Axotomy

For years it has been known that unilateral optic nerve lesions induce a bilateral response that causes an inflammatory and microglial response in the contralateral un-injured retinas. Whether this contralateral response involves retinal ganglion cell (RGC) loss is still unknown. We have analyzed the population of RGCs and the expression of several genes in both retinas of pigmented mice after a unilateral axotomy performed close to the optic nerve head (0.5 mm), or the furthest away that the optic nerve can be accessed intraorbitally in mice (2 mm). In both retinas, RGC-specific genes were down-regulated, whereas caspase 3 was up-regulated. In the contralateral retinas, there was a significant loss of 15% of RGCs that did not progress further and that occurred earlier when the axotomy was performed at 2 mm, that is, closer to the contralateral retina. Finally, the systemic treatment with minocycline, a tetracycline antibiotic that selectively inhibits microglial cells, or with melo...

Axonal injury is a common feature of central nervous system insults that culminates with the death of the affected neurons, and an irreversible loss of function. Inflammation is an important component of the neurodegenerative process, where the microglia plays an important role by releasing proinflammatory factors as well as clearing the death neurons by phagocytosis. Here we have identified the purinergic signaling through the P2X7 receptor as an important component for the neuronal death in a model of optic nerve axotomy. We have found that in P2X7 receptor deficient mice there is a delayed loss of retinal ganglion cells and a decrease of phagocytic microglia at early times points after axotomy. In contralateral to the axotomy retinas, P2X7 receptor controlled the numbers of phagocytic microglia, suggesting that extracellular ATP could act as a danger signal activating the P2X7 receptor in mediating the loss of neurons in contralateral retinas. Finally, we show that intravitreal a...

To analyze the long-term effect of optic nerve injury on retinal ganglion cells (RGCs) and melanopsin+RGCs orthotopic and displaced, and on the rest of the ganglion cell layer (GCL) cells. In adult albino rats, the left optic nerve was crushed (ONC) or transected (ONT). Injured and contralateral retinas were analyzed at increasing survival intervals (up to 15 months). To study all GCL cells and RGCs, retinas were immunodetected with Brn3a and melanopsin to identify the general RGC population (Brn3a+) and m+RGCs, and counter-stained with 4',6-diamidino-2-phenylindole (DAPI). Brn3a+RGCs and m+RGCs displaced to the inner nuclear layer were analyzed as well. In additional retinas, glial cells in the GCL were identified with glial fibrillary acidic protein (GFAP) or Iba1, and in some retinas, Brn3a, calretinin, and γ-synuclein were immunodetected. Orthotopic and displaced RGCs behave similarly within the RGC and m+RGC populations. Both lesions cause an exponential loss of RGCs (4%-1%...

Axotomized central neurons of most invertebrate species demonstrate a strong regenerative capacity, and as such may provide valuable molecular insights and new tools to promote axonal regeneration in injured mammalian neurons. In this study, we identified a novel molluscan protein, caltubin, ubiquitously expressed in central neurons ofLymnaea stagnalisand locally synthesized in regenerating neurites. Reduction of caltubin levels by gene silencing inhibits the outgrowth and regenerative ability of adultLymnaeaneurons and decreases local α- and β-tubulin levels in neurites. Caltubin binds to α- and/or β-tubulin in bothLymnaeaand rodent neurons. Expression of caltubin in PC12 cells and mouse cortical neurons promotes NGF-induced axonal outgrowth and attenuates axonal retraction after injury. This is the first study illustrating that a xenoprotein can enhance outgrowth and prevent degeneration of injured mammalian neurons. These results may open up new avenues in molecular repair strate...

