Px Animals Research Articles

Effects of pinealectomy on the neuroendocrine reproductive system and locomotor activity in male European sea bass, Dicentrarchus labrax

The seasonally changing photoperiod controls the timing of reproduction in most fish species, however, the transduction of this photoperiodic information to the reproductive axis is still unclear. This study explored the potential role of two candidate neuropeptide systems, gonadotropin-inhibitory hormone (Gnih) and kisspeptin, as mediators between the pineal organ (a principle transducer of photoperiodic information) and reproductive axis in male European sea bass, Dicentrarchus labrax. Two seven-day experiments of pinealectomy (Px) were performed, in March (end of reproductive season) and August (resting season). Effects of Px and season on the brain expression of gnih (sbgnih) and its receptor (sbgnihr), kisspeptins (kiss1, kiss2) and their receptors (kissr2, kissr3) and gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) and the main brain receptor (gnrhr-II-2b) genes, plasma melatonin levels and locomotor activity rhythms were examined. Results showed that Px reduced night-time plasma melatonin levels. Gene expression analyses demonstrated a sensitivity of the Gnih system to Px in March, with a reduction in sbgnih in the mid-hindbrain, a region with bilateral connections to the pineal organ. In August, kiss2 levels increased in Px animals but not in controls. Significant differences in expression were observed for diencephalic sbgnih, sbgnihr, kissr3 and tegmental gnrh2 between seasons. Recordings of locomotor activity following surgery revealed a change from light-synchronised to free-running rhythmic behavior. Altogether, the Gnih and Kiss2 sensitivity to Px and seasonal differences observed for Gnih and its receptor, Gnrh2, and the receptor for Kiss2 (Kissr3), suggested they could be mediators involved in the relay between environment and seasonal reproduction.

Effects of melatonin on ovarian follicles

To evaluate the histomorphometry and expression of Ki-67 and c-kit in ovarian follicles of pinealectomized or melatonin-treated pinealectomized rats. Forty adult rats were randomly divided into four groups of 10 animals: Group I - control; Group II - sham-pinealectomized; Group III - pinealectomized (Px), and Group IV - Px treated with melatonin (10μg/night, per animal). After two months' treatment, on the night of proestrous, the animals were placed in metabolic cages for night urine collection and subsequent measurement of 6-sulfatoxymelatonin (6-SMT). The rats were anesthetized, blood samples were taken for estrogen and progesterone determinations, and they were then euthanized. The ovaries were dissected out for further histological and immunohistochemical analyses. Data were first submitted to analysis of variance (ANOVA) complemented with the Tukey-Kramer test for multiple comparisons (P<0.05). The urinary levels of 6-SMT and serum progesterone were lower in the Px group (GIII). Exogenous melatonin treatment restored both blood melatonin and 6-SMT urinary levels. The histomorphometric data in Group III revealed a significant increase of degenerating antral and non-antral follicles with regard to the other groups. In addition no corpora lutea were observed in this group. No significant differences were noticed regarding the number of corpora lutea among the other groups (I, II and IV), but the number of cells and the thickness of the theca interna of Px animals (Group III) were higher than in the other groups. Conversely, the density of progesterone receptors (fmol/g) in the ovaries of Group III was significantly lower than in the other groups. Our data indicate that melatonin exerts a role on the maintenance of a proper follicular function, and is thus important for ovulation and progesterone production.

Oreocnide integrifoliaFlavonoids Augment Reprogramming for Islet Neogenesis andβ-Cell Regeneration in Pancreatectomized BALB/c Mice

Agents which can either trigger proliferation of β-cells or induce neogenesis of β-cells from precursors would be of pivotal role in reversing diabetic manifestations. We examined the role of flavonoid rich fraction (FRF) of Oreocnide integrifolia leaves using a mice model of experimental regeneration. BALB/c mice were subjected to ~70% pancreatectomy (Px) and supplemented with FRF for 7, 14, and 21 days after pancreatectomy. Px animals displayed increased blood glucose levels and decreased insulin titres which were ameliorated by FRF supplementation. FRF-treated mice demonstrated prominent newly formed islets budding off from ducts and depicting increased BrdU incorporation. Additionally, transcripts levels of Ins1/2, Reg-3α/γ, Ngn-3, and Pdx-1 were upregulated during the initial 1 week. The present study provides evidence of a nutraceutical contributing to islet neogenesis from ductal cells as the mode of β-cell regeneration and a potential therapeutic for clinical trials in management of diabetic manifestations.

