Article Figures & Data
Figures
- Figure 1.
Peripheral inflammation induced an increased BDNF expression in both the cingulate and primary sensory cortices. A–C, ELISA titration showing that BDNF concentration was increased markedly in the cingulate cortex (pool of bilateral sites; A) and to a lesser extent in the primary sensory cortex (B) and unchanged in the insulate and motor cortices (C) 24 h after the induction of peripheral inflammation by unilateral intraplantar injection of CFA. E–J, Immunohistochemical staining of BDNF (G–J) and its quantification (E, F) reveals an increased number of BDNF-positive cells in the right and left cingulate cortex (E) in inflamed animals. There is also an increase in the contralateral somatosensory cortex, hindlimb part (F). D, Schematic showing the area used to quantify the number of BDNF-positive cells in the cingulate cortex (D, top) and the S1HL (D, bottom). G, H, Representative BDNF immunostaining in the ACC of a control (G) and a CFA-injected rat (H). I, J, Representative BDNF immunostaining in the S1HL of a control (I) and a CFA-injected rat (J). Scale bars: G, H, 200 μm; I, J, 100 μm. All data are expressed as mean ± SEM; N = 5 CFA-treated animals, n = 5 control animals. Two-way ANOVA followed by Student-Newman–Keuls comparison post hoc test. B: F(1,18) = 2017, p = 0.176; E: F(1,25) = 16.62, p < 0.001; F: F(1,25) = 17.25, p < 0.001; interaction: F(1,25) = 4.95, p = 0.037; *p < 0.05, **p < 0.01, ***p < 0.001 versus control rats.
- Figure 2.
BDNF expression induced by peripheral inflammation occurred in all types of cells. A–C, Double-immunostaining shows that BDNF immunostaining colocalized with the astrocyte marker GFAP (A, arrows), but also with the microglial marker Iba1 (B, arrows), and NeuN (C, arrows), a neuronal marker. Cells positive for BDNF, but negative for the relevant marker, were also observed and are indicated here with arrowheads. Scale bars: A, B, 20 μm; C, 50 μm.
- Figure 3.
Time course and cell-specific expression of GFP or BDNF induced by intracerebral injection of recombinant lentiviral vectors. A, Representative examples of GFP lentiviral expression in the hindlimb part of the S1HL at 1, 2, 3, and 4 weeks after local injection. GFP fluorescence was strictly localized in the S1HL and was maintained throughout the 4 weeks. B, ELISA titration performed on cortical samples 7 or 28 d after intracortical injection of GFP- or [GFP + BDNF]-expressing lentiviral vectors, showing that BDNF was significantly upregulated at both times. All data are expressed as mean ± SEM; **p < 0.01, ***p < 0.001 GFP-transfected versus BDNF-transfected rats (n = 4 GFP, n = 4 BDNF). C–E, Double-immunofluorescent staining showed that GFP (C–E, green) colocalized with GFAP (astrocyte cell; C), Iba1 (microglial cell; D, purple), and Cutl1 (pyramidal cortical neuron; E, purple). Scale bars: A, 1 mm; C, D, 10 μm; E, 20 μm.
- Figure 4.
Upregulation of BDNF expression by injection of lentiviral vector expressing BDNF in the S1 HL or in ACC induced sustained cold hypersensitivity and neuronal hyperexcitability. A–C, Behavioral studies showed that upregulation of BDNF in S1HL induced a cold thermal allodynia trend (A) and cold thermal hyperalgesia (B), but no modification of mechanical sensitivity (C). D–F, Electrophysiological experiments showed a significant increase in LTP in the S1HL of rats injected locally with BDNF expressing vector or in inflamed animal. Representative FPs recorded before (1) and after (2) TBS (D), mean relative FP amplitude of TBS-induced LTP for each experimental group (E, F). G–I, Behavioral studies reveal that upregulation of BDNF in ACC induced a significant cold allodynia (G) and a trend toward cold hyperalgesia (H), but no modification of mechanical sensitivity (I). All data are expressed as mean ± SEM. Statistics: A–C: n = 8 GFP; n = 8 BDNF, two-way ANOVA followed by a Student-Newman–Keuls post hoc test; A: F(1,62) = 3.089, p = 0.085; B: F(1,62) = 3.059, p = 0.018; C: F(1,54) = 0.49, p = 0.487; F, n = 7 GFP, n = 6 BDNF, n = 6 CFA, Mann–Whitney rank sum test, *p < 0.05, **p < 0.01, versus control rats; G–I, n = 6 GFP, n = 6 BDNF, two-way ANOVA followed by a Student-Newman–Keuls post hoc test; G: F(1,50) = 6140, p = 0.017; H: F(1,50) = 5.941, p = 0.019; I: F(1,50) = 0.809, p = 0.373, *p < 0.05 versus control rats. Note that the numerical value of the p value given in the legends of figures is the value of the variance analysis.
- Figure 5.
Local injection of recombinant BDNF protein into the cingulate cortex induced sustained cold hypersensitivity. A–C, Behavioral studies indicate that rBDNF injection into ACC induced a significant cold allodynia (A) and a cold hyperalgesia (B) that was maintained for (at least) 13 d postinjection, but no modification of mechanical sensitivity (C); N = 7 GFP, n = 6 rBDNF, two-way ANOVA followed by a Student-Newman–Keuls post hoc test; A: F(1,85) = 53.318, p < 0.001; B: F(1,85) = 44.451, p < 0.001; C: F(1,85) = 1.167, p = 0.282; *p < 0.05, **p < 0.01, ***p < 0.001 versus control rats; #p < 0.05, ###p < 0.001 comparison between different time points.
