Nerve Axotomy In Mice Research Articles

Sexual Dimorphism of Early Transcriptional Reprogramming in Dorsal Root Ganglia After Peripheral Nerve Injury.

Peripheral nerve injury induces genome-wide transcriptional reprogramming of first-order neurons and auxiliary cells of dorsal root ganglia (DRG). Accumulating experimental evidence suggests that onset and mechanistic principles of post-nerve injury processes are sexually dimorphic. We examined largely understudied aspects of early transcriptional events in DRG within 24 h after sciatic nerve axotomy in mice of both sexes. Using high-depth RNA sequencing (>50 million reads/sample) to pinpoint sexually dimorphic changes related to regeneration, immune response, bioenergy, and sensory functions, we identified a higher number of transcriptional changes in male relative to female DRG. In males, the decline in ion channel transcripts was accompanied by the induction of innate immune cascades via TLR, chemokine, and Csf1-receptor axis and robust regenerative programs driven by Sox, Twist1/2, and Pax5/9 transcription factors. Females demonstrated nerve injury-specific transcriptional co-activation of the actinin 2 network. The predicted upstream regulators and interactive networks highlighted the role of novel epigenetic factors and genetic linkage to sex chromosomes as hallmarks of gene regulation post-axotomy. We implicated epigenetic X chromosome inactivation in the regulation of immune response activity uniquely in females. Sexually dimorphic regulation of MMP/ADAMTS metalloproteinases and their intrinsic X-linked regulator Timp1 contributes to extracellular matrix remodeling integrated with pro-regenerative and immune functions. Lexis1 non-coding RNA involved in LXR-mediated lipid metabolism was identified as a novel nerve injury marker. Together, our data identified unique early response triggers of sex-specific peripheral nerve injury regulation to gain mechanistic insights into the origin of female- and male-prevalent sensory neuropathies.

Facial nerve axotomy in mice: a model to study motoneuron response to injury.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Autophagy in retina and axonal degeneration

AbstractAutophagy is a catabolic pathway that degrades and recycles intracellular components inside lysosomes to maintain cell homeostasis. This pathway is essential to keep postmitic cells such are neurons free of garbaje and damaged cell components. There are several types of autophagy. During macroautophagy, simply called autophagy, parts of the cytosol and even entire organelles are engulfed in an autophagosome that will finally fuse with a lysosome where degradation will take place. In chaperone mediated autophagy (CMA) proteins harbouring a specific aminoacid sequence will be recognized by a chaperone Hsc70 and delivered inside lysosomes thanks to the lysosomal receptor LAMP‐2A. The role of autophagy in the physiology and pathology of the retina is just starting to be investigated. We have demonstrated the cytoprotective role of autophagy in a model of optic nerve axotomy in mice. We show that autophagy‐deficient animals have reduced number of surviving RGCs after ONT and that pharmacological induction of autophagy delays neuronal cell death. We have also observed a reduction in the levels of autophagy during retinal aging and Atg5 deficient animals in the retina show increased photoreceptor death and reduced scotopic vision. Unexpectedly CMA seems to increase in these conditions and we postulate that this is a compensatory response to sustain cell survival. Thus balance in autophagic pathways plays important roles to maintain retinal homestasis.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin+ and calcitonin gene-related peptide (CGRP)+ sprouts peaking at day 14, matching both large, neuropeptide+ subpopulations of axotomized facial motoneurons, but with CGRP+ sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin+ sprouts revealed a stronger response following cut compared with crush (day 7–14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12–48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting. J. Comp. Neurol. 518:699–721, 2010. © 2009 Wiley-Liss, Inc.

IL-15 and IL-15Rα gene deletion: Effects on T lymphocyte trafficking and the microglial and neuronal responses to facial nerve axotomy

IL-15 is a potent T cell chemoattractant, and this cytokine and its unique α subunits, IL-15Rα, can modify immune cell expression of several T cell chemokines and their receptors. Facial nerve axotomy in mice leads to T cell migration across an intact blood–brain-barrier (BBB), and under certain conditions T cells can provide neuroprotection to injured neurons in the facial motor nucleus (FMN). Although chemokines and chemoattractant cytokines are thought to be responsible for T cell migration to the injured cell bodies, data addressing this question are lacking. This study tested the hypothesis that T cell homing to the axotomized FMN would be impaired in knockout (KO) mice with the IL-15 and IL-15Rα genes deleted, and sought to determine if microglial responsiveness and motoneuron death are affected. Both IL-15KO and IL-15RαKO mice exhibited a marked reduction in CD3 + T cells and had fewer MHC2 + activated microglia in the injured FMN than their respective WT controls at day 14 post-axotomy. Although there was a relative absence of T cell recruitment into the axotomized FMN in both knockout strains, IL-15RαKO mice had five times more motoneuron death (characterized by perineuronal microglial clusters engulfing dead motoneurons) than their WT controls, whereas dead neurons in IL-15KO did not differ from their WT controls. Further studies are needed to dissect the mechanisms that underlie these observations (e.g., central vs. peripheral immune contributions).

