Histochemistry and Cell Biology最新文献

Our sense of hearing starts in the inner ear organ, the cochlea, which contains two types of auditory hair cells for signal transduction. Earlier research showed that the complex cochlear physiology is regulated in part by purinergic signalling through activation of purine-mediated P2X, P2Y and adenosine receptors expressed in the cochlea. This study aims to extend our knowledge of purinergic signalling in the cochlea by comprehensively characterising the expression of the P2X4 receptor subtype. Wistar rat cochlea (embryonic day 20.5-6 weeks, both sexes) were collected, and the P2X4 expression was examined by immunohistochemistry. Robust P2X4 expression was found in the organ of Corti (OoC) in the inner hair cells (IHCs) and outer hair cells (OHCs), confirmed by double-labelling with hair cells (HCs) marker myosin VIIa. In IHCs, a robust cytoplasmic P2X4 expression occurred throughout the cell body, with the most intense signal at the medial side. In OHCs, P2X4 formed puncta near the apical and basal ends of the cell body. Using markers for subcellular organelles, P2X4 immunoreactivity was predominately associated with the trans-Golgi network apparatus and early endosomes in IHC and with early endosomes and lysosomes in OHC in the mature cochlea. In both cell types, some co-localisation of P2X4 with presynaptic marker was also observed. Taken together, these observations suggest unique roles for P2X4 in mature IHCs and OHCs as a purinergic receptor subtype responsible for the homeostatic regulation of hair cells and auditory sensory transduction.

Immunocytochemistry and immunohistochemistry are among the most widely used and valuable methods for localizing and quantifying proteins in cells and tissues. However, these methods have several limitations, and they are still being improved. A notable problem in this area is the restricted accessibility of some dense intracellular structures, such as nucleoli, to specific antibodies. This reduced accessibility led to peripheral staining of the nucleoli even in cases of relatively homogeneous distribution of the antigen within the nucleoli. This review aims to elucidate the factors impeding antibody accessibility to the internal regions of the nucleolus in somatic cells and nucleolus-like bodies in oocytes and to explore the methods used to circumvent this limitation.

Inflammation is a key contributor to the development of preeclampsia. Recent studies suggest that circular RNAs (circRNAs) may serve as potential therapeutic targets for this disease, though their specific functions remain incompletely understood. In this study, we investigated the role of hsa_circ_0003314 in preeclampsia pathogenesis. The interaction between hsa_circ_0003314 and microRNA (miR)-1827 was validated using RNA pull-down and luciferase reporter assays, while the binding of miR-1827 to the 3'-UTR of caspase-5 was confirmed by RNA immunoprecipitation and luciferase reporter assays. Pyroptotic cells were quantified by flow cytometry based on the percentage of caspase-1/propidium iodide (PI) double-positive cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure interleukin (IL)-1β concentrations in the culture supernatant. The migration and invasion abilities of HTR-8/SVneo cells were evaluated using Transwell assays. We found that hsa_circ_0003314 expression was upregulated in HTR-8/SVneo cells subjected to hypoxia/reoxygenation (H/R) treatment. Silencing hsa_circ_0003314 enhanced cell migration, invasion, and epithelial-mesenchymal transition (EMT), while reducing the expression of pyroptosis-related proteins, GSDMD-N and HMGB1. The proportion of pyroptotic cells was significantly decreased upon hsa_circ_0003314 knockdown in H/R-treated cells. Mechanistically, hsa_circ_0003314 functions as a molecular sponge for miR-1827, thereby regulating caspase-5 expression. Notably, caspase-5 overexpression rescued the effects of hsa_circ_0003314 knockdown, restoring pyroptosis markers and suppressing the enhanced migratory and invasive behavior of HTR-8/SVneo cells. In conclusion, silencing hsa_circ_0003314 promotes migration, invasion, and EMT in H/R-treated HTR-8/SVneo cells by inhibiting caspase-5-mediated pyroptosis through the sequestration of miR-1827. These findings identify hsa_circ_0003314 as a promising therapeutic target in the treatment of preeclampsia.

