Agustín Arce

Twenty-Four-Hour Rhythms in Immune Responses in Rat Submaxillary Lymph Nodes and Spleen: Effect of Cyclosporine

Twenty-four-hour variations in cellularity, lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced cell proliferation, and natural killer (NK) activity were examined in submaxillary lymph nodes and spleen of rats injected with Freunds complete adjuvant or its vehicle and kept under light from 08:00 to 20:00 h daily. A significant daily variation in cellularity was detected, exhibiting maxima at 09:00 h in submaxillary lymph nodes (nonimmunized and immunized rats) and at 13:00 h in spleen (immunized rats only). Submaxillary lymph node LPS- and Con A-mitogenic effect displayed maximal activity during daytime (peak at 13:00-17:00 h). In spleen, the maxima for 24-h rhythm in LPS-induced cell proliferation and NK activity occurred at midnight and at early morning (09:00 h), respectively. Con A-induced spleen cell proliferation peaked at midday in nonimmunized rats only. Injection of the immunosuppressive drug cyclosporine decreased Freunds adjuvant-induced augmentation of LPS and Con A mitogenic effect in both tissues and diminished spleen cell number. Cyclosporine blunted circadian rhythms in submaxillary lymph node Con A response and cell number, while it shifted the maximum in LPS effect to peak at 01:00 h. Cyclosporine also suppressed the circadian changes in LPS- and Con A-induced spleen cell proliferation, but not those found in NK activity. The results indicate the existence of 24-h rhythms in immune responses of rat submaxillary lymph nodes and spleen with maxima at different times of the day and that were significantly affected by cyclosporine injection.

Twenty-four-hour rhythms of serum ACTH, prolactin, growth hormone, and thyroid-stimulating hormone, and of median-eminence norepinephrine, dopamine, and serotonin, in rats injected with Freund's adjuvant.

The effect of Freunds adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6-10 animals received Freunds complete adjuvant or its vehicle at 1100 h 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvants vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freunds adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freunds adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvants vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600-2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freunds adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freunds adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freunds adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freunds adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine.

Piribedil affects dopamine turnover in cochleas stimulated by white noise

The presence of dopamine (DA) within the cochlea has been previously reported, indicating that its turnover increases under noise stimulation. In the present report, piribedil, a dopaminergic D2 agonist, was used in order to provide evidence of the activity of D2 receptors in the turnover of DA under noise stimulation. Long-Evans rats were intraperitoneally injected with distilled water or with a solution of piribedil one hour previously to either noise or silence exposure. Noise stimulation was performed in an anechoic chamber at 70, 90 or 110 dB SPL for one hour. The animals were then sacrificed and the cochlear contents of DA and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were quantified by HPLC with electrochemical detection. The administration of piribedil to animals kept in silence did not modify the cochlear DA, DOPAC and HVA content. Noise stimulation resulted in a decrease of the cochlear DA content and an increase of the cochlear DOPAC and HVA contents in vehicle treated animals. The administration of piribedil resulted in a blockade of this noise induced cochlear DA turnover. These results suggest that piribedil stimulates cochlear D2 receptors controlling the cochlear DA release. Piribedil action on D2 receptors could explain the improvement observed in some cochleo-vestibular diseases signs after piribedil treatment.

Circadian Rhythms in Adenohypophysial Hormone Levels and Hypothalamic Monoamine Turnover in Mycobacterial-Adjuvant-Injected Rats

The effect of Freund’s adjuvant injection on 24-hour variation in circulating ACTH, prolactin, growth hormone (GH) and thyroid-stimulating hormone (TSH) levels, and of hypothalamic norepinephrine (NE) content and dopamine (DA) and serotonin (5HT) turnover was examined in adult rats. In control rats, serum ACTH and prolactin exhibited peak values at the light-dark transition while the maximum in TSH was found in the late afternoon. GH levels did not vary on a 24-hour basis. In Freund’s-adjuvant-injected rats, 24-hour variations in TSH levels became blunted while 24-hour variations in prolactin and ACTH persisted. Freund’s adjuvant treatment augmented serum ACTH and prolactin levels, and decreased GH and TSH levels. Hypothalamic NE content, and turnover of DA and 5HT varied on a 24-hour basis in rats receiving adjuvant’s vehicle. The NE content of the anterior, medial and posterior hypothalamus peaked at 04.00 h, while that of the median eminence attained its maximum at 16.00–20.00 h. Maxima in hypothalamic DA and 5HT turnover occurred at 04.00 h regardless of the region examined. In Freund’s-adjuvant-injected rats, reduced amplitude of daily variations of NE content in the median eminence and anterior and medial hypothalamus, as well as a phase advance in the 24-hour rhythm of the posterior hypothalamic NE content were seen. Mycobacterial adjuvant injection also reduced the amplitude of circadian rhythm in hypothalamic 5HT turnover, shifted the maximum in median eminence DA turnover towards light-dark transition, and decreased the amplitude of DA turnover rhythm in the anterior, medial and posterior hypothalamus. Administration of the immunosuppressant drug cyclosporine restored the augmented ACTH and prolactin levels and the depressed GH and TSH levels found in Freund’s-adjuvant-injected rats. Cyclosporine was also effective to restore 24-hour rhythmicity of serum ACTH and TSH, but not of prolactin levels. Immunosuppression restored rhythmicity of NE content and of DA and 5HT turnover in anterior, medial and posterior hypothalamic regions. Cyclosporine did not modify the effect of Freund’s adjuvant on median eminence but in was able to counteract the changes in the DA and 5HT turnover in the median eminence found after immunization. The results are in accord with a significant effect of immune-mediated inflammatory response at an early phase after Freund’s adjuvant injection on ACTH, GH, prolactin and TSH release mechanisms, which was partially sensitive to immunosuppression induced by cyclosporine.

Effect of superior cervical ganglionectomy on catecholamine concentration in rat cochlea

Both noradrenergic and dopaminergic nerve terminals have been described in the cochlea. The present report focused on the effect of superior cervical ganglionectomy (SCGx) on monoamine concentration in adult rat cochlea. In homogenates of whole cochleas, we measured the concentrations of norepinephrine (NE), dopamine (DA) and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by HPLC coupled to electrochemical detection. Measurements were carried out 4 h, 24 h or 6 days after unilateral SCGx. Most of the NE (approximately 82%) was lost after sympathectomy on the ipsilateral side, indicating that the principal localization of cochlear NE is in peripheral sympathetic fibers. Since about 18% of NE remained detectable 6 days after SCGx, a second origin of cochlear noradrenergic fibers may exist. Cochlear concentrations of DA or its metabolites did not change after SCGx. Therefore, DA and NE are located in two different populations of fibers within the cochlea, and are presumably related to distinct functional roles.

Tachykinins in the Pineal Gland: Effect of Castration and Ganglionectomy

In this investigation, the presence of NKA-immunoreactive substances was determined in pineal glands from intact, castrated and castrated, testosterone-treated male rats. The effect of environmental light, melatonin treatment and superior cervical ganglionectomy on pineal NKA-immunoreactive substances was also investigated. The results obtained show that NKA is present in measurable amounts in the rat pineal, and NPK is probably also present, Orchidectomy was followed by an increase in the content of NKA-immunoreactive substances in the pineal gland. The replacement treatment with testosterone propionate in castrated rats blocked this effect. NKA-immunoreactive substances were not significantly different quantitatively in pineals from rats killed under light or under darkness. The removal of the superior cervical ganglia was followed by a significant increase in the NKA-immunoreactive substance content in the pineal gland of male rats. These results indicate that NKA and other tachykinins are present in the pineal gland of the male rat, and they seem to be regulated by gonadal hormones and the innervation originated from the superior cervical ganglia.

Age-Dependent Effect of Pituitary Transplants on Immune Responses in Rat Spleen: Modulatory Effect of Cyclosporine

Male rats were grafted an anterior pituitary within breast muscles on day 5 or under the kidney capsule on day 30 or 60 of life. On the 70th day of life (rats operated on the 5th or 30th day) or on the 100th day of life (rats operated on the 60th day), rats were injected subcutaneously with Freunds complete adjuvant, being killed 2 days later. Rats that had received a pituitary graft on the 30th day showed a greater degree of hyper-prolactinemia than rats grafted on the 5th or 60th day. Analyzed as main factors in a factorial analysis of variance (ANOVA), pituitary transplants augmented splenic natural killer (NK) activity and lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced cell proliferation, and decreased splenic cell number. As indicated by significant interactions between treatment and age of transplantation in a factorial ANOVA, splenic NK activity augmented in rats grafted on the 30th day of life, while LPS and Con A splenic cell proliferation augmented in rats grafted neonatally. Spleen cellularity decreased after pituitary transplants in 30- and 60-day-old rats. In a second study, the effect of cyclosporine on spleen immune responses was tested by administering cyclosporine (5 mg/kg) or vehicle to rats grafted as in experiment 1 for 5 days before sacrifice. Cyclosporine decreased splenic NK activity and LPS- and Con A-induced cell proliferation regardless of the presence of a pituitary graft. In rats grafted on the 30th day of life, cyclosporine reversed the effect of pituitary grafts on splenic NK activity, and ectopic pituitary augmenting NK activity in vehicle-treated rats while decreasing it in cyclosporine-injected rats. Cyclosporine reversed the inhibitory effect of pituitary transplants on spleen cell number. The high circulating prolactin levels found in rats with pituitary grafts were decreased by cyclosporine administration. The results are compatible with age-dependent promoting and inhibitory effects of hyperprolactinemia on the immune responses of the spleen, which were antagonized by cyclosporine immunosuppression.

Changes in Serum Growth Hormone and Prolactin Levels, and in Hypothalamic Growth Hormone-Releasing Hormone, Thyrotropin-Releasing Hormone and Somatostatin Content, after Superior Cervical Sympathectomy in Rats

After bilateral superior cervical ganglionectomy (SCGx) of adult male rats, norepinephrine (NE) content of the medial basal hypothalamus (MBH) decreased significantly by 39-47% from 16 h to 7 days after surgery. During this time the levels of serum growth hormone (GH) and prolactin (PRL) and of MBH GH-releasing hormone (GRH), thyrotropin-releasing hormone (TRH) and somatostatin were measured by RIA. In sham-operated controls, serum PRL increased and serum GH decreased 16-24 h after surgery, attaining pre-surgical levels later on. In SCGx rats, significantly lower serum GH and PRL and higher MBH GRH and TRH content as compared to controls was observed 16-24 h after surgery, during the wallerian degeneration phase after SCGx. MBH somatostatin concentration decreased in SCGx rats 20 h after surgery. Two injections of the alpha 1-adrenoceptor blocker prazosin 45 and 90 min before sacrifice, alone or together with the beta-blocker propranolol, prevented the changes in MBH hypophysiotropic hormone content, as well as in serum GH and PRL levels, found in SCGx rats 20 h after surgery. Propranolol treatment did not affect hormone levels. Neither drug modified the decrease in MBH NE content observed after SCGx. The results argue in favor of the existence of physiologically relevant projections from superior cervical ganglion neurons to the MBH controlling hypophysiotropic hormone release.

Interferon-Gamma Release in Sympathetically Denervated Rat Submaxillary Lymph Nodes

Objective: To examine the regulation of interferon (IFN)-γ release by cells derived from submaxillary lymph nodes of rats subjected to an acute or chronic superior cervical ganglionectomy (SCGx). Methods: A unilateral SCGx and a contralateral sham operation were performed. Twenty hours or 7 days later cells from submaxillary lymph nodes were incubated for 24 h without any additional treatment (experiment 1), after adding lipopolysaccharide or concanavalin A (experiment 2) or after adding norepinephrine (NE, 10–8M; experiment 3). IFN-γ concentration in the culture media was measured by ELISA. Results: Compared to controls, cells obtained from lymph nodes at a time of degeneration of sympathetic nerve terminals released more IFN-γ, whereas those derived from chronically SCGx lymph nodes released less IFN-γ. Stimulation of IFN-γ release by mitogens was detectable in the innervated or acutely denervated lymph nodes, but not in chronically denervated lymph nodes. When the effect of 10–8M NE on IFN-γ release was tested, the neurotransmitter augmented cytokine release in cells prepared from chronically denervated lymph nodes only. Conclusion: The microenvironment provided by local sympathetic nerves is essential to enable an appropriate IFN-γ release by submaxillary lymph node cells to occur.

Changes in mediobasal hypothalamic dopamine and indoleamine metabolism after superior cervical ganglionectomy of rats

SummaryEight days after bilateral superior cervical ganglionectomy (Gx) of rats, norepinephrine content of medial basal hypothalamus (MBH) decreased significantly by 44–50%. To obtain information on other possible neurochemical sequela of Gx in MBH, we examined the metabolism of dopamine and serotonin in MBH of Gx rats by employing a high pressure liquid chromatography procedure. Eight days after Gx, MBH dopamine levels augmented significantly. Assessment of dopamine metabolism by measuring dihydroxyphenylacetic acid (DOPAC)/dopamine and homovanillic acid (HVA)/ dopamine indexes indicated a significant decrease of MBH DOPAC/dopamine ratio after Gx. MBH serotonin levels increased, and 5-hydroxyindoleacetic acid (5-HIAA)/serotonin index decreased significantly in Gx rats. To examine the interaction Gx-induced changes on MBH dopamine and serotonin with the modified hormonal milieu produced by an ectopic pituitary transplant, adult male rats bearing an ectopic pituitary within the pectoral muscles from day 5 of life were submitted to Gx on day 60 of life and were studied 8 days later. MBH dopamine content increased significantly after pituitary grafting, an effect counteracted by a subsequent Gx, while Gx alone augmented MBH dopamine levels. DOPAC and HVA contents augmented in pituitary-grafted animals, an effect counteracted by Gx. Gx increased MBH serotonin content in control but not in pituitary-grafted rats. After pituitary grafting a decrease in MBH 5-HIAA levels was found, an effect reversed by Gx. Pituitary transplants brought about a significant increase of MBH DOPAC/dopamine index, and a significant decrease in 5-HIAA/serotonin index, both effects being counteracted by Gx. Gx of control rats resulted in a significant decrease of MBH 5-HIAA/serotonin index. Analyzed as a main effect in a factorial analysis of variance, Gx decreased MBH DOPAC/dopamine and HVA/dopamine indexes significantly. Plasma prolactin increased in pituitary-grafted rats, an effect further increased by a subsequent Gx. In pituitary-grafted, Gx rats plasma GH levels augmented significantly. The data suggest that superior cervical ganglion removal affects differentially dopamine and indoleamine metabolism in MBH of control and pituitary-grafted rats.