Types Of Tissue Preparations Research Articles

Comparison of trace element concentrations in paired formalin-fixed paraffin-embedded and frozen human placentae

IntroductionThere is increasing interest in measuring metals concentrations in human placentas to better understand physiology, disease, and toxic and diagnostic exposures. For these purposes, formalin-fixed paraffin embedded (FFPE) tissues obtained at clinical pathology examination represent a valuable potential store of well-characterized tissues for analysis. However, the limited data that exist comparing metal concentrations in FFPE tissue to recently collected frozen tissues paints a confusing picture, and there is no published data directly comparing frozen and FFPE placental villus tissues. MethodsPaired samples of fresh frozen and FFPE tissue from 22 rapidly processed human singleton placentae were weighed and digested using standard clean laboratory procedures and subsequently analyzed for a suite of 13 metals using a PerkinElmer DRC II ICP-MS. The analytical results were compared using either a paired t-test or a sign test depending on data normality. ResultsConcentrations of metals (aluminum (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), gadolinium (Gd), mercury (Hg), manganese (Mn), lead (Pb), strontium (Sr), and zinc (Zn)) measured in both types of tissue preparations (frozen and FFPE) displayed a consistent range with other studies and did not display significantly different values from each of the paired specimens for any of the 13 specific metals analyzed. DiscussionWithin placentae, metals concentrations of measured trace, toxic and diagnostic elements (Al, As, Ba, Cd, Cr, Cu, Fe, Gd, Hg, Mn, Pb, Sr, and Zn) are consistent between FFPE and fresh placental villus tissue, without indications of systematic element loss or bias. FFPE from archived pathology specimens may offer an important and convenient alternative for measuring trace metals in human frozen placental tissues.

Effects of ischemia on sarcoplasmic reticulum Ca2+uptake and Ca2+release in rat skeletal muscle

In this study, we investigated the hypothesis that prolonged ischemia would impair both sarcoplasmic reticulum (SR) Ca(2+) uptake and Ca(2+) release in skeletal muscle. To induce total ischemia (I), a tourniquet was placed around the upper hindlimb in 30 female Sprague-Dawley rats [wt = 256 +/- 6.7 (SE) g] and inflated to 350 mmHg for 4 h. The contralateral limb served as control (C). Immediately after the 4 h of ischemia, mixed gastrocnemius and tibialis anterior muscle was sampled from both limbs, and both crude muscle homogenates and SR vesicles were prepared. In another 10 control animals (CC), muscles were sampled and prepared exactly the same way, but immediately after anesthetization. Ca(2+) uptake and Ca(2+) release were measured in vitro with Indo-I on both homogenates and SR vesicles. As hypothesized, submaximal Ca(2+) uptake was lower (P < 0.05) in I compared with CC and C, by 25 and 45% in homogenates and SR vesicles, respectively. Silver nitrate (AgNO(3))-induced Ca(2+) release, which occurred in two phases (phase 1 and phase 2), was also altered in I compared with CC and C, in both muscle homogenates and SR vesicles. With ischemia, phase 1 peak Ca(2+) release was 26% lower (P < 0.05) in SR vesicles only. For phase 2, peak Ca(2+) release was 54 and 24% lower (P < 0.05) in SR vesicles and homogenates, respectively. These results demonstrate that prolonged skeletal muscle ischemia leads to a reduced SR Ca(2+) uptake in both homogenates and SR vesicles. The effects of ischemia on SR Ca(2+) release, however, depend on both the phase examined and the type of tissue preparation.

Tissue and concentration-dependent effects of sodium selenite on muscle contraction.