Melatonin augments expression of the procollagen α1 (I) and α1 (III) genes in the infarcted heart scar of pinealectomized rats

The pineal gland is involved in the regulation of collagen accumulation in peripheral wounds and scars of the infarcted heart. This study is aimed to provide an explanation of whether the pineal gland and melatonin (MLT) is involved in the regulation of α1 (I) and α1 (III) procollagen gene expression. A secondary aim is the investigation of whether the mechanism of changes could be explained by the direct influence of MLT on myofibroblasts isolated from the scar. Myocardial infarction was induced by left coronary artery ligation in all rats. Animals were divided into groups: control, vehicle-treated rats, those injected with MLT, sham-operated animals, pinealectomized (Px) rats, and Px rats injected with vehicle or treated with MLT. In the second part of the study, cells from the scar of the infarcted heart were isolated and cultured with MLT at concentrations of 10−7 and 10−9 M. Both α1 (I) and α1 (III) procollagen gene expressions were evaluated by reverse transcription-polymerase chain reaction. Neither MLT given to intact animals nor pinealectomy alone have an influence on procollagen gene expression. However, administration of MLT to the Px animals increased the expression of α1 (I) and α1 (III) procollagen genes. Cells isolated from the heart scar were identified as myofibroblasts. MLT did not influence collagen gene expression in cultured myofibroblasts. The results indicate that MLT has an influence on procollagen gene expression in Px animals. Because the pineal product does not have an influence on the myofibroblast of the scar, the indirect mechanism of MLT action is suggested. This study may have practical implications in patients with a low level of MLT (elderly subjects, patients treated with β-adrenergic blockers).

Inhibitory action of the pineal gland on the nuclear volume of thyroid follicular cells in male gerbils (Meriones unguiculatus).

Karyometric investigations of thyroid follicular cells were performed in four groups of adult male gerbils [I. sham-operated (Sham PX) + short photoperiod (SPP)-exposed; II. Sham PX + long photoperiod (LPP)-exposed; III. pinealectomized (PX) + SPP-exposed; IV. PX + LPP-exposed]. The mean nuclear volume (MNV) of thyroid follicular cells was significantly higher in both PX groups (III and IV) when compared to Sham PX controls (I and II). In Sham PX gerbils the maintenance of animals in SPP (10 h light: 14 h darkness) for 13 weeks resulted in significant decrease of MNV of thyroid follicular cells in comparison to that in Sham PX animals kept in LPP (14 h light: 10 h darkness). Lighting conditions did not significantly affect the MNV of thyroid follicular cells in PX gerbils. These data suggest that the pineal gland itself and not light deprivation is the most important factor depressing the activity of the thyroid in male gerbils. Lighting conditions seem to affect the function of thyroid follicular cells inasmuch as they activate or block the action of the pineal gland.

Physical exercise program reverts the effects of pinealectomy on the amygdala kindling development

Several studies have demonstrated the anticonvulsant effect of melatonin. In view of the positive effects of physical exercise in epilepsy, this study analyzed the influence of physical exercise program on the amygdala kindling development in pinealectomized rats. Animals were divided into six groups: pinealectomized rats (PX), sham rats (SHAM), control rats (CTL), pinealectomized rats submitted to an aerobic exercise program (PX ATL), sham rats submitted to an aerobic exercise program (SHAM ATL) and control rats submitted to an aerobic exercise program (CTL ATL). The stimulus parameters consisted of 60 Hz frequency, diphasic square pulses of 1 ms duration applied for 2 s. The mean number of stimulations and the after-discharge (AD) duration for each stage of kindling were similar among CTL and SHAM animals. PX animals showed particular characteristics during kindling development. They did not present stage 1 and spent a shorter time in stage 2 in relation to the CTL and SHAM animals. Consequently, the AD duration and number of stimulations required to reach stage 5 was lower for the PX group when compared to the CTL and SHAM groups. Concerning the exercising groups, CTL ATL and SHAM ATL animals spent a higher time in stage 1 compared to CTL and SHAM groups. Thus, CTL ATL animals also presented a higher number of stimulations in stage 5 compared to CTL animals. The stage 1 not observed in PX animals was present in PX ATL. Consequently, the number of stimulations required to reach stage 5 was statistically higher for the PX ATL group in relation to the PX group. Our results demonstrate that the acceleration in the kindling development of pinealectomized animals can be reverted by physical exercise.

