Cortical Slice Cultures Research Articles

Prolonged Activity Deprivation Causes Pre- and Postsynaptic Compensatory Plasticity at Neocortical Excitatory Synapses.

Homeostatic plasticity stabilizes firing rates of neurons, but the pressure to restore low activity rates can significantly alter synaptic and cellular properties. Most previous studies of homeostatic readjustment to complete activity silencing in rodent forebrain have examined changes after 2 d of deprivation, but it is known that longer periods of deprivation can produce adverse effects. To better understand the mechanisms underlying these effects and to address how presynaptic as well as postsynaptic compartments change during homeostatic plasticity, we subjected mouse cortical slice cultures to a more severe 5 d deprivation paradigm. We developed and validated a computational framework to measure the number and morphology of presynaptic and postsynaptic compartments from super-resolution light microscopy images of dense cortical tissue. Using these tools, combined with electrophysiological miniature excitatory postsynaptic current measurements, and synaptic imaging at the electron microscopy level, we assessed the functional and morphological results of prolonged deprivation. Excitatory synapses were strengthened both presynaptically and postsynaptically. Surprisingly, we also observed a decrement in the density of excitatory synapses, both as measured from colocalized staining of pre- and postsynaptic proteins in tissue and from the number of dendritic spines. Overall, our results suggest that cortical networks deprived of activity progressively move toward a smaller population of stronger synapses.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Preclinical translational platform of neuroinflammatory disease biology relevant to neurodegenerative disease

Neuroinflammation is a key driver of neurodegenerative disease, however the tools available to model this disease biology at the systems level are lacking. We describe a translational drug discovery platform based on organotypic culture of murine cortical brain slices that recapitulate disease-relevant neuroinflammatory biology. After an acute injury response, the brain slices assume a chronic neuroinflammatory state marked by transcriptomic profiles indicative of activation of microglia and astrocytes and loss of neuronal function. Microglia are necessary for manifestation of this neuroinflammation, as depletion of microglia prior to isolation of the brain slices prevents both activation of astrocytes and robust loss of synaptic function genes. The transcriptomic pattern of neuroinflammation in the mouse platform is present in published datasets derived from patients with amyotrophic lateral sclerosis, Huntington’s disease, and frontotemporal dementia. Pharmacological utility of the platform was validated by demonstrating reversal of microglial activation and the overall transcriptomic signature with transforming growth factor-β. Additional anti-inflammatory targets were screened and inhibitors of glucocorticoid receptors, COX-2, dihydrofolate reductase, and NLRP3 inflammasome all failed to reverse the neuroinflammatory signature. Bioinformatics analysis of the neuroinflammatory signature identified protein tyrosine phosphatase non-receptor type 11 (PTPN11/SHP2) as a potential target. Three structurally distinct inhibitors of PTPN11 (RMC-4550, TN0155, IACS-13909) reversed the neuroinflammatory disease signature. Collectively, these results highlight the utility of this novel neuroinflammatory platform for facilitating identification and validation of targets for neuroinflammatory neurodegenerative disease drug discovery.

A novel organotypic cortical slice culture model for traumatic brain injury: molecular changes induced by injury and mesenchymal stromal cell secretome treatment.

Traumatic brain injury (TBI) is a major worldwide neurological disorder with no neuroprotective treatment available. Three-dimensional (3D) in vitro models of brain contusion serving as a screening platform for drug testing are lacking. Here we developed a new in vitro model of brain contusion on organotypic cortical brain slices and tested its responsiveness to mesenchymal stromal cell (MSC) derived secretome. A focal TBI was induced on organotypic slices by an electromagnetic impactor. Compared to control condition, a temporal increase in cell death was observed after TBI by propidium iodide incorporation and lactate dehydrogenase release assays up to 48 h post-injury. TBI induced gross neuronal loss in the lesion core, with disruption of neuronal arborizations measured by microtubule-associated protein-2 (MAP-2) immunostaining and associated with MAP-2 gene down-regulation. Neuronal damage was confirmed by increased levels of neurofilament light chain (NfL), microtubule associated protein (Tau) and ubiquitin C-terminal hydrolase L1 (UCH-L1) released into the culture medium 48 h after TBI. We detected glial activation with microglia cells acquiring an amoeboid shape with less ramified morphology in the contusion core. MSC-secretome treatment, delivered 1 h post-injury, reduced cell death in the contusion core, decreased NfL release in the culture media, promoted neuronal reorganization and improved microglia survival/activation. Our 3D in vitro model of brain contusion recapitulates key features of TBI pathology. We showed protective effects of MSC-secretome, suggesting the model stands as a tractable medium/high throughput, ethically viable, and pathomimetic biological asset for testing new cell-based therapies.

