Reactive Astrocytes Research Articles

Glial reactivity in a mouse model of beta-amyloid deposition assessed by PET imaging of P2X7 receptor and TSPO using [11C]SMW139 and [18F]F-DPA

BackgroundP2X7 receptor has emerged as a potentially superior PET imaging marker to TSPO, the gold standard for imaging glial reactivity. [11C]SMW139 is the most recently developed radiotracer to image P2X7 receptor. The aim of this study was to image reactive glia in the APP/PS1-21 transgenic (TG) mouse model of Aβ deposition longitudinally using [11C]SMW139 targeting P2X7 receptor and to compare tracer uptake to that of [18F]F-DPA targeting TSPO at the final imaging time point. TG and wild type (WT) mice underwent longitudinal in vivo PET imaging using [11C]SMW139 at 5, 8, 11, and 14 months, followed by [18F]F-DPA PET scan only at 14 months. In vivo imaging results were verified by ex vivo brain autoradiography, immunohistochemical staining, and analysis of [11C]SMW139 unmetabolized fraction in TG and WT mice.ResultsLongitudinal change in [11C]SMW139 standardized uptake values (SUVs) showed no statistically significant increase in the neocortex and hippocampus of TG or WT mice, which was consistent with findings from ex vivo brain autoradiography. Significantly higher [18F]F-DPA SUVs were observed in brain regions of TG compared to WT mice. Quantified P2X7-positive staining in the cortex and thalamus of TG mice showed a minor increase in receptor expression with ageing, while TSPO-positive staining in the same regions showed a more robust increase in expression in TG mice as they aged. [11C]SMW139 was rapidly metabolized in mice, with 33% of unmetabolized fraction in plasma and 29% in brain homogenates 30 min after injection.Conclusions[11C]SMW139, which has a lower affinity for the rodent P2X7 receptor than the human version of the receptor, was unable to image the low expression of P2X7 receptor in the APP/PS1-21 mouse model. Additionally, the rapid metabolism of [11C]SMW139 in mice and the presence of several brain-penetrating radiometabolites significantly impacted the analysis of in vivo PET signal of the tracer. Finally, [18F]F-DPA targeting TSPO was more suitable for imaging reactive glia and neuroinflammatory processes in the APP/PS1-21 mouse model, based on the findings presented in this study and previous studies with this mouse model.

LNC000152 Mediates Aluminum-Induced Proliferation of Reactive Astrocytes.

Aluminum is a metal element with significant neurotoxicity, and there is a substantial correlation between aluminum exposure and cognitive dysfunction. Glial fibrillary acidic protein (GFAP) is widely used as a marker of reactive astrocyte proliferation in response to pathological injury of the central nervous system. Studies of various neurodegenerative diseases have confirmed that the expression changes in GFAP are associated with nerve injury. We investigated the role of LNC000152 in the aluminum-induced reactive proliferation of astrocytes. By establishing two aluminum-exposed cell models of rat primary astrocytes and CTX-TNA2 cell lines, we examined the expression of LNC000152 and GFAP and detected cell proliferation with EdU and cell cycle changes with flow cytometry. The role of aluminum in promoting glial cell proliferation was verified; the expression levels of LNC000152 and GFAP increased with the concentration of aluminum exposure. Intervention of LNC000152 expression by siRNA technology revealed that LNC000152 affected glial cell responsive proliferation by influencing GFAP expression. These results suggest that LNC000152 plays a role in the reactive proliferation of astrocytes induced by aluminum.

GelMA hydrogel as a scaffold to enhance the survival and differentiation of human induced lateral ganglionic eminence precursor cells

