Pattern of Porcine Intervertebral Disk by Sensory Innervation-A Preliminary Retrograde Neuronal Tract-Tracing Study

Abstract

Introduction The problem of intervertebral disk degeneration has been approached from many sides, from orthopedic surgery to molecular biology, and the scientific literature on the subject is particularly diverse. For many intervertebral disk diseases, model system plays an important role in clarifying pathomechanisms and testing novel therapeutic techniques. The goal of this study was to elucidate the detailed origin pattern of sensory nerve fibres within the porcine lumbar intervertebral disk, to provide the basis for further experiments focusing on the physiology, pathophysiology and neurogenically-driven regenerative potential of this organ in a new animal model. Materials and Methods Six juvenile female pigs were injected with 350 µmL of a mixture composed of 30 µmL of 5% aqueous solution of a fluorescent retrograde tracer Fast Blue (FB) and 320 µmL of a radiologic contrasting agent (Omnipaque; 300 mg Iohexolum/mL; Nycomed, Ireland) into the intervertebral disk located between the L4 and L5 lumbar vertebra. The position of the needle was checked radiologically before the deposition of the solution into the pulpous nucleus of the disk under study. After 2 weeks survival period (an optimal time for FB transport to the parent perikarya of nerve terminals supplying the disk) all animals were sacrificed, transcardially perfused with freshly prepared 4% buffered paraformaldehyde and bilateral dorsal root ganglia (DRGs). Th13 to Cq1 were collected, cryoprotected and then sectioned on a cryostat into 10-µmL-thick serial sections. The presence, distribution, and the diameter of retrogradely labelled DRG neurons were investigated under the BX61 fluorescence microscope (Olympus Poland) in each section. Results After application of the tracer into the nucleus pulpous of the L4/L5 intervertebral disk, FB-positive sensory neurons were found in bilateral ThL1-6 DRGs. The average number of FB-positive cells supplying the injected disk yielded approximately 930 cells per animal (ranging from 713 to 1148). The relative frequency of FB-labelled neurons in particular DRGs studied was as follow: L1 0.9%, L2 4.5%, L3-11.5%, L4-63%, L5-11.8, and L6-3.3 (taking all traced neurons found as 100%). Furthermore, it has been found that traced cells belonged to two morphologically distinct classes of DRG neurons: a vast majority of them were small diameter, whereas only few belonged to the class of large-diameter (up to 50µm) neurons. For example, the most of FB + /CGRP + small-size neurons were also immunoreactive to P Conclusion The present study clearly shows that the pattern of porcine intervertebral disk sensory innervation is very complicated and originated from numerous DRGs along the spinal cord. As the present data are preliminary one, and we don't know, as to whether there are any differences in the chemical coding ( = neurotransmitter(s) synthetized and used) of disk-projecting neurons in particular DRGs, it is hard to predict any physiological relevance of the presence of traced sensory cells in such a distant DRG as, Th13 or S2. However, as DRG neurons of different diameter are thought to fulfill different physiological functions, one may suggest that the “small” FB-positive perikarya are involved in conveying nociceptive information from the disk, while the “large” ones are mechanoceptors rather. However, further studies are needed to clarify both the chemical coding of the disk-projecting DRG neurons, as well as to elucidate their physiological relevance for maintaining the disk homeostasis. Moreover, this study provides for the first time data concerning the sources and distribution pattern of sensory neurons supplying an intervertebral disk in the domestic pig, an animal that may be used as a new animal model for studying physiological and pathophysiological processes within this organ. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared