Synaptic Cell Adhesion Molecule 3 (SynCAM3) Deletion Promotes Recovery from Spinal Cord Injury by Limiting Glial Scar Formation.

Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.

Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury.

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca2+ levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca2+, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line in vitro, and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female Trpv4 KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in Trpv4 KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI.SIGNIFICANCE STATEMENT TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.

Fluorescence Probe for Imaging N-Methyl-d-aspartate Receptors and Monitoring GSH Selectively Using Two-Photon Microscopy.

N-Methyl-d-aspartate (NMDA) is an excitotoxic amino acid used to identify a specific subset of glutamate receptors. The activity of NMDA receptors is closely related to the redox level of the biological system. Glutathione (GSH) as an antioxidant plays a key role with regard to modulation of the redox environment. In this work we designed and developed a GSH-specific fluorescent probe with the capability of targeting NMDA receptors, which was composed of a two-photon naphthalimide fluorophore, a GSH-reactive group sulfonamide, and an ifenprodil targeting group for the NMDA receptor. This probe exhibited high selectivity toward GSH in comparison to other similar amino acids. Two-photon fluorescence microscopy allowed this probe to successfully monitor GSH in neuronal cells and hippocampal tissues with an excitation at 750 nm. It could serve as a potential practical imaging tool to explore the function of GSH and related biological processes in the brain.

Efficacy for Whitlockite for Augmenting Spinal Fusion.

Whitlockite (WH) is the second most abundant inorganic component of human bone, accounting for approximately 25% of bone tissue. This study investigated the role of WH in bone remodeling and formation in a mouse spinal fusion model. Specifically, morphology and composition analysis, tests of porosity and surface area, thermogravimetric analysis, an ion-release test, and a cell viability test were conducted to analyze the properties of bone substitutes. The MagOss group received WH, Group A received 100% beta-tricalcium phosphate (ß-TCP), Group B received 100% hydroxyapatite (HAp), Group C received 30% HAp/70% ß-TCP, and Group D received 60% HAp/40% ß-TCP (n = 10 each). All mice were sacrificed 6 weeks after implantation, and micro-CT, hematoxylin and eosin (HE) staining, and Masson trichome (MT) staining and immunohistochemistry were performed. The MagOss group showed more homogeneous and smaller grains, and nanopores (<500 nm) were found in only the MagOss group. On micro-CT, the MagOss group showed larger fusion mass and better graft incorporation into the decorticate mouse spine than other groups. In the in vivo experiment with HE staining, the MagOss group showed the highest new bone area (mean: decortication group, 9.50%; A, 15.08%; B, 15.70%; C, 14.76%; D, 14.70%; MagOss, 22.69%; p < 0.0001). In MT staining, the MagOss group demonstrated the highest new bone area (mean: decortication group, 15.62%; A, 21.41%; B, 22.86%; C, 23.07%; D, 22.47%; MagOss, 26.29%; p < 0.0001). In an immunohistochemical analysis for osteocalcin, osteopontin, and CD31, the MagOss group showed a higher positive area than other groups. WH showed comparable bone conductivity to HAp and ß-TCP and increased new bone formation. WH is likely to be used as an improved bone substitute with better bone conductivity than HAp and ß-TCP.

Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Discrimination between Human Colorectal Neoplasms with a Dual-Recognitive Two-Photon Probe.

Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and ß-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and ß-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and ß-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of ß-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).

Ratiometric Detection of γ-Glutamyltransferase in Human Colon Cancer Tissues Using a Two-Photon Probe.

γ-Glutamyltransferase (GGT) plays a role in cleaving the γ-glutamyl bond of glutathione. The GGT is known to be overexpressed in some tumors and has been recognized as a potential biomarker for malignant tumors. Colon cancer is one of the most common cancers worldwide; however, there is no quantitative method for detecting cancer cells in human colon tissues. In this study, we report a ratiometric two-photon probe for GGT that can be applied in human colon tissues. The probe (Probe 2) showed high fluorescence efficiency, marked fluorescence changes, excellent kinetics, and selectivity for the GGT in live colon cells. Additionally, we obtained ratiometric two-photon microscopy images of GGT activity in human colon tissue. We used this method to compare normal and cancer tissues based on their ratio values; the ratio value was higher in cancer tissue than in normal tissue. This study provides a method for quantitative analysis of GGT, particularly in human colon cancer, which will be useful for studying GGT-related diseases and diagnosing colon cancer.

A Two-Photon Fluorescent Probe for Imaging Endogenous ONOO- near NMDA Receptors in Neuronal Cells and Hippocampal Tissues.

In this study, we developed a two-photon fluorescent probe for detection of peroxynitrite (ONOO-) near the N-methyl-d-aspartate (NMDA) receptor. This naphthalimide-based probe contains a boronic acid reactive group and an ifenprodil-like tail, which serves as an NMDA receptor targeting unit. The probe displays high sensitivity and selectivity, along with a fast response time in aqueous solution. More importantly, the probe can be employed along with two-photon fluorescence microscopy to detect endogenous ONOO- near NMDA receptors in neuronal cells as well as in hippocampal tissues. The results suggest that the probe has the potential of serving as a useful imaging tool for studying ONOO- related diseases in the nervous system.

Carboxylate-Containing Two-Photon Probe for the Simultaneous Detection of Extra- and Intracellular pH Values in Colon Cancer Tissue.

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p Ka values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.

Highly Selective and Sensitive Two-Photon Fluorescence Probe for Endogenous Peroxynitrite Detection and Its Applications in Living Cells and Tissues.

A new two-photon fluorescence probe for endogenous peroxynitrite (ONOO-) detection was designed and synthesized. The probe exhibits good selectivity and sensitivity for ONOO- in phosphate-buffered saline solution with a low detection limit (3.5 × 10-8 M). Furthermore, the probe displays good performance in detecting endogenous ONOO-, not only in RAW 264.7 cells but also in rat hippocampal tissue, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 µm.

Two-Photon Tracer for Human Epidermal Growth Factor Receptor-2: Detection of Breast Cancer in a Live Tissue.

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10-50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90-130 µm in live mouse tissue by TPM.

Readily Accessible and Predictable Naphthalene-Based Two-Photon Fluorophore with Full Visible-Color Coverage.

Herein we report 22 acedan-derived, two-photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two-photon excitation with single wavelength, and to design a turn-on, two-photon fluorescence sensor for endogenous H2 O2 in Raw 264.7 macrophage and rat brain hippocampus ex vivo.

Two-photon probe for Cu²âº with an internal reference: quantitative estimation of Cu²âº in human tissues by two-photon microscopy.

Copper ions play a crucial role in living systems as cofactors of numerous metalloenzymes. To quantitatively estimate the Cu(2+) concentration in human tissue, we have developed a two-photon (TP) probe with an internal reference (ACCu2) that shows significant TP action cross-section and high selectivity for Cu(2+) and can quantitatively estimate the Cu(2+) concentration in human colon tissues by dual-color two-photon microscopy (TPM) imaging with minimum interference from other competing metal ions or pH and minimum cytotoxicity and photostability problems. The Cu(2+) concentrations in human normal colon, polyp, and colon cancer tissues were found to be 8.3 ± 0.3, 13 ± 2, and 22 ± 3 µM, respectively. This result suggests that ACCu2 may be useful for the diagnosis of human colon cancer.

Dual-color imaging of cytosolic and mitochondrial zinc ions in live tissues with two-photon fluorescent probes.

We report two-photon probes for Zn(2+) ions that can simultaneously detect cytosolic and mitochondrial Zn(2+) ions in live cells and living tissues at 115 mm depth by dual-color TPM imaging with minimum interference from other biologically relevant species.

Quantitative estimation of the total sulfide concentration in live tissues by two-photon microscopy.

Hydrogen sulfide (H2S) is a newly recognized transmitter, which protects various organs from oxidative stress. In this article, we report a ratiometric two-photon probe, TFCA, which can be excited by 750 nm femtosecond pulses, shows a 110-fold increase in the intensity ratio upon reaction with HS(-) and high selectivity for HS(-) and can visualize the total sulfide ([H2S] + [HS(-)]) distribution in live tissue by two-photon microscopy (TPM). We also developed a kinetic method to quantitatively estimate the total sulfide concentration ([H2S] + [HS(-)]) in live tissues. The kinetic method allowed us to measure the observed rate constants (kobs) for the sulfide-induced deazidation reaction of TFCA in live cells and tissues using TPM. The total sulfide concentration was calculated by using kobs = k2[HS(-)], with the k2 value determined in HEPES/EtOH (1/1, pH = 7.2), and [H2S]/[HS(-)] = [H(+)]/Ka. The total sulfide concentration was found to be nearly zero in HeLa cells and 4-7 µM in rat colon tissues.

Shape-memory collagen scaffold combined with hyaluronic acid for repairing intervertebral disc.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a common cause of chronic low back pain (LBP) and a socioeconomic burden worldwide. Conservative therapies and surgical treatments provide only symptomatic pain relief without promoting intervertebral disc (IVD) regeneration. Therefore, the clinical demand for disc regenerative therapies for disc repair is high. METHODS: In this study, we used a rat tail nucleotomy model to develop mechanically stable collagen-cryogel and fibrillated collagen with shape-memory for use in minimally invasive surgery for effective treatment of IVDD. The collagen was loaded with hyaluronic acid (HA) into a rat tail nucleotomy model. RESULTS: The shape-memory collagen structures exhibited outstanding chondrogenic activities, having completely similar physical properties to those of a typical shape-memory alginate construct in terms of water absorption, compressive properties, and shape-memorability behavior. The treatment of rat tail nucleotomy model with shape-memory collagen-cryogel/HA alleviated mechanical allodynia, maintained a higher concentration of water content, and preserved the disc structure by restoring the matrix proteins. CONCLUSION: According to these results, the collagen-based structure could effectively repair and maintain the IVD matrix better than the controls, including HA only and shape-memory alginate with HA.

Development of a COX-2-Selective Fluorescent Probe for the Observation of Early Intervertebral Disc Degeneration.

Cyclooxygenase-2 (COX-2) is a biomolecule known to be overexpressed in inflammation. Therefore, it has been considered a diagnostically useful marker in numerous studies. In this study, we attempted to assess the correlation between COX-2 expression and the severity of intervertebral disc (IVD) degeneration using a COX-2-targeting fluorescent molecular compound that had not been extensively studied. This compound, indomethacin-adopted benzothiazole-pyranocarbazole (IBPC1), was synthesized by introducing indomethacin-a compound with known selectivity for COX-2-into a phosphor with a benzothiazole-pyranocarbazole structure. IBPC1 exhibited relatively high fluorescence intensity in cells pretreated with lipopolysaccharide, which induces inflammation. Furthermore, we observed significantly higher fluorescence in tissues with artificially damaged discs (modeling IVD degeneration) compared to normal disc tissues. These findings indicate that IBPC1 can meaningfully contribute to the study of the mechanism of IVD degeneration in living cells and tissues and to the development of therapeutic agents.

Multimodal therapy strategy based on a bioactive hydrogel for repair of spinal cord injury.

Traumatic spinal cord injury results in permanent and serious neurological impairment, but there is no effective treatment yet. Tissue engineering approaches offer great potential for the treatment of SCI, but spinal cord complexity poses great challenges. In this study, the composite scaffold consists of a hyaluronic acid-based hydrogel, decellularized brain matrix (DBM), and bioactive compounds such as polydeoxyribonucleotide (PDRN), tumor necrosis factor-α/interferon-γ primed mesenchymal stem cell-derived extracellular vesicles (TI-EVs), and human embryonic stem cell-derived neural progenitor cells (NPC). The composite scaffold showed significant effects on regenerative prosses including angiogenesis, anti-inflammation, anti-apoptosis, and neural differentiation. In addition, the composite scaffold (DBM/PDRN/TI-EV/NPC@Gel) induced an effective spinal cord regeneration in a rat spinal cord transection model. Therefore, this multimodal approach using an integrated bioactive scaffold coupled with biochemical cues from PDRN and TI-EVs could be used as an advanced tissue engineering platform for spinal cord regeneration.

Detection of nickel in fish organs with a two-photon fluorescent probe.

Molecular imaging by two-photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more-versatile tool, a large variety of two-photon probes are needed. Herein, we report a new two-photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni(2+) ions in fresh fish organs at 90-175 µm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni(2+) ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two-photon-excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP-MS), thereby allowing the quantitative measurement of Ni(2+) ions in live tissue.

Measurement of pH values in human tissues by two-photon microscopy.

pH values go live! A ratiometric two-photon (TP) probe (NP1, see scheme) that has a significant TP action cross-section, high photostability, negligible toxicity, and can estimate pH values in live cells and human tissues by two-photon microscopy is described. NP1 can detect the difference in pH between live cells from the gastroesophageal junction (GEJ) and the lower esophageal sphincter of patients with and without esophagitis.