Membrane palmitoylated protein 6 (MPP6), a membrane skeletal protein, is expressed not only in the peripheral nervous system (PNS) but also in the central nervous system (CNS). In this study, we investigated the localization of MPP6 and its associated protein complexes in the mouse cerebrum, as well as its effects on behavior using MPP6 protein-deficient (Mpp6 -/-) mice. MPP6 was detected in mouse cerebral lysates and synaptic membrane fractions, where it formed protein complexes with other MPP family members, including MPP1, MPP2, and calcium/calmodulin-dependent serine protein kinase (CASK). However, the amounts of these complexes did not differ between Mpp6 -/- and wild-type (Mpp6 +/+) mice. Immunohistochemistry revealed that MPP6 was localized at synapses throughout the cerebrum, particularly in the postsynaptic regions. Ultrastructural analysis showed that synaptic cleft distances and postsynaptic density thickness were slightly reduced in Mpp6 -/- mice compared with Mpp6 +/+ mice. In the elevated plus-maze test, a Mpp6 -/- mouse exhibited unusual behavior not observed in Mpp6 +/+ mice, although there was no statistically significant difference in the time spent in the open and closed arms between the two groups. Locomotor activity measurements revealed that MPP6 -/- mice were more active at midnight and less active from morning to noon than Mpp6 +/+ mice, implying alterations in sleep-wake regulation. These findings suggest that MPP6 plays a role in synaptic function by forming protein complexes with other MPP family members and signaling proteins.

The coronavirus disease 2019 (COVID-19) pandemic has passed; however, its long-term effects are yet to be determined. Pregnant women and their neonates faced a higher risk for complications during this pandemic as COVID-19 was reported to result in oxidative and inflammatory stress and the cytokine storm, which would impact pregnancy, namely the trophoblast invasion and placental development and functioning. Therefore, this study aims to determine the effect of COVID-19 on the placental functioning in South African pregnancies through the analysis of kisspeptin and placental morphology. Immunohistochemical analyses of placental samples were performed to detect the expression of kisspeptin. Histopathological analysis was conducted to identify vascular and inflammatory alterations. This study demonstrated that COVID-19 results in a significantly increased expression of placental kisspeptin in both the central (p = 0.001) and peripheral (p < 0.0001) regions as compared with the placentae from control pregnancies. Upon further analysis, the placentae from COVID-19 pregnancies also presented with severe inflammation and maternal and fetal vascular malperfusion compared with the control placentae. A significantly increased expression of placental kisspeptin was observed in COVID-19 positive pregnancies, implying impaired placental functioning. This was further supported by vascular and inflammatory alterations observed in COVID-19-positive placentae, which may suggest that trophoblast invasion was compromised. To date, there still exists small clusters of COVID-19 outbreaks, and our findings highlight the importance of the future surveillance of these mothers and neonates in COVID-19 pregnancies in South Africa, as neonates from other countries have presented with abnormalities.

Estrogen affects a variety of brain functions, including higher functions. The estrogen level is persistently high during pregnancy and then declines rapidly with delivery, which causes the brain to be subjected to marked effects during this period. Thus, pregnant and parturient women are prone to altered mental activity and have a higher probability of anxiety and depressive disorders, which may be caused by marked changes in estrogen levels. The central nucleus (CeA) and basolateral nucleus (BLA) of the amygdala and the anterior division (BNSTa) and principal nucleus (BNSTp) of the bed nucleus of the stria terminalis are regions involved in emotional processing, including anxiety, and also target brain areas of estrogenic action. However, the expression of estrogen receptors during this period is not clear. In this study, we analyzed the distribution of estrogen receptor α (ERα)-immunoreactive cells at gestational days 7 (G7) (early gestation), 14 (G14) (mid-gestation), 21 (G21) (late gestation), and 4 days after delivery (P4) (early postpartum) in rats. In the CeA, the number of nuclear ERα-positive cells decreased significantly at G7, G14, and G21. In the BLA, distribution of ERα was detected in the cell body (indicating membrane-associated ERα) with a trend toward decreased immunoreactivity during pregnancy. The BNSTp showed no changes, but in the BNSTa, nuclear ERα-positive cells were significantly reduced at G14, G21, and P4. These regional differences in changes of ERα-positive cells may be involved in the symptoms of anxiety and depression during pregnancy and the postpartum period.

