Identification of an adverse outcome pathway (AOP) for chemical-induced craniofacial anomalies using the transgenic zebrafish model.

Craniofacial anomalies are one of the most frequent birth defects worldwide and are often caused by genetic and environmental factors such as pharmaceuticals and chemical agents. Although identifying adverse outcome pathways (AOPs) is a central issue for evaluating the teratogenicity, the AOP causing craniofacial anomalies has not been identified. Recently, zebrafish has gained interest as an emerging model for predicting teratogenicity because of high throughput, cost-effectiveness and availability of various tools for examining teratogenic mechanisms. Here, we established zebrafish sox10-EGFP reporter lines to visualize cranial neural crest cells (CNCCs) and have identified the AOPs for craniofacial anomalies. When we exposed the transgenic embryos to teratogens that were reported to cause craniofacial anomalies in mammals, CNCC migration and subsequent morphogenesis of the first pharyngeal arch were impaired at 24 hours post-fertilization. We also found that cell proliferation and apoptosis of the migratory CNCCs were disturbed, which would be key events of the AOP. From these results, we propose that our sox10-EGFP reporter lines serve as a valuable model for detecting craniofacial skeletal abnormalities, from early to late developmental stages. Given that the developmental process of CNCCs around this stage is highly conserved between zebrafish and mammals, our findings can be extrapolated to mammalian craniofacial development and thus help in predicting craniofacial anomalies in human.

Unexpectedly Large Contribution of Oxygen to Charge Compensation Triggered by Structural Disordering: Detailed Experimental and Theoretical Study on a Li3NbO4-NiO Binary System.

Dependence on lithium-ion batteries for automobile applications is rapidly increasing. The emerging use of anionic redox can boost the energy density of batteries, but the fundamental origin of anionic redox is still under debate. Moreover, to realize anionic redox, many reported electrode materials rely on manganese ions through π-type interactions with oxygen. Here, through a systematic experimental and theoretical study on a binary system of Li3NbO4-NiO, we demonstrate for the first time the unexpectedly large contribution of oxygen to charge compensation for electrochemical oxidation in Ni-based materials. In general, for Ni-based materials, e.g., LiNiO2, charge compensation is achieved mainly by Ni oxidation, with a lower contribution from oxygen. In contrast, for Li3NbO4-NiO, oxygen-based charge compensation is triggered by structural disordering and σ-type interactions with nickel ions, which are associated with a unique environment for oxygen, i.e., a linear Ni-O-Ni configuration in the disordered system. Reversible anionic redox with a small hysteretic behavior was achieved for LiNi2/3Nb1/3O2 with a cation-disordered Li/Ni arrangement. Further Li enrichment in the structure destabilizes anionic redox and leads to irreversible oxygen loss due to the disappearance of the linear Ni-O-Ni configuration and the formation of unstable Ni ions with high oxidation states. On the basis of these results, we discuss the possibility of using σ-type interactions for anionic redox to design advanced electrode materials for high-energy lithium-ion batteries.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine.

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity.

Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10-7 bp) and G:C > C:G (2 × 10-7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10-7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10-7 bp and 0.12 × 10-7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10-7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.

Hawk-Seq™ differentiates between various mutations in Salmonella typhimurium TA100 strain caused by exposure to Ames test-positive mutagens.

A precise understanding of differences in genomic mutations according to the mutagenic mechanisms detected in mutagenicity data is required to evaluate the carcinogenicity of environmental mutagens. Recently, we developed a highly accurate genome sequencing method, 'Hawk-Seq™', that enables the detection of mutagen-induced genome-wide mutations. However, its applicability to detect various mutagens and identify differences in mutational profiles is not well understood. Thus, we evaluated DNA samples from Salmonella typhimurium TA100 exposed to 11 mutagens, including alkylating agents, aldehydes, an aromatic nitro compound, epoxides, aromatic amines and polycyclic aromatic hydrocarbons (PAHs). We extensively analysed mutagen-induced mutational profiles and studied their association with the mechanisms of mutagens. Hawk-Seq™ sensitively detected mutations induced by all 11 mutagens, including one that increased the number of revertants by approximately 2-fold in the Ames test. Although the sensitivity for less water-soluble mutagens was relatively low, we increased the sensitivity to obtain high-resolution spectra by modifying the exposure protocol. Moreover, two epoxides indicated similar 6- or 96-dimensional mutational patterns; likewise, three SN1-type alkylating agents indicated similar mutational patterns, suggesting that the mutational patterns are compound category specific. Meanwhile, an SN2 type alkylating agent exhibited unique mutational patterns compared to those of the SN1 type alkylating agents. Although the mutational patterns induced by aldehydes, the aromatic nitro compound, aromatic amines and PAHs did not differ substantially from each other, the maximum total base substitution frequencies (MTSFs) were similar among mutagens in the same structural groups. Furthermore, the MTSF was found to be associated with the carcinogenic potency of some direct-acting mutagens. These results indicate that our method can generate high-resolution mutational profiles to identify characteristic features of each mutagen. The detailed mutational data obtained by Hawk-Seq™ can provide useful information regarding mutagenic mechanisms and help identify its association with the carcinogenicity of mutagens without requiring carcinogenicity data.

