Neuropeptide Y in rat sympathetic neurons is altered by genetic hypertension and by age.

We have used immunocytochemistry to quantitate neuronal neuropeptide Y in superior cervical ganglia of a strain of normotensive Wistar-Otago rats and a related genetically hypertensive strain over the age range 1-60 weeks. The numbers of neuropeptide Y-immunoreactive cells and total ganglionic cell numbers were both greater in ganglia of young normotensive than in those of hypertensive rats. Between 10 and 60 weeks of age, peptide immunoreactivity and total cell numbers both fell in normotensive rat ganglia but remained constant in ganglia from hypertensive rats. Densitometric analysis showed that the concentrations of neuropeptide Y were similar in neurons of age-matched individuals of both strains, but during aging there was a substantial decline in neuronal peptide content that was similar in both strains and that was not accompanied by any decline in neuronal immunoreactivity for tyrosine hydroxylase. Our results suggest that there is a developmental abnormality of neuropeptide Y in sympathetic neurons of this strain of genetically hypertensive rat and that, furthermore, the aging process is accompanied by a selective loss of neuronal neuropeptide Y that is independent of blood pressure status.

The influence of testosterone on the sex-dependent structural asymmetry of the medial habenular nucleus in the chicken.

The volume of the left and right medial habenular nuclei of male and female chicks was analysed for evidence of structural asymmetry. The influence of exogenous testosterone on habenular asymmetry was also examined. Male and female chickens were injected intramuscularly with either testosterone enanthate or an oil vehicle on day 2 (15 animals per group) and were perfused at days 5, 12, and 19 respectively. Paraffin sections (8 microns) containing the medial habenular nucleus were stained with cresyl violet and both left and right medial habenular nuclei were measured by planimetry. In control chicks, males had structural asymmetry at day 12, whereas females did not show any structural asymmetry. Testosterone did not appear to influence asymmetry in the male chick but induced structural asymmetry at all three ages in the female to favour the right hemisphere. This study demonstrates a role for testosterone in influencing structural asymmetry of a nucleus in the vertebrate brain.

Inhibin-related proteins in rat prostate.

Inhibin and activin are members of the transforming growth factor beta (TGF beta) family which can regulate cell proliferation in a number of tissues. The presence of inhibins and the related proteins, activins, in the prostate has been implicated by the detection of activin type II receptors. The aim of this study was to determine whether or not immunoactive (ir) inhibin and ir-activin are present in the rat prostate and to study the acute regulation by androgens. The results showed that mRNAs for the alpha and beta inhibin subunits were detected in rat prostate by reverse transcription-PCR together with ir-inhibin and ir-activin in prostate cytosols. The levels of ir-activin in the prostate (223 +/- 44 ng/gland) were greater than the levels of ir-inhibin (6.89 ng/gland), and activin immunoreactivity was localised to the epithelial cells. The presence of these proteins and the subunit mRNAs suggests that these proteins are produced in the prostate and may have a role in prostate function. The study of the effect of androgen withdrawal on the levels of ir-activin and ir-inhibin in these tissues showed no change in the content of ir-inhibin or ir-activin (ng/g tissue) after 3 days of castration or following the administration of the cytotoxic drug ethane dimethane sulphonate (EDS), although there was a significant (P < 0.01) decline in prostate weight. Fourteen days after EDS treatment, as the prostate weight fell significantly lower, the amount of ir-inhibin and ir-activin per prostate gland was significantly (P < 0.01) reduced although the concentration was unaffected. These data demonstrate, for the first time, that inhibin alpha and beta subunit mRNA and ir-inhibin and ir-activin are present in the prostate; the role of these proteins in prostate function remains to be established.

Growth inhibitory response to activin A and B by human prostate tumour cell lines, LNCaP and DU145.

Activins are growth and differentiation factors which have been shown to have proliferative and antiproliferative actions in many tissues. In addition, they have been implicated in tumourigenesis in reproductive tissues. Although activin and inhibin are present in rat ventral prostate, inhibin beta, but not alpha, subunit proteins have been detected in the human prostate epithelial tumour cell lines LNCaP, DU145 and PC3. With this absence of capacity to produce inhibins, the aims of this study were to determine the effect of activin A and B and follistatin on DNA synthesis by these human prostate tumour cell lines. The results demonstrate a differential response to exogenously added activin A and B on DNA synthesis in vitro by the tumour cell lines. The inhibitory effects were observed on LNCaP cells in the absence or presence of stimulation with 1 nM 5 alpha-dihydrotestosterone and on the androgen-independent DU145 cells, but not the PC3 cells. Activin A caused a dose-dependent inhibition of DNA synthesis and proliferation by LNCaP and androgen-independent DU145 cells which was maximal at 8 ng/ml. The effect of exogenously added activin A was completely reversed by follistatin, but not by inhibin A. The addition of human recombinant FS 288 alone (400 ng/ml) did not have any effect on DNA synthesis, whereas inhibin A alone (400 ng/ml) caused a significant inhibition of DNA synthesis. The capacity of all three cell lines to produce activins and follistatins was demonstrated by the expression of the mRNAs and confirmed by the localisation of immunoreactivity for these ligands to the cytoplasm of the tumour cells. The growth inhibitory response to activins A and B by LNCaP and DU145 cells, and the ability of follistatin to block these effects, suggest that the autocrine interactions between activins and follistatins have a role in the regulation of LNCaP and DU145 prostate tumour cell growth.

