Electrophysiological responses to seizurogenic compounds dependent on E/I balance in human iPSC-derived cortical neural networks.

Construction of in vitro functional assay systems using human-induced pluripotent stem cells (iPSCs) as indicators for evaluating seizure liability of compounds has been anticipated. Imbalance of excitation/inhibition (E/I) inputs triggers seizure; however, the appropriate ratio of E/I neurons for evaluating seizure liability of compounds in a human iPSC-derived neural network is unknown. Here, five neural networks with varying E/I ratios (88/12, 84/16, 74/26, 58/42, and 48/52) were constructed by altering the ratios of glutamatergic (E) and GABA (I) neurons. The responsiveness of each network against six seizurogenic compounds and two GABA receptor agonists was then examined by using six representative parameters. The 52% GABA neuron network, which had the highest ratio of GABA neurons, showed the most marked response to seizurogenic compounds, however, it suggested the possibility of producing false positives. Moreover, analytical parameters were found to vary with E/I ratio and to differ for seizurogenic compounds with different mechanism of action (MoA) even at the same E/I ratio. Clustering analysis using six parameters showed the balance of 84/16, which is the closest to the biological balance, was the most suitable for detection of concentration-dependent change and classification of the MoA of seizurogenic compounds. These results suggest the importance of using a human-iPSC-derived neural network similar to the E/I balance of the living body in order to improve the prediction accuracy in the in vitro seizure liability assessment.

Analysis of signal components < 500 Hz in brain organoids coupled to microelectrode arrays: A reliable test-bed for preclinical seizure liability assessment of drugs and screening of antiepileptic drugs.

Brain organoids with three-dimensional structure and tissue-like function are highly demanded for brain disease research and drug evaluation. However, to our knowledge, methods for measuring and analyzing brain organoid function have not been developed yet. This study focused on the frequency components of an obtained waveform below 500 Hz using planner microelectrode array (MEA) and evaluated the response to the convulsants pentylenetetrazol (PTZ) and strychnine as well as the antiepileptic drugs (AEDs) perampanel and phenytoin. Sudden and persistent seizure-like firing was observed with PTZ administration, displaying a concentration-dependent periodic activity with the frequency component enhanced even in one oscillation characteristic. On the other hand, in the administration of AEDs, the frequency of oscillation decreased in a concentration-dependent manner and the intensity of the frequency component in one oscillation also decreased. Interestingly, at low doses of phenytoin, a group of synchronized bursts was formed, which was different from the response to the perampanel. Frequency components contained information on cerebral organoid function, and MEA was proven useful in predicting the seizure liability of drugs and evaluating the effect of AEDs with a different mechanism of action. In addition, frequency component analysis of brain organoids using MEA is an important analysis method to perform in vitro to in vivo extrapolation in the future, which will help explore the function of the organoid itself, study human brain developments, and treat various brain diseases.

Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system.

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However, differences in responsiveness depending on convulsant type and antiepileptic drugs, and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ, but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses, frequency analysis of below 250 Hz, excluding the spike component, was performed. The in vivo convulsive firing enhancement of the high γ wave and ß wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods, including frequency analysis, appear effective for predicting convulsion toxicity, side effects, and their mechanism of action as well as the comparison of convulsions induced in vivo.

Detection of synchronized burst firing in cultured human induced pluripotent stem cell-derived neurons using a 4-step method.

Human induced pluripotent stem cell-derived neurons are promising for use in toxicity evaluations in nonclinical studies. The multi-electrode array (MEA) assay is used in such evaluation systems because it can measure the electrophysiological function of a neural network noninvasively and with high throughput. Synchronized burst firing (SBF) is the main analytic parameter of pharmacological effects in MEA data, but an accurate method for detecting SBFs has not been established. In this study, we present a 4-step method that accurately detects a target SBF confirmed by the researcher's interpretation of a raster plot. This method calculates one set parameter per step, in the following order: the inter-spike interval (ISI), the number of spikes in an SBF, the inter-SBF interval, and the number of spikes in an SBF again. We found that the 4-step method is advantageous over the conventional method because it determines the preferable duration of an SBF, accurately distinguishes continuous SBFs, detects weak SBFs, and avoids false detection of SBFs. We found also that pharmacological evaluations involving SBF analysis may differ depending on whether the 4-step or conventional threshold method is used. This 4-step method may contribute to improving the accuracy of drug toxicity and efficacy evaluations using human induced pluripotent stem cell-derived neurons.

Effects of operating conditions on selectivity of a plasma fractionator in double filtration plasmapheresis.

In a typical double filtration plasmapheresis treatment, plasma fractionation between albumin and some immunoglobulins associated with toxins is limited because none of the currently available plasma fractionators has a strict cutoff property for these proteins. Selectivity of immunoglobulins over albumin depends not only on the cutoff properties of the membrane but on the operating conditions such as the flow rate of the supplied plasma (Q(P)) and retained plasma to be discarded (Q(D)) in the plasma fractionator. We carried out an in vitro study using human plasma harvested by single plasma exchange treatments to assess the selectivity of a plasma fractionator, Evaflux 2A-F (Kawasumi Laboratories, Inc., Tokyo, Japan), under various operating conditions. The results of rate-constant filtration experiments showed that the concentrations in the feed tank and the sieving coefficient (SC) values of every protein were decreased slightly within 2 h after the start of the experiment because of membrane trapping, adsorption, and/or plugging. The time-averaged SC value of albumin increased with flow rate ratio (Q(P)/Q(D)) due to increasing filtration fraction (FF), but relative removal efficiency (mD/mP*) for albumin decreased with Q(P)/ Q(D) due to decreasing Q(D). For immunoglobulins, on the other hand, the SC values were almost unchanged, and the mD/mP* values increased with Q(P)/Q(D) due to an increase in FF. Both increasing Q(P) and decreasing Q(D) are effective means of improving selectivity between these proteins in the plasma fractionator. Membrane fouling is, however, obvious beyond a Q(P)/Q(D) value that is thought to be a critical point. Operation should be conducted below the critical Q(P)/Q(D) value, which depends on the patient's plasma components and the cutoff property of the membrane.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 15). Two-week and 4-week administration study of methyl methanesulfonate (MMS).

This study was conducted to assess whether a 2-week treatment period is as effective as 4-week treatment for detection of drug-induced toxicity on the male rat reproductive organs using methyl methanesulfonate (MMS). A two-week study at dose levels of 20 or 40 mg/kg and a 4-week study with 20 mg/kg were conducted. The results can be summarized as follows. No deaths and no apparent clinical signs were observed. Body weights and food consumption were decreased at 40 mg/kg in the 2-week study along with testis and epididymis weights. In the 4-week study, epididymis weights were decreased at 20 mg/kg. The rats treated with 20 mg/kg in the 4-week study and those treated with 40 mg/kg in the 2-week study showed decrease of germ cells, exfoliation of germ cells, vacuolar degeneration of Sertoli cell and cell debris in epididymal ducts on histopathological observation. MMS impairment of spermatogenesis was confirmed by stage analysis. It was concluded that a treatment period of 2 weeks is sufficient to allow evaluation of toxic effects of MMS on the male reproductive organs.

Continuous monitoring of blood volume in double filtration plasmapheresis.

A continuous hematocrit (HCT) monitor, Crit-Line, was introduced to examine the change in patients' blood volume (BV) due to albumin loss during double filtration plasmapheresis (DFPP) treatments. Nine patients with autoimmune diseases or ABO incompatible renal transplantation received 15 DFPP treatments under Crit-Line monitoring. In these patients, plasma albumin concentration (C(P)) changed from 3.7 +/- 0.6 g/dl to 3.5 +/- 0.5 g/dl and HCT from 28.7% +/- 3.3% to 31.3% +/- 4.3% (change ratio [CR] of BV = -8.1%) during treatment with albumin concentrations (C(S)) of 9.5 +/- 1.0 g/dl and 500 ml volumes (V(S)) of supplementation fluid. Although the apparent CR value of C(P) was -5.3%, on average, the CR of albumin in the patients' plasma (M(P)) was -16.1%, which means a corrected CR value of C(P) by the HCT value to eliminate the influence of the patient's blood volume contraction during treatment. Albumin loss usually occurred in DFPP treatments. The decrease in BV was induced by an oncotic pressure drop due to albumin loss, and often resulted in a blood pressure drop. The amount of albumin loss during DFPP treatments strongly depends on sieving coefficients of the plasma separator (SC(PS)) and the plasma fractionator (SC(PF)), the filtration fraction of the plasma fractionator (FF(PF)), pretreatment C(P) value, and C(S) and V(S) values of the supplementation fluid. To determine the optimum C(S) and V(S) values for each patient, the authors introduced a variable blood volume model for albumin transport in DFPP. In this model, changes in C(P), HCT, and BV values could be estimated during treatment. For example, a patient with an HCT of 31.2%, body weight of 61.1 kg, and pretreatment C(P) of 4.4 g/dl received a DFPP treatment using a plasma separator, OP-05 (SC(PS) of 0.99), and a plasma fractionator, Evaflux 2A (SC(PF) of 0.40), under FF(PF) of 0.8 with a V(S) of 500 ml. A value for C(S) of about 10 g/dl is required for the patient to maintain a normal C(P) level during treatment by an estimation from the model. As a result of the treatment with a C(S) of 10 g/dl, the patient had no adverse reactions, such as a blood pressure decrease, during treatment under these conditions.

Transfer of male or female primordial germ cells of quail into chick embryonic gonads.

Blood from an individual quail embryo at stages 13-16, when primordial germ cells (PGCs) were in circulation, was taken from its marginal vein and transfused into the marginal vein of a chick embryo at stages 13-16. Both donor and recipient embryos were cultured in vitro until day 8 of development and their sex was determined by morphological and histological observations of the gonads. Sections of recipient gonads were stained immunohistochemically with QCR1 monoclonal antibody positive for quail PGCs but negative for chick PGCs. Donor and recipient embryos were sexed in 17 pairs which included all four sex combinations. Transferred PGCs, either female-derived ZW type or male-derived ZZ type, were observed in the gonads of both sexes of 15 recipient embryos. The population of donor PGCs ranged from 20 to over 2500. In all four sex combinations, there was a higher population in the left than the right gonad of the embryos.

Development of murine monoclonal antibodies against an enterotoxin produced by Bacillus cereus.

Three murine monoclonal antibodies (MAbs) were prepared against an enterotoxin (ET) produced by Bacillus cereus. Although these MAbs were found to react with the ET, their specificities appeared to be different in competitive binding assays. One of the MAbs (D-8), which was highly reactive with the ET, will be useful in developing immunological methods to detect crude ET and to isolate the ET in high yield.

Basic studies on visible light-curing resin for denture base. Part 8. The hardening depth of denture relining material after penetration of light through the denture base resin.

A study was performed to examine how the duration of irradiation at the time of relining with visible light-curing material influences the depth of hardening of the relining material, in relation to the thickness of the denture base placed between the denture and the relining material. It was found that for a range of denture base thickness of 1-2.5 mm, a desirable degree of hardening could be obtained using an irradiation time of 20 s for relining material with a thickness of up to 4 mm used for the basal surface.