Post-fertilization Zebrafish Larvae Research Articles

Diphenylbutadiene Fluorescent Analogues in Sub-Cellular Imaging and Monitoring Mitophagy.

A series of donor-acceptor (D-π-A) substituted diphenylbutadienes exhibiting solvatochromic emission and a large Stokes shift (100-200 nm) were designed and synthesized for distinctive organelle labelling, enabling real-time monitoring of organelle behaviour such as lysosomal dynamics, mitophagy monitoring, and stress responses. The morpholine-substituted D-A-D diphenylbutadiene (M2) was employed to investigate selective imaging of lysosomes, the uptake of damaged mitochondria through mitophagy, and monitoring lysosomal viscosity or pH changes. Other diphenylbutadiene derivatives (M1, M3, M4) selectively accumulated in lipid droplets. All the synthesized derivatives demonstrated significant uptake in 5-day post-fertilization zebrafish larvae, with M2 showing maximum uptake in the enterocyte-containing heart and intestinal regions, which include the lysosomes.

Development of Liquid Chromatography-Tandem Mass Spectrometry Analytical Methods for the Study of Whole-Body, Head, and Trunk Uptake and Elimination of Methamphetamine in 5-Day Postfertilization Zebrafish Larvae Using the Quick, Easy, Cheap, Effective, Rugged, and Safe Technique.

Synthetic cathinones are drugs of abuse substituted for amphetamine-like stimulant drugs such as methamphetamine. In this study, methamphetamine was studied as a prototypical amphetamine-like drug as a first step toward establishing methods to study this entire drug class. The internal concentration of methamphetamine in zebrafish larvae was determined using matrix-matched calibration along with extraction and purification of samples using the quick, easy, cheap, effective, rugged, and safe technique in liquid chromatography-tandem mass spectrometry. Whole-body and head/trunk uptake and elimination in 5-day postfertilization zebrafish larvae were determined. A gradient method was developed using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases, 10 min of total run time, and a 0.3 mL/min flow rate. The limit of quantification was 60 ng/mL, linearity with r2 = 0.9991, and recovery values from 92% to 120%. The internal concentration of methamphetamine was quantifiable in whole-body homogenates within 15 min of uptake analysis. The internal concentration increased with time, whereas a biphasic elimination pattern was shown. With increasing length of exposure, a higher accumulation of drugs was found in the head than in the trunk.

Simultaneous screening of zebrafish larvae cardiac and respiratory functions: a microfluidic multi-phenotypic approach.

Multi-phenotypic screening of multiple zebrafish larvae plays an important role in enhancing the quality and speed of biological assays. Many microfluidic platforms have been presented for zebrafish phenotypic assays, but multi-organ screening of multiple larvae, from different needed orientations, in a single device that can enable rapid and large-sample testing is yet to be achieved. Here, we propose a multi-phenotypic quadruple-fish microfluidic chip for simultaneous monitoring of heart activity and fin movement of 5-7-day postfertilization zebrafish larvae trapped in the chip. In each experiment, fin movements of four larvae were quantified in the dorsal view in terms of fin beat frequency (FBF). Positioning of four optical prisms next to the traps provided the lateral views of the four larvae and enabled heart rate (HR) monitoring. The device's functionality in chemical testing was validated by assessing the impacts of ethanol on heart and fin activities. Larvae treated with 3% ethanol displayed a significant drop of 13.2 and 35.8% in HR and FBF, respectively. Subsequent tests with cadmium chloride highlighted the novel application of our device for screening the effect of heavy metals on cardiac and respiratory function at the same time. Exposure to 5$\mu$g/l cadmium chloride revealed a significant increase of 8.2% and 39.2% in HR and FBF, respectively. The device can be employed to monitor multi-phenotypic behavioral responses of zebrafish larvae induced by chemical stimuli in various chemical screening assays, in applications such as ecotoxicology and drug discovery.

