Imaging Characteristics of Non-Rheumatoid Arthritis Patients With Retro-Odontoid Pseudotumor: A Multicenter Case-Control Study.

STUDY DESIGN: Multicenter case-control study. OBJECTIVE: To identify imaging characteristics of the cervical spine in patients with retro-odontoid pseudotumor (ROP) without rheumatoid arthritis (RA) and determine the pathogenesis of ROP. SUMMARY OF BACKGROUND DATA: ROP results from proliferative changes in the soft tissue of the atlantoaxial junction surrounding the region of the transverse ligament, and is commonly seen in RA patients. However, the pathogenesis of ROP caused by mechanical instability of the upper cervical spine in patients without RA is yet to be explained. METHODS: We collected imaging data (preoperative radiographs, magnetic resonance imaging [MRI], and computed tomography [CT]) of patients who underwent surgery between April 2011 and March 2022 at the three university hospitals for ROP (cases) and cervical spondylotic myelopathy (as age, sex, and institution matched controls). The two groups were compared for different parameters on cervical dynamic radiographs, MRI, and CT. RESULTS: The ROP group consisted of 42 patients, and the control group comprised 168 patients. C2-C7 range of motion was significantly smaller in the ROP group (25.8 ± 2.6°) compared to the control group (33.0 ± 1.0°). C2-C7 sagittal vertical axis was significantly larger in the ROP group than the control group (39.3 ± 3.6 mm versus 32.2 ± 1.3 mm). MRI and CT assessment showed progressive degeneration at all intervertebral levels in the ROP group. A significant positive correlation was observed between the thickness of the soft tissue posterior to the dental process and the atlantodental interval in the flexion position (r = 0.501). CONCLUSION: The development of ROP was associated with degeneration of facet joints and intervertebral discs in the middle and lower cervical spine. Our findings suggest that decreased mobility of the middle and lower cervical spine may cause instability in the upper cervical spine, leading to the formation of ROP.

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1ß treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Regenerative effects of platelet-rich plasma releasate injection in rabbit discs degenerated by intradiscal injection of condoliase.

BACKGROUND: Intradiscal condoliase injection is an alternative therapeutic option for lumbar disc herniation (LDH). However, it is often associated with disc degeneration. Several in vivo studies have demonstrated the regenerative potential of platelet-rich plasma (PRP) in disc degeneration. Thus, we hypothesized that the intradiscal injection of PRP releasate (PRPr), a soluble releasate isolated from PRP, has the potential to regenerate degenerated intervertebral discs (IVDs) induced by condoliase. This study examined the regenerative effects of PRPr on rabbit IVDs degenerated following condoliase injection. METHODS: Eleven New Zealand white rabbits were used in this study. Condoliase (12.5 mU/10 µl) was injected into two non-contiguous discs (L2-L3 and L4-L5), and L3-L4 disc was left intact as a non-injection control. Saline (20 µl) or PRPr (20 µl) was randomly injected into L2-L3 and L4-L5 discs 4 weeks after the condoliase injection. Disc height (DH) was radiographically monitored biweekly from the day of condoliase injection to 16 weeks post-injection. Changes in DH were expressed as percentage DH (%DH) normalized to the baseline DH. Sixteen weeks after condoliase injection, all rabbits were euthanized, and subjected to MRI and histological analyses. RESULTS: Intradiscal injection of condoliase induced a significant decrease in %DH (L2-L3 and L4-L5) to 52.0% at week 4. However, the %DH began to return to normal after saline injection and reached 76.3% at week 16. In the PRPr group, %DH began to recover to normal after the PRPr injection and was restored to 95.5% at week 16. The MRI-modified Pfirrmann grade of the PRPr group was significantly lower than that of the saline group (P < 0.01). Histological analyses showed progressive degenerative changes, including reduction of the NP area and condensation of the matrix in the saline and PRPr groups. The histological score of the PRPr group was significantly lower than that of the saline group (P < 0.01). CONCLUSIONS: PRPr has great potential to enhance the regeneration of degenerated rabbit IVDs induced by condoliase. The results of this preclinical study suggest that PRPr injection therapy may be indicated for patients with LDH who have poor recovery from disc degeneration after chemonucleolysis treatment with condoliase.

