visualizing-gatekeeper-evan-s-blue-dye-based-assessment-blood-brain

Visualizing the Gatekeeper: Evan's Blue Dye-Based Assessment of Blood-Brain Barrier Permeability in Adult Zebrafish

The Blood-Brain Barrier (BBB) is a semi-permeable interface that maintains central nervous system (CNS) homeostasis by regulating the movement of ...

International Islamic University Malaysia (IIUM)

The second most prevalent neurodegenerative disorder in the elderly is Parkinson's disease (PD). Its etiology is unclear and there are no available disease-modifying medicines. Therefore, more evidence is required concerning its pathogenesis. The use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the basis of most animal models of PD. MPTP is metabolized by monoamine oxidase B (MAO B) to MPP + and induces the loss of dopaminergic neurons in the substantia nigra in mammals. Zebrafish have been commonly used in developmental biology as a model organism, but owing to its perfect mix of properties, it is now emerging as a model for human diseases. Zebrafish () are cheap and easy to sustain, evolve rapidly, breed transparent embryos in large amounts, and are readily manipulated by different methods, particularly genetic ones. Furthermore, zebrafish are vertebrate species and mammalian findings obtained from zebrafish may be more applicable than those derived from genetic models of invertebrates such as  and . The resemblance cannot be taken for granted, however. The goal of the present review article is to highlight the promise of zebrafish as a PD animal model. As its aminergic structures, MPTP mode of action, and PINK1 roles mimic those of mammalians, zebrafish seems to be a viable model for studying PD. The roles of zebrafish MAO, however, vary from those of the two types of MAO present in mammals. The benefits unique to zebrafish, such as the ability to perform large-scale genetic or drug screens, should be exploited in future experiments utilizing zebrafish PD models.

The Promise of the Zebrafish Model for Parkinson's Disease: Today's Science and Tomorrow's Treatment.

 has been traditionally used to treat several medical disorders including inflammation. However, the traditional claims of this plant as an anti-inflammatory agent has not been substantially evaluated using modern scientific techniques. The main objective of this study was to evaluate the anti-inflammatory effect of  leaf extract (ALEX-M) and potentiate its activity through nano-encapsulation. The extract-loaded nanocapsules were fabricated using water-in-oil-in-water () emulsion method and characterized via multiple techniques including DLS, TEM, FTIR, and TGA. The toxicity and the anti-inflammatory activity of ALEX-M and the extract-loaded nanocapsules (ALEX-M-PNCs) were evaluated in-vitro on RAW 264.7 macrophages and  on zebrafish embryos. The nanocapsules demonstrated spherical shape with mean particle diameter of 167.13 ± 1.24 nm, narrow size distribution (PDI = 0.29 ± 0.01), and high encapsulation efficiency (87.36 ± 1.81%). ALEX-M demonstrated high viability at high concentrations in RAW 264.7 cells and zebrafish embryos, however, ALEX-M-PNCs showed relatively higher cytotoxicity. Both free and nanoencapsulated extract expressed anti-inflammatory effects through significant reduction of the pro-inflammatory mediator nitric oxide (NO) production in LPS/IFNγ-stimulated RAW 264.7 macrophages and zebrafish embryos in a concentration-dependent manner. The findings highlight that ALEX-M can be recognized as a potential anti-inflammatory agent, and its anti-inflammatory activity can be potentiated by nano-encapsulation. Further studies are warranted toward investigation of the mechanistic and immunomodulatory roles of ALEX-M.

Fabrication and characterization of Agarwood extract-loaded nanocapsules and evaluation of their toxicity and anti-inflammatory activity on RAW 264.7 cells and in zebrafish embryos.

The emergence of resistance to pathogenic bacteria has resulted from the misuse of antibiotics used in wound treatment. Therefore, nanomaterial-based agents can be used to overcome these limitations. In this study, polycaprolactone (PCL)/gelatin/graphene oxide electrospun nanofibers (PGO) are functionalized via plasma treatment with the monomeric groups diallylamine (PGO-M1), acrylic acid (PGO-M2), and -butyl acrylate (PGO-M3) to enhance the action against bacteria cells. The surface functionalization influences the morphology, surface wettability, mechanical properties, and thermal stability of PGO nanofibers. PGO-M1 and PGO-M2 exhibit good antibacterial activity against  and , whereas PGO-M3 tends to reduce their antibacterial properties compared to PGO nanofibers. The highest proportion of dead bacteria cells is found on the surface of hydrophilic PGO-M1, whereas live cells are colonized on the surface of hydrophobic PGO-M3. Likewise, PGO-M1 shows a good interaction with L929, which is confirmed by the high levels of adhesion and proliferation with respect to the control. All the results confirm that surface functionalization can be strategically used as a tool to engineer PGO nanofibers with controlled antibacterial properties for the fabrication of highly versatile devices suitable for different applications (e.g., health, environmental pollution).

PCL/Gelatin/Graphene Oxide Electrospun Nanofibers: Effect of Surface Functionalization on In Vitro and Antibacterial Response.