Vitamin D regulates transepithelial acid secretion in zebrafish (Danio rerio) larvae

Maintenance of an acid-base balance is essential for normal physiological processes in vertebrates. Freshwater fishes live in an aquatic environment with variable pH, and their buffering capacity for acid-base balance in body fluids is weak. Thus, after acid exposure, fishes secrete excess acid to prevent internal acidosis. Acid-secreting ionocytes present in the adult gills and embryonic skin are primarily responsible for acid secretion, and H+-ATPase and Na+/H+ exchanger 3 (NHE3) are the two main transporters responsible for apical acid secretion. Vitamin D is a well-known hormone involved in the maintenance of Ca2+ homeostasis and is suggested to be involved in acid-base regulation by modulating the activity and/or mRNA expression of NHE3 in mammalian models. It remains unclear whether vitamin D is involved in acid secretion in fishes. The aim of the present study was to use zebrafish as a model to determine whether vitamin D and its receptors influence acid secretion. Our results indicated that the levels of 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3), the bioactive vitamin D, were significantly increased in 3 days post-fertilization zebrafish larvae after exposure to acidic freshwater (AFW, pH 4.0). Exogenous 1α,25(OH)2D3 (20 μg/L) incubation substantially enhanced the mRNA expression of acid-secreting transporters and acid secretion at the skin of the entire body and each H+-ATPase-rich cell (HRC), a type of acid-secreting ionocyte. Furthermore, the expression of vitamin D receptors (VDRs) was identified in HRCs of zebrafish. When both VDRa and VDRb were knocked down, acid secretion and the mRNA expression of acid-secreting transporters were significantly decreased. Moreover, double knockdown of VDRa/b prevented the increase in acid secretion induced by AFW and 1α,25(OH)2D3 treatment. This study is the first to indicate that vitamin D is involved in acid secretion in fish.

Mesembryanthemum tortuosum L. alkaloids modify anxiety-like behaviour in a zebrafish model

Ethnopharmacological relevanceMesembryanthemum tortuosum L. (previously known as Sceletium tortuosum (L.) N.E. Br.) is indigenous to South Africa and traditionally used to alleviate anxiety, stress and depression. Mesembrine and its alkaloid analogues such as mesembrenone, mesembrenol and mesembranol have been identified as the key compounds responsible for the reported effects on the central nervous system. Aim of the studyTo investigate M. tortuosum alkaloids for possible anxiolytic-like effects in the 5-dpf in vivo zebrafish model by assessing thigmotaxis and locomotor activity. Materials and methodsLocomotor activity and reverse-thigmotaxis, recognised anxiety-related behaviours in 5-days post fertilization zebrafish larvae, were analysed under simulated stressful conditions of alternating light-dark challenges. Cheminformatics screening and molecular docking were also performed to rationalize the inhibitory activity of the alkaloids on the serotonin reuptake transporter, the accepted primary mechanism of action of selective serotonin reuptake inhibitors. Mesembrine has been reported to have inhibitory effects on serotonin reuptake, with consequential anti-depressant and anxiolytic effects. ResultsAll four alkaloids assessed decreased the anxiety-related behaviour of zebrafish larvae exposed to the light-dark challenge. Significant increases in the percentage of time spent in the central arena during the dark phase were also observed when larvae were exposed to the pure alkaloids (mesembrenone, mesembrenol, mesembrine and mesembrenol) compared to the control. However, mesembrenone and mesembranol demonstrated a greater anxiolytic-like effect than the other alkaloids. In addition to favourable pharmacokinetic and physicochemical properties revealed via in silico predictions, high-affinity interactions characterized the binding of the alkaloids with the serotonin transporter. ConclusionsM. tortuosum alkaloids demonstrated an anxiolytic-like effect in zebrafish larvae providing evidence for its traditional and modern day use as an anxiolytic.

Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model

ObjectiveIn traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish. MethodsThe characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 μmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio. ResultsAccording to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 μmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 μmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and β-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1. ConclusionRAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

An Endeavor to Find Starter Feed Alternatives and Techniques for Zebrafish First-Feeding Larvae: The Effects on Viability, Morphometric Traits, Digestive Enzymes, and Expression of Growth-Related Genes.

