Background High glucose-induced injury to human retinal endothelial cells (HRCCs) is closely associated with retinal disorders, and melatonin has potential protective effects. However, its underlying mechanism remains unclear. Methods human retinal endothelial cells (HRCCs) were exposed to high glucose and treated with melatonin. Cell viability, apoptosis, and intracellular reactive oxygen species (ROS) levels were assessed using CCK-8, TUNEL staining, and DCFH-DA probe assays, respectively. The expression of PANoptosis-related proteins and components of the Hippo pathway were analyzed by Western blot. LncSNHG5 and miR-26a-5p expression were detected via RT-PCR. Luciferase reporter and RNA-pulldown assays, along with FISH, were employed to confirm direct interactions. Overexpression of LncSNHG5 and co-transfection with miR-26a-5p mimic were utilized to elucidate their roles. Results Melatonin dose-dependently improved HRCC viability, inhibited apoptosis, ROS accumulation and PANoptosis-related proteins (N-GSDMD, p-MLKL, p-RIP3) expression induced by high glucose. High glucose upregulated LncSNHG5, which was dose-dependently downregulated by melatonin. LncSNHG5 directly interacted with miR-26a-5p, and melatonin regulated the Hippo pathway (LATS1/YAP phosphorylation) and PANoptosis via the LncSNHG5/miR-26a-5p axis. Overexpression of LncSNHG5 reversed melatonin’s protective effects, which was attenuated by miR-26a-5p mimic. Conclusion Melatonin alleviates high glucose-induced HRCC injury by regulating PANoptosis, ROS accumulation and Hippo pathway via the LncSNHG5/miR-26a-5p axis.
Mineralocorticoid receptor (MR) overactivation and high salt intake drive vascular dysfunction, characterized by endothelial impairment and stiffening. While the novel non-steroidal MR antagonist (MRA), finerenone improves cardiovascular outcomes in patients with heart failure, its specific effects on vascular sodium accumulation remain unknown. We investigated the effects of finerenone on vascular function, remodeling, and aortic sodium content. Uninephrectomized (UNx) Sprague Dawley rats were subjected to chronic aldosterone infusion (0.75 μg/h) and salt-loading (1% NaCl in drinking water) for 4 weeks, with concurrent oral administration of finerenone (10 mg/kg). Finerenone intervention improved survival and significantly decreased systolic blood pressure. Vascular function analyses revealed that both endothelium-dependent and -independent relaxation were impaired in the model group; however, finerenone significantly preserved these functions. Importantly, sodium content in the aortic tissues as well as plasma was markedly elevated in aldosterone/salt-loaded UNx rats but was significantly reduced by finerenone, concurrent with the downregulation of the MR target gene serum glucocorticoid-regulated kinase 1 (Sgk1). In addition, finerenone attenuated vascular fibrosis and hypertrophy, and restored the aortic nitric oxide level. Altogether, these findings suggest that finerenone ameliorates vascular dysfunction by targeting MR/Sgk1-mediated vascular sodium accumulation and maladaptive remodeling, in concert with systemic blood pressure reduction.
Acute-on-chronic liver failure (ACLF) is a life-threatening condition associated with systemic inflammation, hepatic necroptosis, and disrupted gut-liver axis signaling. Wenyang Jiedu Huayu Formula (WYJDHY), a traditional Chinese medicine formulation, was investigated for its potential therapeutic effects on ACLF and its relationship with gut microbiota modulation. In the ACLF rat model, WYJDHY significantly alleviated liver histopathological damage, reduced neutrophil infiltration and necroptosis, suppressed hepatocyte apoptosis, and improved liver function markers, including alanine aminotransferase (ALT), aspartate transferase (AST), albumin, and total protein. WYJDHY also inhibited inflammation by reducing interleukin-6 (IL-6), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and c-reactive protein (CRP) expression. 16S rRNA gene sequencing revealed that WYJDHY showed a trend toward improving gut microbiota diversity and evenness, partially reversing the altered composition observed in ACLF, including key taxa such as Lachnospiraceae_NK4A136_group, Turicibacter, and Allobaculum. The therapeutic benefits of WYJDHY were significantly diminished in pseudo-germ-free (PG) rats and were transferable through fecal microbiota transplantation (FMT) from WYJDHY-treated rats. These findings demonstrate that WYJDHY alleviates ACLF in a gut microbiota-dependent manner, highlighting the gut–liver axis as a therapeutic target and supporting microbiota-based interventions in ACLF treatment.
