Localization of the glucocorticoid receptor mRNA in cartilage and bone cells of the rat. An in situ hybridization study.

The in vivo localization of glucocorticoid receptor (GR) mRNA expression was studied in the cartilage and bone cells of the femur of young adult rats to compare its distribution with that of the GR protein, which had previously been shown histochemically in the same areas. To achieve this, we used a synthetic oligodeoxynucleotide as a probe, in line with the published human GR (hGR) cDNA sequence. The probe was coupled to fluorescein (FL), applying a rapid Fast-Tag TM FL nucleic acid labeling method. Negative controls were achieved by using sense sequences of the hGR oligoprobe, similarly coupled by using the Fast-Tag TM FL labeling kit. Dewaxed sections were treated for in situ hybridization (ISH) histochemistry with the antisense and sense oligoprobes. The ISH reaction product was more intense in the cytoplasm of proliferative and maturative chondrocytes of the growth plate cartilage than in that shown in the hypertrophic ones. In the metaphyseal secondary ossification zone, osteoblasts (OBs) and osteocytes (OCs) were variably labeled, whereas osteoclasts (OCLs) were always intensely stained. The labeling was also visible in some bone marrow cells, in articular chondrocytes, in the cells of tendon-bone junctions, and in the perichondrium and periosteal cells. Our results confirm a cellular co-location of GR protein and mRNA. In agreement with GR immunolocalization, the variability of labeling appeared to be related to the cell cycle, the stage of differentiation and cell-type differences.

Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats.

The expression of Bcl-2 and Bax has been evaluated by immunohistochemistry in normal rats, and in rats after treatment with high-dose corticosterone (CORT). Proliferative (PC) and maturative/hypertrophic (MaHC) chondrocytes of the growth plate have been examined, as well as osteoblasts (Obs), osteocytes (Ots) and osteoclasts (Ocs) of the metaphyseal secondary spongiosa. For each cell type, the Bcl-2 and Bax immunopositive cells were expressed as a percentage of the total number of cells. Bcl-2 and Bax expression was considered to be enhanced when the percentage of positive cells rose. Bcl-2 and Bax were expressed in all cell types, and two main kinds of labeling distribution, both suggestive of association with intracellular organelles, were observed in the cytoplasm: scarce and spotty labeling (type 1) or abundant, granular and diffuse labeling (type 2). In some cases, nuclear membranes could also be seen to be positive. Positive PCs and Obs generally showed a labeling of type 1, MaHCs and Ocs of type 2, while Ots varied with labeling of type 1 or type 2. CORT administration induced a fall in the percentage of Bcl-2 immunopositive cells, and a rise in that of Bax immunopositive cells, in PCs and Ots. The same trend was observed in MaHCs, although the Bcl-2 decrease was not significant. The percentage of Bcl-2 and Bax immunopositive Obs rose, and their labeling distribution shifted from type 1- to type 2-labeled cells. Ocs showed the highest immunopositivity for both Bcl-2 and Bax, which did not change after CORT administration. These data suggest that CORT treatment, by lowering Bcl-2, and raising Bax expression, may promote the apoptotic process in PCs, MaHCs and Ots. Obs, however, do not undergo the same variations. This finding, together with the results of a previous study showing that CORT administration raises the frequency of apoptotic Obs, does not support a direct relationship between apoptosis and Bax overexpression, at least in Obs. The CORT effect might be related to cell types and their state of differentiation, so that Bcl-2 and Bax might regulate not only the machinery of cell death, but also cell proliferation and differentiation.

Insulin-stimulated cardiac glucose uptake is impaired in spontaneously hypertensive rats: role of early steps of insulin signalling.