Low and variable growth and survival rates (SR) of 6-10 days postfertilization zebrafish larvae are a problem. This problem seems to be linked to starter feed characteristics. This study is an attempt to find alternatives to address these requests. For this, larvae were fed fresh and lyophilized microalgae (Chlorella, Scenedesmus, and Haematococcus), egg yolk (YOLK), lyophilized Artemia nauplii (LAN), and a combination of them. The lowest SR was observed in algae-fed larvae. All died on day 11 showing an emaciated appearance, similar to starved larvae. The highest SR was observed in YOLK- and LAN-fed larvae, which also showed an elongated anterior part of the body. Negative correlations of SR with vegfaa (vascular endothelial growth factor) and morphometric traits with igf2a (insulin-like growth factor) were also found and supported by changes at the molecular level. The presence of algae in the digestive tract of the larvae and the observation of fecal droppings indicate that the algae have an appropriate size and are palatable. The increase in the digestive enzyme activity shows the larval effort to digest the algae. The fact that the algae-fed larvae died even before the larvae were kept in starvation indicates the dramatic amount of energy that the larvae spent in microalgae digestion. Although both YOLK- and LAN-fed larvae had the highest SR, LAN group started to feed on Artemia nauplii sooner. This can be linked to the delayed growth in YOLK-fed larvae and an accelerated growth in the case of LAN-fed group. LAN is an expensive feed with negative effects on water quality, whereas YOLK is a cheap and nutritionally balanced feed with fine granular texture that contributes to a larval SR similar to LAN without affecting water quality. In conclusion, microalgae cannot be considered a suitable starter food for zebrafish, whereas LAN and YOLK can be considered good starter feeds.

Multi-phenotypic and bi-directional behavioral screening of zebrafish larvae.

Multi-phenotypic screening of zebrafish larvae, such as monitoring the heart and tail activities, is important in biological assays. Microfluidic devices have been developed for zebrafish phenotypic assays, but simultaneous lateral-dorsal screening of the same larva in a single chip is yet to be achieved. We present a multi-phenotypic microfluidic device for monitoring of tail movement and heart rate (HR) of 5-7-day postfertilization zebrafish larvae. Tail movements were stimulated using electric current and quantified in terms of response duration (RD) and tail beat frequency (TBF). The positioning of a right-angle prism provided a lateral view of the larvae and enabled HR monitoring. Investigations were performed on zebrafish larvae exposed to 3% ethanol, 250μM 6-hydroxydopamine (6-OHDA) or 1mM levodopa. Larvae exposed to ethanol showed a significant drop in HR, whereas electric stimulation increased the HR temporarily. Larvae experienced a significant drop in RD, TBF and HR when exposed to 6-OHDA. HR was not affected by levodopa post-treatment, whereas RD and TBF were restored to normal levels. The results showed potential for applications that involve monitoring of cardiac and behavioral parameters in zebrafish larvae. Tests can be done using the same chip, without changing the larvae's orientation. This eliminates undue stress caused by reorientation, which may affect their behavior, and the use of separate devices to obtain dorsal and lateral views. The device can be implemented to improve multi-phenotypic and quantitative screening of zebrafish larvae in response to chemical and physical stimuli in different zebrafish disease models.

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPIassay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

Phenotypic chemical and mutant screening of zebrafish larvae using an on-demand response to electric stimulation.

Behavioral responses of zebrafish larvae to environmental cues are important functional readouts that should be evoked on-demand and studied phenotypically in behavioral, genetical and developmental investigations. Very recently, it was shown that zebrafish larvae execute a voluntary and oriented movement toward the positive electrode of an electric field along a microchannel. Phenotypic characterization of this response was not feasible due to larva's rapid movement along the channel. To overcome this challenge, a microfluidic device was introduced to partially immobilize the larva's head while leaving its mid-body and tail unrestrained in a chamber to image motor behaviors in response to electric stimulation, hence achieving quantitative phenotyping of the electrically evoked movement in zebrafish larvae. The effect of electric current on the tail-beat frequency and response duration of 5-7days postfertilization zebrafish larvae was studied. Investigations were also performed on zebrafish exposed to neurotoxin 6-hydroxydopamine and larvae carrying a pannexin1a (panx1a) gene knockout, as a proof of principle applications to demonstrate on-demand movement behavior screening in chemical and mutant assays. We demonstrated for the first time that 6-hydroxydopamine leads to electric response impairment, levodopa treatment rescues the response and panx1a is involved in the electrically evoked movement of zebrafish larvae. We envision that our technique is broadly applicable as a screening tool to quantitatively examine zebrafish larvae's movements in response to physical and chemical stimulations in investigations of Parkinson's and other neurodegenerative diseases, and as a tool to combine recent advances in genome engineering of model organisms to uncover the biology of electric response.

Imaging neural events in zebrafish larvae with linear structured illumination light sheet fluorescence microscopy.