Tinospora cordifolia (Willd.) Hook. F. & Thomson (T. cordifolia), commonly known as Giloy or Guduchi, is widely recognized traditional medicinal herb for its potent nootropic and therapeutic properties. While extensive preclinical studies have demonstrated its diverse pharmacological potential, further in-depth clinical research is necessary to elucidate its precise molecular mechanisms of action. The current review aims to provide a rigorous research gap analysis while showcasing the phytochemical profile, nutritive profile, pharmacological attributes, safety, and medicinal information. Additionally, study looked into a network pharmacology-based analysis integrating publicly available biological databases to explore T. cordifolia's potential molecular mechanisms underlying its neuroprotective effects. To gather all research on T. cordifolia's neurobiological functions, a thorough search was done across multiple databases, including Google Scholar, Scopus, the Web of Science, and PubMed till 30th of January, 2026. Globally, several studies have shed light on T. cordifolia's neuro-pharmacological profile. Network pharmacology results suggest that these drugs interact with proteins that are present in Serotonin, Dopaminergic, Vesicular Monoamine Transporters, Cholinergic, Nicotinic Pathways, Opioid Pathways, Histaminergic, Glucocorticoid, Mineralocorticoid, and Hormonal Regulation pathway. Future scientific advancements in the study of T. cordifolia are expected to drive a paradigm shift in medical research and practice, particularly in the development of phytopharmaceuticals targeting the central nervous system.
Purpose To investigate the role of Frizzled-7 (FZD7) in diabetic retinopathy (DR) and elucidate the molecular mechanism by which FZD7 regulates ferroptosis during disease progression. Methods Core DR-associated genes were identified through integrated bioinformatic analyses and validated by RT-qPCR. Gain- and loss-of-function experiments were performed in high glucose (HG)-treated ARPE-19 cells to investigate the role of the β-catenin/DHODH-FSP1 pathway in FZD7-mediated ferroptosis. In vivo, a streptozotocin-induced DR mouse model was used to assess the effects of pharmacological FZD7 inhibition on retinal ferroptotic injury and pathological changes. Results Bioinformatic screening identified four hub genes associated with DR, among which FZD7 was significantly upregulated and further validated in HG-treated ARPE-19 cells. Mechanistically, FZD7 promoted ferroptosis through β-catenin-dependent suppression of the ferroptosis-defense proteins DHODH and FSP1, leading to iron accumulation, oxidative stress, lipid peroxidation, and mitochondrial dysfunction. Conversely, FZD7 silencing restored DHODH and FSP1 expression, attenuated ferroptotic injury, and preserved mitochondrial integrity. Consistent with these findings, pharmacological inhibition of FZD7 in DR mice alleviated retinal ferroptosis, reduced oxidative damage, and improved retinal ultrastructural abnormalities. Conclusions FZD7 drives ferroptotic retinal injury in DR through β-catenin-dependent suppression of the DHODH/FSP1 ferroptosis-defense pathway. Inhibition of FZD7 restores endogenous anti-ferroptotic mechanisms and confers retinal protection, establishing FZD7 as a previously unrecognized therapeutic target for diabetic retinopathy.
Sunitinib is associated with a higher incidence of thrombocytopenia than other molecularly targeted drugs. However, the underlying mechanism remains unclear. Because platelets are derived from megakaryocytes originating from hematopoietic progenitor cells in the bone marrow, sunitinib is presumed to interfere with megakaryocyte proliferation, maturation, or platelet production. Therefore, megakaryocytes play a crucial role in elucidating the mechanism of sunitinib-induced thrombocytopenia. However, in vivo sampling of megakaryocytes is challenging due to the invasiveness of bone marrow collection and the limited cell yield. Accordingly, we investigated the mechanism of thrombocytopenia induced by sunitinib and its active metabolite, N-desethyl sunitinib, using human induced pluripotent stem cell-derived immortalized megakaryocyte cell lines (imMKCLs).Both sunitinib and N-desethyl sunitinib suppressed the proliferation and cell division of imMKCLs. Furthermore, RNA sequencing revealed downregulation of cell cycle-related genes. Although the expression of cyclin-dependent kinase inhibitors, CDKN1A and CDKN1B, was significantly increased, polyploidization in imMKCLs remained unaffected.In conclusion, sunitinib and N-desethyl sunitinib induce thrombocytopenia by inhibiting the proliferation of immature megakaryocytes through suppression of cell cycle progression.
