Ells to generate four types of BM chimeric mice (TRPM2BM+/Rec+, TRPM2BM? Rec+ , TRPM2BM+/Rec? TRPM2BM?Rec?mice) (see Materials and Methods for details). Flow cytometric analysis revealed that, 6 weeks after BM transplantation, more than 90 of the BMderived cells in the blood of the chimeric mice were replaced with GFP+ cells (Fig. 1). In TRPM2BM+/Rec+ mice, partial sciatic nerve ligation (pSNL) surgery significantly decreased the 50 withdrawal threshold to mechanical stimulation in the ipsilateral paw (p,0.001, Kruskal-Wallis test). Significant decreases were observed 3, 7 and 14 days after pSNL surgery, compared with presurgery (Day 0). In TRPM2BM?Rec+, TRPM2BM+/Rec? and TRPM2BM?Rec?mice, pSNL-induced mechanical allodynia was attenuated. In TRPM2BM?Rec+ mice, although pSNL 24195657 surgery significantly decreased the 50 withdrawal threshold in the ipsilateral paw (p,0.001, Kruskal-Wallis test), a significant decrease was observed only 3 days, but not 7 and 14 days, after pSNL surgery, compared with pre-surgery (Day 0). Compared with TRPM2BM+/Rec+ mice, pSNL-induced mechanical allodynia was significantly attenuated on Day 7 and 14. In TRPM2BM+/Rec?and TRPM2BM?Rec?mice, pSNL surgery had no effect on the 50 withdrawal threshold in the ipsilateral paw, and no significant mechanical allodynia was observed during the observed period, compared with pre-surgery (Day 0). Compared with TRPM2BM+/ Rec+ mice, pSNL-induced mechanical allodynia was significantly attenuated on Day 3 and 7 in TRPM2BM+/Rec?mice, and on Day 3 in TRPM2BM?Rec?mice. On the other hand, the 50 withdrawal thresholds in the contralateral paws were not changed in any chimeric mice (Fig. 2).Figure 2. Peripheral nerve injury-induced mechanical allodynia in WT/TRPM2-KO BM chimeric mice. In the pSNL model of neuropathic pain, the 50 withdrawal threshold to mechanical Title Loaded From File stimuli was determined in the ipsilateral (A) and contralateral paws (B) of TRPM2BM+/Rec+, TRPM2BM?Rec+, TRPM2BM+/Rec? and TRPM2BM+/Rec+ mice. *p , 0.05; **p , 0.01; ***p , 0.001, compared with TRPM2BM+/Rec+ mice. #p ,0.05; ##p ,0.01, compared with Day 0 in each BM chimeric mouse group. n = 5?. Data are expressed as the mean 6 SEM. doi:10.1371/journal.pone.0066410.gImmunohistochemistryMice were deeply anesthetized with sodium pentobarbital and Title Loaded From File perfused through the ascending aorta with PBS followed by 4 (W/V) paraformaldehyde in phosphate buffer. The sciatic nerve was cut 5 mm on either side of the ligation site, and the L3 5 spinal cord was extirpated from pSNL-induced neuropathic pain model mice. Then samples were postfixed for 4 h and cryoprotected overnight at 4uC in 15 sucrose. The tissues were frozen and sectioned with a cryostat (Leica, Nussloch, Germany). The sections (20 mm) were treated with 4 normal goat serum for 1 h at room temperature. After washing with PBS, the sections were incubated with a primary antibody directed against Iba-1 (rabbit anti-Iba-1 antibody, 1:500; Wako Pure Chemical Industries, Osaka, Japan) at 4uC overnight. The sections were washed three times in PBS and labeled with fluorescence-labeled secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG, 1:500; Molecular Probes, Invitrogen, Life Technologies, Carlsbad, 17460038 CA) at room temperature for 1 h in the dark. After washing three times in PBS, the sections were mounted in the anti-fading medium Vectashield (Vector Laboratories, Burlingame, CA). Five nonadjacent sections of the sciatic nerve and the L3 5 spinal