MRI Findings of Immune Checkpoint Inhibitor-Induced Hypophysitis: Possible Association with Fibrosis.

BACKGROUND AND PURPOSE: Hypophysitis is one of the well-known adverse effects of immune checkpoint inhibitors. Immune checkpoint inhibitor-induced hypophysitis frequently causes irreversible hypopituitarism, which requires long-term hormone replacement. Despite the high frequency and clinical significance, characteristic MR imaging findings of immune checkpoint inhibitor-induced hypophysitis have not been established. In the present study, we aimed to review and extract the MR imaging features of immune checkpoint inhibitor-induced hypophysitis. MATERIALS AND METHODS: This retrospective international multicenter study comprised 20 patients with melanoma who were being treated with immune checkpoint inhibitors and clinically diagnosed with immune checkpoint inhibitor-induced hypophysitis. Three radiologists evaluated the following MR imaging findings: enlargement of the pituitary gland and stalk; homogeneity of enhancement of the pituitary gland; presence/absence of a well-defined poorly enhanced area and, if present, its location, shape, and signal intensity in T2WI; and enhancement pattern in contrast-enhanced dynamic MR imaging. Clinical symptoms and hormone levels were also recorded. RESULTS: Enlargement of the pituitary gland and stalk was observed in 12 and 20 patients, respectively. Nineteen patients showed poorly enhanced lesions (geographic hypoenhancing lesions) in the anterior lobe, and 11 of these lesions showed hypointensity on T2WI. Thyrotropin deficiency and corticotropin deficiency were observed in 19/20 and 12/17 patients, respectively, which persisted in 12/19 and 10/12 patients, respectively, throughout the study period. CONCLUSIONS: Pituitary geographic hypoenhancing lesions in the anterior lobe of the pituitary gland are characteristic and frequent MR imaging findings of immune checkpoint inhibitor-induced hypophysitis. They reflect fibrosis and are useful in distinguishing immune checkpoint inhibitor-induced hypophysitis from other types of hypophysitis/tumors.

Low prevalence of maternal microchimerism in peripheral blood of Japanese children with type 1 diabetes.

AIM: To clarify the prevalence and degree of maternal microchimerism in Japanese children with type 1 diabetes, as well as its effect on phenotypic variation. METHODS: We studied 153 Japanese children with type 1 diabetes, including 124 children positive for ß-cell autoantibodies, and their 71 unaffected siblings. The number of circulating microchimeric cells per 105 host cells was estimated by the use of quantitative-polymerase chain reaction targeting non-transmitted maternal human leukocyte antigen alleles. The results were compared to previous data from white European people. Phenotypic comparison was performed between maternal microchimerism carriers and non-carriers with diabetes. RESULTS: Maternal microchimerism was detected in 15% of children with autoantibody-positive type 1 diabetes, 28% of children with autoantibody-negative type 1 diabetes, and 16% of unaffected siblings. There were no differences in the prevalence or levels of maternal microchimerism among the three groups or between the children with type 1 diabetes and their unaffected siblings. Furthermore, maternal microchimerism carriers and non-carriers exhibited similar phenotypes. CONCLUSIONS: Maternal microchimerism appears to be less common in Japanese children with type 1 diabetes than in white European people. Our data indicate that maternal microchimerism is unlikely to be a major trigger or a phenotypic determinant of type 1 diabetes in Japanese children and that the biological significance of maternal microchimerism in type 1 diabetes may differ among ethnic groups.

Estimation of glycaemic control in the past month using ratio of glycated albumin to HbA1c.

AIMS: To evaluate comprehensively the use of the glycated albumin to HbA1c ratio for estimation of glycaemic control in the previous month. METHODS: A total of 306 children with Type 1 diabetes mellitus underwent ≥10 simultaneous measurements of glycated albumin and HbA1c . Correlation and concordance rates were examined between HbA1c measurements taken 1 month apart (ΔHbA1c ) and glycated albumin/HbA1c ratio fluctuations were calculated as Z-scores from the cohort value at enrolment of this study cohort (method A) or the percent difference from the individual mean over time (method B). RESULTS: Fluctuations in glycated albumin/HbA1c ratio (using both methods) were weakly but significantly correlated with ΔHbA1c , whereas concordance rates were significant for glycaemic deterioration but not for glycaemic improvement. Concordance rates were higher using method B than method A. CONCLUSIONS: The glycated albumin/HbA1c ratio was able to estimate glycaemic deterioration in the previous month, while estimation of glycaemic improvement in the preceding month was limited. Because method B provided a better estimate of recent glycaemic control than method A, the individual mean of several measurements of the glycated albumin/HbA1c ratio over time may also identify individuals with high or low haemoglobin glycation phenotypes in a given population, such as Japanese children with Type 1 diabetes, thereby allowing more effective diabetes management.

