Characterization of novel, severely immunodeficient PrkdcΔex57/Δex57 mice.

Severely immunodeficient mice are useful for understanding the pathogenesis of certain tumors and for developing therapeutic agents for such tumors. In addition, engraftment of these mice with human hematopoietic cells can yield information that helps us understand the in vivo molecular mechanisms underlying actual human viral infections. In our present research, we discovered a novel, severely immunodeficient strain of mice having a mutation in exon 57 of the Prkdc gene (PrkdcΔex57/Δex57) in an inbred colony of B10.S/SgSlc mice. Those PrkdcΔex57/Δex57 mice showed thymic hypoplasia and lack of mature T cells and B cells in peripheral lymphoid tissues, resulting in very low levels of production of serum immunoglobulins. In addition, those mice were highly susceptible to influenza viruses due to the lack of acquired immune cells. On the other hand, since they had sufficient numbers of NK cells, they rejected tumor transplants, similarly to Prkdc+/+ mice. Next, we generated Foxn1nu/nuPrkdcΔex57/Δex57Il2rg-/- (NPG) mice on the BALB/cSlc background, which lack all lymphocytes such as T cells, B cells and innate lymphoid cells, including NK cells. As expected, these mice were able to undergo engraftment of human tumor cell lines. These findings suggest that PrkdcΔex57/Δex57 mice will be useful as a novel model of immunodeficiency, while NPG mice will be useful for xenografting of various malignancies.

Unmasked insulinoma occasioned by severe hypoglycemic coma immediately postpartum: a case report.

BACKGROUND: Insulinoma in women during pregnancy and postpartum is very rare; approximately 65% of cases are diagnosed early in pregnancy and ~ 35% immediately after delivery, few being found in middle or late pregnancy, likely due to increased insulin resistance seen after early-stage pregnancy. We successfully treated a case of insulinoma in which severe hypoglycemic coma immediately after delivery occasioned detailed investigation and diagnosis. CASE PRESENTATION: Our patient experienced hypoglycemic coma in the 3rd month of pregnancy (initially considered due to her hyperemesis gravidarum) that improved spontaneously during the gestational period. No abnormalities of plasma glucose or body weight were found in regular checkups during her pregnancy; however, recurrence of hypoglycemic coma after delivery led us to suspect insulinoma. While contrast enhanced computer tomography and endoscopic ultrasonography (EUS) initially failed to detect a tumor in the pancreas, selective arterial calcium stimulation test revealed an insulin-secreting tumor localized in the pancreatic body. She then underwent spleen-preserving distal pancreatectomy; a 10-mm tumor positive for chromogranin A, synaptophysin and insulin was identified. CONCLUSIONS: Although pregnancy can mask insulinoma-associated symptoms and make diagnosis challenging, hypoglycemic episodes during early pregnancy, which were observed in this case, are suggestive of insulinoma. Importantly, in this case, accurate preoperative localization of the tumor enabled prompt curative surgery after delivery. Thus, clinical vigilance for the occurrence of insulinoma and its localization is appropriate for pregnant women suffering severe hypoglycemia.

Refinement of decision tree to assess the consequences of increased serum ALP in dogs: Additional analysis on toxicity studies of pesticides evaluated recently in Japan.

Recently we provided a new interpretation that increased serum ALP in dogs is not adverse if no hepatotoxic finding coexists in the analysis of toxicity studies of over 200 pesticides evaluated in Japan (Yokoyama et al., 2019). We also proposed a decision tree to evaluate the adversity of the increased ALP. The present analysis was conducted to validate the reliability of this interpretation with 129 pesticides more recently evaluated. Before applying, the decision tree was revised to be consistent in all steps. The pesticides showed similar characterization of increased ALP to the previous analysis in that the increase was more frequent than in rats and that liver hypertrophy and hepatotoxicity commonly coexisted with an increase in ALP in dogs. When short- and long-term studies of 58 pesticides inducing ALP activity in dogs were applied to the revised tree, the increased ALP in 8 pesticides was judged not adverse in either study. The revision of the tree did not affect the NOAEL judgment of these pesticides; however, the revised routes contributed to the judgment more robustly. This study showed the reliability of our interpretation and applicability of the decision tree to evaluate the adversity of increased ALP in dogs.

Reduction of Kiss1 expression in the anteroventral periventricular nucleus is associated with atrazine-induced attenuation of the luteinizing hormone surge in female rats.