In this study, we demonstrated that sodium selenite with high doses (> or = 10(-3) M) were potent in inducing a contracture type effect on heart and smooth muscles. Selenite (Se), at a concentration of 10(-3) M, caused a contracture effect in heart preparations. Also, low Se concentrations did not have any significant effect. Although low concentrations of Se (> or = 10(-5) M) had a biphasic effects on acetylcholine (ACh) induced and spontaneous ileum contractions, 10(-3) M selenite enhanced once more a contracture effect similar to that of the heart preparations. Replacing Ca2+ concentration of the bathing solution by twofold Ca2+ or Ca2+-free did not change the effects of selenite (10(-5) M) on contractility of ileum preparations. In vascular smooth muscle, low concentration of selenite (< 10(-4)) had no significant effects on KCl, and phenylephrine-induced contractions and acetylcholine-induced endothelium-dependent relaxations of isolated rabbit aorta. However, the contractions induced by phenylephrine and the relaxations induced by acetylcholine in rabbit aorta were depressed significantly by high concentration of selenite (10(-3) M). The results obtained by selenite exposure from these three different types of tissue preparations first suggest that the high concentration of selenite exposure induces some alterations in the functions of muscles and endothelium in a tissue- and dose-dependent manner. Second, this observed irreversible type of dysfunction of tissues induced by 10(-3) M selenite is not directly dependent on the Ca2+ entrance into the cytosol, but might be induced by the increase of intracellular Ca2+ with the disturbance of Ca2+ regulation.

Heterogenous Binding of 3H‐Remoxipride to Membranes of Rat Liver and Brain

The binding of the antipsychotic agent 3H-remoxipride to membranes of rat liver and brain (whole brain and cerebellum) was studied with filtration technique. Saturable high affinity binding was obtained for all three types of tissue preparations. Heterogenous binding sites were found since various types of compounds inhibited the binding of 3H-remoxipride with shallow dose-response curves. In the liver preparation it was possible to differentiate between two different binding sites. One site, called rem1, was defined with 100 nM alaproclate and bound sigma 2 ligands with high affinity, e.g. haloperidol, GBR 12909, DTG and (+)-3-PPP. High correlation (r = 0.93) was obtained between the inhibition of the binding of 3H-remoxipride to the rem1 site and the inhibition of the binding of the sigma ligand 3H-DTG reported previously (Ross 1991), indicating that the rem1 site is identical to a sigma 2-like binding site. The other 3H-remoxipride binding site in the rat liver, rem2 appears to be identical to the site binding alaproclate, proadifen and cocaine with high affinity. Cd2+ and Zn2+ were potent inhibitors of both binding sites, Cd2+ particularly of rem2 binding and Zn2+ preferably of rem1 binding. The apparent Bmax (pmol/g original tissue) and KD (nM) values for rem1 binding were 441 +/- 43 and 80 +/- 14, and for rem2 binding 727 +/- 116 and 95 +/- 6. The binding of 3H-remoxipride to the brain preparations was also inhibited with shallow dose-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)

Is poor prognosis really related to HLA-DR expression by malignant melanoma cells?

HLA-DR expression was examined in 50 consecutive primary cutaneous malignant melanomas with a Breslow depth greater than 2 mm using two well-characterized monoclonal antibodies which detect fixation-resistant epitopes. In 31 of these cases (62%) a subpopulation of tumour cells was reactive, although there was considerable heterogeneity. Positive labelling did not correlate with depth but was associated with a reduced likelihood of developing early metastatic disease and a tendency for better overall survival, particularly in male patients. These findings contrast with earlier studies using cryostat sections and one study on paraffin-embedded tissue in which HLA-DR expression was shown to be a poor prognostic factor, but are consistent with the findings in other malignant tumours studied. The significance of HLA-DR expression as a marker of prognosis may depend on the type of tissue preparation, the sensitivity of the immunocytochemical techniques used and the method of assessment.

Endothelial-dependent sexual dimorphism in vascular smooth muscle: role of Mg2+ and Na+.

