Direct evidence for ultrastructures of the α-synuclein-associated synaptic vesicle pool in presynaptic terminals.

SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson's disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson's disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.

Ultrastructural analysis of whole glomeruli using array tomography.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.

Protein glycosylation plays a vital role in various cellular functions, many of which occur within the Golgi apparatus. The Golgi pH regulator (GPHR) is essential for the proper functioning of the Golgi apparatus. The lysosomal membrane contains highly glycosylated membrane proteins in abundance. This study investigated the role of the Golgi luminal pH in N-glycosylation of lysosomal membrane proteins and the effect of this protein modification on membrane stability using Gphr-deficient MEFs. We showed that Gphr deficiency causes an imbalance in the Golgi luminal pH, resulting in abnormal protein N-glycosylation, indicated by a reduction in sialylated glycans and markedly reduced molecular weight of glycoproteins. Further experiments using FRAP and PLA revealed that Gphr deficiency prevented the trafficking dynamics and proximity condition of glycosyltransferases in the Golgi apparatus. In addition, incomplete N-glycosylation of lysosomal membrane proteins affected lysosomal membrane stability, as demonstrated by the increased susceptibility to lysosomal damage. Thus, this study highlights the critical role of Golgi pH regulation in controlling protein glycosylation and the impact of Golgi dysfunction on lysosomal membrane stability.

Distribution of peak frequency and omnipolar voltage in electrograms across the atrial body and thoracic veins in a normal heart.

BACKGROUND: The innovative peak frequency mapping facilitates the quantification of electrogram sharpness. However, reference values for normal atrial tissue are currently undefined. In this study, we explored the distribution of peak frequency and omnipolar peak-to-peak voltage (V-max) in a normal heart. METHODS: Twenty-two patients with structurally normal heart were included. Either the right atrium (RA) and superior vena cava (SVC) or the left atrium (LA) and pulmonary veins (PVs) were mapped during sinus rhythm. RESULTS: In total, 13,654 points in the RA and 4143 points in the SVC from 15 patients and 4662 points in the LA and 2761 points in PVs from 7 patients were analyzed. The correlation between peak frequency and V-max was weak (R = 0.223). The median peak frequency was larger in the SVC than in the RA (441 [358-524] Hz vs. 358 [291-441] Hz, P < 0.0001) and in PVs than in the LA (346 [253-441] Hz vs. 323 [262-397] Hz, P < 0.0001). Conversely, the median V-max was smaller in the SVC than in the RA (1.96 [0.77-3.75] mV vs. 4.11 [2.10-6.83] mV, P < 0.0001) and in PVs than in the LA (1.16 [0.33-3.17] mV vs. 4.42 [2.63-6.84] mV, P < 0.0001). More than 95% of peak frequencies were > 174 Hz in the RA and > 185 Hz in the LA, and > 95% of V-maxes were > 0.52 and > 1.07 mV in the RA and LA, respectively. CONCLUSION: Given the limited correlation between peak frequency and V-max, and recognizing their potential to provide distinct information, they can be used complementarily. Employing these parameters to extract varied insights can provide comprehensive understandings of tissue characteristics.

An optimized approach for increasing lesion size in temperature-controled setting using a catheter with a surface thermocouple and efficient irrigation.