OBJECTIVE: Although the heart is one of the target organs of insulin, it is still unknown whether the effect of insulin on cardiac muscle is preserved in essential hypertension, where insulin resistance has been observed in skeletal muscle. METHODS: We evaluated cardiac glucose uptake and the early steps of insulin signalling in spontaneously hypertensive (SHR, 10-12 weeks old) and in age-matched normotensive Wistar-Kyoto (WKY) rats. Cardiac glucose uptake (micromol/100 g per min) was assessed by 2-[14C]deoxyglucose method. After an overnight fast, 16 WKY rats and 17 SHR underwent a hyperinsulinemic euglycemic clamp. In particular, 2-h intravenous (i.v.) infusion of insulin (10 mU/kg per min) or saline (NaCl 0.9%) was administered, followed by an i.v. bolus injection of 2-[14C]deoxyglucose (100 microCi/kg) to measure cardiac glucose uptake. RESULTS: During saline infusion, cardiac glucose uptake was significantly higher in SHR compared to WKY rats (85 +/- 18 versus 8 +/- 3 mg/kg per min, P < 0.01). Furthermore, insulin was able to markedly increase cardiac glucose uptake in WKY rats whereas this insulin action was entirely abolished in SHR; thus, the cardiac glucose uptake became similar in the two rat strains (76 +/- 16 versus 82 +/- 16 mg/kg per min, not significant). More importantly, during saline infusion SHR showed a significantly higher phosphorylation of insulin receptor substance-1 (IRS-1) coupled to enhanced association of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and to an increased PI 3-kinase activity compared to WKY rats. As expected, insulin exposure evoked an activation of its signalling cascade in WKY rats. In contrast, in SHR, the hormone failed to activate post-receptor molecular events. CONCLUSIONS: Our data indicate that the heart of SHR shows an overactivity of the proximal steps of insulin signalling which cannot be further increased by the exposure to the hormone. This abnormality may account for the marked increase of basal cardiac glucose uptake and the loss of insulin-stimulated glucose uptake observed in SHR.

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone.

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.

Lack of effect of insulin on glucose utilization of the hypothalamus in normotensive and hypertensive rats.

Hypertension is frequently associated with insulin resistance and enhanced sympathetic activity supposedly mediated by an effect of the hormone on the hypothalamus. In this study we sought to determine whether insulin modifies the functional activity of the hypothalamus and other brain areas of spontaneously hypertensive (SHR) and normotensive WKY rats. The study was carried out in control and hyperinsulinemic, normoglycemic rats. Insulin plasma levels were increased to 198 +/- 10 (WKY) or 220 +/- 10 microunits/ml (SHR). Brain functional activity was evaluated by the 2-[14C]deoxyglucose method for measuring local rates of glucose utilization. The results show that insulin has no effect on any of the brain areas examined including the hypothalamus, of both WKY and SHR rats. The two strains of rats have comparable cerebral metabolic rates also under basal conditions.

Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlu1a and mGlu5 receptors.

(+)-2-Methyl-4-carboxyphenylglycine (LY367385), a potent and selective antagonist of mGlu1a metabotropic glutamate receptors, was neuroprotective in the following in vitro and in vivo models of excitotoxic death: (i) mixed cultures of murine cortical cells transiently exposed to N-methyl-D-aspartate (NMDA); (ii) rats monolaterally infused with NMDA into the caudate nucleus; and (iii) gerbils subjected to transient global ischemia. We have compared the activity of LY367385 with that of the novel compound (+/-)-alpha-thioxantylmethyl-4-carboxyphenylglycine (LY367366), which antagonizes both mGlu1a and -5 receptors at low micromolar concentrations, but also recruits other subtypes at higher concentrations. Although LY367366 was neuroprotective, it was in general less efficacious than LY357385, suggesting that inhibition of mGlu1 receptors is sufficient to confer significant neuroprotection. We conclude that endogenous activation of mGlu1a receptors (or perhaps other mGlu1 receptor splice variants) contributes to the development of neuronal degeneration of excitotoxic origin.

Cytochemical demonstration of the glucocorticoid receptor in skeletal cells of the rat.

