Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice.

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons.

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-KATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15 s reperfusion and 15 s re-ischemia, and was started 30 s after the 7.5 min ischemic load. We recorded N-methyl-D-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca2+ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-KATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca2+ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca2+ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-KATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

Melatonin-Induced Postconditioning Suppresses NMDA Receptor through Opening of the Mitochondrial Permeability Transition Pore via Melatonin Receptor in Mouse Neurons.

Mitochondrial membrane potential regulation through the mitochondrial permeability transition pore (mPTP) is reportedly involved in the ischemic postconditioning (PostC) phenomenon. Melatonin is an endogenous hormone that regulates circadian rhythms. Its neuroprotective effects via mitochondrial melatonin receptors (MTs) have recently attracted attention. However, details of the neuroprotective mechanisms associated with PostC have not been clarified. Using hippocampal CA1 pyramidal cells from C57BL mice, we studied the involvement of MTs and the mPTP in melatonin-induced PostC mechanisms similar to those of ischemic PostC. We measured changes in spontaneous excitatory postsynaptic currents (sEPSCs), intracellular calcium concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents after ischemic challenge, using the whole-cell patch-clamp technique. Melatonin significantly suppressed increases in sEPSCs and intracellular calcium concentrations. The NMDAR currents were significantly suppressed by melatonin and the MT agonist, ramelteon. However, this suppressive effect was abolished by the mPTP inhibitor, cyclosporine A, and the MT antagonist, luzindole. Furthermore, both melatonin and ramelteon potentiated depolarization of mitochondrial membrane potentials, and luzindole suppressed depolarization of mitochondrial membrane potentials. This study suggests that melatonin-induced PostC via MTs suppressed the NMDAR that was induced by partial depolarization of mitochondrial membrane potential by opening the mPTP, reducing excessive release of glutamate and inducing neuroprotection against ischemia-reperfusion injury.

Lipid Core Burden Index Assessed by Near-Infrared Spectroscopy of Symptomatic Carotid Plaques: Association with Magnetic Resonance T1-Weighted Imaging.

INTRODUCTION: Vulnerable plaques are a strong predictor of cerebrovascular ischemic events, and high lipid core plaques (LCPs) are associated with an increased risk of embolic infarcts during carotid artery stenting (CAS). Recent developments in magnetic resonance (MR) plaque imaging have enabled noninvasive assessment of carotid plaque vulnerability, and the lipid component and intraplaque hemorrhage (IPH) are visible as high signal intensity areas on T1-weighted MR images. Recently, catheter-based near-infrared spectroscopy (NIRS) has been shown to accurately distinguish LCPs without IPH. This study aimed to determine whether the results of assessment of high LCPs by catheter-based NIRS correlate with the results of MR plaque imaging. METHODS: We recruited 82 consecutive symptomatic carotid artery stenosis patients who were treated with CAS under NIRS and MR plaque assessment. Maximum lipid core burden index (max-LCBI) at minimal luminal areas (MLA), defined as max-LCBIMLA, and max-LCBI for any 4-mm segment in a target lesion, defined as max-LCBIAREA, were assessed by NIRS. Correlations were investigated between max-LCBI and MR T1-weighted plaque signal intensity ratio (T1W-SIR) and MR time-of-flight signal intensity ratio (TOF-SIR) in the same regions as assessed by NIRS. RESULTS: Both T1W-SIRMLA and T1W-SIRAREA were significantly lower in the high LCP group (max-LCBI >504, p < 0.001 for both), while TOF-SIRMLA and TOF-SIRAREA were significantly higher in the high LCP group (p < 0.001 and p = 0.004, respectively). A significant linear correlation was present between max-LCBIMLA and both TIW-SIRMLA and TOF-SIRMLA (r = -0.610 and 0.452, respectively, p < 0.0001 for both). Furthermore, logistic regression analysis revealed that T1W-SIRMLA and TOF-SIRMLA were significantly associated with a high LCP assessed by NIRS (OR, 44.19 and 0.43; 95% CI: 6.55-298.19 and 0.19-0.96; p < 0.001 and = 0.039, respectively). CONCLUSIONS: A high LCP assessed by NIRS correlates with the signal intensity ratio of MR imaging in symptomatic patients with unstable carotid plaques.

Three-dimensional reconstruction imaging by C-arm computed tomography accurately visualizes in-stent neointimal formation in patients with stent-assisted coil embolization.