Ischemic stroke (IS) triggers neuroinflammation that drives neuronal dysfunction and tissue injury. Resident central nervous system macrophages critically contribute to IS-induced injury through inflammatory infiltration and cytokine secretion. Methylophiopogonone A (MOA), a flavonoid from Ophiopogon japonicus, protects against heart and liver injury, but its role in IS-induced brain injury remains unclear. Here, we investigated whether MOA attenuates neuroinflammation and brain injury in a mouse IS model and explored its molecular mechanism in macrophage inflammatory activation. MOA treatment reduced brain infarct volume and neurological deficits in transient middle cerebral artery occlusion (tMCAO) mice. It also significantly decreased the secretion of pro-inflammatory cytokine (IL-2, IL-6, IL-1β, IL-18, TNF-α) and TGF-β, and suppressed M1 activation marker expression (IL-6, TNF-α, iNOS) in brain tissue. Immunofluorescence revealed that MOA reduced neutrophil and monocyte-derived macrophage infiltration into ischemic lesions, inhibited macrophage iNOS expression, and suppressed NF-κB p65 phosphorylation. Mechanistically, MOA directly bound the NF-κB p65 subunit, inhibiting its phosphorylation and nuclear translocation in macrophages. This suppressed M1 activation gene expression (IL-6, TNF-α, iNOS) and reduced pro-inflammatory cytokine secretion (IL-6, IL-1β, IL-18, TNF-α). Collectively, these findings demonstrate that MOA protects against tMCAO-induced ischemic injury by inhibiting NF-κB signaling-mediated M1 activation of macrophages and reducing the infiltration of both macrophages and neutrophils. These results position MOA as a potential therapeutic agent for neuroinflammation and brain injury associated with IS.
Friedelin (FD), a natural pentacyclic triterpene, exerts neuroprotective effects, but its role in cerebral ischemic stroke remains unknown. In this study, FD attenuated middle cerebral artery occlusion and reperfusion (MCAO/R)-induced neurological deficits, reduced infarct volume, and inhibited neuronal apoptosis in mice. Additionally, FD suppressed proinflammatory microglial phenotype and proinflammatory cytokine secretion in oxygen-glucose deprivation and reoxygenation (OGD/R)-exposed BV2 cells and in mice after MCAO/R. FD alleviated HT22 cell injury after exposure to conditioned medium from BV2 cells subjected to OGD/R. A mechanistic exploration using network pharmacology and the finding that FD concentration-dependently suppressed nitric oxide (NO) overproduction induced by the Toll-like receptor 4 (TLR4)-specific ligand ultrapure LPS, identified TLR4 as a potential target of FD. FD suppressed proinflammatory microglial phenotype, proinflammatory cytokine secretion and ferroptosis by reducing TLR4 protein expression in mice after MCAO/R and in OGD/R-exposed BV2 cells. Inhibiting ferroptosis in BV2 cells ameliorated OGD/R-induced proinflammatory microglial phenotype and proinflammatory cytokine secretion. Thus, FD alleviated cerebral ischemic injury by repressing proinflammatory microglial phenotype through the regulation of TLR4-induced ferroptosis.
Osteoarthritis is the most common form of arthritis, for which effective and safer treatments are needed. While chondroitin sulfate (CS) has been reported to exert analgesic effects, its cellular targets and immunomodulatory mechanisms remain unclear. This study aimed to clarify the effects of low-molecular-weight CS-rich extract (LMW-CSE) derived from shark cartilage on the hyperalgesia observed in adjuvant-induced arthritis (AIA) mice and to investigate the role of peripheral macrophage (MΦ) subsets in its analgesic action. LMW-CSE was orally administered to mice at doses up to 300 mg/kg twice daily, starting 14 days before AIA with an injection of complete Freund's adjuvant (CFA), and continuing until the completion of behavioral testing. LMW-CSE significantly attenuated hyperalgesia in AIA mice. In addition, the increase in the number of activated excitatory neurons in the spinal superficial dorsal horn of AIA mice was attenuated by LMW-CSE treatment. Flow cytometric analysis revealed that LMW-CSE suppressed the accumulation of proinflammatory M1 MΦs and increased the proportion of immunoregulatory M2 MΦs at inflammatory sites, accompanied by reduced expression of proinflammatory cytokines. These findings suggest that LMW-CSE exerts anti-hyperalgesic effects, at least in part through modulation of peripheral macrophage subsets, supporting its potential as a therapeutic strategy for inflammatory pain.