Quantitative Analysis of Lumbar Disc Bulging in Patients with Lumbar Spinal Stenosis: Implication for Surgical Outcomes of Decompression Surgery.

This study aimed to quantitatively assess disc bulging using computed tomography (CT) in patients with lumbar spinal stenosis (LSS) and to examine whether disc bulging affects the surgical outcomes of patients with LSS after posterior decompression surgery. Sixty-three patients who underwent posterior decompression surgery for LSS were included. The extent of disc bulging was evaluated as the percentage of the extended area of the disc against the endplate area (%EAD) on axial CT images. The participants completed the following clinical outcome assessments (COAs) preoperatively and 12 months postoperatively: the JOA Back Pain Evaluation Questionnaire (JOABPEQ), Oswestry Disability Index (ODI), and Roland-Morris Disability Questionnaire (RDQ). The mean %EAD of 315 intervertebral discs was 18.9 ± 8.0. The %EAD was highest at L4/L5, followed by L3/L4, L2/L3, L1/L2, and L5/S1. The %EAD of the surgical level showed no significant correlation with all the preoperative COAs, but it had significant correlation with lumbar function, walking ability, social function domains of the JOABPEQ, ODI score, and RDQ score 12 months postoperatively. %EAD was significantly associated with the postoperative score in the walking ability domain of the JOABPEQ. %EAD affects postoperative clinical outcomes, including low back pain-related quality of life after decompression surgery.

Daily Physical Training Improved Coronal Imbalance of Adult Degenerative Scoliosis: A Case Report.

Background and Objectives: Adult (de novo) degenerative scoliosis (ADS) develops through degenerative changes in the lumbar spine, leading to spinal malalignment, which usually progresses with age. Strong evidence for non-operative care in patients with ADS is lacking, and whether physical exercise can improve the scoliosis curve remains unknown. Materials and Methods: We present a case of early stage ADS in which the coronal imbalance was improved by daily training. A 65-year-old female patient complained of lower back pain (LBP) and bilateral leg pain. She was diagnosed with early stage ADS with lumbar degenerative spondylolisthesis by imaging. She completed six months of daily physical training, including swimming, aerobic bikes, stretching, yoga, and Taijiquan. Results: Her LBP and neurological symptoms improved, and coronal-spinal balance was restored, which was maintained for four years by continued daily physical training. Conclusions: This is the first case of a 65-year-old ADS patient whose coronal balance was significantly restored through daily physical training. Substantial physical training focused on trunk muscle strength is important for spinal stabilization and for improving spinal malalignment in patients with early stage ADS.

Expression of GADD45G and CAPRIN1 in Human Nucleus Pulposus: Implications for Intervertebral Disc Degeneration.

Marked cellular changes occur in human intervertebral disc (IVD) degeneration during disc degeneration with biochemical changes. Genome-wide analysis of the DNA methylation profile has identified 220 differentially methylated loci associated with human IVD degeneration. Among these, two cell-cycle-associated genes, growth arrest and DNA damage 45 gamma (GADD45G) and cytoplasmic activation/proliferation-associated protein-1 (CAPRIN1), were focused on. The expression of GADD45G and CAPRIN1 in human IVDs remains unknown. We aimed to examine the expression of GADD45G and CAPRIN1 in human nucleus pulposus (NP) cells and evaluate those in human NP tissues in the early and advanced stages of degeneration according to Pfirrmann magnetic resonance imaging (MRI) and histological classifications. Human NP cells were cultured as monolayers after isolation from NP tissues by sequential enzyme digestion. Total RNA was isolated, and the mRNA expression of GADD45G and CAPRIN1 was quantified using real-time polymerase chain reaction. To examine the effects of pro-inflammatory cytokines on mRNA expression, human NP cells were cultured in the presence of IL-1ß. Protein expression was evaluated using Western blotting and immunohistochemistry. GADD45G and CAPRIN1 expression was identified in human NP cells at both mRNA and protein levels. The percentage of cells immunopositive for GADD45G and CAPRIN1 significantly increased according to the Pfirrmann grade. A significant correlation between the histological degeneration score and the percentage of GADD45G-immunopositive cells was identified, but not with that of CAPRIN1-immunopositive cells. The expression of cell-cycle-associated proteins (GADD45G and CAPRIN1) was enhanced in human NP cells at an advanced stage of degeneration, suggesting that it may be regulated during the progression of IVD degeneration to maintain the integrity of human NP tissues by controlling cell proliferation and apoptosis under epigenetic alteration.

Intradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model.

BACKGROUND: Establishing an optimal animal model for intervertebral disc (IVD) degeneration is essential for developing new IVD therapies. The intra-articular injection of monosodium iodoacetate (MIA), which is commonly used in animal models of osteoarthritis, induces cartilage degeneration and progressive arthritis in a dose- and time-dependent manner. The purpose of this study was to determine the effect of MIA injections into rabbit IVDs on the progression of IVD degeneration evaluated by radiographic, micro-computerized tomography (micro-CT), magnetic resonance imaging (MRI), and histological analyses. METHODS: In total, 24 New Zealand White (NZW) rabbits were used in this study. Under general anesthesia, lumbar discs from L1-L2 to L4-L5 had a posterolateral percutaneous injection of MIA in contrast agent (CA) (L1-L2: CA only; L2-L3: MIA 0.01 mg; L3-L4: 0.1 mg; L4-L5: 1.0 mg; L5-L6: non-injection (NI) control). Disc height was radiographically monitored biweekly until 12 weeks after injection. Six rabbits were sacrificed at 2, 4, 8, and 12 weeks post-injection and processed for micro-CT, MRI (T2-mapping), and histological analyses. Three-dimensional (3D) disc height in five anatomical zones was evaluated by 3D reconstruction of micro-CT data. RESULTS: Disc height of MIA-injected discs (L2-L3 to L4-L5) gradually decreased time-dependently (P < 0.0001). The disc height of MIA 0.01 mg-injected discs was significantly higher than those of MIA 0.1 and 1.0 mg-injected discs (P < 0.01, respectively). 3D micro-CT analysis showed the dose- and time-dependent decrease of 3D disc height of MIA-injected discs predominantly in the posterior annulus fibrosus (AF) zone. MRI T2 values of MIA 0.1 and 1.0 mg-injected discs were significantly decreased compared to those of CA and/or NI controls (P < 0.05). Histological analyses showed progressive time- and dose-degenerative changes in the discs injected with MIA (P < 0.01). MIA induced cell death in the rabbit nucleus pulposus with a high percentage, while the percentage of cell clones was low. CONCLUSIONS: The results of this study showed, for the first time, that the intradiscal injection of MIA induced degenerative changes of rabbit IVDs in a time- and dose-dependent manner. This study suggests that MIA injection into rabbit IVDs could be used as an animal model of IVD degeneration for developing future treatments.

Cervical schwannoma in the early stage of pregnancy: a case report.

BACKGROUND: Although spinal schwannomas generally grow very slowly, it has been reported that these clinical growths and their associated neurological symptoms accelerate during pregnancy. Because these cases are rare, surgical intervention for this tumor during pregnancy poses a significant challenge. The change of pregnancy-related hormones, such as estrogen and progesterone, is considered to have an effect on the clinical symptoms of spinal tumors. Expressions of the receptors for estrogen and progesterone in orbital and vestibular schwannomas have been reported; however, those expressions in spinal schwannomas have not been examined. CASE PRESENTATION: A 36-year-old woman at 8 weeks' gestation suffered from developing neck pain and neurological symptoms in the right upper extremity. Magnetic resonance imaging (MRI) confirmed the presence of a cervical intradural extramedullary tumor. Under general anesthesia, using intraoperative neurophysiological monitoring of motor-evoked potentials (MEPs), spinal tumor resection following a hemi-laminoplasty was performed in a prone position at 12 weeks gestation. The pathological diagnosis following surgery was spinal schwannoma. Her neurological symptoms were significantly improved after surgery and she delivered a healthy baby in her 40th week of pregnancy. At a 12-month follow-up, no abnormalities were observed during medical examinations of both mother and child. An immunohistochemical study identified the expression of estrogen receptors, but not progesterone receptors, in the spinal schwannoma. CONCLUSIONS: A cervical spinal schwannoma was successfully removed under general anesthesia at 12 weeks gestation by coordination between orthopaedic, obstetric and anesthesia teams. For the first time, an immunohistochemical analysis showed that the expression of estrogen receptors was identified in spinal schwannoma cells, suggesting the possibility that these hormone receptors in spinal schwannoma might contribute to the worsening of neurological symptoms during pregnancy.

