Details of Research Program
The research program of the laboratory is focused on the pathogenesis of neuropathic pain. Focal injury to peripheral nerve that leaves it intact can produce painful hyperalgesia states referred to as neuropathic pain or causalgia. The mechanisms by which an acutely painful event can progress towards chronic pain are not known but there are certainly structural, physiologic, and biochemical changes in nerve that influence dorsal root ganglia and spinal cord neurons. Nerve injuries that give rise to hyperalgesia include the severe inflammatory neuropathies, and compression and traumatic neuropathies, especially those associated with a rapid onset of axonal degeneration. Common pathophysiologic features in these neuropathies seem to include reductions in nerve blood flow associated with endoneurial edema, increased endoneurial fluid pressure, and nerve fiber injury to both large and small fibers. These events are associated with activation of glial cells and monocytes/macrophages.The role of cytokines are especially interesting in the context of the pathogenesis of the neuropathy and the instigation of pain. Our working hypothesis is that focal nerve lesions resulting in axonal injury and Wallerian degeneration of the nerve fiber are key pathogenic events in the development of hyperalgesia. Cytokine factors liberated by macrophages and Schwann cells in the course of Wallerian degeneration may be the signal that alters neuronal function, predisposing the sensory system to hyperalgesia. Recent work is focused on the role of tumor necrosis factor alpha (TNF) in neuropathy and pain. Studies include analysis of TNF receptor binding and release by matrix mettaloproteinases. We have observed that a) inhibition of macrophage recruitment to the site of nerve injury reduces the onset and duration of hyperalgesia, b) blockade of TNF production by macrophages reduces hyperalgesia, c) Schwann cells normally produce TNF protein and upregulate its production following nerve injury, d) TNF applied to nerve causes neuropathic pain behaviors, e) TNF causes ectopic firing of pain fibers at the site of nerve injury, f) TNF produces neuropathologic features of painful neuropathies, and g) changes in TNF receptor binding by matrix metalloproteinases precede TNF protein upregulation.
Major Laboratory Methods
The laboratory is organized to integrate results from different methodologies, each of which provide complementary insight into the pathogenesis of disease states. Techniques include in vivo measurements of physiologic variables and behavioral states, in vitro studies of neurons and Schwann cells in culture, and extensive capabilities in histology and molecular biology.
Nerve Blood Flow - Studies of blood flow in peripheral nerves, dorsal root ganglia (DRG), and spinal nerve roots have been pioneered in the laboratory using several techniques. Presently we use laser Doppler methodology to assess changes in blood flow and 14C-iodoantipyrine autoradiography for more quantitative studies.
Endoneurial Fluid Pressure - EFP methodology was developed in the laboratory using microcirculatory equipment developed at UCSD. The technique uses 4-micron diameter micropipettes to quantify tissue (interstitial) fluid pressure. These studies have identified a biomechanical link between increases in EFP and reduced blood flow to the nerve. The technique has also been used to support the hypothesis that the proximo-distal transport of endoneurial fluid is driven by a gradient in EFP from the DRG to the nerve terminals.
Electrophysiology - Routine electrophysiological studies (conduction velocity) are performed in rodent preparations. Collaborative studies with Dr. Linda Sorkin extend these methods to allow for single fiber recording.
Axonal Transport - The laboratory has a standing interest in retrograde axonal transport of substances that influence the function and/or anatomy of the DRG and dorsal horn centers for processing of sensory information.Thermal Hyperalgesia - The thermal nociceptive threshold is routinely characterized using our modification of the Hargreaves method.
Mechanical Allodynia - von Frey hairs are used to characterize the mechanical sensitivity of receptive fields.Tissue Processing - The laboratory is fully equipped to process tissue for light- and electron-microscopy using frozen sections or sections from specimens embedded in plastic. Paraffin embedded sections are also routinely processed for GLP and immunohistochemistry studies. Microwave fixation techniques are currently being developed.
Immunohistochemistry - Immunohistochemical detection of proteins and receptor complexes are routinely performed in frozen or paraffin sections. EM immunohistochemistry and IHC of semi-thin sections embedded in plastic are also performed. Non-radioactive in situ hybridization techniques are available.
ELISA and Western Blotting - We use these antibody specific methods to visualize and quantify the presence of proteins or other compounds of interest. These techniques are facilitated by quantitative PCR (Stratagene) and other molecular biology tools in the laboratory.
Cell Culture - We maintain a cell culture facility within the laboratory for study of sensory neurons and Schwann cells.
Gelatin Zymography - Zymography techniques are used to identify matrix metalloproteases. Of special interest is the MMP control of TNF receptors.
Image Analysis - The laboratory is fully equiped for digital image analysis using Silicon Graphics and Macintosh computers coupled to sensitive digital cameras mounted on Leica and Olympus microscopes.Quantitative morphometry is performed with statistical sampling techniques.