Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

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Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among such genes was neuronatin, whose expression was significantly up-regulated. Neuronatin was identified as a gene selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development, and markedly reduced in adult brains. The present study investigated whether the expression of neuronatin correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve. Adult male Sprague-Dawley rats weighting 230 to 280 g were used. The rats were grouped into two: those that were sham operated and those that had sciatic nerve axotomy. The specimens-L4,5 dorsal root ganglians(DRG) and their corresponding spinal cords-were collected at post-axotomy day 1, day 3, and day 5. The neuronatin protein contents were analyzed by western blotting and immunohisto- chemistry. Changes in the mRNA levels were evaluated using RT-PCR. Randall and Selitto test was performed to reveal changes in the animal behaviors. The subcellular co-localization of neuronatin with neuronal cell type specific markers were also investigated in correlation with pain-related animal behavior. It was found that after sciatic nerve injury, the expression of neuronatin in dorsal root ganglians was increased in protein extracts. Furthermore, the results of immunohistochemistry revealed that the cell numbers of DRGs were relatively increased. Unmyelinated C-fiber and thinly myelinated A-£_ fiber in adult DRGs were also among the principal sub-population of primary afferent neurons with distributed neuronatin. The increased expression of neuronatin and its subcellular localization were related to mechanical hyperalgesia. The results indicated that there was a following significant correlation between mechanical allodynia axotomy of sciatic nerve and the increased expression of neuronatin in C-fiber and A-£_ fiber of DRG neurons.

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The nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut. / text

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Indiana University-Purdue University Indianapolis (IUPUI) / Facial nerve axotomy (FNA) in immunodeficient mice causes significantly more facial motoneuron (FMN) loss relative to wild type (WT), indicating that the immune system is neuroprotective. Further studies reveal that both CD4+ T cells and interleukin 10 (IL-10) act centrally to promote neuronal survival after injury. This study first investigated the roles of IL-10 and CD4+ T cells in neuroprotection after axotomy. CD4+ T cell-mediated neuroprotection requires centrally-produced IL-10, but the source of IL-10 is unknown. Using FNA on IL-10 reporter mice, immunohistochemistry was employed to identify the IL-10 source. Unexpectedly, axotomy induced astrocyte production of IL-10. To test if microglia- or astrocyte-specific IL-10 is needed for neuroprotection, cell-specific conditional knockout mice were generated. Neither knockout scenario affected FMN survival after FNA, suggesting that coordinated IL-10 production by both glia contributes to neuroprotection. The effect of immune status on the post-FNA molecular response was studied to characterize CD4+ T cell-mediated neuroprotection. In the recombinase-activating gene2 knockout (RAG-2-/-) mouse model of immunodeficiency, glial microenvironment responses were significantly impaired. Reconstitution with CD4+ T cells restored glial activation to normal levels. Motoneuron regeneration responses remained unaffected by immune status. These findings indicate that CD4+ T cell-mediated neuroprotection after injury occurs indirectly via microenvironment regulation. Immunodysregulation is evident in amyotrophic lateral sclerosis (ALS), and FMN survival after FNA is worse in the mutant superoxide dismutase (mSOD1) mouse model of ALS. Further experiments reveal that mSOD1 CD4+ T cells are neuroprotective in RAG-2-/- mice, whereas mSOD1 whole splenocytes (WS) are not. The third aim examined if the mSOD1 WS environment inhibits mSOD1 CD4+ T cell glial regulation after axotomy. Unexpectedly, both treatments were equally effective in promoting glial activation. Instead, mSOD1 WS treatment induced a motoneuron-specific death mechanism prevalent in ALS. In conclusion, the peripheral immune system regulates the central glial microenvironment utilizing IL-10 to promote neuronal survival after axotomy. Astrocytes, specifically, may be responsible for transducing peripheral immune signals into microenvironment regulation. Additionally, the immune system in ALS may directly participate in disease pathology.

Indiana University-Purdue University Indianapolis (IUPUI) / The adaptive arm of the immune system is necessary for facial motoneuron (FMN) survival after facial nerve axotomy (FNA). CD4+ T cells mediate FMN survival after FNA in an interleukin-10 (IL-10) dependent manner, but are not themselves the cellular source of neuroprotective IL-10. The aims of this study are to elucidate the neuroprotective capacity of cell-specific IL-10 expression, and to investigate the manner in which CD4+ T cells participate in IL-10 signaling after FNA. Immunohistochemistry revealed that FMN themselves were constitutive producers of IL-10, and astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 is critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 was necessary for FMN survival. Gene expression analysis of wild-type, immunodeficient, and immune cell-reconstituted animals was performed to determine the role of the immune system in modulating the central IL-10 signaling cascade. This revealed that CD4+ T cells were necessary for full upregulation of central IL-10 receptor (IL-10R) expression after FNA, regardless of their own IL-10R beta (IL-10RB) expression or IL-10R signaling capability. Surprisingly, the ability of CD4+ T cells to respond to IL-10 was critical for their ability to mediate neuroprotection. Adoptive transfer of IL-10RB-deficient T cells resulted in increased central expression of genes associated with microglial activation, antigen presentation, T cell co-stimulation, and complement deposition in response to injury. These data suggest that IL-10RB functions on the T cell to prevent non-neuroprotective immune activation after axotomy. The conclusions drawn from this study support a revised hypothesis for the mechanisms of IL-10-mediated neuroprotection, in which IL-10 serves both trophic and immune-modulating roles after axotomy. This research has implications for the development of immune-modifying therapies for peripheral nerve injury and motoneuron diseases. / 2 years (2021-05-24)

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