Protein-altering variants of PTPN2 in childhood-onset Type 1A diabetes.

AIM: To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. METHODS: PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. RESULTS: One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. CONCLUSIONS: The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.

FUT2 non-secretor status is associated with Type 1 diabetes susceptibility in Japanese children.

AIM: To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. METHODS: We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. RESULTS: The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. CONCLUSIONS: The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children.

Variants associated with autoimmune Type 1 diabetes in Japanese children: implications for age-specific effects of cis-regulatory haplotypes at 17q12-q21.

AIMS: The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. METHODS: We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. RESULTS: Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5 ; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. CONCLUSIONS: The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

Noradrenergic modulation of vicarious trial-and-error behavior during a spatial decision-making task in rats.

Deliberation between possible options before making a decision is crucial to responding with an optimal choice. However, the neural mechanisms regulating this deliberative decision-making process are still unclear. Recent studies have proposed that the locus coeruleus-noradrenaline (LC-NA) system plays a role in attention, behavioral flexibility, and exploration, which contribute to the search for an optimal choice under uncertain situations. In the present study, we examined whether the LC-NA system relates to the deliberative process in a T-maze spatial decision-making task in rats. To quantify deliberation in rats, we recorded vicarious trial-and-error behavior (VTE), which is considered to reflect a deliberative process exploring optimal choices. In experiment 1, we manipulated the difficulty of choice by varying the amount of reward pellets between the two maze arms (0 vs. 4, 1 vs. 3, 2 vs. 2). A difficulty-dependent increase in VTE was accompanied by a reduction of choice bias toward the high reward arm and an increase in time required to select one of the two arms in the more difficult manipulation. In addition, the increase of c-Fos-positive NA neurons in the LC depended on the task difficulty and the amount of c-Fos expression in LC-NA neurons positively correlated with the occurrence of VTE. In experiment 2, we inhibited LC-NA activity by injection of clonidine, an agonist of the alpha2 autoreceptor, during a decision-making task (1 vs. 3). The clonidine injection suppressed occurrence of VTE in the early phase of the task and subsequently impaired a valuable choice later in the task. These results suggest that the LC-NA system regulates the deliberative process during decision-making.

Role of surgery, radiotherapy and chemotherapy in papillary tumors of the pineal region: a multicenter study.

Papillary tumor of the pineal region (PTPR), recently described as a distinct clinicopathological entity, can show aggressive biological behavior. The optimal therapeutic approach of PTPR has not been well defined. The role of surgery, radiotherapy, and chemotherapy in the treatment of PTPR was analyzed in a large multicenter series. In order to determine factors that influence prognosis, outcome data of a series of 44 patients with histopathologically proven PTPR were retrospectively analyzed. Of the 44 patients, 32 were still alive after a median follow-up of 63.1 months. Twelve patients experienced progressive disease, with seven undergoing two relapses and five more than two. Median overall survival (OS) was not achieved. Median progression-free survival (PFS) was 58.1 months. Only gross total resection and younger age were associated with a longer OS, radiotherapy and chemotherapy having no significant impact. PFS was not influenced by gross total resection. Radiotherapy and chemotherapy had no significant effect. This retrospective series confirms the high risk of recurrence in PTPR and emphasizes the importance of gross total resection. However, our data provide no evidence for a role of adjuvant radiotherapy or chemotherapy in the treatment of PTPR.

Nocturnal blood glucose and IGFBP-1 changes in type 1 diabetes: Differences in the dawn phenomenon between insulin regimens.