Cell reprogramming holds enormous potential to revolutionize our understanding of neurological and neurodevelopmental disorders, as well as enhance drug discovery and regenerative medicine. We have developed a direct cell reprogramming technology that allows us to generate lineage-specific neural cells. To extend our technology, we have investigated the incorporation of directly reprogrammed human lateral ganglionic eminence precursor cells (hiLGEPs) in a 3-dimensional (3D) matrix. Hydrogels are one of the most promising bio-scaffolds for 3D cell culture, providing cells with a supportive environment to adhere, proliferate, and differentiate. In particular, gelatin methacryloyl (GelMA) hydrogels have been used for a variety of 3D biomedical applications due to their biocompatibility, enzymatic cleavage, cell adhesion and tunable physical characteristics. This study therefore investigated the effect of GelMA hydrogel encapsulation on the survival and differentiation of hiLGEPs, both in vitro and following ex vivo transplantation into a quinolinic acid (QA) lesion rat organotypic slice culture model. We demonstrate, for the first time, that the encapsulation of hiLGEPs in GelMA hydrogel significantly enhances the survival and generation of DARPP32+ striatal neurons both in vitro and following ex vivo transplant. Furthermore, GelMA-encapsulated hiLGEPs were predominantly located away from the reactive astrocyte network that forms following QA lesioning, suggesting GelMA provides a protective barrier for cells in regions of inflammatory activation. Overall, these results indicate that GelMA hydrogel has the potential to act as a 3D bio-scaffold to augment the viability and differentiation of hiLGEPs for research and translation of pharmaceutical development and regenerative medicine.

Complement C3 From Astrocytes Plays Significant Roles in Sustained Activation of Microglia and Cognitive Dysfunctions Triggered by Systemic Inflammation After Laparotomy in Adult Male Mice

Aberrant activation of complement cascades plays an important role in the progress of neurological disorders. Complement C3, the central complement component, has been implicated in synaptic loss and cognitive impairment. Recent study has shown that wound injury-induced systemic inflammation can trigger the increase of C3 in the brain. Our previous studies have demonstrated that laparotomy-triggered systemic inflammation could induce neuroinflammation and cognitive dysfunctions. Furthermore, sustained activation of microglia was observed even 14 days after laparotomy, while most of cytokines had returned to basal levels rapidly at the earlier time point. Although we have demonstrated that anti-inflammatory intervention successfully attenuated cognitive dysfunction by preventing increase of cytokines and activation of microglia, how sustained activation of microglia and cognitive dysfunction occur is still a mystery. In this study, we investigated the role of C3 in mediating activation of microglia and cognitive dysfunction by using laparotomy in adult male mouse only as the experimental model of systemic inflammation and AAV9-C3shRNA. Our data observed that laparotomy induced neurotoxic reactive astrocytes with an increase of C3 in the hippocampus. Furthermore, inhibition of C3 by AAV9-C3shRNA prevented synaptic engulfment by microglia and attenuated cognitive dysfunctions after laparotomy. Inhibition of C3 did not modulate activation of astrocytes and expression of various cytokines. Current findings demonstrated that C3 plays significant roles in sustained activation of microglia and cognitive dysfunctions, which suggests that C3 is the valuable molecule target to attenuate in neurological conditions characterised by neuroinflammation and cognitive dysfunction.Graphical

Unravelling the Road to Recovery: Mechanisms of Wnt Signalling in Spinal Cord Injury.

Spinal cord injury (SCI) is a complex neurodegenerative pathology that consistently harbours a poor prognostic outcome. At present, there are few therapeutic strategies that can halt neuronal cell death and facilitate functional motor recovery. However, recent studies have highlighted the Wnt pathway as a key promoter of axon regeneration following central nervous system (CNS) injuries. Emerging evidence also suggests that the temporal dysregulation of Wnt may drive cell death post-SCI. A major challenge in SCI treatment resides in developing therapeutics that can effectively target inflammation and facilitate glial scar repair. Before Wnt signalling is exploited for SCI therapy, further research is needed to clarify the implications of Wnt on neuroinflammation during chronic stages of injury. In this review, an attempt is made to dissect the impact of canonical and non-canonical Wnt pathways in relation to individual aspects of glial and fibrotic scar formation. Furthermore, it is also highlighted how modulating Wnt activity at chronic time points may aid in limiting lesion expansion and promoting axonal repair.

AAV8 vector induced gliosis following neuronal transgene expression.

