The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. Here, to explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3 and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely, thymosin beta 4, thymosin beta 10 and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

GhAGL16 (AGAMOUS-LIKE16) Negatively Regulates Tolerance to Water Deficit in Transgenic Arabidopsis and Cotton.

Cotton is one of the most economically important crops in the world, and drought is a key abiotic factor that can significantly reduce cotton yield. MADS-box transcription factors play essential roles in various aspects of plant growth and development as well as responses to biotic and abiotic stress. However, the use of MADS-box transcription factors to regulate water stress responses has not been fully explored in cotton. Here, we showed that GhAGL16 acts as a negative regulator of water deficit in cotton, at least in part by regulating ABA signaling. GhAGL16-overexpressing (GhAGL16-OE) transgenic Arabidopsis had lower survival rates and relative water contents (RWCs) under water stress. Isolated leaves of GhAGL16-OE Arabidopsis had increased water loss rates, likely attributable to their increased stomatal density. GhAGL16-OE Arabidopsis also showed reduced primary root lengths in response to mannitol treatment and decreased sensitivity of seed germination to ABA treatment. By contrast, silencing GhAGL16 in cotton enhanced tolerance to water deficit by increasing proline (Pro) content, increasing superoxide dismutase (SOD) and peroxidase (POD) activities, and reducing malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents under water stress. Subcellular localization and transcriptional activation assays confirmed that GhAGL16 is a nuclear protein that lacks transcriptional self-activation activity. The expression of ABA biosynthesis-related genes (GhNCED3/7/14), a catabolism-related gene (GhCYP707A), and a gene related to the ABA signaling pathway (GhABF4) was altered in GhAGL16-silenced plants. Taken together, our data demonstrate that GhAGL16 plays an important role in cotton resistance to water stress.

Combination of fMRI and PET reveals the beneficial effect of three-phase enriched environment on post-stroke memory deficits by enhancing plasticity of brain connectivity between hippocampus and peri-hippocampal cortex.

AIM: The three-phase enriched environment (EE) intervention paradigm has been shown to improve learning and memory function after cerebral ischemia, but the neuronal mechanisms are still unclear. This study aimed to investigate the hippocampal-cortical connectivity and the metabolic interactions between neurons and astrocytes to elucidate the underlying mechanisms of EE-induced memory improvement after stroke. METHODS: Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) or sham surgery and housed in standard environment or EE for 30 days. Memory function was examined by Morris water maze (MWM) test. Magnetic resonance imaging (MRI) was conducted to detect the structural and functional changes. [18 F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) was conducted to detect brain energy metabolism. PET-based brain connectivity and network analysis was performed to study the changes of hippocampal-cortical connectivity. Astrocyte-neuron metabolic coupling, including gap junction protein connexin 43 (Cx43), glucose transporters (GLUTs), and monocarboxylate transporters (MCTs), was detected by histological studies. RESULTS: Our results showed EE promoted memory function improvement, protected structure integrity, and benefited energy metabolism after stroke. More importantly, EE intervention significantly increased functional connectivity between the hippocampus and peri-hippocampal cortical regions, and specifically regulated the level of Cx43, GLUTs and MCTs in the hippocampus and cortex. CONCLUSIONS: Our results revealed the three-phase enriched environment paradigm enhanced hippocampal-cortical connectivity plasticity and ameliorated post-stroke memory deficits. These findings might provide some new clues for the development of EE and thus facilitate the clinical transformation of EE.

Three-phase Enriched Environment Improves Post-stroke Gait Dysfunction via Facilitating Neuronal Plasticity in the Bilateral Sensorimotor Cortex: A Multimodal MRI/PET Analysis in Rats.