Transient receptor potential Ankyrin-1 (TRPA1) agonists suppress myelination and induce demyelination in organotypic cortical slices.

Oligodendrocytes are highly specialized glial cells characterized by their production of multilayer myelin sheaths that wrap axons to speed up action potential propagation. It is due to their specific role in supporting axons that impairment of myelin structure and function leads to debilitating symptoms in a wide range of degenerative diseases, including Multiple Sclerosis and Leukodystrophies. It is known that myelin damage can be receptor-mediated and recently oligodendrocytes have been shown to express Ca2+ -permeable Transient Receptor Potential Ankyrin-1 (TRPA1) channels, whose activation can result in myelin damage in ischemia. Here, we show, using organotypic cortical slice cultures, that TRPA1 activation, by TRPA1 agonists JT010 and Carvacrol for varying lengths of time, induces myelin damage. Although TRPA1 activation does not appear to affect oligodendrocyte progenitor cell number or proliferation, it prevents myelin formation and after myelination causes internodal shrinking and significant myelin degradation. This does not occur when the TRPA1 antagonist, A967079, is also applied. Of note is that when TRPA1 agonists are applied for either 24 h, 3 days or 7 days, axon integrity appears to be preserved while mature myelinated oligodendrocytes remain but with significantly shortened internodes. These results provide further evidence that TRPA1 inhibition could be protective in demyelination diseases and a promising therapy to prevent demyelination and promote remyelination.

A transcriptional constraint mechanism limits the homeostatic response to activity deprivation in mammalian neocortex.

Healthy neuronal networks rely on homeostatic plasticity to maintain stable firing rates despite changing synaptic drive. These mechanisms, however, can themselves be destabilizing if activated inappropriately or excessively. For example, prolonged activity deprivation can lead to rebound hyperactivity and seizures. While many forms of homeostasis have been described, whether and how the magnitude of homeostatic plasticity is constrained remains unknown. Here, we uncover negative regulation of cortical network homeostasis by the PARbZIP family of transcription factors. In cortical slice cultures made from knockout mice lacking all three of these factors, the network response to prolonged activity withdrawal measured with calcium imaging is much stronger, while baseline activity is unchanged. Whole-cell recordings reveal an exaggerated increase in the frequency of miniature excitatory synaptic currents reflecting enhanced upregulation of recurrent excitatory synaptic transmission. Genetic analyses reveal that two of the factors, Hlf and Tef, are critical for constraining plasticity and for preventing life-threatening seizures. These data indicate that transcriptional activation is not only required for many forms of homeostatic plasticity but is also involved in restraint of the response to activity deprivation.

Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex.

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly or indirectly, through the production of intermediate basal progenitors. The balance between these major progenitor types is critical for the production of the proper number and types of neurons, and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that, in the developing mouse neocortex of both sex, deleting CDC25B in apical progenitors leads to a transient increase in the production of TBR1+ neurons at the expense of TBR2+ basal progenitors. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is because of the G2 phase lengthening in CDC25B mutants. Together, this study identifies a new role for CDC25B and G2 phase length in direct versus indirect neurogenesis at early stages of cortical development.SIGNIFICANCE STATEMENT This study is the first analysis of the function of CDC25B, a G2/M regulator, in the developing neocortex. We show that removing CDC25B function leads to a transient increase in neuronal differentiation at early stages, occurring simultaneously with a decrease in basal intermediate progenitors (bIPs). Conversely, a CDC25B gain of function promotes production of bIPs, and this is directly related to CDC25B's ability to regulate CDK1 activity. This imbalance of neuron/progenitor production is linked to a G2 phase lengthening in apical progenitors; and using pharmacological treatments on cortical slice cultures, we show that shortening the G2 phase is sufficient to enhance bIP production. Our results reveal the importance of G2 phase length regulation for neural progenitor fate determination.

