Axonal degeneration in an in vitro model of ischemic white matter injury.

Ischemic white matter injuries underlie cognitive decline in the elderly and vascular dementia. Ischemia in the subcortical white matter is caused by chronic reduction of blood flow due to narrowing of small arterioles. However, it remains unclear how chronic ischemia leads to white matter pathology. We aimed to develop an in vitro model of ischemic white matter injury using organotypic slice cultures. Cultured cerebellar slices preserved fully myelinated white matter tracts that were amenable to chronic hypoxic insult. Prolonged hypoxia caused progressive morphological evidence of axonal degeneration with focal constrictions and swellings. In contrast, myelin sheaths and oligodendrocytes exhibited remarkable resilience to hypoxia. The cytoskeletal degradation of axons was accompanied by mitochondrial shortening and lysosomal activation. Multiple pharmacological manipulations revealed that the AMPA glutamate receptor, calpain proteolysis, and lysosomal proteases were independently implicated in hypoxia-induced axonal degeneration in our model. Thus, our in vitro model would be a novel experimental system to explore molecular mechanisms of ischemic white matter injury. Furthermore, we verified that the in vitro assay could be successfully utilized to screen for molecules that can ameliorate hypoxia/ischemia-induced axonal degeneration.

High-mobility group box-1 as an autocrine trophic factor in white matter stroke.

Maintenance of white matter integrity in health and disease is critical for a variety of neural functions. Ischemic stroke in the white matter frequently results in degeneration of oligodendrocytes (OLs) and myelin. Previously, we found that toll-like receptor 2 (TLR2) expressed in OLs provides cell-autonomous protective effects on ischemic OL death and demyelination in white matter stroke. Here, we identified high-mobility group box-1 (HMGB1) as an endogenous TLR2 ligand that promotes survival of OLs under ischemic stress. HMGB1 rapidly accumulated in the culture medium of OLs exposed to oxygen-glucose deprivation (OGD). This conditioned medium exhibited a protective activity against ischemic OL death that was completely abolished by immunodepletion of HMGB1. Knockdown of HMGB1 or application of glycyrrhizin, a specific HMGB1 inhibitor, aggravated OGD-induced OL death, and recombinant HMGB1 application reduced the extent of OL death in a TLR2-dependent manner. We confirmed that cytosolic translocation of HMGB1 and activation of TLR2-mediated signaling pathways occurred in a focal white matter stroke model induced by endothelin-1 injection. Animals with glycyrrhizin coinjection showed an expansion of the demyelinating lesion in a TLR2-dependent manner, accompanied by aggravation of sensorimotor behavioral deficits. These results indicate that HMGB1/TLR2 activates an autocrine trophic signaling pathways in OLs and myelin to maintain structural and functional integrity of the white matter under ischemic conditions.

CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.

CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

The intervention mechanism of Tanshinone IIA in alleviating neuronal injury induced by HMGB1 or TNF-α-mediated microglial activation.

Tanshinone IIA (Tan IIA), a neuroprotective natural compound extracted from Salvia miltiorrhiza, is used in stroke treatment. However, elucidating Tan IIA's neuroprotective mechanisms remains challenging due to limitations in assessing drug efficacy and biochemical parameters in clinical studies. This study investigated Tan IIA's impact on neuroinflammatory responses and its neuroprotective mechanisms using HMGB1- or TNF-α-stimulated BV2 microglia in a co-culture system with primary neuron cells. The results indicated that Tan IIA significantly reduced microglial activation induced by TNF-α or HMGB1. Concurrently, Tan IIA disrupted the interactions between HMGB1 and toll-like receptor 4 (TLR4), and between TNF-α and TNF receptor 1 (TNFR1), modulating the HMGB1/TLR4/nuclear factor-kappa B (NF-κB) and TNF-α/TNFR1/NF-κB signaling pathways and related protein expressions. Moreover, co-culture experiments showed that neuronal apoptosis induced by microglial activation was reversed by Tan IIA. In conclusion, Tan IIA provides neuroprotection by modulating signaling pathways in microglia, thus preventing neuronal apoptosis. This study offers new insights into therapeutic targets for ischemic stroke.

Arctiin Mitigates Neuronal Injury by Modulating the P2X7R/NLPR3 Inflammasome Signaling Pathway.