The three-phase Enriched Environment (EE) paradigm has been shown to promote post-stroke functional improvement, but the neuronal mechanisms are still unclear. In this study, we applied a multimodal neuroimaging protocol combining magnetic resonance imaging (MRI) and positron emission tomography (PET) to examine the effects of post-ischemic EE treatment on structural and functional neuroplasticity in the bilateral sensorimotor cortex. Rats were subjected to permanent middle cerebral artery occlusion. The motor function of the rats was examined using the DigiGait test. MRI was applied to investigate the EE-induced structural modifications of the bilateral sensorimotor cortex. [18F]-fluorodeoxyglucose PET was used to detect glucose metabolism. Blood oxygen level-dependent (BOLD)-functional MRI (fMRI) was used to identify the regional brain activity and functional connectivity (FC). In addition, the expression of neuroplasticity-related signaling pathways including neurotrophic factors (BDNF/CREB), axonal guidance proteins (Robo1/Slit2), and axonal growth-inhibitory proteins (NogoA/NgR) as well as downstream proteins (RhoA/ROCK) in the bilateral sensorimotor cortex were measured by Western blots. Our results showed the three-phase EE improved the walking ability. Structural T2 mapping imaging and diffusion tensor imaging demonstrated that EE benefited structure integrity in the bilateral sensorimotor cortex. PET-MRI fused images showed improved glucose metabolism in the corresponding regions after EE intervention. Specifically, the BOLD-based amplitude of low-frequency fluctuations showed that EE increased spontaneous activity in the bilateral motor cortex and ipsilateral sensory cortex. In addition, FC results showed increased sensorimotor connectivity in the ipsilateral hemisphere and increased interhemispheric motor cortical connectivity and motor cortical-thalamic connectivity following EE intervention. In addition, a strong correlation was found between increased functional connectivity and improved motor performance of limbs. Specifically, EE regulated the expression of neuroplasticity-related signaling, involving BDNF/CREB, Slit2/Robo1, as well as the axonal growth-inhibitory pathways Nogo-A/Nogo receptor and RhoA/ROCK in the bilateral sensorimotor cortex. Our results indicated that the three-phase enriched environment paradigm enhances neuronal plasticity of the bilateral sensorimotor cortex and consequently ameliorates post-stroke gait deficits. These findings might provide some new clues for the development of EE and thus facilitate the clinical translation of EE.

Inhibition of Prostaglandin-Degrading Enzyme 15-PGDH Mitigates Acute Murine Lung Allograft Rejection.

PURPOSE: Acute rejection is a frequent complication among lung transplant recipients and poses substantial therapeutic challenges. 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme responsible for the inactivation of prostaglandin E2 (PGE2), has recently been implicated in inflammatory lung diseases. However, the role of 15-PGDH in lung transplantation rejection remains elusive. The present study was undertaken to examine the expression of 15-PGDH in rejected lung allografts and whether inhibition of 15-PGDH ameliorates acute lung allograft rejection. METHODS: Orthotopic mouse lung transplantations were performed between donor and recipient mice of the same strain or allogeneic mismatched pairs. The expression of 15-PGDH in mouse lung grafts was measured. The efficacy of a selective 15-PGDH inhibitor (SW033291) in ameliorating acute rejection was assessed through histopathological examination, micro-CT imaging, and pulmonary function tests. Additionally, the mechanism underlying the effects of SW033291 treatment was explored using CD8+ T cells isolated from mouse lung allografts. RESULTS: Increased 15-PGDH expression was observed in rejected allografts and allogeneic CD8+ T cells. Treatment with SW033291 led to an accumulation of PGE2, modulation of CD8+ T-cell responses and mitochondrial activity, and improved allograft function and survival. CONCLUSION: Our study provides new insights into the role of 15-PGDH in acute lung rejection and highlights the therapeutic potential of inhibiting 15-PGDH for enhancing graft survival. The accumulation of PGE2 and modulation of CD8+ T-cell responses represent potential mechanisms underlying the benefits of 15-PGDH inhibition in this model. Our findings provide impetus for further exploring 15-PGDH as a target for improving lung transplantation outcomes.

Tetramethylpyrazine promotes axonal remodeling and modulates microglial polarization via JAK2-STAT1/3 and GSK3-NFκB pathways in ischemic stroke.

Ischemic stroke results in demyelination that underlies neurological disfunction. Promoting oligodendrogenesis will rescue the injured axons and accelerate remyelination after stroke. Microglia react to ischemia/hypoxia and polarize to M1/M2 phenotypes influencing myelin injury and repair. Tetramethylpyrazine (TMP) has neuroprotective effects in treating cerebrovascular disorders. This study aims to evaluate whether TMP promotes the renovation of damaged brain tissues especially on remyelination and modulates microglia phenotypes following ischemic stroke. Here magnetic resonance imaging (MRI)-diffusion tensor imaging (DTI) and histopathological evaluation are performed to characterize the process of demyelination and remyelination. Immunofluorescence staining is used to prove oligodendrogenesis and microglial polarization. Western blotting is conducted to examine interleukin (IL)-6, IL-10, transforming growth factor ß (TGF-ß) and Janus protein tyrosine kinase (JAK) 2-signal transducer and activator of transcription (STAT) 1/3-glycogen synthase kinase (GSK) 3-nuclear transcription factor κB (NFκB) signals. Results show TMP alleviates the injury of axons and myelin sheath, increases NG2+, Ki67+/NG2+, CNPase+, Ki67+/CNPase+, Iba1+/Arg-1+ cells and decreases Iba1+ and Iba1+/CD16+ cells in periinfarctions of rats. Particularly, TMP downregulates IL-6 and upregulates IL-10 and TGF-ß expressions, besides, enhances JAK2-STAT3 and suppresses STAT1-GSK3-NFκB activation in middle cerebral artery occlusion (MCAo) rats. Then we demonstrate that TMP reverses M1/M2 phenotype via JAK2-STAT1/3 and GSK3-NFκB pathways in lipopolysaccharide (LPS) plus interferon-γ (IFN-γ)-stimulated BV2 microglia. Blocking JAK2 with AG490 counteracts TMP's facilitation on M2 polarization of microglia. This study warrants the promising therapy for stroke with TMP treatment.

Disruption of extracellular redox balance drives persistent lung fibrosis and impairs fibrosis resolution.

Lung fibrosis is a devastating outcome of various diffuse parenchymal lung diseases. Despite rigorous research efforts, the mechanisms that propagate its progressive and nonresolving nature remain enigmatic. Oxidative stress has been implicated in the pathogenesis of lung fibrosis. However, the role of extracellular redox state in disease progression and resolution remains largely unexplored. Here, we show that compartmentalized control over extracellular reactive oxygen species (ROS) by aerosolized delivery of recombinant extracellular superoxide dismutase (ECSOD) suppresses an established bleomycin-induced fibrotic process in mice. Further analysis of publicly available microarray, RNA-seq and single-cell RNAseq datasets reveals a significant decrease in ECSOD expression in fibrotic lung tissues that can be spontaneously restored during fibrosis resolution. Therefore, we investigate the effect of siRNA-mediated ECSOD depletion during the established fibrotic phase on the self-limiting nature of the bleomycin mouse model. Our results demonstrate that in vivo knockdown of ECSOD in mouse fibrotic lungs impairs fibrosis resolution. Mechanistically, we demonstrate that transforming growth factor (TGF)-ß1 downregulates endogenous ECSOD expression, leading to the accumulation of extracellular superoxide via Smad-mediated signaling and the activation of additional stores of latent TGF-ß1. In addition, depletion of endogenous ECSOD during the fibrotic phase in the bleomycin model induces an apoptosis-resistant phenotype in lung fibroblasts through unrestricted Akt signaling. Taken together, our data strongly support the critical role of extracellular redox state in fibrosis persistence and resolution. Based on these findings, we propose that compartment-specific control over extracellular ROS may be a potential therapeutic strategy for managing fibrotic lung disorders.

Tetramethylpyrazine promotes stroke recovery by inducing the restoration of neurovascular unit and transformation of A1/A2 reactive astrocytes.

2,3,5,6-Tetramethylpyrazine (TMP) as an active ingredient extracted from a traditional Chinese herbal medicine Ligusticum chuanxiong Hort. has been proved to penetrate blood-brain barrier (BBB) and show neuroprotective effects on cerebral ischemia. However, whether TMP could regulate astrocytic reactivity to facilitate neurovascular restoration in the subacute ischemic stroke needs to be urgently verified. In this research, permanent occlusion of the middle cerebral artery (MCAO) model was conducted and TMP (10, 20, 40 mg/kg) was intraperitoneally administrated to rats once daily for 2 weeks. Neurological function was evaluated by motor deficit score (MDS). Magnetic resonance imaging (MRI) was implemented to analyze tissue injury and cerebral blood flow (CBF). Magnetic resonance angiography (MRA) was applied to exhibit vascular signals. Transmission electron microscopy (TEM) was performed to detect the neurovascular unit (NVU) ultrastructure. Haematoxylin and eosin (HE) staining was utilized to evaluate cerebral histopathological lesions. The neurogenesis, angiogenesis, A1/A2 reactivity, aquaporin 4 (AQP4) and connexin 43 (Cx43) of astrocytes were observed with immunofluorescent staining. Then FGF2/PI3K/AKT signals were measured by western blot. Findings revealed TMP ameliorated neurological functional recovery, preserved NVU integrity, and enhanced endogenous neurogenesis and angiogenesis of rats with subacute ischemia. Shifting A1 to A2 reactivity, suppressing excessive AQP4 and Cx43 expression of astrocytes, and activating FGF2/PI3K/AKT pathway might be potential mechanisms of promoting neurovascular restoration with TMP after ischemic stroke.

