Ivanusic Lab ~ Current Projects

1. Central projections of primary afferents fibres of bony origin

It is generally assumed that the afferents of bone follow the same path into the CNS as do afferents of cutaneous and muscular tissues. However, no studies have been undertaken to identify the central projections of afferents from bone or periosteum. It is also unclear whether the neurotransmitters and neuropeptides contained within bone-associated afferents are the same as in other fibre types, such as cutaneous, joint or muscle afferents. The aim of this project is to determine the location of the central projections of bone-associated afferents, and to investigate the neurochemistry of these neurons. This will be achieved by placing small volumes of neuro-anatomical tracers into a number of bony tissue locations (e.g., cortical, medullary and periosteal). After an appropriate survival time and subsequent processing, the distribution of labeled fibres will be traced within the central nervous system using light microscopy. Co-localization of markers for common neurotransmitters and neuropeptides with the neuro-anatomical tracer will be examined using immuno-histochemistry.

Project 2 – Central pathways that mediate bone pain

Whilst Project 1 will provide valuable data regarding the first part of the pathways that relay sensory information from bony tissues to the brain, it will not allow for investigation of the second and third order neurons of the pathways because the neuro-anatomical tracers used are incapable of moving across synapses. The specific aim of project 2 is to determine which parts of the CNS are involved, beyond the primary afferent fibres, in the relay of sensory information from bone to the brain. This will be achieved by examining the distribution of FOS expression in the CNS following stimulation of the rat tibia with electrical and mechanical stimuli, as well as known irritants such as mustard oil. FOS is a protein marker for neuronal activity and its distribution following activation of neuronal pathways with stimuli such as those discussed above can be examined following a standard immuno-histochemical staining procedure. This animal model further provides a unique opportunity to examine the central activity patterns of neurons that are specifically activated by painful stimuli. We have results which indicate that increased FOS expression is produced in a number of dorsal horn lamina in the spinal cord following noxious mechanical stimulation of bone, and intend to combine this with retrograde tracing techniques to determine the target of these activated cells. Co-localization of markers for common neurotransmitters and neuropeptides with the FOS protein will also be examined with the aim of identifying putative local neuronal circuits involved in the transmission of information about bone pain.

Project 3 - The spinal segmental innervation of bone (the sclerotomes)

The ‘sclerotome’ is defined as a region of the bony skeleton that is innervated by a single spinal nerve, much like a dermatome and myotome for skin and musculature respectively. Knowledge of the dermatomes and myotomes is used regularly by surgeons, medical clinicians, physiotherapists, chiropractics and osteopaths in discussions of a variety of presenting problems, but sclerotomes have been largely ignored in such discussions due to lack of a definitive description in the past. The specific aim of this project is to produce a definitive map of the spinal segmental innervation of the bony skeleton. To provide an accurate representation of the sclerotomes, small volumes of neuro-anatomical tracers will be injected into a single dorsal root ganglion (DRG) of rats. After an appropriate survival time and subsequent processing to visualize the tracer, the distribution of labeled fibres in bony tissue will be identified, therefore delineating the sensory innervation of bone associated with a given DRG and spinal segmental nerve.  The spinal segmental innervation of the whole of the bony skeleton will be reconstructed after injections are made in single DRG all the way along the vertebral column. The project will provide a clear basis for understanding the segmental patterns of bone pain and guide the development of therapies to treat it.

Project 4 – The response characteristics of primary afferent fibres of bony origin

The electrophysiological response characteristics of single sensory neurons in skin, and to a lesser degree muscle, have been studied extensively in the past. Although preliminary observations have revealed that primary afferent fibres arising from the periosteum are responsive to focal mechanical stimulation, the extent to which this mechano-sensitivity is innocuous or nociceptive, and the extent to which individual primary afferent fibres display the capacity to respond to many different stimuli, remains untested. It is also unclear whether the sensitivity of these fibres can be altered during inflamed and pathological conditions. In order to examine these questions, the responses of individual fibres arising from the periosteum will be examined using mechanical, thermal and chemical stimuli, during normal, inflamed and pathological conditions.
To achieve this, electrophysiological investigations of individual, bone-associated primary afferent fibres will be made in a nerve-periosteum preparation. The activity of single fibres arising from the periosteum will be selectively recorded in response to precisely controlled, reproducible mechanical stimuli, local application of known algesic substances (eg. potassium ions, hydrogen ions, bradykinin, substance P and capsaicin) and noxious and innocuous thermal stimulation. The response properties and receptive fields of the fibres will be assessed under normal and experimentally-induced conditions such as inflammation.