Are breast tumours innervated? Immunohistological investigations using antibodies against the neuronal marker protein gene product 9.5 (PGP 9.5) in benign and malignant breast lesions.

The aim of the present study was to assess the innervation pattern of benign and malignant breast lesions using the neuronal marker protein gene product (PGP) 9.5. An unlabelled antibody technique (using streptavidin biotin complex formation) was used on paraffin wax sections of tissues fixed in neutral buffered formalin. In 2/4 cases of chronic mastopathy, PGP 9.5 immunoreactivity was seen in relation to blood vessels and the ductal system. No immunoreactivity for PGP 9.5 was seen in the affected tissues of 9/10 cases of fibroadenomata. In 9/16 breast cancers, PGP 9.5-labelled perivascular nerve fibres were detected in connective tissue stroma supporting carcinoma tissue, though not in the immediate vicinity of such tumour tissue. Labelled nerve fibres were detected in large bundles at the periphery of tumours, possibly unrelated to the latter. Our results indicate that the newly formed blood vessels within a tumour are not innervated, though major blood vessels which supply the tumour are innervated.

An immunohistochemical and electron microscopic study of the peri-ureteric ganglia of the guinea-pig.

The neuropeptide- and catecholamine-synthesizing enzyme content and ultrastructure of the peri-ureteric ganglia of guinea-pigs were investigated. Small numbers of neuronal perikarya were present at frequent intervals forming ganglia close to, and along the entire length of, the ureter. Each of these ganglia was surrounded by a connective tissue capsule, and was located in the peri-ureteric connective tissues. Within each ganglion were typical nerve terminals and varicosities containing small, clear synaptic vesicles or synaptic vesicles with an electron-dense core, or a mixture of the two. In the ganglia, immunoreactivity to tyrosine hydroxylase, dopamine beta hydroxylase, neuropeptide tyrosine, or vasoactive intestinal peptide was present in neuronal perikarya; immunoreactivity to substance P or leucine enkephalin was present in nerve terminals and varicosities. Electron-microscopic immunogold studies indicated that there was no coexistence of substance P and enkephalin in the nerve terminals, unlike related ganglia in the pelvis of guinea-pigs.

Localization of substance P and leucine enkephalin in the nerve terminals of the guinea pig paracervical ganglion.

Immunoreactivities (IR) of substance P and leucine enkephalin have been demonstrated in the guinea-pig paracervical ganglion by an immunogold electron microscope method. Both substance P-IR and leucine enkephalin-IR were detected in large synaptic vesicles with electron-dense cores. The former neuropeptide was detected in nerve terminals and varicosities comprised mainly of large vesicles with electron-dense cores; the latter was detected in nerve terminals and varicosities that also included small, clear synaptic vesicles. In a minority of nerve terminals and varicosities coexistence of both immunoreactivities could be demonstrated within vesicles with an electron-dense core. Also present in these nerve terminals and varicosities were small, clear synaptic vesicles, though these were unreactive.

Projections of the guinea-pig paracervical ganglion to pelvic viscera.

The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine beta-hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters.

Morphological, histochemical and immunohistological studies of the paracervical ganglion in prepubertal, pregnant and adult, non-pregnant guinea-pigs.

The morphology and ultrastructure of the paracervical ganglion were examined in prepubertal and pregnant guinea-pigs using the light and electron microscope. The neuropeptide and acetylcholinesterase content of the neuronal perikarya and SIF cells, nerve fibres and nerve terminals of the ganglion were examined in prepubertal, adult, non-pregnant and pregnant guinea-pigs. The ganglion consisted of up to seven different sized clusters of neuronal perikarya lying parallel to the long axis of the uterovaginal junction, with 60 or more neuronal perikarya in any one cluster in the paracervical connective tissues. The number of neuronal perikarya within each cluster was related to the total area of each cluster. The light and electron microscopy of the clusters was typical of autonomic ganglia, though no vacuolated neurons were observed as has been reported in the rat. The neuropeptide and acetylcholinesterase content of the ganglionic clusters was not different in prepubertal and adult, non-pregnant guinea-pigs. In pregnant guinea-pigs there was an apparent small decrease in numbers of neuronal perikarya containing the neuropeptides VIP, NPY or TH immunoreactivities, though no quantitative studies were undertaken. In pregnancy no SIF cells were detected, though in prepubertal and non-pregnant, adult guinea-pigs these cells contained TH, NPY or SP immunoreactivity.