Potential protective function of the sterol regulatory element binding factor 1-fatty acid desaturase 1/2 axis in early-stage age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of vision loss in elderly individuals throughout the developed world. Inhibitors of vascular endothelial growth factor have been successfully used to treat choroidal neovascularization in late-stage AMD. The pathogenesis of early-stage AMD, however, remains largely unknown, impairing efforts to develop effective therapies that prevent progression to late-stage AMD. To address this, we performed comparative transcriptomics of macular and extramacular retinal pigmented epithelium-choroid (RPE-choroid) tissue from early-stage AMD patients. We found that expression of fatty acid desaturase 1 (FADS1), FADS2, and acetyl-CoA acetyltransferase 2 (ACAT2) is increased in macular but not extramacular tissue, possibly through activation of sterol regulatory element binding factor 1 (SREBF1). Consistent with this, we also found that expression of Fads1 is increased in RPE-choroid in a mouse model of early-stage AMD. In zebrafish, deletion of fads2, which encodes a protein that functions as both Fads1 and Fads2 in other species, enhanced apoptosis in the retina upon exposure to intense light. Similarly, pharmacological inhibition of Srebf1 enhanced apoptosis and reduced fads2 expression in zebrafish exposed to intense light. These results suggest that the SREBF1-FADS1/2 axis may be activated in macular RPE-choroid as a protective response during early-stage AMD and could thus be a therapeutic target for early-stage AMD.

Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish.

Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish with another PPARα agonist, gemfibrozil, also increased expression of the mbp promoter-driven fluorescent reporter in an SREBF-dependent manner. These results suggest that activation of SREBFs by small molecular weight compounds may be a feasible therapeutic approach to stimulate myelination.

Downregulation of GSTK1 Is a Common Mechanism Underlying Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and is associated with a number of potential outcomes, including impaired diastolic function, heart failure, and sudden cardiac death. Various etiologies have been described for HCM, including pressure overload and mutations in sarcomeric and non-sarcomeric genes. However, the molecular pathogenesis of HCM remains incompletely understood. In this study, we performed comparative transcriptome analysis to identify dysregulated genes common to five mouse HCM models of differing etiology: (i) mutation of myosin heavy chain 6, (ii) mutation of tropomyosin 1, (iii) expressing human phospholamban on a null background, (iv) knockout of frataxin, and (v) transverse aortic constriction. Gene-by-gene comparison identified five genes dysregulated in all five HCM models. Glutathione S-transferase kappa 1 (Gstk1) was significantly downregulated in the five models, whereas myosin heavy chain 7 (Myh7), connective tissue growth factor (Ctgf), periostin (Postn), and reticulon 4 (Rtn4) were significantly upregulated. Gene ontology comparison revealed that 51 cellular processes were significantly enriched in genes dysregulated in each transcriptome dataset. Among them, six processes (oxidative stress, aging, contraction, developmental process, cell differentiation, and cell proliferation) were related to four of the five genes dysregulated in all HCM models. GSTK1 was related to oxidative stress only, whereas the other four genes were related to all six cell processes except MYH7 for oxidative stress. Gene-gene functional interaction network analysis suggested correlative expression of GSTK1, MYH7, and actin alpha 2 (ACTA2). To investigate the implications of Gstk1 downregulation for cardiac function, we knocked out gstk1 in zebrafish using the clustered regularly interspaced short palindromic repeats/Cas9 system. We found that expression of the zebrafish homologs of MYH7, ACTA2, and actin alpha 1 were increased in the gstk1-knockout zebrafish. In vivo imaging of zebrafish expressing a fluorescent protein in cardiomyocytes showed that gstk1 deletion significantly decreased the end diastolic volume and, to a lesser extent, end systolic volume. These results suggest that downregulation of GSTK1 may be a common mechanism underlying HCM of various etiologies, possibly through increasing oxidative stress and the expression of sarcomere genes.