1. In isolated aortae of the male rat [Mg2+]o withdrawal and concomitant reduction in [Na+]o (to 84 mM) induced significant increases of basal tone, but, surprisingly, this did not occur in intact aortae removed from female rats. Such tension development, however, was observed in endothelium-denuded aortic preparations from both sexes. These observed gender-related differences were not dependent on animal strain or types of tissue preparations. 2. No tension development was observed in aortae obtained from castrated males treated with oestradiol. Aortic tissues of sexually-immature male and female rats exhibited marked tension development when exposed to 0 mM [Mg2+]o and low [Na+]o. 3. Tension development in Mg(2+)-free, low-Na+ media was not tachyphylactic and completely dependent on extracellular Ca2+; addition of 1.2 mM Mg2+ to the Mg2+ and Na(+)-deficient incubation media relaxed the increase in tension to a normal basal level. 4. Two known endothelial-derived relaxant factor (EDRF) inhibitors, methylene blue and haemoglobin, induced tension development in female aortae with intact endothelium exposed to Mg(2+)-Na+ deficient media, while use of a specific inhibitor of EDRF-derived nitric oxide, viz., NG-monomethyl-L-arginine (L-NMMA), resulted in potentiation of tension development in male, but not in female, aortae. This effect of L-NMMA was antagonized by L-arginine. 5. The Ca ionophore, A23187, partially relaxed contractile responses in male aortae (with intact endothelium) which were followed by potentiated contractions. Endothelium-dependent vasodilator responses to A23187 (10(-10)-10(-6) M) of aortic rings from male or female rats in normal Krebs-Ringer bicarbonate solution were not different.6. These results suggest that: (a) as in vascular smooth muscle cells, Mg2+ plays an important role in Ca2 + homeostasis in endothelial cells, probably via Na+-Ca2+ exchange; and (b) sex steroid hormones, probably the female sex hormone, 17-beta-oestradiol, may regulate contractile responses of intact vascular smooth muscle by modifying endothelium functions through such Mg2 '-regulated internal Natdependent Ca2+ entry. These data may help to explain why female subjects, despite Mg deficiency, unlike male subjects, are protected against ischaemic heart disease and cerebrovascular disease until menopause.

Glutathione S-transferases in endosulfan-treated red sword grass moth ( Xylena vetusta Hb.) and hebrew character moth ( Orthosia gothica L.) Reared on leaves from apple ( Malus domestica cult.) or willow ( Salix caprea L.)

1. In the final instar of red sword grass moth ( Xylena vetusta Hb.) (RSGM) and Hebrew character moth (Orthosia gothica L.) (HCM) reared on leaves from apple ( Malus domestica cult.) or willow ( Salix caprea L.), glutathione S-transferase (GST) activities in midgut and soft tissues, excluding the digestive tract, varied with a factor of 900. 2. In RSGM and HCM midgut and RSGM soft tissues, where GST activities were found to be low, the type of food, rather than the type of species, determined the GST activities towards CDNB and DCNB. 3. In contrast, in HCM soft tissues with high GST activities, food did not seem to have any influence. Furthermore, endosulfan (Thiodan 35) significantly ( P < 0.02) increased GST activities in midgut of HCM reared on both types of food. 4. SDS-PAGE of GSH-affinity purified proteins revealed that in both types of tissue preparations, and on both types of food, RSGM and HCM contained a single protein band corresponding to a molecular weight of 26.2 and 25.0 kDa, respectively. 5. Endosulfan treatment of RSGM led to the appearance on SDS-PAGE of a new band corresponding to a molecular weight of 24.6 kDa in midgut of larvae on both types of food, and in soft tissues of larvae on apple. 6. Isoelectric focusing of GSH-affinity purified GSTs from RSGM indicated the presence of 2 GST molecules in midgut and soft tissues, one with a similar (pI 8.8), and the other with a different, isoelectric point in midgut (pI 4.1) and soft tissues (pI 3.8). 7. In HCM, the isoelectric point of GST in midgut and soft tissues was above 9.3.

Comparison of GABA uptake by brain and kidney preparations

The uptake ofγ-aminobutyric acid (GABA) was measured in rat cerebral cortical synaptosomes and rat kidney brush-border membrane vesicles (BBMV). Three GABA uptake systems (Km = 1.3, 50 and 3246µM respectively) were present in synaptosomes, but only two uptake systems (Km = 11 and 1203µM respectively) were detectable in BBMV. The uptake systems in the two types of tissue preparations were similar in that every system was inhibited byp-hydroxymercuribenzoate and by the action of neuraminidase, thereby indicating that, irrespective of the tissue, both sulfhydryl and sialyl groups were necessary components of all the GABA uptake systems. In contrast, differences were observed between synaptosomal and BBMV uptake systems with respect to their sensitivity to inhibition by GABA structural analogs. While the synaptosomal GABA uptake systems were strongly inhibited by nipecotic acid, diaminobutyric acid,β-alanine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), none of these compounds significantly inhibited the BBMV uptake systems. It is therefore concluded that the GABA uptake systems in brain and kidney tissues are quite different entities. While the role of GABA transport in the inactivation of the neurotransmitter function of GABA is well documented, the role of the kidney GABA uptake system is unclear, and the possibility exists that the latter systems are present in kidney tissues primarily to transport compounds other than GABA. The present study does however highlight a difference in drug susceptibility of the brain and kidney uptake systems, a phenomenon which may have some therapeutic relevance with respect to the use of GABA analogs as anticonvulsant agents.