Somatic variants of MAP3K3 are sufficient to cause cerebral and spinal cord cavernous malformations.

Cerebral cavernous malformations (CCMs) and spinal cord cavernous malformations (SCCMs) are common vascular abnormalities of the CNS that can lead to seizure, haemorrhage and other neurological deficits. Approximately 85% of patients present with sporadic (versus congenital) CCMs. Somatic mutations in MAP3K3 and PIK3CA were recently reported in patients with sporadic CCM, yet it remains unknown whether MAP3K3 mutation is sufficient to induce CCMs. Here we analysed whole-exome sequencing data for patients with CCM and found that ∼40% of them have a single, specific MAP3K3 mutation [c.1323C>G (p.Ile441Met)] but not any other known mutations in CCM-related genes. We developed a mouse model of CCM with MAP3K3I441M uniquely expressed in the endothelium of the CNS. We detected pathological phenotypes similar to those found in patients with MAP3K3I441M. The combination of in vivo imaging and genetic labelling revealed that CCMs were initiated with endothelial expansion followed by disruption of the blood-brain barrier. Experiments with our MAP3K3I441M mouse model demonstrated that CCM can be alleviated by treatment with rapamycin, the mTOR inhibitor. CCM pathogenesis has usually been attributed to acquisition of two or three distinct genetic mutations involving the genes CCM1/2/3 and/or PIK3CA. However, our results demonstrate that a single genetic hit is sufficient to cause CCMs.

Fluoxetine shows neuroprotective effects against LPS-induced neuroinflammation via the Notch signaling pathway.

AIM: To determine the neuroprotective effects of fluoxetine on depression-like and motor behaviors in rats treated with lipopolysaccharide (LPS) and the mechanisms involved. METHODS: A rat model of depression in Parkinson's disease (dPD) was established by administering LPS (0.5 mg/kg, i.p.) for 4 days. The sucrose preference test (SPT), open field test (OFT), and rotarod test evaluated depression-like and motor behaviors. White matter fiber integrity and intrinsic activity in the brain were assessed using magnetic resonance imaging. For pathological and molecular expression detection, hematoxylin-eosin staining, immunohistochemistry, Luminex technology, western blotting, and quantitative real-time PCR were used. RESULTS: Fluoxetine increased the sucrose preference in the SPT, the horizontal and center distances in the OFT, and the standing time in the rotarod test. Fluoxetine also improved intrinsic activities and white matter fiber damage in the brain, increased c-Fos expression, reduced Iba-1 expression in the prefrontal cortex, hippocampus, and substantia nigra, and increased TH expression in the substantia nigra. Fluoxetine reduced the concentration of inflammatory cytokines (IL-1α, IL-6, TNF-α, and IFN-γ). The gene and protein expression of Notch1, Jagged1, Hes1, and Hes5 were significantly lower than the LPS group after treatment with fluoxetine. CONCLUSION: Fluoxetine plays neuroprotective effects in relieving LPS-induced depression-like and motor behaviors. The underlying mechanisms may be related to inhibiting microglial activation, regulating the Notch signaling pathway, and inhibiting the inflammatory response.

Efficient virus-mediated genome editing in cotton using the CRISPR/Cas9 system.

Plant virus-mediated sgRNA delivery and expression have great advantages; sgRNA expression can rapidly expand and accumulate along with virus replication and movement, resulting in efficient gene editing efficiency. In this study, a VIGE system based on cotton leaf crumple virus (CLCrV) was established using cotton overexpressing Cas9 (Cas9-OE) as the VIGE receptor. CLCrV-mediated VIGE could not only target and knock out the GhMAPKKK2, GhCLA1 and GhPDS genes subgroup A and D genome sequences but also achieve double mutation of GhCLA1 and GhPDS genes at the same time. These results verified the effectiveness and efficiency of this system. In addition, the off-target effect assay demonstrated that the CLCrV-mediated VIGE system not only has high gene editing efficiency but also high gene editing specificity in cotton. We further explored whether the FT-sgRNA strategy could transport sgRNA to cotton apical meristem (SAM) over long distances to avoid using tissue culture to obtain stable genetic mutants. The results showed that the sgRNA fused with FT mRNA at the 5' end could also efficiently achieve targeted editing of endogenous genes in cotton, but it was difficult to detect heritable mutant progeny. The above results showed that the CLCrV-mediated VIGE system provided an accurate and rapid validation tool for screening effective sgRNAs in cotton.