Background: Stent apposition to the vessel wall and in-stent neointimal formation after stent-assisted coil embolization for intracranial aneurysm are important factors associated with postoperative thromboembolic complications. No assessment methods have been established to depict 3-dimensional (3D) all-round in-stent neointimal formation. Objective: To demonstrate the superiority of Dyna-3D imaging assessment as a modality for all-round ISNF in comparison with conventional two-dimensional digital subtraction angiography (2D-DSA). Methods: Consecutive patients who underwent braided stent-assisted coil embolization for unruptured aneurysm between November 2016 and September 2021 were enrolled. Radiological assessments for stent apposition to the parent vessel after stent deployment and in-stent neointimal formation after 3 months were obtained. Dyna-3D was reconstructed by overlapping a plain image showing stent struts with a rotational DSA image showing the vessel lumen. Reconstructed Dyna-3D images can be rotated to any angle on the screen to evaluate to stent apposition around the vessel and in-stent neointimal formation in 3D, for comparison with 2D-DSA evaluations. Results: Among the 73 patients enrolled, 70 patients (96%) showed complete stent wall apposition on Dyna-3D. Higher intra-rater agreement was confirmed on assessment of in-stent neointimal formation with Dyna-3D (Cohen's κ = 0.811) than with conventional 2D-DSA (Cohen's κ = 0.517). in-stent neointimal formation could not be confirmed on conventional imaging in 9 cases (16%) and on Dyna-3D in 2 cases (3%). The number of in-stent neointimal formations rated as stent wire completely outside the endothelial line was significantly higher with Dyna-3D than with 2D-DSA (p = 0.0001). Conclusion: All-round 3D evaluation by Dyna-3D imaging appears useful for confirming in-stent neointimal formation after braided stent deployment in patients after stent-assisted coil embolization.

Dual-layered stents reduce cerebral embolism compared with first-generation stents during carotid stenting of high lipid core plaque lesions.

BACKGROUND: Periprocedural lipid core plaque (LCP) has been detected in carotid arteries assessed by catheter-based near-infrared spectroscopy (NIRS). High LCP is associated with cerebral embolism after carotid artery stenting (CAS) using a first-generation stent. We aimed to evaluate whether dual-layered stents reduce embolic infarcts in patients with high LCP and change of lipid signal as assessed by NIRS during CAS. METHODS: Participants comprised 210 consecutive patients undergoing CAS. The study was divided into two distinct periods, with first-generation closed-cell stents used in the earlier period and dual-layered stents used in the later period. NIRS was performed at baseline, after stent implantation, and after balloon post-dilatation to analyze maximal lipid core burden index at minimal luminal area (max-LCBIMLA). RESULTS: The ipsilateral cerebral embolism rate was significantly lower with dual-layered stents (9%) than with first-generation stents (33%, p<0.001), particularly with highly lipidic lesions (12% vs 60%, p<0.001). On multivariate logistic regression analysis, high LCP and first-generation stent usage were factors related to ipsilateral cerebral embolism (both p<0.001; OR 8.28 (95% CI 3.49 to 19.64) and OR 8.07 (95% CI 2.33 to 27.93), respectively). Max-LCBIMLA decreased significantly after stenting in both groups (both p<0.01) and max-LCBIMLA after balloon post-dilatation was significantly lower with dual-layered stents (22.4±65.6) than with first-generation stents (124.2±208.2; p=0.006). CONCLUSIONS: Dual-layered stents reduce embolic infarcts in patients with highly lipidic plaque lesions as assessed by NIRS who undergo CAS. Dual-layered stents significantly reduced NIRS-derived lipid signals after stenting.

Long-Term Survival after Linac-Based Stereotactic Radiosurgery and Radiotherapy with a Micro-Multileaf Collimator for Brain Metastasis.

BACKGROUND: this study aimed to evaluate the prognostic factors associated with long-term survival after linear accelerator (linac)-based stereotactic radiosurgery (SRS) and fractionated stereotactic radiotherapy (fSRT) with a micro-multileaf collimator for brain metastasis (BM). METHODS: This single-center retrospective study included 226 consecutive patients with BM who were treated with linac-based SRS or fSRT with a micro-multileaf collimator between January 2011 and December 2018. Long-term survival (LTS) was defined as survival for more than 2 years after SRS/fSRT. RESULTS: The tumors originated from the lung (n = 189, 83.6%), breast (n = 11, 4.9%), colon (n = 9, 4.0%), stomach (n = 4, 1.8%), kidney (n = 3, 1.3%), esophagus (n = 3, 1.3%), and other regions (n = 7, 3.1%). The median pretreatment Karnofsky performance scale (KPS) score was 90 (range: 40-100). The median follow-up time was 13 (range: 0-120) months. Out of the 226 patients, 72 (31.8%) were categorized in the LTS group. The median survival time was 43 months and 13 months in the LTS group and in the entire cohort, respectively. The 3-year, 4-year, and 5-year survival rate in the LTS group was 59.1%, 49.6%, and 40.7%, respectively. Multivariate regression logistic analysis showed that female sex, a pre-treatment KPS score ≥ 80, and the absence of extracranial metastasis were associated with long-term survival. CONCLUSIONS: female sex, a favorable pre-treatment KPS score, and the absence of extracranial metastasis were associated with long-term survival in the current cohort of patients with BM.

