Protocol for gene knockdown using siRNA in primary cultured neonatal murine microglia.

In this protocol, we describe the small interfering RNA (siRNA)-mediated gene knockdown in primary mouse microglia, providing an approach to investigate functions such as phagocytosis and chemotaxis. The approach includes siRNA design, establishment of mixed glial cultures, microglia isolation, and siRNA transfection. Validation of knockdown efficacy employs quantitative immunoblot analysis. This technique empowers the investigation of specific molecular and cellular functions within the intricate microenvironment of the brain, comprising diverse cell types. For complete details on the use and execution of this protocol, please refer to Iguchi et al. (2023).1.

Pathological Roles of INPP5D in Alzheimer's Disease.

Current hypothesis of Alzheimer's disease (AD) postulates that amyloid ß (Aß) deposition in the brain causes tau inclusion in neurons and leads to cognitive decline. The discovery of the genetic association between triggering receptor expressed on myeloid cells 2 (TREM2) with increased AD risk points to a causal link between microglia and AD pathogenesis, and revealed a crucial role of TREM2-dependent clustering of microglia around amyloid plaques that prevents Aß toxicity to facilitate tau deposition near the plaques. Here we review the physiological and pathological roles of another AD risk gene expressed in microglia, inositol polyphosphate-5-polyphosphatase D (INPP5D), which encodes a phosphoinositide phosphatase. Evidence suggests that its risk polymorphisms alter the expression level and/or function of INPP5D, while concomitantly affecting tau levels in cerebrospinal fluids. In ß-amyloidosis mice, INPP5D was upregulated upon Aß deposition and negatively regulated the microglial clustering toward amyloid plaques. INPP5D seems to exert its function by acting antagonistically at downstream of the TREM2 signaling pathway, suggesting that it is a novel regulator of the protective barrier by microglia. Further studies to elucidate INPP5D's role in AD may help in developing new therapeutic targets for AD treatment.

Carbamazepine, a beta-cell protecting drug, reduces type 1 diabetes incidence in NOD mice.

Pancreatic beta-cells are selectively destroyed by the host immune system in type 1 diabetes. Thus, drugs that preserve beta-cell mass and/or function have the potential to prevent or slow the progression of this disease. We recently reported that the use-dependent sodium channel blocker, carbamazepine, protects beta-cells from inflammatory cytokines in vitro. Here, we tested the effects of carbamazepine treatment in female non-obese diabetic (NOD) mice by supplementing LabDiet 5053 with 0.5% w/w carbamazepine to achieve serum carbamazepine levels of 14.98 ± 3.19 µM. Remarkably, diabetes incidence over 25 weeks, as determined by fasting blood glucose, was ~50% lower in carbamazepine treated animals. Partial protection from diabetes in carbamazepine-fed NOD mice was also associated with improved glucose tolerance at 6 weeks of age, prior to the onset of diabetes in our colony. Less insulitis was detected in carbamazepine treated NOD mice at 6 weeks of age, but we did not observe differences in CD4+ and CD8+ T cell composition in the pancreatic lymph node, as well as circulating markers of inflammation. Taken together, our results demonstrate that carbamazepine reduces the development of type 1 diabetes in NOD mice by maintaining functional beta-cell mass.

Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods.

This study is to investigate the change of morphology of the meiotic spindle and the extent of zona hardening relating to the morphological survival and developmental competence of thawed oocytes. Four- to 8-week-old female mice (C57BL/6) primed with an intraperitoneal injection of pregnant mare's serum gonadotropin and human chorionic gonadotropin. Cryopreserved oocytes using two protocols: vitrificaton using ethylene glycol (EG) and slow freezing using propanediol (PROH). The freezing oocytes were thawed and were fertilized and subsequently cultured in vitro. Spindle/chromosome imagery, dissolution of zona pellucida, and post-thawing survival and development were comparable between two groups. The vitrification cryopreservation method proved to be better than the slow-freezing protocol when comparing the frequency of normal-shaped spindle development post-thawing. The difference in the time required for the dissolution of the zona pellucida under treatment of pronase that was determined to exist between the two cryopreservation methods was statistically significant (P<0.005). The survival rate of post-thawed mature oocytes was significantly greater for the vitrification group than it was for the slow-freezing cryopreservation group (P=0.005). The vitrification cryopreservation of mature murine oocytes would appear to be more satisfactory than the slow controlled-rate freezing method as regards the post-thawing oocyte survival and also the incidence of the normal spindle apparatus in the ooplasm.

Apoptosis and kinematics of ejaculated spermatozoa in patients with varicocele.

Increased DNA fragmentation is found in sperm from infertile men. Varicocele is an important cause of male infertility, even though it is present in 15% of men who father children. Semen analysis does not always identify infertility in these patients. Sperm motility is strongly correlated with male fertility potential. The goal of this study was to determine the correlation between apoptosis and kinematics in the ejaculated spermatozoa of patients affected by varicocele. Fresh semen samples were obtained from 30 patients with varicocele and 15 fertile controls. These samples were compared using computer-assisted semen analysis and were assayed to determine the degree of sperm apoptosis. The apoptotic index (AI) was calculated by dividing the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) stained spermatozoa by the total number of Hoechst 33258-stained sperm cells for 300 sperm. Five microscopic fields were analyzed to obtain 5 AIs for each individual. Results demonstrated no significant difference in semen quality and sperm motion characteristics; however, a significantly higher AI (23.05% +/- 4.07%: mean difference +/- SE, 95% CI, 15.06%-31.03%, P <.0001) was identified in the varicocele group than in the fertile controls. We concluded that sperm apoptosis does not seem to correlate with semen quality and sperm kinematics and that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared to normal fertile men.