Postoperative abdominal adhesions can cause several complications, such as intestinal obstruction, female infertility, and chronic pelvic pain. By alkaline cauterization of the cecum, we established a mouse abdominal adhesion model to investigate the roles of secreted protein acidic and rich in cysteine (SPARC) in adhesion formation. Alkaline cauterization led to elevation of adhesion scores and mRNA levels of SPARC, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and collagen I on Days 3, 7, and 14, with a positive correlation between SPARC levels and MCP-1, TNF-α, and collagen I during Days 3-14. Histological assessment revealed the presence of fibrillar collagen in the adhesive tissue between the cecum and other organs/tissues and SPARC at the fibrotic sites. Matrix metalloproteinase (MMP)-9 and MMP-2 were upregulated in the adhesion group on Days 3-7 and 7-14, respectively, with these expressions correlating with SPARC on Days 3, 7, and 14. A disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1), which colocalized with SPARC, was increased in the adhesion model, and showed a positive correlation with SPARC, MCP-1, TNF-α, and collagen I on Days 3 and 7, but not on Day 14. In conclusion, SPARC induction might play important roles in adhesion formation and development.
Background: Ginsenoside RD2, a bioactive compound derived from ginseng, has been shown to possess various biological activities, including potential anti-cancer effects. However, its therapeutic efficacy and molecular mechanisms, particularly in gastric cancer (GC), remain inadequately understood. Methods: The impact of RD2 on GC cells' malignant behavior, epithelial-mesenchymal transition (EMT), and tumor growth in vivo were analyzed in vitro and in vivo. Hub genes were identified using bioinformatics analysis of the Cancer Genome Atlas (TCGA)-Stomach Adenocarcinoma (STAD) dataset and RNA sequencing (RNA-Seq) analysis of AGS cells treated with 80 mu M RD2. After treating cells with RD2, cell viability, colony formation, and transwell assays were carried out to assess the functions of TRIM46 and DUSP1 in mediating the effects of RD2. Results: In a dose-dependent fashion, RD2 dramatically reduced the proliferation, migration, and invasion of GC cells. RD2 treatment reduced the expression of N-cadherin, Vimentin, and Snail and increased that of E-cadherin. Gene expression analysis showed that six key genes (MUC1, MMP9, COL18A1, GPC1, TRIM46, JUP) were downregulated by RD2. In addition, RD2 decreased TRIM46 expression and increased DUSP1 levels, suggesting a potential association with ERK signaling. TRIM46 overexpression reversed the inhibitory effect of RD2 on GC progression, while DUSP1 overexpression counteracted the effects of TRIM46 on EMT and cell viability. Conclusion: Our results suggest RD2 inhibits GC progression through modulation of TRIM46 and DUSP1, with potential involvement of ERK signaling. TRIM46 promotes GC cell growth and EMT, while DUSP1 overexpression counteracts these effects, highlighting the potential therapeutic target of RD2 in GC treatment.
Radiation-induced intestinal injury is a major complication of abdominal radiotherapy; however, effective protective strategies remain limited. To overcome the systemic limitations of total body irradiation models, we developed a localized abdominal irradiation (ABI) mouse model, enabling targeted evaluation of intestinal damage. Using this model, we investigated the protective effects of valproic acid (VPA), a histone deacetylase inhibitor with known anti-inflammatory and cytoprotective properties, against radiation-induced intestinal injury. VPA administration before irradiation mitigated weight loss, preserved villus structure, and improved the villus-to-crypt ratio. Immunohistochemical analyses revealed enhanced epithelial regeneration, with increased Ki-67–positive cell counts in the intestinal mucosa. The inflammatory and oxidative stress marker levels were significantly lower in the VPA group than in the vehicle group. Additionally, intestinal barrier function was preserved, as evidenced by decreased fluorescein isothiocyanate–dextran permeability. These protective effects were most pronounced when VPA was administered prior to irradiation, suggesting that early intervention is crucial for mitigating acute radiation damage. Our findings highlight the usefulness of the ABI model for assessing intestinal injury with high specificity. The results support the potential of VPA as an effective radioprotective agent in abdominal radiotherapy and warrant further investigation into its clinical application, optimal dosing, and underlying action mechanisms.
