Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies

Incidence, level and distribution of PACAP mRNA expression in DRG neurons after delayed antibody infusion.

<p>Representative scatterplots depicting relative changes in PACAP mRNA hybridization signal over individual neurons in relation to neuronal size after delayed control IgG (A) or anti-BDNF (B) infusions. Each dot represents hybridization signal intensity over a quantified neuron. Solid lines divide the plots into unlabeled (shaded) and labeled (≥6X background labeling) populations, and dashed lines separate moderately from highly labeled populations (≥32 X background). Numbers and percentages of DRG neurons expressing detectable PACAP mRNA after delayed intrathecal control (C) or anti-BDNF (D) antibody treatment for 3 days following 7 days of injury (n = 6, 3 animals analysed per treatment group). Note, delayed intrathecal anti-BDNF infusion did not have any significant effect on the injury-induced increase in PACAP mRNA expression after axotomy.</p

Alternate approaches supporting BDNF regulation of injury-associated PACAP expression.

<p>(A) Impact of BDNF siRNA infusion on PACAP mRNA expression in injured DRG neurons. Darkfield photomicrographs of L5 DRG sections processed for in situ hybridization to detect PACAP mRNA in intact sensory neurons (Intact), in association with 3d sciatic spinal nerve injury (Injury), 3d injury + infusion of either non-targeting control siRNA (Injury + NT siRNA) or siRNA targeting BDNF mRNA (Injury + siBDNF) as indicated. Note, there is no detectable difference in injury-induced PACAP expression after infusion of non-targeting control siRNA as compared to the Injury alone group, whereas animals treated with siBDNF, display decreased neuronal PACAP expression. Scale bar 100 µm. (B) qRT-PCR analysis of mRNA samples extracted from L4,5 DRG having undergone unilateral sciatic spinal nerve injury with or without either immediate or one week delayed 3 day intrathecal infusion of anti-BDNF or control IgG (Ctrl IgG) as indicated (performed in triplicate). Note that immediate anti-BDNF treatment significantly impairs the induction of injury-associated PACAP expression, while control IgG infusion has no effect. Delayed anti-BDNF does not significantly impact maintenance of PACAP expression in injured sensory neurons (***p<0.001).</p

Neuronal injury-induced upregulation of PACAP mRNA expression is not discernibly impacted by delayed anti-BDNF infusion.

<p>Darkfield photomicrographs depicting PACAP mRNA expression in DRG neurons (A, B) and spinal cord motor neurons (C, D) after 10 day sciatic spinal nerve transection in conjunction with delayed intrathecal control IgG (A, C) or anti-BDNF (B, D) infusions for 3 days, 7 days after injury. Note, delayed anti-BDNF infusion does not visibly affect PACAP mRNA signal intensity over DRG or spinal cord motor neurons. Scale bar 200 µm.</p

Incidence, level and distribution of PACAP mRNA expression in DRG neurons after immediate antibody infusion.

<p>Representative scatterplots depicting relative changes in PACAP mRNA hybridization signal over individual neurons in relation to neuronal size after sciatic spinal nerve transection and immediate intrathecal control IgG (A), or anti-BDNF (B) infusion. Each dot represents signal intensity over an individual neuron as a function of neuronal size. Solid lines divide the plots into unlabeled (shaded), and labeled (≥6 X background labeling) populations, and dashed lines separate moderately from highly labeled (≥32 X background) neuronal populations. (C, D) STEP I -incidence of PACAP expression was determined for all neurons analysed for the Control IgG infused group (C; n = 4 animals or 1369 neurons) or anti-BDNF infused group (D; n = 4 animals or 1168 neurons). STEP II –impact of treatment on the incidence of PACAP expression in small (<35 µm) versus medium-large (>35 µm) DRG neurons was determined for immediate intrathecal infusions of either Control IgG (C; n = 2 or 482 neurons) or anti-BDNF (D; n = 2 or 416 neurons) for the animals that had undergone computer-assisted image analysis. Note: the injury-induced increase in PACAP mRNA expression is significantly prevented by immediate intrathecal anti-BDNF infusion (p<0.001, chi-square test). A reduction is observed both in the number of small and medium-large diameter DRG neurons expressing detectable PACAP mRNA, as well as at the level of hybridization signal/neuron.</p

PACAP mRNA expression is reduced in injured sensory and motor neurons after immediate anti-BDNF infusion.

<p>Darkfield photomicrographs of PACAP mRNA hybridization signal over sensory neurons in L5 DRG sections (A, B) and spinal cord motor neurons (C, D) in response to 3 days sciatic spinal nerve transection in conjunction with immediate 3 days infusion of intrathecal control IgG (A, C) or anti-BDNF (B, D) infusions. The increase in PACAP mRNA expression observed after nerve transection is markedly reduced by anti-BDNF infusion. Both the incidence of neurons expressing detectable PACAP mRNA and the level of expression in individual neurons are decreased. Scale bar (A, B) 100 µm, (C, D) 200 µm.</p

Intrathecally infused anti-BDNF and control IgGs effectively penetrate DRG tissue.

<p>Brightfield photomicrographs of L5 DRG sections with or without antibody infusions, depicting antibody penetration into intact DRG or 3 days following sciatic spinal nerve injury. Both control sheep IgGs and sheep anti-BDNF antibodies readily penetrate the tissue after intrathecal delivery showing immunoreactivity both in the parenchyma and the surrounding connective tissue in intact (B, C) and injured (E, F) animals. Note, no staining for sheep IgGs is found in DRGs from animals not receiving antibody infusions (A, D). Scale bar 100 µm.</p