Ivanusic Lab ~ Current Projects
Project 1 - Neuro-chemical phenotype of primary afferent neurons that innervate bone
 Figure 1 |
It is generally assumed that the primary afferents neurons that innervate bone have a similar distribution and neuro-chemical phenotype as primary afferents neurons that innervate cutaneous and muscular tissues. However, it is 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 investigate the distribution and neuro-chemical phenotype of primary afferent neurons that innervate bone and to compare them to neurons that innervate skin. 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) or skin. After an appropriate survival time and subsequent processing, the distribution of labelled neurons in the peripheral sensory ganglia (DRG) will be determined using fluorescence microscopy, and the neuro-chemical phenotype of these neurons will be examined by using immuno-histochemical labelling of common neurotransmitters and neuropeptides. Changes in the neuro-chemical phenotype of these neurons will also be examined following inflammation of bone, as such changes may reflect mechanisms that are (at least in part) responsible for chronic or persistent pain associated with inflammation. Students can expect to gain experience in animal handling, anaesthesia and surgery, dissection, histological and immuno-histochemical processing, and fluorescence microscopy. |
Figure 1 - Photomicrographs of DRG sections following injections of Fast Blue in the rat tibia. The left panel shows retrograde labeled soma (A-D) and the adjacent photographs show the same fields viewed in order to reveal neuronal SP-IR (E), CGRP-IR (F), IB4 binding (G) or NF200-IR (H). Asterisks indicate retrograde labeled, SP-IR, CGRP-IR, IB4 binding or NF200-IR neurons. Arrows indicate examples of double-labeled cells.
Project 2 - Investigation of the central nervous system areas associated with bone pain
Whilst Project 1 will provide valuable data regarding the first part of the pathways that relay sensory information from bone 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 aim of project 2 is to determine which parts of the CNS are involved, beyondthe primary afferent neurons, 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 mechanical stimuli, and following inflammation. 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. We have results which show increased FOS expression in neurons of the dorsal horn of the spinal cord following noxious mechanical stimulation of bone, have combined this approach with retrograde tracing techniques to determine the target of these activated neurons, and plan to continue using these approaches to further define brain areas associated with noxious stimulation of bone. 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. Students can expect to gain experience in animal handling, anaesthesia and surgery, dissection, histological and immunohistochemical processing, and fluorescence microscopy.
 Figure 2 |
Figure 2 - (A, B, C) Photomicrographs of the same field of the superficial dorsal horn of the lumbar spinal cord, presented in pseudo-colour, showing Fos-LI nuclei (arrowheads in A), Fluorogold labelling (arrowheads in B), and colocalization of Fos-LI and Fluorogold (arrowhead in C). Scale bars in A, B and C = 20 µm. (D) Low power image of Fos-LI in the dorsal horn. Dorsal is at the top and lateral to the left of the image. SDH, superficial dorsal horn (lamina I and II). DDH, deep dorsal horn (lamina III-VI). Scale bar = 200 µm. (E) Graph of the percentage of retrogradely labelled neurons containing Fos-LI nuclei in the sham surgery and bone drilling groups following injections of Fluorogold into the lateral parabrachial nucleus. There was a significant increase (asterisk; P<0.05) in the percentage of Fluorogold-labelled cells that contained Fos-LI nuclei in the lumbar dorsal horn in the bone drilling group relative to the sham surgery group following spinoparabrachial injections. |
Project 3 - Developing an animal model of inflammatory bone pain
The broad objective of the work done in this laboratory is to determine the neural mechanisms that mediate bone pain, particularly through an investigation of the central pathways that relay information about pain from bone to the brain. This is an area that has received little attention in the past, probably because bone is viewed as a difficult tissue to work with, and also due to the lack of an appropriate behavioural model for investigation of pain associated with noxious stimulation of bone in experimental animals. The aim of this project is to develop a robust and consistent animal model of inflammatory bone pain that can be used to better understand inflammation induced changes in neurons that relay information about pain from bone to the brain. An important feature of this model will be the ability to measure indirect indices of pain to confirm that the animals are sensitive to the inflammation. Data from the behavioral experiments will be correlated with data collected in the experiments outlined in projects 1 and 2 above, and could provide a crucial platform to test targeted strategies to assist with the difficult task of alleviating bone pain. Students can expect to gain experience in animal handling, anaesthesia and surgery, dissection, and behavioural testing protocols.