Light sheet fluorescence microscopy (LSFM) is a powerful tool for investigating model organisms including zebrafish. However, due to scattering and refractive index variations within the sample, the resulting image often suffers from low contrast. Structured illumination (SI) has been combined with scanned LSFM to remove out-of-focus and scattered light using square-law detection. Here, we demonstrate that the combination of LSFM with linear reconstruction SI can further increase resolution and contrast in the vertical and axial directions compared to the widely adopted root-mean square reconstruction method while using the same input images. We apply this approach to imaging neural activity in 7-day postfertilization zebrafish larvae. We imaged two-dimensional sections of the zebrafish central nervous system in two colors at an effective frame rate of 7 frames per second.

Modelling acrylamide acute neurotoxicity in zebrafish larvae

Acrylamide (ACR), a type-2 alkene, may lead to a synaptopathy characterized by ataxia, skeletal muscles weakness and numbness of the extremities in exposed human and laboratory animals. Currently, only the mildly affected patients undergo complete recovery, and identification of new molecules with therapeutic bioactivity against ACR acute neurotoxicity is urgently needed. Here, we have generated a zebrafish model for ACR neurotoxicity by exposing 5 days post-fertilization zebrafish larvae to 1 mM ACR for 3 days. Our results show that zebrafish mimics most of the pathophysiological processes described in humans and mammalian models. Motor function was altered, and specific effects were found on the presynaptic nerve terminals at the neuromuscular junction level, but not on the axonal tracts or myelin sheath integrity. Transcriptional markers of proteins involved in synaptic vesicle cycle were selectively altered, and the proteomic analysis showed that ACR-adducts were formed on cysteine residues of some synaptic proteins. Finally, analysis of neurotransmitters profile showed a significant effect on cholinergic and dopaminergic systems. These data support the suitability of the developed zebrafish model for screening of molecules with therapeutic value against this toxic neuropathy.

A non-toxic dose of cobalt chloride blocks hair cells of the zebrafish lateral line

Experiments on the flow-sensitive lateral line system of fishes have provided important insights into the function and sensory transduction of vertebrate hair cells. A common experimental approach has been to pharmacologically block lateral line hair cells and measure how behavior changes. Cobalt chloride (CoCl2) blocks the lateral line by inhibiting calcium movement through the membrane channels of hair cells, but high concentrations can be toxic, making it unclear whether changes in behavior are due to a blocked lateral line or poor health. Here, we identify a non-toxic treatment of cobalt that completely blocks lateral line hair cells. We exposed 5-day post fertilization zebrafish larvae to CoCl2 concentrations ranging from 1 to 20 mM for 15 min and measured 1) the spiking rate of the afferent neurons contacting hair cells and 2) the larvae's health and long-term survival. Our results show that a 15-min exposure to 5 mM CoCl2 abolishes both spontaneous and evoked afferent firing. This treatment does not change swimming behavior, and results in >85% survival after 5 days. Weaker treatments of CoCl2 did not eliminate afferent activity, while stronger treatments caused close to 50% mortality. Our work provides a guideline for future zebrafish investigations where physiological confirmation of a blocked lateral line system is required.

Protective effect of p38 mitogen-activated protein kinase inhibitors on zebrafish larvae brain after hypoxia/reoxygenation injury

