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

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

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

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

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

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

Calpain II in rat peripheral nerve

We used a polyclonal antiserum directed against calpain II to study the distribution of that enzyme in rat sciatic nerve. Western blot of nerve homogenate showed that the antibody reacted with a single protein band of 80 kDa, corresponding to the catalytic subunit of calpain II. By light microscopy, immunoreactivity appeared predominantly in Schwann cell cytoplasm. By electron microscopy, calpain II was especially dense along the plasmalemma of Schwann cells, and was also seen in axoplasm.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29028/1/0000060.pd

Phosphorylation-dependent neurofilament epitopes are reduced at the node of Ranvier

Neurofilaments in axons are highly phosphorylated at multiple sites on the 200 kDa neurofilament (neurofilament-H) and 160 kDa (neurofilament-M) subunit peptides. We used a panel of monoclonal and polyclonal antibodies against distinct neurofilament epitopes to study the distribution of these epitopes along the axons of large myelinated fibres in rat sciatic nerve using quantitative electron microscopic immunocytochemistry with colloidal gold. Antibodies specific for phosphorylated epitopes on neurofilament-H showed a 60% reduction in density of immunoreactivity at the node of Ranvier, compared to the internodal axon. Antibodies directed against neurofilament-M, which recognized phosphorylated epitopes preferentially, showed a 40% reduction in density of immunoreactivity at the node. Following dephosphorylation of the neurofilaments in tissue sections by alkaline phosphatase treatment, antibodies which recognized dephosphorylated forms of neurofilament-H showed no reduction in density of immunoreactivity at the node. Similarly, an antibody directed against the 70 kDa subunit (neurofilament-L), showed no reduction in density of immunoreactivity at the node.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47426/1/11068_2005_Article_BF01194978.pd

Reduction of Voltage Gated Sodium Channel Protein in DRG by Vector Mediated miRNA Reduces Pain in Rats with Painful Diabetic Neuropathy

BackgroundPainful 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.ResultsSubcutaneous 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.ConclusionsThese 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