Chemogenetic inhibition of pain-related neurons in the posterior insula cortex reduces mechanical hyperalgesia and anxiety-like behavior during acute pain.

Pain is a complex phenomenon that involves sensory, emotional, and cognitive components. The posterior insula cortex (pIC) has been shown to integrate multisensory experience with emotional and cognitive states. However, the involvement of the pIC in the regulation of affective behavior in pain remains unclear. Here, we investigate the role of pain-related pIC neurons in the regulation of anxiety-like behavior during acute pain. We combined a chemogenetic approach with targeted recombination in active populations (TRAP) in mice. Global chemogenetic inhibition of pIC neurons attenuates chemically-induced mechanical hypersensitivity without affecting pain-related anxiety-like behavior. In contrast, inhibition of pain-related pIC neurons reduces both mechanical hypersensitivity and pain-related anxiety-like behavior. The present study provides important insights into the role of pIC neurons in the regulation of sensory and affective pain-related behavior.

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and ß repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and ß of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

Cilostazol improves the prognosis after hepatectomy in rats with sinusoidal obstruction syndrome.

BACKGROUND AND AIM: Safe radical hepatectomy is important for patients with colorectal liver metastases complicated by sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy. This study aimed to investigate the impact of preoperative administration of cilostazol (CZ), an oral selective phosphodiesterase III inhibitor, on hepatectomy in rat SOS model. MATERIAL AND METHODS: Rats were divided into NL (normal liver), SOS (monocrotaline [MCT]-treated), and SOS + CZ (MCT + CZ-treated) groups. MCT or CZ was administered orally, and a 30% partial hepatectomy was performed 48 h after MCT administration. Postoperative survival rates were evaluated (n = 9, for each). Other rats were sacrificed on postoperative days (POD) 1 and 3 and evaluated histologically, immunohistochemically, biochemically, and using transmission electron microscopy (TEM), focusing particularly on SOS findings, liver damage, and liver sinusoidal endothelial cell (LSEC) injury. RESULTS: The cumulative 10-day postoperative survival rate was significantly higher in the SOS + CZ group than in the SOS group (88.9% vs 33.3%, P = 0.001). Total SOS scores were significantly lower in the SOS + CZ group than in the SOS group on both POD 1 and 3. Serum biochemistry and immunohistochemistry showed that CZ reduced liver damage after hepatectomy. TEM revealed that LSECs were significantly preserved morphologically in the SOS + CZ group than in the SOS group on POD 1 (86.1 ± 8.2% vs 63.8 ± 9.3%, P = 0.003). CONCLUSION: Preoperative CZ administration reduced liver injury by protecting LSECs and improved the prognosis after hepatectomy in rats with SOS.

Combined Experiments with in Vivo Fiber Photometry and Behavior Tests Can Facilitate the Measurement of Neuronal Activity in the Primary Somatosensory Cortex and Hyperalgesia in an Inflammatory Pain Mice Model.

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

Factors Influencing Discontinuation of Clazosentan Therapy in Elderly Patients with Aneurysmal Subarachnoid Hemorrhage: A Retrospective Study from a Japanese Single Center.

BACKGROUND Clazosentan is an endothelin receptor antagonist approved in Japan for preventing cerebral vasospasm and vasospasm-associated cerebral ischemia and infarction. This study included elderly patients aged ≥75 years with aneurysmal subarachnoid hemorrhage (SAH) and aimed to evaluate the factors associated with discontinuing anti-vasospasm therapy with clazosentan. MATERIAL AND METHODS In this single-center retrospective observational study, we extracted diagnostic and therapeutic work-up data of consecutive 40 patients with SAH treated with clazosentan infusion (10 mg/h) as first-line anti-vasospasm therapy between May 2022 and August 2023. Patient data were compared between the discontinued and completed groups, and related factors for the discontinuation were further analyzed. RESULTS Clazosentan was discontinued in 22% (n=9) of patients due to intolerable dyspnea accompanied by hypoxemia at 5±3 days after therapy initiation, in which 44% (n=4) were elderly (≥75 years). Patients who discontinued clazosentan therapy showed significantly lower urine volumes compared with those who completed the therapy (P<0.05). Multivariate regression analysis revealed that day-to-day urine volume variance and older age were independent risk factors for drug cessation (P<0.05). The cut-off value for predicting clazosentan discontinuation was -0.7 mL/kg/h with sensitivity of 86% and specificity of 75% (area under the curve: 0.76±0.10; 95% confidence interval: 0.56-0.96; P=0.035). CONCLUSIONS Our results suggest that approximately 20% of SAH patients suffered from intolerable respiratory symptoms attributable to hypoxemia. We found that both reduced day-to-day urine volume variation and older age are independent risk factors for drug discontinuation.