Atrazine, a commonly used herbicide, suppresses the luteinizing hormone (LH) surge in female rats, although the underlying mechanism remains unclear. Kisspeptin, encoded by the Kiss1 gene, is a hypothalamic peptide that controls gonadotropin-releasing hormone (GnRH) release from the GnRH neurons. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are involved in regulating pre-ovulatory GnRH and LH surge. To clarify the effect of atrazine on the LH surge in female rats, we investigated its effects on hypothalamic GnRH and kisspeptin. Ovariectomized female rats in a high-dose estradiol supplementation model were orally administered vehicle or 100 mg/kg of atrazine once daily for 5 days. This attenuated the LH surge but did not affect baseline LH levels, with no difference in hypothalamic GnRH levels between the vehicle-treated and atrazine-treated animals. After the fifth treatment, subcutaneous administration of kisspeptin (at 0, 0.1, 1, and 10 nmol/kg) induced a dose-dependent LH release almost equivalent in the vehicle- and atrazine-treated animals, suggesting that GnRH neurons maintain normal responsiveness to kisspeptin. However, Kiss1 mRNA expression levels in the AVPV were significantly reduced in the atrazine-treated animals. Given the normal response of GnRH neurons to exogenously administered kisspeptin, the suppressive effect of atrazine may be explained by suppression of Kiss1 expression in the AVPV leading to the attenuation of kisspeptin release from kisspeptin neurons in the AVPV. Further studies are warranted to elucidate more precisely the mechanism of atrazine's involvement in the suppression of Kiss1 mRNA expression in the AVPV.

Toxicological significance of increased serum alkaline phosphatase activity in dog studies of pesticides: Analysis of toxicological data evaluated in Japan.

Increased serum alkaline phosphatase (ALP) activity is an indicator of hepatobiliary damage in humans and experimental animals. Practically, increased ALP accompanied by no other hepatotoxic changes is often encountered in toxicity studies of pesticides in dogs. Here, we analyzed the toxicological significance of increased ALP in response to 206 pesticides evaluated by the Food Safety Commission of Japan as toxicological evaluation reports in their risk assessment process. Our analysis indicated that increased ALP was more frequent in dogs (108/206) than in rats (36/206). In 87 of 108 pesticides, increased ALP was observed with hepatotoxicity in dogs. However, increased ALP had no specific relationship with certain types of hepatotoxicity and was not a sensitive marker of hepatotoxicity. Approximately 50% of 87 pesticides showing hepatotoxicity also induced liver hypertrophy. No hepatotoxic changes were seen with the remaining 21 pesticides, other than increases in liver weight and/or liver hypertrophy. Most of these 21 pesticides were phenobarbital-like liver metabolism enzyme inducers in rodents. These results suggested that increased ALP was not an indicator of hepatotoxicity in dogs if hepatotoxic findings were absent. This analysis provided a new interpretation of the toxicological significance of ALP in dogs and could contribute to toxicological evaluation of pesticides.

Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency.

Collagen is one of the most important components of the extracellular matrix that is involved in the strength of tissues, cell adhesion and cell proliferation. Mutations in several collagen and post-translational modification enzyme genes cause Ehlers-Danlos syndrome (EDS) characterized by joint and skin hyperextensibility as well as fragility of various organs. Carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase-1 (CHST14/D4ST1) is a critical enzyme for biosynthesis of dermatan sulfate, a side chain of various proteoglycans including biglycan that regulates collagen fibrils through their interaction. Mutations in CHST14 were found to cause a new form of EDS, named musculocontractural type EDS (mcEDS-CHST14). Large subcutaneous hematomas are one of the most serious complications accompanied by decreased quality of life and potential lethality. In this study, Chst14 gene-deleted mice were expected to be an animal model of the vascular abnormalities of mcEDS-CHST14. However, only limited numbers of adult mice were generated because of perinatal lethality in most Chst14 gene-deleted homozygote (Chst14-/-) mice. Therefore, we investigated the placentas of these fetuses. The placentas of Chst14-/- fetuses showed a reduced weight, alterations in the vascular structure, and ischemic and/or necrotic-like changes. Electron microscopy demonstrated an abnormal structure of the basement membrane of capillaries in the placental villus. These findings suggest that Chst14 is essential for placental vascular development and perinatal survival of fetuses. Furthermore, placentas of Chst14-/- fetuses could be a useful model for vascular manifestations in mcEDS-CHST14, such as the large subcutaneous hematomas.