Objective To examine the effects of p38 mitogen-activated protein kinase (MAPK) inhibitor on the behavioral response to zebrafish larvae after hypoxia/reoxygenation brain injury and to identify whether the protective effect is mediated by inhibiting apoptosis and protein, and mRNA related to apoptosis. Methods The 5-day post-fertilization zebrafish larvae were randomly assigned to 3 groups: control group, model group and intervention group.Fishes in the intervention group were separated into 3 subgroups according to p38 MAPK inhibitor concentration (5, 10, 20 μmol/L). The activity levels of the larvae were analyzed by using quantization mode of ZebraLab software, swimming distance and moving speed were recorded.Terminal transferase dUTP nick end labeling (TUNEL) assays of brain assays were performed.The protein levels of phosphorylation of p38 MAPK, apoptosis related proteins of B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax) and Caspase-3 were determined by Western blot.The mRNA expressions of Bcl-2, Bax, and caspase-3 were also analyzed by reverse transcription-quantitative PCR (RT-qPCR). Results The activity movement analysis of the intervention group 2 and 3 demonstrated a significantly increase in the swimming distance compared with the model group(P<0.05). After irradiation under strong light, all groups showed dramatically increasing in the moving speed.After removal of strong light, a significant decrease in moving speed was found in the control group and intervention group 2 and intervention group 3.The TUNEL assay showed that apoptosis index decreased in the intervention group (21.7±2.0, 12.8±1.9, 17.7±2.6) compared with model group (46.8±5.3) (all P<0.01). Western blot assays demonstrated a significant increase protein level of phosphorylation of p38 MAPK after hypoxia and reoxygenation, and the inhibitor reduced the p-p38 MAPK expression.Compared with the model group, p38 MAPK inhi-bitor increased the protein and mRNA expression level of Bcl-2, whereas reduced the Bax and caspase-3 expression in the brain. Conclusions Under the influences of p38 MAPK inhibitor, zebrafish larvae improved the behavioral changes after hypoxia-induced brain injury.The inhibitor (10 μmol/L) optimally reduces hypoxia-induced apoptosis in brain by up-regulating Bcl-2, down-regulating Bax/caspase-3 protein and their mRNA level. Key words: Hypoxia-ischemia; Mitogen-activated protein kinase; Apoptosis

A sensitive biomarker for the detection of aquatic contamination based on behavioral assays using zebrafish larvae

An effective biological early warning system for the detection of water contamination should employ undemanding species that rapidly react to the presence of contaminants in their environment. The demonstrated reaction should be comprehensible and unambiguously evidential of the contamination event. This study utilized 96h post fertilization zebrafish larvae and tested their behavioral response to acute exposure to low concentrations of cadmium chloride (CdCl2) (5.0, 2.5, 1.25, 0.625mg/L) and permethrin (0.05, 0.029, 0.017, 0.01μg/L). We hypothesize that the number of larvae that show advanced trajectories in a group corresponds with water contamination, as the latter triggers avoidance behavior in the organisms. The proportion of advanced trajectories in the control and treated groups during the first minute of darkness was designated as a segregation parameter. It was parametrized and a threshold value was set using one CdCl2 trial and then applied to the remaining CdCl2 and permethrin replicates. For all cases, the method allowed distinguishing between the control and treated groups within two cycles of light: dark. The calculated parameter was statistically significantly different between the treated and control groups, except for the lowest CdCl2 concentration (0.625mg/L) in one replicate. This proof-of-concept study shows the potential of the proposed methodology for utilization as part of a multispecies biomonitoring system.

Effect of JNK inhibitor SP600125 on hair cell regeneration in zebrafish (Danio rerio) larvae.

The c-Jun amino-terminal kinase (JNK) proteins are a subgroup of the mitogen-activated protein kinase family. They play a complex role in cell proliferation, survival, and apoptosis. Here, we report a novel role of JNK signalling in hair cell regeneration. We eliminated hair cells of 5-day post-fertilization zebrafish larvae using neomycin followed by JNK inhibition with SP600125. JNK inhibition strongly decreased the number of regenerated hair cells in response to neomycin damage. These changes were associated with reduced proliferation. JNK inhibition also increased cleaved caspase-3 activity and induced apoptosis in regenerating neuromasts. Finally, JNK inhibition with SP600125 decreased the expression of genes related to Wnt. Over-activation of the Wnt signalling pathway partly rescued the hair cell regeneration defects induced by JNK inhibition. Together, our findings provide novel insights into the function of JNK and show that JNK inhibition blocks hair cell regeneration by controlling the Wnt signalling pathway.

Effect of Taurine on the expression of glucose regulated protein 78 and growth arrest and DNA damage-inducible protein 34 in zebrafish larvae brain after hypoxia/reoxygenation brain injury

