Brain Barrier Permeability Research Articles

Erythrocyte membrane coated with nitrogen-doped quantum dots and polydopamine composite nano-system combined with photothermal treatment of Alzheimer's disease

Mitochondrial dysfunction and metal ion imbalance are recognized as pathological hallmarks of Alzheimer's Disease (AD), leading to deposition of β-amyloid (Aβ) thereby and inducing neurotoxicity, activating apoptosis, eliciting oxidative stress, and ultimately leading to cognitive impairment. In this study, the red blood cell membrane (RBC) was used as a vehicle for encapsulating carbon quantum dots (CQD) and polydopamine (PDA), creating a nanocomposite (PDA-CQD/RBC). This nanocomposite was combined with near-infrared light (NIR) for AD treatment. The RBC offers anti-immunorecognition properties to evade immune clearance, PDA exhibits enzyme-mimicking activity to mitigate oxidative stress damage, and CQD acts as a chelating agent for metal ions (Cu2+), effectively preventing Cu2+-mediated aggregation of Aβ. Furthermore, the local heating induced by near-infrared laser irradiation can dismantle the formed Aβ fibers and enhance the blood–brain barrier's permeability. Both in vitro and animal experiments have shown that PDA-CQD/RBC, in combination with NIR, mitigates neuroinflammation, and ameliorates behavioral deficits in mice. This approach targets multiple pathological pathways, surpassing the limitations of single-target treatments and enhancing therapeutic efficacy while decelerating disease progression.

Vagus nerve stimulation in cerebral stroke: biological mechanisms, therapeutic modalities, clinical applications, and future directions.

Stroke is a major disorder of the central nervous system that poses a serious threat to human life and quality of life. Many stroke victims are left with long-term neurological dysfunction, which adversely affects the well-being of the individual and the broader socioeconomic impact. Currently, post-stroke brain dysfunction is a major and difficult area of treatment. Vagus nerve stimulation is a Food and Drug Administration-approved exploratory treatment option for autism, refractory depression, epilepsy, and Alzheimer's disease. It is expected to be a novel therapeutic technique for the treatment of stroke owing to its association with multiple mechanisms such as altering neurotransmitters and the plasticity of central neurons. In animal models of acute ischemic stroke, vagus nerve stimulation has been shown to reduce infarct size, reduce post-stroke neurological damage, and improve learning and memory capacity in rats with stroke by reducing the inflammatory response, regulating blood-brain barrier permeability, and promoting angiogenesis and neurogenesis. At present, vagus nerve stimulation includes both invasive and non-invasive vagus nerve stimulation. Clinical studies have found that invasive vagus nerve stimulation combined with rehabilitation therapy is effective in improving upper limb motor and cognitive abilities in stroke patients. Further clinical studies have shown that non-invasive vagus nerve stimulation, including ear/cervical vagus nerve stimulation, can stimulate vagal projections to the central nervous system similarly to invasive vagus nerve stimulation and can have the same effect. In this paper, we first describe the multiple effects of vagus nerve stimulation in stroke, and then discuss in depth its neuroprotective mechanisms in ischemic stroke. We go on to outline the results of the current major clinical applications of invasive and non-invasive vagus nerve stimulation. Finally, we provide a more comprehensive evaluation of the advantages and disadvantages of different types of vagus nerve stimulation in the treatment of cerebral ischemia and provide an outlook on the developmental trends. We believe that vagus nerve stimulation, as an effective treatment for stroke, will be widely used in clinical practice to promote the recovery of stroke patients and reduce the incidence of disability.

Breast-to-brain metastasis is exacerbated with chemotherapy through blood-cerebrospinal fluid barrier and induces Alzheimer's-like pathology.

Control of breast-to-brain metastasis remains an urgent unmet clinical need. While chemotherapies are essential in reducing systemic tumor burden, they have been shown to promote non-brain metastatic invasiveness and drug-driven neurocognitive deficits through the formation of neurofibrillary tangles (NFT), independently. Now, in this study, we investigated the effect of chemotherapy on brain metastatic progression and promoting tumor-mediated NFT. Results show chemotherapies increase brain-barrier permeability and facilitate enhanced tumor infiltration, particularly through the blood-cerebrospinal fluid barrier (BCSFB). This is attributed to increased expression of matrix metalloproteinase 9 (MMP9) which, in turn, mediates loss of Claudin-6 within the choroid plexus cells of the BCSFB. Importantly, increased MMP9 activity in the choroid epithelium following chemotherapy results in cleavage and release of Tau from breast cancer cells. This cleaved Tau forms tumor-derived NFT that further destabilize the BCSFB. Our results underline for the first time the importance of the BCSFB as a vulnerable point of entry for brain-seeking tumor cells post-chemotherapy and indicate that tumor cells themselves contribute to Alzheimer's-like tauopathy.

In silico analysis of molecular descriptors for quercetin analogues: a way to improve blood–brain barrier permeation

In silico analysis of molecular descriptors for quercetin analogues: a way to improve blood–brain barrier permeation

The validation of predictive potential via the system of self-consistent models: the simulation of blood-brain barrier permeation of organic compounds.

To apply the quantitative relationships "structure-endpoint" approach, the reliability of prediction is necessary but sometimes challenging to achieve. In this work, an attempt is made to accomplish the reliability of forecasts by creating a set of random partitions of data into training and validation sets, followed by constructing random models. A system of random models for a helpful approach should be self-consistent, giving a similar or at least comparable statistical quality of the predictions for models obtained using different splits of available data into training and validation sets. The carried out computer experiments aimed at obtaining blood-brain barrier permeation models showed that, in principle, can be used such an approach (the Monte Carlo optimization of the correlation weights for different molecular features) for the above purpose taking advantage of specific algorithms to optimize the modelling steps with applying of new statistical criteria such as the index of ideality of correlation (IIC) and the correlation intensity index (CII). The results so obtained are good and better than what was reported previously. The suggested approach to validation of models is non-identic to traditionally applied manners of the checking up models. The concept of validation can be used for arbitrary models (not only for models of the blood-brain barrier).

Pharmacokinetic and pharmacodynamic profiling of compounds similar to paracetamol from zinc database: an in silico analysis

Introduction: Paracetamol is the most used drug for the management of pain and as an antipyretic through its mechanism of action on Cox 1,2 and 3 receptors. Paracetamol is a lipid-soluble molecule that can pass through the Blood Brain Barrier. Paracetamol has been formulated differently to ensure the optimal onset and duration of action as both analgesic and as antipyretic. Paracetamol overdose is associated with major side effects such as liver damage through its metabolite N-acetyl-p-benzoquinone Imine. Methods: This study generated zinc compounds that are similar in structure to Paracetamol through Ligand-based virtual screening. Molecular docking of these compounds to Cox 1, 2, and 3 receptors followed through Structure-based virtual screening. Compounds with better docking scores to these receptors were analyzed for pharmacokinetics and toxicity profiles. Results: ZINC01714506; 0.986; ZINC01714507; 0.986; and ZINC00394165; 0.987 showed the highest docking scores to cox 3 receptor with probability scores of -6.7kcal/mol, -6.4 and -6.2 kcal/ mol as compared to Paracetamol with -5.3kcal/mol. ZINC01714507; 0.986; ZINC01714506; 0.986; and ZINC00394165; 0.987; showed higher docking scores to Cox 2 with docking scores of -8.3kcal.mol, -8.1kcal/mole and -8.0 kcal/mol compared to paracetamol with -6.6kcal/mol. ZINC00394165; 0.987; ZINC00406627; 0.980; and ZINC01714506; 0.986; showed highest docking scores to Cox-1 than paracetamol with scores of -7.7kcal/mol, -7.6 and-7.6kcal/mol. ZINC01714506; 0.986 was predicted the safest with oral LD50 of 2000mg/kg as compared to paracetamol’s 338mg/kg. ZINC00294715; 0.980, ZINC01747085; 0.985, ZINC00394165; 0.987, ZINC00406627; 0.980, ZINC01557001; 0.987 and ZINC19281575; 0.992 were predicted hepatoactive. ZINC00294715; 0.980; ZINC01557001; 0.987; and ZINC19281575; 0.992; lack Blood Brain Barrier permeation. All compounds showed high GIT absorption and all conform to Lipinski’s rule of five. Conclusion: ZINC01557001; 0.987; ZINC01714506; 0.986; ZINC34120167; 0.994; ZINC00394165; 0.987, ZINC01714507; 0.986; and ZINC01747085; 0.985; are promising in drug discovery for new analgesic and antipyretic drugs, based on better docking scores and better oral LD50