Aim: Neurodegeneration caused by the axonal injury is a widely seen phenomenon in spinal cord and traumatic brain injuries. Due to the disintegration of the synaptic connection neurotrophic factors could not be transported retrogradely towards the cell body and the deprivation of the trophic factors lead to the degeneration and death of the injured neuron. Rifampicin is an antibiotic exhibiting several neuroprotective functions in various neurodegenerative conditions. Here we aim to investigate the acute neuroprotective effect of rifampicin in primary cortical neuron culture in which neurons are axotomized by laser axotomy.Methods: Neonatal male mice were used in order to isolate cortical neurons. Isolated primary cortical neurons were cultured. After 24 hours three different rifampicin concentrations (1 μM, 10 μM and 100 μM) were applied to the neurons and after 15 minutes of rifampicin addition, neurons were laser axotomized. Viability of the cells was evaluated by propidium iodid...

Agonists of the vanilloid receptor transient vanilloid potential 1 (TRPV1) are emerging as highly efficacious nonopioid analgesics in preclinical studies. These drugs selectively lesion TRPV1+ primary sensory afferents, which are responsible for the transmission of many noxious stimulus modalities. Resiniferatoxin (RTX) is a very potent and selective TRPV1 agonist and is a promising candidate for treating many types of pain. Recent work establishing intrathecal application of RTX for the treatment of pain resulting from advanced cancer has demonstrated profound analgesia in client-owned dogs with osteosarcoma. The present study uses transcriptomics and histochemistry to examine the molecular mechanism of RTX action in rats, in clinical canine subjects, and in 1 human subject with advanced cancer treated for pain using intrathecal RTX. In all 3 species, we observe a strong analgesic action, yet this was accompanied by limited transcriptional alterations at the level of the dorsal roo...

Tetanus neurotoxin (TeNT) cleaves synaptobrevin, a protein involved in synaptic vesicle docking and fusion, thereby preventing neurotransmitter release and causing a functional deafferentation. We injected TeNT into the lateral rectus muscle of adult cats at 0.5 or 5 ng/kg (low and high dose, respectively). In the periphery, TeNT slightly slowed motor axon conduction velocity, and at high doses, partially blocked neuromuscular transmission. TeNT peripheral actions displayed time courses different to the more profound and longer-lasting central actions. Central effects were first observed 2 days postinjection and reversed after 1 mo. The low dose induce depression of inhibitory inputs, whereas the high dose produce depression of both inhibitory and excitatory inputs. Simultaneous recordings of eye movement and neuronal firing revealed that low-dose injections specifically reduced inhibition of firing during off-directed saccadic movements, while high-dose injections of TeNT affected ...

Sympathetic neurons in the superior cervical ganglion (SCG) of adult rats depend on target-derived nerve growth factor (NGF) for maintenance of tyrosine hydroxylase (TH) levels and the noradrenergic neurotransmitter system. Axotomy of a SCG results in NGF deprivation, causing a decline in TH activity; continuous local application of NGF can prevent this decline in TH activity. We now report that injection of a defective herpes simplex virus 1 vector that expresses NGF (pHSVngf) into a SCG can prevent the decline in TH activity that follows axotomy. SCG of adult rats were injected with either pHSVngf virus or pNFlac virus, which expresses Escherichia coli beta-galactosidase. Analysis of RNA from pHSVngf-infected SCG indicated that the NGF gene was efficiently transcribed and processed. Furthermore, 4 days after pHSVngf injection animals underwent axotomy of the virus-injected SCG. After another 10 days, animals were sacrificed and both the injected-axotomized and contralateral contro...

Distal to a peripheral nerve transection, myelin degradation and Schwann cell (SC) proliferation are accompanied by a marked upregulation of brain-derived neurotrophic factor (BDNF) and a decrease of ciliary neurotrophic factor (CNTF) in non-neuronal cells. To investigate the role of SC differentiation in trophic factor regulation, we studied BDNF and CNTF expression in sciatic nerves fromTrembler-J(Tr-J) mice. In these animals, a mutation in thepmp-22gene causes a failure of myelination and continuous SC proliferation, but axonal continuity is preserved. In spite of the severe abnormalities inTr-Jnerves, BDNF levels remained as low as in the intact controls. Thus, the primary SC disorder inTr-Jproduces a different pattern of BDNF expression from that caused by axonal breakdown due to nerve transection. Furthermore, the upregulation of BDNF mRNA triggered by transection was 70-fold in control nerves, but only 30-fold inTr-Jsciatic nerves. Because these results raised the possibility...

Galanin, one of the most inducible neuropeptides, is widely present in developing brains, and its expression is altered by pathologic events (e.g., epilepsy, ischemia, and axotomy). The roles of galanin in brain development under both normal and pathologic conditions have been hypothesized, but the question of how galanin is involved in fetal and early postnatal brain development remains largely unanswered. In this study, using granule cell migration in the cerebellum of early postnatal mice (both sexes) as a model system, we examined the role of galanin in neuronal cell migration during normal development and after brain injury. Here we show that, during normal development, endogenous galanin participates in accelerating granule cell migration via altering the Ca2+and cAMP signaling pathways. Upon brain injury induced by the application of cold insults, galanin levels decrease at the lesion sites, but increase in the surroundings of lesion sites. Granule cells exhibit the following...

Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Tα1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors.In situhybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, ...

Autocrine trophic functions of brain-derived neurotrophic factor (BDNF) have been proposed for many central neurons because this neurotrophin displays striking colocalization with its receptor trkB within the CNS. In the cortex, the distribution patterns of BDNF and trkB expression are almost identical. Corticospinal neurons (CSNs) are a major cortical long-distance projecting system. They are localized in layer V of the somatosensory cortex, and their axons project into the spinal cord where they contribute to the innervation of spinal motoneurons. We have shown recently that adult CSNs express trkB mRNA and are rescued from axotomy-induced death by BDNF treatment. Half of the axotomized CSNs survived without BDNF infusions. These findings raise the possibility that endogenous cortical BDNF is involved in the trophic support of this neuronal population. To test the hypothesis that endogenous cortical BDNF promotes survival of adult CSNs, we infused the BDNF-neutralizing affinity-purified antibody RAB to axotomized and unlesioned CSNs for 7 d. This treatment resulted in increased death of axotomized CSNs. Survival of unlesioned CSNs was not affected by RAB treatment. In situ hybridizations for BDNF and trkB mRNA revealed that virtually all CSNs express trkB, whereas only half of them express BDNF. Thus, autocrine/paracrine mechanisms are likely to contribute to the endogenous BDNF protection of axotomized CSNs. We have demonstrated previously that, in addition to BDNF, glial cell line-derived neurotrophic factor (GDNF) and neurotrophin 3 (NT-3) also rescue CSNs from axotomy-induced death. We now show that the rescuing by GDNF requires the presence of endogenous cortical BDNF, implicating a central role of this neurotrophin in the trophic support of axotomized CSNs and a trophic cross-talk between BDNF and GDNF regarding the maintenance of lesioned CSNs. In contrast, NT-3 promotes survival of axotomized CSNs even when endogenous cortical BDNF is neutralized by RAB, indicating a potential of compensatory mechanisms for the trophic support of CSNs.

The Nogo-66 receptor (NgR) plays a critical role in restricting axon regeneration in the central nervous system. This inhibitory action is in part mediated by a neuronal receptor complex containing p75NTR, a multifunctional receptor also well known to trigger cell death upon binding to neurotrophins such as NGF. In the present study, we show that Pep4 and NEP1–40, which are two peptides derived from the Nogo-66 sequence that modulate NgR-mediated neurite outgrowth inhibition, prevent NGF-stimulated p75NTR-dependent death of cultured embryonic motor neurons. They also confer protection on spinal cord motor neurons after neonatal sciatic nerve axotomy. These findings demonstrate an as-yet-unknown function of NgR in maintaining neuronal survival that may be relevant for motor neuron development and degeneration.