Neuroprotective Effects on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons: A Role for Microglia?

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone protects motoneurons from induced dendritic atrophy. We explored a potential mechanism for this induced atrophy and protection by testosterone, examining the microglial response to partial depletion of motoneurons. Motoneurons innervating the vastus medialis muscles of adult male rats were killed by intramuscular injection of cholera toxin‐conjugated saporin; some saporin‐injected rats were treated with testosterone. Microglia were later visualized via immunohistochemical staining, classified as monitoring or activated, and counted stereologically. Partial motoneuron depletion increased the number of activated microglia in the quadriceps motor pool, and this increase was attenuated with testosterone treatment. The attenuation in microglial response could reflect an effect of testosterone on suppressing microglia activation, potentially sparing motoneuron dendrites. Alternatively, testosterone could be neuroprotective, sparing motoneuron dendrites, secondarily resulting in reduced microglial activation. To discriminate between these hypotheses, following partial motoneuron depletion, rats were treated with minocycline to inhibit microglial activation. Motoneurons innervating the ipsilateral vastus lateralis muscle were later labeled with cholera toxin‐conjugated horseradish peroxidase, and dendritic arbors were reconstructed. Reduction of microglial activation by minocycline did not prevent induced dendritic atrophy following partial motoneuron depletion. Further, reduction of microglial activation by minocycline treatment resulted in dendritic atrophy in intact animals. Together, these findings indicate that the neuroprotective effect of testosterone on dendrites following motoneuron death is not due to inhibiting microglial activation, and that microglial activity contributes to the normal maintenance of dendritic arbors.     

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in peripheral nerve repair

Introduction: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxi?ed by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats. Materials and methods: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were ?xed in paraformaldehyde and cut in 20 micrometer slices by cryostat. Results: The expression of NGAL signi?cantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene. Discussion: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.     

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4⁺ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4⁺ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3^−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2^−/− (recombination activating gene-2-deficient) mice adoptively transferred CD4⁺ T-cells isolated from CCR3^−/− mice, but not in CCR3^−/− mice adoptively transferred CD4⁺ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4⁺ T-cell- and CCR3-mediated neuroprotection after FMN injury.     

Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg²⁺ or Mn²⁺ was essential for maximum phosphorylation although Zn²⁺, Co²⁺, and Ca²⁺, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.     

Time Course and Characteristics of the Capacity of Sensory Nerves to Reinnervate Skin Territories outside Their Normal Innervation Zones

Factors involved in the outcome of regeneration of the saphenous nerve after a cut or crush lesion were studied in adult rats with electrophysiological recordings of low-threshold mechanoreceptor activity and plasma extravasation of Evans blue after electrical nerve stimulation that activated C fibers.

In the first series of experiments, saphenous and sciatic nerve section was combined with anastomosis of the transected proximal end of the saphenous nerve to the distal end of the cut tibial nerve. Regeneration of saphenous nerve fibers involved in plasma extravasation and low-threshold mechanoreceptor activity in the glabrous skin was observed 13 weeks after nerve anastomosis. Substance P-, calcitonin gene-related peptide-, and protein gene product 9.5 (PGP-9.5)-immunoreactive (IR) thin epidermal and dermal nerve endings, as well as coarse dermal PGP-9.5-IR nerve fibers and Meissner corpuscles and Merkel cell-neurite-like complexes, were observed in the reinnervated glabrous skin at this time.

In a second series of experiments, the time course of the regeneration of saphenous nerve axons to the permanently sciatic-nerve-denervated foot sole was examined. Saphenous-nerve-induced plasma extravasation and low-threshold mechanoreceptor activity in the saphenous nerve were found in the normal saphenous nerve territory 2, 3, 4, and 6 weeks after sciatic nerve cut combined with saphenous nerve crush in the left hindlimb. Saphenous-nerve-induced plasma extravasation was also present in the glabrous skin normally innervated by the sciatic nerve 3, 4, and 6 weeks after the sciatic cut/saphenous crush lesion. However, no low-threshold mechanoreceptor activity was detected in the saphenous nerve when the glabrous skin area was stimulated.

In a third series of experiments, the fate of the expansion of the saphenous nerve territory after saphenous nerve crush was examined when the crushed sciatic nerve had been allowed to regenerate. Nerve fibers involved in plasma extravasation were observed in the glabrous skin of the hindpaw after saphenous nerve, as well as after tibial nerve, C-fiber stimulation 3, 12, and 43 weeks after the saphenous crush/sciatic crush lesion.

Low-threshold mechanoreceptors from the regenerated saphenous nerve, which primarily innervates hairy skin, seem to be functional in the glabrous skin if the axons are guided by the transected tibial nerve by anastomosis. Furthermore, the results indicate that fibers from the regenerating saphenous nerve that have extended into denervated glabrous skin areas can exist even if sciatic nerve axons are allowed to grow back to their original territory.     

A novel inositol-containing glycosphingolipid isolated from human peripheral nerve

An inositol-phosphate-containing glycosphingolipid, not reported earlier, was isolated from human cauda equina. Structural characterization showed the glycosphingolipid to be inosital-phosphoryl-2(3) galactosylceramide. The concentration varied between 25 and 30 nmol/g fresh tissue.