FT-Raman Spectroscopy Study of the Remineralization of Microwave-Exposed Artificial Caries.

Dental caries is a microbially mediated disease that can result in significant tooth structure degradation. Although the preponderance of lesions is treated by surgical intervention, various strategies have been developed for its noninvasive management. Here, we use a novel approach for noninvasive treatment based on killing Streptococcus mutans with high-frequency microwave energy (ME). The rationale for this approach is based on modulating the pH of caries to a physiological state to enable spontaneous tooth remineralization from exogenous sources. In the present study, after demonstrating that ME kills >99% of S. mutans in planktonic cultures, 8 enamel slabs were harvested from a single tooth. Baseline mineral concentration at each of 12 points per slab was obtained using Fourier transform (FT)-Raman spectroscopy. Surface demineralization was subsequently promoted by subjecting all samples to an S. mutans acidic biofilm for 6 d. Half of the samples were then exposed to high-frequency ME, and the other half were used as controls. All samples were next subjected to a remineralization protocol consisting of two 45-min exposures per 24-h period in tryptic soy broth followed by immersion in a remineralizing solution for the remaining period. After 10 d, samples were removed and cleaned. FT-Raman spectra were again obtained at the same 12 points per sample, and the mineral concentration was determined. The effect of the remineralization protocol on the demineralized slabs was expressed as a percentage of mineral loss or gain relative to baseline. The mineral concentration of the microwave-exposed group collectively approached 100% of baseline values, while that of the control group was in the order of 40%. Differences between groups were significant (P = 0.001, Mann-Whitney U test). We concluded that killing of S. mutans by ME promotes effective remineralization of S. mutans-demineralized enamel compared with controls.

Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus.

The actions of androgens in both peripheral and central tissues are linked in part to their ability to specifically bind and activate androgen receptors (ARs). ARs have been well studied in the rat hypothalamus and peripheral reproductive tissues, where they are directly involved in endocrine feedback mechanisms and reproduction. Previous studies revealed relatively high levels of AR and AR messenger RNA (mRNA) in the rat hippocampus; however, the action of androgen in this brain region remains unclear. To begin to address this issue, we used a multidisciplinary approach to quantitate hippocampal AR and AR mRNA levels and investigate their regulation after various hormonal manipulations. In vitro binding assays revealed a single, saturable, high affinity binding site for androgen in hippocampal cytosols. The expression of AR mRNA in the intact adult male rat hypothalamus and hippocampus was demonstrated using reverse transcription-polymerase chain reaction and quantified using a ribonuclease protection assay. Comparable levels of AR mRNA were found in the hippocampus and hypothalamus. In addition, in situ hybridization analysis revealed a unique distribution of AR mRNA in the hippocampus. AR mRNA was found predominately in the CA1 pyramidal cells, which form the major signal output of the hippocampal trisynaptic circuit. Reverse transcription-polymerase chain reaction of total RNA from microdissected hippocampal regions confirmed this distribution. Ribonuclease protection assay demonstrated a significant decrease in the AR mRNA content of the hippocampus in animals killed 4 days after castration or in intact rats after four daily injections of the AR antagonist, flutamide (15 mg/animal), compared to that in intact controls (P < 0.01). In contrast, a 35% increase (P < 0.05) in the hippocampal AR mRNA content was found in old (22-month-old) compared to young (5-month-old) male rats. In both cases, [3H]dihydrotestosterone binding to the cytosolic preparation did not parallel the changes observed in the AR mRNA content. Taken together, these data demonstrate that hippocampal cells containing AR can respond to circulating androgen to alter AR gene expression. Furthermore, AR mRNA autoregulation appears to be both age and tissue specific and does not directly follow the regulatory patterns described for other steroid hormone receptors found in the hippocampus.

Prior chronic exposure to cocaine inhibits the serotonergic stimulation of ACTH and secretion of corticosterone.

The effect of long-term pretreatment with cocaine on serotonergic regulation of ACTH (adrenocorticotropic hormone; corticotropin) and secretion of corticosterone in rats was investigated. The following observations were made: (1) Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. However, cocaine caused a reduction in the ability of the 5-HT (5-hydroxytryptamine, serotonin) releaser p-chloroamphetamine (PCA) to increase corticosterone in plasma, 42 hr after the last injection of cocaine. (2) Exposure to cocaine for 7 days was sufficient to produce a maximal inhibition of the PCA-induced increase in ACTH in plasma. (3) The inhibitory effect of cocaine on PCA-induced release of ACTH was more marked than on corticosterone. (4) Conversely, the dose-dependent stimulatory effect of two 5-HT1 agonists, RU 24969 (5-methoxy-3-(1,2,3,4-tetrahydro-4-pyridinyl)-1H-indole) and m-CPP (m-chlorophenylpiperazine), on ACTH and corticosterone was not reduced by 7 days of exposure to cocaine. Taken together, these findings indicate that pretreatment with cocaine reduced the function of serotonergic nerve-terminals but not postsynaptic receptors, that stimulate ACTH and secretion of corticosterone.

Androgens selectively modulate C-fos messenger RNA induction in the rat hippocampus following novelty.

We have previously shown that androgen receptors are found in high concentrations in hippocampal CA1 pyramidal cells. To begin to explore the possible roles for androgen receptors in this area of the brain, we studied the effects of endogenous and exogenous androgen on the behaviourally induced expression of cellular immediate early gene messenger RNAs. Adult male Fischer 344 rats were either gonadectomized, gonadectomized and given two Silastic capsules of dihydrotestosterone propionate at the time of surgery, or left intact. Three weeks later, animals were placed into a novel open field for 20 min. This behavioural paradigm caused region- and gene-specific increases of c-fos, jun-B, c-jun and zif268 messenger RNA in the hippocampus as determined by semi-quantitative in situ hybridization histochemistry. The removal of circulating androgen by gonadectomy potentiated, whereas dihydrotestosterone treatment of castrates attenuated, the behaviourally induced expression of c-fos messenger RNA in the CA1 region of the hippocampus. No changes in c-fos messenger RNA expression were detected in the CA3 or dentate gyrus regions where androgen receptor levels are low. Androgen status did not affect either the basal or stimulated expression of Jun-B, c-Jun or zif268 messenger RNA in any of the three cellular regions of the hippocampus examined. These results implicate androgen receptors in modulating the active response of hippocampal neurons to a behaviourally relevant stimulus. Since the products of cellular immediate genes can function to alter an array of downstream genes, the modulation of these genes in the hippocampus by gonadal hormones may have important ramifications for hippocampal function.

Antiphospholipid antibody syndrome presenting as a refractory noninflammatory pulmonary vasculopathy.

The clinical manifestations of antiphospholipid antibody syndrome (APLAS) are protean. Pulmonary manifestations are often thromboembolic in origin; ARDS and pulmonary hypertension have been reported as features of a widespread vasculopathy associated with systemic lupus or Sjögren's syndrome. This is the report of a woman with primary APLAS who died of a noninflammatory pulmonary vasculopathy. The case is unusual in its pulmonary manifestations, its initial response to corticosteroids and antithrombotic medications, its failure to stabilize with high-intensity warfarin sodium and aspirin treatment, and finally its fulminant progression despite multiple interventions.

The treadmill test--where to stop and what does it mean?

The prognostic utility of an exercise ECG test depends upon having an adequate workload to stress the cardiac system. A negative stress test, in which there are no adverse clinical or ECG findings, and in which an adequate workload is achieved, stratifies patients into a low-risk group. The 1997 American Heart Association guidelines imply that any index of workload--heart rate, rate-pressure product, or exercise duration in multiples of resting O2 consumption (METS)--could be used to indicate that adequate stress was achieved. However, while there is considerable evidence supporting the use of METS as a strong independent prognostic variable, there is less support for the use of rate-pressure product or heart rate. Indeed, there is evidence that a high heart rate at a low workload carries an adverse prognosis. Further research is needed to identify the number of METS achieved that would define an adequate workload. In the meantime, a review of the literature suggests that 7 to 10 METS is a reasonable ballpark figure of the minimum workload in patients with a negative stress test that would imply a favorable outcome.

Distribution of NMDAR1 receptor subunit mRNA and [125I]MK-801 binding in the hypothalamus of intact, castrate and castrate-DHTP treated male rats.

This study examines NMDAR1 receptor subunit mRNA expression and [125I]MK-801 binding in hypothalamic and limbic nuclei of intact, castrate and castrate-dihydrotestosterone propionate (DHTP)-treated male rats. In intact rats, the highest levels of NMDAR1 mRNA were observed in the supraoptic, suprachiasmatic, ventromedial and arcuate nuclei. Low levels of hybridization were observed in the bed nucleus of the stria terminalis, lateral preoptic area, lateral hypothalamic area and lateral septum. In castrated rats both NMDAR1 mRNA and [125I]MK-801 binding are significantly decreased in the lateral septum compared to castrate rats treated with DHTP, a non-aromatizable androgen. NMDAR1 mRNA was also significantly decreased in the supraoptic nucleus of castrate rats when compared to castrate rats treated with DHTP. These data suggest that androgens may modulate NMDA receptor function in some parts of the central nervous system.

Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat.

In the adult male rat, androgen and estrogen synergize in the regulation of male reproductive behaviors. To explore some of the molecular mechanisms underlying this synergism we examined the distribution and hormonal regulation of androgen receptor (AR) and estrogen receptor (ER) mRNAs in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BST) of the adult male rat. Using in situ hybridization, AR and ER mRNAs were found to be distributed in overlapping but unique patterns. The highest density of AR mRNA was found in the central part of the medial preoptic n. and the principal n. of the BST. Gonadectomy (GDX) of adult male rats caused an increase in hybridization density in both brain areas after 4 days followed by a decrease after 2 months. In contrast, ER mRNA was increased following GDX and remained high regardless of length of time. Treatment of adult GDX'd males with dihydrotestosterone (DHT) reversed the effects of GDX on AR mRNA at both the short and long-term castrate but had no effect on ER mRNA in both the MPOA and BST. Estrogen treatment increased AR mRNA in the long-term castrate only and decreased ER mRNA in both long- and short-term castrates. Immunocytochemical detection of AR revealed a similar distribution to AR mRNA; however, AR immunoreactivity was reduced in the MPOA and BST after both short- and long-term GDX. In vitro [3H]DHT binding in cytosols of the preoptic area showed appreciable binding but there was no effect of length of time following GDX. These data show that the pattern of regulation of AR mRNA is unique to this receptor type and does not follow the pattern of regulation of the ER mRNA. Furthermore, although the distribution of AR mRNA and AR protein coincide within the MPOA, changes in mRNA levels as a result of castration or hormone treatment do not result in corresponding changes in binding. This mismatch between mRNA and binding suggests a complex regulation of AR beyond simply changes in transcription.

Androgens modulate glucocorticoid receptor mRNA, but not mineralocorticoid receptor mRNA levels, in the rat hippocampus.

Androgen, mineralocorticoid and glucocorticoid receptors (AR, MR and GR, respectively) are ligand-activated transcription factors that alter gene expression and have a wide variety of effects in the central nervous system. High levels of AR, MR and GR mRNA have been found in the CA1 pyramidal cell region of the rat hippocampus and all 3 of these proteins bind to a similar hormone response element in DNA suggesting the possibility of common receptor function or cross-talk between these receptors at the level of transcription. To begin to investigate this hypothesis, we examined the regulation of AR, MR and GR mRNA expression in the rat hippocampus following treatment with androgens in combination with gonadectomy and/or adrenalectomy. Three-month-old male Sprague-Dawley rats were either castrated for 3 weeks, castrated and immediately implanted with 2 Silastic capsules filled with the non-aromatizable androgen, dihydrotestosterone, or left gonadally intact. Four days prior to sacrifice, these animals were either adrenalectomized or sham operated. GR, MR and AR mRNA were measured in the hippocampal subfields using in situ hybridization. In the CA1 region, dihydrotestosterone treatment of castrates decreased GR mRNA levels to 69 percent of levels found in gonadally intact rats and prevented the adrenalectomy-induced increases in GR mRNA observed in the gonadally intact and castrated animals. No changes in GR mRNA were observed in the CA3 region or dentate gyrus, where AR expression is low or absent. There was no effect of androgen treatment on MR mRNA levels nor did gonadectomy or androgen replacement alter the increases in MR mRNA following adrenalectomy. AR mRNA levels in the CA1 region were unchanged across all treatment groups. In vitro binding studies revealed almost complete nuclear occupancy of hippocampal AR in dihydrotestosterone-treated castrates. No appreciable in vitro binding of dihydrotestosterone to hippocampal MR or GR (Ki approximately 1500 nM) was observed which suggests that androgen regulation of GR mRNA in the hippocampus is occurring through AR binding. These data demonstrate a functional similarity of androgens and glucocorticoids in the regulation of GR mRNA levels in an area where AR and GR are colocalized. Androgen-mediated downregulation of GR expression may prove to be an important event in the adaptive responses of CA1 pyramidal cells to hormonal stimuli.

Repeated injections of cocaine inhibit the serotonergic regulation of prolactin and renin secretion in rats.

Alterations in serotonergic function following repeated cocaine injections were examined using neuroendocrine responses to a serotonin (5-HT) releaser and 5-HT agonists. Forty-two hours following administration of cocaine (1-15 mg/kg i.p.) twice daily for 7 or 30 days, male Sprague-Dawley rats were injected with the 5-HT releaser p-chloroamphetamine (PCA; 8 mg/kg i.p.) and blood samples were collected 1 h later for radioimmunoassays of plasma prolactin, plasma renin activity (PRA) and plasma renin concentration (PRC). PCA significantly increased secretion of prolactin and renin. These responses were attenuated in rats pretreated with cocaine for 30 days. In rats receiving cocaine for 7 days, the attenuation of PCA-induced secretion of prolactin and renin was less consistently observed. To determine whether these alterations were due to pre- or postsynaptic effects, rats were injected with cocaine (15 mg/kg i.p.) twice daily for 7 days, and the neuroendocrine responses to the direct 5-HT agonists RU 24969 and m-CPP were examined, 42 h after the last cocaine injection. Pretreatment with cocaine potentiated RU 24969-induced stimulation of plasma prolactin concentration. However, cocaine did not alter the ability of m-CPP to increase plasma prolactin concentrations. The stimulation of renin secretion in response to both 5-HT agonists was not altered by cocaine pretreatment. The data suggest that repeated cocaine impairs the function of serotonergic nerve terminals that regulate these endocrine responses. Furthermore, the 5-HT receptors that mediate prolactin secretion may exhibit supersensitivity.

Neuroendocrine responses to cocaine do not exhibit sensitization following repeated cocaine exposure.

Cocaine-induced enhancement of motor activity and extracellular dopamine concentrations exhibits sensitization upon repeated exposure. In this study, the neuroendocrine responses to cocaine were examined following cocaine pretreatment regimens which have been shown to produce behavioral sensitization. Adult male rats were injected with cocaine (15 mg/kg, IP) once daily for 14 days, followed by a dose-response challenge with cocaine (1-15 mg/kg, IP) either 18 hours or 7 days after the final pretreatment injection. Blood was collected 15 minutes following injections for radioimmunoassay of ACTH, corticosterone, prolactin, and renin. Cocaine increased plasma ACTH and corticosterone, while it decreased prolactin and renin concentrations. Pretreatment with cocaine for 2 weeks did not alter any of these endocrine responses after either the 18 hour or 7 day interval between pretreatment and challenge injections. In contrast, sensitization to the locomotor stimulant effects of cocaine was observed on the final day of pretreatment injections, and 7 days later. These data suggest that these endocrine effects of cocaine do not exhibit sensitization following repeated cocaine exposure.

Cocaine-induced suppression of renin secretion is partially mediated by serotonergic mechanisms.

Acute cocaine reduces renin secretion. To determine whether serotonergic neurons mediate this effect, male Sprague-Dawley rats received the serotonin (5-HT) neurotoxin 5,7-dihydroxytryptamine (75 micrograms/side, ICV) 2 weeks prior to cocaine injections (3.75-15 mg/kg, IP). 5-HT lesions attenuated the cocaine-induced reduction of plasma renin concentration (PRC), suggesting a partial 5-HT role. To determine which receptors mediate this response, rats were pretreated with the partial 5-HT1A agonist 8-[2-[4-(2-methoxyphenyl)-l-piperazinyl]ethyl]-8-azaspirol-[4,5]- decane-7,9-dione (BMY 7378) (1 mg/kg, SC), the 5-HT1C/5-HT2 antagonist ritanserin (0.1 mg/kg, SC), or the alpha 2/5-HT1A antagonist yohimbine (1 mg/kg, SC) prior to cocaine. None of the antagonists altered the cocaine-induced suppression of PRC, although BMY 7378 and yohimbine elevated PRC. The data suggest that cocaine's effect is partially mediated by a serotonergic mechanism, but do not support a role for 5-HT1A receptors, 5-HT2/5-HT1C receptors, or alpha 2-adrenoceptors in mediating the suppressive effect of cocaine on renin secretion.

Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis.

The rapid activation of stress-responsive neuroendocrine systems is a basic reaction of animals to perturbations in their environment. One well-established response is that of the hypothalamo-pituitary-adrenal (HPA) axis. In rats, corticosterone is the major adrenal steroid secreted and is released in direct response to adrenocorticotropin (ACTH) secreted from the anterior pituitary gland. ACTH in turn is regulated by the hypothalamic factor, corticotropin-releasing hormone. A sex difference exists in the response of the HPA axis to stress, with females reacting more robustly than males. It has been demonstrated that in both sexes, products of the HPA axis inhibit reproductive function. Conversely, the sex differences in HPA function are in part due to differences in the circulating gonadal steroid hormone milieu. It appears that testosterone can act to inhibit HPA function, whereas estrogen can enhance HPA function. One mechanism by which androgens and estrogens modulate stress responses is through the binding to their cognate receptors in the central nervous system. The distribution and regulation of androgen and estrogen receptors within the CNS suggest possible sites and mechanisms by which gonadal steroid hormones can influence stress responses. In the case of androgens, data suggest that the control of the hypothalamic paraventricular nucleus is mediated trans-synaptically. For estrogen, modulation of the HPA axis may be due to changes in glucocorticoid receptor-mediated negative feedback mechanisms. The results of a variety of studies suggest that gonadal steroid hormones, particularly testosterone, modulate HPA activity in an attempt to prevent the deleterious effects of HPA activation on reproductive function.

The King's College Hospital Acute Stroke Unit.

The King's College Hospital (KCH) Acute Stroke Unit (ASU) was set up in January 1994 in order to provide acute management for patients admitted with stroke and to undertake biomedical research. Of 206 patients admitted to KCH with a stroke or suffering an in-hospital stroke, 141 (68%) patients were admitted to the ASU over its first 6 months of operation: 120 (85%) were from the Accident and Emergency Department and 21 (15%) from other wards. Management included resuscitation and medical stabilisation, investigation, prevention of stroke complications (including aspiration, venous thrombosis, and pressure sores), rehabilitation (physiotherapy, occupational therapy, speech and language therapy), nutrition (dietetics) and initiation of secondary prevention measures (aspirin or anticoagulation, blood pressure and lipid lowering, and carotid endarterectomy). All aspects of management are driven by agreed guidelines. Patients remain under the care of the admitting physician but specific stroke management and guidance is provided by two research doctors and the unit's nurses, therapists and dietician. The unit also facilitates research into stroke pathophysiology and acute therapeutic interventions. Our experience suggests that an ASU is relatively easy to set up and may contribute to improved care. Whether ASUs improve patient survival and functional outcome, and are cost-effective, requires further study.

Cocaine-induced elevation of plasma adrenocorticotropin hormone and corticosterone is mediated by serotonergic neurons.

We investigated the hypothesis that cocaine-induced elevations of plasma adrenocorticotropin hormone (ACTH) and corticosterone are mediated by brain serotonin (5-HT) neurons. Adult male rats were pretreated with the 5-HT depleting agent p-chlorophenylalanine, the 5-HT neurotoxin 5,7-dihydroxytryptamine, the partial 5-HT1A agonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8- azaspirol[4,5]-decane-7,9-dione (BMY 7378) or the 5-HT1C/2 antagonist ritanserin. The effects of cocaine (2-15 mg/kg, i.p.) on plasma ACTH and corticosterone were then examined. Cocaine dose-dependently increased ACTH and corticosterone concentration. This increase was prevented by 5-HT depletion with PCPA and by destruction of 5-HT neurons with i.c.v. injections of 5,7-dihydroxytryptamine. The cocaine-induced elevation of ACTH and corticosterone was not significantly modified by administration of the partial 5-HT1A agonist BMY 7378, suggesting that 5-HT1A receptors probably do not mediate ACTH and corticosterone secretion. However, pretreatment with the 5-HT2/5-HT1C antagonist ritanserin virtually eliminated the cocaine-induced elevation of corticosterone. To determine whether these effects of cocaine are centrally mediated, conscious rats received cocaine injections into the cerebral ventricle through chronically implanted cannulas. Plasma ACTH concentrations were dose-dependently increased, whereas low doses (50 micrograms/kg, i.c.v.) produced a maximal increase in corticosterone concentration. These data indicate that the cocaine-induced stimulation of ACTH and corticosterone secretion is mediated by 5-HT neurons in brain, and furthermore, that 5-HT2 or 5-HT1C receptors are responsible for this effect.