Expression of light sensitive ion channels by selected neurons has been achieved by viral mediated transduction with gene constructs, but for this to have therapeutic uses, for instance in treating epilepsy, any adverse effects of viral infection on the cerebral cortex needs to be evaluated. Here, we assessed the impact of adeno-associated virus 8 (AAV8) carrying DNA code for a soma targeting light activated chloride channel/FusionRed (FR) construct under the CKIIa promoter. Viral constructs were harvested from transfected HEK293 cells in vitro and purified. To test functionality of the opsin, cultured rodent neurons were transduced and the light response of transduced neurons was assayed using whole-cell patch-clamp recordings. In vivo expression was confirmed by immunofluorescence for FR. Unilateral intracranial injections of the viral construct were made into the mouse neocortex and non-invasive fluorescence imaging of FR expression made over 1-4 weeks post-injection using an IVIS Spectrum system. Sections were also prepared from injected mouse cortex for immunofluorescence staining of FR, alongside glial and neuronal marker proteins. In vitro, cortical neurons were successfully transduced, showing appropriate physiological responses to light stimulation. Following injections in vivo, transduction was progressively established around a focal injection site over a 4-week period with spread of transduction proportional to the concentration of virus introduced. Elevated GFAP immunoreactivity, a marker for reactive astrocytes, was detected near injection sites associated with, and proportional to, local FR expression. Similarly, we observed reactive microglia around FR expressing cells. However, we found that the numbers of NeuN+ neurons were conserved close to the injection site, indicating that there was little or no neuronal loss. In control mice, injected with saline only, astrocytosis and microgliosis was limited to the immediate vicinity of the injection site. Injections of opsin negative viral constructs resulted in comparable levels of astrocytic reaction as seen with opsin positive constructs. We conclude that introduction of an AAV8 vector transducing expression of a transgene under a neuron specific promotor evokes a mild inflammatory reaction in cortical tissue without causing extensive short-term neuronal loss. The expression of an opsin in addition to a fluorescent protein does not significantly increase neuroinflammation.

Reactive gliosis in traumatic brain injury: a comprehensive review.

Traumatic brain injury (TBI) is one of the most common pathological conditions impacting the central nervous system (CNS). A neurological deficit associated with TBI results from a complex of pathogenetic mechanisms including glutamate excitotoxicity, inflammation, demyelination, programmed cell death, or the development of edema. The critical components contributing to CNS response, damage control, and regeneration after TBI are glial cells-in reaction to tissue damage, their activation, hypertrophy, and proliferation occur, followed by the formation of a glial scar. The glial scar creates a barrier in damaged tissue and helps protect the CNS in the acute phase post-injury. However, this process prevents complete tissue recovery in the late/chronic phase by producing permanent scarring, which significantly impacts brain function. Various glial cell types participate in the scar formation, but this process is mostly attributed to reactive astrocytes and microglia, which play important roles in several brain pathologies. Novel technologies including whole-genome transcriptomic and epigenomic analyses, and unbiased proteomics, show that both astrocytes and microglia represent groups of heterogenic cell subpopulations with different genomic and functional characteristics, that are responsible for their role in neurodegeneration, neuroprotection and regeneration. Depending on the representation of distinct glia subpopulations, the tissue damage as well as the regenerative processes or delayed neurodegeneration after TBI may thus differ in nearby or remote areas or in different brain structures. This review summarizes TBI as a complex process, where the resultant effect is severity-, region- and time-dependent and determined by the model of the CNS injury and the distance of the explored area from the lesion site. Here, we also discuss findings concerning intercellular signaling, long-term impacts of TBI and the possibilities of novel therapeutical approaches. We believe that a comprehensive study with an emphasis on glial cells, involved in tissue post-injury processes, may be helpful for further research of TBI and be the decisive factor when choosing a TBI model.

Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain.

Astrocytes, the most abundant glial cell type in the brain, are underrepresented in traditional cortical organoid models due to the delayed onset of cortical gliogenesis. Here we introduce a new glia-enriched cortical organoid model that exhibits accelerated astrogliogenesis. We demonstrated that induction of a gliogenic switch in a subset of progenitors enabled the rapid derivation of astroglial cells, which account for 25-31% of the cell population within 8-10 weeks of differentiation. Intracerebral transplantation of these organoids reliably generated a diverse repertoire of cortical neurons and anatomical subclasses of human astrocytes. Spatial transcriptome profiling identified layer-specific expression patterns among distinct subclasses of astrocytes within organoid transplants. Using an in vivo acute neuroinflammation model, we identified a subpopulation of astrocytes that rapidly activates pro-inflammatory pathways upon cytokine stimulation. Additionally, we demonstrated that CD38 signaling has a crucial role in mediating metabolic and mitochondrial stress in reactive astrocytes. This model provides a robust platform for investigating human astrocyte function.

