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Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells
摘要： Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/β-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.
关键词： retina,Ripk1,axon,retinal ganglion cell,neurite growth,Wnt signaling
更新于2025-11-21 11:24:58
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Microfluidic Long-Term Gradient Generator with Axon Separation Prototyped by 185 nm Diffused Light Photolithography of SU-8 Photoresist
摘要： We have developed a cast micro?uidic chip for concentration gradient generation that contains a thin (~5 μm2 cross-sectional area) microchannel. The diffusion of diffused 185 nm ultraviolet (UV) light from an inexpensive low-pressure mercury lamp exposed a layer of the SU-8 photoresist from the backside and successfully patterned durable 2 μm-high microchannel mold features with smooth bell-shaped sidewalls. The thin channel had appropriate ?ow resistance and simultaneously satis?ed both the rapid introduction of test substance and long-term maintenance of gradients. The average height and width at the half height of the channel, de?ned by a 2 μm-wide line mask pattern, were 2.00 ± 0.19 μm, and 2.14 ± 0.89 μm, respectively. We were able to maintain the concentration gradient of Alexa Fluor 488 ?uorescent dye inside or at the exit of the thin microchannel in an H-shaped micro?uidic con?guration for at least 48 h. We also demonstrated the cultivation of chick embryo dorsal root ganglion neuronal cells for 96 h, and the directional elongation of axons under a nerve growth factor concentration gradient.
关键词： prototyping,axon elongation,micro?uidic gradient generator,SU-8,microchannel
更新于2025-09-23 15:22:29
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Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration
摘要： Purpose: The present study examines the role of Sox11 in the initial response of retinal ganglion cells (RGCs) to axon damage and in optic nerve regeneration in mouse. Methods: Markers of retinal injury were identified using the normal retina database and optic nerve crush (ONC) database on GeneNetwork2 (www.genenetwork.org). One gene, Sox11, was highly upregulated following ONC. We examined the role of this transcription factor, Sox11, following ONC and optic nerve regeneration in mice. In situ hybridization was performed using the Affymetrix 2-plex Quantigene View RNA In Situ Hybridization Tissue Assay System. Sox11 was partially knocked out by intravitreal injection of AAV2-CMV-Cre-GFP in Sox11f/f mice. Optic nerve regeneration model used Pten knockdown. Mice were perfused and the retinas and optic nerves were dissected and examined for RGC survival and axon growth. Results: Sox11 was dramatically upregulated in the retina following ONC injury. The level of Sox11 message increased by approximately eightfold 2 days after ONC. In situ hybridization demonstrated low-level Sox11 message in RGCs and cells in the inner nuclear layer in the normal retina as well as a profound increase in Sox11 message within the ganglion cells following ONC. In Sox11f/f retinas, partially knocking out Sox11 significantly increased RGC survival after ONC as compared to the AAV2-CMV-GFP control group; however, it had little effect on the ability of axon regeneration. Combinatorial downregulation of both Sox11 and Pten resulted in a significant increase in RGC survival as compared to Pten knockdown only. When Pten was knocked down there was a remarkable increase in the number and the length of regenerating axons. Partially knocking out Sox11 in combination with Pten deletion resulted in a fewer regenerating axons. Conclusion: Taken together, these data demonstrate that Sox11 is involved in the initial response of the retina to injury, playing a role in the early attempts of axon regeneration and neuronal survival. Downregulation of Sox11 aids in RGC survival following injury of optic nerve axons, while a partial knockout of Sox11 negates the axon regeneration stimulated by Pten knockdown.
关键词： optic nerve crush,AAV2,retinal ganglion cells,axon regeneration,Sox11
更新于2025-09-23 15:22:29
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Return of function after CNS axon regeneration: Lessons from injury-responsive intrinsically photosensitive and alpha retinal ganglion cells
摘要： This review addresses issues relating to the survival and axon regeneration of both intrinsically photosensitive retinal ganglion cells (ipRGC) and αRGC, and possible ensuing patterns of functional recovery after optic nerve crush, all of which are broadly relevant to recovery from injury in the central nervous system (CNS) as whole. Although much needs to be clarified about the connectivity, function and patterns of myelination of regenerated CNS axons, the results of recent research on activity-induced αRGC axon regeneration associated with functional restitution have highlighted key focal obstacles to recovery including neurotrophic support, axon misguidance, target recognition failure and dysmyelination. Pan RGC survival/axon regeneration requires receptor binding and downstream signalling by a cocktail of growth factors, more generally defined in the CNS by the individual trophic requirements of neuronal subsets within a given disconnected centre. Resolution of the problem of failed axon guidance and target recognition is complicated by a confounding paradox that axon growth inhibitory ligand disinhibition required for axon regeneration may mask axon guidance cues that are essential for accurate re-innervation. The study of the temporal parameters of remyelination of regenerated αRGC axons may become feasible if they establish permanent homologous connections, allowing time for new myelin sheaths to fully form. Unless near complete re-innervation of denervated targets is re-instated in the CNS, debilitating dysfunctional neurological sequelae may ensue from the resulting imbalance in connectivity.
关键词： CNS axon regeneration,recovery of function,ipRGC,CNS trauma
更新于2025-09-23 15:21:21
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Optic Nerve Head Astrocytes Display Axon-Dependent and -Independent Reactivity in Response to Acutely Elevated Intraocular Pressure
摘要： PURPOSE. Optic nerve head (ONH) astrocytes provide support for axons, but exhibit structural and functional changes (termed reactivity) in a number of glaucoma models. The purpose of this study was to determine if ONH astrocyte structural reactivity is axon-dependent. METHODS. Using rats, we combine retrobulbar optic nerve transection (ONT) with acute controlled elevation of intraocular pressure (CEI), to induce total optic nerve axon loss and ONH astrocyte reactivity, respectively. Animals were euthanized immediately or 1 day post CEI, in the presence or absence of ONT. ONH sections were labeled with fluorescent-tagged phalloidin and antibodies against b3 tubulin, phosphorylated cortactin, phosphorylated paxillin, or complement C3. ONH label intensities were quantified after confocal microscopy. Retrobulbar nerves were assessed for axon injury by light microscopy. RESULTS. While ONT alone had no effect on ONH astrocyte structural orientation, astrocytes demonstrated significant reorganization of cellular extensions within hours after CEI, even when combined with ONT. However, ONH astrocytes displayed differential intensities of actin (phosphorylated cortactin) and focal adhesion (phosphorylated paxillin) mediators in response to CEI alone, ONT alone, or the combination of CEI and ONT. Lastly, label intensities of complement C3 within the ONH were unchanged in eyes subjected to CEI alone, ONT alone, or the combination of CEI and ONT, relative to controls. CONCLUSIONS. Early ONH astrocyte structural reactivity to elevated IOP is multifaceted, displaying both axon dependent and independent responses. These findings have important implications for pursuing astrocytes as diagnostic and therapeutic targets in neurodegenerative disorders with fluctuating levels of axon injury.
关键词： elevated intraocular pressure,glaucoma,optic nerve head,axon,astrocyte reactivity
更新于2025-09-19 17:15:36
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Quantification of Trigeminovascular Hypersensitivity in a Chronic Migraine Patient Using Laser Speckle Contrast Analysis (LASCA)
摘要： Background: Activation of peripheral and/ or central trigeminovascular pain pathways are implicated in the pathogenesis of migraine. Small fibers mediate pain, thermal sensation and autonomic functions. Axon-flare response is correlated with local C-fiber activation and CGRP release. LASCA detects very subtle microcirculatory changes which is not visible by naked eye. Case: Axon-flare response was elicited by intradermal (i.d.) 0.01 ml histamine introduced to the left forehead, trigeminal nerve ophtalmic branch (V1) skin area. Skin microvascular blood flow data was recorded using a LASCA, real-time microcirculation imaging system. In the healthy control, prick stimulus slightly elevated focal skin microcirculation only at the stimulated focal area. However, in our chronic migraine patient, the unilateral prick stimulation transiently (10-12 seconds) increased ipsilateral skin microcirculation at all 3 branches of the trigeminal nerve with a slight expansion across the midline. Besides, left V1 stimulation by histamine i.d. induced not only prominent but also long lasting (10-15 minutes of recording time) axon flare response at ipsilateral V1, V2, V3, and even with an expansion to the contralateral V1 without any report of allodynia or hyperalgesia. The treatment decreased axon-flare characteristics probably by inhibiting the neurogenic inflammation. Discussion: The clinical characteristics and individual response to treatment varies widely across pain patients. Here, we demonstrated the presence of transient spread of increased microcirculation at ipsilateral trigeminal, and also across the midline after prick stimulus, whereas a more prominent, widespread, and long-lasting, histamine-induced axon flare response in a rare subclass of chronic migraine patient with autonomic symptoms. The modulatory effect of the pharmacological intervention has also been objectively quantified by LASCA.
关键词： treatment,ophthalmic branch,C-fiber,axon-flare response,method
更新于2025-09-11 14:15:04
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High-resolution imaging of distinct human corpus callosum microstructure and topography of structural connectivity to cortices at high field
摘要： Characterization of the microstructural properties and topography of the human corpus callosum (CC) is key to understanding interhemispheric neural communication and brain function. In this work, we tested the hypothesis that high-resolution T1 relaxometry at high field has adequate sensitivity and specificity for characterizing microstructural properties of the human CC, and elucidating the structural connectivity of the callosal fibers to the cortices of origin. The high-resolution parametric T1 images acquired from healthy subjects (N = 16) at 7 T clearly showed a consistent T1 distribution among individuals with substantial variation along the human CC axis, which is highly similar to the spatial patterns of myelin density and myelinated axon size based on the histology study. Compared to the anterior part of the CC, the posterior midbody and splenium had significantly higher T1 values. In conjunction with T1-based classification method, the splenial T1 values were decoded more reliably compared to a conventional partitioning method, showing a much higher T1 value in the inferior splenium than in the middle/superior splenium. Moreover, the T1 profile of the callosal subdivision represented the topology of the fiber connectivity to the projected cortical regions: the fibers in the posterior midbody and inferior splenium with a higher T1 (inferring a larger axon size) were mainly connected to motor–sensory and visual cortical areas, respectively; in contrast, the fibers in the anterior/posterior CC with a lower T1 (inferring a smaller axon size) were primarily connected to the frontal/parietal–temporal areas. These findings indicate that high-resolution T1 relaxometry imaging could provide a complementary and robust neuroimaging tool, useful for exploring the complex tissue properties and topographic organization of the human corpus callosum.
关键词： Myelin density,Structural connectivity,Topography,Parametric T1 MRI,Corpus callosum,Axon size
更新于2025-09-09 09:28:46
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Discordant Responses to MAPK Pathway Stimulation Include Axonal Growths in Adult Drosophila Photoreceptors
摘要： Wallenda (WND) is the Drosophila member of a conserved family of dual leucine-zipper kinases (DLK) active in both neuronal regeneration and degeneration. We examined the role of WND over-expression on sensory neuron morphology by driving WND in multiple subtypes of Drosophila photoreceptors. WND overexpression under control of the pan-retinal GAL4 driver GMR causes multiple photoreceptor defects including cell death, rhabdomere degeneration, and axonal sprouting. Individual photoreceptor subtypes were assayed using GAL4 drivers speci?c for each photoreceptor class. Many R7 and R8 cells exhibit axonal sprouting while some show cell degeneration. Delaying the onset of WND overexpression until 20 days of age showed that older adult R7 cells retain the ability to initiate new axon growth. R1–6 photoreceptor cells degenerate in response to WND expression and exhibit rhodopsin loss and rhabdomere degeneration. RNAi knockdown of the MAPK signaling components Kayak (KAY) and Hemipterous (HEP) attenuates the WND-induced loss of Rh1 rhodopsin. UAS-induced HEP expression is similar to WND expression, causing degeneration in R1–6 photoreceptors and axonal sprouting in R7 photoreceptors. These results demonstrate that WND in adult Drosophila photoreceptor cells acts through MAPK signaling activity with both regenerative and degenerative responses. These photoreceptors provide a tractable experimental model to reveal cellular mechanisms driving contradictory WND signaling responses.
关键词： degeneration,dual leucine kinase,Drosophila,axon regeneration,photoreceptors
更新于2025-09-04 15:30:14