Chronic morphine potentiates the inflammatory response by disrupting interleukin-1beta modulation of the hypothalamic-pituitary-adrenal axis.

Interleukin-1-beta (IL-1beta) can promote inflammation by up-regulating vascular adhesion molecules and inhibit inflammation by activating the hypothalamic-pituitary-adrenal (HPA) axis to produce anti-inflammatory glucocorticoids. In this study, chronic morphine was shown to suppress IL-1beta-induction of corticotropin releasing factor (CRF) mRNA and plasma corticosterone levels. Leukocyte-endothelial adhesion (LEA) in rat mesenteric venules increased during IL-1beta- and FMLP-induced inflammation. Chronic morphine potentiated the LEA response to either IL-1beta or FMLP alone, and greatly enhanced LEA in response to combined IL-1beta and FMLP. Thus, it appears that chronic morphine exposure may promote a potentially damaging inflammatory reaction by disrupting the balance between IL-1beta-mediated local inflammation and the anti-inflammatory effects of the HPA axis.

An insulin-dependent hypoglycaemia induced by electroacupuncture at the Zhongwan (CV12) acupoint in diabetic rats.

Acupuncture at the Zhongwan acupoint has been widely used in traditional Chinese medicine to relieve symptoms of diabetes mellitus. Our study investigated the effect on plasma glucose of electroacupuncture applied at the Zhongwan acupoint in rat diabetic models. Plasma concentrations of insulin, glucagon and beta-endorphin- were also determined using radioimmunoassay. A decrease in plasma glucose was observed in rats after electroacupuncture (15 Hz, 10 mA) for 30 min at the Zhongwan acupoint. This was observed in normal rats and rat models with Type II (non-insulin-dependent) diabetes mellitus. No significant effect on plasma glucose was observed in rat models with Type I (insulin-dependent) diabetes mellitus: neither the streptozotocin (STZ)-induced diabetic rats nor the genetic (BB/W) rats. Further, the hypoglycaemic action of electroacupuncture stimulation disappeared in rats with insulin-resistance induced by an injection of human long-acting insulin repeated daily to cause the loss of tolbutamide-induced hypoglycaemia. An insulin-related action can thus be hypothesised. This hypothesis is supported by an increase in plasma insulin-like immunoreactivity after electroacupuncture stimulation in normal rats. Participation of glucagon was ruled out because there was no change in plasma glucagon-like immunoreactivity resulting from electroacupuncture stimulation. In addition to an increase in plasma beta-endorphin-like immunoreactivity, the plasma glucose lowering action of electroacupuncture stimulation at Zhongwan acupoint was abolished by naloxone in a sufficient dose to block opioid receptors. Thus we suggest that electroacupuncture stimulation at the Zhongwan acupoint induces secretion of endogenous beta-endorphin which reduces plasma glucose concentration in an insulin-dependent manner.

FOS expression induced by interleukin-1 or acute morphine treatment in the rat hypothalamus is attenuated by chronic exposure to morphine.

Interleukin-1 (IL-1) is a cytokine involved in a variety of biological activities. It has been hypothesized that the immunomodulatory effects of IL-1 are the result of both direct action on immune cells and indirect action on a regulatory cascade mediated through the hypothalamus. Chronic exposure to substances of abuse, such as morphine, appears to modulate immunoresponsiveness by mechanisms not yet defined. The expression of FOS, the protein product of the c-fos proto-oncogene, has been widely used as an anatomical marker for monitoring neuronal activity. We have previously shown that acute treatment with either morphine or IL-1 induces FOS immunoreactivity in the rat brain, including the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In this study, using immunocytochemical staining of FOS, we demonstrate that chronic exposure to morphine attenuates the cellular responsiveness to IL-1 and to morphine in the PVN and SON, whereas pretreatment with naloxone, an opiate receptor antagonist, does not reverse the effect of IL-1 on FOS expression. The results not only confirm that the PVN and SON are neuroanatomical sites where the actions of both morphine and IL-1 converge, but also indicate that chronic exposure to morphine may desensitize the cellular response involved in hypothalamic functions through an IL-1-dependent pathway.

Antisense oligodeoxynucleotide attenuates in vivo expression of c-fos in the paraventricular hypothalamic nucleus of the rat brain.

The proto-oncogene, c-fos, is expressed in neurons in response to stimulation by growth factors, neurotransmitters, and immunocytokines. In order to determine whether interleukin-1 beta (IL-1 beta)-induced Fos protein expression in the paraventricular hypothalamic nucleus (PVN) can be reduced or eliminated by a c-fos antisense oligodeoxynucleotide, rats were injected with c-fos antisense and Fos expression was examined immunocytochemically. Our results indicate that c-fos antisense oligodeoxynucleotide treatment decreases the density of Fos immuno-labeled nuclei in the PVN following IL-1 beta administration. These data suggest that, with the use of antisense technology, it may be possible to determine the role of proto-oncogene proteins in specific brain areas.