The co-localisation of substance P and VIP in cholinergic-type terminals of the rat parotid gland.

Parotid glands from the rat were examined for substance P (SP) and vasoactive intestinal polypeptide (VIP)-like immunoreactivity with the peroxidase-antiperoxidase (PAP) and immunogold methods. The majority of nerve terminals associated with the acinar secretory cells contained numerous small agranular vesicles measuring 40-60 nm in diameter and a few larger vesicles which had an electron-dense core and measured 90-120 nm in diameter. Electron-dense peroxidase reaction product indicative of SP and/or VIP-like immunoreactivity was found within the larger dense-cored vesicles and attached to the outer membrane of the small agranular vesicles. Some nerve terminals associated with the acinar cells contained no reaction product irrespective of whether sections were incubated for SP or VIP. With the immunogold method gold particles indicative of SP and/or VIP-like immunoreactivity were found associated with the larger dense-cored vesicles with very little gold labelling over the small agranular vesicles. When ultrathin sections were incubated for both SP and VIP-like immunoreactivity all of the labelled terminals examined contained gold particles indicative of the presence of both peptides. In several terminals individual dense-cored vesicles contained gold particles of different sizes which indicates co-existence of SP and VIP within the same vesicle. Several nerve terminals associated with the acinar cells contained no gold labelling of their synaptic vesicles. Occasionally nerve terminals were found around blood vessels that were positive for SP-like immunoreactivity and VIP-like immunoreactivity but none was found, using the immunogold method, that contained both peptides. Very few nerve terminals were found associated with ducts and none contained reaction product or gold particles indicative of SP or VIP-like immunoreactivity. The ultrastructural features of the nerve terminals containing SP and/or VIP-like immunoreactivity could not be distinguished from those that have been described as representing cholinergic terminals. The fact that the postganglionic parasympathetic secretomotor neurons contain, in addition to acetylcholine, two neuropeptides and the possible functional implications thereof are discussed.

Axonal regeneration through a peripheral nerve implanted into a brain cavity.

A cavity was prepared in the rat parietal cortex by suction, filled with gel foam and left for 3 weeks during which time it became highly vascularised. Into this 3-week-old capillary bed a 5 mm length of autologous common peroneal nerve was implanted. Animals were killed at various time intervals up to 7 months after implantation of the nerve segment. The ultrastructural features of the vascular bed before and after implantation of the nerve segment were compared. In the absence of a peripheral nerve implant no axons were found within the cavity. However, at 5 weeks after implantation numerous axon-like profiles and capillaries containing fenestrations were observed within the implant. Eight weeks after implantation of the peripheral nerve both myelinated and non-myelinated axons were observed within the implant and in the surrounding capillary bed. No obvious increase in the number of axons was observed with increasing time periods. To investigate the origin of the axons within the vascular bed and/or implant the fluorochrome true blue was injected into the cavity 7 months after implantation of the nerve. Three days later selected areas of the brain, the trigeminal, superior cervical and otic ganglia were examined for retrogradely labelled fluorescent cells. Labelled cells were found adjacent to the cavity and in the ipsilateral trigeminal and superior cervical ganglia. The significance of these results in relation to the enhancement of axonal regeneration from the damaged central nervous system (CNS) is discussed.

An ultrastructural study of the early stages of axonal regeneration through rat nerve grafts.

Segments of rat sciatic nerve 5 mm long were removed and either maintained alive in tissue culture medium or killed by freeze-drying. Twenty-four h later the nerve segments were replaced as autografts. Animals were killed 3-14 days after grafting. Grafts of cultured nerves (C-grafts) always contained many living cells. Grafts of freeze-dried nerves (FD-grafts) contained few living cells at 3 days, but were repopulated by 7 days. A few regenerating axons were identified in the most proximal parts of 3 day C-grafts and by 14 days many myelinated axons extended to the distal ends. Axons were absent from 3 and 7 day FD-grafts, but by 14 days some non-myelinated axons extended to the distal end of such grafts. Regenerating axons were always associated with Schwann cells. Small perineurial compartments were formed at the junctional zones of all grafts and throughout the FD-grafts. Revascularization of the FD-grafts was delayed when compared to that in C-grafts. Fenestrated capillaries were observed in both types of graft. These experiments demonstrate that axons regenerate through FD-grafts that have been repopulated by cells and the grafts probably lack the normal perineurial and blood/nerve diffusion barriers. The significance of these results is discussed in relation to the requirements for successful axonal regeneration.