Background: We explore an optimized approach for increasing lesion size using a novel ablation catheter with a surface thermocouple and efficient irrigation in a temperature-control setting. Methods: We conducted radiofrequency applications at various power levels (35 W, 40 W, and 45 W), contact forces (CFs, 10 g/20 g), and durations (60 s/120 s/180 s) in perpendicular/parallel catheter orientations, with normal saline irrigation (NS-irrigation) and Half NS-irrigation (HNS-irrigation) in an ex-vivo model (Step 1). In addition, we performed applications (35 W/40 W/45 W for 60 s/120 s/180 s in NS-irrigation and 35 W/40 W for 60 s/120 s/180 s in HNS-irrigation) in four swine (Step 2), evaluating lesion characteristics and the occurrence of steam pops. Results: In Step 1, out of 288 lesions, we observed 47 (16.3%) steam pops, with 13 in NS-irrigation and 34 in HNS-irrigation (p = .001). Although steam pops were mostly observed with the most aggressive setting (45 W/180 s, 54%) with NS-irrigation, they happened in less aggressive settings with HNS irrigation. Lesion size significantly increased with longer-duration ablation but not with HNS-irrigation. The optimal %impedance-drop cutoff to predict steam pops was 20% with a negative-predictive-value (NPV) = 95.1% including NS- and HNS-irrigation groups, and 22% with an NPV = 96.1% in NS-irrigation group. In Step 2, similar to the ex-vivo model, lesion size significantly increased with longer-duration ablation but not with HNS-irrigation. Steam pops were absent with NS-irrigation (0/35) even with the largest %impedance-drop reaching 31% at 45 W/180 s. All steam pops were observed with HNS-irrigation (6/21, 29%). The optimal %impedance-drop cutoff predicting steam pops was 24% with an NPV = 96.3% including both NS- and HNS-irrigation groups. Conclusions: Rather than using HNS-irrigation, very long-duration of radiofrequency applications up to 45 W/180 s may be recommended to safely and effectively increase lesion dimensions using this catheter with NS-irrigation.

Severe behavioral and psychological symptoms of dementia successfully treated at home with a blonanserin transdermal patch: A case report.

AIM: Behavioral psychological symptoms of dementia (BPSD) are sometimes difficult to treat due to severe psychiatric symptoms such as delusions of poisoning and violent behavior. Moreover, in cases of parental neglect, the management of these psychiatric symptoms becomes more difficult. Therefore, home-visiting doctors sometimes have to manage patients with BPSD and severe psychiatric symptoms, and a new approach is needed. In this case report, the effect of blonanserin transdermal patch on these patients is to be highlighted. METHODS: The patient is a 91-year-old woman diagnosed with Alzheimer's disease. She had severe BPSD such as delusion of robbery and violent behavior, and refused oral medications including memantine and yokukansan. Then she was treated with blonanserin transdermal patch (20 mg/day). The severity of psychiatric symptoms of BPSD was assessed over time using the Neuropsychiatric Inventory (NPI) score. Moreover, the patient's cognitive function was also assessed over time by Mini-Mental State Examination (MMSE). RESULTS: After the introduction of blonanserin patch, the patient's psychiatric symptoms were stabilized markedly, and both NPI and MMSE scores improved. The patient was able to stay at home calmly and was mentally well stabilized to the extent that she did not require hospitalization. No apparent side effects were admitted. CONCLUSIONS: The blonanserin transdermal patch may be able to manage BPSD at home and is effective in patients who refuse oral medications. Home-visiting doctors may consider the use of blonanserin patches at home for patients with severe BPSD, manifesting as delusions of poisoning and refusing oral drugs.

Impact of contact force on the lesion characteristics of very high-power short-duration ablation using a QDOT-MICRO catheter.

Background: Lesion size is reported to become larger as contact force (CF) increases. However, this has not been systematically evaluated in temperature-guided very high-power short-duration (vHPSD) ablation, which was therefore the purpose of this study. Methods: Radiofrequency applications (90 W/4 s, temperature-control mode) were performed in excised porcine myocardium with four different CFs of 5, 15, 25, and 35 g using QDOT-MICRO™ catheter. Ten lesions for each combination of settings were created, and lesion metrics and steam-pops were compared. Results: A total of 320 lesions were analyzed. Lesion depth, surface area, and volume were smallest for CF of 5 g than for 15, 25, and 35 g (depth: 2.7 mm vs. 2.9 mm, 3.0 mm, 3.15 mm, p < .01; surface area: 38.4 mm2 vs. 41.8 mm2, 43.3 mm2, 41.5 mm2, p < .05; volume: 98.2 mm3 vs. 133.3 mm3, 129.4 mm3, 126.8 mm3, p < .01 for all pairs of groups compared to CF = 5 g). However, no significant differences were observed between CFs of 15-35 g. Average power was highest for CF of 5 g, followed by 15, 25, and 35 g (83.2 W vs. 82.1 W vs. 77.1 W vs. 66.1 W, p < .01 for all pairs), reflecting the higher incidence of temperature-guided power titration with greater CFs (5 g:8.8% vs. 15 g:52.5% vs. 25 g:77.5% vs. 35 g:91.2%, p < .01 for all pairs except for 25 g vs. 35 g). The incidence of steam-pops did not significantly differ between four groups (5 g:3.8% vs. 15 g:10% vs. 25 g:6.2% vs. 35 g:2.5%, not significant for all pairs). Conclusions: For vHPSD ablation, lesion size does not become large once the CF reaches 15 g, and the risk of steam-pops may be mitigated through power titration even in high CFs.

