Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis

Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA

Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

BACKGROUND: In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. METHODOLOGY/PRINCIPAL FINDINGS: We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. CONCLUSIONS/SIGNIFICANCE: We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord

Spatio-temporal pattern of induction of bradykinin receptors and inflammation in rat dorsal root ganglia after unilateral nerve ligation

Expression of bradykinin receptors was analyzed in freshly isolated dorsal root ganglion neurons of the ipsi- and contralateral segments L4/L5, L2/L3, and T12/T13 two to twenty days after unilateral injury of the adult rat sciatic nerve using gold labeled bradykinin. The number of infiltrating leucocytes was investigated by flow cytometry. Sciatic nerve injury transiently increased the proportion of neurons expressing bradykinin receptors not only in the ipsilateral ganglia L4/L5, but also in the homonymous contralateral ganglia and also bilaterally in the adjacent ganglia L2/L3. Neurons of the ganglia T12/T13 were not affected. The time course of upregulation was different between neurons of the injured nerve and uninjured ones. Furthermore, the proportion of neurons expressing a high density of receptors increased also bilaterally in ganglia L4/L5 and L2/L3. As on the ipsilateral side, the increase in neurons expressing bradykinin receptors in the contralateral homonymous ganglia was due to an induction of the B1 receptor subtype and an upregulation of the B2 subtype. As a possible source for stimulating factors for induction of bradykinin receptors the number of macrophages and lymphocytes was investigated two to twenty days after nerve ligation. No increase was observed prior to day ten and only in ipsilateral ganglia L4/L5, not contralaterally and not in adjacent ganglia L2/L3 and T12/T13. The experiments show that the induction of bradykinin receptors following a unilateral nerve lesion is not restricted to neurons projecting into the damaged nerve but is (i) bilateral, (ii) different in time course between injured and uninjured neurons, and (iii) locally confined to neurons of the adjacent ganglia. Macrophages and lymphocytes are increased after ten day ligation only in the affected ganglia and are probably not involved in the induction of bradykinin receptors

Research consortium Neuroimmunology and pain in the research network musculoskeletal diseases

The research consortium Neuroimmunology and Pain (Neuroimpa) explores the importance of the relationships between the immune system and the nervous system in musculoskeletal diseases for the generation of pain and for the course of fracture healing and arthritis. The spectrum of methods includes analyses at the single cell level, in vivo models of arthritis and fracture healing, imaging studies on brain function in animals and humans and analysis of data from patients. Proinflammatory cytokines significantly contribute to the generation of joint pain through neuronal cytokine receptors. Immune cells release opioid peptides which activate opioid receptors at peripheral nociceptors and thereby evoke hypoalgesia. The formation of new bone after fractures is significantly supported by the nervous system. The sympathetic nervous system promotes the development of immune-mediated arthritis. The studies show a significant analgesic potential of the neutralization of proinflammatory cytokines and of opioids which selectively inhibit peripheral neurons. Furthermore, they show that the modulation of neuronal mechanisms can beneficially influence the course of musculoskeletal diseases. Interventions in the interactions between the immune system and the nervous system hold a great therapeutic potential for the treatment of musculoskeletal diseases and pain