Impact of prior axonal injury on subsequent injury during brain tissue stretching – A mesoscale computational approach

Epidemiology studies of traumatic brain injury (TBI) show individuals with a prior history of TBI experience an increased risk of future TBI with a significantly more detrimental outcome. But the mechanisms through which prior head injuries may affect risks of injury during future head insults have not been identified. In this work, we show that prior brain tissue injury in the form of mechanically induced axonal injury and glial scar formation can facilitate future mechanically induced tissue injury. To achieve this, we use finite element computational models of brain tissue and a history-dependent pathophysiology-based mechanically-induced axonal injury threshold to determine the evolution of axonal injury and scar tissue formation and their effects on future brain tissue stretching. We find that due to the reduced stiffness of injured tissue and glial scars, the existence of prior injury can increase the risk of future injury in the vicinity of prior injury during future brain tissue stretching. The softer brain scar tissue is shown to increase the strain and strain rate in its vicinity by as much as 40% in its vicinity during dynamic stretching that reduces the global strain required to induce injury by 20% when deformed at 15 s−1 strain rate. The results of this work highlight the need to account for patient history when determining the risk of brain injury.

Transformation of A1/A2 Astrocytes Participates in Brain Ischemic Tolerance Induced by Cerebral Ischemic Preconditioning via Inhibiting NDRG2.

Cerebral ischemic preconditioning (CIP) has been shown to improve brain ischemic tolerance against subsequent lethal ischemia. Reactive astrocytes play important roles in cerebral ischemia-reperfusion. Recent studies have shown that reactive astrocytes can be polarized into neurotoxic A1 phenotype (C3d) and neuroprotective A2 phenotype (S100A10). However, their role in CIP remains unclear. Here, we focused on the role of N-myc downstream-regulated gene 2 (NDRG2) in regulating the transformation of A1/A2 astrocytes and promoting to brain ischemic tolerance induced by CIP. A Sprague Dawley rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used. Rats were divided into the following six groups: (1) sham group; (2) CIP group: left middle cerebral artery was blocked for 10min; (3) MCAO/R group: left middle cerebral artery was blocked for 90min; (4) CIP + MCAO/R group: CIP was performed 72h before MCAO/R; (5) AAV-NDRG2 + CIP + MCAO/R group: adeno-associated virus (AAV) carrying NDRG2 was administered 14 days before CIP + MCAO/R; (6) AAV-Ctrl + CIP + MCAO/R group: empty control group. The rats were subjected to neurological evaluation 24h after the above treatments, and then were sacrificed for 2, 3, 5-triphenyltetraolium chloride staining, thionin staining, immunofluorescence and western blot analysis. In CIP + MCAO/R group, the neurological deficit scores decreased, infarct volume reduced, and neuronal density increased compared with MCAO/R group. Notably, CIP significantly increased S100A10 expression and the number of S100A10+/GFAP+ cells, and also increased NDRG2 expression. MCAO/R significantly decreased S100A10 expression and the number of S100A10+/GFAP+ cells yet increased C3d expression and the number of C3d+/GFAP+ cells and NDRG2 expression, and these trends were reversed by CIP + MCAO/R. Furthermore, over-expression of NDRG2 before CIP + MCAO/R, the C3d expression and the number of C3d+/GFAP+ cells increased, while S100A10 expression and the number of S100A10+/GFAP+ cells decreased. Meanwhile, over-expression of NDRG2 blocked the CIP-induced brain ischemic tolerance. Taken together, these results suggest that CIP exerts neuroprotective effects against ischemic injury by suppressing A1 astrocyte polarization and promoting A2 astrocyte polarization via inhibiting NDRG2 expression.

Sirtuin 3 regulates astrocyte activation by reducing Notch1 signaling after status epilepticus.

