460 Progress Towards A Phase 1/2 Trial Of Hsv‐Mediated Gene Transfer Of Gad In Patients With Painful Diabetic Neuropathy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90202/1/S1754-3207_10_70465-X.pd

Changes in intra-axonal calcium distribution following nerve crush

We used the oxalate-pyroantimonate method to demonstrate the ultrastructural distribution of calcium within rat sciatic nerve 4 h after a crush injury. In normal nerve there are discrete gradients of axoplasmic calcium precipitate with the amount of precipitate decreasing in the axoplasm beneath the Schmidt Lantermann clefts and in the paranodal regions at the node of Ranvier. Near the crush site a marked increase in endoneurial and intra-axonal calcium precipitate correlated with morphologic evidence of axonal degeneration. More distant from the crush site, both in the distal segment destined to degenerate and in the proximal segment destined to regenerate, the most prominent finding was a loss of the normal gradient of precipitate beneath the Schmidt Lantermann clefts. The calcium influx at the crush site corresponds to the known role of calcium in triggering degeneration. The alterations in the distal axon may be an early stage leading to degeneration. Alteration in calcium distribution in the proximal nerve stump may play a role in the regulation of the response to injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50077/1/480170508_ftp.pd

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy

Abstract Background Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms NaV1.7 and NaV1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in NaV1.7 protein levels in DRG in vivo. To further evaluate the role of NaVα subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against NaVα subunits. Results Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in NaVα subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia. Conclusions These data support the role of increased NaVα protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.http://deepblue.lib.umich.edu/bitstream/2027.42/112553/1/12990_2011_Article_484.pd

Immunocytochemical Demonstration of Na + ,K + -ATPase in Internodal Axolemma of Myelinated Fibers of Rat Sciatic and Optic Nerves

We used postembedding electron microscopic immunocytochemistry with colloidal gold to determine the ultrastructural distribution of Na + ,K + -ATPase in the sciatic and optic nerves of the rat. Using a polyclonal antiserum raised against the denatured catalytic subunit of brain Na + ,K + -ATPase, we found immunoreactivity along the internodal axolemma of myelinated fibers in both nerves. This antiserum did not produce labeling of nodal axolemma. These results suggest that an important site of energy-dependent sodium-potassium exchange is along the internodal axolemma of myelinated fibers in the mammalian CNS and PNS and that there may be differences between the internodal and nodal forms of the enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66444/1/j.1471-4159.1991.tb02114.x.pd

Tumor necrosis factor–Α contributes to below-level neuropathic pain after spinal cord injury

Objective Our objective was to elucidate the mechanisms responsible for below-level pain after partial spinal cord injury (SCI). Methods We used lateral hemisection to model central neuropathic pain and herpes simplex viral (HSV) vector–mediated transfer of the cleaved soluble receptor for tumor necrosis factor–Α (TNF-Α) to evaluate the role of TNF-Α in the pathogenesis of below-level pain. Results We found activation of microglia and increased expression of TNF-Α below the level of the lesion in the lumbar spinal cord after T13 lateral hemisection that correlated with emergence of mechanical allodynia in the hind limbs of rats. Lumbar TNF-Α had an apparent molecular weight of 27kDa, consistent with the full-length transmembrane form of the protein (mTNF-Α). Expression of the p55 TNF soluble receptor (sTNFRs) by HSV-mediated gene transfer resulted in reduced pain behavior and a decreased number of ED1-positive cells, as well as decreased phosphorylation of the p38 MAP kinase (p-p38) and diminished expression of mTNF-Α in the dorsal horn. Interpretation These results suggest that expression of mTNF-Α after injury is related to development of pain, and that reverse signaling through mTNF-Α by sTNFR at that level reduces cellular markers of inflammatory response and pain-related behavior. Ann Neurol 2006;59:843–851Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50655/1/20855_ftp.pd

Engineered gp120 immunogens that elicit VRC01-like antibodies by vaccination

Background: One of the great challenges for an HIV vaccine is to elicit broadly neutralizing antibodies specific for conserved epitopes from which the virus cannot easily escape. The CD4 binding site is one such epitope against which several antibodies (e.g. b12, VRC01) have been isolated. In macaques infected with SHIV, passive immunization with these CD4-directed neutralizing antibodies fails to control the virus, but prophylactic administration is highly protective. Similarly, patients who generate neutralizing antibodies over the course of an HIV infection derive no clinical benefit from them, but eliciting such antibodies prophylactically by vaccination may prevent the virus from establishing its lethal foothold

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7 or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially sightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26644/1/0000186.pd

A quantitative study of retrograde axonal transport in motor and sensory neurons

We used 3H N-succinimidyl propionate to covalently label in vivo proteins of the rat sciatic nerve, and studied the accumulation of radioactively labeled proteins in the cell bodies of the ipsilateral dorsal root ganglion and ventral horn of spinal cord to assess retrograde axonal transport in sensory and motor neurons respectively. In each case the early accumulation of a small amount of radioactively labeled protein is followed by the later accumulation of a larger amount, which subsequently declines to lower levels. The differences between accumulation in the motor neuron and sensory neuron are discussed. Quantitative assessment of retrograde axonal transport will allow future determination of alterations in that transport after nerve injury and in toxic states, which will help elucidate the role of retrogradely transported proteins in neuronal cell biology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26223/1/0000303.pd

Post-transcriptional regulation of GABAB receptor and GIRK1 channels by Nogo receptor 1

Abstract Background Type B GABA receptors (GABA Rs) play a critical role in synaptic transmission. We carried out studies to determine whether neuronal cell surface expression of GABAB-Rs might be regulated by the Nogo receptor 1 (NgR1). Results siRNA knock-down of NgR1 resulted in a selective increase of GABAB R1 and GABAB R2 protein without altering the expression of GABAA receptor or GAD65. The increase in GABAB receptor subunits was unaccompanied by a change in mRNA, but inhibition of mTOR by rapamycin blocked the increase in GABAB protein. NgR1 siRNA also caused an increase in G protein coupled inwardly rectifying potassium channel (GIRK1). The increase in GABAB receptor and GIRK1 channel proteins was in the plasma membrane, determined by cell surface biotinylation. In NgR1 knockout mice, the amount of GABAB R2 and GIRK1 in hippocampus-derived synaptosomes was increased. Conclusions Together these findings suggest that NgR1 mediated modulation of synaptic transmission may be accomplished, at least in part, through modulation of G protein coupled receptors and channels.http://deepblue.lib.umich.edu/bitstream/2027.42/112645/1/13041_2013_Article_223.pd

Cytochemical localization of Ca2+-ATPase activity in peripheral nerve

We used an electron microscopic cytochemical method to determine the localization of Ca2+-ATPase in rat peripheral nerve. We found that reaction product occurred along most cytoplasmic membranes in the dorsal root ganglia (DRG). Unmyelinated axons demonstrated reaction product on the axolemma diffusely along their length. Myelinated fibers, in contrast, had reaction product limited to the axolema in the paranodal region. Internodal axolemma never showed reaction product and nodal axolemma was only occasionally stained, usually in sections reacted for the maximum times. Schwann cell plasma membranes uniformly showed reaction product. The restricted localization of Ca2+-ATPase to the paranodal region of myelinated fibers suggests that calcium efflux may occur principally at those sites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27368/1/0000394.pd