Journal of Histochemistry & Cytochemistry最新文献

Although perineurium has an important role in maintenance of the blood-nerve barrier, understanding of perineurial cell-cell junctions is insufficient. The aim of this study was to analyze the expression of junctional cadherin 5 associated (JCAD) and epidermal growth factor receptor (EGFR) in the perineurium of the human inferior alveolar nerve (IAN) and investigate their roles in perineurial cell-cell junctions using cultured human perineurial cells (HPNCs). In human IAN, JCAD was strongly expressed in endoneurial microvessels. JCAD and EGFR were expressed at various intensities in the perineurium. In HPNCs, JCAD was clearly expressed at cell-cell junctions. EGFR inhibitor AG1478 treatment changed cell morphology and the ratio of JCAD-positive cell-cell contacts of HPNCs. Therefore, JCAD and EGFR may have a role in the regulation of perineurial cell-cell junctions.

Gastrointestinal symptoms are common health problems found during aging and neurodegenerative diseases. Trimethyltin-induced rat is known as an animal model of hippocampal degeneration with no data on enteric neurodegeneration. This study aimed to investigate the effect of trimethyltin (TMT) induction on the gastrointestinal tract. A 28-day animal study with male Sprague-Dawley rats (3 months old, 150-200 g) given a single TMT injection (8 mg/kg body weight, intraperitoneal) was conducted. The number of neurons in the colonic myenteric plexus was measured using stereological estimation. Histological scoring of colon inflammation, immunohistochemistry of tumor necrosis factor-α (TNF-α), and quantitative PCR were conducted. This study showed neuronal loss in the colonic myenteric plexus of TMT-induced rat model of neurodegeneration. Minor colon inflammation characterized by inflammatory cell infiltration and slightly higher expression of TNF-α in the colon mucosa were observed in the TMT-induced rat. However, the gut microbiota composition of the TMT-induced rat was not different from that of the control rats. This study demonstrates that TMT induces colonic myenteric plexus neurodegeneration and minor colon inflammation, which suggests the potential of this animal model to elucidate the communication between the gastrointestinal tract and central nervous system in neurodegenerative diseases.

In multi-analyte extractions, tissue is typically homogenized in a lysis buffer, and then DNA, RNA, and protein are purified from the supernatant. However, yields are typically lower than in dedicated, single-analyte extractions. In a two-part experiment, we assessed whether yields could be improved by revisiting the normally discarded, post-homogenized tissue debris. We initially performed additional homogenizations, each followed by a simultaneous extraction. These yielded no additional RNA, 13% additional DNA (which became progressively more degraded), and 161.7% additional protein (which changed in proteome when analyzed using SDS-PAGE). We then digested post-homogenized tissue debris from a simultaneous extraction using proteinase K and extracted DNA using silica spin columns or alcohol precipitation. An average additional DNA yield of 27.1% (silica spin columns) or 203.9% (alcohol precipitation) was obtained with/without compromising DNA integrity (assessment by long-range PCR, DNA Integrity Numbers, and size at peak fluorescence of electropherogram). Validation using a cohort of 65 tissue blocks returned an average additional DNA yield of 31.6% (silica columns) and 54.8% (alcohol precipitation). Users can therefore refreeze the homogenized remnants of tissue blocks rather than disposing of them and then perform additional DNA extractions if yields in the initial multi-analyte extractions were low.

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O₂) or normoxia (21% O₂). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

This commentary reflects on the significance and impact of the highly cited companion article that was published in the Journal of Histochemistry and Cytochemistry in 1997 (Gijlswijk RPM et al. Fluorochrome-labeled Tyramides: Use in Immunocytochemistry and Fluorescence In Situ Hybridization. Journal of Histochemistry & Cytochemistry. 1997;45(3):375-382).

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.

Preeclampsia is a complication of pregnancy that affects 3-5% of pregnancies and is one of the major causes of maternal/neonatal mortality and morbidities worldwide. We aimed to investigate the distribution of Foxp3+ regulatory T-cells and CD68+ Hofbauer cells in the placenta of preeclamptic and healthy pregnant women with a special focus on correlating these findings with placental histology. Decidua and chorionic villi of the placenta obtained from healthy and preeclamptic pregnancies were evaluated in full-thickness sections. Sections were stained with hematoxylin and eosin and Masson's trichrome and immunostained for Foxp3 and CD68 for histological analyses. The total histomorphological score for placentas was found to be higher in preeclamptic placentas than that in the controls. The CD68 immunoreactivity was higher in the chorionic villi of preeclamptic placentas than that in the controls. The immunoreactivity of Foxp3 was found widely distributed within the decidua in both the groups and did not differ significantly. Interestingly, Foxp3 immunoreactivity in the chorionic villi was found mainly in the villous core and, to a lesser extent, in the syncytiotrophoblasts. We found no significant relation between Foxp3 expressions and morphological changes observed in preeclamptic placentas. Although extensive research is being carried out regarding the pathophysiology of preeclampsia, the findings are still controversial.

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 RNA has been found in the human testis on occasion, but subgenomic SARS-CoV-2 and infectious SARS-CoV-2 virions have not been found. There is no direct evidence of SARS-CoV-2 infection of testicular cells. To better understand this, it is necessary to determine whether SARS-CoV-2 receptors and proteases are present in testicular cells. To overcome this limitation, we focused on elucidating with immunohistochemistry the spatial distribution of the SARS-CoV-2 receptors angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147), as well as their viral spike protein priming proteases, transmembrane protease serine 2 (TMPRSS2) and cathepsin L (CTSL), required for viral fusion with host cells. At the protein level, human testicular tissue expressed both receptors and proteases studied. Both ACE2 and TMPRSS2 were found in interstitial cells (endothelium, Leydig, and myoid peritubular cells) and in the seminiferous epithelium (Sertoli cells, spermatogonia, spermatocytes, and spermatids). The presence of CD147 was observed in all cell types except endothelium and peritubular cells, while CTSL was exclusively observed in Leydig, peritubular, and Sertoli cells. These findings show that the ACE2 receptor and its protease TMPRSS2 are coexpressed in all testicular cells, as well as the CD147 receptor and its protease CTSL in Leydig and Sertoli cells, indicating that testicular SARS-CoV-2 infection cannot be ruled out without further investigation.

Skeletal muscle atrophy is represented by a dramatic decrease in muscle mass, and it is related to a lower life expectancy. Among the different causes, chronic inflammation and cancer promote protein loss through the effect of inflammatory cytokines, leading to muscle shrinkage. Thus, the availability of safe methods to counteract inflammation-derived atrophy is of high interest. Betaine is a methyl derivate of glycine and it is an important methyl group donor in transmethylation. Recently, some studies found that betaine could promote muscle growth, and it is also involved in anti-inflammatory mechanisms. Our hypothesis was that betaine would be able to prevent tumor necrosis factor-α (TNF-α)-mediated muscle atrophy in vitro. We treated differentiated C2C12 myotubes for 72 hr with either TNF-α, betaine, or a combination of them. After the treatment, we analyzed total protein synthesis, gene expression, and myotube morphology. Betaine treatment blunted the decrease in muscle protein synthesis rate exerted by TNF-α, and upregulated Mhy1 gene expression in both control and myotube treated with TNF-α. In addition, morphological analysis revealed that myotubes treated with both betaine and TNF-α did not show morphological features of TNF-α-mediated atrophy. We demonstrated that in vitro betaine supplementation counteracts the muscle atrophy led by inflammatory cytokines.