Effect of reference electrode on intracardiac electrograms: Close indifferent electrode vs Wilson central terminal.

BACKGROUND: Unipolar electrograms (uni-EGMs) are an essential part of intracardiac mapping. Although Wilson central terminal (WCT) is conventionally used as a reference for signals, avoidance of contamination by far-field and nonphysiologic signals is challenging. OBJECTIVE: The aim of the study was to explore the impact of an intracardiac indifferent reference electrode close to the recording electrodes, in lieu of WCT, on electrograms. METHODS: Sinus node activation was mapped in patients undergoing catheter ablation by a multielectrode array with a close indifferent electrode (CIE) embedded in the distal end of the catheter shaft. An equal number of points was sequentially acquired at each site with use of CIE as a reference first and subsequently with WCT. Uni-EGMs, bipolar EGMs, and the earliest activation area (defined as the area activated in the first 10 ms of the beat) were compared between CIE- and WCT-based activation maps. RESULTS: Seventeen patients (61 ± 18 years; 76% male) were studied. Uni-EGM voltages acquired with CIE were significantly larger than (n = 11) or comparable to (n = 4) those acquired with WCT. When points from the entire cohort were analyzed altogether, unipolar voltages and their maximum negative dV/dT and bipolar voltages recorded with CIE were significantly larger than those recorded with WCT (2.36 [1.42-3.79] mV vs 1.96 [1.25-3.03] mV, P < .0001; 0.40 [0.18-0.77] mV/s vs 0.35 [0.15-0.71] mV/s, P < .0001; and 1.46 [0.66-2.81] mV vs 1.33 [0.54-2.64] mV, P < .0001, respectively). The earliest activation area was significantly smaller in CIE-based activation maps than in WCT-based ones (0.3 [0.7-1.4] cm2 vs 0.6 [1.0-1.8] cm2, P = .01). CONCLUSION: CIE-based maps were associated with an approximately 20% increase in unipolar voltage and may highlight the origin of a focal activation more clearly than WCT-based ones.

Safety verification of a novel irrigation catheter with flexible tip of laser-cut kerfs and contact force sensor.