Endogenous T lymphocytes and microglial reactivity in the axotomized facial motor nucleus of mice: Effect of genetic background and the RAG2 gene

Following facial nerve axotomy in mice, peripheral T cells home to the injured facial motor nucleus (FMN) where they may influence the glial response. Interactions between T cells and microglia, which proliferate in response to axotomy, appear to confer neuroprotection to injured motoneurons. The primary objective of this study was to determine whether T lymphocytes could influence the microglial reaction to motoneuron injury. These experiments tested the hypotheses that (1) C57BL/6 (B6) and 129 mice, inbred strains which have high and low levels of astroglial reactivity in the axotomized FMN, respectively, would also exhibit high and low levels of T cell infiltration, and (2) that these differences would correspond with levels of microglial reactivity and neuronal regeneration. Thus, we compared the response to facial nerve axotomy in B6, 129, and immunodeficient RAG2 knockout (RAG2 KO) mice on these two backgrounds at 14 day post-axotomy for differences in levels of 1) CD3 + T cell infiltration; (2) major histocompatibility complex II (MHC2) expression by microglia; (3) perineuronal microglial phagocytic clusters, an indirect measure of neuronal death; and (4) overall microglial activity as assessed by CD11b expression. To examine the inheritance pattern of the abovementioned neuroimmune measures, we also made assessments in B6x129 F1 generation mice. B6 and 129 mice displayed high and low levels of T cell infiltration to the affected FMN and low and high MHC2 expression, respectively. Levels of microglial activity did not differ between the two strains. In immunodeficient RAG2 KO mice on both backgrounds, the number of MHC2 + microglia did not differ from their immunologically normal background controls. Moreover, deletion of either the RAG2 or RAG1 genes in B6 mice was not associated with increased neuronal death at day 14 post-axotomy, as we had previously found in B6 mice with the severe combined immunodeficiency (SCID) mutation. Contrary to our hypothesis, the paucity of T cells in the affected FMN of the 129 mice was associated with less neuronal death when compared to B6 mice, which showed a robust T cell response. Moreover, the data suggest that parameters of the central and peripheral immune responses to axotomy are independently regulated. Assessments in B6x129 F1 generation mice revealed dominant phenotypes for both T cell infiltration and neurodegeneration, whereas both strains contributed significantly to the phenotype for MHC2 expression. Our findings suggest that (1) T cells do not appear to modify measures of microglial reactivity in the axotomized FMN; and (2) the impact of T cells on injured motoneurons in immunologically intact mice and in immunodeficient mice grafted with T cells by adoptive transfer may be different. Further study is required to understand the role of T cells following motoneuron injury in immunologically intact mice and how the seemingly divergent effects of T cells in intact and immunodeficient mice might provide insight into their role in neuronal injury and repair.

The expression of a motoneuron-specific serine protease, motopsin (PRSS12), after facial nerve axotomy in mice

Motopsin (PRSS12) is a mosaic serine protease that is preferentially expressed in motor neurons. To study the relationship between motopsin and motoneuron function, we investigated the expression of motopsin mRNA in facial nerve nuclei after facial nerve axotomy at the anterior margin of the parotid gland in mice. Neuronal function was monitored by assessing vibrissal motion in 3 months. Vibrissal behaviour on the injured side disappeared until the day 14 post-operation, and then recovered between the day 21 and 35. Motopsin expression decreased at the day 14, but markedly recovered by the day 21. In contrast, expression of growth-associated protein-43 (GAP-43) was induced at the day 3. These results suggest that the recovery of motopsin expression is correlated with the recovery of the facial motor neuronal function.

IL-2 gene knockout affects T lymphocyte trafficking and the microglial response to regenerating facial motor neurons

Following facial nerve axotomy in mice, T cells cross the intact blood–brain barrier (BBB), home to nerve cell bodies in the facial motor nucleus (FMN), and augment neuroregenerative processes. The pivotal T cell immunoregulatory cytokine, IL-2, appears to have bidirectional effects on neuronal and microglial cell function, suggesting rival hypotheses that IL-2 could either enhance or disrupt processes associated with regeneration of axotomized facial motor neurons. We tested these competing hypotheses by comparing the effect of facial nerve axotomy on C57BL/6-IL-2 −/− knockout and C57BL/6-IL-2 +/+ wild-type littermates. Since IL-2 may also be produced endogenously in the brain, we also sought to determine whether differences between the knockout and wild-type mice were attributable to loss of IL-2 gene expression in the CNS, loss of peripheral sources of IL-2 and the associated effects on T cell function, or a combination of these factors. To address this question, we bred novel congenic mice with the SCID mutation (mice lacking T cell derived IL-2) that were homozygous for either the IL-2 knockout or wild-type gene alleles (C57BL/6 scid-IL-2 −/− and C57BL/6 scid-IL-2 +/+ littermates, respectively). Groups were assessed for differences in (1) T lymphocytes entering the axotomized FMN; (2) perineuronal CD11b + microglial phagocytic clusters, a measure of motor neuron death; and (3) activated microglial cells as measured by MHC-II positivity. C57BL/6-IL-2 −/− knockout mice had significantly higher numbers of T cells and lower numbers of activated MHC-II-positive microglial cells in the regenerating FMN than wild-type littermates, although the number of CD11b + phagocytic microglia clusters did not differ. Thus, despite the significant impairment of T cell function known to be associated with loss of peripheral IL-2, the increased number of T cells entering the axotomized FMN appears to have sufficient activity to support neuroregenerative processes. Congenic C57BL/6 scid-IL-2 −/− knockout mice had lower numbers of CD11b + microglial phagocytic clusters than congenic C57BL/6 scid-IL-2 +/+ wild-type littermates, suggesting that loss of the IL-2 gene in the CNS (and possibly the loss of other unknown sources of the gene) enhanced neuronal regeneration. Further study of IL-2's complex actions in neuronal injury may provide greater understanding of key variables that determine whether or not immunological processes in the brain are proregenerative.