Vitamin B Complex Treatment Attenuates Local Inflammation after Peripheral Nerve Injury

Abstract

Peripheral nerve injury (PNI) leads to a series of cellular and molecular events necessary for axon regeneration and reinnervation of target tissues, among which inflammation is crucial for the orchestration of all these processes. Macrophage activation underlies the pathogenesis of PNI and is characterized by morphological/phenotype transformation from proinflammatory (M1) to an anti-inflammatory (M2) type with different functions in the inflammatory and reparative process. The aim of this study was to evaluate influence of the vitamin B (B1, B2, B3, B5, B6, and B12) complex on the process of neuroinflammation that is in part regulated by l-type CaV1.2 calcium channels. A controlled transection of the motor branch of the femoral peripheral nerve was used as an experimental model. Animals were sacrificed after 1, 3, 7, and 14 injections of vitamin B complex. Isolated nerves were used for immunofluorescence analysis. Treatment with vitamin B complex decreased expression of proinflammatory and increased expression of anti-inflammatory cytokines, thus contributing to the resolution of neuroinflammation. In parallel, B vitamins decreased the number of M1 macrophages that expressed the CaV1.2 channel, and increased the number of M2 macrophages that expressed this channel, suggesting their role in M1/M2 transition after PNI. In conclusion, B vitamins had the potential for treatment of neuroinflammation and neuroregeneration and thereby might be an effective therapy for PNI in humans.

Keywords: M1/M2 macrophages; calcium channels; neuroinflammation; peripheral nerve injury; regeneration; vitamin B complex therapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Effect of peripheral nerve injury (PNI) and treatment with B vitamins on macrophage morphology. Cross sections of femoral nerve obtained from the sham (S), operated (O, transection of motor branch and immediate reconstruction using termino–terminal anastomosis), and operated and treated with vitamin B complex (B1, B2, B3, B5, B6, and B12) (OT) groups were stained for ED1 (red) which is a common marker of activated macrophages. (A) The representative images showed morphological changes of ED1+ macrophages during the postoperative period (1, 3, 7, and 14 days) and after 1, 3, 7, and 14 injections of complex of B vitamins. Transition from the M1 (arrow heads) to M2 (arrows) morphology type in the O group is seen at day 7 and 14 post-injury. After treatment with B vitamins the appearance of M2 macrophages began after the third injection. Scale bar: 50 µm. (B) Total quantification of ED1-positive cells/mm2 from experimental groups is depicted in the graphs (black bars). The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 vs. control, or vs. O group, as indicated at the graphs).

Figure 2

Treatment with B vitamins reduced PNI-induced expression of proinflammatory mediators in M1 macrophages. (A,C) Cross sections of femoral nerves obtained from the operated (O) and operated and treated with vitamin B complex (OT) groups were counterstained with (A) anti-TNF-α (M1 marker, green) and anti-ED1 (red) or with (C) anti-iNOS (M1 marker, green) and anti-ED1 (red) antibodies. The quantification of double-positive ED1+/TNF-α+ cells (B) and ED1+/iNOS + cells (D) is presented as number of double-positive cells/mm2 and the percentage of double-positive cells in the ED1+ cell population. The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 OT vs. O group, as indicated at the graphs). At day 1 and 3 days post operation (dpo) ED1+/TNF-α+, as well as ED1+/iNOS+ macrophages in both the O and OT groups, had oval and round morphology and showed complete overlapping (yellow fluorescence)(inserts). Treatment with B vitamins reduced TNF-α and iNOS staining and the majority of macrophages were only ED1+ (red arrow head). At day 7 post-injury most of macrophages were only ED1+ and were polarized toward M2 type (white arrows, insert), while only a few ED1+/TNF-α+ (white arrow head, insert) macrophages were noticed. Some ED1− cells (yellow arrows) that were both TNF-α+ and iNOS+ were also noticed. # indicates where the high magnification images in inserts are taken from. Scale bar: 100 µm. PNI: peripheral nerve injury; TNF: tumor necrosis factor; iNOS: inducible nitric oxide synthase.