Depression, recognized globally as a primary cause of disability, has its pathogenesis closely related to neuroinflammation and neuronal damage. Arctiin (ARC), the major bioactive component of Fructus arctii, has various pharmacological activities, such as anti-inflammatory and neuroprotective effects. Building on previous findings that highlighted ARC's capability to mitigate depression by dampening microglial hyperactivation and thereby reducing neuroinflammatory responses and cortical neuronal damage in mice, the current study delves deeper into ARC's therapeutic potential by examining its impact on hippocampal neuronal damage in depression. Utilizing both chronic unpredictable mild stress (CUMS)-induced depression model in mice and corticosterone (CORT)-stimulated PC12 cell model of neuronal damage, the techniques including Nissl staining, immunohistochemistry, western blotting, ELISA, lactate dehydrogenase assays, colony formation assays, immunofluorescence staining and molecular docking were employed to unravel the mechanisms behind ARC's neuroprotective effects. The findings revealed that ARC not only mitigates hippocampal neuropathological damage and reduces serum CORT levels in CUMS-exposed mice but also enhances cell activity while reducing lactate dehydrogenase release in CORT-stimulated PC12 cells. ARC attenuated neuroinflammatory responses and neuronal apoptosis by inhibiting the overactivation of the P2X7 receptor (P2X7R)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling pathway, similar to the effect of A438079 (P2X7R antagonist). Interestingly, pretreatment with A438079 blocked the neuroprotective effect of ARC. Computer modeling predicted that both ARC and A438079 have strong binding with P2X7R and they have the same binding site. These results suggested that ARC may exert a neuroprotective role by binding to P2X7R, thereby inhibiting the P2X7R/NLRP3 inflammasome signaling pathway.

Modeling subcortical ischemic white matter injury in rodents: unmet need for a breakthrough in translational research.

Subcortical ischemic white matter injury (SIWMI), pathological correlate of white matter hyperintensities or leukoaraiosis on magnetic resonance imaging, is a common cause of cognitive decline in elderly. Despite its high prevalence, it remains unknown how various components of the white matter degenerate in response to chronic ischemia.This incomplete knowledge is in part due to a lack of adequate animal model. The current review introduces various SIWMI animal models and aims to scrutinize their advantages and disadvantages primarily in regard to the pathological manifestations of white matter components. The SIWMI animal models are categorized into 1) chemically induced SIWMI models, 2) vascular occlusive SIWMI models, and 3) SIWMI models with comorbid vascular risk factors. Chemically induced models display consistent lesions in predetermined areas of the white matter, but the abrupt evolution of lesions does not appropriately reflect the progressive pathological processes in human white matter hyperintensities. Vascular occlusive SIWMI models often do not exhibit white matter lesions that are sufficiently unequivocal to be quantified. When combined with comorbid vascular risk factors (specifically hypertension), however, they can produce progressive and definitive white matter lesions including diffuse rarefaction, demyelination, loss of oligodendrocytes, and glial activation, which are by far the closest to those found in human white matter hyperintensities lesions. However, considerable surgical mortality and unpredictable natural deaths during a follow-up period would necessitate further refinements in these models. In the meantime, in vitro SIWMI models that recapitulate myelinated white matter track may be utilized to study molecular mechanisms of the ischemic white matter injury. Appropriate in vivo and in vitro SIWMI models will contribute in a complementary manner to making a breakthrough in developing effective treatment to prevent progression of white matter hyperintensities.

Arctigenin protects against depression by inhibiting microglial activation and neuroinflammation via HMGB1/TLR4/NF-κB and TNF-α/TNFR1/NF-κB pathways.

BACKGROUND AND PURPOSE: Arctigenin, a major bioactive component of Fructus arctii, has been reported to have antidepressant-like effects. However, the mechanisms underlying these effects are still unclear. Neuroinflammation can be caused by excessive production of proinflammatory cytokines in microglia via high-mobility group box 1 (HMGB1)/TLR4/NF-κB and TNF-α/TNFR1/NF-κB signalling pathways, leading to depression. In this study, we have investigated the antidepressant mechanism of arctigenin by conducting in vitro and in vivo studies. EXPERIMENTAL APPROACH: The effects of chronic unpredictable mild stress (CUMS) on wild-type (WT) and TLR4-/- mice were examined. Antidepressant-like effects of arctigenin were tested using the CUMS-induced model of depression in WT mice. The effects of arctigenin were assessed on the HMGB1/TLR4/NF-κB and TNF-α/TNFR1/NF-κB signalling pathways in the prefrontal cortex (PFC) of mouse brain and HMGB1- or TNF-α-stimulated primary cultured microglia. The interaction between HMGB1 and TLR4 or TNF-α and TNFR1 with or without arctigenin was examined by localized surface plasmon resonance (LSPR) and co-immunoprecipitation assays. KEY RESULTS: The immobility times in the tail suspension test (TST) and forced swimming test (FST) were reduced in TLR4-/- mice, compared with WT mice. Arctigenin exhibited antidepressant-like effects. Arctigenin also inhibited microglia activation and inflammatory responses in the PFC of mouse brain. Arctigenin inhibited HMGB1 and TLR4 or TNF-α and TNFR1 interactions, and suppressed both HMGB1/TLR4/NF-κB and TNF-α/TNFR1/NF-κB signalling pathways. CONCLUSIONS AND IMPLICATIONS: Arctigenin has antidepressant-like effects by attenuating excessive microglial activation and neuroinflammation through the HMGB1/TLR4/NF-κB and TNF-α/TNFR1/NF-κB signalling pathways. This suggests that arctigenin has potential as a new drug candidate suitable for clinical trials to treat depression.