The blood-brain barrier (BBB), primarily formed by brain microvascular endothelial cells (BMECs), restricts substance entry into the brain. BBB inflammation, involving barrier dysfunction and immune cell recruitment, contributes to neurological diseases and represents a therapeutic target. In this study, we characterized human immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) models as a tool for evaluating effects of BBB inflammation-targeted drugs. First, we examined the responses of the hiMCS-BBB models to the pro-inflammatory cytokine (TNF-alpha and IFN gamma) exposure. The cytokine treatment reduced claudin-5 protein expression levels at the BBB, accompanied by a 1.4 +/- 0.1-fold increase in lucifer yellow permeability. Consistently, E-selectin protein expression was upregulated, accompanied by a 22.1-fold increase in THP-1 cell adhesion to the BBB. Then, we tested whether natalizumab (a monoclonal antibody used for multiple sclerosis treatment) and JPH203 (an inhibitor of L-type amino acid transporter 1) could modulate the immune cell adhesion. Both agents remarkably suppressed the cytokineinduced THP-1 cell adhesion to 0.2-fold relative to the respective controls. To summarize, our results demonstrate that the hiMCS-BBB models exhibit key features of BBB inflammation in response to inflammation stimuli and recapitulate clinically relevant responses to anti-inflammatory drugs, highlighting their potential to accelerate BBB-targeting drug development.
Acetaminophen (APAP) overdose is a leading cause of acute liver injury, yet effective protective therapies remain limited. Isoschaftoside (Iso) is a flavonoid phytochemical with reported antioxidant and anti-inflammatory activities, but its role in APAP-induced liver injury remains unclear. Here, we evaluated the hepatoprotective effect and mechanism of Iso in vivo and in vitro. BALB/c mice received APAP (300 mg/kg, i.g.) to induce acute liver injury, followed 1.5 h later by Iso (8 mg/kg, i.p.); samples were collected at 6 h. Liver pathology was examined by hematoxylin-eosin (H&E) staining; serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) were measured using commercial assay kits; and cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Signaling-related mRNA and protein levels were assessed by quantitative real-time PCR (RT-qPCR) and western blot. Network pharmacology and molecular docking suggested involvement of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-kappa B) signaling, which was further tested in AML12 hepatocytes. Iso markedly alleviated APAP-induced histological damage and reduced serum liver enzymes and pro-inflammatory cytokines. In AML12 cells, Iso suppressed APAP-triggered TLR4/NF-kappa B activation and apoptosis. These findings indicate that Iso mitigates APAP-induced acute liver injury largely by inhibiting TLR4/NF-kappa B-mediated inflammatory and apoptotic responses, supporting its potential as a therapeutic candidate.
Hepatic steatosis and fibrosis represent major complications of type 2 diabetes mellitus (T2DM) with limited therapeutic options. Ginsenoside Rg1, a bioactive constituent of Panax ginseng, exhibits hepatoprotective properties, but its mechanism in diabetic liver injury remains unclear. The transient receptor potential canonical 6 (TRPC6) channel is activated in metabolic disorders and promotes fibrosis. We hypothesized that Rg1 alleviates T2DM-associated liver injury by inhibiting TRPC6-mediated calcium signaling. In vivo, T2DM mice were treated with Rg1 and/or Trpc6 knockout. In vitro, HepG2 cells were exposed to high glucose plus palmitic acid (HG + PA) with Rg1, the TRPC6 inhibitor BI-749327, or the activator OAG. Both Rg1 and Trpc6 knockout ameliorated T2DM-induced liver damage, reducing steatosis and collagen deposition. Mechanistically, they downregulated hepatic TRPC6, calcineurin, and thioredoxin-interacting protein (TXNIP), and inhibited nuclear translocation of carbohydrate response element-binding protein (ChREBP), thereby suppressing NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation. In vitro, Rg1 and BI-749327 attenuated HG + PA-induced calcium overload, lipid accumulation, inflammation, mitochondrial dysfunction, and fibrotic activation, which were significantly abrogated by OAG. Our findings identify Rg1 as a promising agent for T2DM-associated hepatic complications via inhibiting TRPC6-mediated calcium signaling, highlighting TRPC6 as a novel therapeutic target.
