Acta Medica Okayama volume59 issue4

We describe a novel method for immunofluorescent detection of multiple antigens in a single paraffin-embedded tissue section. We hypothesized that if fluorescent dyes are resistant to heat treatment, then thermal inactivation of immunoglobulins during antigen detection procedures might make it possible to use multicolor immunofluorescence detection even if the primary antibodies are from the same species. We found that several fluorescent dyes, including fluorescein isothiocyanate (FITC), Cy3 and Cy5, were resistant to heating at 90 degrees Celsius for 15 min, whereas the antigenicities of the primary antibodies were lost completely. This novel method, which uses heat treatment between staining steps, has great advantages for multicolor immunofluorescence because unlabeled primary antibodies from the same species can be used. Therefore, by using this method not only 3 unlabeled mouse monoclonal antibodies but also 3 unlabeled rabbit antisera can be used as primary antibodies for multicolor immunofluorescence.

A 67-year-old male visited his physician because of a 2-month history of cough and sputum. An abnormal shadow at the left upper mediastinum on chest x-ray film was detected, and the patient was referred to our department for further examination. Chest x-ray film revealed a round shadow at the left upper posterior mediastinum. Computed tomography(CT)revealed a uniform iso density mass about 4 cm in diameter, with a well-defined border. After the intravenous contrast administration, a slight peripheral enhancement was seen around the mass. On magnetic resonance imaging, the mass was hypointense in T1-weighting and hyperintense in T2-weighting. The contrast pattern was the same as that observed in the CT scan. On sagittal and coronal sections, the mass was adjacent to the aortic arch. Although a benign tumor was mostly suspected based on imaging findings, a malignant tumor was also possible. Accordingly, we resected this mass with video-assisted thoracoscopy. Findings at operation were a cystic mass. The pathological findings were compatible with benign parathyroid cyst, which was suspected to be the cystic degeneration of a parathyroid adenoma.

The nuclear matrix is an operationally defined nuclear skeletal structure that is believed to be involved in many nuclear functions including DNA replication, transcription, repair, and prem RNA processing/transport. Until relatively recently, the nuclear matrix was thought to be a rigid and static structure, but it is now thought to be dynamic. This paradigm shift was based in part on the tracking of the intranuclear movement of proteins tagged with fluorochromes. In this review, we attempt to redefine the nuclear matrix in light of recent findings and describe some useful techniques for the dynamic analysis of nuclear function.

Changes in brain vascularity in adult rats during adaptation to chronic normobaric hypoxia with or without elevated CO(2) were morphometrically investigated. Immunohistochemistry with anti-rat endothelial cell antigen (RECA-1) antibody was carried out for the vascular analysis. After the rats were subjected to hypoxia for 2 to 8 weeks (wks)(10 percent O(2) in N(2)), the total area of blood vessels was measured in 6 brain regions. After 2 wks of hypoxia, the blood vessel area was found to be significantly increased in the frontal cortex, striatum, hippocampus, thalamus, cerebellum, and medulla oblongata, by 44% , 96% , 65% , 50% , 102% and 97% , respectively. The ratio of large vessels with an area > 500 micro m(2) was also increased in all brain regions. Hypoxic adaptation in brain vascularity did not change during 8 wks of hypoxia, and the hypoxia-induced levels measured in the vasculature returned to control levels 2 wks after the termination of hypoxia in areas of the brain other than the cortex and thalamus. In addition, hypoxia-induced changes in terms of the total vascular area and vessel size distribution were significantly inhibited by the elevation in CO(2), whereas chronic hypercapnia without hypoxia had no effect on brain vascularity. These findings suggested that adaptations in brain vascularity in response to hypoxia are rapidly induced, and there are regional differences in the reversibility of such vascular changes. Carbon dioxide is a potent suppressor of hypoxia-induced vascular changes, and may play an important role in vascular remodeling during the process of adaptation to chronic hypoxia.

We evaluated the visceral adipose tissue accumulation in university students in Okayama prefecture, Japan. Fifty-eight Japanese university students (10 men and 48 women, age 18.4 +/- 0.6 years)were enrolled in this study. Fat distribution was evaluated by visceral fat (V) and subcutaneous fat (S) areas measured with computed tomography (CT) scanning at umbilical levels. Anthropometric parameters,i.e., height, weight, waist circumference, hip circumference, and body fat percentage; blood examination; and blood pressure (BP) were also measured. In 58 subjects, the V area was 23.4 +/- 21.0 cm(2) and the S area was 122.5 +/- 57.9 cm(2). V areas were significantly correlated with hepatic enzymes, uric acid, triglyceride, and BP in men, while they were weakly correlated with hepatic enzymes, triglyceride, and high density lipoprotein (HDL) cholesterol in women. Correlation coefficients between V areas and clinical parameters were comparatively higher than those between other body composition parameters,i.e., S areas, weight, body mass index (BMI), body fat percentage, and clinical parameters. The present study suggests that visceral adipose tissue accumulation is important for hepatic enzymes, uric acid, triglyceride, and BP in university students.

Macrophages and microglia are implicated in spinal cord injury, but their precise role is not clear. In the present study, activation of these cells was examined in a spinal cord injury model using 2 different antibodies against ED1 clone and ionized calcium binding adaptor molecule 1 (Iba1). Activation was observed at 1, 4, 8, and 12 weeks after contusion injury and was compared with sham operated controls. Our results indicate that activation could be observed in both the dorsal funiculus and the ventral white matter area in the spinal cord at 5 mm rostral to the epicenter of injury. For both cells, there was a gradual increase in activation from 1-4 weeks, followed by down-regulation for up to 12 weeks. As a result, we could stain macrophages by ED1 and microglia by Iba1. We concluded that macrophages may play a role in the phagocytosis of denatured dendrites after spinal cord injury, while microglia may have some cooperative functions, as they were found scattered near the macrophages.

Primary aorto-enteric fistula (PAEF)is a serious complication of abdominal aortic aneurysm(AAA). We report a patient with PAEF associated with inflammatory AAA who underwent emergent surgery. A 52-year-old male presented with recurrent hematemesis. A computer tomography scan showed a sealed rupture of the AAA adjacent to the duodenum. At surgery, a coin-sized PAEF was noted. The aorta was replaced with a Dacron graft in situ . Histological examination revealed the characteristics of an inflammatory AAA. The postoperative course was uneventful, and there has been no evidence of infection during a follow-up period of 3 years. We discuss the etiologic and surgical considerations regarding this unusual entity.

The effects of irradiation on different cell compartments in the submandibular gland were analyzed in adult C57BL/6 mice exposed to X-ray irradiation and followed up for 10 days. Apoptosis was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method (TUNEL). Cell proliferation was detected using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Radiation-induced apoptosis occurred rapidly, reaching a maximum 3 days post-irradiation. The percentage of apoptotic cells increased with the irradiation dose. At day 1 post-irradiation, cell proliferation was significantly reduced in comparison to sham-irradiated controls. After post-irradiation arrest of the cell cycle, proliferation increased in all gland compartments, reaching a maximum at day 6 post-irradiation. The proliferation response corresponded to the dose of irradiation. We suggest that the reason for gland dysfunction could be the coexistence of high apoptotic and proliferative activity in the irradiated gland.