OBJECTIVE: Insulin-like growth factor binding protein-1 (IGFBP-1) is known to regulate the bioavailability of insulin-like growth factor (IGF) and the levels of IGFBP-1 are increased in the morning in patients with type 1 diabetes mellitus. We investigated the nocturnal fluctuations of glucose, IGFBP-1, and free IGF-1 levels with three insulin regimens. RESEARCH DESIGN AND METHODS: Forty-eight type 1 diabetes patients were divided into three groups according to their basal insulin therapy (continuous subcutaneous insulin infusion [CSII], insulin glargine, NPH insulin). Blood samples were obtained every 2 h between 2 300 h and 0700 h to measure plasma glucose, IGFBP-1 and free IGF-1 levels. RESULTS: The dawn phenomenon was more frequent with NPH (62.1%) than with glargine (16.6%, p<0.05) and CSII (14.3%, p<0.05). In the NPH group, the serum IGFBP-1 levels were markedly increased from 21.0+/-3.6 ng/ml at 2 300 h to 200.3+/-21.8 ng/ml at 0700 h and free IGF-1 levels were inversely decreased; these changes were partially suppressed in the CSII and glargine groups. CONCLUSIONS: The use of insulin regimens that provide sufficient insulin levels in the early morning can suppress the dawn phenomenon, leading to improved glycemic control. The increase in circulating IGFBP-1 in the morning, as a result of waning of insulin action, lowers free IGF-1 levels and may cause insulin resistance.

Short-term plasticity of central benzodiazepine receptors in status epilepticus: case report.

(123)I-iomazenil SPECT is of value in determining an epileptogenic focus, however, transient uptake change has been rarely reported in epileptic disorders. A 78-year-old woman diagnosed as status epilepticus (SE) showed transient reduction in (123)I-iomazenil uptake within the epileptic foci on SPECT images during a couple of weeks. It suggests a seizure-related 'short-term' plasticity in the central benzodiazepine receptors and dynamic change in the regulatory mechanisms of inhibitory neurotransmitter system within the epileptic foci in patients with SE.

Malignant transformation of endometriosis and genetic alterations of K-ras and microsatellite instability.

OBJECTIVES: To clarify the role of specific genetic alterations in the multi-step process of malignant transformation of endometriosis. METHODS: In cases of ovarian endometrioid carcinoma, we separated regions of normal endometriosis, atypical endometriosis and ovarian endometrioid carcinoma by laser microdissection, and examined K-ras mutation and microsatellite instability in each separated tissue sample. RESULTS: We detected K-ras mutation and microsatellite instability in endometrioid carcinoma tissue, but not in normal or atypical endometriosis bordering the cancerous region. CONCLUSIONS: The present findings suggest that K-ras mutation and microsatellite instability are associated with malignant transformation from atypical endometriosis to ovarian endometrioid carcinoma.

Detection of p53 mutations in the plasma DNA of patients with ovarian cancer.

Mutation of p53 is one of the most common genetic abnormalities detected in up to 81% of cases of ovarian cancer. To evaluate the use of plasma DNA analysis as a method for somatic mutation screening, we measured the presence of p53 mutations in DNA isolated from plasma and cancer tissue from patients with ovarian cancer. We analyzed the plasma DNA for the presence of p53 mutations (exons 5-8). Of 27 cases of ovarian cancer, 12 cases (44%) had mutations of p53 in cancer tissue. In two of the 12 cases (16.7%), identical mutations were detected in DNA of their preoperative plasma. In our follow-up of the two patients with p53 mutations in their plasma, mutant DNA was undetectable in their plasma after surgery. In one case, the p53 mutation re-surfaced in their plasma 16 months after surgery, and the patient died 2 months later. We have shown that tumor-derived DNA can be detected in the plasma of some patients with ovarian cancer, particularly in those with more advanced stage.

A case of micrographia after subcortical infarction: possible involvement of frontal lobe function.

Most reports of micrographia associated with focal brain lesions have related this finding to damage in the left basal ganglia. Here we describe the case of a 68-year-old man presenting with reversible micrographia accompanied by hypophonia in the absence of extrapyramidal signs after cerebral infarction in the left subcortical region. At the time of the patient's admission, diffusion-weighted magnetic resonance imaging sequence showed the lesion to principally involve the corona radiata, with some involvement of the putamen. Neurologically, mild right-sided brachiofacial hemiparesis and grasp reflexes - a frontal lobe sign - were observed. As his micrographia and hypophonia improved, the patient's grasp reflexes improved in parallel. In addition, recovery of regional cerebral blood flow in the left frontal lobe was confirmed by single photon emission computed tomography (technetium-99 m HMPAO). The present case suggests the possibility that the function of frontal-subcortical circuit might also be involved in the production and improvement of micrographia and that micrographia and hypophonia may share a common pathophysiology.