Objective To investigate the expression of endoplasmic reticulum stress-related factors, glucose-regulated protein 78 (GRP78)and growth arrest and DNA damage-inducible protein 34 (GADD34)and neuronal apoptosis in the brain of zebrafish larvae after hypoxia/reoxygenation brain injury, and the neuroprotective role of Taurine. Methods The 5 day post-fertilization zebrafish larvae were randomly assigned to 3 groups: control group, hypoxia/reoxygenation model group (model group) and Taurine treatment group(Taurine group). According to the different time points for observation, each group was subdivided into 5 subgroups (1 h, 3 h, 6 h, 24 h, 48 h)with 100 zebrafish larvae each.The pathological changes in the brain tissues and cell apoptosis were detected by fluorescence terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL). The expression of GRP78 and GADD34 mRNA in the brain of zebrafish larvae were detected by real-time quantitative reverse transcription PCR(qRT-PCR). The changes in GRP78 and GADD34 protein were detected by Western blot. Results (1)TUNEL: apoptosis index(AI)was increased after hypoxia/reoxygenation, and reached the peak at 3 h in model group, the AI in Taurine group was decreased compared with that in the model group at the same time point (1 h: 22.83±1.80 vs 30.18±1.81, 3 h: 23.22±2.46 vs 42.97±4.01, 6 h: 16.80±1.69 vs 22.97±1.91, all P<0.05). (2) qRT-PCR: the expression of GRP78 and GADD34 mRNA was increased at 1 h after hypoxia/reoxygenation, and reached the peak at 3 h in the model group, the expression in Taurine group was decreased compared with that in the model group at the same time point (GRP78 mRNA: 1 h: 2.35±0.13 vs 5.36±0.35, 3 h: 3.08±0.33 vs 4.27±0.52, 6 h: 1.57±0.12 vs 3.00±0.13, all P<0.05; GADD34 mRNA: 1 h: 5.14±0.55 vs 7.45±0.67, 3 h: 2.79±0.58 vs 5.83±0.51, 6 h: 1.79±0.22 vs 3.67±0.30, all P<0.05). (3) Western blot: the expression of GRP78 and GADD34 protein was increased at 1 h, reached the peak at 6 h, but it was decreased in Taurine group (GRP78 protein: 1 h: 1.12±0.11 vs 1.37±0.13, 3 h: 0.79±0.11 vs 1.25±0.10, 6 h: 0.55±0.10 vs 1.52±0.14, all P<0.05; GADD34 protein: 1 h: 0.92±0.11 vs 1.11±0.13, 3 h: 0.96±0.11 vs 1.52±0.09, 6 h: 0.76±0.05 vs 1.89±0.06, all P<0.05). (4) The expression of GRP78 and GADD34 protein was positively correlated with AI in the model group(r=0.53, 0.56 respectively, all P<0.05). Conclusion One of neuroprotective mechanisms of Taurine against hypoxia/reoxygenation brain injury may down-regulate GRP78 and GADD34 expression. Key words: Endoplasmic reticulum stress; Hypoxic-ischemic brain damage; Glucose-regulated protein 78; Growth arrest and DNA damage-inducible protein 34; Apoptosis

Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish

ObjectiveCaffeic acid phenethyl ester (CAPE) is known to reduce the generation of oxygen-derived free radicals, which is a major mechanism of aminoglycoside-induced ototoxicity. The objective of the present study was to evaluate the effects of CAPE on neomycin-induced ototoxicity in zebrafish (Brn3c: EGFP). MethodsFive-day post-fertilization zebrafish larvae (n=10) were exposed to 125μM neomycin and one of the following CAPE concentrations for 1h: 50, 100, 250, 500, or 1000μM. Ultrastructural changes were evaluated using scanning electron microscopy (SEM). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay and 2-[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. ResultsCAPE decreased neomycin-induced hair cell loss in the neuromasts (500μM CAPE: 12.7±1.1 cells, 125μM neomycin only: 6.3±1.1 cells; n=10, P<0.05). In the ultrastructural analysis, structures of mitochondria and hair cells were preserved when exposed to 125μM neomycin and 500μM CAPE. CAPE decreased apoptosis and mitochondrial damage. ConclusionIn the present study, CAPE attenuated neomycin-induced hair cell damage in zebrafish. The results of the current study suggest that neomycin induces apoptosis, and the apoptotic cell death can be prevented by treatment with CAPE in zebrafish.

Inhibition of SULT4A1 Expression Induces Up-Regulation of Phototransduction Gene Expression in 72-Hour Postfertilization Zebrafish Larvae

Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression.

Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish

ObjectiveEdaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). MethodsFive day post-fertilization zebrafish larvae were exposed to 1000μM cisplatin and 50μM, 100μM, 250μM, 500μM, 750μM, and 1000μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. ResultsEdaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750μM: 8.7±1.5 cells, cisplatin 1000μM only: 3.7±0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000μM cisplatin and 750μM edaravone for 4h. ConclusionsEdaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish.