Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43)

Abstract

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.

Fig. 1

GD17. PGP-immunonegative (a) and GAP-immunoreactive (IR) (b) fibres (arrows) in the perichondrium of the proximal epiphysis of the cartilagineous primordium. PE – proximal epiphysis, PC – perichondrium, V – vein, SF – subpopliteal fossa.

Fig. 5

PD1. CGRP-IR (a) and GAP-IR (b) fibres in the periosteum (PO) of the diaphysis: thick fibres and bundles in the outer fibrous layer (arrows), thin fibres and terminals in the deep cellular lining (arrowheads). Note some very weakly GAP-IR fibres in the inner layer (asterisk). PO – periosteum, D – diaphysis, M – skeletal muscles.

Fig. 9

PD1. CGRP-IR (a) and GAP-IR (b) fibres accompanying a blood vessel in the intercondylar eminence of the tibia (arrows). TPE – tibial proximal epiphysis, FDE – femoral distal epiphysis, IE – intercondylar eminence.