Regulation of Pancreatic β-Cell Regeneration in the Normoglycemic 60% Partial-Pancreatectomy Mouse

beta-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory beta-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was approximately 68% of the shams by 15 days, partly due to enhanced beta-cell proliferation. A transient approximately 2.8-fold increase in the prevalence of beta-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory beta-cell growth by activating beta-cell proliferation with an increase in the number of beta-cell clusters/small islets.

Pinealectomy Reduces Optic Nerve But Not Intergeniculate Leaflet Input to the Suprachiasmatic Nucleus at Night

The suprachiasmatic nucleus (SCN) of the hypothalamus regulates circadian rhythms in mammals. It receives, among others, direct inputs from the retina and from the thalamic intergeniculate leaflet (IGL). The former sends photic signals to the SCN, whereas the latter probably integrates photic and nonphotic information. To characterise these inputs in vivo, extracellular single-unit recordings were made from the SCN of rats under urethane anaesthesia during electrical stimulation of the optic nerve (OptN) or the IGL region. Cell responses were evaluated by creating peri-stimulus time histograms. Because humoral signals such as melatonin might modulate the activity of the SCN in addition to neural inputs, recordings were also made using pinealectomised (Px) rats to test for a possible role of this hormone in regulating inputs to the SCN. A significantly greater number of cells responded to IGL (60 of 90, 67%) than to OptN (35 of 75, 47%) stimulation in intact animals (chi(2) = 5.905, P = 0.015). The same was true when Px animals were tested (IGL, 82 of 131, 63%; OptN, 31 of 111, 28%; chi(2) = 27.637, P < 0.001). In intact animals, the proportion of cells responsive to IGL stimulation during the day and during the night was not significantly different from the proportion responsive in Px animals. The same was true for OptN stimulation during the day. However, during the night, the proportion of cells responsive to OptN stimulation in intact animals was significantly greater than the proportion responsive in Px animals (chi(2) = 7.127, P = 0.008). Our findings suggest that a lack of melatonin modulates OptN but not IGL inputs to the SCN.

Pinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: gross morphological, histological and stereological analysis

At present, an intensive effort for prevention of neuroma formation following peripheral nerve section continues. It has been recently suggested that surgical pinealectomy (Px) induces elevation of the collagen content in the granulation tissue of a wound, while melatonin application after Px suppresses elevation of the collagen accumulation in the tissue. The aim of the present study was to assess whether melatonin had the ability to suppress collagen production and neuroma formation following peripheral nerve transection. A total of 40 male rats (four groups of 10) were left intact (intact controls) or sham operated (sham group), were Px, or were Px and given melatonin (Px + melatonin group). All animals underwent a surgical intervention consisting of right sciatic nerve neurectomy. After 4 wk, the animals were killed following intracardiac perfusion. Gross morphology of neuroma formation in the proximal nerve segment was examined and proximal neuroma evaluated. Macroscopic and microscopic findings revealed that Px caused a proliferation of connective tissue and large neuroma formation at the proximal ends of transected nerves. Stereological analysis showed that there was a statistically significant reduction in connective tissue content of the same region in Px animals treated with melatonin (P < 0.005). The results achieved in a rodent model of sciatic nerve neuroma formation showed that there was a positive correlation between macroscopic and microscopic observations, and that melatonin enhanced axonal regeneration presumably due to its inhibitory effect on neuroma formation.

Amikacin-induced acute renal injury in rats: protective role of melatonin.