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13-day-old) rat cerebellum was conducted by means of binding experiments in membrane-enriched preparations and autoradiography, using two radioligands, [125I-Tyr0,D-Trp8]SRIF-14 [( 125I-Tyr0,D-Trp8]S14) and 125I-SMS 204-090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13-day-old rat cerebellum showed that the KD values for [125I-Tyr0,D-Trp8]S14 and 125I-SMS 204-090 binding were 0.35 +/- 0.04 and 0.39 +/- 0.01 nM, respectively. The corresponding Bmax values were 52.7 +/- 4.8 and 49.9 +/- 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SS1). Competition studies showed that different D-Trp-substituted analogs displaced [125I-Tyr0,D-Trp8]S14 binding with Hill coefficients less than 1, a finding indicating the existence of different subtypes of binding sites. When [Tyr0,D-Trp8]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13-day-old cerebellum and adult cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of slow waves in the isolated proximal colon of the cat

Slow oscillations of the membrane potential difference of circular smooth muscle cells of the colon, slow waves, arise at the interface between the submucosa and muscularis externa of the colon. These studies test the hypothesis that the generation of slow waves in the circular muscle layer of the colon of the cat is dependent upon an intact interface between the submucosa and muscularis externa. Electrical recordings were made from 3 types of tissue preparations from the proximal colon: (a) submucosa and muscularis externa intact, (b) submucosa isolated from the circular muscle, and (c) muscularis externa isolated from the submucosa. The cellular make-up of each tissue was determined by the osmic acid-zinc iodide method (Champy-Maillet), and the Masson's trichrome stain. Continuous slow waves were recorded from intact tissues. Removal of the submucosa abolished slow waves from the underlying muscularis externa. Either irregular electrical transients or waxing and waning slow wave-like activity were recorded from the isolated submucosa. Submucosal tissues removed from the muscularis externa contained no circular smooth muscle, but did contain elements of the plexus submucosus extremus.

Antibody Response following Implantation of Xenogenic Lenticules

Rabbits underwent implantation of either lyophilized or fresh porcine lenticules into the central or peripheral cornea. All animals were followed for up to four months by slit-lamp examination and macrophotography to determine implant rejection. Serum antibody levels to soluble porcine cornea extract were determined by an enzyme-linked immunosorbant assay. Only those animals receiving lenticules into the peripheral cornea experienced a rejection and developed an antibody response to the porcine cornea extract. The production of antibody preceded the appearance of vascularization of the implanted lenticules. Thus, the site of lenticule implantation, not the type of tissue preparation, determined the outcome of the graft.

THE DISTRIBUTION OF GLUTAMATE DECARBOXYLASE IN RAT TISSUES; ISOTOPIC VS FLUORIMETRIC ASSAYS

Abstract—The activity of glutamate decarboxylase (GAD, EC 4.1.1.15) in normal and neoplastic rat tissues was determined by two assay methods, one based on the production of 14CO2 from [14C]glutamic acid and the other on the fluorimetric measurement of γ‐aminobutyric acid (GABA) formation. Activities obtained with the isotopic assay were high in every tissue (ranging from over 800 in liver and brain to 107nmol CO2/min/g in lung). They were drastically diminished by Triton X‐100, by an oxygen‐free atmosphere or by the mitochondrial electron transport inhibitors, rotenone and antimycin A. Activities measured fluorimetrically were significant in only a few tissues and were stimulated by Triton (e.g. from 299 to 569 nmol GABA/min/g brain) but were unaffected by rotenone. For several tissues after Triton treatment the fluorimetric and isotopic assays (in air) gave the same results (i.e. the two end products, CO2 and GABA were in stoichiometric agreement); however, the fluorimetric assay remains the more reliable measure of GAD activity since Triton may not inhibit completely the non‐GAD dependent decarboxylation of glutamate in all types of tissue preparations. The hepatic, renal and mammary tumours tested were devoid of GAD; among non‐neural normal tissues, kidney, liver and, possibly, adrenal gland contained significant GAD activity. In kidney and liver the activity was 15 and 10 per cent of that in brain.