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Cardiac myocytes and dendritic cells harbor human immunodeficiency virus in infected patients with and without cardiac dysfunction: detection by multiplex, nested, polymerase chain reaction in individually microdissected cells from right ventricular endomyocardial biopsy tissue.

Two hundred fifteen patients infected with human immunodeficiency virus (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged electrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the virus on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the virus in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

Adult

Effects of castration and adrenalectomy on in vitro rates of tryptophan hydroxylation and levels of serotonin in microdissected brain nuclei of adult male rats.

Rates of 5-hydroxytryptophan (5-HTP) synthesis and levels of serotonin (5-HT) were measured in microdissected brain nuclei following castration or adrenalectomy of adult male rats. Fourteen days following gonadectomy, 5-HTP synthesis decreased in the nucleus raphe dorsalis (DR) and nucleus centralis superior (NCS), while levels of 5-HT were unchanged in the 7 brain nuclei examined. Administration of testosterone to castrated rats not only did not reverse the castration-induced decrease in 5-HTP synthesis in the DR and NCS, but also decreased 5-HT synthesis in the nucleus amygdaloideus centralis (AGC) and the nucleus septalis lateralis (LS). Following administration of testosterone, 5-HT levels were unchanged. 10 days following bilateral adrenalectomy, 5-HTP synthesis increased in the NCS and the median eminence. Levels of 5-HT increased only in the median eminence. The increased 5-HTP synthesis and 5-HT levels following adrenalectomy were not reversed by corticosterone administration. In addition, these selective changes in 5-HT metabolism did not result from hormonal effects on the availability of tryptophan to the brain. We conclude that there are subsets of serotonergic neurons in rat central nervous system which respond uniquely to removal of the gonads and adrenals. Furthermore, the dissociation between serum and brain tryptophan concentrations and changes in rates of 5-HTP synthesis argue against tryptophan availability as being a primary determinant of 5-HT biosynthesis and for a direct endocrine central nervous system interaction with serotonergic neurons.

5-Hydroxytryptophan

Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized Long Evans rats.

The ability of several stimuli which augment central catecholamine (CA) neuronal activity to reinitiate estrous cycles in old constant estrous (CE) rats suggests CA neuronal function is impaired with advanced age. We examined the effects of age on dopamine (DA) and norepinephrine (NE) levels and turnover rates within microdissected brain regions of previously normally cycling young (3-4 months old) and middle-aged (10 months old) and CE old (20-22 months old) Long Evans 2 weeks after ovariectomy. Steady-state DA concentrations were significantly decreased in old compared to young rats in the nucleus accumbens (34%), anterior hypothalamic nucleus (54%, NHA ), neurointermediate pituitary lobe (51%, NIL) and median eminence (74%, ME). The rate constant of DA loss, an estimate of neuronal activity, decreased in old versus young rats only in the preoptic area suprachiasmatica (60%, POAs ) and NHA (60%) and was unchanged or augmented in the 7 other regions. In contrast, a decline in DA turnover rate of 29-67% was observed in 6 of 9 regions in middle-aged rats and 45-81% in 5 of 9 regions in old rats. Steady-state NE concentrations similarly were significantly decreased in old versus young rats in the POAs (54%), medial forebrain bundle (44%), nucleus suprachiasmatica (49%) and ME (59%). The rate constant of NE loss progressively decreased with increasing age only in the POAs and was unchanged or augmented in other regions. Turnover rate of NE was decreased from 21 to 98% in 4 of 8 regions from old animals. A strong positive correlation was noted between the rate constant of NE (but not DA) loss measured in young rats and the magnitude of the age-related depletion in NE concentrations within specific brain regions. Collectively these data indicate that with increasing age: CA neuronal function is differentially altered in nuclei located along the preoptico-tuberal pathway; substantial declines in both DA and NE concentrations are the primary contributor to the reduced amine turnover noted in several of these regions; and the observed age-related alterations in CA turnover may contribute to impaired LH response and the persistent hyperprolactinemia in old CE rats.

Aging

Development of methionine-enkephalin in microdissected areas of the rabbit brain.

Microdissected areas of the rabbit brain were isolated at prenatal day E-29, postnatal days P-3, 7, 14, 21, 2 months and adults. Methionine-enkephalin (ME) was assayed by RIA and ME concentration [ME] was expressed relative to the protein content of the extracted brain tissues. In brain nuclei with important roles in respiratory control [ME] was higher in prenatal and early postnatal life than in adults. In contrast, the prenatal and early postnatal [ME] levels in other nuclei were lower than or equal to adult values. These data suggest an important and changing role for ME in respiratory control throughout development. Early high [ME] levels within brainstem respiratory control nuclei may contribute to the newborn's increased susceptibility to respiratory depression.

Age Factors

Distribution of androgen receptor in microdissected brain areas of the female baboon (Papio cynocephalus).

We measured androgen receptors in the brain and pituitary of 4 female baboons (Papio cynocephalus) by the in vitro binding of methyltrienolone (R1881) to cytosols from 17 brain subregions as well as anterior and posterior pituitaries. High levels of AR were detected in anterior (22.1 +/- 7.1 (S.E.M.) fmol/mg protein) and posterior pituitary (12.6 +/- 3.3 fmol/mg protein). In brain tissue, the highest androgen receptor levels were found in the infundibular nucleus/median eminence (9.4 +/- 2.3 fmol/mg protein), ventromedial nucleus (6.3 +/- 1.7 fmol/mg protein) and periventricular area (4.9 +/- 1.3 fmol/mg protein). Saturation analysis of anterior pituitary and brain tissue (pool of hypothalamic, preoptic area, amygdala and septum remaining after microdissection of brain nuclei) showed that [3H]R1881 binds to the androgen receptor with high specificity and affinity (Kd = 1.25 x 10(-10) M, 0.45 x 10(-10) M, in anterior pituitary and HPA cytosol, respectively). Serum testosterone levels were low in all animals (0.59 +/- 0.26 ng/ml). With these data we described the quantitative distribution of androgen receptor in the pituitary and in specific brain nuclei in a species of nonhuman primate. The distribution is similar in many respects to that described in the male rat and the data suggest a conservation of androgen receptor distribution across species.

Animals

Sex differences in cytosolic progestin receptors in microdissected regions of the hypothalamus/preoptic area of guinea pigs.

Cytosolic progestin receptors (CPRs) were measured in microdissected nuclei of the hypothalamus and preoptic area of male and female guinea pigs. Adult gonadectomized animals were given 3 daily injections of 20 micrograms/day estradiol benzoate (EB) or oil vehicle. 24 h later, animals were sacrificed and cytosolic progestin receptors were measured using the synthetic progestin 3H-R5020. CPR levels did not differ significantly between oil treated males and oil treated females in any brain areas examined. With EB treatment, males showed significant increases in CPRs in most of the brain areas in which females showed increases, i.e. in the medial preoptic area, the periventricular part of the preoptic area, the periventricular part of the anterior hypothalamus, the ventromedial nucleus of the hypothalamus, the periventricular part of the medial hypothalamus and the arcuate-median eminence. However, EB treated males showed significantly lower CPR levels than EB treated females in both the periventricular part of the preoptic area and the periventricular part of the medial hypothalamus.

Animals

Aging in male rats modifies castration and testosterone-induced neuropeptide Y response in various microdissected brain nuclei.

Neuropeptide Y (NPY) is localized in several hypothalamic sites which are implicated in the control of hypothalamic luteinizing hormone-releasing hormone (LHRH) and pituitary luteinizing hormone (LH) release. We have observed previously that in young rats castration decreases and testosterone (T) replacement restores NPY levels in selected hypothalamic sites. However, in aged male rats, NPY levels were decreased in all hypothalamic sites studied. Since testicular function is diminished in aged rats, we reasoned that decreased T feedback may be responsible for the reduction of NPY in the hypothalamus. Therefore, we compared the effects of castration and T-replacement on NPY levels in microdissected hypothalamic sites of 2.5-month- (young) and 15-month-old (aged) male rats. Serum LH and T levels were markedly reduced in aged as compared to those observed in young rats. In association with the decreased hormone levels, NPY levels were significantly reduced in each of the 7 hypothalamic sites of aged as compared to young rats. Further, in young rats, castration reduced and T-replacement prevented the castration-induced depletion in only 3 sites, viz. the ventromedial hypothalamic nucleus (VMN), arcuate nucleus (ARC) and median eminence (ME). In contrast, castration in aged rats reduced NPY levels not only in the VMN as in young rats, but also in the medial preoptic area (MPOA) and dorsomedial nucleus (DMN). However, the marked reduction in the ME and ARC NPY levels of young rats following castration was not observed in the ME and ARC of aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging

Prazosin treatment does not affect progestin receptor induction in microdissected regions of the rat hypothalamus.