Thoracolumbar intraosseous spinal epidural arteriovenous fistulas after vertebral compression fracture: A case report and literature review.

BACKGROUND: The pathophysiology of spinal epidural arteriovenous fistulas (SEAVFs) with perimedullary venous drainage remains to be elucidated. This report describes a case of intraosseous SEAVF in a patient with a history of a thoracolumbar vertebral fracture at the same level 10 years before presenting with progressive myelopathy secondary to retrograde venous reflux into the perimedullary vein. CASE DESCRIPTION: A 71-year-old man presenting with progressive paraparesis was diagnosed with a SEAVF involving a previous Th12 and L1 vertebral compression fracture on which feeders from multiple segmental arteries converged. The interesting feature of this case was that the fistula was located in the fractured vertebral body. The fistula was totally obliterated by transarterial embolization of the segmental arteries followed by symptom improvement. CONCLUSION: We presented a rare case of an intraosseous SEAVF secondary to a thoracolumbar compression fracture with perimedullary venous reflux causing progressive myelopathy. The fistula was located in the fractured vertebral body.

Active management of the target P2Y12 reaction unit range in patients undergoing stent-assisted coil embolization for unruptured cerebral aneurysms.

BACKGROUND: Platelet function tests have been increasingly adopted to measure patient responses to antiplatelet drugs, and to predict complications. However, no established optimal antiplatelet management for stent-assisted coil embolization (SAC) have been established. The purpose of the present study was to investigate the efficacy and feasibility of clopidogrel dose adjustment for active target P2Y12 reaction unit (PRU). METHODS: A total of 202 consecutive patients undergoing SAC to treat unruptured intracranial aneurysms were prospectively recruited. All patients were given two antiplatelet agents starting 7 days prior to the procedure, and platelet function was measured with the VerifyNow test. Clopidogrel hyper-responsive patients received reduced dosing according to the values of follow-up PRUs before and 7, 14, 30, and 90 days after the procedure. Patients were divided into three groups according to clopidogrel responsiveness before treatment, and clinical outcomes and time in target PRU ranges (TTR) were analyzed. RESULTS: No delayed ischemic or hemorrhagic events occurred that were associated with out-of-range PRU. PRU values in the hypo-responsive and hyper-responsive groups significantly improved 7 days after treatment with active target PRU management (p=0.05,<0.001, respectively). PRU values were controlled within the target PRU range with drug adjustment (p=0.034), and the time in TTR for all patients was 97% (4.8%-100%), which showed the feasibility of optimal control of PRU values with the protocol. CONCLUSION: Active target PRU management can achieve control of optimal PRU values and may decrease perioperative ischemic and hemorrhagic events among patients undergoing SAC.

Near-infrared spectroscopy carotid plaque characteristics and cerebral embolism in carotid artery stenting.

BACKGROUND: Perioperative thromboembolism is the main consideration in carotid artery stenting (CAS). Precise evaluation of carotid plaque components is clinically important to reduce ischaemic complications since CAS mechanically pushes plaque outwards, which releases plaque debris into the bloodstream. AIMS: This study aimed to determine whether high lipid core plaque (LCP) assessed by catheter-based near-infrared spectroscopy (NIRS) is associated with ipsilateral cerebral embolism by diffusion-weighted magnetic resonance imaging during CAS using a first-generation stent. METHODS: Carotid stenosis magnetic resonance (MR) T1-weighted plaque signal intensity ratio (T1W-SIR) followed by NIRS assessment at the time of CAS (using the carotid artery Wallstent) was performed in 117 consecutive patients. RESULTS: The maximum lipid core burden index (max-LCBI) at minimal luminal areas (MLA; max-LCBIMLA) and the max-LCBI for any 4 mm segment in a target lesion defined as max-LCBIarea were significantly higher for the post-procedural new ipsilateral diffusion-weighted magnetic resonance imaging (DWI)-positive than negative patients (p<0.001 for all). There was a significant linear correlation between max-LCBIarea and the number of new emboli (r=0.544, p<0.0001). We also found that the second quantile (Q2) of T1W-SIRMLA had a significantly higher max-LCBIMLA and a higher incidence of DWI positivity than Q1 and Q3 (p<0.001 for all). Furthermore, max-LCBIMLA appeared to distinguish between patients with and without postoperative new ipsilateral DWI positivity (AUC 0.91, 95% CI: 0.86-0.96; p<0.0001). CONCLUSIONS: High LCP assessed by NIRS is associated with cerebral embolism by diffusion-weighted imaging in CAS using a first-generation stent.

