Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways

Abstract

Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair.

Fig 1. Body weight variation and bone healing progression in femur-defect vs sham-operated mice.

The body weight variation (a) and bone healing progression (b) were evaluated in the femur-defect and sham-operated animals at day 1, 3 and 7 post-surgery. (a) Femur-defect and sham-operated mice presented no differences in body weight evolution. (b) The progression of bone healing after non-critical femur defect was evaluated in histological sections of femur stained with Masson’s trichrome. Day 1 was characterized by the presence of a high number of polimorphonucleated cells (black arrowhead) in a dense network of fibers (white arrow) in the defect (dashed circle). At day 3, the defect was filled by the granulation tissue, and at day 7 high amounts of collagen (*) were observed in the extracellular matrix. Bone trabeculae (TB) were observed in the defect area at day 21 post-defect. Upper line, Scale bar = 100 μm; Middle line, Scale bar = 50 μm; Lower line, Scale bar = 10 μm.

Fig 2. NPY system within bone microenvironment is responsive to bone defect.

NPY (a) and Y1R (b) mRNA expression levels were assessed at days 1, 3 and 7 post-surgery in femurs from femur-defect and sham-operated mice. (a) Femur-defect mice displayed 2.5-fold higher NPY mRNA expression levels at day 1 and (b) 2-fold higher Y1R mRNA expression levels at day 3 as compared to sham-operated animals. In (a) and (b) each column represents the mean + SEM, for 8 animals per group. *p<0.050; ** p<0.010. NPY (c) and Y1R (d) immunoreactivity were assessed in femoral histological sections from femur-defect and sham-operated mice. Within the defect, polymorphonuclear cells stained for NPY (c) and Y1R (d) were observed at day 1 post-defect. NPY-positive and Y1R-positive cells were also observed within the granulation tissue at day 3. At day 7 a high number of NPY- and Y1R-positive osteoblasts (c and d, respectively) were observed surrounding the newly formed bone. Panel (e) shows NPY and Y1R immunoreactivity in the areas adjacent to the defect. NPY-positive nerve fibers were observed alongside blood vessels in the bone (1; white arrowhead) and in the bone marrow (2; white arrowhead), and also scattered in the bone marrow (3; white arrowhead); NPY and Y1R immunoreactivity was also observed for osteoblasts (4 and 6, respectively; thin white arrow), osteocytes (1 and 6, respectively; thick white arrow) and bone marrow cells (5 and 6, respectively; dashed arrow). * indicates bone tissue. Panel (c) and (d)- scale bar = 10 μm; Panel (e)- scale bar = 50 μm.

Fig 3. Impact of bone defect on the NPY neuronal pathways in the sensory nervous system.

The mRNA expression levels of NPY (a), Y1R (b) and Y2R (c) in the DRG from femur-defect and sham-operated mice were analysed at day 1, 3 and 7 post-surgery. (a) NPY mRNA expression presented a 4-fold increase from day 1 to day 3 post-surgery in both femur-defect and sham-operated mice, which was sustained at day 7. (b) At day 3, femur-defect mice displayed a decrease in the Y1R mRNA expression to half of the expression levels found in sham-operated animals. (c) Y2R mRNA expression showed no differences between femur-defect and sham-operated mice. In (a), (b) and (c) each column represents the mean + SEM, for 8 animals per group. *p<0.050; ** p<0.010; *** p<0.005. Panel (c) and (d) show NPY and Y1R immunoreactivity in DRG sections, respectively. The quantification of NPY and Y1R staining intensity was performed and is shown in (e) and (h), respectively. Femur-defect mice displayed higher NPY immunoreactivity in the DRG at day 1 and 3 as compared to sham-operated (d and e). The Y1R immunoreactivity was also higher in femur-defect mice at day 1, and increased between day 1 and day 3 in both femur-defect and sham-operated mice (g and h). Panel (d) scale bar = 50 μm; Panel (g) scale bar = 25 μm In (e) and (h) each column represents the mean + SEM, for 3 animals per group. *p<0.050; ** p<0.010; *** p<0.005.

Fig 4. The hypothalamic NPY neuronal pathway is targeted during bone repair.

The mRNA expression levels of NPY (a), Y1R (b) and Y2R (c) in the hypothalamus of femur-defect and sham-operated mice were assessed at days 1, 3 and 7 post-surgery. (a) Femur-defect mice presented a 2.5-fold higher NPY mRNA expression at day 1 and (c) a 2-fold lower Y2R mRNA expression at day 7, as compared to sham-operated mice. (b) No differences were observed in the Y1R mRNA expression between femur-defect and sham-operated mice. In (a), (b) and (c) each column represents the mean + SEM, for 8 animals per group. *p<0.050, *** p<0.005. Panel (d) shows NPY immunoreactivity in the hypothalamic section and in (e) the data from the quantitative analysis of the NPY staining intensity is provided. Femur-defect mice presented an increase in the NPY staining intensity in the arcuate nucleus from day 1 to day 3 post-surgery. Scale bar = 200 μm. In (e) each column represents the mean + SEM, for 3 animals per group. ** p<0.010;.