Sex differences in estrogen-induced progestin receptors have been described in specific regions of the rat brain. To determine if alpha 1-neurotransmission plays a role in the expression of the sex differences in progestin receptor induction, the effects of the alpha 1-antagonist, prazosin, on progestin binding in microdissected regions of the rat brain was determined. Adrenalectomized/gonadectomized male or female rats were administered various doses of estradiol benzoate (EB) in combination with prazosin. With all treatment paradigms, and in both sexes, no significant effect of prazosin treatment on progestin receptor levels was observed. These results are consistent with the idea that sex differences in the estrogen-induction of progestin receptors in the rat hypothalamus are not due to sex differences in the alpha 1-adrenergic regulation of progestin receptor synthesis.

Adrenergic Fibers

Microdissection of the mouse egg.

A rapid and efficient method for microdissection of the mouse egg is described. The dissection is carried out in hanging drops of medium surrounded by heavy liquid paraffin oil at room temperature. Eggs are first deformed into a cylindrical shape and then dissected at a predetermined site with a glass needle on a Leitz micromanipulator. The survival rate of the dissected fragments is 75-90% and between 20 and 30 eggs can be dissected in an hour. Development of the dissected eggs is at least as good as that described after other types of manipulation. Cytoplasts and karyoplasts of various sizes can be prepared, as well as gynogenetic and androgenetic eggs with different amounts of cytoplasm. This procedure may help to examine nuclear-cytoplasmic interactions in eggs reconstituted from a variety of fragments.

Animals

Vitamin E distribution in ocular tissues following long-term dietary depletion and supplementation as determined by microdissection and gas chromatography-mass spectrometry.

Vitamin E is thought to be important for protection of polyunsaturated fatty acids (PUFA) from oxidative damage. A microbiochemical procedure using microdissection and gas chromatography-mass spectrometry was developed to determine vitamin E distribution in ocular tissues in a rodent model, with the eventual goal of using it in a study of phototoxic degeneration of the retina, where PUFA oxidation is potentially the causal mechanism. Sample preparation was achieved by freeze-drying the retina followed by micro-dissection to obtain the desired structures for analysis. A deuterated alpha-tocopherol internal standard is added to the tissue sample before extraction and derivatization which are achieved in a single step. The data presented show the vitamin-E content in various structures of the retina, particularly the outer segments and retinal pigment epithelium (RPE); however, the vitamin E content of other ocular tissues is also included. Data were obtained from albino and pigmented rats receiving vitamin E-depleted, supplemented, and regular chow diets, and from rabbits and cats receiving regular chow diets formulated for each species. Within all dietary groups the highest concentration of vitamin E was located in the RPE followed by the outer segments of the photoreceptor cells. Other ocular tissues consistently contained lower amounts of vitamin E. Different tissues were depleted of vitamin E at different rates and this points out the importance of determining vitamin E levels in tissues of interest in studies on the consequences of dietary depletion.

Animals

Measurement of immunoreactive angiotensin II levels in microdissected brain nuclei from developing spontaneously hypertensive and Wistar Kyoto rats.

Levels of immunoreactive angiotensin II (ANG II) were measured in specific microdissected nuclei from the brains of newborn (NB; less than 1 week of age), 4-, 8-, and 12-week-old spontaneously hypertensive rats (SHR) and their age-matched normotensive controls, Wistar Kyoto (WKY) rats, using a sensitive radioimmunoassay. The structures investigated included the paraventricular nucleus of the hypothalamus (PVH), the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMN of X), the locus coeruleus (LC), and the A1 region of the medulla. A section of cerebellar cortex was used as a control. Although ANG II was detected in each of the nuclei examined, there were no differences in the ANG II contents of any of these structures between young (NB and 4 week old) SH and WKY rats. However, by 8 weeks of age, the SHR had significantly higher ANG II levels in the PVH, NTS, and DMN of X than its normotensive control, and at 12 weeks of age, significantly higher ANG II levels were observed in the PVH, NTS, DMN of X, and LC of the SHR compared to those in the WKY. During the developmental period under investigation, both strains revealed increases in the ANG II content of all nuclei except for the LC, where the ANG II levels decreased with age. No detectable ANG II was found in the cerebellar cortex of either strain at any age.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II

Immune responses of sheep to microdissected parts of Haemonchus contortus.