Elective carotid stenting after urgent best medical treatment suppresses recurrent stroke in patients with symptomatic carotid artery severe stenosis.

OBJECTIVE: Urgent carotid endarterectomy and carotid artery stenting (CAS) for symptomatic advanced carotid artery stenosis is controversial because carry risks of hemorrhagic and thromboembolic complications. As treatments for preventing recurrent stroke have recently advanced, this study evaluated whether elective CAS with urgent best medical therapy reduces recurrent stroke for symptomatic severe carotid artery stenosis. PATIENTS AND METHODS: A total of 131 consecutive patients who underwent CAS for severe stenosis of the carotid artery between 2013-2017 were divided into acute ischemic minor stroke (AIMS) and Asymptomatic groups. The AIMS group comprised 59 patients presenting with minor stroke who underwent elective CAS with oral dual antiplatelet therapy, statin therapy, and add-on oral omega-3 fatty acid ethyl esters from 4 weeks before CAS. The Asymptomatic group comprised 72 patients treated with best medical therapy for 4 weeks before CAS. RESULTS: No recurrent ischemic stroke was observed under urgent best medical treatment before elective CAS in the AIMS group. Although the frequency of vulnerable plaque and degree of stenosis were much higher in the AIMS group, no significant differences were seen in perioperative complications. Baseline serum eicosapentaenoic acid (EPA) levels and EPA/ arachidonic acid (AA) were significantly lower in the AIMS group (p = 0.04, 0.04, respectively) and serum EPA/AA was significantly increased a day before CAS and 3 months after CAS compared with baseline. CONCLUSION: Urgent best medical treatment reduces recurrent stroke and facilitates safe elective CAS in patients with symptomatic and severe carotid artery stenosis.

Lipid Core Plaque Distribution Using Near-infrared Spectroscopy Is Consistent with Pathological Evaluation in Carotid Artery Plaques.

Carotid artery stenting (CAS) is performed as a treatment for carotid artery stenosis. However, lipid-rich plaques cause embolic complications and sequelae. Near-infrared spectroscopy (NIRS) can identify lipid components by applying a near-infrared absorption pattern, and the distribution of lipid components can be evaluated as the maximum lipid core burden index (maxLCBI). Intravascular ultrasound (IVUS) equipped with NIRS has been clinically applied recently, and its diagnostic usefulness and validation have been reported for coronary arteries; however, its consistency with actual pathological diagnosis in carotid artery lesions has not been validated. In this study, we investigated the consistency between the maxLCBI values and histopathological diagnoses. Patients with cervical carotid artery stenosis who underwent carotid endarterectomy (CEA) were examined in this prospective study. Pathological diagnosis was determined after NIRS evaluation, which was performed on the extracted plaques ex vivo. The histological slices of decalcified and paraffin-embedded sections were stained by hematoxylin-eosin (HE) and Elastica van Gieson (EVG), and for low-density lipoprotein (LDL), C-reactive protein (CRP), CD68, and glycophorin A. The correlation between maxLCBI values and histological findings. Seventy lesions assessed by NIRS were pathologically analyzed. There was a positive linear correlation between maxLCBI values and pathological findings as determined by HE (angle), HE (area%), EVG, CRP, and CD68 staining (respectively, r = 0.624, p <0.001; r = 0.578, p <0.001; r = 0.534, p <0.001; r = 0.723, p <0.001; r = 0.653, p <0.001). In conclusion, the maxLCBI values assessed by NIRS showed a significant positive linear correlation with pathological evaluations in carotid lesions. The maxLCBI values in carotid arteries are consistent with pathological evaluations.

Production of antibody fragments using the baculovirus-insect cell system.

The baculovirus-insect cell system is effective in the production of large quantities of biologically active recombinant proteins. The secretory production of an antibody Fab fragment in lepidopteran insect cells infected with a recombinant baculovirus that contains both the heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of the Fab fragment is described in this chapter. The recombinant baculovirus can be generated rapidly and efficiently through site-specific transposon-mediated insertion of foreign genes into a baculovirus genome called a bacmid in Escherichia coli. Factors influencing the Fab fragment production are discussed.

Efficient production of an antibody Fab fragment using the baculovirus-insect cell system.

The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (>600 µg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus-insect cell system may allow efficient production of antibody Fab fragments.