Interleukin-1 activation of FOS proto-oncogene protein in the rat hypothalamus.

The activation of FOS proto-oncogene protein has been used as an anatomical marker of activated brain areas. Immunocytochemical detection of FOS can provide information about the sites of action of extracellular stimuli, in spite of the relative absence of specific receptors, at the level of single cell resolution. Following the intracerebroventricular (i.c.v.) injection of recombinant human interleukin-1 (alpha) the c-fos mRNA levels isolated from rat hypothalamus were activated rapidly. In association with c-fos mRNA activation, the i.c.v. injection of interleukin-1 (alpha and beta) markedly induced the FOS immunoreactivity in the hypothalamus including periventricular (PE), paraventricular (PVN), supraoptic (SON), arcuate (ARC), and supramammillary (SuM) nuclei. Within the magnocellular neurons of the SON and PVN, activation of FOS by IL-1 appeared to be greater in areas known to have a high proportion of oxytocin-containing cells than in those of vasopressin-containing cells. Parvocellular neurons were also activated in the PVN. These data suggest sites of action of interleukin-1 in the rat hypothalamic areas reported to have relative absence of interleukin-1 receptor expression.

Equine urine pH: normal population distributions and methods of acidification.

Our investigation of the urine of grazing horses at the University of Kentucky shows that the mean pH level is about 7.9, and if their diet is supplemented with grain, it is about 7.4. There appears to be no significant effect of time of day or year on urine pH levels in horses. However, horses taken from pasture and supplemented with grain in a stalled environment show a slight decrease in urine pH. Additionally, we investigated the effects of storage on pH levels. Equine urine samples appear to be quite stable with regard to pH for 48h, but then show a marked increase. Urine pH can have a great effect on the urine concentration of some drugs and therefore, uncertainties can arise when data generated in grazing horses are compared or extrapolated to racing horses whose urine pH can be quite low. In an effort to simulate the drop in urine pH seen in some racing horses, we examined the effects of ammonium chloride, ascorbic acid, lactic acid and methionine on urine pH in research horses. Both oral and intravenous routes of administration were used. Although all agents tested showed varying degrees of efficacy, oral administration of ascorbic acid proved to be the safest and most effective agent to model the rapid acidification of urine seen in post race samples.

The fos proto-oncogene protein:regulation by morphine in the rat hypothalamus.

Mechanisms by which opiates alter neuronal functions, including neuroendocrine functions, are not well defined. We have previously demonstrated that morphine rapidly and transiently increases expression of the proto-oncogene c-fos in the rat caudate-putamen. This regulation of the c-fos gene by morphine may represent a portion of the intracellular cascade coupling activation of opiate receptors on the cell surface to subsequent alterations in neuropeptide gene expression. In the present study, we have focussed on effects of morphine on c-fos expression in the ventromedial hypothalamus, which contains estrogen-concentrating neurons and a large number of neurons expressing the opioid proenkephalin and Proopiomelanocortin. The hypothalamus has been identified as a "final common pathway" between the remainder of the central nervous system and the pituitary gland. As a marker for c-fos expression, we have detected pp50 c-fos (FOS) protein immunocytochemically, using a polyclonal antibody to the M peptide of FOS, and revealed an intense nuclear stain in many neurons. Labeled nuclei were drawn by camera lucida from 12 matched sections (one side only) covering the rostral and middle levels of the ventromedial nucleus of six rats given morphine and six given phosphate buffered saline. Morphine treatment significantly increased the number and density of immuno-labeled nuclei in the ventromedial nucleus, but not in the arcuate nucleus. These results suggest effects of morphine (directly or indirectly) on neurons in the ventromedial hypothalamic nucleus, despite the relative absence of morphine receptors in this nucleus. These results may also provide an anatomical basis for neuroendocrine alterations following morphine treatment.

The effect of protracted tetracycline treatment on bone growth and maturation.

Mature female rhesus monkeys were used to evaluate the effects of a one-year course of tetracycline (50 mg/kg/day, intramuscularly) on the formation, maturation, and mineralization of mandibular bone. The bones from the treated group contained normal concentrations of calcium (Ca), inorganic phosphorus (Pi), and hydroxyproline (HO-Pr), and the treatment schedule did not alter the distribution (percentage) of total osteons into slightly, moderately, and highly mineralization classes. Tetracycline impairs bone mineralization and the subsequent maturation of the mineral and matrix moieties. The percentage of highly mineralized osteons labeled with tetracycline is subnormal. Density gradient fractionation studies indicate the presence of abnormally high Ca/Pi ratios in the temporally young newly formed bone mineral and somewhat higher ratios in the most mature bone fraction. Protracted tetracycline treatment at high dosages impairs bone growth and maturation in adult rhesus monkeys.