BACKGROUND AND AIMS: The safety evaluation of TactiFlex, a novel contact-force sensing catheter with a flexible 4-mm tip irrigated through laser-cut kerfs, has been ongoing. This study aimed to verify the safety of this type of catheter. METHODS: Study 1: Radiofrequency (RF) applications at a range of powers (30-50 W), contact forces (10-20 g), and durations (10-60 s) using perpendicular/parallel catheter orientation with half-normal (HNS) or normal saline irrigation were compared between TactiFlex (4-mm tip) and TactiCath (3.5-mm tip) with temperature-controlled mode in excised porcine hearts. Study 2: The relation between RF applications using TactiFlex and the incidence of steam-pops in the real clinical cases were examined. RESULTS: Study-1: 576 RF lesions were examined. TactiFlex demonstrated a significantly lower risk of steam-pops (5[1.7%] vs. 59[20.5%], p < .0001). Compared to 3.5-mm-tip catheter (TactiCath), 4-mm-tip catheter (TactiFlex) produced smaller lesion volume at perpendicular (193[98-554]mm3 vs. 263[139-436]mm3 , p < .0001), but relatively similar lesion volume at parallel contact (243[105-443]mm3 vs. 278[180-440]mm3 , p = .06). HNS-irrigation tended to increase the lesion volume in both catheters and to increase the incidence of steam-pops with TactiCath, but not with TactiFlex. The cut-off value of %impedance-drop ( = absolute impedance-drop/initial impedance) of 20% predicted steam-pops with a sensitivity = 100% and specificity = 89.6% in TactiFlex. Study-2: 5496 RF applications in 84 patients (51AFs/8ATs/3AVNRTs/4AVRTs/17PVCs/4VTs) using TactiFlex were analyzed. Four steam-pops (0.07%) in three patients with pericardial effusion were observed (%impedance-drop = 24%/26%/29%/35%, respectively). The cut-off value of %impedance-drop = 20%, derived from ex-vivo study, showed sensitivity = 100% and specificity = 90.1% in detecting steam-pops. CONCLUSION: TactiFlex reduced the risk of steam-pops than TactiCath. %impedance-drop ≤ 20% may be reasonable for safely use with a sufficient safety margin. For 4-mm-tip catheter, parallel-contact may be recommended for larger lesion creation.

The effect of half-normal saline irrigation on lesion characteristics in temperature-flow-controlled ablation.

PURPOSE: Radiofrequency (RF) ablation with half-normal saline (HNS) irrigation is reported to potentially enlarge local lesion compared to normal saline (NS) in power-controlled ablation (PC-Abl). However, the effect of HNS-irrigation in temperature-flow-controlled ablation (TFC-Abl) on lesion characteristics is unknown. We compared this between TFC-Abl with QDOT-Micro™ catheter and PC-Abl with Thermocool SmartTouch SF™ catheter (STSF). METHODS: RF-application with NS (n = 480) and HNS (n = 480) irrigation were performed on swine myocardium placed in a circulating saline bath. Lesion characteristics without steam-pops under various conditions (target AI, 400/550; ablation power, 30/50 W; contact force, 10/20/30 g; catheter orientation, perpendicular/parallel) were assessed and compared between two irrigants. RESULTS: After matching, 343 lesions without steam-pops in each group were evaluated. In PC-Abl, lesion size did not differ between two groups (NS, 188 ± 97 vs. HNS, 200 ± 95 mm3, p = 0.28 in volume; 33.9 ± 7.3 vs. 34.8 ± 9.5 mm2, p = 0.34 in surface area; and 4.0 ± 1.0 vs. 4.0 ± 1.0 mm, p = 0.81 in depth), but steam-pops were more frequently observed with HNS-irrigation (23.8% vs. 37.9%, p = 0.001). Contrary, in TFC-Abl, HNS-irrigation produced significantly larger (214 ± 106 vs. 243 ± 128 mm3, p = 0.017) and deeper (4.0 ± 1.0 vs. 4.3 ± 1.1 mm, p = 0.002) lesions without increasing the risk of steam-pops (15.0% vs 15.0%, p = 0.99). Automatic temperature-guided titration was more frequently observed in HNS-irrigation (54.8% vs. 78.5%, p < 0.001). CONCLUSIONS: TFC-Abl with QDOT-Micro™ catheter utilizing HNS-irrigation might increase volume and depth of local lesion without increasing the risk of stem-pops compared to NS-irrigation. Power-controlled ablation with HNS-irrigation showed similar focal lesion with higher incidence of steam-pops (SPs) compared to normal saline (NS) irrigation. Contrary, temperature-flow-controlled ablation with HNS-irrigation provided larger and deeper lesion than NS-irrigation with similar incidence of SPs. ns, p > 0.05; *, 0.01 < p ≤ 0.05; **, 0.005 < p ≤ 0.01. HNS, half-normal saline; NS, normal saline.

Superiority of the Combination of Input and Output Parameters to the Single Parameter for Lesion Size Estimation.