Chemical-Induced Cleft Palate Is Caused and Rescued by Pharmacological Modulation of the Canonical Wnt Signaling Pathway in a Zebrafish Model.

Cleft palate is one of the most frequent birth defects worldwide. It causes severe problems regarding eating and speaking and requires long-term treatment. Effective prenatal treatment would contribute to reducing the risk of cleft palate. The canonical Wnt signaling pathway is critically involved in palatogenesis, and genetic or chemical disturbance of this signaling pathway leads to cleft palate. Presently, preventative treatment for cleft palate during prenatal development has limited efficacy, but we expect that zebrafish will provide a useful high-throughput chemical screening model for effective prevention. To achieve this, the zebrafish model should recapitulate cleft palate development and its rescue by chemical modulation of the Wnt pathway. Here, we provide proof of concept for a zebrafish chemical screening model. Zebrafish embryos were treated with 12 chemical reagents known to induce cleft palate in mammals, and all 12 chemicals induced cleft palate characterized by decreased proliferation and increased apoptosis of palatal cells. The cleft phenotype was enhanced by combinatorial treatment with Wnt inhibitor and teratogens. Furthermore, the expression of tcf7 and lef1 as a readout of the pathway was decreased. Conversely, cleft palate was prevented by Wnt agonist and the cellular defects were also prevented. In conclusion, we provide evidence that chemical-induced cleft palate is caused by inhibition of the canonical Wnt pathway. Our results indicate that this zebrafish model is promising for chemical screening for prevention of cleft palate as well as modulation of the Wnt pathway as a therapeutic target.

Progression of glaucomatous optic neuropathy associated with chorioretinal microvascular embolism after intranasal injection of a corticosteroid suspension.

A patient with glaucoma developed sudden blurred vision immediately after the nasal mucosal injection of a betamethasone acetate solution into the inferior turbinate. The fundus examination revealed several white emboli in the choroidal vessels of the temporal region of the optic disc. After vigorous massage, her visual acuity recovered from counting fingers to 20/32. Six days after the initial examination, Goldmann perimetry showed expansion of the superior and inferior arcuate scotomas. In this case, temporary ischemia of the central retinal and short posterior ciliary arteries involving the arterial circle of Zinn-Haller led to the deterioration of the preexisting glaucomatous optic neuropathy.

Chemical-induced craniofacial anomalies caused by disruption of neural crest cell development in a zebrafish model.

BACKGROUND: Craniofacial anomalies are among the most frequent birth defects worldwide, and are thought to be caused by gene-environment interactions. Genetically manipulated zebrafish simulate human diseases and provide great advantages for investigating the etiology and pathology of craniofacial anomalies. Although substantial advances have been made in understanding genetic factors causing craniofacial disorders, limited information about the etiology by which environmental factors, such as teratogens, induce craniofacial anomalies is available in zebrafish. RESULTS: Zebrafish embryos displayed craniofacial malformations after teratogen treatments. Further observations revealed characteristic disruption of chondrocyte number, shape and stacking. These findings suggested aberrant development of cranial neural crest (CNC) cells, which was confirmed by gene expression analysis of the CNC. Notably, these observations suggested conserved etiological pathways between zebrafish and mammals including human. Furthermore, several of these chemicals caused malformations of the eyes, otic vesicle, and/or heart, representing a phenocopy of neurocristopathy, and these chemicals altered the expression levels of the responsible genes. CONCLUSIONS: Our results demonstrate that chemical-induced craniofacial malformation is caused by aberrant development of neural crest. This study indicates that zebrafish provide a platform for investigating contributions of environmental factors as causative agents of craniofacial anomalies and neurocristopathy.