Different patterns of immunolocalization of calcitonin gene-related peptide and substance P in sympathetic ganglia of normotensive and genetically hypertensive rats.

Calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity were investigated in the superior cervical ganglion of normotensive and genetically hypertensive Otago Wistar rats by an immunoperoxidase method. CGRP- and SP-positive varicose axons invested separate subpopulations of ganglion cells, neither of which contained neuropeptide Y. The densities of CGRP axons were similar in normotensive and hypertensive rats while the numbers of SP axons were several times higher in the hypertensive strain. Decentralization of the ganglion or chronic capsaicin treatment removed all immunoreactive terminals, indicating that both axon populations are likely to be collaterals from thoracic sensory afferents.

Aspects of current and future inhibin research.

Inhibin was first isolated in 1985. Major progress has been made in defining various aspects of its structure and physiology, using a heterologous radioimmunoassay. Current research is aimed at characterizing the nature of the circulating forms of inhibin and is examining whether there are sex-specific roles for inhibins A and B. It has been recognized that various forms of epithelial and stromal ovarian cancer produce members of the inhibin peptide family but the precise nature of these products is not yet clear. The recognition that the inhibin subunits together with follistatin are expressed locally within the pituitary has lead to an investigation of their possible roles in intrapituitary regulation. It is clear that these peptides also have intragonadal roles. Of particular current interest is the nature of the signals that control the specificity of cellular peptide production and that determine whether a particular cell produces inhibin or activin. The inhibins are members of a complex family with many potential roles in physiology and pathophysiology. The role of the inhibins in feedback control of follicle stimulating hormone in the male, particularly, remains unclear. New applications for inhibin and related peptides are likely to be developed.

Sex-dependent structural asymmetry of the medial habenular nucleus of the chicken brain.

An investigation of structural asymmetry in the avian brain was conducted on the epithalamic medial habenular nucleus of the chicken. Twelve male and ten female two-day-old chickens were used for a morphometric evaluation of asymmetry. The medial habenular nucleus was measured from paraffin-wax-embedded, 8 micron-thick sections by use of a semiautomatic image analyser. The volumes of the right and left medial habenula of each animal were statistically analysed ('within animal experimental design'). The right medial habenula in males showed significant group asymmetry. In contrast, females failed to demonstrate group bias in favour of either hemisphere. However, individual females were lateralised, with either a larger right or left medial habenula. Although individuals of both sexes were lateralised, there was no significant sex difference in volume in either the right or left medial habenula. We propose that sex-linked structural asymmetry may be influenced by steroid hormonal effects in the central nervous system, and that such asymmetry could be more prevalent in the non-mammalian vertebrate brain than previously considered.

Light microscopic observations of an intraepithelial granular cell type in the bovine parotid gland.

Bovine parotid gland intralobular ducts were frequently found to contain an intraepithelial granular cell type, adjacent to the basement membrane. Granular cells were non-migratory and polymorphic in appearance, with strong cytoplasmic basophilia and acidophil metachromatic granules. Such cells were not found in the bovine mandibular and sublingual glands. It is proposed that the granular cell may be similar or identical to the globule leucocyte cell type and functionally associated with the parotid gland. A possible role for parotid gland granular cells in ruminant mucosal immunity is also discussed.

Substance P immunoreactivity in the superior cervical ganglia of normotensive and genetically hypertensive rats.

Substance P-like immunoreactivity (SPI) was investigated in the superior cervical ganglion of normotensive and genetically hypertensive (GH) Otago Wistar rats aged 1, 2, 8-10 and 50-60 weeks, by used of an indirect immunoperoxidase method. SPI was not seen in neuronal cell bodies but a subpopulation of ganglion cells was supplied by SP-positive terminals which closely invested the cell surface. This subpopulation showed no particular topographical distribution. The number of SP-positive terminal varicosities per unit area was several times higher in GH rats than in normotensive rats at all ages over 2-60 weeks. The proportion of neurons supplied by SP-positive terminals (sampled in 8-10 week-old rats) was also greater in GH than in normotensive rats. Decentralization of the ganglion or chronic capsaicin treatment removed all SP-immunoreactive terminals around the cell bodies, indicating that the SP-positive terminals are collaterals of thoracic sensory afferents. As SP has been reported to have an excitatory effect in sympathetic ganglia, intraganglionic release of SP might contribute to the development of hypertension in the GH strain.