BACKGROUND: For lesion size prediction, each input parameter, including ablation energy (AE), and output parameter, such as impedance, is individually used. We hypothesize that using both parameters simultaneously may be more optimal.Methods and Results: Radiofrequency applications at a range of power (30-50 W), contact force (10 g and 20 g), duration (10-60 s), and catheter orientation with normal saline (NS)- or half-normal saline (HNS)-irrigation were performed in excised porcine hearts. The correlations, with lesion size of AE, absolute impedance drop (∆Imp-drop), relative impedance drop (%Imp-drop), and AE*%Imp-drop were examined. Lesion size was analyzed in 283 of 288 lesions (NS-irrigation, n=142; HNS-irrigation, n=141) without steam pops. AE*%Imp-drop consistently showed the strongest correlations with lesion maximum depth (NS-irrigation, ρ=0.91; HNS-irrigation, ρ=0.94), surface area (NS-irrigation, ρ=0.87; HNS-irrigation, ρ=0.86), and volume (NS-irrigation, ρ=0.94; HNS-irrigation, ρ=0.94) compared with the other parameters. Moreover, compared with AE alone, AE*%Imp-drop significantly improved the strength of correlation with lesion maximum depth (AE vs. AE*%Imp-drop, ρ=0.83 vs. 0.91, P<0.01), surface area (ρ=0.73 vs. 0.87, P<0.01), and volume (ρ=0.84 vs. 0.94, P<0.01) with NS-irrigation. This tendency was also observed with HNS-irrigation. Parallel catheter orientation showed a better correlation with lesion depth and volume using ∆Imp-drop, %Imp-drop, and AE*%Imp-drop than perpendicular orientation. CONCLUSIONS: The combination of input and output parameters is more optimal than each single parameter for lesion prediction.

Impact of an Electronic Medical Record-Connected Questionnaire on Efficient Nursing Documentation: Usability and Efficacy Study.

BACKGROUND: Documentation tasks comprise a large percentage of nurses' workloads. Nursing records were partially based on a report from the patient. However, it is not a verbatim transcription of the patient's complaints but a type of medical record. Therefore, to reduce the time spent on nursing documentation, it is necessary to assist in the appropriate conversion or citation of patient reports to professional records. However, few studies have been conducted on systems for capturing patient reports in electronic medical records. In addition, there have been no reports on whether such a system reduces the time spent on nursing documentation. OBJECTIVE: This study aims to develop a patient self-reporting system that appropriately converts data to nursing records and evaluate its effect on reducing the documenting burden for nurses. METHODS: An electronic medical record-connected questionnaire and a preadmission nursing questionnaire were administered. The questionnaire responses entered by the patients were quoted in the patient profile for inpatient assessment in the nursing system. To clarify its efficacy, this study examined whether the use of the electronic questionnaire system saved the nurses' time entering the patient profile admitted between August and December 2022. It also surveyed the usability of the electronic questionnaire between April and December 2022. RESULTS: A total of 3111 (78%) patients reported that they answered the electronic medical questionnaire by themselves. Of them, 2715 (88%) felt it was easy to use and 2604 (85%) were willing to use it again. The electronic questionnaire was used in 1326 of 2425 admission cases (use group). The input time for the patient profile was significantly shorter in the use group than in the no-use group (P<.001). Stratified analyses showed that in the internal medicine wards and in patients with dependent activities of daily living, nurses took 13%-18% (1.3 to 2 minutes) less time to enter patient profiles within the use group (both P<.001), even though there was no difference in the amount of information. By contrast, in the surgical wards and in the patients with independent activities of daily living, there was no difference in the time to entry (P=.50 and P=.20, respectively), but there was a greater amount of information in the use group. CONCLUSIONS: The study developed and implemented a system in which self-reported patient data were captured in the hospital information network and quoted in the nursing system. This system contributes to improving the efficiency of nurses' task recordings.

TUNEL-positive structures in activated microglia and SQSTM1/p62-positive structures in activated astrocytes in the neurodegenerative brain of a CLN10 mouse model.