A Small Gtp-Binding Protein GhROP3 Interacts with GhGGB Protein and Negatively Regulates Drought Tolerance in Cotton (Gossypium hirsutum L.).

As a plant-specific Rho-like small G protein, the ROP (Rho-related GTPase of plants) protein regulates the growth and development of plants and various stress responses in the form of molecular switches. Drought is a major abiotic stress that limits cotton yield and fiber quality. In this study, virus-induced gene silencing (VIGS) technology was used to analyze the biological function of GhROP3 in cotton drought stress tolerance. Meanwhile, we used yeast two-hybrid and bimolecular fluorescence complementation assays to examine the interaction between GhROP3 and GhGGB. GhROP3 has a high expression level in cotton true leaves and roots, and responds to drought, high salt, cold, heat stress, and exogenous abscisic acid (ABA) and auxin (IAA) treatments. Silencing GhROP3 improved the drought tolerance of cotton. The water loss rates (WLR) of detached leaves significantly reduced in silenced plants. Also, the relative water content (RWC) and total contents of chlorophyll (Chl) and proline (Pro) of leaves after drought stress and the activities of three antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) significantly increased, whereas the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) significantly reduced. In the leaves of silenced plants, the expression of genes related to ABA synthesis and its related pathway was significantly upregulated, and the expression of decomposition-related GhCYP707A gene and genes related to IAA synthesis and its related pathways was significantly downregulated. It indicated that GhROP3 was a negative regulator of cotton response to drought by participating in the negative regulation of the ABA signaling pathway and the positive regulation of the IAA signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhROP3 protein interacted with the GhGGB protein in vivo and in vitro. This study provided a theoretical basis for the in-depth investigation of the drought resistance-related molecular mechanism of the GhROP3 gene and the biological function of the GhGGB gene.

Magnetic Resonance Imaging Investigation of Neuroplasticity After Ischemic Stroke in Tetramethylpyrazine-Treated Rats.

Ischemic stroke elicits white matter injury typically signed by axonal disintegration and demyelination; thus, the development of white matter reorganization is needed. 2,3,5,6-Tetramethylpyrazine (TMP) is widely used to treat ischemic stroke. This study was aimed to investigate whether TMP could protect the white matter and promote axonal repair after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and treated with TMP (10, 20, 40 mg/kg) intraperitoneally for 14 days. The motor function related to gait was evaluated by the gait analysis system. Multiparametric magnetic resonance imaging (MRI) was conducted to noninvasively identify gray-white matter structural integrity, axonal reorganization, and cerebral blood flow (CBF), followed by histological analysis. The expressions of axonal growth-associated protein 43 (GAP-43), synaptophysin (SYN), axonal growth-inhibitory signals, and guidance factors were measured by Western blot. Our results showed TMP reduced infarct volume, relieved gray-white matter damage, promoted axonal remodeling, and restored CBF along the peri-infarct cortex, external capsule, and internal capsule. These MRI findings were confirmed by histopathological data. Moreover, motor function, especially gait impairment, was improved by TMP treatment. Notably, TMP upregulated GAP-43 and SYN and enhanced axonal guidance cues such as Netrin-1/DCC and Slit-2/Robo-1 but downregulated intrinsic growth-inhibitory signals NogoA/NgR/RhoA/ROCK-2. Taken together, our data indicated that TMP facilitated poststroke axonal remodeling and motor functional recovery. Moreover, our findings suggested that TMP restored local CBF, augmented guidance cues, and restrained intrinsic growth-inhibitory signals, all of which might improve the intracerebral microenvironment of ischemic areas and then benefit white matter remodeling.

The Impact of Ischemic Stroke on Gray and White Matter Injury Correlated With Motor and Cognitive Impairments in Permanent MCAO Rats: A Multimodal MRI-Based Study.