Antidepressive Effect of Arctiin by Attenuating Neuroinflammation via HMGB1/TLR4- and TNF-α/TNFR1-Mediated NF-κB Activation.

Inflammation is a potential factor in the pathophysiology of depression. A traditional Chinese herbal medicine, arctiin, and its aglycone, arctigenin, are the major bioactive components in Fructus arctii and exhibit neuroprotective and anti-inflammatory activities. Arctigenin has been reported to have antidepressant-like effects. However, the antidepressant-like effects of arctiin, its precursor, remain unknown. In this study, we investigated the antidepressant-like effects of arctiin and its underlying mechanisms by in vivo and in vitro experiments in mice. Our results showed that arctiin significantly attenuated sucrose consumption and increased the immobility time in tail suspension and forced swimming tests. Arctiin decreased neuronal damage in the prefrontal cortex (PFC) of the brain. Arctiin also attenuated the levels of three inflammatory mediators, indoleamine 2,3-dioxygenase, 5-hydroxytryptamine, and dopamine, that were elevated in the PFC or serum of chronic unpredictable mild stress (CUMS)-exposed mice. Arctiin reduced excessive activation of microglia and neuroinflammation by reducing high mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4)- and tumor necrosis factor-α (TNF-α)/TNF receptor 1 (TNFR1)-mediated nuclear factor-kappa B (NF-κB) activation in the PFC of CUMS-exposed mice and HMGB1- or TNF-α-stimulated primary cultured microglia. These findings demonstrate that arctiin ameliorates depression by inhibiting the activation of microglia and inflammation via the HMGB1/TLR4 and TNF-α/TNFR1 signaling pathways.

Insulin-like Growth Factor-1 Receptor Dictates Beneficial Effects of Treadmill Training by Regulating Survival and Migration of Neural Stem Cell Grafts in the Injured Spinal Cord.

Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/-) for Igf1r, the gene encoding IGF-1 receptor, into the mouse spinal cord after injury, with or without combining TMT. We analyzed the influence of genotype and TMT on locomotor recovery and survival and migration of NSC grafts. In vitro experiments were performed to examine the potential roles of IGF-1 signaling in the migratory ability of NSCs. Mice receiving +/- NSC grafts showed impaired locomotor recovery compared with those receiving wild-type (+/+) NSCs. Locomotor improvement by TMT was more pronounced with +/+ grafts. Deficiency of one allele of Igf1r significantly reduced survival and migration of the transplanted NSCs. Although TMT did not significantly influence NSC survival, it substantially enhanced the extent of migration for only +/+ NSCs. Cultured neurospheres exhibited dynamic motility with cytoplasmic protrusions, which was regulated by IGF-1 signaling. IGF-1 signaling in transplanted NSCs may be essential in regulating their survival and migration. Furthermore, TMT may promote NSC graft-mediated locomotor recovery via activation of IGF-1 signaling in transplanted NSCs. Dynamic NSC motility via IGF-1 signaling may be the cellular basis for the TMT-induced enhancement of migration.

An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling.

The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle for tissue repair in central nervous system (CNS). Here we report that injection of imidazole-poly(organophosphazenes) (I-5), a hydrogel with thermosensitive sol-gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model. Cystic cavities are bridged by fibronectin-rich extracellular matrix. The fibrotic extracellular matrix remodeling is mediated by matrix metalloproteinase-9 expressed in macrophages within the fibrotic extracellular matrix. A poly(organophosphazenes) hydrogel lacking the imidazole moiety, which physically interacts with macrophages via histamine receptors, exhibits substantially diminished bridging effects. I-5 injection improves coordinated locomotion, and this functional recovery is accompanied by preservation of myelinated white matter and motor neurons and an increase in axonal reinnervation of the lumbar motor neurons. Our study demonstrates that dynamic interactions between inflammatory cells and injectable biomaterials can induce beneficial extracellular matrix remodeling to stimulate tissue repair following CNS injuries.The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle. Here the authors show an injection of imidazole poly(organophosphazenes), a hydrogel with thermosensitive sol-gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model.