Adult specimens of Haemonchus contortus were microdissected into four body fragments: oesophagus, cuticle of oesophagus, gut and cuticle with adjacent muscle layer. The antigenicity of these different body fragments was analysed in comparison to total (whole) worm extracts with immunoblotting and ELISA using sera of H. contortus-infected sheep. In particular, oesophagus-derived antigens appeared to be specifically recognized and may prove valuable in diagnostic assays.

Animals

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei.

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.

Animals

The structure of the wall of the rat intraacinar pulmonary artery: an electron microscopic study of microdissected preparations.

Rat intraacinar arterial segments that by light microscopy lack a medial muscle layer are capable of constriction and, during pulmonary hypertension, acquire morphologically differentiated smooth muscle. These facts suggest that effector cells of smooth muscle type are present in the normal vessel wall. Studies in the literature, however, fail to agree on their location or even existence. By combining microdissection, step sectioning, and electron microscopy, we have now performed a serial study of six arterial pathways. At its proximal end, the artery has a circumferentially continuous single layer of smooth muscle cells, separated from the endothelium by a fenestrated internal elastic lamina. Myoendothelial junctions are frequent and incorporate basal laminae of both cell types. More distally, the internal elastic lamina is discontinuous and the smooth muscle cells lose myofilaments and dense bodies so as to resemble intermediate cells. They still form a continuous layer, however. At mid-alveolar-duct level, this layer is discontinuous, and in the most distal arteries investigated, the cells are often solitary. They lie close to the endothelial cell, but, except for localized regions of contact, are separated from it by a single basal lamina that is continuous with one covering their abluminal surface.

Animals

Nuclear estrogen receptor binding in microdissected brain regions of female rats during pregnancy: implications for maternal and sexual behavior.

Previously, we showed that changes in nuclear estrogen receptor (NER) concentrations within large dissections of the preoptic area and hypothalamus of pregnant rats might underlie changes in both maternal and sexual behaviors. To more precisely localize these biochemical changes, NER concentrations were measured in microdissected nuclei within the preoptic area and hypothalamus of pregnant rats on days 8, 16 and 22. Results indicated that NER levels changed significantly throughout pregnancy and that the magnitude and direction of change was site specific. Although a different pattern of change was found in each nucleus, NER levels in most preoptic area nuclei were significantly higher by the 16th day of pregnancy compared to those on day 8, while receptor levels in most hypothalamic nuclei did not increase until day 22 of pregnancy. The higher NER levels found in preoptic area nuclei of female rats as pregnancy advances can be correlated with growing maternal responsiveness during pregnancy, while elevated concentrations of NER in hypothalamic nuclei at the end of pregnancy may indicate preparation for postpartum estrus.

Amygdala

A tissue culture model for the study of peptide synthesis and secretion from microdissected hypothalamic explants.

Using established tissue culture methodology, a protocol has been developed for the culture of specific hypothalamic regions. A microdissection technique is used to remove the paraventricular and supraoptic nuclear regions from fresh tissue. These explants are maintained in culture and appear to be viable on the basis of several criteria: the continuing presence of peptides in both tissue and media, the ability to incorporate a labeled amino acid precursor into proteins and peptides, the histological appearance using phase contrast microscopy, and the immunocytochemical identification of peptide neurons. This tissue culture model may prove useful in the study of neuroendocrine mechanisms in specific brain regions.

Catecholamines

Cystic changes in adult human kidneys after ureteral obstruction: microdissection studies.

Five kidneys from patients suffering from ureteral obstruction were selected for the study; from each, 100 nephrons and 20 collecting ducts were studied using Darmady's microdissection technique. Whereas three kidneys showed macroscopic cysts, microscopically, all kidneys displayed dilation and diverticulation of the nephrons particularly of the loop of Henlé and of distal convoluted tubules. Some dilatation of the collecting ducts was also noticed.

Aged

Microdissection findings of medullary sponge kidney.

Findings on microdissection of specimens of medullary sponge kidney showed uniform enlargement of collecting tubules in part of the papillae. Nephrons were normal in fragments without calculi and infection.

Dissection