Cardiopulmonary Events of the Elderly (≥75 Years) during Clazosentan Therapy after Subarachnoid Hemorrhage: A Retrospective Study from a Tertiary Stroke Center in Japan.

Clazosentan has been shown to prevent vasospasm and reduce mortality in patients after aneurysmal subarachnoid hemorrhage (SAH) and has been approved for clinical use in Japan; however, its systemic events in the elderly (aged ≥ 75 years) have not been well-documented. Here, we report serious/intolerable cardiopulmonary complications requiring discontinuation of drug therapy in elderly SAH patients. In this single-center case series study, medical records of consecutive SAH patients treated postoperatively with clazosentan (10 mg/h) between June 2022 and May 2023 were reviewed retrospectively. Thirty-three patients received clazosentan therapy, of whom six were elderly with a mean age of 80.3 ± 5.2 (range 75-89) years. Among them, despite no obvious medical history of systemic abnormalities, clazosentan was discontinued in three (50%) patients due to pleural effusion and hypoxemia with or without hypotension at 5 ± 3 days after therapy initiation, which was higher than the incidence for younger patients (15%). The elderly patients had significantly lower urine output (1935 ± 265 vs. 1123 ± 371 mL/day, p = 0.03) and greater weight gain (2.1 ± 1.1 vs. 4.2 ± 1.9 kg from baseline, p = 0.04) than patients who completed the therapy. One 89-year-old female developed congestive heart failure and hydrostatic pulmonary edema associated with increased intravascular and lung volumes even after therapy was discontinued, while the remaining two cases recovered within 2 days after drug cessation. These results suggest that elderly patients are more vulnerable to fluid retention and have a higher risk of cardiopulmonary complications during clazosentan therapy than younger patients. Careful monitoring of urine volume and weight gain and caution regarding age- and therapy-related hemodynamic insufficiencies are required.

Differences in the microstructural and mechanical qualities of semitendinosus tendon grafts between skeletally immature and mature patients in anterior cruciate ligament reconstruction.

BACKGROUND: This study aimed to investigate the microstructural and mechanical properties of semitendinosus tendon graft tissues during anterior cruciate ligament reconstruction and the clinical outcomes in skeletally immature and mature patients. METHODS: Twenty-two patients who underwent primary anterior cruciate ligament reconstruction using a hamstring tendon graft were analyzed and divided into skeletally immature (n = 7) and mature groups (n = 15) based on magnetic resonance imaging findings of the epiphyseal plate of the distal femur. Tissue samples were collected from the mid-portion of the semitendinosus tendon. The collagen fibril diameter, maximum stress, and strain at maximum stress point in the semitendinosus tendon tissues were calculated for comparison of the microstructural and mechanical properties between the two groups. Postoperative outcomes were also assessed between the two groups. RESULTS: The mean and 60th and 80th percentiles of fibril diameters in the skeletally immature group were significantly smaller than those in the mature group (65.9 ± 13.0, 73.5 ± 19.3, and 91.3 ± 27.4 nm in the skeletally immature group; and 90.3 ± 14.7, 94.0 ± 18.4, and 125.3 ± 19.9 nm in the skeletally immature group; p = 0.001, 0.024, and 0.004, respectively). Additionally, the strain at maximum stress was higher in the skeletally immature group (237.2 ± 102.4% vs. 121.5 ± 51.9%, p = 0.024). However, there was no difference in maximum stress between the skeletally immature and mature groups (19.9 ± 14.3 MPa vs. 24.5 ± 23.4 MPa, p = 0.578). Strain was negatively correlated with the mean fibril diameter and the 60th and 80th percentiles of fibril diameters, whereas stress was positively correlated with the mean fibril diameter. The skeletally immature group had a higher pivot shift test-positive rate than the mature group at the last follow-up (p = 0.023). CONCLUSION: Semitendinosus tendon graft tissues differed microstructurally and mechanically between skeletally immature and mature patients. LEVEL OF EVIDENCE: Level Ⅳ.

Hedgehog signaling plays a crucial role in hyperalgesia associated with neuropathic pain in mice.

Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. In Drosophila, the Hedgehog (Hh) signaling pathway is related to increased pain sensitivity (hyperalgesia) but does not affect the baseline nociceptive threshold. In general, the contribution of the Hh signaling pathway to neuropathic pain in vertebrates is a highly debated issue. Alternatively, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve-transection (SNT) surgery, microglial activation increased in the ipsilateral spinal dorsal horn compared with that in the sham group; however, 21 days after surgery, microglial activation decreased. Contrastingly, astrocyte activation in the spinal cord did not differ between the groups. On day 21 of postsurgery, the SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in dorsal root ganglion (DRG) neurons. Intrathecal administration of the Hh signaling inhibitor vismodegib attenuated the mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment with the Hh signaling activator smoothened agonist in naive mice induced mechanical allodynia, which was abolished by the ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling by pretreatment with vismodegib significantly reduced ATP secretion and the frequency/number of spontaneous elevations of intracellular calcium ion levels in cultured DRG cells. Thus, the Hh signaling pathway appears to modulate the neural activity of DRG neurons via ATP release, and it plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus.

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Prediction of Cross-Clamp-Induced Cerebral Ischemia During Carotid Endarterectomy Using Arterial Blood Flow Assessment.

BACKGROUND: Cross-clamping during carotid endarterectomy (CEA) is associated with the risk of cerebral ischemia. Various studies have evaluated different criteria for detecting cerebral ischemia, but difficulties arise when ischemic changes appear after the carotid artery is cross-clamped and incised. Here, we explored a parameter that can predict cerebral ischemia prior to cross-clamping during CEA using a blood-flow meter. METHODS: The carotid arterial blood flow was measured directly (direct ABF) in the common carotid artery prior to cross-clamping. The anatomical information in preoperative magnetic resonance imaging, cerebral blood flow in xenon-enhanced computed tomography, and carotid peak systolic flow velocity by carotid echo from the skin surface were also evaluated. A decrease in the short-latency somatosensory evoked potentials (SSEP) during cross-clamping to insert a shunt was assessed, and a decrease in amplitude of ≥ 50% was considered an indicator for cerebral ischemia. Surgery was performed under general anesthesia, and a shunt was inserted in all cases. RESULTS: Of 156 CEA patients between April 2013 and March 2020, 30 had decreased SSEP during cross-clamping. The baseline characteristics and intra- and postoperative findings were not significantly different between patients with and without a decrease in SSEP. Among the evaluated parameters, only the direct-ABF ratio (ABF-internal carotid artery/ABF-common carotid artery) differed significantly between the 2 groups (P = 0.011). The direct-ABF ratio ≤ 0.58 was predictive of cerebral ischemia during CEA. CONCLUSIONS: Direct-ABF measurement with an ultrasonic blood-flow meter can be useful for predicting cerebral ischemia prior to carotid artery cross-clamping during CEA.

Differences in cellular and microstructural properties of the semitendinosus muscle tendon between young and adult patients.

BACKGROUND: Poor outcomes associated with anterior cruciate ligament reconstruction in paediatric patients are a major concern. The tendon structure and its cellular characteristics are key factors that affect the mechanical properties of tendons. This study aimed to evaluate the effects of growth on the cellular and microstructural properties of the tendon of the semitendinosus muscle in humans. METHODS: Semitendinosus muscle tendon samples from 76 patients who underwent ligament reconstruction were examined and divided into three groups: immature (10.8 ± 2.7 years old), young (16.5 ± 1.8 years old), and adult (35.2 ± 8.6 years old), based on age and the state of the epiphyseal plate in the distal femur. The number of tendon cells per unit area was assessed, and the major-to-minor-length ratio of the tendon cell nuclei was calculated to evaluate the shape of the nuclei using haematoxylin and eosin staining. The collagen fibril diameter and distribution were determined using electron microscopy. RESULTS: The major-to-minor-length ratio of the tendon cell nuclei significantly increased with age (p-value; immature vs. young: 0.018, young vs adult: 0.001, immature vs adult: 0.001). The shape of the tendon cell nuclei was rounder in the immature group and more elongated in the adult group. A significant decrease in the number of tendon cells was observed with age (immature: 565 ± 134/mm2, young: 356 ± 105/mm2, adult: 272 ± 81/mm2; p-value: immature vs young: 0.001, young vs adult: 0.012, immature vs adult: 0.001). The mean fibril diameter in the immature group was significantly smaller (p-value: immature vs young: 0.018, young vs adult: 0.001, immature vs adult: 0.001). The distribution of the collagen fibrils changed from right skewed in the immature group to flat in the adult group. CONCLUSIONS: The characteristics of the tendon cells and the microstructure of collagen in muscle tendons significantly changed with age.

Triple bypass for multisystem smooth muscle dysfunction syndrome due to Arg179His ACTA2 mutation.

Missense mutations in the smooth muscle-specific isoform of the alpha-actin (ACTA2) gene, which encodes smooth muscle actin, congenitally cause systemic smooth muscle dysfunction, leading to multiple systemic smooth muscle dysfunction syndrome. This disease is often diagnosed through the development of congenital mydriasis, patent ductus arteriosus, or thoracic aortic aneurysm at a young age. Some patients develop cerebrovascular lesions, also known as ACTA2 cerebral arteriopathy, which cause ischemic stroke and require surgical revascularization. However, an effective and safe treatment has not yet been established owing to the rarity of the disease. Furthermore, most reports of this disease involve children, with only a few reports on adults and few detailed reports on treatment outcomes published to date. We report a 46-year-old woman with ACTA2 cerebral arteriopathy caused by Arg179His, the most common mutation in this disease; she is the oldest patient reported with this disease to the best of our knowledge. The patient was diagnosed with multiple systemic smooth muscle dysfunction syndrome and ACTA2 cerebral arteriopathy after experiencing a stroke in the right cingulate gyrus. She underwent direct triple bypass with three anastomoses of the right superficial temporal artery to the middle and anterior cerebral arteries. She developed an ischemic stroke as a postoperative complication.The efficacy and safety of this procedure have not been clearly confirmed owing to the frailty of the donor superficial temporal artery and the poor development of collateral circulation; however, direct bypass should be considered a treatment option for patients experiencing progressive multiple strokes.

Heptamer-type small guide RNA that can shift macrophages toward the M1 state.

Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology. We thought that some heptamer-type sgRNAs for TRUE gene silencing would be able to transform TAM toward the M1 state and might become therapeutic drugs for myeloma. Here, we searched for heptamer-type sgRNAs that can shift macrophages toward the M1 state. We screened a heptamer-type sgRNA library for the ability to up-regulate IL-12b gene expression in human macrophage-like cell lines, and found three such sgRNAs. One of the sgRNAs, H12960, which also showed such ability in human fresh macrophages and mouse macrophage-like cell lines, efficiently suppressed human myeloma cell growth in SCID/NOD mice.

A case of idiopathic extracranial carotid artery pseudoaneurysm with a rare clinical course and pathological features.

Extracranial carotid artery aneurysms (ECAAs) are rare, with the etiology mainly classified as degeneration or dissection. Pseudoaneurysms in the region are even rarer and are seen following trauma, iatrogenic injury, or infection. We report a case of extracranial carotid artery pseudoaneurysm (pseudo-ECAA) with a rare clinical course and pathological features. A 58-year-old man presented with swelling and purpura on the left side of his neck after sneezing. Radiological examinations suggested a ruptured left common carotid artery aneurysm. The operative findings were consistent with a pseudoaneurysm. Pathological examination revealed disarrangement and degeneration of smooth muscle fibers in the media, in addition to scattered foci of mucoid accumulation and irregular-shaped cavitation in the medial extracellular matrix, raising the possibility of an intrinsic dysfunction of the vascular wall in the pathological process of pseudoaneurysm formation.