Background: Identifying the alterations of the cerebral gray and white matter is an important prerequisite for developing potential pharmacological therapy for stroke. This study aimed to assess the changes of gray and white matter after permanent middle cerebral artery occlusion (pMCAO) in rats using magnetic resonance imaging (MRI), and to correlate them with the behavior performance. Methods: Rats were subjected to pMCAO or sham surgery and reared for 30 days. Motor and cognitive function of the rats were examined by gait and Morris water maze (MWM) tests, respectively. Multimodal MRI was conducted to examine the functional and structural changes of the gray and white matter followed with luxol fast blue (LFB) staining. Results: The gait and MWM tests revealed significant motor and cognitive dysfunction in pMCAO rats, respectively. Magnetic resonance angiography presented abnormal intracranial arteries in pMCAO rats with reduced signal intensity of the anterior cerebral artery, anterior communicating cerebral artery, internal carotid artery, and increased basilar artery vessel signal compared with sham rats. Arterial spin labeling confirmed the decreased cerebral blood flow in the infarcted sensorimotor cortex and striatum. Structural T2-weighted imaging and T2 mapping showed brain atrophy and elevation of T2 value in the gray (sensorimotor cortex, striatum) and white (external capsule, internal capsule) matter of pMCAO rats. The results from diffusion tensor imaging (DTI) corresponded well with LFB staining showing reduced relative FA accompanied with increased relative AD and RD in the gray and white matter of pMCAO rats compared with sham rats. Fiber tracking derived from DTI further observed significantly reduced fiber density and length in the corresponding brain regions of pMCAO rats compared with sham rats. Specially, the DTI parameters (especially FA) in the relevant gray matter and white matter significantly correlated with the behavior performance in the gait and MWM tests. Conclusion: Collectively, the gray and white matter damages could be non-invasively monitored in pMCAO rats by multimodal MRI. DTI-derived parameters, particularly the FA, might be a good imaging index to stage gray and white matter damages associated with post-stroke motor and cognitive impairments.

Hypothermia selectively protects the anterior forebrain mesocircuit during global cerebral ischemia.

Hypothermia is an important protective strategy against global cerebral ischemia following cardiac arrest. However, the mechanisms of hypothermia underlying the changes in different regions and connections of the brain have not been fully elucidated. This study aims to identify the metabolic nodes and connection integrity of specific brain regions in rats with global cerebral ischemia that are most affected by hypothermia treatment. 18F-fluorodeoxyglucose positron emission tomography was used to quantitatively determine glucose metabolism in different brain regions in a rat model of global cerebral ischemia established at 31-33°C. Diffusion tensor imaging was also used to reconstruct and explore the brain connections involved. The results showed that, compared with the model rats established at 37-37.5°C, the rat models of global cerebral ischemia established at 31-33°C had smaller hypometabolic regions in the thalamus and primary sensory areas and sustained no obvious thalamic injury. Hypothermia selectively preserved the integrity of the anterior forebrain mesocircuit, exhibiting protective effects on the brain during the global cerebral ischemia. The study was approved by the Institutional Animal Care and Use Committee at Capital Medical University (approval No. XW-AD318-97-019) on December 15, 2019.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. AIM OF THE STUDY: We aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. METHODS: Male Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. RESULTS: TSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3ß, and down-regulated phosphorylated ß-catenin and CRMP-2 expression. CONCLUSION: Taken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/ß-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

Characterizing the seizure onset zone and epileptic network using EEG-fMRI in a rat seizure model.

Accurate epileptogenic zone (EZ) or seizure onset zone (SOZ) localization is crucial for epilepsy surgery optimization. Previous animal and human studies on epilepsy have reported that changes in blood oxygen level-dependent (BOLD) signals induced by epileptic events could be used as diagnostic markers for EZ or SOZ localization. Simultaneous electroencephalography and functional magnetic resonance imaging (EEG-fMRI) recording is gaining interest as a non-invasive tool for preoperative epilepsy evaluation. However, EEG-fMRI studies have reported inconsistent and ambiguous findings. Therefore, it remains unclear whether BOLD responses can be used for accurate EZ or SOZ localization. In this study, we used simultaneous EEG-fMRI recording in a rat model of 4-aminopyridine-induced acute focal seizures to assess the spatial concordance between individual BOLD responses and the SOZ. This was to determine the optimal use of simultaneous EEG-fMRI recording in the SOZ localization. We observed a high spatial consistency between BOLD responses and the SOZ. Further, dynamic BOLD responses were consistent with the regions where the seizures were propagated. These results suggested that simultaneous EEG-fMRI recording could be used as a noninvasive clinical diagnostic technique for localizing the EZ or SOZ and could be an effective tool for mapping epileptic networks.

Heritable gene editing using FT mobile guide RNAs and DNA viruses.

BACKGROUND: The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virus-mediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. RESULTS: We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the deficiency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant offspring. Our results showed that sgRNAs fused with FT mRNA at the 5' end (FT strategy) effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efficiency of 4.35-8.79%. In addition, gene-edited offspring by FT-sgRNAs did not contain any components of the CLCrV genome. CONCLUSIONS: FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana.