Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of βIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased βIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model

Role of Mesencephalic Astrocyte-Derived Neurotrophic Factor in Alcohol-Induced Liver Injury

Consumption of alcohol in immoderate quantity induces endoplasmic reticulum (ER) stress response (alcohol-induced ER stress). Mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER stress-inducible protein, works as an evolutionarily conserved regulator of systemic and liver metabolic homeostasis. In this study, the effects of MANF on alcohol-induced liver injury were explored by using hepatocyte-specific MANF-knockout mice (MANFΔHep) in a chronic-plus-binge alcohol feeding model. We found that alcohol feeding upregulated MANF expression and MANFΔHep mice exhibited more severe liver injury with extra activated ER stress after alcohol feeding. In addition, we found that MANF deficiency activated iNOS and p65 and increased the production of NO and anti-inflammatory cytokines, which was further enhanced after alcohol treatment. Meanwhile, MANF deletion upregulated the levels of CYP2E1, 4-HNE, and MDA and downregulated the levels of GSH and SOD. These results indicate that MANF has potential protection on alcohol-induced liver injury, and the underlying mechanisms may be associated with meliorating the overactivated ER stress triggered by inflammation and oxidative stress via inhibiting and reducing NO/NF-κB and CYP2E1/ROS, respectively. Therefore, MANF might be a negative regulator in alcohol-induced ER stress and participate in the crosstalk between the NF-κB pathway and oxidative stress in the liver. Conclusions. This study identifies a specific role of MANF in alcohol-induced liver injury, which may provide a new approach for the treatment of ALI

Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

<div><p>Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized <i>in vitro</i> model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of <i>β</i>III tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased <i>β</i>III tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model.</p></div

Graphs showing the mean percentages of collapse growth cone and the extent of colocalizatuon of F actin and <i>β</i>III tubulin in growth cones of control cultures and cultures after 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point.

<p><b>(A)</b> Stretch injury induced increased axonal growth cone collapsed at both 24 h and 72 h PI compared to the control. In addition, repetitive very mild (2×0.5%) stretch injury induced more collapsed growth cones when compared to single 0.5% stretched axon at 72 h PI. <b>(B)</b> The growth cones in 5% stretched axon had significantly higher colocalization value of <i>β</i>III tubulin and F-actin compared to both the growth cones in control and 0.5% stretched axon at 72 h PI. However, there was no significant difference between the growth cones in control, 0.5% stretched or 5% stretched axon at 24 h PI. The growth cones in 2×0.5% repetitive stretched axon has significantly higher colocalization value of <i>β</i>III tubulin and F-actin if compare to both the growth cones in control and single 0.5% stretched axon at 72 h PI. *p<0.05. Error bar = mean ± SEM.</p

Tau (microtubule marker) immunofluorescence labeling demonstrating the effect of EpoD exposure on 5% stretched axons at 7 DIV.

<p><b>(A)</b> Representative fluorescence images of unstretched and stretched culture 24 h after the indicated treatment. <b>(B)</b> EpoD (100 nM) applied to axon compartment alone for 24 h induced a significant decrease in degenerative index in the axon compartment when compared to vehicle- treated cultures following injury. The arterisk indicates the difference from paired control. *<i>p</i><0.05. Error bar = mean ± SEM. Scale bar = 30 <i>μ</i>m.</p

Graphs showing average growth cone area of control, 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point.

<p>There was a significant decrease in growth cone size after single stretch injury at both 24 h and 72 h PI and after repetitive very mild (2×0.5%) stretch injury at 72 h PI compared to control. *<i>p</i><0.05. Error bar = mean ± SEM.</p