View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Acta Orthopaedica 2010; 81 (5): 639–646 639 Neuropeptide Y innervation during fracture healing and remodeling A study of angulated tibial fractures in the rat Hua Long, Mahmood Ahmed, Paul Ackermann, André Stark, and Jian Li Section of Orthopaedics, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Corresponding author: [email protected] Submitted 09-02-20. Accepted 10-03-26 Background and purpose Autonomic neuropeptide Y (NPY) is notype of the neurotransmitters (Bjurholm et al. 1988, Hill et involved in local bone remodeling via the central nervous system. al. 1991). This strongly supports the peripheral role of neu- However, the role of peripheral neuronal NPY in fracture healing ropeptides in bone physiology. Recently, the role of neural is not known. We investigated the relationship between bone heal- signaling by the central nervous system in bone remodeling ing and side-specific occurrence of NPY in angular and straight through various pathways has been given some attention. One fractures. such pathway is the neuropeptide Y (NPY) system. NPY, a Methods Tibial fractures in Sprague-Dawley rats were fixed 36 amino acid peptide, belongs to a class of peptides that with intramedullary pins in straight alignment and anterior includes pancreatic polypeptide (PP) and peptide-YY (PYY), angulation. The samples were analyzed by radiography, histol- which are expressed in the central and peripheral autonomic ogy, and immunohistochemistry (IHC) between 3 and 56 days nervous system. This system signals through 5 distinct recep- postfracture. tors (Y1, Y2, Y4, Y5, and Y6) expressed in various tissues. Results In the angular fractures, radiography and histology NPY has several functions: among these are effects on energy showed a 3.5-fold increase in callus thickness on the concave side homeostasis, food intake, immunity, and the cardiovascular compared to the convex side at day 21, whereas a 0.2-fold reduc- system (Lin et al. 2004). Recently, an important role of NPY tion in callus thickness was seen on the convex side between days in bone homeostasis has been identified through its effect 21 and 56. IHC showed regenerating NPY fibers in the callus and on Y2 and Y1 receptors: a study in mice with hypothalamic woven bone in both fractures at day 7. In angular fractures, a deletion of Y2 receptors showed a generalized increase in 5-fold increase in NPY fibers was observed on the concave side bone formation (Baldock et al. 2002). In contrast to this cen- compared to the convex side at 7 days, whereas a 6-fold increase tral action of Y2 receptors, the presence of Y1 receptors on in NPY fibers was seen on the convex side between 21 and 56 days; osteoblasts suggests a direct local effect on these cells (Lun- only a 0.1-fold increase in NPY fibers was seen on the concave side dberg et al. 2007). It has been shown that mice deficient in during the same time period. In straight fractures, similar bony Y1 receptors have a high bone mass (Baldock et al. 2007). and neuronal changes were observed on both sides. Whether it occurs through NPY-ergic pathways emanating Interpretation The increase in NPY innervation on the convex from the hypothalamus or through skeletal NPY innervation side appears to correlate with the loss of callus thickness on the has not been determined. same side in angular fractures. Our results highlight the probable In the bone, NPY-immunoreactive fibers have been identi- function of the peripheral NPY system in local bone remodeling. fied in areas of high osteogenic potential such as bone marrow and the periosteum (Bjurholm et al. 1988, Hill et al. 1991). These nerve fibers were found to be arranged as networks, mainly around blood vessels. Experimental studies in vivo The ability of bone to maintain its structures and proper- have shown that NPY acts as a potent vasoconstrictor of blood ties or to remodel after trauma under different conditions is vessels in bone (Lindblad et al. 1994), but its role in local bone well known, but the underlying neurobiological mechanisms turnover is not clear. Moreover, under conditions of trauma it are poorly understood. The bone has a rich supply of nerve is not known whether NPY participates in different biological fibers, which can be classified as belonging to the sensory, events such as inflammation, cell proliferation, angiogenesis, autonomic, and opioid nervous systems according to the phe- vasoregulation, and callus formation. Open Access - This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the source is credited. DOI 10.3109/17453674.2010.504609 640 Acta Orthopaedica 2010; 81 (5): 639–646 Figure 1. Lateral view of tibia at day 0 and day 35 postfracture, fixed in straight alignment (A) and anterior angulation (B). The broken squares in the X-ray images (panels A and B) depict the dis- sected sample for microscopic analysis of longitudinal sections shown in the schematic illustration (C). The small dotted squares in the drawing of the dissected sample represent the 8 micro- scopic fields studied in each tissue sec- tion for morphological and semi-quanti- tative analysis of NPY immunoreactivity. D = day. Recently, we reported a rat model of angulated tibial fracture and 56 after fracture (Figure 1A and B). The radiographic that showed side-specific changes in bone formation on the set-up was adjusted to result in size enhancement of the image concave side and bone resorption on the convex side of defor- by 33%. Callus size was then measured manually on a 300% mities in the same bone (Li et al. 2004, 2007). The process magnified printout. The total callus size was assessed by mea- of bone formation/resorption in this animal model, leading to suring the transverse diameter (in mm) of the callus and the bone healing and remodeling, might be affected by peripheral thickness of cortical bone anterior and posterior to the nail at expression of NPY because of its role in skeletal homeostasis. the level of the apex of the angulated needle. We therefore assessed the temporal and side-specific expres- sion of NPY at the fracture site during healing and remodeling Sampling by morphological and semiquantitative analysis using immu- The rats were anesthetized with sodium pentobarbitone (60 nohistochemistry. mg/kg, i.p.). 4 rats in each fracture group were killed on days 3, 7, 21, 35, and 56, while the 4 control rats with intact tibias were killed on day 21. In vivo intra-aortic perfusion was car- ried out with phosphate-buffered saline (PBS), followed by Materials and methods fixation with Zamboni’s buffered 4% paraformaldehyde solu- 44 male Sprague-Dawley rats with a mean weight of 200 g tion containing 0.2% picric acid in 0.2 M Sörensen phosphate were housed 3 per cage with free access to standard rat chow buffer, pH 7.2. The right tibia was dissected and then demin- and water under controlled temperature and a 12-hour light/ eralized at room temperature in a solution containing 7% dark cycle. All animal experiments were performed with AlCl3, 5% formic acid, and 8.5% HCl for 12 h. The samples approval from the Ethics Committee for Animal Research, were soaked in 20% sucrose for at least 2 days. Each demin- Stockholm North. The rats were allocated to 3 groups as eralized tibia was divided sagittally in two halves and 2-cm follows: 20 rats with a tibial fracture in anterior angulation long samples of the medial half of the tibial diaphysis, which (group A); 20 rats with a tibial fracture in straight alignment included the proximal, middle, and distal parts of the healing (group B); and 4 control rats with intact tibia (group C). fracture, were collected for analysis. Subsequently, the 2-cm long samples were sectioned at a thickness of 15 µm using Surgery a cryostat. Thus, the regions undergoing endochondral and The rats were anesthetized with an intraperitoneal (i.p.) injec- intramembranous ossification were all included in the same tion of fentanyl-fluanisone (Hypnorm, 0.5 mL/kg). The right section. tibia was first weakened at the mid-diaphysis by percutaneous drilling with an 18-G needle. A small anteromedial skin inci- Immunohistochemistry sion was made proximal to the tibial tubercle and a 17-G can- Serial sections were numbered consecutively from the middle nula needle was inserted through the proximal metaphyseal to the medial aspect. Two-interval sections, i.e. one close to cortex into the medullary canal as described previously (Li the middle part and another close to the medial part of the et al. 2004). The tibia was then fractured by manual 3-point tibia at each time point, were chosen for H & E staining and bending and fixed in either 40° anterior angulation or straight for immunostaining according to the biotin-avidin system. alignment, which in both cases included the normal 13° ana- Thus, the sections were hydrated in 0.01 M PBS for 10 min tomical curve of the diaphysis. and then incubated with 10% normal goat serum at room tem- perature for 30 min. Subsequently, the sections were incu- Radiography bated at room temperature overnight in a humid atmosphere The progress of fracture healing was monitored on lateral with polyclonal antibody to NPY (1:5,000; Peninsula Labo- radiographs under Hypnorm anesthesia on day 0, 7, 21, 35, ratories, San Carlos, CA). After rinsing in PBS (2 × 5 min), Acta Orthopaedica 2010; 81 (5): 639–646 641 the sections were incubated with biotinylated goat anti-rabbit data was skewed, the results are presented as mean, median, antibody (1:250; Vector Laboratories, Burlingame, CA) for 40 and range (minimum and maximum).