Automated Detection of Cerebral Microbleeds on Two-dimensional Gradient-recalled Echo T2* Weighted Images Using a Morphology Filter Bank and Convolutional Neural Network.

PURPOSE: We present a novel algorithm for the automated detection of cerebral microbleeds (CMBs) on 2D gradient-recalled echo T2* weighted images (T2*WIs). This approach combines a morphology filter bank with a convolutional neural network (CNN) to improve the efficiency of CMB detection. A technical evaluation was performed to ascertain the algorithm's accuracy. METHODS: In this retrospective study, 60 patients with CMBs on T2*WIs were included. The gold standard was set by three neuroradiologists based on the Microbleed Anatomic Rating Scale guidelines. Images with CMBs were extracted from the training dataset comprising 30 cases using a morphology filter bank, and false positives (FPs) were removed based on the threshold of size and signal intensity. The extracted images were used to train the CNN (Vgg16). To determine the effectiveness of the morphology filter bank, the outcomes of the following two methods for detecting CMBs from the 30-case test dataset were compared: (a) employing the morphology filter bank and additional FP removal and (b) comprehensive detection without filters. The trained CNN processed both sets of initial CMB candidates, and the final CMB candidates were compared with the gold standard. The sensitivity and FPs per patient of both methods were compared. RESULTS: After CNN processing, the morphology-filter-bank-based method had a 95.0% sensitivity with 4.37 FPs per patient. In contrast, the comprehensive method had a 97.5% sensitivity with 25.87 FPs per patient. CONCLUSION: Through effective CMB candidate refinement with a morphology filter bank and FP removal with a CNN, we achieved a high CMB detection rate and low FP count. Combining a CNN and morphology filter bank may facilitate the accurate automated detection of CMBs on T2*WIs.

HI-FISH: WHOLE BRAIN IN SITU MAPPING OF NEURONAL ACTIVATION IN DROSOPHILA DURING SOCIAL BEHAVIORS AND OPTOGENETIC STIMULATION.

Monitoring neuronal activity at single-cell resolution in freely moving Drosophila engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here we describe a method, called HI-FISH, for brain-wide mapping of active cells expressing the Immediate Early Gene hr38 using a high-sensitivity/low background amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1a cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH) we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally we compare the activity of P1a neurons during sequential performance of courtship and aggression, using intronic vs. exonic hr38 probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1a neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in Drosophila.

Pulmonary varix clearly demonstrated by 3D-CT before pulmonary angiography.

Pulmonary varix is a rare and usually asymptomatic localized dilation of a pulmonary vein. This disease should be distinguished from other pulmonary and mediastinal diseases, particularly pulmonary arteriovenous malformations. Herein, we encountered a case of pulmonary varix clearly demonstrated by 3-dimensional reconstructed computed tomography (3D-CT) which proved useful in its diagnosis. The 3D-CT enabled easy understanding of the vascular connections and confirmation of the absence of an inflow pulmonary artery. We also performed angiography which showed findings consistent with those obtained by the 3D-CT, thus confirming the diagnosis of pulmonary varix. After the diagnosis, the patient was followed up for several years without any treatment and she remained asymptomatic. On follow-up CT, the lesion remained unchanged.

Adrenocortical carcinoma with multiple liver metastases controlled by bland transarterial embolization and surgery resulting in long-term survival.

Adrenocortical carcinoma (ACC) is a rare malignant tumor with a poor prognosis. Local recurrence or distant metastases occur in more than 50% of cases. Patients with metastases have limited treatment options, and <15% have a 5-year survival time. Herein, we describe a 44-year-old woman with ACC and who underwent retroperitoneal tumor resection. Multiple liver and lung metastases were found 1-year postresection. Mitotane therapy started as systemic treatment. Lung metastases were controlled but liver metastases were progressive. The liver metastases were treated by performing 2 resections and 6 bland transarterial embolization (bland TAE), and are presently controlled with only 2 liver metastases of <20 mm. The present case showed that bland TAE can achieve long-term prevention of the progression of liver metastases of ACC. The ultraselective bland TAE for selective embolization supported by the latest computed tomography analysis techniques during arteriography could minimize liver damage caused by embolization and allowed multiple treatments which prolonged survival. We conclude that bland TAE can be effective for controlling liver metastases of ACC.

Potential of PALBI-T score as a prognostic model for hepatocellular carcinoma in alcoholic liver disease.