Effects of posterior corneal astigmatism on the accuracy of AcrySof toric intraocular lens astigmatism correction.

AIM: To evaluate the effects of posterior corneal surface measurements on the accuracy of total estimated corneal astigmatism. METHODS: Fifty-seven patients with toric intraocular lens (IOL) implantation and posterior corneal astigmatism exceeding 0.5 diopter were enrolled in this retrospective study. The keratometric astigmatism (KA) and total corneal astigmatism (TA) were measured using a Pentacam rotating Scheimpflug camera to assess the outcomes of AcrySof IOL implantation. Toric IOLs were evaluated in 26 eyes using KA measurements and in 31 eyes using TA measurements. Preoperative corneal astigmatism and postoperative refractive astigmatism were recorded for statistical analysis. The cylindrical power of toric IOLs was estimated in all eyes. RESULTS: In all cases, the difference of toric IOL astigmatism magnitude between KA and TA measurements for the estimation of preoperative corneal astigmatism was statistically significant. Of a total of 57 cases, the 50.88% decreased from Tn to Tn-1, and 10.53% decreased from Tn to Tn-2. In all cases, 5.26% increased from Tn to Tn+1. The mean postoperative astigmatism within the TA group was significantly lower than that in the KA group. CONCLUSION: The accuracy of total corneal astigmatism calculations and the efficacy of toric IOL correction can be enhanced by measuring both the anterior and posterior corneal surfaces using a Pentacam rotating Scheimpflug camera.

Optical performance of toric intraocular lenses in the presence of decentration.

AIM: To evaluate the optical performance of toric intraocular lenses (IOLs) after decentration and with different pupil diameters, but with the IOL astigmatic axis aligned. METHODS: Optical performances of toric T5 and SN60AT spherical IOLs after decentration were tested on a theoretical pseudophakic model eye based on the Hwey-Lan Liou schematic eye using the Zemax ray-tracing program. Changes in optical performance were analyzed in model eyes with 3-mm, 4-mm, and 5-mm pupil diameters and decentered from 0.25 mm to 0.75 mm with an interval of 5° at the meridian direction from 0° to 90°. The ratio of the modulation transfer function (MTF) between a decentered and a centered IOL (MTFDecentration/MTFCentration) was calculated to analyze the decrease in optical performance. RESULTS: Optical performance of the toric IOL remained unchanged when IOLs were decentered in any meridian direction. The MTFs of the two IOLs decreased, whereas optical performance remained equivalent after decentration. The MTFDecentration/MTFCentration ratios of the IOLs at a decentration from 0.25 mm to 0.75 mm were comparable in the toric and SN60AT IOLs. After decentration, MTF decreased further, with the MTF of the toric IOL being slightly lower than that of the SN60AT IOL. Imaging qualities of the two IOLs decreased when the pupil diameter and the degree of decentration increased, but the decrease was similar in the toric and spherical IOLs. CONCLUSIONS: Toric IOLs were comparable to spherical IOLs in terms of tolerance to decentration at the correct axial position.

Role of toll-like receptor 2 in ischemic demyelination and oligodendrocyte death.

White matter is frequently involved in ischemic stroke, and progressive ischemic white matter injuries are associated with various neurologic dysfunctions in the elderly population. Demyelination and oligodendrocyte (OL) loss are prominent features of ischemic white matter injury. Endothelin-1 injection into the internal capsule resulted in a localized demyelinating lesion in mice, where loss of OL lineage cells and inflammatory cell infiltration were observed accompanied by upregulation of toll-like receptor 2 (TLR2). Intriguingly, the extent of demyelinating pathology was markedly larger in TLR2 deficient mice than that of wild-type (WT) mice. TLR2 deficient mice showed enhanced OL death and decreased phosphorylation of ERK1/2 compared with WT animals. Cultured OLs from TLR2 deficient mice were more vulnerable to oxygen-glucose deprivation than WT OLs. Applying TLR2 agonists Pam3CSK4 or Zymosan after oxygen-glucose deprivation substantially rescued WT OL death with augmentation of ERK1/2 phosphorylation. Treatment with Pam3CSK4 also reduced the extent of endothelin-1 induced ischemic demyelination in vivo. Our data indicate TLR2 may provide endogenous protective effects on ischemic demyelination and OL degeneration.