Mild hypothermia enhances the neuroprotective effects of a selective thrombin inhibitor following transient focal ischemia in rats.

The aim of this study is to determine whether a selective thrombin inhibitor, Argatroban, would prevent neuronal cell death and whether extra-mild hypothermia (35 degrees C) would enhance the neuroprotective effect of a selective thrombin inhibitor following transient focal ischemia in rats. Sprague-Dawley rats were subjected to MCAo using an intraluminal suture technique for 2 hrs. The rats were reperfused for 24 h and decapitated for infarct and edema analysis. Argatroban-treated animals received a continuous injection of argatroban (3.0 mg/kg) for 24 hrs after onset of ischemia, while vehicle-treated groups received same dose of vehicle. During ischemia, temporal muscle and rectal temperatures were monitored and maintained at 37 degrees C in the normothermic animals and at 35 degrees C in the hypothermic animals. Argatroban ameliorated the cortical ischemic damage significantly (p < 0.05). Moreover, argatroban with mild hypothermia decreased the cortical infarct or edema volume significantly compared with those of groups I and III (p < 0.05). Argatroban improved neurological symptoms significantly and also improved survival rate. These results demonstrate that extra-mild hypothermia (35 degrees C) enhances neuroprotective effects of a selective thrombin inhibitor, argatroban, suggesting that this combined therapy may be a new therapeutic strategy for the treatment of acute stroke.

The neuroprotective effect of a free radical scavenger and mild hypothermia following transient focal ischemia in rats.

Edaravone, a novel free radical scavenger, has been reported to reduce ischemic damage in rats subjected to transient focal ischemia. The aim of this study is, therefore, to investigate the effect of a combined therapy with edaravone and mild hypothermia of 35 degrees C. Sprague-Dawley rats were subjected to MCA occluding an intraluminal suture technique for 2 hrs. The rats were reperfused for 24 h and decapitated for infarct and edema analysis. Animals were randomly devided into four groups: (I) vehicle + normothermia (control) (II) vehicle + mild hypothermia (III) Edaravone + normothermia (IV) Edaravone + mild hypothermia. Mild hypothermia alone had no reduction of the brain damage. The edaravone alone significantly reduced edema volume. The combined treatment with edaravone and mild hypothermia reduced both infarct and edema volume. In addition, this treatment provided for the best functional outcome. These results demonstrate that free radical scavenger, edaravone attenuates brain edema and that the combined therapy with edaravone and mild hypothermia significantly reduces not only edema but also infarct on transient focal cerebral ischemia in rats. The neuroprotective effects seen in this study may be due to the combined interaction of antiedema activity between edaravone and mild hypothermia, suppressing free radical production.

Cerebral blood flow index image as a simple indicator for the fate of acute ischemic lesion.

BACKGROUND AND PURPOSE: To evaluate the feasibility of utilizing cerebral blood flow (CBF) index images, we attempted to investigate 1) whether CBF index images can reveal the resulting infracted area, 2) whether the CBF index can correlate other modality (SPECT). METHODS: DWI and DPI were obtained in 17 patients within 12 hours of stroke onset and follow up MRI. On three DPI delivered images, namely relative regional cerebral blood volume (rrCBV), uncorrected mean transit time (MTTu) and CBF index images, correlations between initial lesion volume of and follow up infarction volume of three images and rCBF images delivered with singular value decomposition (SVD) methods were assessed. Then 99mTc-ECD SPECT was taken immediately after MRI to correlate to MRI data. RESULTS: Among the three images, lesion volume of CBF index images against follow up infarct volume had the highest correlation (r = 0.995) to a linear fit and the slope was closest to 1.0 (0.91) and had identical accuracy to the regression coefficient of rCBF images. CBF index well correlated to SPECT delivered CBF. CONCLUSION: CBF index images can accurately predict final infarct volume. Evaluating CBF index images together with DWI can guide the initial assessment in the acute stage of cerebral ischemia.

Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side.

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.

Influence of natural, electrically neutral lipids on the potentiometric responses of cation-selective polymeric membrane electrodes.

Ionophore-free ion exchanger electrodes were found to exhibit quite a high selectivity for the creatininium ion; however, measurements in diluted urine samples revealed large emf drifts. Potentiometric, chromatographic, NMR, and mass spectrometric evidence did not reveal any major cationic interfering agents, and anionic interfering agents cannot trivially explain the consistently positive emf drifts. Ultrafiltration of urine samples showed that the interfering agents have molecular weights below 1000 u. The drifts are apparently caused by electrically neutral lipophilic compounds of low molecular weight that are easily extracted into organic phases. Follow-up experiments showed that p-cresol and cholesterol cause no significant emf responses but that coproporphyrin, phosphatidylserine, taurocholic acid, cholic acid, phosphatidylethanolamine, and octanoic acid cause positive emf drifts of the type that was observed with the urine samples. The extent of the responses and the response time depend not only on the specific compound but also on the cation in the sample solution. These results suggest that the emf drifts are due to extraction of such natural lipids into the organic membrane phase where they interact in an ionophore-like fashion with the analyte and interfering ions. Changes in the potentiometric selectivities after contact with natural lipids support this interpretation. The same effect of natural lipids is also expected for ionophore-based electrodes. Indeed, exposure of a valinomycin-based electrode to a methylene chloride extract of urine resulted in a significant reduction of the Na+ discrimination, increasing log Kpot(K,Na) from -3.9 to -3.1.

Micromere descendants at the blastula stage are involved in normal archenteron formation in sea urchin embryos.

Several lines of evidence suggest that micromere signaling plays a key role in endo-mesoderm differentiation along the animal-vegetal (A-V) axis in sea urchin embryos. A recent study has suggested that the activity of micromeres of inducing endoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula stage in the echinoids Scaphechinus mirabiris and Hemicentrotus pulcherrimus. In the present study, to confirm the inductive capacity of the micromere descendants in normal development, the timing of initiation of gastrulation and the elongation rate of the archenteron were examined in both micromereless embryos and in micromereless embryos cultured until the hatching blastula stage and then recombined with micromere descendants of the same age. The micromereless embryos consistently exhibited a delay in the initiation of gastrulation and a decrease in elongation rate of the archenteron, as compared with those in controls. In contrast, when the micromereless embryos cultured until the hatching blastula stage were recombined with micromere descendants of the same age, the recombinant embryos exhibited rescue of both the delay in initiation of gastrulation and a decrease in elongation rate of the archenteron. The delayed expression of alkaline phosphatase activity, an endoderm-specific marker, in the micromereless embryos was also rescued in the recombinant embryos. The recombined micromere descendants formed the larval spicules in the same schedule as that observed in the controls. These results indicate that at the hatching blastula stage, micromere descendants emanate a signal(s) required for normal gastrulation of the presumptive endo-mesodermal region.

Scanning electrochemical microscopy. 41. Theory and characterization of ring electrodes.

Ring ultramicroelectrodes, which are of particular interest as probes for scanning electrochemical microscopy (SECM), combined with near-field scanning optical microscopy, were investigated. Theoretical SECM tip current-distance (approach) curves for a ring electrode were calculated by numerical (finite element) analysis. The SECM curves obtained were a function of the geometry of the tips including the thickness of the ring and the insulating sheath. Theoretical approach curves over conductive substrates showed a strong dependence on the ratio of inner to outer radii of ring microelectrodes (a/b) and were relatively insensitive to the thickness of the insulating sheath (r(g)). For insulating substrates, however, the approach curves varied significantly with r(g), but much less with the a/b ratio. Comparison of experimental and theoretical SECM curves provided a good method of evaluating the size and shape of ring electrodes. Good agreement of the experimental and theoretical curves was found with a ring microelectrode with a nominal 200-nm ring thickness, yielding values of 1.7, 1.9, and 5.7 microm for the inner (a) and outer (b) radii of a ring and the outermost radius of insulating sheath (r(g)), respectively.