Neuronal ceroid lipofuscinosis is a group of pediatric neurodegenerative diseases. One of their causative genes, CLN10/CtsD, encodes cathepsin D, a major lysosomal protease. Central nervous system (CNS)-specific CtsD-deficient mice exhibit a neurodegenerative disease phenotype with accumulation of ceroid lipofuscins, granular osmiophilic deposits, and SQSTM1/p62. We focused on activated astrocytes and microglia in this neurodegenerative mouse brain, since there are few studies on the relationship between these accumulators and lysosomes in these glial cells. Activated microglia and astrocytes in this mouse thalamus at p24 were increased by approximately 2.5- and 4.6-fold compared with the control, while neurons were decreased by approximately half. Granular osmiophilic deposits were detected in microglial cell bodies and extended their processes in the thalamus. LAMP1-positive lysosomes, but not SQSTM1/p62 aggregates, accumulated in microglia of this mouse thalamus, whereas both lysosomes and SQSTM1/p62 aggregates accumulated in its astrocytes. TUNEL-positive signals were observed mainly in microglia, but few were observed in neurons and astrocytes. These signals were fragmented DNA from degenerated neurons engulfed by microglia or in the lysosomes of microglia. Abnormal autophagic vacuoles also accumulated in the lysosomes of microglia. Granular osmiophilic deposit-like structures localized to LAMP1-positive lysosomes in CtsD-deficient astrocytes. SQSTM1/p62-positive but LAMP1-negative membranous structures also accumulated in the astrocytes and were less condensed than typical granular osmiophilic deposits. These results suggest that CtsD deficiency leads to intracellular abnormalities in activated microglia and astrocytes in addition to neuronal degeneration.

Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells.

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λem ∼550 nm) and far-red (λem ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 µm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.

Impact of baseline pool impedance on lesion metrics and steam pops in catheter ablation.

INTRODUCTION: Little is known about the impact of blood-pool local impedance (LI) on lesion characteristics and the incidence of steam pops. METHODS: Radiofrequency applications at a range of powers (30, 40, and 50 W), contact forces (CF) (5, 15, and 25 g), and durations (15, 30, 45, and 120 s) using perpendicular/parallel catheter orientation were performed in 40 excised porcine preparations, using a catheter capable of monitoring LI (StablePoint©, Boston Scientific). To simulate the variability in blood-pool impedance, the saline-pool LI was modulated by calibrating saline concentrations. Lesion characteristics were compared under three values of saline-pool LI: 120, 160, and 200 Ω. RESULTS: Of 648 lesions created, steam pops occurred in 175 (27.0%). When power, CF, time, and catheter orientation were adjusted, ablation at a saline-pool impedance of 160 or 200 Ω more than doubled the risk of steam pops compared with a saline-pool impedance of 120 Ω (Odds ratio = 2.31; p = .0002). Lesions in a saline-pool impedance of 120 Ω were significantly larger in surface area (50 [38-62], 45 [34-56], and 41 [34-60] mm2 for 120, 160, and 200 Ω, p < .05), but shallower in depth (4.0 [3-5], 4.4 [3.2-5.3], and 4.5 [3.8-5.5] mmfor 120, 160, and 200 Ω, respectively, p < .05) compared with the other two settings. The correlation between the absolute LI-drop and lesion size weakened as the saline-pool LI became higher (e.g., 120 Ω group (r2 = .30, r2 = .18, and r2 = .16, respectively for 120, 160, and 200 Ω), but the usage of %LI-drop (= absolute LI-drop/initial LI) instead of absolute LI-drop may minimize this effect. CONCLUSIONS: In an experimental model, baseline saline-pool impedance significantly affects the lesion metrics and the risk of steam pops.

Recent advances in in-resin correlative light and electron microscopy of Epon-embedded cells.

Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.

Comparison of lesion characteristics using temperature-flow-controlled versus conventional power-controlled ablation with fixed ablation index.