It is well established that some agents such as aminoglycosides generate free oxygen radicals, leading to an increased oxireductase production, which in turn increases tissue toxicity. The aim of this study is to test whether melatonin, the chief secretory product of the pineal gland and a highly effective antioxidant and free radical scavenger, reduces the nephrotoxicity caused by amikacin (AK). Herein, we investigated the physiologic and pharmacological role of melatonin in influencing AK-induced nephrotoxicity. For this, pinealectomized (Px) and sham operated (non-Px) rats were used. Both AK and melatonin were administered to all groups. We investigated the effects of melatonin on AK-induced changes in levels of malondialdehyde (MDA), a lipid peroxidation product, glutathione (GSH), an antioxidant whose levels are influenced by oxidative stress, and blood urea nitrogen (BUN) and serum creatine (Cr) levels. Morphologic changes in the kidney were also examined by using light microscopy. MDA levels were found to be higher in Px than in non-Px AK-treated animals. Melatonin administration to Px rats reduced MDA levels. In relative to non-Px rats, Px animals treated with AK had significantly lower GSH concentrations while melatonin administration elevated GSH levels in the kidney; however, this stimulatory effect of melatonin was not observed in non-Px AK-treated rats. Treatment with AK alone resulted in significantly higher plasma Cr and BUN levels. Repeated administration of melatonin prevented the AK-induced elevation of plasma Cr and BUN levels. Morphologic damage to renal tubules as a result of AK was more severe in the renal cortex than in the medulla. The damage to the kidney induced by AK was reversed by melatonin in the Px rats. In conclusion, these results show that physiologic melatonin concentrations are important in reducing AK-induced renal damage, while pharmacologic concentrations of melatonin did not add to the beneficial effect.

Influence of the pineal gland on the physiology, morphometry and morphology of pancreatic islets in rats.

To investigate the influence of the pineal gland through melatonin secretion on the physiological and morphological parameters of pancreatic islets, we studied the plasma biochemistry and morphological and morphometric characteristics of the endocrine pancreas of male Wistar rats. The animals were distributed into five groups of ten rats each: NC - normal control group; NS -- sham-operated group; Px (25) -- pinealectomised group, studied 15--25 days after surgery; Px (70) -- pinealectomised group, studied 60-70 days after surgery; ALX - alloxan monohydrate-treated group. Data are analyzed statistically by ANOVA and by the Kruskal-Wallis test. Although there was no significant difference in plasma glucose or insulin levels between the Px (25), Px (70) and NC groups, Px (25) animals showed a tendency to increased glucose and reduced insulin levels. The ALX group showed a clear elevation of plasma glucose and a reduction of plasma insulin compared to the other groups. Morphometric analysis showed a larger pancreatic islet area and a lower pancreatic islet density in the pancreas of Px (70) animals and an increase in degenerative pathological processes in the pancreatic islets of the Px (25) and ALX groups. The present results suggest that melatonin, in addition to acting on tissue sensitivity to insulin (as reported in other studies), affects the secretory action of beta cells, as demonstrated by the morphological and morphometric changes observed in pinealectomised animals.

Pineal, Photoperiod and Gonadal Function in the Indian Palm Squirrel, Funambulus pennanti

Effects of morning and evening injections of pineal 5-methoxyindoles (MI), melatonin (aMT) and 5-methoxytryptamine (MT), for 60 continuous days, were observed on the testes of sham-operated (SO) and pinealectomized (Px) Indian palm squirrel, Funambulus pennanti maintained under different photoperiods during the gonad active phase. Long photoperiod (LP) of 14L:10D appeared stimulatory to the testes and caused a significant increase in the weight and seminiferous tubule diameter of both SO and Px animals, as compared to the animals under natural day-length (NDL). Short photoperiod (SP) of 10L:14D had an inhibitory influence and reduced the testes weight and its tubule diameter. aMT and MT injections during evening hours significantly reduced testes weight and tubule diameter of SO and Px animals under NDL, LP and SP. However morning injections, under all conditions, were without any significant effect. The results suggest an inhibitory effect of aMT and MT, under above photoperiodic conditions, on the testes of this tropical mammal. The time of administration of the MI is important in the expression of the effect.

