Influence of histochemical and immunohistochemical stains on polymerase chain reaction.

The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3'-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLE(min)) and the maximum amount of PCR products (CONC(max)). The hematoxylin stain showed a significantly prolonged CYCLE(min) (P < .01) and lower CONC(max) (P < .05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLE(min) (P < .01), although the CONC(max) was not significantly different from that of the control (P = .051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONC(max) (P < .05), whereas the CYCLE(min) was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE(min) (P < .01), and the PCR amplification did not reach the "plateau" level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLE(min) nor CONC(max) being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker.     

Inhibition of passive cutaneous anaphylaxis-associated scratching behavior by mu-opioid receptor antagonists in ICR mice

BACKGROUND: Itching in humans is attenuated by mu-opioid receptor antagonists. ICR mice display increases in scratching behavior upon induction of IgE-mediated passive cutaneous anaphylaxis (PCA), or intradermal injection of compound 48/80 or histamine. METHODS: Cutaneous reactions were induced in ICR mice by IgE-mediated PCA, compound 48/80 and histamine, and the scratching behavior associated with the cutaneous reactions was evaluated. RESULTS: Naloxone and nalmefene reduced the incidence of scratching behavior associated with PCA. Naloxone also inhibited the induction of scratching behavior caused by compound 48/80 and histamine. Naloxone did not affect the increase in vascular permeability caused by PCA and injection of compound 48/80. CONCLUSION: Scratching behavior in mice may be induced by a sensation or a mechanism similar to itching in humans and should become a useful model for examining itching in humans.     

Closed-loop identification of carotid sinus baroreflex transfer characteristics using electrical stimulation

Although random aortic pressure (AOP) perturbation according to a binary white noise sequence enables us to estimate open-loop dynamic characteristics of the carotid sinus baroreflex under closed-loop conditions, the necessity of arterial catheter implantation limits the applicability of this method in freely moving animal experiments. Thus, we explored a closed-loop system identification method using electrical stimulation. In 6 anesthetized and vagotomized rabbits, we stimulated the aortic depressor nerve with a binary white noise sequence (0-10 Hz) under baroreflex closed-loop conditions while measuring cardiac sympathetic nerve activity (SNA) and AOP. We used a closed-loop identification method to estimate the peripheral arc transfer function from SNA to AOP. The peripheral arc transfer function approximated a second-order low-pass filter and its fitted parameters did not differ from those obtained by an open-loop identification method (dynamic gain: 1.16+/-0.32 vs. 1.02+/-0.11; natural frequency: 0.08+/-0.03 vs. 0.09+/-0.03 Hz; damping ratio: 1.53+/-0.15 vs. 1.57+/-0.21). In 6 different rabbits, we applied intermittent rapid pacing (396 beats/min) under baroreflex closed-loop conditions to estimate the neural arc transfer function from AOP to SNA. The neural arc transfer function approximated a first-order high-pass filter and its fitted parameters did not differ from those obtained by an open-loop identification method (dynamic gain: -1.15+/-0.45 vs. -1.06+/-0.05; corner frequency: 0.12+/-0.05 vs. 0.13+/-0.03 Hz). In conclusion, the closed-loop identification method using electrical stimulation is effective to estimate the neural and peripheral arc transfer functions.     

Enkephalin-like immunoreactive neurons and fibers in the ventromedial hypothalamic nucleus

Enkephalin-like immunoreactive (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10-25 microns in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (60-230 nm with a predominance in the 60-80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from unlabeled axonal boutons containing many small, clear vesicles and occasional LCV. The ultrastructural features of the ENK-IR cells in the VMH seemed to correspond to the "common cells" described by Millhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synaptic contacts with the cell bodies and dendrites of unlabeled neurons. The findings suggest that opioid peptides directly influence VMH neurons through synaptic contacts.     

Effects of Daily Matcha and Caffeine Intake on Mild Acute Psychological Stress-Related Cognitive Function in Middle-Aged and Older Adults: A Randomized Placebo-Controlled Study

Matcha, a type of green tea, has a higher amino acid content than other types of tea. We previously examined the ability of matcha to improve cognitive function in older adults and determined that continuous matcha intake improves attention and executive function. This study aimed to compare the effects of matcha and caffeine and clarify the differences between these effects. The study was registered at the University Hospital Medical Information Network (UMIN000036578). The effect of single and continuous intake was compared, and the usefulness of continuous intake was evaluated under the stress condition. The Uchida–Kraepelin test (UKT) was used to induce mild acute stress, and the Cognitrax was used to evaluate cognitive function. A single dose of caffeine improved attentional function during or after stress loading. The reduced reaction time in the Cognitrax, observed following a single dose of matcha, was likely due to caffeine. The matcha group showed an increase in the amount of work after continuous intake, whereas the caffeine group only showed an increase in the amount of work for the UKT after a single dose. Ingesting matcha with caffeine improves both attention and work performance when suffering from psychological stress compared with caffeine alone.     

The relation between superoxide dismutase in cancer tissue and clinico pathological features in breast cancer

The localization of Cu/Zn- and Mn-superoxide dismutase (SOD) in breast cancer tissue (12 papillotubular carcinomas, 21 solid-tubular carcinomas, 16 scirrhous carcinomas, 1 medullary carcinoma, 1 secreting carcinoma, 1 lobular carcinoma, 1 Paget's disease) was investigated via an immunohistochemical technique using antihuman Cu/Zn- and Mn-SOD antibodies in 10%formalin fixed-paraffin embedded thin sections. Both SODs stained strongly in the normal breast gland, but not clearly in many cancer tissues. Furthermore, Cu/Zn-SOD stained more strongly in well differentiated tubular carcinomas than in poorly differentiated tubular carcinomas. It tended to stain less in tumors which recurred or had a poor outcome, and in tumors with a diploid pattern on DNA flow cytometry. Mn-SOD staining was similar to that of Cu/Zn-SOD, but no significant differences among subgroups was found, since the incidence of positively staining tumors was too small in all groups. The intensity of SOD staining seems to change in relation to cell proliferation and differentiation in breast carcinoma, and may be a prognostic indicator, since SOD decreased in poorly differentiated carcinoma and in tumors which developed distant metastasis. Thus, the localization of SOD in breast cancer tissue can provide useful information for cancer treatment.