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Combined retrograde tracing (using fluorescent tracer Fast blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons projecting to the trapezius muscle in mature male rats (n=9). As revealed by retrograde tracing, Fast blue-positive (FB+) neurons were located within the ambiguous nucleus and accessory nucleus of the grey matter of the spinal cord. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (ChAT)-positive]. Retrogradely labelled neurons displayed also immunoreactivities to calcitonin gene-related peptide (CGRP; approximately 60% of FB+ neurons), nitric oxide synthase (NOS; 50%), substance P (SP; 35%), Leu5-Enkephalin (LEnk; 10%) and vasoactive intestinal polypeptide (VIP; 5%). The analysis of double-stained tissue sections revealed that all CGRP-, VIP- and LEnk-immunoreactive FB+ perikarya were simultaneously ChAT-positive. The vast majority of the neurons expressing SP- or NOS-immunoreactivity were also cholinergic in nature; however, solitary somata were ChAT-negative. FB+ perikarya were surrounded by numerous varicose nerve fibres (often forming basket-like structures) immunoreactive to LEnk or SP. They were also associated with some CGRP-, NOS- and neuropeptide Y-positive nerve terminals.
The present study was carried out on sexually mature boars. All the animals were injected with fast blue into right testis and then divided into four groups (G1- control animals, G2 - hemicastrated, G3 - castrated, and G4 - castrated and injected with testosterone). After 3 weeks, G1 pigs were transcardially perfused. In G2 pigs right testes, whereas in G3 and G4 animals both testes were removed. G4 pigs were injected with testosterone. After 2 weeks, the pigs were transcardially perfused and then their caudal mesenteric ganglia (CaMG) and anterior pelvic ganglia (APG) were collected. The ganglia were cut into 12 µm-thick cryostat sections. Sections were stained using antisera against TH or DßH, VACHT or CHAT, NPY, VIP and GAL, and androgen receptor (AR). Immunohistochemical staining of CaMG-sections revealed that approximately 74% of FB-positive (FB⁺) neurons contained immunoreactivity to TH or DßH, whereas 4% of FB⁺ cells were VACHT-positive. Among FB⁺/DßH⁺ neurons, 72% contained NPY and 2% stained for GAL. All FB⁺/VACHT⁺ neurons were also VIP⁺. 62% of FB⁺ somata were NPY⁺, whereas 6% stained for VIP. In all experimental animals, numbers of FB⁺perikarya immunoreactive to TH (approx. 30%) and DßH (approx. 50%) were smaller than those found in G1 animals, whereas numbers of neurons displaying immunoreactivity to other substances studied were higher. The most significant increases regarded those expressing GAL (approx. 30%) and VIP (approx. 20%) whereas less distinct changes dealt with NPY⁺ and VAChT⁺ or ChAT⁺ neurones. In APG, 60% of FB⁺ neurons contained immunoreactivity to TH or DßH, whereas 12% of FB⁺ cells were VACHT-positive. Among FB⁺/DßH⁺ neurons, 55% contained NPY and 3% stained for GAL. All FB⁺/VACHT⁺ neurons were also VIP⁺. 46% of FB⁺ somata were NPY-IR, whereas 19% stained for VIP. In all experimental pigs, the immunohistochemical properties of the APG FB⁺ neurones were similar to those found in relation to CaMG-perikarya. Neurones of both studied ganglia were surrounded by dense networks of VACHT-positive nerve fibres. The most apparent changes in the immunohistochemical features of the FB⁺ neurons evoked by bilateral castration were observed in G3 pigs; whereas changes found in G4 were very similar to those observed in G2 animals.
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