In silico systems for predicting chemical-induced side effects using known and potential chemical protein interactions, enabling mechanism estimation.

In silico models for predicting chemical-induced side effects have become increasingly important for the development of pharmaceuticals and functional food products. However, existing predictive models have difficulty in estimating the mechanisms of side effects in terms of molecular targets or they do not cover the wide range of pharmacological targets. In the present study, we constructed novel in silico models to predict chemical-induced side effects and estimate the underlying mechanisms with high general versatility by integrating the comprehensive prediction of potential chemical-protein interactions (CPIs) with machine learning. First, the potential CPIs were comprehensively estimated by chemometrics based on the known CPI data (1,179,848 interactions involving 3,905 proteins and 824,143 chemicals). Second, the predictive models for 61 side effects in the cardiovascular system (CVS), gastrointestinal system (GIS), and central nervous system (CNS) were constructed by sparsity-induced classifiers based on the known and potential CPI data. The cross validation experiments showed that the proposed CPI-based models had a higher or comparable performance than the traditional chemical structure-based models. Moreover, our enrichment analysis indicated that the highly weighted proteins derived from predictive models could be involved in the corresponding functions of the side effects. For example, in CVS, the carcinogenesis-related pathways (e.g., prostate cancer, PI3K-Akt signal pathway), which were recently reported to be involved in cardiovascular side effects, were enriched. Therefore, our predictive models are biologically valid and would be useful for predicting side effects and novel potential underlying mechanisms of chemical-induced side effects.

Genome-wide somatic mutation analysis via Hawk-Seq™ reveals mutation profiles associated with chemical mutagens.

It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/107-108 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

Safety Pharmacological Evaluation of the Coffee Component, Caffeoylquinic Acid, and Its Metabolites, Using Ex Vivo and In Vitro Profiling Assays.

Although coffee components have gained interest for use as pharmaceuticals, little is known about their safety pharmacological effects. Hence, we aimed to evaluate the safety pharmacological effects of a chlorogenic acid (CGA)-related compound contained in coffee, 5-O-caffeoylquinic acid (5-CQA), and its metabolites, 5-O-feruloylquinic acid (5-FQA), caffeic acid (CA), and ferulic acid (FA). Langendorff perfused heart assay, electrophysiological assay of acute rat hippocampal slices, and in vitro Magnus assay of gastrointestinal tracts were conducted at 1-100 µM. Moreover, in vitro profiling assays against 38 major targets were conducted. In the Langendorff assay, no significant adverse effects were observed. In the electrophysiological assay, although epileptiform discharge rates were increased at 10 µM CA with 4-aminopyridine, and area under the curve (AUC) and number of population spike were increased at 10 µM FA with bicuculline, dose dependency was not confirmed, and no significant changes were observed at 1 µM and by CGAs alone. In the Magnus assay, a slight increase in contraction activity was observed at >1 µM FA in the stomach fundi and 100 µM 5-CQA in the ileum, suggesting enterokinesis promotion. No significant interactions were observed in the in vitro profiling assays. Therefore, CGAs could have a fundamental function as safe pharmaceuticals.

Effects of dietary alpha-linolenic acid-enriched diacylglycerol oil on embryo/fetal development in rats.

Recent studies suggest that diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil provide potential health benefits in preventing or managing obesity. However, available safety information about reproductive and developmental toxicities of ALA-DAG oil is limited. This study was conducted to clarify the effect, if any, of ALA-DAG oil on embryo-fetal development, following maternal exposure during the critical period of major organogenesis. ALA-DAG oil was administered via gavage to pre-mated female Sprague Dawley rats from gestation day 6 through 19, at dose levels of 0, 1.25, 2.5, and 5.0 mL/kg/day (equivalent to 0, 1149, 2325, and 4715 mg/kg/day, respectively), with total volume adjusted to 5 mL/kg/day with rapeseed oil. All females survived to the scheduled necropsy. There were no treatment-related changes in clinical or internal findings, maternal body weights, feed consumption, intrauterine growth, survival, and number of implantations. No ALA-DAG oil-related fetal malformations or developmental variations were noted. A maternal maximum tolerated dose for ALA-DAG oil could not be achieved in this study. Based on these results, a dose level of 5.0 mL/kg (4715 mg/kg/day), the highest dose tested, was considered as the no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity.

A 90-day repeated-dose toxicity study of dietary alpha linolenic acid-enriched diacylglycerol oil in rats.

Diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil-which mainly consists of oleic and linolenic, linoleic acids-have potential health benefits in terms of preventing or managing obesity. Although safety of DAG oil has been extensively investigated, toxicity of ALA-DAG oil has not been well understood. Hence, the present study was conducted to clarify the potential adverse effects, if any, of ALA-DAG oil in rats (10/sex/group) fed diets containing 1.375%, 2.75%, or 5.5% ALA-DAG oil for 90 days. Compared to control rats fed rapeseed oil or ALA-triacylglycerol oil (flaxseed oil), rats receiving ALA-DAG oil did not reveal any toxicologically significant treatment-related changes as evaluated by clinical signs, functional observational battery, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weight, necropsy and histopathology. The no observed adverse effect levels for dietary exposure to ALA-DAG oil for male and female rats were 2916 and 3326 mg/kg body weight/day, respectively, the highest dose tested. The findings from this study suggest that consumption of ALA-DAG oil is unlikely to cause adverse effects.

Indocyanine Green Dye Filling Time for Polypoidal Lesions in Polypoidal Choroidal Vasculopathy Affects the Visibility of the Lesions on OCT Angiography.

PURPOSE: To elucidate the factors that affect the visibility of polypoidal lesions on OCT angiography (OCTA) based on indocyanine green angiography (ICGA) findings in eyes with polypoidal choroidal vasculopathy (PCV). DESIGN: Retrospective, consecutive case series. PARTICIPANTS: Thirty-one eyes of 31 patients with PCV diagnosed on ICGA. METHODS: We compared the numbers of polyps visible on ICGA and on OCTA. The following time points and durations were determined: first appearance of dye in the choroidal arteries, first appearance of dye within a polyp, and the time it took the dye to fill entire lesions. Then we compared visible and invisible polyps on OCTA in these respects. MAIN OUTCOME MEASURES: The visibility of polypoidal lessons on OCTA and, in relation to this, the time it took the indocyanine green dye to fill the polypoidal lesions. RESULTS: The total number of polyps detected on ICGA was 62, and 49 of these (79.0%) were imaged on OCTA. The time points determined were not significantly different, but elapsed time from the first appearance of the dye in the choroidal arteries to the first appearance of the dye within a polyp was significantly longer for the polyps that were not detected on OCTA than for those that were (5.15±2.30 seconds vs. 2.08±1.08 seconds; P < 0.001). CONCLUSIONS: The visibility of polyps on OCTA decreases when blood flow is slower as manifested by a longer choroid-to-polyp dye infusion time on ICGA.

A surviving 24-month-old patient with neonatal-onset carnitine palmitoyltransferase II deficiency.

The early-onset form of carnitine palmitoyltransferase (CPT) II deficiency has severe outcomes; patients typically die during the newborn period. We report a case of neonatal-onset CPT II deficiency with prolonged survival, exceeding 24 months. The patient was successfully treated by continuous hemodialysis (CHD), which enabled her to overcome repeated crises. We suggest that early intensive treatment, including CHD, is a key for prolonged survival in patients with neonatal-onset CPT II deficiency.

Alpha-linolenic acid-enriched diacylglycerol oil does not promote tumor development in tongue and gastrointestinal tract tissues in a medium-term multi-organ carcinogenesis bioassay using male F344 rat.

Alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil is an edible oil enriched with DAG (>80%) and ALA (>50%). The present study investigated whether ALA-DAG oil promotes tumorigenesis in the tongue and gastrointestinal tract, using a rat medium-term multi-organ carcinogenesis bioassay model. Rats were treated with five genotoxic carcinogens to induce multi-organ tumorigenesis until week 4, and from 1 week after withdrawal, fed a semi-synthetic diet (AIN-93G) containing ALA-DAG oil at concentrations of 0, 13,750, 27,500, and 55,000 ppm. Rats fed AIN-93G containing 55,000 ppm ALA-triacylglycerol or a standard basal diet served as reference and negative control groups, respectively. Animals were euthanized at week 30. ALA-DAG oil was shown to have no effects on survival, general condition, body weight, food consumption, or organ weight. More discolored spots were observed in the stomachs of the 13,750- and 55,000-ppm ALA-DAG groups than in those of the control groups; however, there were no differences in the frequency of histopathological findings across groups. There were no meaningful increases in the incidence of pre-neoplastic and neoplastic lesions in the tongue and gastrointestinal tract among the groups. We therefore conclude that ALA-DAG oil does not promote tumor development in the digestive system.

Detection of Asymptomatic Choroiditis by Fundus Examination in Vogt-Koyanagi-Harada Disease.

PURPOSE: To describe the ophthalmoscopic changes of the choroidal vessels in the posterior pole of Vogt-Koyanagi-Harada disease patients whose choroid is thickened due to asymptomatic choroiditis. METHODS: Three patients whose choroidal vessels are indistinct or not visible in the posterior pole by ophthalmoscopy were examined by fundus photography and enhanced-depth imaging optical coherence tomography (EDI-OCT). RESULTS: They did not complain of vision problems, and visual acuity was good. Serous retinal detachment was not observed by ophthalmoscopy or by OCT. However, choroidal thickening was observed in the EDI-OCT, while choroidal vessels became blurred or invisible on ophthalmoscopy. After steroid therapy, choroidal thickness decreased to normal levels and the small and large choroidal vessels previously indiscernible became visible by ophthalmoscopy and fundus photography. CONCLUSIONS: Asymptomatic choroiditis not accompanied by serous retinal detachment can be detected and assessed by observing the changes in the choroidal vessels by ophthalmoscopy and fundus photography.

In toto extraction of spontaneous conjunctival cysts without incision under slit-lamp microscopic view.

OBJECTIVE: We describe a technique for the in toto extraction of spontaneous conjunctival cysts without incision under slit-lamp microscopic view. DESIGN: Interventional case series. PARTICIPANTS: Eleven eyes of 11 patients. METHODS: This was a multicentre study. Selection criterion was symptomatic conjunctival spontaneous cysts. Topical anaesthesia was applied, and the conjunctival cyst was extracted with a 26-gauge × 0.5-inch needle attached to a 1-mL disposable syringe. The bevel of the needle was oriented toward the surgeon, and it was inserted into the conjunctival cyst. The surgeon then pulled the plunger to produce negative pressure and sucked the fluid from the cyst. After the fluid was drained, the negative pressure also sucked the inner walls of the cysts into the needle tip. The surgeon slowly pulled the needle tip from the subconjunctival space. In most cases, a stump of the cyst remained at the entry site; it was grasped with forceps and totally extracted. RESULTS: This technique was used on 11 eyes that were followed for 23.2 months. The conjunctival cysts were extracted in toto from all eyes, and a recurrence was observed in 1 eye during the follow-up period. In the 1 eye, a small residual cyst recurred, and it was extracted by the same technique. CONCLUSIONS: This method of extracting conjunctival cysts is indicated for spontaneous conjunctival cysts that are considered to be free from adhesion to the surrounding tissue. It is an easy and minimally invasive method of extraction under a slit-lamp microscope without incision.

Traumatic myopia secondary to ciliary spasm after blunt eye trauma and reconsideration of its pathogenesis.

PURPOSE: To reconsider the pathogenesis of traumatic myopia by describing two cases and literature review. METHODS: Refraction was measured, A-scan ultrasonography was performed, and ultrasound biomicroscopy (UBM) was used to observe the ciliary body in the acute and recovery stages. RESULTS: The first patient had a myopic shift of -6.12 diopters (D) on the initial examination (day 2) compared with the recovered stage. UBM showed ciliochoroidal effusion in one half of the circumference and severe edema in three eighths of the ciliary body, which led to an anterior rotation of the ciliary body. Immediately after the cycloplegia, the myopic shift decreased by -3.9 D. On day 11, the refraction was -0.63 D, and the UBM findings were normalized. The second patient had a myopic shift of -4.38 D on the first examination compared with the recovered stage. UBM showed an annular ciliochoroidal effusion leading to anterior rotation of the ciliary body. Immediately after the cycloplegia, there was a decrease of -2.75 D. Biometric measurements showed an increased anterior chamber depth and a decreased thickness of the lens. On day 16, the refraction was -1.0 D, and the UBM findings were normalized. In both cases, biometric measurements in the acute phase suggested a anterior chamber shallowing, a thickening of the lens, and an anterior shift of the lens-iris diaphragm CONCLUSIONS: Cycloplegia decreased the myopic shift by more than 60 %. These findings indicate a significant contribution of ciliary spasm combined with ciliochoroidal effusion, anterior shift of the lens-iris diaphragm, and thickening of the lens.