Maximum Nidus Depth as a Risk Factor of Surgical Morbidity in Eloquent Brain Arteriovenous Malformations.

BACKGROUND: Eloquent location of a brain arteriovenous malformation (BAVM) is known to increase the surgical risk. Surgical removal of such BAVMs is challenging. Useful indicators for the safe removal of eloquent BAVMs are needed. The aim of this study was to determine the surgical risk factors for these challenging entities. METHODS: The authors retrospectively reviewed 29 motor and/or sensory BAVM patients who underwent surgeries. The risk factors for surgical morbidity were analyzed. As a new risk factor, maximum nidus depth, was evaluated. RESULTS: Complete obliteration was achieved in 28 patients (96.6%). Postoperative transient and permanent neurological deteriorations were seen in nine patients (31.0%) and five patients (17.2%), respectively. In univariate analysis, maximum nidus depth (p = 0.0204) and asymptomatic onset (p = 0.0229) were significantly correlated with the total morbidity. In multivariate analysis, only maximum nidus depth was significantly correlated with total morbidity (p = 0.0357; odds ratio, 2.78598; 95% confidence interval, 0.8866-8.7535). The cut-off value for the maximum nidus depth was 36 mm for total morbidity (area under the curve [AUC], 0.7428) and 41 mm for permanent morbidity (AUC, 0.8833). The cutoff value of the maximum nidus size was 30 mm for total morbidity (AUC, 0.5785) and 30 mm for permanent morbidity (AUC, 0.7625). AUC was higher for the maximum nidus depth than it was for the maximum nidus size. CONCLUSIONS: Maximum nidus depth was significantly associated with surgical morbidity of eloquent BAVMs. The maximum nidus depth is a novel and a simpler indicator of the risk of surgical morbidity.

[Intraoperative Rupture from Cerebral Aneurysm: Tactics for Management and Strategy for Prevention].

Rupture of a cerebral aneurysm during surgery carries risks that may lead to poor patient outcomes. Rupture often occurs during the separation procedure of the aneurysm from the surrounding structure. Knowledge regarding the basic principles of operation of surgical apparatus, for example suction devices, before and after the aneurysm rupture event is of paramount importance. It is desirable to deal with these complicated situations automatically in a non-heuristic manner, although some experience and learning are necessary to obtain this ability. When we necessarily apply temporary occlusion of the parent arteries, we need to consider the merits and demerits of temporary occlusion, as well as the limits of the occlusion time in order to prevent ischemic complications.

Self-glycerophospholipids activate murine phospholipid-reactive T cells and inhibit iNKT cell activation by competing with ligands for CD1d loading.

Glycosphingolipids and glycerophospholipids bind CD1d. Glycosphingolipid-reactive invariant NKT-cells (iNKT) exhibit myriad immune effects, however, little is known about the functions of phospholipid-reactive T cells (PLT). We report that the normal mouse immune repertoire contains αß T cells, which recognize self-glycerophospholipids such as phosphatidic acid (PA) in a CD1d-restricted manner and don't cross-react with iNKT-cell ligands. PA bound to CD1d in the absence of lipid transfer proteins. Upon in vivo priming, PA induced an expansion and activation of T cells in Ag-specific manner. Crystal structure of the CD1d:PA complex revealed that the ligand is centrally located in the CD1d-binding groove opening for TCR recognition. Moreover, the increased flexibility of the two acyl chains in diacylglycerol ligands and a less stringent-binding orientation for glycerophospholipids as compared with the bindings of glycosphingolipids may allow glycerophospholipids to readily occupy CD1d. Indeed, PA competed with α-galactosylceramide to load onto CD1d, leading to reduced expression of CD1d:α-galactosylceramide complexes on the surface of dendritic cells. Consistently, glycerophospholipids reduced iNKT-cell proliferation, expansion, and cytokine production in vitro and in vivo. Such superior ability of self-glycerophospholipids to compete with iNKT-cell ligands to occupy CD1d may help maintain homeostasis between the diverse subsets of lipid-reactive T cells, with important pathogenetic and therapeutic implications.

Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.

Genotype-Phenotype Correlation in Long-Term Cohort of Japanese Patients with Moyamoya Disease.

BACKGROUND: Homozygosity of this p.R4810K founder variant of RNF213moyamoya disease (MMD) susceptibility gene is known to influence the severity of the clinical disease phenotype at disease onset. However, the association between this genotype and long-term clinical manifestations has remained unclear. OBJECTIVES: The principal goal of this study was to investigate whether and how the p.R4810K variant of RNF213influences the long-term phenotype in Japanese patients with MMD. METHOD: This retrospective cohort study included 94 Japanese patients with MMD who underwent direct or combined bypass for revascularization with the p.R4810K genotype determined in our hospital. The following phenotypic parameters were analyzed at disease onset and over a long-term period: age and initial presentation at onset, recurrent stroke after initial revascularization, and final modified Rankin Scale. RESULTS: The p.R4810K genotype was significantly associated with the phenotype at onset, especially in younger patients. Over a median follow-up period of 100 months, recurrent stroke occurred in 6 out of 94 patients: none out of 5 patients with the homozygous variant, 5 out of 64 with the heterozygous variant, and 1 out of 25 in the wild-type group. There were no significant differences among the genotypes. In particular, recurrent cerebral hemorrhage occurred in 5 patients, all possessing the heterozygous variant. The log-rank test showed no difference between the genotypes in the stroke-free survival rate. Furthermore, the p.R4810K genotype was not associated with a poor functional condition. CONCLUSIONS: The p.R4810K founder variant of RNF213 affects the phenotype at disease onset. However, the optimal revascularization may be effective, regardless of the genotype, even for the homozygous variant, which has been thought to be the most pathogenic. This genotype may not strongly influence the long-term clinical manifestations or poor prognosis in MMD.

Successful Reperfusion with Endovascular Therapy Has Beneficial Effects on Long-Term Outcome Beyond 90 Days.

BACKGROUND AND PURPOSE: Recent studies have demonstrated that endovascular reperfusion therapy improves clinical outcomes at 90 days after ischemic stroke. However, the effects on long-term outcomes are not well known. We hypothesized that successful reperfusion might be associated with long-term improvement beyond 90 days after endovascular therapy. To assess the long-term effects beyond 90 days, we analyzed the association of successful reperfusion with a temporal change in modified Rankin Scale (mRS) score from 90 days to 1 year after endovascular therapy. METHODS: We retrospectively analyzed a database of consecutive patients with acute ischemic stroke who received endovascular therapy between April 2006 and March 2016 at 4 centers. We compared the incidences of improvement and deterioration in patients with successful reperfusion (i.e., modified thrombolysis in cerebral infarction score of 2b or 3) with those in patients with unsuccessful reperfusion. We defined improvement and deterioration as decrease and increase on the mRS score by 1 point or more from 90 days to 1 year after endovascular therapy respectively. RESULTS: A total of 268 patients were included in the current study. The rate of patients with improvement tended to be higher in patients with successful reperfusion than in patients with unsuccessful reperfusion (20% [34/167 patients] vs. 12% [12/101], p = 0.07). The rate of patients with deterioration was lower in patients with successful reperfusion than in patients with unsuccessful reperfusion (25% [42/167] vs. 42% [42/101], p < 0.01). After adjustment for confounders, successful reperfusion was associated with improvement (adjusted OR 2.65; 95% CI 1.23-5.73; p < 0.05) and deterioration (adjusted OR 0.33; 95% CI 0.18-0.62; p < 0.01), independent of the 90-day mRS score. CONCLUSIONS: Successful reperfusion has further beneficial legacy effects on long-term outcomes beyond 90 days after stroke.