Sirtuin3 (Sirt3) is a nicotinamide adenine dinucleotide enzyme that contributes to aging, cancer, and neurodegenerative diseases. Recent studies have reported that Sirt3 exerts anti-inflammatory effects in several neuropathophysiological disorders. As epilepsy is a common neurological disease, in the present study, we investigated the role of Sirt3 in astrocyte activation and inflammatory processes after epileptic seizures. We found the elevated expression of Sirt3 within reactive astrocytes as well as in the surrounding cells in the hippocampus of patients with temporal lobe epilepsy and a mouse model of pilocarpine-induced status epilepticus (SE). The upregulation of Sirt3 by treatment with adjudin, a potential Sirt3 activator, alleviated SE-induced astrocyte activation; whereas, Sirt3 deficiency exacerbated astrocyte activation in the hippocampus after SE. In addition, our results showed that Sirt3 upregulation attenuated the activation of Notch1 signaling, nuclear factor kappa B (NF-κB) activity, and the production of interleukin-1β (IL1β) in the hippocampus after SE. By contrast, Sirt3 deficiency enhanced the activity of Notch1/NF-κB signaling and the production of IL1β. These findings suggest that Sirt3 regulates astrocyte activation by affecting the Notch1/NF-κB signaling pathway, which contributes to the inflammatory response after SE. Therefore, therapies targeting Sirt3 may be a worthy direction for limiting inflammatory responses following epileptic brain injury.

Modulation of GPER1 alleviates early brain injury via inhibition of A1 reactive astrocytes activation after intracerebral hemorrhage in mice

BackgroundEarly brain injury (EBI) caused by inflammatory responses in acute phase of Intracerebral hemorrhage (ICH) plays a vital role in the pathological progression of ICH. Increasing evidences demonstrate A1 reactive astrocytes are associated with the severity of EBI. G-protein coupled estrogen receptor 1 (GPER1) has been proved mediating the neuroprotective effects of estrogen in central nervous system (CNS) disease. However, whether GPER1 plays a protective effect on ICH and A1 reactive astrocytes activation is not well studied. MethodsICH model was established by infused the autologous whole blood into the right basal ganglia in wild type and GPER1 knockout mice. GPER1 specific agonist G1 and antagonist G15 were administered by intraperitoneal injection at 1 h or 0.5 h after ICH. Neurological function was detected on day 1 and day 3 by open field test and corner turn test following ICH. Besides, A1 reactive astrocytes were determined by immunofluorescence staining after ICH on day 3. To further identify the possible mechanism of GPER1 mediated neuroprotective effect, Western blot assays was performed after ICH on day 3. ResultsAfter ICH, G1 treatment alleviated mice neurobehavior deficits on day 1 and day 3. Meanwhile, G1 treatment also significantly reduced the GFAP positive astrocytes and the C3 positive cells after ICH. Interestingly, G15 reversed the protective effect of G1 on the neurobehavior of ICH mice. Meanwhile, the expression of GFAP+C3+ A1 reactive astrocytes were also reduced by activation of GPER1. Mechanistic studies indicated TLR4 and NF-κB mediated the neuroprotective effect of GPER1. ConclusionGenerally, activation of GPER1 alleviated the EBI through inhibiting A1 reactive astrocytes activation via TLR4/NF-κB pathway after ICH in mice. Additionally, GPER1may be a promising target for ICH treatment.

Neural Circuitry Polarization in the Spinal Dorsal Horn (SDH): A Novel Form of Dysregulated Circuitry Plasticity during Pain Pathogenesis.

Pathological pain emerges from nociceptive system dysfunction, resulting in heightened pain circuit activity. Various forms of circuitry plasticity, such as central sensitization, synaptic plasticity, homeostatic plasticity, and excitation/inhibition balance, contribute to the malfunction of neural circuits during pain pathogenesis. Recently, a new form of plasticity in the spinal dorsal horn (SDH), named neural circuit polarization (NCP), was discovered in pain models induced by HIV-1 gp120 and chronic morphine administration. NCP manifests as an increase in excitatory postsynaptic currents (EPSCs) in excitatory neurons and a decrease in EPSCs in inhibitory neurons, presumably facilitating hyperactivation of pain circuits. The expression of NCP is associated with astrogliosis. Ablation of reactive astrocytes or suppression of astrogliosis blocks NCP and, concomitantly, the development of gp120- or morphine-induced pain. In this review, we aim to compare and integrate NCP with other forms of plasticity in pain circuits to improve the understanding of the pathogenic contribution of NCP and its cooperation with other forms of circuitry plasticity during the development of pathological pain.

Lithium-loaded GelMA-Phosphate glass fibre constructs: Implications for astrocyte response.

Combinations of different biomaterials with their own advantages as well as functionalization with other components have long been implemented in tissue engineering to improve the performance of the overall material. Biomaterials, particularly hydrogel platforms, have shown great potential for delivering compounds such as drugs, growth factors, and neurotrophic factors, as well as cells, in neural tissue engineering applications. In central the nervous system, astrocyte reactivity and glial scar formation are significant and complex challenges to tackle for neural and functional recovery. GelMA hydrogel-based tissue constructs have been developed in this study and combined with two different formulations of phosphate glass fibers (PGFs) (with Fe3+ or Ti2+ oxide) to impose physical and mechanical cues for modulating astrocyte cell behavior. This study was also aimed at investigating the effects of lithium-loaded GelMA-PGFs hydrogels in alleviating astrocyte reactivity and glial scar formation offering novel perspectives for neural tissue engineering applications. The rationale behind introducing lithium is driven by its long-proven therapeutic benefits in mental disorders, and neuroprotective and pronounced anti-inflammatory properties. The optimal concentrations of lithium and LPS were determined in vitro on primary rat astrocytes. Furthermore, qPCR was conducted for gene expression analysis of GFAP and IL-6 markers on primary astrocytes cultured 3D into GelMA and GelMA-PGFs hydrogels with and without lithium and in vitro stimulated with LPS for astrocyte reactivity. The results suggest that the combination of bioactive phosphate-based glass fibers and lithium loading into GelMA structures may impact GFAP expression and early IL-6 expression. Furthermore, GelMA-PGFs (Fe) constructs have shown improved performance in modulating glial scarring over GFAP regulation.

Hemorrhagic stroke-induced subtype of inflammatory reactive astrocytes disrupts blood-brain barrier.

Astrocytes undergo disease-specific transcriptomic changes upon brain injury. However, phenotypic changes of astrocytes and their functions remain unclear after hemorrhagic stroke. Here we reported hemorrhagic stroke induced a group of inflammatory reactive astrocytes with high expression of Gfap and Vimentin, as well as inflammation-related genes lipocalin-2 (Lcn2), Complement component 3 (C3), and Serpina3n. In addition, we demonstrated that depletion of microglia but not macrophages inhibited the expression of inflammation-related genes in inflammatory reactive astrocytes. RNA sequencing showed that blood-brain barrier (BBB) disruption-related gene matrix metalloproteinase-3 (MMP3) was highly upregulated in inflammatory reactive astrocytes. Pharmacological inhibition of MMP3 in astrocytes or specific deletion of astrocytic MMP3 reduced BBB disruption and improved neurological outcomes of hemorrhagic stroke mice. Our study demonstrated that hemorrhagic stroke induced a group of inflammatory reactive astrocytes that were actively involved in disrupting BBB through MMP3, highlighting a specific group of inflammatory reactive astrocytes as a critical driver for BBB disruption in neurological diseases.

A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

Fabrication of multifunctional silk nanofibril/hyaluronic acid scaffold for spinal cord repair

Bioactive scaffolds accurately mimicking the structure and composition of the extracellular matrix have garnered significant interest in tissue engineering. In this study, we developed a platform utilizing natural silk nanofibrils, hyaluronic acid, and basic fibroblast growth factor for the purpose of promoting spinal cord regeneration by creating an optimal microenvironment. The bioactive scaffold exhibited notable characteristics such as high porosity and hydrophilicity, attributed to its unique nanostructure, high connectivity, and polysaccharide composition. Furthermore, the pore size of the scaffold can be adjusted within the range of 90 μm to 120 μm by varying the content of hyaluronic acid. In vitro, human umbilical vein endothelial cells were seeded into the scaffold, demonstrating enhanced cell viability. The scaffold facilitated cell proliferation and migration. In vivo experiments on rats indicated that the scaffold had a beneficial impact on spinal cord regeneration, creating a conducive environment for motor function recovery of the rats. This effect may be attributed to the scaffold's ability to stimulate axon growth and neuronal survival, as well as inhibit the formation of glial scars, as evidenced by the decreased expression of growth associated protein-43, microtubule-associated protein 2, and neurofilament-200. This study presents a promising method to develop a feasible bioscaffold for the treatment of spinal cord injury.