This research investigated the effects of benzimidazole (BZ) and electrical stimulation (ES) on peripheral nerve regeneration after short- and long-term injury and assessed functional recovery by means of stereological, histological, and electrophysiological analyses. Fifty-four male albino Wistar rats were divided into nine groups of six animals each. No treatment or surgery was applied to the control (CONT) group. The sciatic nerve was crushed for 5 s in the short-term injury (STI) and for 60 s in the long-term injury (LTI) groups. In the STI + BZ group and the LTI + BZ group, the rats received 25 mg/kg/day of BZ via oral gavage for 28 days. In the STI + ES and LTI + ES groups, a 3-V current was applied for 20 min daily for 28 days. In the STI + BZ + ES group and the LTI + BZ + ES groups, 3-V ES was applied for 20 min per day for 28 days following oral administration of BZ at 25 mg/kg/day for 28 days. All groups were subjected to electrophysiological, electron microscopic, stereological, and statistical analyses. The stereological analyses revealed a significant increases in the numbers of myelinated axons in the STI + ES groups compared with the STI (p < 0.01). BZ treatment yielded no significant differences in the numbers of myelinated axons in the groups (p > 0.05). Histological evaluation of the STI and LTI groups showed that ES and BZ treatment positively affect the histological structure of the nerve.

Salamander Pleurodeles waltl is an emerging animal model for developmental and regenerative biology studies. However, the exploration of skeletal muscle regeneration has been hindered by the absence of suitable in vitro cell systems for in-depth mechanism research. In this study, we established a protocol for the cultivation of muscle stem cells derived from Pleurodeles waltl for cell biology experiments. Trunk and limb muscles were minced and digested with collagenase. Cells with a high nucleoplasmic ratio were isolated from the muscle tissue. Immunofluorescence and RT-PCR analysis revealed that these proliferating cells expressed the typical muscle stem cell markers. Furthermore, these cells demonstrated effective myogenic differentiation in vitro, as evidenced by the expression of the myogenic differentiation marker protein, myosin heavy chain. Additionally, it was observed that cultured myotubes derived from these cells initiated DNA synthesis and upregulate cell cycle genes upon stimulation with a high concentration of serum. Notably, the muscle stem cells (Pw-1) maintained a steady proliferation rate even after undergoing 35 subcultures. In conclusion, this study has successfully established a method for isolating and cultivating muscle stem cells from salamanders, confirming the dedifferentiation potential of the myotubes derived from these cells. This methodology provides a valuable tool for exploring the molecular mechanisms that govern skeletal muscle regeneration.

Pulmonary fibrosis (PF) is an insidious, progressive, and fatal age-associated disease that occurs primarily in older adults and has a poor prognosis. Alveolar epithelial cell (AEC) senescence is the critical pathological mechanism of PF. The accumulation of oxygen radicals, commonly referred to as reactive oxygen species (ROS), strongly contributes to cellular senescence. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor. Triggering via TREM-1 results in ROS, leading to the amplification of inflammation. However, whether TREM-1 is involved in PF by inducing oxidative stress to exacerbate AEC senescence remains unclear. We first observed that blockade of TREM-1 during the fibrotic phase attenuated bleomycin (BLM)-induced PF in mice, with decreased expression of senescence-related proteins, including p16, p21, p53, and γ-H2AX, in the lung tissue. Moreover, TREM-1 blockade during the fibrosis stage restored antioxidant levels by increasing the percentage of Nrf2- and HO-1-positive cells in mice with PF. Notably, TREM-1 was highly expressed in surfactant-associated protein (SPC)-positive AECs in mice with PF. In vitro, blocking TREM-1 activated Nrf2 antioxidant signaling, thereby decreasing intracellular ROS levels and diminishing BLM-induced senescence in AECs. Furthermore, inhibition of Nrf2/HO-1 partially counteracted the anti-senescence effect of blocking TREM-1 in BLM-treated AECs. In this study, we reported that TREM-1 stimulated the senescence of AECs, induced ROS and exacerbated PF. We also provide compelling evidence suggesting that the Nrf2/HO-1 signaling pathway underpins TREM-1-triggered senescence. Therefore, our findings provide new insights into the molecular mechanisms associated with TREM-1 and AEC senescence in the pathogenesis of PF.