cord were randomly sele.Ells to generate four types of BM chimeric mice (TRPM2BM+/Rec+, TRPM2BM? Rec+ , TRPM2BM+/Rec? TRPM2BM?Rec?mice) (see Materials and Methods for details). Flow cytometric analysis revealed that, 6 weeks after BM transplantation, more than 90 of the BMderived cells in the blood of the chimeric mice were replaced with GFP+ cells (Fig. 1). In TRPM2BM+/Rec+ mice, partial sciatic nerve ligation (pSNL) surgery significantly decreased the 50 withdrawal threshold to mechanical stimulation in the ipsilateral paw (p,0.001, Kruskal-Wallis test). Significant decreases were observed 3, 7 and 14 days after pSNL surgery, compared with presurgery (Day 0). In TRPM2BM?Rec+, TRPM2BM+/Rec? and TRPM2BM?Rec?mice, pSNL-induced mechanical allodynia was attenuated. In TRPM2BM?Rec+ mice, although pSNL 24195657 surgery significantly decreased the 50 withdrawal threshold in the ipsilateral paw (p,0.001, Kruskal-Wallis test), a significant decrease was observed only 3 days, but not 7 and 14 days, after pSNL surgery, compared with pre-surgery (Day 0). Compared with TRPM2BM+/Rec+ mice, pSNL-induced mechanical allodynia was significantly attenuated on Day 7 and 14. In TRPM2BM+/Rec?and TRPM2BM?Rec?mice, pSNL surgery had no effect on the 50 withdrawal threshold in the ipsilateral paw, and no significant mechanical allodynia was observed during the observed period, compared with pre-surgery (Day 0). Compared with TRPM2BM+/ Rec+ mice, pSNL-induced mechanical allodynia was significantly attenuated on Day 3 and 7 in TRPM2BM+/Rec?mice, and on Day 3 in TRPM2BM?Rec?mice. On the other hand, the 50 withdrawal thresholds in the contralateral paws were not changed in any chimeric mice (Fig. 2).Figure 2. Peripheral nerve injury-induced mechanical allodynia in WT/TRPM2-KO BM chimeric mice. In the pSNL model of neuropathic pain, the 50 withdrawal threshold to mechanical stimuli was determined in the ipsilateral (A) and contralateral paws (B) of TRPM2BM+/Rec+, TRPM2BM?Rec+, TRPM2BM+/Rec? and TRPM2BM+/Rec+ mice. *p , 0.05; **p , 0.01; ***p , 0.001, compared with TRPM2BM+/Rec+ mice. #p ,0.05; ##p ,0.01, compared with Day 0 in each BM chimeric mouse group. n = 5?. Data are expressed as the mean 6 SEM. doi:10.1371/journal.pone.0066410.gImmunohistochemistryMice were deeply anesthetized with sodium pentobarbital and perfused through the ascending aorta with PBS followed by 4 (W/V) paraformaldehyde in phosphate buffer. The sciatic nerve was cut 5 mm on either side of the ligation site, and the L3 5 spinal cord was extirpated from pSNL-induced neuropathic pain model mice. Then samples were postfixed for 4 h and cryoprotected overnight at 4uC in 15 sucrose. The tissues were frozen and sectioned with a cryostat (Leica, Nussloch, Germany). The sections (20 mm) were treated with 4 normal goat serum for 1 h at room temperature. After washing with PBS, the sections were incubated with a primary antibody directed against Iba-1 (rabbit anti-Iba-1 antibody, 1:500; Wako Pure Chemical Industries, Osaka, Japan) at 4uC overnight. The sections were washed three times in PBS and labeled with fluorescence-labeled secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG, 1:500; Molecular Probes, Invitrogen, Life Technologies, Carlsbad, 17460038 CA) at room temperature for 1 h in the dark. After washing three times in PBS, the sections were mounted in the anti-fading medium Vectashield (Vector Laboratories, Burlingame, CA). Five nonadjacent sections of the sciatic nerve and the L3 5 spinal cord were randomly sele.