Migration and differentiation of neural cell lines transplanted into mouse brains

In the past few years, the plasticity of the regional specification of the CNS has been widely debated on the results from in utero transplantation. Two different results are reported with this transplantation method. One is that the distribution of transplanted cells is dependent on the donor origin, and the other is that the distribution is independent on the donor cell origin. The present study attempted to examine closely the plasticity of the regional specification by in utero transplantation method with clonal neural cell lines, 2Y-3t and 2Y-5o2b. These lines were established from a cerebellum of an adult p53-deficient mouse. Our results showed that transplanted cells migrated into various regions of the CNS and supported the independent distribution. Moreover, different distribution patterns of transplanted cells were observed between host sexes. Labeled cells were localized around the ventricle of neonatal host brains, where they were undifferentiated. In 2-3 weeks after birth, labeled cells were found in the brain parenchyma and some of them took neuronal morphology. In the rostral migratory stream (RMS), cells with unipolar or bipolar morphology were still undifferentiated. In other regions, labeled cells were often associated with blood vessels; the soma were on the surface of vessels, extending processes or neurites into surrounding brain parenchyma. Time-lapse imaging demonstrated that they were migrating with blood vessels.     

Dynamic changes in conjunctival dendritic cell numbers, anatomical position and phenotype during experimental allergic conjunctivitis

Dendritic cells (DCs) are a subset of antigen-presenting cells (APCs) that are involved in the initiation and control of the immune response to antigens present at the interface with the environment. A limited number of groups have studied DCs in human and animal conjunctiva but no data is available concerning the different DC subsets present in the conjunctival tissue. The aims of this study are to characterize the phenotypes and numbers of DCs present in the murine model of allergic conjunctivitis using the technique of immunohistochemistry so as to aid the understanding of the mechanisms involved in allergic eye disease. A double immunofluorescence method was used to analyze the phenotypic distribution and density of DC subsets in the mouse conjunctival tissues of the allergic model using a panel of antibodies: CD11c, as a general marker of DCs, coupled with another DC subset marker such as Langerin for Langerhans cells (LCs), CD11b for myeloid DCs (mDCs) and mPDCA-1 for plasmacytoid DCs (pDCs). In the na?ve conjunctiva, mDCs were consistently detected in the subepithelial layer and substantia propria. In the epithelium and the subepithelial layer, very few LCs and virtually no pDCs were observed. Following allergen challenge, there was a marked influx of mDCs and pDCs, but no LCs, into the subepithelial layer and throughout the substantia propria. These results indicate that conjunctival DC subsets may play an important role in the immune-regulatory processes involved in the inflammatory component of allergic conjunctivitis.     

Impact of turbulent blood flow in the aortic root on de novo aortic insufficiency during continuous-flow left ventricular-assist device support

Severe aortic insufficiency (AI) after implantation of continuous-flow left ventricular-assist device (LVAD) affects device performance and outcomes. However, the mechanism for the occurrence and progression of AI has not been elucidated. We investigated the impact of nonphysiological retrograde blood flow in the aortic root on AI after LVAD implantation. Blood flow pattern was analyzed in patients with and without AI (n = 3 each) who underwent LVAD implantation, by computational fluid dynamics with patient-specific geometries, which were reproduced using electrocardiogram-gated 320-slice computed tomographic images. The total volume of retrograde blood flow during one cardiac cycle (716 ± 88 mL) was higher and the volume of slow blood flow (<0.1 cm/s) (0.16 ± 0.04 cm³) was lower in patients with AI than in those without AI (360 ± 111 mL, P = .0495, and 0.49 ± 0.08 cm³, P = .0495, respectively). No significant difference in wall shear stress on the aortic valve was observed between the groups. Patients with AI had a perpendicular anastomosis at the distal ascending aorta and the simulation in the modified anastomosis model of patients with AI showed that the retrograde blood flow pattern depended on the angle and position of anastomosis. Computational fluid dynamics revealed strong retrograde blood flow in the ascending aorta and aortic root in patients with AI after LVAD implantation. The angle and position of LVAD outflow anastomosis might impact retrograde blood flow and de novo AI after LVAD implantation.     

Antimicrobial susceptibility surveillance of obligate anaerobic bacteria in the Kinki area

Obligate anaerobes exist as resident flora in various sites in humans, but they are also emphasized as endogenous causative microorganism of infections. We performed surveillance to understand the trend of drug susceptibility in obligate anaerobic bacteria in the Kinki area of Japan. In the experiment, we used 156 obligate anaerobe isolates collected from 13 institutions that participated in the Study of Bacterial Resistance Kinki Region of Japan. MALDI Biotyper was used to identify the collected strains, and among the 156 test strains, those that could be identified with an accuracy of Score Value 2.0 or more included 6 genera, 30 species, and 144 strains (Bacteroides spp. 77 strains, Parabacteroides sp. 2 strains, Prevotella spp. 29 strains, Fusobacterium spp. 14 strains, Porphyromonas spp. 2 strains, and Clostridioides difficile 20 strains), and they were assigned as subject strains for drug susceptibility testing. The drug susceptibility test was carried out by broth microdilution method using Kyokuto Opt Panel MP ANA (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and judged according to CLSI criteria. As a result, Bacteroides and Parabacteroides species showed good sensitivities to tazobactam-piperacillin, imipenem, metronidazole and chloramphenicol, and low sensitivities to ampicillin, cefoperazone and vancomycin. Prevotella species showed good sensitivities to sulbactam-ampicillin, tazobactam-piperacillin, cefmetazole, imipenem, doripenem and metronidazole. Susceptibility rates to other drugs were slightly different depending on the bacterial species. Both Fusobacterium spp. and Porphyromonas spp. showed high sensitivities to many drugs. C. difficile was highly sensitive to vancomycin and metronidazole, having MIC₉₀s of 0.5 μg/mL and ≤2 μg/mL, respectively.     

Ventricular fibrillation threshold measured by continuous 50 cps stimulation for the evaluation of the antifibrillatory effect of the drugs

The role of electrophysiologic parameters on the induction of ventricular fibrillation (VF) by continuous 50 cycle per second (cps) electrical stimulation was studied in 21 open chest dogs. The current strength of the 50 cps stimulation required to induce VF when applied to the ventricle for 2 seconds was defined as the ventricular fibrillation threshold (VFT). The intravenous injection of antiarrhythmic drugs raised the VFT in a dose dependent manner. The changes in VFT were associated with a rise in excitation threshold. The slopes of the regression equations relating the excitation threshold to VFT were almost identical, that is, 2.8 with lidocaine, 3.4 with procainamide and 3.2 with disopyramide. Prolongation of refractory period increased the cycle length of ventricular excitations just prior to VF but was not correlated with the changes in VF. Localized myocardial ischemia induced by coronary ligation also resulted in the elevation of VFT. The slope of the regression equation between excitation threshold and VFT was 1.9 which was slightly lower than that observed at the administration of the drugs. The fact that the VFT was mainly attributed to the changes in excitation threshold at the site where the test stimulus was applied would limit the usefulness of the 50 cps continuous stimulation method for the evaluation of vulnerability to VF.     

Usefulness of rapid bedside cardiac troponin T assay for the diagnosis of acute myocardial infarction

Cardiac troponin T(cTnT) is one of the most myocardial-specific markers for the diagnosis of acute myocardial infarction(AMI). Recently, the rapid bedside cTnT assay(Trop T rapid assay sensitive version), which can provide qualitative determinations within 15 min, has been developed for the emergency clinical setting. To evaluate the usefulness of rapid bedside cTnT assay, we performed the Trop T test and measured serum levels of myoglobin(Mb), creatine kinase MB isoenzyme(CK-MB) and cTnT in 256 consecutive emergency patients with suspected AMI(65 found to have AMI and 191 without AMI). The diagnostic sensitivities for AMI of Trop T, Mb and CK-MB measurements were 66%, 92% and 52%, respectively, whereas the specificities were 80%, 18% and 74%, respectively. The diagnostic accuracy for AMI of Trop T(77%) was significantly higher than that of Mb(37%, p < 0.001) and CK-MB(69%, p < 0.05). The sensitivity for AMI of Mb(86%) was significantly(p < 0.001) higher than that of Trop T (31%) and CK-MB(31%) in patients admitted < or = 3 hr after the onset of AMI. In contrast, the sensitivities of Trop T(80% and 100%) in patients admitted at 3-6 hr and > 6 hr showed no significant differences from those of Mb(100% and 96%). Furthermore, Trop T in patients admitted > 6 hr had significantly(p < 0.01) higher sensitivity compared with CK-MB(69%). The mortality rate in the non-AMI group during hospitalization in patients with positive Trop T test(39%) was significantly(p < 0.001) higher than that in patients with negative test(9%). When the positive Trop T test was regarded as > or = 0.10 ng/ml of serum cTnT, Trop T test had the best concordance of 92% with a quantitative of cTnT assay.     

Aromatase-knockout mouse carrying an estrogen-inducible enhanced green fluorescent protein gene facilitates detection of estrogen actions in vivo

Aromatase is an enzyme that converts androgen to estrogen in the gonads and also at extragonadal sites, including the brain. In this study we developed a transgenic mouse that carries an enhanced green fluorescent protein (EGFP) gene inducible by estrogen through an estrogen response element to facilitate detection of estrogen actions in vivo. The expression of EGFP in aromatase-deficient (Ar(-/-)) female mice was significantly suppressed at the pituitary gland, ovary, uterus, and gonadal fat pad and was induced by dietary 17beta-estradiol to wild-type (Ar(+/+)) levels or higher. These results demonstrate that the expression of the EGFP gene is tissue selective and estrogen dependent in vivo. Employing this transgenic mouse, we examined whether estrogen synthesis in the extragonadal sites is necessary for reproduction in female mice. When ovaries of Ar(-/-) mice were replaced with Ar(+/+) ovaries, a significant induction of EGFP expression in the pituitary gland and uterus was observed. Histological examinations showed the presence of antral follicles in the replaced ovaries, indicating that the transplants are functional in Ar(-/-) mice. After crossing with males, three of 10 Ar(-/-)females with Ar(+/+) ovaries became pregnant and fed their pups. Collectively, these observations indicate that estrogen synthesis in the ovary is sufficient for supporting female reproduction, and that infertility of Ar(-/-) females is primarily due to a defect in estrogen synthesis in the ovary.     

Failure to express the P-selectin gene or P-selectin blockade confers early pulmonary protection after lung ischemia or transplantation

Endothelial P-selectin expression contributes to the first wave of neutrophil (polymorphonuclear leukocyte; PMN) influx in several inflammatory conditions. Although remote tissue ischemia, such as a crush injury to the hindlimb, may result in P-selectin-mediated pulmonary leukosequestration, it is not known whether the lungs exhibit a similar response after hypothermic preservation or when subjected to a direct ischemic insult. To determine if P-selectin may mediate early primary graft failure, left lungs harvested from male Lewis rats were preserved for 6 hr at 4°C and transplanted orthotopically into isogeneic recipients. Recipients immunodepleted of PMNs before transplantation demonstrated improved graft function; pulmonary vascular resistance was reduced ≈6-fold, arterial oxygenation was increased ≈3-fold, and recipient survival was increased ≈4-fold (P < 0.05, 0.05, and 0.005, respectively). Administration of a blocking anti-P-selectin IgG 10 min before reperfusion diminished graft PMN infiltration and resulted in improved graft function and recipient survival compared with controls. To establish the role of P-selectin in normothermic pulmonary ischemia, mice were subjected to temporary left pulmonary artery ligation. After functional removal of the nonischemic right lung, mice deletionally mutant for the P-selectin gene (P-selectin −/−) exhibited reduced PMN infiltration (≈2-fold), improved arterial oxygenation (≈2-fold), and improved survival (≈3-fold) compared with P-selectin +/+ control mice (P < 0.05, 0.01, and 0.05, respectively). These studies isolate and identify the central role of a single gene product (P-selectin) in early PMN recruitment and tissue injury after frank pulmonary ischemia and in the setting of lung transplantation after hypothermic preservation.     

Vestibular and cochlear nerve enhancement on MRI and its correlation with vestibulocochlear functional deficits in patients with Ramsay Hunt syndrome

ObjectiveThe correlation between enhancement of the vestibulocochlear nerves on gadolinium-enhanced magnetic resonance imaging (MRI) and vestibulocochlear functional deficits was examined in patients with Ramsay Hunt syndrome (RHS).MethodsNineteen patients with RHS who showed herpes zoster oticus, peripheral facial palsy, and vertigo were enrolled. Canal paresis (CP) in the caloric test, abnormal response to ocular and cervical vestibular myogenic potentials (oVEMP and cVEMP), and refractory sensorineural hearing loss were evaluated. MRI images perpendicular to the internal auditory canal were reconstructed to identify the superior (SVN) and inferior vestibular nerves (IVN) and the cochlear nerve (CV). The signal intensity increase (SIinc) of the four-nerve enhancement was calculated as an index.ResultsAmong RHS patients, 79%, 53%, 17% and 26% showed CP in the caloric test, abnormal responses to oVEMP and cVEMP, and refractory sensorineural hearing loss, respectively. SIinc rates of the SVN were significantly increased in RHS patients with CP in the caloric test, and with abnormal responses to oVEMP and cVEMP. SIinc rates of the SVN tended to increase in RHS patients with refractory sensorineural hearing loss (p = 0.052). SIinc rates of the IVN were significantly increased in RHS patients with abnormal responses to oVEMP and cVEMP, and refractory sensorineural hearing loss, but not in those with CP in the caloric test. SIinc rates of the CN were significantly increased in RHS patients with CP in the caloric test, abnormal response to oVEMP and refractory sensorineural hearing loss, but not in those with abnormal response to cVEMP.ConclusionIn patients with RHS, the origin of vertigo may be superior vestibular neuritis, which is affected by reactive varicella-zoster virus from the geniculate ganglion of the facial nerve through the faciovestibular anastomosis. The results also suggested that in some RHS patients, inferior vestibular neuritis contributes to the development of vertigo and that the origin of refractory sensorineural hearing loss is cochlear neuritis.