INTRODUCTION: The QDOT-MicroTM catheter is a novel irrigated contact force (CF) sensing catheter which benefits from thermocouples for temperature monitoring, allowing temperature-flow-controlled (TFC) ablation. We compared lesion metrics at fixed ablation index (AI) value during TFC-ablation and conventional power-controlled (PC)-ablation. METHODS: A total of 480 RF-applications were performed on ex-vivo swine myocardium with predefined AI targets (400/550) or until steam-pop occurred, using the QDOT-MicroTM (TFC-ablation) and Thermocool SmartTouch SFTM (PC-ablation). RESULTS: Both TFC-ablation and PC-ablation produced similar lesions in volume (218 ± 116 vs. 212 ± 107 mm3 , p = .65); however, lesions using TFC-ablation were larger in surface area (41.3 ± 8.8 vs. 34.8 ± 8.0 mm2 , p < .001) and shallower in depth (4.0 ± 1.0 vs. 4.2 ± 1.1 mm, p = .044). Average power tended to be lower in TFC-alation (34.2 ± 8.6 vs. 36.9 ± 9.2, p = .005) compared to PC-ablation due to automatic regulation of temperature and irrigation-flow. Although steam-pops were less frequent in TFC-ablation (24% vs. 15%, p = .021), they were particularly observed in low-CF (10 g) and high-power ablation (50 W) in both PC-ablation (n = 24/240, 10.0%) and TFC-ablation (n = 23/240, 9.6%). Multivariate analysis revealed that high-power, low-CF, long application time, perpendicular catheter orientation, and PC-ablation were risk factors for steam-pops. Furthermore, activation of automatic regulation of temperature and irrigation-flow was independently associated with high-CF and long application time while ablation power had no significant relationship. CONCLUSIONS: With a fixed target AI, TFC-ablation reduced the risk of steam-pops, producing similar lesions in volume, but with different metrics in this ex-vivo study. However, lower CF and higher power in fixed-AI ablation may increase the risk of steam-pops.

Comparison of three different approaches to very high-power short-duration ablation using the QDOT-MICRO catheter.

BACKGROUND/OBJECTIVES: The QDOT-MICRO™ catheter allows very high-power and short-duration (vHPSD) ablation. This study aimed to investigate lesion characteristics using different ablation settings. METHODS: Radiofrequency applications (90 W/4 s, temperature-control mode with 55°C or 60°C target) were performed in excised porcine myocardium using three different approaches: single (SA), double nonrepetitive (DNRA), and double repetitive applications (DRA). Applications were performed with an interval of 1 min for DNRA, and without interval for DRA. RESULTS: A total of 480 lesions were analyzed. Lesion depth and volume were largest for DRA followed by DNRA and SA regardless of catheter direction (depth: 3.8 vs. 3.3 vs. 2.6 mm, p < .001 for all comparisons; volume: 176.6 vs. 145.1 vs. 97.0 mm3 , p < .001 for all comparisons). Surface area was significantly larger for DRA than for SA (45.1 vs. 38.3 mm2 , p < .001) and larger for DNRA than for SA (44.5 vs. 38.3 mm2 , p < .001), but was similar between DRA and DNRA (45.1 vs. 44.5 mm2 , p = .54). Steam-pops more frequently occurred for DRA than for SA (15.6% vs. 4.4%, p = .004) and DNRA (15.6% vs. 6.9%, p = .061), but the incidence was similar between SA and DNRA (4.4% vs. 6.9%, p = 1). Although surface area and lesion volume were larger in lesions with steam-pops than without steam-pops (46.5 vs. 38.1 mm2 , p = .018 and 128.3 vs. 96.8 mm3 , p = .068, respectively), lesions were not deeper (pop(+): 2.5 mm vs. pop(-): 2.6 mm, p = .75). CONCLUSIONS: DNRA produces larger lesions than SA without increasing the risk of steam-pops. DRA produces the largest lesions among the three groups, but with an increased risk of steam-pops. Even with steam-pops, lesions do not become deeper in vHPSD ablation.

Osmium-Resistant Fluorescent Proteins and In-Resin Correlative Light-Electron Microscopy of Epon-Embedded Mammalian Cultured Cells.

Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins.