. Data are expressed as mean ± SEM. *p < 0.05 versus before L5 SPNL (pre) by one-way ANOVA followed by Fisher's PLSD.

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