BACKGROUND AND AIM: With the control of viral hepatitis, alcoholic hepatocellular carcinoma (HCC) is becoming increasingly important in Japan. In alcoholic cirrhosis, the impact of portal hypertension is significant. Thus, it may be difficult to predict prognosis accurately with the reported prognostic scores. Here we propose the platelet-albumin-bilirubin tumor nodes metastasis (TNM) score (PALBI-T score) as a prognostic model for HCC in alcoholic liver disease, and investigate its usefulness. The PALBI-T score is an integrated score based on the TNM stage and PALBI grade including platelets, reflecting portal hypertension. METHODS: This study included 163 patients with alcoholic HCC treated at our Center from 1997 to 2018. We compared the prognostic prediction abilities of the Japan Integrated Staging (JIS) score, ALBI-T score, and PALBI-T score. The PALBI-T score was calculated similarly to the JIS and ALBI-T scores. Areas under the receiver operating characteristic curve (AUC) were calculated for predicting overall survival (OS). RESULTS: In predicting the 1-year survival, the JIS score had a larger AUC (AUC = 0.925) than the ALBI-T score (AUC = 0.895) and PALBI-T score (AUC = 0.891). On the other hand, there was no significant difference in predicting OS among the integrated scores. The PALBI-T score (AUC = 0.740) had the largest AUC, and the JIS score (AUC = 0.729) and ALBI-T score (AUC = 0.717) were not significantly different from the PALBI grade (AUC = 0.634). The PALBI grade reflected the degree of portal hypertension. CONCLUSION: In patients with alcoholic HCC, the Japan Integrated Staging score is useful for predicting short-term prognosis. The PALBI-T score, which reflects portal hypertension, appears to be a more valid prognostic score for predicting long-term prognosis.

A Circuit Node that Integrates Convergent Input from Neuromodulatory and Social Behavior-Promoting Neurons to Control Aggression in Drosophila.

Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.

Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila.

Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. Here, we identify a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM(+) neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction.

The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.

Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.

Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases.

A ROCK inhibitor permits survival of dissociated human embryonic stem cells.

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix.

Here we report a human-derived material with potent inductive activity that selectively converts ES cells into neural tissues. Both mouse and human ES cells efficiently differentiate into neural precursors when cultured on the matrix components of the human amniotic membrane in serum-free medium [amniotic membrane matrix-based ES cell differentiation (AMED)]. AMED-induced neural tissues have regional characteristics (brainstem) similar to those induced by coculture with mouse PA6 stromal cells [a common method called stromal cell-derived inducing activity (SDIA) culture]. Like the SDIA culture, the AMED system is applicable to the in vitro generation of various CNS tissues, including dopaminergic neurons, motor neurons, and retinal pigment epithelium. In contrast to the SDIA method, which uses animal cells, the AMED culture uses a noncellular inductive material derived from an easily available human tissue; therefore, AMED should provide a more suitable and versatile system for generating a variety of neural tissues for clinical applications.

Generation of cerebellar neuron precursors from embryonic stem cells.

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.

Fluorescence-activated cell sorting-based purification of embryonic stem cell-derived neural precursors averts tumor formation after transplantation.

The differentiation of dopaminergic (DA) neurons from mouse embryonic stem cells (ESCs) can be efficiently induced, making these neurons a potential source for transplantation as a treatment for Parkinson's disease, a condition characterized by the gradual loss of midbrain DA neurons. One of the major persistent obstacles to the successful implementation of therapeutic ESC transplantation is the propensity of ESC-derived grafts to form tumors in vivo. To address this problem, we used fluorescence-activated cell sorting to purify mouse ESC-derived neural precursors expressing the neural precursor marker Sox1. ESC-derived, Sox1+ cells began to express neuronal cell markers and differentiated into DA neurons upon transplantation into mouse brains but did not generate tumors in this site. In contrast, Sox1- cells that expressed ESC markers frequently formed tumors in vivo. These results indicate that Sox1-based cell sorting of neural precursors prevents graft-derived tumor formation after transplantation, providing a promising strategy for cell transplantation therapy of neurodegenerative disorders.

Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells.

We report directed differentiaion of retinal precursors in vitro from mouse ES cells. Six3+ rostral brain progenitors are generated by culturing ES cells under serum-free suspension conditions (SFEB culture) in the presence of Wnt and Nodal antagonists (Dkk1 and LeftyA), and subsequently steered to differentiate into Rx+ cells (16%) by treatment with activin and serum. Consistent with the characteristics of early neural retinal precursors, the induced Rx+ cells coexpress Pax6 and the mitotic marker Ki67, but not Nestin. The ES cell-derived precursors efficiently generate cells with the photoreceptor phenotype (rhodopsin+, recoverin+) when cocultured with embryonic retinal cells. Furthermore, organotypic culture studies demonstrate the selective integration and survival of ES cell-derived cells with the photoreceptor phenotype (marker expression and morphology) in the outer nuclear layer of the retina. Taken together, ES cells treated with SFEB/Dkk1/LeftyA/serum/activin generate neural retinal precursors, which have the competence of photoreceptor differentiation.

Directed differentiation of telencephalic precursors from embryonic stem cells.

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.