Long term results of submandibular gland transfer for the management of xerophthalmia.

Free submandibular gland transfer is potentially the best surgical solution to total xerophthalmia. Experimentally, gland function has only previously been followed for 2 months. This study was designed to investigate the long term effects of free transfer on gland function. Simultaneous creation of experimental xerophthalmia and free microvascular transfer of the submandibular gland to the orbit was completed in 10 rabbits. Two died before the end of the experimental period and the study was based on the remaining eight. Six months after transfer, five of the eight transferred glands were histologically normal and contained healthy neurones and nerve terminals. In these animals, tear secretion in the transfer eye was significantly greater than that of the control eye at 2 (p less than 0.05) and 6 months (p less than 0.02) after transfer. In the transfer eye, tear secretion was also found to have significantly increased from preoperative levels at 2 (p less than 0.05) and 6 months (p less than 0.01). The observed innervation of the transferred glands may play a role in maintaining secretion in the long term.

Quantitation of tyrosine hydroxylase and neuropeptide Y immunoreactivity in single rat sympathetic neurons: effects of preganglionic nerve activity.

Using computerised densitometry to measure immunocytochemical reaction product in a model system, we established conditions that produced a linear relationship between the logarithm of antigen concentration and the measured intensity of staining. We then applied the densitometric technique to assess the changes in tyrosine hydroxylase (TH) and neuropeptide tyrosine (NPY) within sympathetic neurons of rat superior cervical ganglion following chronic decentralization and following reserpine treatment. One week after surgical or pharmacological decentralization, there was appreciable reduction of neuronal levels of both TH and NPY. However, there remained considerable variation in the immunoreactivities of individual cells. Three days of treatment with reserpine elevated TH levels but substantially reduced NPY. Both these effects were prevented by prior decentralization of the ganglia. No differences were seen between normotensive and the Otago strain of genetically hypertensive rats, either in basal TH or NPY immunoreactivities or in responses to the maneuvers performed. Comparison of our findings with previous biochemical data indicate that densitometric immunocytochemistry provides an accurate index of neuronally localised antigen concentrations but also allows analysis of interneuronal differences that are not otherwise apparent.

Inhibin and activin are demonstrable by immunohistochemistry in ovarian tumor tissue.

Elevated serum immunoreactive inhibin concentrations have been reported in patients with mucinous and granulosa cell tumors of the ovary. The present study aimed to determine whether the inhibins and/or the related peptides, the activins, were demonstrable within ovarian tumor tissue. Immunohistochemical analyses were performed on 11 ovarian tumors, 5 mucinous, 3 serous, 1 granulosa, 1 clear cell, and 1 metastatic colonic cancer. Both monoclonal and polyclonal antisera specific for inhibin-A, activin-A, and activin-B, and their alpha-, beta A- and beta B-subunits were used. The mucinous cells of all five mucinous tumors showed positive staining for activin-A and activin-B, and their beta A- and beta B-subunits, and three stained positive for inhibin-A and the alpha-subunit. The granulosa cell tumor also showed positive staining for inhibin-A and the activins. The remaining tumors were negative. The findings are consistent with the hypersecretion of inhibin (and possibly activin) by some ovarian malignancies and suggest that immunohistochemistry for the inhibins and the activins should be explored further in the classification of ovarian malignancies.

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue.

An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.

The functional and structural effects of hypothermic storage on ischaemic arterial grafts.

The effects of hypothermic ischaemia on blood vessels are unknown. This study aimed to determine the 3 week patency rate and the pathology of 9 experimental groups of hypothermically stored ischaemic arteries and one control group in a rabbit femoral artery model. Ischaemia times were 0 h, 24 h, 1, 2, 4, 6, 8 and 10 weeks (Groups 1-8). Patency was over 80% in all groups after 3 weeks reinsertion. Following reinsertion control grafts maintained normal arterial structure, but cellular degeneration had occurred in all ischaemic grafts and appeared complete after 4 weeks ischaemia. The graft connective tissue framework frequently remained intact. Repair was evident in central graft regions after 2 weeks ischaemia and 3 weeks reinsertion, but occurred only adjacent to the anastomosis in 4-10 week ischaemic arteries. Four week ischaemic arteries (Groups 9 and 10) reinserted for 6 and 12 weeks respectively exhibited near complete repair but patency dropped to 60% in the 12 week group.