- Figure 6.
Blockade of BDNF/TrkB signaling by intra-ACC injection of TrkB antagonist prevented the cold hypersensitivity and cortical hyperexcitability induced by peripheral inflammation. A–D, Blockade of the BDNF/TrkB signaling pathway by the antagonist CTX-B prevented the occurrence of cold allodynia in CFA-injected rats (A). It also temporarily prevented the development of cold hyperalgesia for 2 d (B), but had no effect on the mechanical allodynia observed in the hindpaw ipsilateral to CFA injection (C). Neither intraplantar injection of CFA into the right paw nor intra-ACC injection of CTX-B modified the withdrawal threshold for the contralateral hindpaw (D, left). E–F, Representative examples of pERK staining in the cingulate cortex in a control (E) and a CFA-treated (F) rat. Arrows indicate pERK positive neurons. Scale bar, 90 μm. G, Quantification of pERK-positive neurons, showing that the antagonistic effect of CTX-B on BDNF-TrkB signaling prevented a peripheral inflammation-evoked increase in the number of pERK neurons in the ACC. H, Representative photomicrographs of pERK and BDNF immunostainings and colocalisation (arrows) in neurons of the cingulate cortex. Scale bar, 40 μm. All data are expressed as mean ± SEM; N = 6 animals per group, three-way ANOVA, followed by a Student-Newman–Keuls post hoc test. A, Inflammation treatment parameter: F(1,116) = 9.334, p = 0.003; CTX-B treatment parameter: F(1,116) = 4.213, p = 0.043; B, Inflammation treatment parameter: F(1,116) = 106.608, p < 0.001; CTX-B treatment parameter: F(1,116) = 7.245, p = 0.008; C, Inflammation treatment parameter: F(1,116) = 236.029, p < 0.001; CTX-B treatment parameter: F(1,116) = 1.104, p = 0.296; **p < 0.01 CFA-saline versus saline-saline rats; §p < 0.05 CFA-saline versus CFA-CTX-B rats. D, *p < 0.05, **p < 0.01, ***p < 0.001 CFA-saline versus saline-saline rats; ††p < 0.01, †††p < 0.001 CFA-CTX-B versus saline-CTX-B rats; §p < 0.05, §§§p < 0.001 CFA-saline versus CFA-CTX-B rats. G, F(3,20) = 5.875, p = 0.006.
- Figure 7.
Blockade of BDNF/TrkB signaling by intra-ACC injection of TrkB antagonist prevented the passive avoidance behavior induced by an aversive mechanical stimulation in link to peripheral inflammation. Blockade of the BDNF/TrkB signaling pathway by the antagonist CTX-B prevented passive avoidance behavior induced by static mechanical injection of the right paw previously injected with CFA (inflammation treatment parameter: F(1,99) = 14.995, p < 0.001; CTX-B treatment parameter: F(1,99) = 12.463, p < 0.001; interaction between these parameters: F(1,99) = 6.875, p = 0.01). All data are expressed as mean ± SEM; *p < 0.05, **p < 0.01 CFA-saline versus saline-saline rats; §p < 0.05, §§§p < 0.001 CFA-saline versus CFA-CTX-B rats (saline-saline, n = 8; saline-CTX-B, n = 6; CFA-saline, n = 8; CFA-CTX-B, n = 7 animals).
Tables
- Table 1.
Information regarding the stereotaxic injections of viral vectors, rBDNF or CTX-B
Injected molecule Cingulate cortex S1HL Coordinates Concentrations, volumes Coordinates Concentrations, volumes GFP-expressing lentiviral vector One bilateral injection at AP bregma + 1 mm, lateral: 0.75 mm, depth: 1 mm 0.5 μl of GFP lentiviral vector solution at 1.3 × 106 TU/μl per injection site 2 bilateral injections at AP bregma −0.5 mm and −1.5 mm, lateral: 2.5 mm, depth: 1 mm 0.5 μl of GFP lentiviral vector solution at 1.3 × 106 TU/μl per injection site Mix of GFP- and BDNF-expressing viral vectors One bilateral injection at AP bregma + 1 mm, lateral: 0.75 mm, depth: 1 mm 0.5 μl of solution containing 1.3 × 106 TU/μl of BDNF and 1.3 × 106 TU/μLGFP lentiviral vectors per injection site 2 bilateral injections at AP bregma −0.5 mm and −1.5 mm, lateral: 2.5 mm, depth: 1 mm 0.5 μl of solution containing 1.3 × 106 TU/μl of BDNF and 1.3 × 106 TU/μLGFP lentiviral vectors per injection site rBDNF One bilateral injection at AP bregma + 1 mm, lateral: 0.75 mm, depth: 1 mm using a 26-gauge preimplanted double-guide (Plastic One) 0.5 μl of a solution at 1 μg/μl; controls: 0.5 μl saline CTX-B One bilateral injection at AP bregma + 1 mm, lateral: 0.75 mm, depth: 1 mm using a 26-gauge preimplanted double-guide (Plastic One) 0.5 μl of a 10 μg/μl solution dissolved in saline; controls: 0.5 μl saline Volumes and concentrations of vectors or drugs, and also the coordinates of injections are indicated for each type of experiment.