Comparative Transcriptome Analysis Identifies CCDC80 as a Novel Gene Associated with Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a heterogeneous disorder associated with a progressive increase in pulmonary artery resistance and pressure. Although various therapies have been developed, the 5-year survival rate of PAH patients remains low. There is thus an important need to identify novel genes that are commonly dysregulated in PAH of various etiologies and could be used as biomarkers and/or therapeutic targets. In this study, we performed comparative transcriptome analysis of five mammalian PAH datasets downloaded from a public database. We identified 228 differentially expressed genes (DEGs) from a rat PAH model caused by inhibition of vascular endothelial growth factor receptor under hypoxic conditions, 379 DEGs from a mouse PAH model associated with systemic sclerosis, 850 DEGs from a mouse PAH model associated with schistosomiasis, 1598 DEGs from one cohort of human PAH patients, and 4260 DEGs from a second cohort of human PAH patients. Gene-by-gene comparison identified four genes that were differentially upregulated or downregulated in parallel in all five sets of DEGs. Expression of coiled-coil domain containing 80 (CCDC80) and anterior gradient two genes was significantly increased in the five datasets, whereas expression of SMAD family member six and granzyme A was significantly decreased. Weighted gene co-expression network analysis revealed a connection between CCDC80 and collagen type I alpha 1 (COL1A1) expression. To validate the function of CCDC80 in vivo, we knocked out ccdc80 in zebrafish using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. In vivo imaging of zebrafish expressing a fluorescent protein in endothelial cells showed that ccdc80 deletion significantly increased the diameter of the ventral artery, a vessel supplying blood to the gills. We also demonstrated that expression of col1a1 and endothelin-1 mRNA was significantly decreased in the ccdc80-knockout zebrafish. Finally, we examined Ccdc80 immunoreactivity in a rat PAHmodel and found increased expression in the hypertrophied media and adventitia of the pre-acinar pulmonary arteries (PAs) and in the thickened intima, media, and adventitia of the obstructed intra-acinar PAs. These results suggest that increased expression of CCDC80 may be involved in the pathogenesis of PAH, potentially by modulating the expression of endothelin-1 and COL1A1.

E2F4 Promotes Neuronal Regeneration and Functional Recovery after Spinal Cord Injury in Zebrafish.

Mammals exhibit poor recovery after spinal cord injury (SCI), whereas non-mammalian vertebrates exhibit significant spontaneous recovery after SCI. The mechanisms underlying this difference have not been fully elucidated; therefore, the purpose of this study was to investigate these mechanisms. Using comparative transcriptome analysis, we demonstrated that genes related to cell cycle were significantly enriched in the genes specifically dysregulated in zebrafish SCI. Most of the cell cycle-related genes dysregulated in zebrafish SCI were down-regulated, possibly through activation of e2f4. Using a larval zebrafish model of SCI, we demonstrated that the recovery of locomotive function and neuronal regeneration after SCI were significantly inhibited in zebrafish treated with an E2F4 inhibitor. These results suggest that activation of e2f4 after SCI may be responsible, at least in part, for the significant recovery in zebrafish. This provides novel insight into the lack of recovery after SCI in mammals and informs potential therapeutic strategies.

EP300 Protects from Light-Induced Retinopathy in Zebrafish.

Exposure of rhodopsin to bright white light can induce photoreceptor cell damage and degeneration. However, a comprehensive understanding of the mechanisms underlying light-induced retinopathy remains elusive. In this study, we performed comparative transcriptome analysis of three rodent models of light-induced retinopathy, and we identified 37 genes that are dysregulated in all three models. Gene ontology analysis revealed that this gene set is significantly associated with a cytokine signaling axis composed of signal transducer and activator of transcription 1 and 3 (STAT1/3), interleukin 6 signal transducer (IL6ST), and oncostatin M receptor (OSMR). Furthermore, the analysis suggested that the histone acetyltransferase EP300 may be a key upstream regulator of the STAT1/3-IL6ST/OSMR axis. To examine the role of EP300 directly, we developed a larval zebrafish model of light-induced retinopathy. Using this model, we demonstrated that pharmacological inhibition of EP300 significantly increased retinal cell apoptosis, decreased photoreceptor cell outer segments, and increased proliferation of putative Müller cells upon exposure to intense light. These results suggest that EP300 may protect photoreceptor cells from light-induced damage and that activation of EP300 may be a novel therapeutic approach for the treatment of retinal degenerative diseases.

DNA Damage Response Is Involved in the Developmental Toxicity of Mebendazole in Zebrafish Retina.