Chemistry-intuitive explanation of graph neural networks for molecular property prediction with substructure masking

Graph neural networks (GNNs) have been widely used in molecular property prediction, but explaining their black-box predictions is still a challenge. Most existing explanation methods for GNNs in chemistry focus on attributing model predictions to individual nodes, edges or fragments that are not necessarily derived from a chemically meaningful segmentation of molecules. To address this challenge, we propose a method named substructure mask explanation (SME). SME is based on well-established molecular segmentation methods and provides an interpretation that aligns with the understanding of chemists. We apply SME to elucidate how GNNs learn to predict aqueous solubility, genotoxicity, cardiotoxicity and blood–brain barrier permeation for small molecules. SME provides interpretation that is consistent with the understanding of chemists, alerts them to unreliable performance, and guides them in structural optimization for target properties. Hence, we believe that SME empowers chemists to confidently mine structure-activity relationship (SAR) from reliable GNNs through a transparent inspection on how GNNs pick up useful signals when learning from data.

3a-(4-Chlorophenyl)-1-methyl-3a,4-dihydroimidazo[1,5-a]quinazolin-5(3H)-one: Synthesis and In Silico Evaluation as a Ligand in the µ-Opioid Receptor

In the search for fused heterocycle molecules with potential biological activities, the new title compound was produced in racemic form via a four step-synthetic sequence with an overall yield of 60%. It was structurally characterised via 1H-, 13C-NMR and IR analyses, and the molecular composition was confirmed through a high-resolution MS experiment. After predicting its analgesic activity using PASS online software, wherein a good overlap between its enantiomers and the structure of the natural opioid morphine was observed, the compound was evaluated through docking calculations as a ligand of the µ-opioid receptor. The resulting energy values and interactions were comparable to the data obtained for morphine and its synthetic derivative fentanyl, which is used in the therapeutic treatment of severe forms of pain. Moreover, the title compound displayed favourable predicted blood–brain barrier permeation and drug-likeness.

Computational pharmacology and computational chemistry of 4-hydroxyisoleucine: Physicochemical, pharmacokinetic, and DFT-based approaches.

Computational pharmacology and chemistry of drug-like properties along with pharmacokinetic studies have made it more amenable to decide or predict a potential drug candidate. 4-Hydroxyisoleucine is a pharmacologically active natural product with prominent antidiabetic properties. In this study, ADMETLab 2.0 was used to determine its important drug-related properties. 4-Hydroxyisoleucine is compliant with important drug-like physicochemical properties and pharma giants' drug-ability rules like Lipinski's, Pfizer, and GlaxoSmithKline (GSK) rules. Pharmacokinetically, it has been predicted to have satisfactory cell permeability. Blood-brain barrier permeation may add central nervous system (CNS) effects, while a very slight probability of being CYP2C9 substrate exists. None of the well-known toxicities were predicted in silico, being congruent with wet lab results, except for a "very slight risk" for respiratory toxicity predicted. The molecule is non ecotoxic as analyzed with common indicators such as bioconcentration and LC50 for fathead minnow and daphnia magna. The toxicity parameters identified 4-hydroxyisoleucine as non-toxic to androgen receptors, PPAR-γ, mitochondrial membrane receptor, heat shock element, and p53. However, out of seven parameters, not even a single toxicophore was found. The density functional theory (DFT) study provided support to the findings obtained from drug-like property predictions. Hence, it is a very logical approach to proceed further with a detailed pharmacokinetics and drug development process for 4-hydroxyisoleucine.

Arginine vasopressin hormone receptor antagonists in experimental autoimmune encephalomyelitis rodent models: A new approach for human multiple sclerosis treatment.

