Rotenone-induced PD model of zebrafish
Rotenone is a natural compound found in plant seeds and rhizomes, which achieves insecticidal effect by interfering with the electron transport chain of mitochondria, and is the main active ingredient in many insecticides. Rotenone can be absorbed through the gastrointestinal tract and lungs. Rotenone is more toxic to fish than mammals. Rotenone, due to its high hydrophobicity, can easily cross the blood-brain barrier and biomembranes, leading to mitochondrial dysfunction in dopaminergic neurons. Rotenone is almost insoluble in water, while low concentrations of rotenone can cause most fish to die quickly. In some studies, a 4-month-old wild-type zebrafish was treated with 2 g·L1 rotenone per day for 4 weeks, and the PD model of zebrafish was successfully made, and the expression level of tyrosine hydroxylase in the zebrafish brain was reduced, which was consistent with the loss of dopaminergic neurons in the brain of PD patients. Rotenone modeling can also mimic PD induced by long-term exposure to toxic environmental compounds, such as pesticides, heavy metals, and the environment of related occupations.
6-Hydroxydopamine (6-OHDA)-induced PD model of zebrafish
6-OHDA is a norepinephrine homolog, which can change the sensitivity and specificity of striatal dopamine receptors by damaging the substantia-striatal pathway, and play a certain role in disrupting the synthesis and transport of dopamine, resulting in a series of characteristic changes similar to human PD symptoms and pathology. 6-OHDA does not penetrate the blood-brain barrier and is highly oxidized. In related experiments, 5 g· L 1 6-OHDA injection into the vitreous cavity of zebrafish could significantly reduce the content of TH protein in zebrafish retina, effectively kill dopaminergic cells in the retina, and simulate the motor defects of PD in behavior. In another study, zebrafish were housed in 250 mol·l 1 6-ohda for 14 days, and zebrafish showed significant anxiety and reduced exercise ability.
MPTP-induced PD model of zebrafish
The neurotoxin MPTP is highly lipophilic and can be metabolized in the brain through the blood-brain barrier to produce the toxic substance 1-methyl-4-phenypyridine ion (MPP+), which is the main substance used to establish the PD model of zebrafish. After MPP+ is transported by dopamine transporters to dopaminergic neurons, it is deposited in the first partea, which affects mitochondrial energy metabolism and free radical metabolism, and consumes oxygen to synthesize ATP, but also produces reactive oxygen species and free radicals that are harmful to the body, causing degeneration and necrosis of dopaminergic neurons. It has been found that MPTP modeling can activate the TLR4 NF-B signaling pathway, reduce the expression of BCL-2 in the brain, and enhance the expression of TLR4, MyD88, NF-B, TNF-, IL-1, IL-6, BAX, and CASPASE-3 in mice. In some experiments, 50 mol·l 1 mpTP was selected to acutely model zebrafish with 1 dpf, and the zebrafish was placed under a fluorescence microscope to observe the development of dopamine neurons and cerebral blood vessels, and it was seen that the dopamine neurons were significantly lost, the cerebral blood vessels were severely damaged, and the number was significantly reducedBehavioral tests of zebrafish to 5 dpf showed a decrease in exercise capacity, a significant decrease in the total distance of movement, and a slowdown in the average speed. In addition, 100 mol·ml 1 mptp was added to zebrafish culture water at 3 dpf for modeling, and the behavior trajectory of zebrafish could be detected at 6 dpf, which showed that the zebrafish decreased their moving distance and increased their activity, showing PD-like symptoms. The blood-brain barrier of juvenile zebrafish is more permeable to neurotoxins such as MPTP than rodents. Using MPTP as a PD model of zebrafish is easy to operate and affordable.
Transgenic zebrafish PD model
At present, the PD-related genes mainly include -syn, parkin, uch-l1, pink1, DJ-1, lrrk2, etc., and common transgenic animal models mainly target the above genes.
Knockout A PD model of zebrafish that knocks down the parkin gene
Parkin is the most common recessive mutation in PD, which mainly affects mitochondrial dysfunction, neuroinflammation, oxidative stress, etc. Parkin, as a ligand for ubiquitin proteins, generally directs the degradation of -syn in the brain, so mutations in this gene lead to the accumulation and aggregation of -syn, which in turn affects the course of the disease. Knockdown of the zebrafish Parkin gene with a large fragment and morpholino antisense oligonucleotide using the CRISPR Cas9 system results in impaired mitochondrial function, selective loss of dopaminergic neurons, and increased sensitivity to MPTP in zebrafish.
A PD model of zebrafish knockout of the pink1 gene
PINK1 is a protein kinase, predominantly located in the inner mitochondrial membrane, capable of regulating mitochondrial function by influencing mitochondrial respiration and autophagy processes, and mutations in this gene lead to autosomal recessive early-onset PD. In some studies, the method of MO knockdown of zebrafish pink1 gene was used to obtain a zebrafish PD model with severe developmental defects, which showed spinal and caudal deformities, pericardial edema, and pathological changes such as decreased number of TH-positive neurons, accumulation of reactive oxygen species, and abnormal mitochondrial function, but the above changes disappeared about 5 days after injection. In addition, the TOL2 transposon mediated zebrafish PINK1 transgenic PD model was also studied, and obvious oxidative stress response and related PD-like pathological changes were observed.
PD model of zebrafish knockout of the DJ-1 gene
Defects in the DJ-1 gene lead to the development of autosomal recessive PD. ZDJ-1, a zebrafish homolog of DJ-1, has been cloned and identified to establish a new vertebrate model in which the effect of this gene in PD progression has been examined. The CRISPR Cas9 system was used to knock out the DJ-1 gene in zebrafish to obtain a related model, and the decrease in TH level, skeletal muscle respiratory failure and body weight decreased with age, especially in males.
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