The in vivo localization of the glucocorticoid receptor (GR) was studied in cartilage and bone cells of femurs of young adult rats. Deparaffinized sections were treated with a polyclonal antibody raised against the amino-terminus of human GR; the immunoreaction was detected with the streptavidin-biotin amplification method. Histomorphometric, computer-assisted analysis of GR-positive cells was performed by counting the percentage of GR-immunostained cells in the proliferative and maturative/hypertrophic zone of the epiphyseal growth plate cartilage, and of the percentage of positive osteoblasts (OBs), osteoclasts (OCLS) and osteocytes (OCs) in the metaphyseal secondary ossification zone. Numbers of OBs and OCLs per mm of metaphyseal endosteal perimeter, and numbers of OCs per mm2 of trabecular area were also counted. Immunopositive cells were found both in cartilage and bone, with variable degree of nuclear and/or cytoplasmic immunostaining; immunonegative cells were present among the positive ones. Histomorphometry showed that about 54% of chondrocytes in the proliferative zone, and 55% of chondrocytes in the maturative/hypertrophic zone of the growth plate were labeled; in metaphyseal bone, 68% of OBs, 65% of OCs, and 98% of OCLs were GR-positive. The density of positive cells was 12.06 OBs/mm, 3.32 OCLs/mm, and 520.40 OCs/mm2. These results, for the first time obtained in vivo, show that GR is present in cartilage and bone cells, and that the degree of GR-immunostaining is variable in the same type of cell. This may be dependent on the cell cycle and stage of differentiation, and may reflect a variable cellular sensitivity to the stimulation of the glucocorticoid hormone.

Modifications of local cerebral metabolic rates for glucose and motor behavior in rats with unilateral lesion of the subthalamic nucleus.

Inactivation of the subthalamic nucleus (STN) has attracted interest as a therapeutic tool in Parkinson's disease. The functional consequences of the inactivation, however, are uncertain. In this study definition of the pattern of changes of cerebral functional activity associated with lesion of the STN and dopaminergic stimulation, by using the [14C]deoxyglucose method, was sought. Six or 7 days following unilateral lesion of the STN, the animals were divided into two groups: One group (n = 10) was administered apomorphine (1 mg/kg) subcutaneously; the second group (n = 10) received saline. The [14C]deoxyglucose procedure was initiated 10 minutes following the drug or saline injection. The results show that systemic administration of apomorphine to rats with unilateral lesion of the STN causes ipsiversive rotational behavior and asymmetries of glucose utilization of defined brain areas, including the substantia nigra reticulata, globus pallidus, and entopeduncular nucleus. These nuclei are the main targets of the subthalamic excitatory projections. Lesion of the nucleus per se (without challenge with apomorphine) has no significant consequences on glucose utilization. The findings indicate that the STN is involved in the activation of the basal ganglia output nuclei induced by systemic dopaminergic stimulation.

Selective activation of group-II metabotropic glutamate receptors is protective against excitotoxic neuronal death.

Aminopyrrolidine-2R,4R-dicarboxylated (2R,4R-APDC) has recently been introduced as a potent and highly selective agonist of metabotropic glutamate (mGlu) receptor subtypes mGlu2 and -3. In murine cortical cultures containing both neurons and astrocytes, 2R,4R-APDC attenuated the delayed neuronal degeneration induced by a 10-min pulse of N-methyl-D-aspartate (NMDA). 2R,4R-APDC was maximally neuroprotective in a range of concentrations (0.1-1 microM) comparable to that reported for the activation of mGlu2 or -3 receptors in heterologous expression systems. The action of 2R,4R-APDC was sensitive to the mGlu2/3 receptor antagonists, (2S)-alpha-ethylglutamate and (2S,1S',2S',3R')-2-(2'-carboxy-3'-phenylcyclopropyl)glycine. These results indicate that activation of mGlu2 and/or -3 receptors is sufficient per se to protect neurons against excitotoxic degeneration, and encourage the search for potent, selective and systemically active mGlu2/3 receptor agonists as neuroprotective drugs.

Increased motor response to cocaine administration following recovery from chronic corticosterone treatment in the rat.

Several lines of evidence suggest interaction between glucocorticoids and the rat brain dopaminergic system. Here we demonstrate that a two week recovery from chronic high-dose corticosterone treatment potentiates the behavioral response to acute cocaine challenge in the rat. This effect is associated with significant increases of plasma corticosterone levels in response to cocaine. Then, derangement of the hypothalamus-pituitary-adrenal axis, induced by long-term treatment with corticosterone, facilitates the behavioral response to cocaine.

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia.

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.

Differential effects of acute administration of clozapine or haloperidol on local cerebral glucose utilization in the rat.

We employed the [14C]2-deoxyglucose method in order to map local brain metabolic activity of rats administered 1, 5, or 20 mg/kg of clozapine, or 0.5 mg/kg of haloperidol, as compared to saline. Clozapine produced a dose-dependent reduction of glucose utilization. At the dose of 1 mg/kg, the effects were limited to limbic areas. An additional number of structures were significantly affected following administration of 5 mg/kg (the whole hippocampal formation and septal area, and cortical limbic areas). The dose of 20 mg/kg markedly reduced glucose utilization in most of the areas examined. Haloperidol (0.5 mg/kg) reduced glucose utilization of the orbital cortex, hippocampal formation and septal area, globus pallidus, amygdala, ventral thalamus, and substantia nigra reticulata. The results show that acute administration of clozapine or haloperidol are associated with different distribution patterns of altered cerebral energy metabolism. Clozapine differently from haloperidol, reduces energy metabolism of the nucleus accumbens and other limbic areas. Haloperidol, but not clozapine (1 or 5 mg/kg), affects the substantia nigra reticulata.

Functional correlates of heroin sensitization in the rat brain.

The aim of the study was to measure the changes in cerebral energy metabolism and c-fos mRNA expression following challenge with heroin in drug-naive rats and in animals previously sensitized to the drug. Acute heroin administration to drug naive-rats produced a generalized metabolic depression. In contrast, challenge with heroin in drug-sensitized rats produced selective metabolic increases in structures belonging to the basal ganglia. These changes were accompanied by increased c-fos mRNA expression in the caudate-putamen nucleus. These results demonstrate that the process of sensitization to heroin is coupled to functional changes that are confined to the subcortical motor circuits of the basal ganglia.

Increased functional response to cocaine challenge following recovery from chronic corticosterone in the rat.

Several lines of evidence suggest an interaction between glucocorticoids and the rat brain dopaminergic system. Here we demonstrate that a 14-day period of recovery from chronic corticosterone (10 mg/day for 21 consecutive days) potentiates the functional response to acute cocaine challenge in the rat by producing selective metabolic changes in limbic and motor areas, that are not measurable in vehicle-pretreated rats. These data indicate that chronic corticosterone has a long-term facilitatory role in the central effects of cocaine.

Effects of prolonged autovehicle driving on male reproduction function: a study among taxi drivers.

This study had the purpose of exploring the possible association between the work exposures of professional drivers and their reproductive health, by studying a group of 201 taxi drivers in the city of Rome. Data on work and reproductive history were collected by interviews. Biological markers examined in 72 subjects included salivary testosterone levels, sperm quality (i.e., sperm concentration, sperm morphology, and motility), and fertility experience, including time to pregnancy. Their spermatologic profile was compared with that of a control group of 50 healthy subjects of similar age and smoking habits. The results showed that taxi drivers, compared to the controls, had a significantly lower prevalence of normal sperm forms (45.8% vs. 64.0%); this was particularly true for those with a longer time on this job. This result was confirmed by a multivariate analysis in which confounders such as age, smoking, and alcohol consumption were controlled. The other sperm parameters did not differ in the study and the control groups. Among the life-style factors, we found smoking to be associated with poorer sperm morphology. Moderate alcohol consumption was associated with a better seminologic profile, while the pattern in respect to coffee intake was inconclusive. Subjects with poor semen quality also more frequently exhibited longer time to pregnancy of their partner. The results suggest that prolonged urban automobile driving might be a risk factors for sperm quality, and particularly for sperm morphology, but the finding needs further confirmation.

Intravenous morphine increases glucose utilization in the shell of the rat nucleus accumbens.

The [14C]2-deoxyglucose method was applied to measure the effects of the acute intravenous administration of morphine sulphate (0.2-0.4 mg/kg) on cerebral glucose utilization in rats. Morphine produced dose-dependent increases of glucose metabolism in the shell of the nucleus accumbens, without affecting functional activity in any other brain area. These results provide further evidence for the preferential effects of intravenously abused substances in the shell of the nucleus accumbens.

Differential regulation of adrenocorticoid receptors in the hippocampus and spinal cord of adrenalectomized rats.

Using multiple polymerase chain reaction assay and cytosolic receptor binding assay we studied type I, mineralocorticoid receptor (MR), and type II, glucocorticoid receptor (GR), adrenocorticoid receptors expression in rat hippocampus and spinal cord, at various times after adrenalectomy: 12 hr, 24 hr, 3 days, and 1 week. Analysis of the data demonstrates that in hippocampus the expression of MR and GR mRNA was not significantly affected by adrenalectomy. On the contrary, Bmax of MR was significantly increased at each time post-surgery, with only slight modifications of Kd. Bmax and Kd for GR showed a significant increase after 3 days and 1 week. In the spinal cord, MR mRNA was increased 12 hr after adrenalectomy, reaching a maximum at 3 days. Bmax of MR was also significantly increased after 3 days, whereas its Kd remained unchanged for the entire duration of the the study. Both GR mRNA and binding parameters were poorly affected by adrenalectomy. The results of the present experiments demonstrate that the absence of adrenocortical hormones influences differentially MR and GR expression in hippocampus and spinal cord, suggesting the existence of various and independent mechanisms of regulation of adrenocorticoid receptor.

Arginine-aspartate and haloperidol-induced neurobehavioral effects in the rat.

This study determined the effects, in the rat, of 8-day treatment with arginine-aspartate on haloperidol-induced catalepsy, decrease of locomotor activity and change of striatal dopamine, homovanillic acid (HVA) and dihydroxy-phenylacetic acid (DOPAC) content. Arginine-aspartate was able to attenuate the haloperidol-induced decrease of locomotor activity and to significantly reduce the catalepsy. Moreover, arginine-aspartate treatment itself increased striatal dopamine content and produced a significant decrease of the HVA/dopamine ratio. Pretreatment with arginine-aspartate was able to partially counteract the haloperidol-induced changes of dopamine metabolism: the haloperidol-induced increases of the DOPAC/dopamine and HVA/dopamine ratios were significantly reduced in arginine-aspartate- pretreated rats. These results suggest that the action of arginine-aspartate on haloperidol-induced neurobehavioral effects is probably mediated by interference with striatal dopaminergic innervation.

[Ultrastructural morphological study of supragingival tartar]. / Studio morfologico ultrastrutturale di tartaro sopragengivale.

Specimens of old supragingival calculus, collected from 5 patients with periodontitis, were examined electron-microscopically. Both intracellular and extracellular calcification were found. Intracellular calcification began as needle-shaped crystals or minute amorphous deposits within microorganisms. Extracellular calcification originated within the interbacterial matrix.

Histochemical properties of the "crystal ghosts" of calcifying epiphyseal cartilage.

The crystal ghosts represent a crystal associated organic material which is stained by acidic phosphotungstic acid, periodic acid-silver methenamine and periodic acid-thiosemicarbazide-osmium, and is reactive with cations and with colloidal iron at pH 2.0, and unreactive after methylation and saponification. These results suggest that crystal ghosts are, or derive from, proteoglycans of calcifying matrix.