Disconnecting prefrontal cortical neurons from the ventral midline thalamus: Loss of specificity due to progressive neural toxicity of an AAV-Cre in the rat thalamus

BackgroundThe thalamic reuniens (Re) and rhomboid (Rh) nuclei are bidirectionally connected with the medial prefrontal cortex (mPFC) and the hippocampus (Hip). Fiber-sparing N-methyl-D-aspartate lesions of the ReRh disrupt cognitive functions, including persistence of certain memories. Because such lesions irremediably damage neurons interconnecting the ReRh with the mPFC and the Hip, it is impossible to know if one or both pathways contribute to memory persistence. Addressing such an issue requires selective, pathway-restricted and direction-specific disconnections. New methodA recent method associates a retrograde adeno-associated virus (AAV) expressing Cre recombinase with an anterograde AAV expressing a Cre-dependent caspase, making such disconnection feasible by caspase-triggered apoptosis when both constructs meet intracellularly. We injected an AAVrg-Cre-GFP into the ReRh and an AAV5-taCasp into the mPFC. As expected, part of mPFC neurons died, but massive neurotoxicity of the AAVrg-Cre-GFP was found in ReRh, contrasting with normal density of DAPI staining. Other stainings demonstrated increasing density of reactive astrocytes and microglia in the neurodegeneration site. Comparison with existing methodsReducing the viral titer (by a 4-fold dilution) and injection volume (to half) attenuated toxicity substantially, still with evidence for partial disconnection between mPFC and ReRh. ConclusionsThere is an imperative need to verify potential collateral damage inherent in this type of approach, which is likely to distort interpretation of experimental data. Therefore, controls allowing to distinguish collateral phenotypic effects from those linked to the desired disconnection is essential. It is also crucial to know for how long neurons expressing the Cre-GFP protein remain operational post-infection.

G-CSF increases calprotectin expression, liver damage and neuroinflammation in a murine model of alcohol-induced ACLF.

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF. Methods: ACLF was triggered by a single alcohol binge (5g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200μg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed. Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1β was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice. Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

BM-MSC-Loaded Graphene-Collagen Cryogels Ameliorate Neuroinflammation in a Rat Spinal Cord Injury Model.

A major obstacle to axonal regeneration following spinal cord injury (SCI) is neuroinflammation mediated by astrocytes and microglial cells. We previously demonstrated that graphene-based collagen hydrogels alone can decrease neuroinflammation in SCI. Their regenerative potential, however, is poorly understood and incomplete. Furthermore, stem cells have demonstrated both neuroprotective and regenerative properties in spinal cord regeneration, although there are constraints connected with the application of stem cell-based therapy. In this study, we have analyzed the regeneration capability of human bone marrow mesenchymal stem cell (BM-MSC)-loaded graphene-cross-linked collagen cryogels (Gr-Col) in a thoracic (T10-T11) hemisection model of SCI. Our study found that BM-MSC-loaded Gr-Col improves axonal regeneration, reduces neuroinflammation by decreasing astrocyte reactivity, and promotes M2 macrophage polarization. BM-MSC-loaded-Gr-Col demonstrated enhanced regenerative potential compared to Gr-Col and the injury group control. Next-generation sequencing (NGS) analysis revealed that BM-MSC-loaded-Gr-Col modulates the JAK2-STAT3 pathway, thus decreasing the reactive and scar-forming astrocyte phenotype. The decrease in neuroinflammation in the BM-MSC-loaded-Gr-Col group is attributed to the modulation of Notch/Rock and STAT5a/b and STAT6 signaling. Overall, Gene Set Enrichment Analysis suggests the promising role of BM-MSC-loaded-Gr-Col in promoting axonal regeneration after SCI by modulating molecular pathways such as the PI3/Akt pathway, focal adhesion kinase, and various inflammatory pathways.