Intestinal helminths cause iron-deficiency anemia in pregnant women, associated with premature delivery, low birth weight, maternal ill health, and maternal death. Although benzimidazole compounds such as mebendazole (MBZ) are highly efficacious against helminths, there are limited data on its use during pregnancy. In this study, we performed in vivo imaging of the retinas of zebrafish larvae exposed to MBZ, and found that exposure to MBZ during 2 and 3 days post-fertilization caused malformation of the retinal layers. To identify the molecular mechanism underlying the developmental toxicity of MBZ, we performed transcriptome analysis of zebrafish eyes. The analysis revealed that the DNA damage response was involved in the developmental toxicity of MBZ. We were also able to demonstrate that inhibition of ATM significantly attenuated the apoptosis induced by MBZ in the zebrafish retina. These results suggest that MBZ causes developmental toxicity in the zebrafish retina at least partly by activating the DNA damage response, including ATM signaling, providing a potential adverse outcome pathway in the developmental toxicity of MBZ in mammals.

In Vivo Detection of Mitochondrial Dysfunction Induced by Clinical Drugs and Disease-Associated Genes Using a Novel Dye ZMJ214 in Zebrafish.

Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various in vivo fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. In vivo imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction in vivo.

Using zebrafish in systems toxicology for developmental toxicity testing.

With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments.

Pharmacological profiling of zebrafish behavior using chemical and genetic classification of sleep-wake modifiers.

Sleep-wake states are impaired in various neurological disorders. Impairment of sleep-wake states can be an early condition that exacerbates these disorders. Therefore, treating sleep-wake dysfunction may prevent or slow the development of these diseases. Although many gene products are likely to be involved in the sleep-wake disturbance, hypnotics and psychostimulants clinically used are limited in terms of their mode of action and are not without side effects. Therefore, there is a growing demand for developing new hypnotics and psychostimulants with high efficacy and few side effects. Toward this end, animal models are indispensable for use in genetic and chemical screens to identify sleep-wake modifiers. As a proof-of-concept study, we performed behavioral profiling of zebrafish treated with chemical and genetic sleep-wake modifiers. We were able to demonstrate that behavioral profiling of zebrafish treated with hypnotics or psychostimulants from 9 to 10 days post-fertilization was sufficient to identify drugs with specific modes of action. We were also able to identify behavioral endpoints distinguishing GABA-A modulators and hypocretin (hcrt) receptor antagonists and between sympathomimetic and non-sympathomimetic psychostimulants. This behavioral profiling can serve to identify genes related to sleep-wake disturbance associated with various neuropsychiatric diseases and novel therapeutic compounds for insomnia and excessive daytime sleep with fewer adverse side effects.

Systems pharmacology of adiposity reveals inhibition of EP300 as a common therapeutic mechanism of caloric restriction and resveratrol for obesity.

Both caloric restriction (CR) and resveratrol (RSV) have beneficial effects on obesity. However, the biochemical pathways that mediate these beneficial effects might be complex and interconnected and have not been fully elucidated. To reveal the common therapeutic mechanism of CR and RSV, we performed a comparative transcriptome analysis of adipose tissues from diet-induced obese (DIO) zebrafish and obese humans. We identified nine genes in DIO zebrafish and seven genes in obese humans whose expressions were regulated by CR and RSV. Although the gene lists did not overlap except for one gene, the gene ontologies enriched in the gene lists were highly overlapped, and included genes involved in adipocyte differentiation, lipid storage and lipid metabolism. Bioinformatic analysis of cis-regulatory sequences of these genes revealed that their transcriptional regulators also overlapped, including EP300, HDAC2, CEBPB, CEBPD, FOXA1, and FOXA2. We also identified 15 and 46 genes that were dysregulated in the adipose tissue of DIO zebrafish and obese humans, respectively. Bioinformatics analysis identified EP300, HDAC2, and CEBPB as common transcriptional regulators for these genes. EP300 is a histone and lysyl acetyltransferase that modulates the function of histone and various proteins including CEBPB, CEBPD, FOXA1, and FOXA2. We demonstrated that adiposity in larval zebrafish was significantly reduced by C646, an inhibitor of EP300 that antagonizes acetyl-CoA. The reduction of adiposity by C646 was not significantly different from that induced by RSV or co-treatment of C646 and RSV. These results indicate that the inhibition of EP300 might be a common therapeutic mechanism between CR and RSV in adipose tissues of obese individuals.