Multiple sclerosis (MS) is a chronic demyelinating and neurodegenerative disease that affects the central nervous system. MS is a heterogeneous disorder of multiple factors that are mainly associated with the immune system including the breakdown of the blood-brain and spinal cord barriers induced by T cells, B cells, antigen presenting cells, and immune components such as chemokines and pro-inflammatory cytokines. The incidence of MS has been increasing worldwide recently, and most therapies related to its treatment are associated with the development of several secondary effects, such as headaches, hepatotoxicity, leukopenia, and some types of cancer; therefore, the search for an effective treatment is ongoing. The use of animal models of MS continues to be an important option for extrapolating new treatments. Experimental autoimmune encephalomyelitis (EAE) replicates the several pathophysiological features of MS development and clinical signs, to obtain a potential treatment for MS in humans and improve the disease prognosis. Currently, the exploration of neuro-immune-endocrine interactions represents a highlight of interest in the treatment of immune disorders. The arginine vasopressin hormone (AVP) is involved in the increase in blood-brain barrier permeability, inducing the development and aggressiveness of the disease in the EAE model, whereas its deficiency improves the clinical signs of the disease. Therefore, this present review discussed on the use of conivaptan a blocker of AVP receptors type 1a and type 2 (V1a and V2 AVP) in the modulation of immune response without completely depleting its activity, minimizing the adverse effects associated with the conventional therapies becoming a potential therapeutic target in the treatment of patients with multiple sclerosis.

Evaluation of pyrimidine/pyrrolidine-sertraline based hybrids as multitarget anti-Alzheimer agents: In-vitro, in-vivo, and computational studies

Alzheimer’s disease (AD) is a complex, multifactorial and most prevalent progressive neurodegenerative ailment. Its multifactorial and complex nature causes the lack of disease modifying drugs. Hence, multi-target drug design strategies have been adopted to halt the progression of AD. In current research, we applied multitarget strategy to tackle multifactorial nature of AD. Rational design and synthesis of framework of hybrids containing Pyrimidine/pyrrolidine-sertraline scaffolds were carried out. The synthesized compounds were further evaluated for their in-vitro enzyme inhibition potential against cholinesterases, monoamine oxidases and β-site amyloid precursor protein cleaving enzyme-1 (BACE-1). Compound 19 emerged as an optimal multipotent hybrid with IC50 values of 0.07 µM, 0.09 µM, 0.63 µM, 0.21 µM and 0.73 µM against AChE, BChE, MAO-A, MAO-B and BACE-1 respectively. After in-vivo cytotoxicity and in-vitro PAMPA blood brain barrier permeation assays, a number of widely used behavioral assessment tests were also performed for the evaluation of memory and learning.Determination of biochemical parameters showed low levels of acetylcholinesterase by the treatment with synthesized compounds. Furthermore, levels of neurotransmitters such as serotonin, dopamine and noradrenaline were also analyzed. Increased neurotransmitter levels showed the improved short and long-term memory as well as enhanced learning behavior. Docking studies on the target enzymes showed correlation with the experimental in-vitro enzyme inhibition results.

Combined Micellar Liquid Chromatography Technique and QSARs Modeling in Predicting the Blood-Brain Barrier Permeation of Heterocyclic Drug-like Compounds.

The quantitative structure-activity relationship (QSAR) methodology was used to predict the blood-brain permeability (log BB) for 65 synthetic heterocyclic compounds tested as promising drug candidates. The compounds were characterized by different descriptors: lipophilicity, parachor, polarizability, molecular weight, number of hydrogen bond acceptors, number of rotatable bonds, and polar surface area. Lipophilic properties of the compounds were evaluated experimentally by micellar liquid chromatography (MLC). In the experiments, sodium dodecyl sulfate (SDS) as the effluent component and the ODS-2 column were used. Using multiple linear regression and leave-one-out cross-validation, we derived the statistically significant and highly predictive quantitative structure-activity relationship models. Thus, this study provides valuable information on the expected properties of the substances that can be used as a support tool in the design of new therapeutic agents.

CNSC-02. BREAST TO BRAIN METASTASIS IS EXACERBATED WITH CHEMOTHERAPY THROUGH BLOOD-CEREBRAL SPINAL FLUID-BARRIER AND INDUCES ALZHEIMER’S-LIKE PATHOLOGY

Abstract Control of breast to brain metastasis remains an urgent unmet clinical need. While chemotherapies are essential in reducing systemic tumor burden, they have also shown to promote non-brain metastatic invasiveness and drug-driven neurocognitive deficits through formation of neurofibrillary tangles (NFT) independently. Now, in this study we investigated the effect of chemotherapy on brain metastatic progression and promoting tumor-mediated NFT. Results show chemotherapies promote increased brain-barrier permeability and facilitate enhanced tumor infiltration, particularly through the blood-cerebrospinal fluid-barrier (BCSFB). This is attributed to increased expression of matrix metalloproteinase 9 (MMP9) which, in turn, mediates loss of Claudin-6 within the choroid plexus cells of the BCSFB. Importantly, increased MMP9 activity in the choroid epithelium following chemotherapy results in cleavage of Tau released from breast cancer cells. This cleaved Tau forms tumor-derived NFT that further destabilize the BCSFB. Our results underline for the first time the importance of the BCSFB as a vulnerable point of entry for brain-seeking tumor cells post-chemotherapy and indicate that tumor cells themselves contribute to Alzheimer’s-like tauopathy.

Pharmacophore Modeling Using Machine Learning for Screening the Blood-Brain Barrier Permeation of Xenobiotics.

Daily exposure to xenobiotics affects human health, especially the nervous system, causing neurodegenerative diseases. The nervous system is protected by tight junctions present at the blood–brain barrier (BBB), but only molecules with desirable physicochemical properties can permeate it. This is why permeation is a decisive step in avoiding unwanted brain toxicity and also in developing neuronal drugs. In silico methods are being implemented as an initial step to reduce animal testing and the time complexity of the in vitro screening process. However, most in silico methods are ligand based, and consider only the physiochemical properties of ligands. However, these ligand-based methods have their own limitations and sometimes fail to predict the BBB permeation of xenobiotics. The objective of this work was to investigate the influence of the pharmacophoric features of protein–ligand interactions on BBB permeation. For these purposes, receptor-based pharmacophore and ligand-based pharmacophore fingerprints were developed using docking and Rdkit, respectively. Then, these fingerprints were trained on classical machine-learning models and compared with classical fingerprints. Among the tested footprints, the ligand-based pharmacophore fingerprint achieved slightly better (77% accuracy) performance compared to the classical fingerprint method. In contrast, receptor-based pharmacophores did not lead to much improvement compared to classical descriptors. The performance can be further improved by considering efflux proteins such as BCRP (breast cancer resistance protein), as well as P-gp (P-glycoprotein). However, the limited data availability for other proteins regarding their pharmacophoric interactions is a bottleneck to its improvement. Nonetheless, the developed models and exploratory analysis provide a path to extend the same framework for environmental chemicals, which, like drugs, are also xenobiotics. This research can help in human health risk assessment by a priori screening for neurotoxicity-causing agents.

Lessons from the Cerebrospinal Fluid Analysis of HTLV-1-Infected Individuals: Biomarkers of Inflammation for HAM/TSP Development.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease that leads to motor impairment due to a chronic inflammatory process in the central nervous system (CNS). However, the HAM/TSP pathogenesis is not completely clear, and biomarkers to define the disease prognosis are still necessary. Thus, we aimed to identify biomarkers for HAM/TSP and potential mechanisms involved in disease development. To that end, the concentrations of VILIP-1, BDNF, VEGF, β-NGF, TGF-β1, fractalkine/CX3CL1, IL-6, IL-18, and TNF-α, and the soluble forms of TREM-1, TREM-2, and RAGE, were assessed using a multiplex bead-based immunoassay in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n = 20), asymptomatic HTLV-1 carriers (AC) (n = 13), and HTLV-1-seronegative individuals (n = 9), with the results analyzed according to the speed of HAM/TSP progression. HAM/TSP patients had elevated fractalkine in the serum but not in the CSF, particularly those with low neuroinflammatory activity (CSF/serum ratio of neopterin <1 and of CXCL10 < 2). HAM/TSP patients with normal CSF levels of neurofilament light chain (NfL) showed elevated β-NGF in serum, and serum BDNF levels were increased in HTLV-1-infected individuals, particularly in HTLV-1 AC. Both HTLV-1 AC and HAM/TSP patients had lower TGF-β1 levels in CSF compared to uninfected individuals, and HAM/TSP patients with active CNS inflammation showed higher CSF levels of IL-18, which correlated with markers of inflammation, neuronal death, and blood–brain-barrier permeability. Although none of the factors evaluated were associated with the speed of HAM/TSP progression, reduced TGF-β1 levels in CSF suggest that suppressive responses to control subclinical and/or active neurodegeneration are impaired, while increased CSF IL-18 indicates the involvement of inflammasome-mediated mechanisms in HAM/TSP development.

Determination of lipophilicity and ionization of fentanyl and its 3‑substituted analogs by reversed-phase thin-layer chromatography

Lipophilicity and ionization constant of sixteen fentanyl analogues were investigated by reversed-phase thin-layer chromatography (RP-TLC). Fourteen compounds have nitrogen containing groups at C-3 position of the piperidine ring and two compounds are 3-carbomethoxy derivatives of fentanyl. Among them, five are diastereomeric cis/trans couples and six novel trans diastereomers. In addition, the lipophilicity and ionization constant of fentanyl were also determined by the same method, as a reference. The physicochemical property, lipophilicity, expressed as retention indices RM0, b, and C0, as well as PC1, was determined and correlated with in silico values. Ionization constants were determined on the basis of the relationships between analyte’s retention expressed as RF and mobile phase pH.Calculated structural descriptors together with the retention indices, were subjected to the principal component analysis – PCA and hierarchical cluster analysis – HCA, in order to provide basic insights into the similarities among the studied compounds. The blood – brain barrier (BBB) permeation was investigated in the function of experimentally obtained values of lipophilicity, polar surface area and molecular weight. In general, results of the present research corroborate well with previously determined antinociceptive activities of the investigated compounds, pointing out that besides the cis/trans isomerism, another set of important parameters should be taken into account when designing new derivatives of C-3 substituted fentanyl, especially lipophilicity, voluminosity and HBD/A character of the substituents. Accordingly, RP-TLC can be considered as a valuable asset in the ligand-based drug design.

Design, synthesis, and biological evaluation of aromatic tertiary amine derivatives as selective butyrylcholinesterase inhibitors for the treatment of Alzheimer's disease

Butyrylcholinesterase (BChE) is recently regarded as a biomarker in progressed Alzheimer's disease (AD), the development of selective BChE inhibitors has attracted a great deal of interest and may be a viable therapeutic strategy for AD. Previously, an aromatic tertiary amine derivative (S17–1001) was screened and validated as a selective BChE inhibitor. Structured-based molecular modification guided the synthesis of 43 analogs. Biological test of cholinesterase inhibition, in vitro blood brain barrier permeation assay, neurotoxicity assay and neuroprotective effects assay indicated two optimal compounds 17c and 19c. Both compounds showed selective BChE inhibitory (hBChE < 20 nM, eeAChE > 10 μM), good BBB permeation and primary cell safety. Besides, 17c can dose-response protect cell from Aβ1−42 induced damage. It also demonstrated that 17c and 19c were able to restore cognitive impairment in vivo test. These data suggest that 17c and 19c represent promising candidate for follow-up in the drug-discovery process against AD.

BSCI-12 BREAST TO BRAIN METASTASIS IS EXACERBATED WITH CHEMOTHERAPY THROUGH BLOOD-CEREBRAL SPINAL FLUID-BARRIER AND INDUCES ALZHEIMER’S-LIKE PATHOLOGY

Abstract Control of breast to brain metastasis remains an urgent unmet clinical need. While chemotherapies are essential in reducing systemic tumor burden, they have also shown to promote non-brain metastatic invasiveness and drug-driven neurocognitive deficits through formation of neurofibrillary tangles (NFT) independently. Now, in this study we investigated the effect of chemotherapy on brain metastatic progression and promoting tumor-mediated NFT. Results show chemotherapies promote increased brain-barrier permeability and facilitate enhanced tumor infiltration, particularly through the blood-cerebrospinal fluid-barrier (BCSFB). This is attributed to increased expression of matrix metalloproteinase 9 (MMP9) which, in turn, mediates loss of Claudin-6 within the choroid plexus cells of the BCSFB. Importantly, increased MMP9 activity in the choroid epithelium following chemotherapy results in cleavage of Tau released from breast cancer cells. This cleaved Tau forms tumor-derived NFT that further destabilize the BCSFB. Our results underline for the first time the importance of the BCSFB as a vulnerable point of entry for brain-seeking tumor cells post-chemotherapy and indicate that tumor cells themselves contribute to Alzheimer’s-like tauopathy.

Spatiotemporal Dynamics of the Molecular Expression Pattern and Intercellular Interactions in the Glial Scar Response to Spinal Cord Injury

Nerve regeneration in adult mammalian spinal cord is poor because of the lack of intrinsic regeneration of neurons and extrinsic factors – the glial scar is triggered by injury and inhibits or promotes regeneration. Recent technological advances in spatial transcriptomics (ST) provide a unique opportunity to decipher most genes systematically throughout scar formation, which remains poorly understood. Here, we first constructed the tissue-wide gene expression patterns of mouse spinal cords over the course of scar formation using ST after spinal cord injury from 32 samples. Locally, we profiled gene expression gradients from the leading edge to the core of the scar areas to further understand the scar microenvironment, such as neurotransmitter disorders, activation of the pro-inflammatory response, neurotoxic saturated lipids, angiogenesis, obstructed axon extension, and extracellular structure re-organization. In addition, we described 21 cell transcriptional states during scar formation and delineated the origins, functional diversity, and possible trajectories of subpopulations of fibroblasts, glia, and immune cells. Specifically, we found some regulators in special cell types, such as Thbs1 and Col1a2 in macrophages, CD36 and Postn in fibroblasts, Plxnb2 and Nxpe3 in microglia, Clu in astrocytes, and CD74 in oligodendrocytes. Furthermore, salvianolic acid B, a blood–brain barrier permeation and CD36 inhibitor, was administered after surgery and found to remedy fibrosis. Subsequently, we described the extent of the scar boundary and profiled the bidirectional ligand-receptor interactions at the neighboring cluster boundary, contributing to maintain scar architecture during gliosis and fibrosis, and found that GPR37L1_PSAP, and GPR37_PSAP were the most significant gene-pairs among microglia, fibroblasts, and astrocytes. Last, we quantified the fraction of scar-resident cells and proposed four possible phases of scar formation: macrophage infiltration, proliferation and differentiation of scar-resident cells, scar emergence, and scar stationary. Together, these profiles delineated the spatial heterogeneity of the scar, confirmed the previous concepts about scar architecture, provided some new clues for scar formation, and served as a valuable resource for the treatment of central nervous system injury.

Quantitative Determination of 5-Aminoisoquinoline, a PARP-1 Inhibitor by UPLC-MS/MS: In Silico ADME Profile and In Vitro Metabolic Stability Study

5-Aminoisoquinoline (5-AIQ) is a water-soluble, potent and selective Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor, widely used as a biochemical and pharmacological tool to study the inhibitory effect of PARPs enzyme. In this study, a simple, selective and reliable ultra-performance liquid chromatography-tandem mass spectrometry assay has been developed for the quantitative analysis of 5-AIQ in plasma using pantoprazole as an internal standard (IS). Both 5-AIQ and IS were separated on an Acquity CSH18 (2.1 × 100 mm; 1.7 µm) column after chromatographic elution of mobile phase comprising of 10 mM ammonium acetate and acetonitrile (35:65; v/v) at a flow rate of 0.3 mL/min. Electrospray ionization in positive mode was used for sample ionization and precursor to product ion transitions of 145.0 &gt; 91.0; 145.0 &gt; 117.4 for 5-AIQ and 384.0 &gt; 138.1 for IS were used for detection and quantification in multiple reaction monitoring mode. The assay was linear in the concentration range of 1.0 to 666 ng/mL with correlation coefficient of ≥0.995. The precision and bias were within the acceptable limits of ≤12.68% and −8.6 to 5.9%, respectively, with mean recovery of 79.1% from plasma and negligible matrix effects (92.4%). In silico ADME prediction, 5-AIQ showed to be very soluble in water and high gastrointestinal absorption along with blood–brain barrier (BBB) permeability. The validated assay was successfully applied in a metabolic stability study, and 5-AIQ was moderately metabolized by human liver microsomes with an in vitro half-life of 14.5 min and intrinsic clearance of 47.6 µL/min/mg. The validated method can be utilized for future pharmacokinetic and bio-distribution studies.