Figure 3

Effects of PNI and B vitamin treatment on expression of anti-inflammatory cytokines in M2 macrophages. (A,C) Cross sections of femoral nerve obtained from the operated (O) and operated and treated with vitamin B complex (OT) groups were counterstained with (A) anti-IL4 (M2a marker, green) and anti-ED1 (red) or with (C) anti-IL10 (M2c marker, green) and anti-ED1 (red) antibodies. The quantification of double-positive ED1+/IL-4+ cells (B) and ED1+/IL-10+ cells (D) is presented as number of double-positive cells/mm2 and the percentage of double positive cells in the ED1+ cell population. The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 OT vs. O group, as indicated at the graphs). At day 3 post-injury ED1+/IL-4+ as well as ED1+/IL-10+ macrophages with oval and round morphology in both the O and OT groups showed complete overlapping (yellow fluorescence, white arrow head) (inserts). Treatment with B vitamins increased IL-4 immunoreactivity in “foamy” M2 macrophages (white arrow), while IL-10 staining was reduced and the majority of M2 macrophages were only ED1+ (white arrow). At 7 and 14 dpo IL-4 and IL-10 staining was seen in ED1− cells in both groups (yellow arrows). M2 macrophages were void of IL-4 at day 7, but IL-4 was abundantly present at day 14 (white arrow). ED1+/IL-10+ M2 macrophages were seen at 7 dpo (white arrows), but were sparsely present at day 14. # indicates where the high magnification images in inserts are taken from. Scale bar: 100 µm. PNI: peripheral nerve injury; IL-4: interleukin -4; IL-10: interleukin-10.

Figure 4

Treatment with vitamin B complex induces time-dependent changes of CaV1.2 channel expression in M1 macrophages after PNI. To evaluate cellular distribution of the CaV1.2 isoform of l-VDCCs (green), cross sections of femoral nerve obtained from the: (A) operated (O); and (B) operated and treated with vitamin B complex (OT) groups were counterstained with anti-TNF-α (M1 marker, blue) and anti-ED1 (red) antibodies. The quantification of single-, double-, and triple-positive cells is presented as number of ED1+ cells/mm2, ED1+/TNF-α+ cells/mm2, and ED1+/TNF-α+/CaV1.2+ cells/mm2 (C), and as the percentage of triple -positive cells (ED1+/TNF-α+/CaV1.2+ cells) in ED1+ and ED1+/TNF-α+ cell populations (D). The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 OT vs. O group, as indicated at the graphs). Intensive CaV1.2 staining, besides in M1 macrophages, was observed in axons (green asterisks) and in some ED1− cells (yellow and green arrows) as well. ED1+/CaV1.2+/TNF-α+ M1 macrophages are marked with a white arrowhead, ED1+ macrophages with oval/round morphology (M1 type) are marked with a red arrowhead, and “foamy” ED1+ macrophages (M2) are indicated with white arrows. # indicates where the high magnification micrographs are taken from. Scale bars: 20 µm and 100 µm. PNI: peripheral nerve injury; TNF: tumor necrosis factor.

Figure 5

Treatment with vitamin B complex induces time-dependent changes in CaV1.2 channel expression in M2 macrophages after PNI. To evaluate cellular distribution of the CaV1.2 isoform of the L type of voltage-dependent calcium channels (l-VDCCs (green)), triple immunofluorescence staining of femoral nerve cross sections obtained from the operated (O) and operated and treated with vitamin B complex (OT) groups at (A) 7 dpo and (B) 14 dpo was performed. Anti-CaV1.2 (green), anti-IL-10 (M2 marker, blue), and anti-ED1 (red) antibodies were used. The quantification of single, double, and triple-positive cells is presented as the number of ED1+ cells/mm2, ED1+/IL-10+ cells/mm2, and ED1+/IL-10+/CaV1.2+ cells/mm2 (C) and as the percentage of triple-positive cells (ED1+/ IL-10+/CaV1.2+ cells) in ED1+ and ED1+/IL-10+ cell populations (D). The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 OT vs. O group, as indicated at the graphs). In the O group at both time-points (7 and 14 dpo) ED1+ macrophages with either oval or “foamy” morphology (inserts, red arrowhead and white arrows, respectively) were not co-stained with CaV1.2 and IL-10. After treatment with 7 and particularly after 14 injections of B vitamins they were mostly ED1+/CaV1.2+/IL-10+ (inserts, white arrowheads and blue arrows, respectively). At 14 dpo in the OT group some of the “foamy” macrophages were ED1+/CaV1.2+/IL-10− (yellow arrowheads), and these IL-10− cells were ED1+/Arg-1+ (yellow, insert in the left lower corner). Intensive CaV1.2 staining was seen in axons (green asterisks) and in some ED1−/IL-10+ cells (yellow and green arrows). # indicates where the high magnification micrographs are taken from. Scale bars: 50 µm and 100 µm. PNI: peripheral nerve injury; IL-10: interleukin-10.