Melatonin is a pineal hormone that regulates circadian rhythms primarily through melatonin receptors. We recently demonstrated that melatonin inhibits the voltage-gated potassium KV4.2 channel (IC50 = 479 μM) through a melatonin receptor-independent mechanism. However, it remains unclear whether this inhibitory effect is specific to melatonin or shared by structurally related compounds. In the present study, we examined the effects of melatonin-related compounds on KV4.2 channels using human embryonic kidney 293 cells stably expressing KV4.2 channels. Whole-cell patch-clamp recordings showed that luzindole, a melatonin receptor antagonist, inhibited KV4.2 currents (IC50 = 33 μM). Notably, ramelteon, a melatonin receptor agonist, also inhibited KV4.2 currents (IC50 = 177 μM). These results indicate that the inhibitory effects of luzindole and ramelteon are mediated by direct interactions with KV4.2 channels rather than through melatonin receptors. In addition, luzindole inhibited KV1.5 channels. In contrast, melatonin biosynthetic precursors failed to inhibit KV4.2 currents, suggesting that the indole or indole-like structural motif alone is insufficient for KV4.2 channel inhibition. Instead, specific structural features, including the methoxy group in melatonin and the furan ring in ramelteon, together with increased hydrophobicity, may be critical determinants of KV4.2 channel blockade. Collectively, these findings reveal previously unrecognized pharmacological properties of melatonin-related compounds.
Sepsis, a life-threatening disorder driven by a dysregulated host response to infection, is frequently accompanied by acute lung injury (ALI), worsening disease severity, and mortality. Excessive macrophage-mediated inflammation is central to its pathogenesis. Given emerging evidence that mitochondria-endoplasmic reticulum (ER) crosstalk drives inflammatory injury, we investigated whether modulating this interaction could mitigate sepsis-induced damage. Using LPS-stimulated THP-1 macrophages and a murine model of LPS-induced sepsis, we evaluated the anti-inflammatory and organ-protective effects of dendrobine, a bioactive alkaloid from Dendrobium nobile Lindl. Dendrobine suppressed glycolysis-induced mitochondria-ER crosstalk, thereby reducing macrophage-driven inflammation and tissue injury. In vivo, dendrobine lowered circulating interleukin (IL)-1β and IL-18 levels and alleviated ALI. Mechanistically, dendrobine decreased reactive oxygen species production and mitochondrial DNA (mtDNA) release, leading to downregulation of NLRP3 and cleaved caspase-1. These effects stemmed from inhibition of hypoxia-inducible factor-1α (HIF-1α) and hexokinase 2 (HK2)-mediated glycolysis, preventing HK2 dissociation from voltage-dependent anion channel 1 (VDAC1) and disrupting IP3R-GRP75-VDAC1 complex formation. Collectively, these findings demonstrate that dendrobine protects against sepsis-induced organ injury by targeting the IP3R-GRP75-VDAC1-HK2 axis in macrophages, highlighting its therapeutic potential for sepsis.
This study evaluated glyoxal fixation for whole-brain serial section imaging using the FAST (block-face serial microscopy tomography) system. Glyoxal-fixed mouse brains exhibited slight shrinkage compared with paraformaldehyde-fixed brains but showed no appreciable variations over time. Notably, glyoxal provided an ∼11-fold higher autofluorescence, facilitating precise focal plane identification and high-contrast label-free structural imaging. We observed that 4 days of fixation ensured tissue rigidity for smooth sectioning and intact section retrieval. Furthermore, the application of an autofluorescence quenching reagent enabled clear post hoc c-Fos immunostaining. Thus, glyoxal fixation is a powerful tool for multiscale label-free 3D brain mapping.