NIR spectrometer using a Schottky photodetector enhanced by grating-based SPR.

We present a near-infrared (NIR) spectrum measurement method using a Schottky photodetector enhanced by surface plasmon resonance (SPR). An Au grating was fabricated on an n-type silicon wafer to form a Schottky barrier and act as an SPR coupler. The resulting photodetector provides wavelength-selective photodetection depending on the SPR coupling angle. A matrix was pre-calculated to describe this characteristic. The spectrum was obtained from this matrix and the measured photocurrents at various SPR coupling angles. Light with single and multiple wavelengths was tested. Comparative measurements showed that our method is able to detect spectra with a wavelength resolution comparable to that of a commercial spectrometer.

Depinning-Induced Capillary Wave during the Sliding of a Droplet on a Textured Surface.

Surfaces covered with hydrophobic micro-/nanoscale textures can allow water droplets to slide easily because of low contact angle hysteresis. In contrast to the case of a droplet sliding on a smooth surface, when a droplet slides on a textured surface, it must recede from the textures at its rear edge and the resultant depinning events induce a capillary wave on the surface of the droplet. Although this depinning-induced capillary wave can be observed to some extent through high-speed imaging, important parameters of the wave, such as the wavelength and frequency, and the factors that determine these parameters are not fully understood. We report direct measurements of this depinning-induced capillary wave using microelectromechanical systems (MEMS)-based force sensors fabricated on a textured surface. Such sensor measurements reveal the frequency of the vibration occurring on the surface of the droplet, from which it is possible to calculate the wavelength of the capillary wave. We show that the frequency and wavelength of the depinning-induced capillary wave during the sliding of a water droplet on a micropillar array depend upon neither the size of the droplet nor its sliding velocity. However, the frequency (wavelength) decreases (increases) as the pitch of the micropillar array increases. We argue that the wavelength of the depinning-induced capillary wave is equal to the maximum length of the liquid bridges that develop at the micropillars before depinning. This hypothesis is confirmed by comparing the wavelengths obtained from the sensor measurements to the maximum liquid-bridge lengths calculated from observations using a high-speed camera.

Characterization of Chromophoric Water-Soluble Organic Matter in Urban, Forest, and Marine Aerosols by HR-ToF-AMS Analysis and Excitation-Emission Matrix Spectroscopy.

Chromophoric water-soluble organic matter in atmospheric aerosols potentially plays an important role in aqueous reactions and light absorption by organics. The fluorescence and chemical-structural characteristics of the chromophoric water-soluble organic matter in submicron aerosols collected in urban, forest, and marine environments (Nagoya, Kii Peninsula, and the tropical Eastern Pacific) were investigated using excitation-emission matrices (EEMs) and a high-resolution aerosol mass spectrometer. A total of three types of water-soluble chromophores, two with fluorescence characteristics similar to those of humiclike substances (HULIS-1 and HULIS-2) and one with fluorescence characteristics similar to those of protein compounds (PLOM), were identified in atmospheric aerosols by parallel factor analysis (PARAFAC) for EEMs. We found that the chromophore components of HULIS-1 and -2 were associated with highly and less-oxygenated structures, respectively, which may provide a clue to understanding the chemical formation or loss of organic chromophores in atmospheric aerosols. Whereas HULIS-1 was ubiquitous in water-soluble chromophores over different environments, HULIS-2 was abundant only in terrestrial aerosols, and PLOM was abundant in marine aerosols. These findings are useful for further studies regarding the classification and source identification of chromophores in atmospheric aerosols.

Effects of Adrenomedullin on Doxorubicin-Induced Cardiac Damage in Mice.

Doxorubicin (DOX) is one of the best known anticancer drugs, and is used in the treatment of lymphoma, lung cancer, stomach cancer, and a number of other cancers. However, DOX has some serious side effects, the worst being lethal heart failure. Occasionally, its side effects result in the cessation of the anticancer treatment, thus having a serious adverse influence on prognosis. Agents that can be administered as alternative prophylactics or to ameliorate the side effects of DOX will be useful in increasing the safety and efficacy of anticancer therapy. Adrenomedullin (AM) is a peptide hormone secreted by many organs, including the heart; it has an organ-protective effect, including antiapoptotic, anti-inflammatory, and antioxidative stress. Blood AM levels increase with heart failure; endogenic AM has been suggested in order to protect the heart. Furthermore, exogenous AM administration has shown therapeutic effects for heart failure in patients. However, it is unclear whether AM can protect the heart against drug-induced cardiac injury in vivo. The present study was performed in order to investigate the effects of AM on DOX-induced cardiac damage. Male BALB/c mice were treated with DOX and/or AM. Exogenous AM improved the survival ratio of DOX-treated mice. In addition, AM reduced serum lactate dehydrogenase (LDH) levels following DOX treatment. On pathological examination, AM was shown to inhibit DOX-induced cardiac tissue damage, mitochondrial abnormality, and cell death. These findings suggest that AM has a protective effect against DOX-induced cardiac damage.

A Tactile Sensor Using Piezoresistive Beams for Detection of the Coefficient of Static Friction.

This paper reports on a tactile sensor using piezoresistive beams for detection of the coefficient of static friction merely by pressing the sensor against an object. The sensor chip is composed of three pairs of piezoresistive beams arranged in parallel and embedded in an elastomer; this sensor is able to measure the vertical and lateral strains of the elastomer. The coefficient of static friction is estimated from the ratio of the fractional resistance changes corresponding to the sensing elements of vertical and lateral strains when the sensor is in contact with an object surface. We applied a normal force on the sensor surface through objects with coefficients of static friction ranging from 0.2 to 1.1. The fractional resistance changes corresponding to vertical and lateral strains were proportional to the applied force. Furthermore, the relationship between these responses changed according to the coefficients of static friction. The experimental result indicated the proposed sensor could determine the coefficient of static friction before a global slip occurs.

Out-of-plane actuation with a sub-micron initial gap for reconfigurable terahertz micro-electro-mechanical systems metamaterials.

We propose a reconfigurable terahertz (THz) metamaterial that can control the transmittance by out-of-plane actuation with changing the sub-micron gap distance between electrically coupled metamaterial elements. By using the out-of-plane actuation, it was possible to avoid contact between the coupled metamaterial elements across the small initial gap during the adjustment of the gap size. THz spectroscopy was performed during actuation, and the transmission dip frequency was confirmed to be tunable from 0.82 to 0.92 THz for one linear polarization state and from 0.80 to 0.91 THz for the other linear polarization; the two polarizations were orthogonal. The proposed approach will contribute to the development of tunable metamaterials based on structural deformations.

Effects of food restriction on toxicological parameters in juvenile rats.

To examine the effects of decreased food consumption on toxicological parameters in juvenile rats, rats on postnatal day 21 were fed 40%, 50% (only four weeks), and 60% less food, compared to that of controls for four or eight weeks, and clinical observations, measurement of body and organ weights, morphological differentiation analysis, clinical pathology, and macroscopic and microscopic examinations were conducted. The body weight decreased depending on the degree of food restriction (FR). Cleavage of the balano-preputial skinfold was delayed, and cell debris in the epididymal lumen was noted as a related finding after four-week FR. Vaginal opening was also delayed, and some histopathological findings, such as absence of corpus luteum in the ovary, mucinous degeneration in the vagina, and immature uterus, were noted after eight-week FR. Erythrocyte count increased after four-week FR, but slightly decreased in males only after eight-week FR, and decreased leukocyte and/or reticulocyte counts, accompanied by related histopathological findings were noted after four- and eight-week FR. In blood chemistry, the levels of total protein including globulin, glucose, triglyceride, and calcium decreased, and sodium and chloride increased after four- and eight-week FR. Increases in activities of aspartate transaminase and lactate dehydrogenase and total bilirubin levels were noted after four-week FR, which were attenuated after eight-week FR. The effects of FR seemed to be more remarkable after four weeks. In drug safety evaluation, findings caused by malnutrition should be considered in juvenile toxicity studies when decreased food consumption is observed.

Measurement method for light transmittance of layered metamaterials.

We propose a method to measure light transmittance of layered metamaterials by placing the metamaterials directly on a Si photodiode. Our measurement method enables the direct detection of transmitted light that appears as an evanescent wave in natural materials. Here, we report the transmittance measurements of a typical metamaterial using this method. The metamaterial was composed of Ag/Al(2)O(3) layers and was fabricated by direct evaporation on the Si photodiode. The measured transmittance agrees with the simulated transmittance. Our results confirmed that this measurement method can determine the transmittance properties of metamaterials and that it is applicable to other types of metamaterials.

Direct physical exfoliation of few-layer graphene from graphite grown on a nickel foil using polydimethylsiloxane with tunable elasticity and adhesion.

We firstly introduce a facile method for the site-specific direct physical exfoliation of few-layer graphene sheets from cheap and easily enlargeable graphite grown on a Ni foil using an optimized polydimethylsiloxane (PDMS) stamp. By decreasing the PDMS cross-linking time, the PDMS elasticity is reduced to ∼52 kPa, similar to that of a typical gel. As a result of this process, the PDMS becomes more flexible yet remains in a handleable state as a stamp. Furthermore, the PDMS adhesion to a graphite/Ni surface, as measured by the peel strength, increases to ∼5.1 N m⁻¹, which is approximately 17 times greater than that of typical PDMS. These optimized properties allow the PDMS stamp to have improved contact with the graphite/Ni surface, including the graphite wrinkles. This process is verified, and changes in surface morphology are observed using a 3D laser scanning microscope. Under conformal contact, the optimized PDMS stamp demonstrates the site-specific direct physical exfoliation of few-layer graphene sheets including mono- and bi-layer graphene sheets from the graphite/Ni substrate without the use of special equipment, conditions or chemicals. The number of layers of the exfoliated graphene and its high quality are revealed by the measured Raman spectroscopy. The exfoliation method using tunable elasticity and adhesion of the PDMS stamp can be used not only for cost-effective mass production of defect-less few-layer graphene from the graphite substrate for micro/nano device arrays but also for nano-contact printing of various structures, devices and cells.

Reduced responsiveness of kisspeptin neurons to estrogenic positive feedback associated with age-related disappearance of LH surge in middle-age female rats.

Age-related disappearance of the LH surge is one of major biomarkers of reproductive aging in female rats. Kisspeptin neurons in the hypothalamic anteroventral periventricular nucleus (AVPV) are proposed as the critical regulator of the preovulatory LH surge in response to estrogenic positive feedback. Here we investigated the possible involvement of the AVPV kisspeptin neurons in the disappearance of the LH surge in middle-age rats. Middle-age rats exhibiting persistent estrus (M-PE) did not show an LH surge although neither Kiss1 mRNA nor peptide in the AVPV was differentially expressed when compared to young rats exhibiting normal estrous cycles (YN). M-PE released LH in response to exogenous kisspeptin in a similar dose-dependent manner as YN, suggesting that their GnRH neurons still maintained responsiveness to kisspeptin. To investigate the estrogenic positive feedback effect on kisspeptin neurons in the AVPV, rats were ovariectomized and supplemented with estradiol (OVX+E2). We performed in situ hybridization and immunohistochemistry for Kiss1 mRNA and cFos, respectively, and found that M-PE exhibited a significantly lower percentage of Kiss1 mRNA positive neurons with cFos immunoreactivity, although the total number of kisspeptin neurons was not different from that in cyclic rats. Furthermore, OVX+E2 M-PE did not show the surge-like LH release under high estradiol administration while YN did. Thus our current study suggests that the reduced responsiveness of the AVPV kisspeptin neurons to estrogenic positive feedback presumably results in the decrease in kisspeptin secretion from neurons and eventually causes the age-related disappearance of the LH surge in middle age female rats.

Ratiometric optical temperature sensor using two fluorescent dyes dissolved in an ionic liquid encapsulated by Parylene film.

A temperature sensor that uses temperature-sensitive fluorescent dyes is developed. The droplet sensor has a diameter of 40 µm and uses 1 g/L of Rhodamine B (RhB) and 0.5 g/L of Rhodamine 110 (Rh110), which are fluorescent dyes that are dissolved in an ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate) to function as temperature indicators. This ionic liquid is encapsulated using vacuum Parylene film deposition (which is known as the Parylene-on-liquid-deposition (PoLD) method). The droplet is sealed by the chemically stable and impermeable Parylene film, which prevents the dye from interacting with the molecules in the solution and keeps the volume and concentration of the fluorescent material fixed. The two fluorescent dyes enable the temperature to be measured ratiometrically such that the droplet sensor can be used in various applications, such as the wireless temperature measurement of microregions. The sensor can measure the temperature of such microregions with an accuracy of 1.9 °C, a precision of 3.7 °C, and a fluorescence intensity change sensitivity of 1.0%/K. The sensor can measure temperatures at different sensor depths in water, ranging from 0 to 850 µm. The droplet sensor is fabricated using microelectromechanical system (MEMS) technology and is highly applicable to lab-on-a-chip devices.

Cyba and Nox2 mutant rats show different incidences of eosinophilia in the genetic background- and sex-dependent manner.

The Matsumoto Eosinophilia Shinshu (MES) is a rat model for hereditary blood eosinophilia. The incidence of eosinophilia is 100% in both female and male MES. The primary cause of the eosinophilia in MES is a loss-of-function mutation in the gene encoding the cytochrome b-245, alpha polypeptide (Cybames mutant allele). CYBA protein is a constituent of the superoxide-generating NADPH oxidase complex, the catalytic subunit of which is either NOX1, NOX2, or NOX4. However, the molecular mechanisms for the loss of CYBA to cause eosinophilia and even which of the three NOX isotypes is causally linked to the disease have been unknown. To resolve the latter issue, we generated F344/N rats knockout for Nox1, Nox2, and Nox4 genes. Also, we bred F344.MES-Cybames congenic rats that have a similar genetic background to the Nox knockout rats. We found that approximately 20% of female F344/N-Nox2em1 rats but none of the males developed blood eosinophilia. Also, we observed that all female F344.MES-Cybames and approximately 50% of male congenic rats developed the disorder. These results revealed that loss of NOX2 is the cause of blood eosinophilia in rats. Meanwhile, the data also indicated that in addition to the loss of NOX2 NADPH oxidase, both the genetic background of F344/N strain and gender influence the development of the disorder. These Nox and Cyba mutant rat strains with different eosinophilia incidences should be useful to elucidate molecular mechanisms and factors involved in the development of the disease.

Optical measurement of directional strain by scattering from nano-disk pairs aligned on an elastomer.

We propose a strain measurement method utilizing light scattered by a pair of nano-disks onto an elastomer sheet. Such nanoparticle pairs exhibit a scattering spectrum that is dependent on the gap distance and the incident light polarization. We utilized this behavior by forming nano-disk pairs with a diameter of 105 nm and a gap of 20-50 nm on a polydimethylsiloxane (PDMS) sheet so that the gap could be altered with strain. The axial direction of the nano-disk pair could be identified by finding the peak wavelength maximum while rotating the polarization angle. Under tensile strain, the peak wavelength decreased from 674 nm at a strain of 0% to 637 nm at a strain of 9.5%, with a peak-shift sensitivity of  - 3.4 nm/% strain. Under compression strain, the peak wavelength increased from 681 nm at a strain of 0% to 705 nm at a strain of  - 3.8%, and the peak-shift sensitivity was  - 16 nm/% strain for strains of 0 to  - 1%. These experimentally determined peak scattering wavelength positions and peak-shift directions are consistent with the simulation results. This method offers wireless measurements of in situ strain on a nanometric scale.

Electrochemical impedance measurement of a carbon nanotube probe electrode.

We measured and analyzed the electrochemical impedance of carbon nanotube (CNT) probe electrodes fabricated through the physical separation of insulated CNT bridges. The fabricated CNT electrodes were free-standing CNTs that were completely covered with an insulator, except for their tips. Typical dimensions of the nanoelectrodes were 1-10 nm in CNT diameter, 80-300 nm in insulator diameter, 0.5-4 µm in exposed CNT length and 1-10 µm in probe length. The electrochemical impedance at frequencies ranging from 40 Hz to 1 MHz was measured in physiological saline. The measured impedance of the CNT electrode was constant at 32 MΩ at frequencies below 1 kHz and was inversely proportional to frequency at frequencies above 10 kHz. By means of comparison with the parasitic capacitive impedance of the insulator membrane, we confirmed that the electrode was sufficiently insulated such that the measured constant impedance was given by the exposed CNT tip. Consequently, we can use the CNT electrode for highly localized electrochemical impedance measurements below 1 kHz. Considering an equivalent circuit and the nanoscopic dimensions of the CNT electrode, we demonstrated that the constant impedance was governed by diffusion impedance, whereas the solution resistance, charge-transfer resistance and double-layer capacitance were negligible.