In vivo luminescent imaging of NF-κB activity and NF-κB-related serum cytokine levels predict pain sensitivities in a rodent model of peripheral neuropathy.

BACKGROUND: Methods for the detection of the temporal and spatial generation of painful symptoms are needed to improve the diagnosis and treatment of painful neuropathies and to aid preclinical screening of molecular therapeutics. METHODS: In this study, we utilized in vivo luminescent imaging of NF-κB activity and serum cytokine measures to investigate relationships between the NF-κB regulatory network and the presentation of painful symptoms in a model of neuropathy. RESULTS: The chronic constriction injury model led to temporal increases in NF-κB activity that were strongly and non-linearly correlated with the presentation of pain sensitivities (i.e. mechanical allodynia and thermal hyperalgesia). The delivery of NEMO-binding domain peptide reduced pain sensitivities through the inhibition of NF-κB activity in a manner consistent with the demonstrated non-linear relationship. Importantly, the combination of non-invasive measures of NF-κB activity and NF-κB-regulated serum cytokines produced a highly predictive model of both mechanical (R(2) = 0.86) and thermal (R(2) = 0.76) pain centred on the NF-κB regulatory network (NF-κB, IL-6, CXCL1). CONCLUSIONS: Using in vivo luminescent imaging of NF-κB activity and serum cytokine measures, this work establishes NF-κB and NF-κB-regulated cytokines as novel multivariate biomarkers of pain-related sensitivity in this model of neuropathy that may be useful for the rapid screening of novel molecular therapeutics.

Effects of morphine and methadone on serum testosterone and luteinizing hormone levels and on the secondary sex organs of the male rat.

The effects of morphine and methadone on the endocrine control of the male rat's sexual function were examined. The results indicate that these narcotics markedly reduce the structural and functional integrity of the secondary sex organs by producing a pronounced reduction in serum testosterone levels. Serum levels of luteinizing hormone (LH) were not detectable in narcotic-treated animals, whereas serum levels of the follicle stimulating hormone (FSH) were unaltered. On the basis of these observations, it seems reasonable to conclude that the narcotics inhibit the secretion of LH, by an action either in the hypothalamus (e.g., suppression of LH-releasing hormone) or directly in the pituitary gland, which leads to a reduction in serum testosterone levels and a subsequent reduction in the wet-tissue weight and secretory activity of the secondary sex organs.

Effect of dopamine agonist (Lergotrile mesylate) therapy on twenty-four hour secretion of prolactin in treated Parkinson's disease.

Plasma PRL was measured at 20-min intervals in six patients with Parkinson's disease under various treatment protocols. In addition, 24-h mean GH levels were measured. The results of these studies showed that two untreated patients with Parkinson's disease had normal 24-h mean PRL levels with the normal increase during sleep. During chronic treatment with L-dopa-carbidopa (Sinemet), the 24-h PRL level was 12.8 +/- 4.9 ng/ml (mean +/- SD) and there was persistence of augmented PRL secretion during sleep. The 24-h mean GH level ranged from 1.5-4.4 ng/ml, with a mean of 2.5 ng/ml. The addition of a dopamine agonist (Lergotrile mesylate) resulted in a significant (P less than 0.01) suppression of the 24-h mean PRL levels and abolition of the normal sleep augmentation after 2 weeks of therapy. This suppression was maintained in one patient who was restudied 4 months after the addition of dopamine agonist therapy to L-dopa-carbidopa. The 24-h mean GH levels did not change significantly after the addition of the dopamine agonist when compared to L-dopa-carbidopa alone. These results suggest a dichotomy between the PRL and GH responses to combined L-dopa-carbidopa and dopamine agonist therapy. In addition, the preservation of normal PRL regulation in the two untreated patients with Parkinson's disease suggests that dopaminergic neurons are not universally affected in this disorder.

Effect of erythromycin on carbamazepine kinetics.

Two recent reports of carbamazepine-induced intoxication during concurrent therapy with macrolide antibiotics prompted us to perform a carefully controlled two-way cross-over study in eight healthy male nonsmokers. Treatment A was 250 mg erythromycin every 6 hr for 5 days before and 3 days after 400 mg carbamazepine. Treatment B was 400 mg of carbamazepine alone. One half of the subjects received treatment A, then B, while the other half received treatment B, then A. There was a 4-wk washout period between treatments. Plasma samples obtained at various times up to 72 hr after the carbamazepine dose were assayed in duplicate by HPLC. The data were fit to a one-compartment open model with first-order absorption and elimination. Clearance of oral carbamazepine was lower in the presence of erythromycin (mean +/- SD, 0.290 +/- 0.074 and 0.360 +/- 0.072 1 X kg-1 X day-1). There were no differences in apparent volume of distribution (1.01 +/- 0.20 and 1.04 +/- 0.12 1 X kg-1), elimination rate constant (0.302 +/- 0.113 and 0.348 +/- 0.079 day-1), or absorption rate constant (14.5 +/- 8.7 and 15.5 +/- 16.6 day-1) between the two treatment groups. The decrease in clearance of oral carbamazepine secondary to erythromycin indicates that further clinical studies are warranted.

The methylphenidate-induced stereotypy in the awake rat: local cerebral metabolism.

The local cerebral metabolic rate for glucose (1=CMRg) was computed in rats with methylphenidate-induced stereotypy using the quantitative 14C-2-deoxyglucose (2-DG) technique. Four rats received methylphenidate 15 mg per kilogram IP. Compared to five control animals, treated rats showed statistically significant (p less than or equal to 0.05) increases in 1-CMRg in globus pallidus, ventral lateral nucleus of the thalamus, subthalamic nucleus, red nucleus, substantia nigra, entopeduncular nucleus, inferior olivary nucleus, and the lateral cerebellar cortex. Significantly, 1-CMRg decreased in area 4 of the motor cortex. The auditory system showed no change in 1-CMRg, demonstrating the specific action of methylphenidate in the rat brain. This technique allows evaluation of the functional anatomy of the entire central nervous system and may be helpful in understanding the mechanisms of methylphenidate-induced stereotypy.

Role of nicotinamide adenine dinucleotide in ethanol-induced depressions in testicular steroidogenesis.

It is rapidly becoming accepted, without direct evidence, that a change in the NAD+/NADH ratio in the testes produced by the metabolism of ethanol is the principal mechanism involved in its now well-established effects on testicular steroidogenesis. The purposes of the present studies were 2-fold: (1) to examine whether, in fact, in vivo or in vitro ethanol exposure alters the NAD+/NADH ratio in the testes; and (2) to examine the validity of previous reports in which it was found that NAD+ prevented the effects of ethanol on testicular steroidogenesis under in vitro conditions. With regard to the first objective, we found that a large dose of ethanol (2.5 g/kg) markedly reduced gonadotropin-stimulated testicular steroidogenesis in vivo in the male rat, but it did not alter the NAD+ and NADH concentrations in the testes. Similarly, extremely high ethanol concentrations (200 mM) substantially suppressed hMG-stimulated testosterone biosynthesis in in vitro Leydig cell preparations but no change in NAD+ concentration occurred; NADH levels were very low in the Leydig cell preparations (less than 2% of NAD+ levels), but did not appear to change as a function of ethanol exposure. Finally, in contrast to previously published results, we found that NAD+ (1 mM) did not prevent the in vitro effects of ethanol on cAMP-stimulated testicular steroidogenesis. Consequently, our results fail to support the hypothesis that acute in vivo or in vitro ethanol administration inhibits the biosynthesis of testosterone by altering the NAD+/NADH ratio in the testes.

The effect of acute and chronic hematocrit changes on cardiovascular hemodynamics in spontaneously hypertensive rats.

Heparin given over a long term by a subcutaneous route consistently lowers blood pressure in the hypertensive rat models. The decrease in blood pressure is accompanied by a parallel decrease in hematocrit suggesting a causal relationship between hematocrit and blood pressure. The aim of this study was to define the relationships between acute and chronic hematocrit changes and blood pressure in the normotensive and hypertensive states. Normotensive Wistar (NWR) and spontaneously hypertensive (SHR) rats were used. Hematocrit was decreased acutely by blood-letting, and chronically by treatment with either heparin (H) or phenylhydrazine (P) for 4 weeks. Acute and chronic hematocrit increase was accomplished by packed cells transfusion. Systolic blood pressure was measured weekly; and at the end of the experimental period, plasma volume, cardiac output, and mean arterial pressure were obtained. Acute hematocrit decrease or increase (hematocrit ranging from 25 to 65%) did not affect blood pressure in either strain of rats; whereas chronic hematocrit changes (hematocrit ranging from 35 to 61%) significantly affected blood pressure only in SHR. Thus, chronic hematocrit decrease induced by H or P resulted in a significant fall in blood pressure compared to control (201 +/- 3 v 175 +/- 4, 167 +/- 4 mm Hg, respectively; P < .05). Conversely, a chronic hematocrit increase resulted in a significant rise in blood pressure (201 +/- 3 v 219 +/- 4 mm Hg; P < .05). Similar hematocrit changes produced in NWR, as in SHR, did not affect blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

A relationship between blood pressure control, hematocrit level, and renal function in treated essential hypertension.

The effect of rigid blood pressure control on renal function is an unsettled issue. This study describes a retrospective analysis on the relationships between blood pressure control, hematocrit levels, and renal function in 97 treated hypertensive patients. Data analysis was done on systolic and diastolic blood pressure, hematocrit levels, renal function assessed by serum urea nitrogen (SUN), serum creatinine (Scr), and hydrochlorothiazide (HCTZ) dose at entry and at four anniversary dates thereafter. The patients were divided into two groups: group I and group II on the basis of HCTZ dose. Group I received an average of 100 mg HCTZ daily, whereas group II received an average of 50 mg HCTZ daily. In group I, the decrement in both systolic and diastolic blood pressure over time was highly significant (P < .0001); however, no change in renal function was noted. In group II, systolic blood pressure decreased significantly (P < .01) from entry to year 1, then leveled off. In year 4, systolic blood pressure was not different from that of entry. Conversely, the difference between entry and year 4 diastolic blood pressure was highly significant (P < .0001). In group II, significant decreases were noted between entry and year 4, SUN (16.5 +/- 5.7 versus 14.9 +/- 4.1 mg/dL; P < .0012) and Scr (1.29 +/- .23 versus 1.24 +/- .19 mg/dL; P < .0192). Hematocrit showed diverse responses; in group I, hematocrit significantly increased from entry to year 4 (44.8 +/- 2.5 versus 47.2 +/- 3.9%; P < .01); whereas, in group II, hematocrit significantly decreased (47.7 +/- 3.8 versus 44.9 +/- 3.4%; P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic haloperidol administration increases GABA binding and enhances neuronal responsiveness to iontophoresed GABA in rat globus pallidus.

Experiments were conducted to assess the effects of chronic haloperidol (CHAL) administration on gamma-aminobutyric acid (GABA) receptor binding within the rat globus pallidus (GP) and on the responsiveness of individual pallidal neurons to microiontophoretically applied GABA and glycine. Rats were administered haloperidol in their food for 30 days in increasing concentrations and the experiments were conducted 2 days after termination of the haloperidol treatment. GABA receptor binding and neuronal responsiveness to GABA were significantly increased within the GP following CHAL treatment. The mean EC50 value for GABA was significantly decreased in the CHAL-treated rats, but there was no change in the EC50 for glycine. Scatchard analysis of [3H]muscimol binding demonstrated a single high-affinity binding site (Kd = 5 nM) within both control and CHAL-treated rats. The binding capacity (Bmax) of this high-affinity site was significantly increased in CHAL-treated rats without any change in the dissociation constant (Kd) for this site. These results suggest that CHAL administration may lead to a decrease in GABA release by striatopallidal efferents. The results of this study, combined with those of our previous study on SNR neurons, have demonstrated that blockade of striatally mediated dopamine (DA) neurotransmission leads to similar changes in GABAergic mechanisms at the level of the GP and SNR and suggest that DA regulation of the striatopallidal and striatonigral GABAergic pathways need not be differentially organized as has previously been postulated.

Morphine exerts testosterone-like effects in the hypothalamus of the castrated male rat.

Previous research has indicated that endogenous opioids participate in the regulation of activity in the hypothalamic-pituitary-luteinizing hormone (LH) axis and mediate the negative feedback control exerted by testosterone. If this assumption is correct, then two predictions can be made. First, the effects of testosterone should be competitively inhibited by narcotic antagonists; and, second, opiates should mimic the acute and chronic effects of testosterone in the castrated male rat. The results of the present investigations support both of these predictions. We found that naloxone competitively antagonized the depressive effects of testosterone on serum LH in the castrated rat and, conversely, that testosterone competitively antagonized the LH-releasing properties of naloxone. In addition, morphine and testosterone both depressed serum LH levels in a dose-dependent fashion in the acutely castrated animal. Moreover, morphine was just as effective as testosterone in reversing the castration-induced fall in hypothalamic-LH-releasing hormone (LH-RH), which occurs in the chronically castrated male rat. On the other hand, morphine failed to reverse the long-term changes in pituitary LH content and increase in serum LH, which is consistent with prior observations that morphine affects only the hypothalamic aspect of the hypothalamic-pituitary-LH axis in the male rat. These results, thus, support the concept that an as yet unidentified opioid-containing neuronal system regulates activity in the hypothalamic-pituitary-LH axis and mediates the effects of testosterone on this axis.

Quantitative measurement of local cerebral metabolic rate for glucose utilizing tritiated 2-deoxyglucose.

The [14C]2-deoxyglucose (2-DG) technique has been widely utilized for quantitative measurement of local cerebral metabolic for glucose (1CMRG) in animals. The technique as presently used is limited by the energy of 14C beta-particles, which can travel relatively great distances in tissue. This results in limited audioradiographic resolution and in computed 14C concentrations which are a function of tissue section thickness. [3H]2-DG has less energetic beta-particles; hence, autoradiographs have better resolution and optical densities are independent of tissue thickness for sections greater than 5 micrometer. We have developed a method for quantitation of 1CMRG in rats using [3H]2-DG and a newly developed ultrasensitive X-ray film. Autoradiographic tissue standards were prepared by injecting rats with [3H]2-DG and assaying micro-samples of brain for 3H concentration. Ten rats were used in this study. Five rats received [3H]2-DG (300 muCi/100 g) and 5 rats received [14C]2-DG (7.5 muCi/100 g). The mean 1CMRG values for selected areas of the central nervous system demonstrated no significant difference (P greater than 0.05) between the [14C]2-DG and the [3H]2-DG groups. Values for 1CMRG from the [3H]2-DG group showed no variation attributable to inadequate microtome precision. The improved resolution obtained by utilizing [3H]2-DG is especially evident where gray matter (high 1CMRG) is immediately adjacent to white matter (low 1CMRG).

Assessment of carotid artery patency on routine spin-echo MR imaging of the brain.

We retrospectively reviewed the routine spin-echo MR studies of the brain in 12 patients with 13 angiographically demonstrated occlusions and in 14 patients with 16 high-grade stenoses of the carotid arteries. Intraluminal signal that was isointense with adjacent brain on long TR/short TE and long TR/long TE images was 100% specific for atherosclerotic occlusion. Of the 13 proved occlusions, six (46%) had significant degrees of hyperintense intraluminal signal indistinguishable from that observed consequent to slow flow distal to high-grade stenoses. MR detected only five (31%) of the 16 proved high-grade stenoses. Normal flow void does not exclude significant extracranial carotid stenosis. Occlusion cannot always be distinguished from high-grade stenosis when hyperintense intraluminal signal is encountered. However, a reliable diagnosis of atherosclerotic occlusion can be made when isointense intraluminal signal is observed.

Preoperative management of the surgical patient with neurologic disease.

This article reviews neurologic problems that have been categorized into two groups: parenchymal and vascular disease. Each disease has a brief summary of key clinical points, followed by recommended management strategies. The neurologic diseases most frequently encountered by the medical consultant are presented.

Local cerebral glucose metabolism after global ischemia: treatment by ventriculocisternal perfusion with a fluorocarbon emulsion.

The local cerebral metabolic rate for glucose (LCMRg) was measured in cats subjected to global cerebral ischemia (GCI). Control (nonperfused) cats showed decreased LCMRg (P less than 0.01) in the frontal, temporal, parietal, and occipital cortex 9.5 hours after a 10-minute exposure to GCI. Cats perfused ventriculocisternally with oxygenated nutrient solution (ONS) for 8 hours showed significant increases in the LCMRg (p less than 0.05) at 9.5 hours postischemia in the parietal and occipital areas over the levels found in untreated ischemic cats. Supplementing the ONS perfusion medium with fluorocarbon (OFNS) increased the LCMRg (P less than 0.05) in the frontal, as well as the parietal and occipital areas, over that seen in untreated ischemic brains. The increase of LCMRg in three (rather than only two) cortical areas may be a result of the ability of the fluorocarbon in OFNS to deliver greater quantities of oxygen to the brain than ONS without fluorocarbon. Perfusion with OFNS without glucose, or with low (50 mg%) glucose, was more effective than OFNS with high (200 mg%) glucose in restoring LCMRg to normal in all four cortical areas affected by GCI. In five brain areas not affected by GCI, perfusion with OFNS having no glucose significantly increased LCMRg as compared to normal animals. This study demonstrates that OFNS perfused by the ventriculocisternal route can restore toward normal the LCMRg following GCI and that different concentrations of glucose in the perfusing fluid will have variable effects on LCMRg in certain brain areas.

Characterization and possible opioid modulation of N-methyl-D-aspartic acid induced increases in serum luteinizing hormone levels in the developing male rat.

It has been previously reported that the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), elicits prompt increases in serum luteinizing hormone (LH) levels in young male rats. The present studies were carried out to determine whether the effects of NMDA on LH were mediated by the release of LHRH from the hypothalamus. We also examined whether NMDA-sensitive neuronal pathways interacted with the endogenous opioid system regulating LHRH release and the ontogeny of NMDA-evoked increases in serum LH. We found that the age-response curve for NMDA-induced increases in LH was an inverted U; at early ages (10 and 15 days) the amino acid was marginally effective in increasing LH levels, it became maximally effective from post-natal days 20-40 and thereafter rapidly lost its efficacy such that it was virtually inactive in adult animals. Dose-response curves revealed that adult animals were more than 10-fold less sensitive to NMDA than their younger counterparts. Our studies also demonstrated that NMDA increased LH via a direct effect on the hypothalamic release of LHRH since a potent LHRH antagonist competitively inhibited the effects of NMDA. Finally, we observed that morphine competitively inhibited the effects of NMDA on LH release, suggesting a relationship between NMDA-sensitive neuronal pathways and those endogenous opioid-containing systems which are known to regulate LH release.

Ethanol inhibits the naloxone-induced release of luteinizing hormone-releasing hormone from the hypothalamus of the male rat.

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.

Ethanol-induced reductions in testicular steroidogenesis: major differences between in vitro and in vitro approaches.

Although it is well established that ethanol suppresses gonadotropin- and cAMP-stimulated testicular steroidogenesis, there is not good agreement on two issues: which is the step in testosterone's biosynthetic pathway affected by ethanol; and the role of alterations in the NAD+/NADH ratio in ethanol's effects. In these studies, we have identified major differences between in vivo and in vitro approaches, which have previously been considered as totally equivalent experimental paradigms, which could explain these discrepancies. Under in vitro conditions, we observed that ethanol selectively inhibited the conversion of androstenedione to testosterone, but that it had a much more general effect under in vivo conditions. In addition, in agreement with other studies, NAD+ overcame ethanol's effects on testicular steroidogenesis in vitro, but only when labeled or unlabeled pregnenolone was added. In the absence of added pregnenolone, NAD+ was not effective in preventing ethanol's effects. Our results, thus, indicate that the differences which currently exist in the literature may be explained by the indiscriminate usage of in vivo and in vitro techniques.

Muscle fiber types and morphometric analysis of skeletal msucle in six-year-old children.

Needle biopsies from the vastus lateralis of 13 six-year-old Swiss children were analyzed for muscle fiber type populations and morphometrical characteristics. No significant differences existed between the males and females for fiber type distribution, maximum oxygen consumption, or any of the ultra-structural parameters investigated. The vastus lateralis muscle consisted of 19.7% fast twitch glycolytic (FG) fibers, 21.5% fast twitch oxidative glycolytic (FOG) fibers, and 5,9,% slow twitch oxidative (SO) fibers. Maximum oxygen consumption averaged 45.2 ml/kg min-1 when the subjects were considered as a single group. Morphometrically, it was found that the mean volume density of the central mitochondria was 5.54%, the mitochondrial/myofibrillar volume ratio was 6.68%, and the intracellular lipid volume was 0.46%. There was a significant correlation (r=0.69) between the mitochondrial volume density and the distribution of SO fibers as determined histochemically. It was concluded that the fiber type distribution pattern and ultrastructure of skeletal muscle in six-year-old children was not different from normal adult tissue.

Effect of lead chromate on chromosome aberration, sister-chromatid exchange and DNA damage in mammalian cells in vitro.

Possible mutagenic activity of lead chromate in mammalian cells was studied using assays for chromosome aberrations and sister-chromatid exchanges in cultured human lymphocytes, and DNA fragmentation as detected by alkaline-sucrose gradient sedimentation in cultured Chinese hamster ovary (CHO) cells. Lead chromate caused dose-related increases in chromosome aberration and sister-chromatid exchange in human lymphocytes. No increase in DNA damage was observed in CHO cells, possibly due to the relative insensitivity of the CHO cells and the limited solubility of lead chromate in tissue culture medium. The mutagenicity of lead chromate in human lymphocytes appears to be entirely due to the chromate ion since chromosome aberrations were induced by potassium chromate but not lead chloride.

Comparative mammalian in vitro and in vivo studies on the mutagenic activity of rhodamine WT.

Rhodamine WT, a xanthene dye used in the tracing of pollutants in water and in related studies, was tested for its mutagenicity in a battery of in vitro and in vivo mammalian assays. Using Chinese hamster ovary cells in the absence of metabolic activation mix, small dose-related increases in cytotoxicity, DNA damage (as detected by alkaline sucrose-gradient sedimentation) and sister-chromatid exchanges were detected, but an increase in the level of chromosomal damage was not seen. In the presence of metabolic activation a small, but statistically significant dose-related increase in sister-chromatid exchanges was evident, with no increase in cytotoxicity, DNA damage or chromosome aberrations. Furthermore, no increase in bone marrow micronuclei or sperm abnormalities was observed in male B6C3F1 mice. The data from all these mammalian assays, although involving different end-points, are in contrast to the mutagenic effects previously seen in Salmonella.