Lysosome is a primary degradative organelle and is crucial in cellular homeostasis. A reduction in its function due to ageing has been associated with the development of Alzheimer's disease (AD), a common neurodegenerative disorder characterised by the deposition of neurotoxic amyloid plaque in the brain and cerebral vessel walls. The breakdown of the blood-brain barrier (BBB) plays a vital role in the pathogenesis of AD. However, the impact of lysosomal dysfunction on brain endothelial cells, the key component of the BBB, in the disease progression is yet to be fully understood. In this study, human brain endothelial cells (HBEC-5i) were exposed to a lysosomotropic compound, chloroquine (CQ) for 24 h. Cell viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay to determine the inhibitory concentration (IC) at IC (17.5 µM), IC (70.5 µM), and IC (125 µM). The morphological changes observed include vacuoles arrested in the cytosols and cell shrinkage that were more prominent at IC and IC. Lysosomal dysfunction was evaluated by measuring the lysosomal-associated membrane protein-1 (LAMP-1) and microtubule-associated protein light chain 3-II (LC3-II) using the capillary-based immunoassay. LC3-II was significantly increased at IC and IC (p < 0.05 and p < 0.001 respectively). The concentration of intracellular and extracellular Aβ42 was quantitated using the enzyme-linked immunosorbent assay, which demonstrated a significant increase (p < 0.05) in intracellular Aβ42 at IC. This study showed that perturbation of lysosomal function impairs autophagy that leads to intracellular increment of Aβ, indicating the important roles of lysosomes in endothelial cells homeostasis and disease progression.

Lysosomal dysfunction induced cytosolic vacuolation and increased intracellular amyloid-beta 42 (Aβ42) in human brain endothelial cells (HBEC-5i).

High-mobility group box 1 (HMGB1) has been implicated as a key player in two critical factors of Parkinson's disease (PD): mitochondrial dysfunction and neuroinflammation. However, the specific role of HMGB1 in PD remains elusive. We investigated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on mitochondrial dysfunction and HMGB1-associated inflammatory genes as well as locomotor activity in zebrafish, aiming to elucidate the role of HMGB1 in PD. Adult zebrafish received MPTP injections, and locomotor activity was measured at 24- and 48-h post-administration. Gene expression levels related to mitophagy (fis1, pink1, and park2) and HMGB1-mediated inflammation (hmgb1, tlr4, and nfkb) were quantified through RT-qPCR analysis. Following MPTP injection, the significant increase in transcript levels of fis1, pink1, and park2 indicated notable changes in PINK1/Parkin mitophagy, while the upregulation of hmgb1, tlr4, and nfkb genes pointed to the activation of the HMGB1/TLR4/NFκB inflammatory pathway. Furthermore, MPTP-injected zebrafish exhibited decreased locomotor activity, evident through reduced distance travelled, mean speed, and increased freezing durations. HMGB1 plays a major role in cellular processes as it is involved in both the mitophagy process and functions as a pro-inflammatory protein. MPTP administration in adult zebrafish activated mitophagy and inflammatory signaling, highlighting the significant role of HMGB1 as a mediator in both processes and further emphasizing its significant contribution to PD pathogenesis.

Mitophagy: A Bridge Linking HMGB1 and Parkinson's Disease Using Adult Zebrafish as a Model Organism.

Lysosomes are an important intracellular organelle that regulates cellular degradation. Dysfunctional lysosomes disrupt this process, leading to the accumulation of toxic proteins that are meant to be degraded inside the cell, leading to cellular stress and potential toxicity. One of the proteins is beta-amyloid which is associated with conditions like cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). To identify its effects on the vascular compartment, the current study explored lysosomal dysfunction's impact on cytosolic vacuole formation and amyloid beta 40 (Aß40) levels in human brain endothelial cells (HBEC-5i). Cells treated with the lysosomotropic compound chloroquine (70.5 µM) exhibited morphological changes, including prominent cytosolic vacuole formation. The vacuole density was recorded at 11.86 ± 1.907 vacuoles per cell (p < 0.05), and its diameter was significantly increased (3.76 ± 0.182 µm, p < 0.05) compared to the negative control group. However, the average cell size remained unchanged despite the vacuole formation in CQ-treated cells. ELISA tests on lysate and supernatant revealed no significant differences between treatment and control groups in intracellular and extracellular Aß40 levels. This suggested that while lysosomal dysfunction induced cytosolic vacuole changes, it did not significantly alter Aß40 levels. Further research is needed to elucidate the pathways involved in Aß40.

Article	Year
The Promise of the Zebrafish Model for Parkinson's Disease: Today's Science and Tomorrow's Treatment. Frontiers in genetics\| PubMed ID: 33936174	2021
Fabrication and characterization of Agarwood extract-loaded nanocapsules and evaluation of their toxicity and anti-inflammatory activity on RAW 264.7 cells and in zebrafish embryos. Drug delivery\| PubMed ID: 34894947	2021
PCL/Gelatin/Graphene Oxide Electrospun Nanofibers: Effect of Surface Functionalization on In Vitro and Antibacterial Response. Nanomaterials (Basel, Switzerland)\| PubMed ID: 36770449	2023
Lysosomal dysfunction induced cytosolic vacuolation and increased intracellular amyloid-beta 42 (Aβ42) in human brain endothelial cells (HBEC-5i). Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 36931027	2023
Mitophagy: A Bridge Linking HMGB1 and Parkinson's Disease Using Adult Zebrafish as a Model Organism. Brain sciences\| PubMed ID: 37509008	2023
The effects of lysosomal dysfunction on cytoplasmic vacuolation and amyloid-beta 40 (Aß40) level in human brain endothelial cells (HBEC-5i). Cellular and molecular biology (Noisy-le-Grand, France)\| PubMed ID: 40285786	2025
"Tamoxifen for residual NF-PitNETs: A Proof-of-Concept study integrating receptor profiling, pilot trial, and target pathway analysis". Endocrine\| PubMed ID: 40889098	2025

Mohd Hamzah Mohd Nasir

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