Routine culture and study of adult human brain cells from neurosurgical specimens.

When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens. The cell types that can be cultured include dissociated cultures of neurons, astrocytes, microglia, pericytes and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells. The initial setup of cultures takes ~2 h, and the cells are ready for further experiments within days to weeks. The resulting cells can be studied as purified or mixed population cultures, slice cultures and explant-derived cultures. This protocol therefore enables the investigation of human brain cells to facilitate translation of neuroscience research to the clinic.

From Euler to the Neocortex – the potential of graph analytical approaches to characterize electrophysiological network properties of human cortical brain slice cultures

From Euler to the Neocortex – the potential of graph analytical approaches to characterize electrophysiological network properties of human cortical brain slice cultures

マウス皮質培養スライスにおけるマイクログリアによるミエリン片の貪食

マウス皮質培養スライスにおけるマイクログリアによるミエリン片の貪食

Dravet Variant SCN1A A1783V Impairs Interneuron Firing Predominantly by Altered Channel Activation.

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in inhibitory interneurons. Recently, a new conditional mouse model expressing the recurrent human p.(Ala1783Val) missense variant has become available. In this study, we provided an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Based on these data, simulated interneuron (IN) firing properties in a conductance-based single-compartment model suggested surprisingly similar firing deficits for NaV1.1A1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaV1.1A1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.Ala1783V in vitro confirmed a predicted IN firing deficit and revealed an accompanying reduction of interneuronal input resistance while demonstrating normal excitability of pyramidal neurons. Altered input resistance was fed back into the model for further refinement. Taken together these data demonstrate that primary loss of function (LOF) gating properties accompanied by altered membrane characteristics may match effects of full haploinsufficiency on the neuronal level despite maintaining physiological peak current density, thereby causing DS.

Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400ng/ml) for 7days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1ng/ml GDF-9. Moreover, 50ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1ng/ml GDF-9. However, the concentration of 50ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

Derivation of Adult Human Cortical Organotypic Slice Cultures for Coculture with Reprogrammed Neuronal Cells.

Adult human cortical organotypic slice culture is an attractive model system to explore mechanisms of human brain pathology as well as to test drug candidates for treatment of neurodegeneration. Acute studies in human brain slices are limited by the lifetime of the tissue and focus mainly on hippocampus slice preparation. Here we describe the derivation of human organotypic slice cultures of cortical origin, which can be kept in culture for up to 6weeks. This method enabled us to test the system in coculture with reprogrammed neurons and show its feasibility in neuronal cell integration experiments in human-to-human grafting situation.

Rho Guanine Nucleotide Exchange Factors Regulate Horizontal Axon Branching of Cortical Upper Layer Neurons.

Axon branching is a crucial process for cortical circuit formation. However, how the cytoskeletal changes in axon branching are regulated is not fully understood. In the present study, we investigated the role of RhoA guanine nucleotide exchange factors (RhoA-GEFs) in branch formation of horizontally elongating axons (horizontal axons) in the mammalian cortex. In situ hybridization showed that more than half of all known RhoA-GEFs were expressed in the developing rat cortex. These RhoA-GEFs were mostly expressed in the macaque cortex as well. An overexpression study using organotypic cortical slice cultures demonstrated that several RhoA-GEFs strongly promoted horizontal axon branching. Moreover, branching patterns were different between overexpressed RhoA-GEFs. In particular, ARHGEF18 markedly increased terminal arbors, whereas active breakpoint cluster region-related protein (ABR) increased short branches in both distal and proximal regions of horizontal axons. Rho kinase inhibitor treatment completely suppressed the branch-promoting effect of ARHGEF18 overexpression, but only partially affected that of ABR, suggesting that these RhoA-GEFs employ distinct downstream pathways. Furthermore, knockdown of either ARHGEF18 or ABR considerably suppressed axon branching. Taken together, the present study revealed that subsets of RhoA-GEFs differentially promote axon branching of mammalian cortical neurons.

Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.

α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na+,K+-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the α2 isoform of NKA (α2-NKA) that has a high affinity for ouabain, we hypothesized that α2-NKA mediates this effect. Knockdown of α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 μM), a Na+,Ca2+ exchanger (NCX) antagonist. α2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca2+ transients ([Ca2+]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca2+]t was observed at DIV5. At DIV6 almost no [Ca2+]t were observed either in control or in α2-siRNA-treated cultures. Immunocytochemical analyses showed that α2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of α2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that α2-NKA activity in OLCs affects NCX-mediated [Ca2+]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K+] changes, might locally influence NCX-mediated [Ca2+]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.

PARP Inhibition Prevents Ethanol-Induced Neuroinflammatory Signaling and Neurodegeneration in Rat Adult-Age Brain Slice Cultures.

Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol's brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day-binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors-olaparib, veliparib, and 4-aminobenzamide-provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol's amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective-thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2→phospholipid→ARA and HMGB1→TLR4→proinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration.

Insulin-like growth factor binding protein-6 released from human mesenchymal stem cells confers neuronal protection through IGF-1R-mediated signaling

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a desirable cell source for cell-based therapy to treat nervous system injuries due to their ability to differentiate into specific cell types. In addition to their multi-potency, hMSCs render the tissue microenvironment more favorable for tissue repair by secreting various growth factors. Our previous study demonstrated that hMSCs secrete several growth factors, including several insulin-like growth factor binding proteins (IGFBPs). Among these, IGFBP-6 binds with high affinity and inhibits insulin growth factor-2 (IGF-2) to inhibit the growth of IGF-2-dependent tumors. However, the function of IGFBP-6 in the nervous system remains to be fully elucidated. The present study investigated the protective effects of IGFBP-6 secreted by hMSCs on H2O2-injured primary cortical neuron cultures and lysolecithin-injured organotypic spinal cord slice cultures. Treatment of the H2O2-injured cortical neurons with conditioned media from hMSCs (hMSC-CM) increased the phosphorylation of Akt, reduced cell death and mitochondrial translocation of Bax, and regulated extracellular levels of IGF-1 and IGF-2. MTT assay, western blot analysis and ELISA were used to detect the cell viability and protein expression levels, respectively. An inhibitory antibody against IGFBP-6 eliminated this hMSC-CM-mediated neuroprotective effect in the injured cortical neuron cultures and spinal cord slice cultures. In addition, treatment with cyclolignan picropodophyllin, an inhibitor of IGF-1 receptor (IGF-1R), significantly inhibited neuronal protection by hMSC-CM. These findings demonstrated that hMSC-CM-mediated neuroprotection was attributed to IGF-1R-mediated signaling, potentiated via the inhibition of IGF-2 by IGFBP-6. The results of the present study provide insight into the mechanism by which hMSC administration may promote recovery from nerve injury.

Effects of Gualou Guizhi Decoction Aqueous Extract on Axonal Regeneration in Organotypic Cortical Slice Culture after Oxygen-Glucose Deprivation.

Gualou Guizhi decoction (GLGZD) is effective for the clinical treatment of limb spasms caused by ischemic stroke, but its underlying mechanism is unclear. Propidium iodide (PI) fluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemistry, western blot, and real-time qPCR were used to observe the axonal regeneration and neuroprotective effects of GLGZD aqueous extract on organotypic cortical slices exposed to oxygen-glucose deprivation (OGD) and further elucidate the potential mechanisms. Compared with the OGD group, the GLGZD aqueous extract decreased the red PI fluorescence intensity; inhibited neuronal apoptosis; improved the growth of slice axons; upregulated the protein expression of tau and growth-associated protein-43; and decreased protein and mRNA expression of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor 1 (NgR1), ras homolog gene family A (RhoA), rho-associated coiled-coil-containing protein kinase (ROCK), and phosphorylation of collapsin response mediator protein 2 (CRMP2). Our study found that GLGZD had a strong neuroprotective effect on brain slices after OGD injury. GLGZD plays a vital role in promoting axonal remodeling and functional remodeling, which may be related to regulation of the expression of Nogo-A and its receptor NgR1, near the injured axons, inhibition of the Rho-ROCK pathway, and reduction of CRMP2 phosphorylation.