Effect of Pineal Extirpation on Daily and Long-Term Variations in Thermal Tolerance in a Tropical Catfish, Clarias batrachus

Thermal tolerance in laboratory acclimated Clarias batrachus was assessed by determining the critical thermal maximum (CTM) every 4 hours for 2 days in intact and pinealectomized (Px) animals during January and in sham-pinealectomized (sham-Px) and Px animals during April. In the intact and sham-Px group a statistically significant (P <0.005) diurnal cycle of thermal tolerance was validated which was asynchronous with that of the daily fluctuations in ambient temperature (Ta). A nocturnal elevation of thermal tolerance was observed in all the groups. Pineal removal resulted in a highly significant (P <0.001) increase of CTM. Additionally, pinealectomy abolished the normal diurnal cycle in CTM or dampened its amplitude considerably. In another experiment the long-term effect of pinealectomy on thermal tolerance was determined in sham-Px and Px fishes. CTM was significantly (P <0.05) elevated during the first three weeks following pinealectomy, pointing to a short-term control from the pineal on thermal tolerance. This study provides the first demonstration of a direct pineal influence on thermal tolerance and its temporal organization.

Effect of aging on B-cell function and replication in rat pancreas after 90% pancreatectomy.

We studied the effect of aging on B-cell function and replication in depancreatized rats. Male Wistar rats, at the ages of 1, 5, and 15 months, underwent 90% pancreatectomy (Px) or a sham operation, and islet function and regeneration were examined 3 weeks later. Plasma glucose levels in 1-month-old rats reached a peak 2 weeks after Px and then declined, while those in 5- and 15-month-old rats reached significant levels as early as the day after Px or 2 days after surgery and continued to increase over the following 3 weeks. Consequently, in contrast to young Px rats, weight loss due to severe diabetes was observed in 5- and 15-month-old Px rats. Plasma glucose responses to intravenous glucose loading (0.5 g/kg body weight) were much greater in older Px rats than in younger rats. There was a marginal insulin response to glucose in 1-month-old Px rats, whereas no insulin response to glucose was observed in older Px animals. The insulin content of the residual pancreas was increased threefold in 1-month-old Px rats, but there was no increase in 5- and 15-month-old Px rats. These data demonstrate that the effect of reducing islet mass is much greater in aged rats than in young rats and that the replicatory capacity of B cells tends to diminish after adulthood has been reached.

Pancreatic A-cell function in the partially pancreatectomized Otsuka Long-Evans Tokushima fatty rat, a model of spontaneous non—insulin-dependent diabetes mellitus

We examined whether a 70% pancreatectomy changes the morphofunctionality of pancreatic A cells in a model rat (Otsuka Long-Evans Tokushima Fatty [OLETF]) with non—insulin-dependent diabetes mellitus. Male OLETF rats aged 6 weeks were assigned to two groups: partial pancreatectomy (Px) and sham pancreatectomy (sham). The Px group was divided into three subgroups based on treatment received after surgery, which included treatment with nicotinamide, phlorhizin, or saline. As a control, their diabetes-resistant counterparts, Long-Evans Tokushima Otsuka (LETO) rats, were similarly treated and grouped. Six weeks after surgery, plasma glucagon responses to arginine- and insulin-induced hypoglycemia were examined. In addition, the glucagon content and morphological features of pancreatic A cells in Px-remnant and remnant-equivalent pancreata were investigated 7 weeks after surgery.A sustained nonfasting hyperglycemia was evident in Px OLETF rats, which was ameliorated by administration of nicotinamide. The glucagon content and A-cell mass were not decreased significantly in the remnant pancreas of saline- and phlorhizin-treated Px animals of either strain but increased in nicotinamide-treated animals compared with those in the remnant equivalent of the respective sham rats. The areas under the response curves of plasma glucagon (ΣIRG) during an arginine infusion test and 90 minutes of insulin-induced hypoglycemia were 1,010.7 ± 72.9, 1,083.1 ± 95.3, 1,029.6 ± 65.0, and 1,779.8 ± 226.9 pmol · L −1 · min −7 versus 1,997.0 ± 283.1, 2,217.0 ± 395.0, 1,479.6 ± 78.0, and 3,466.4 ± 174.0 pmol · L −1 · min −1 in phlorhizin-, nicotinamide-, and saline-treated Px OLETF and sham OLETF rats, respectively. A similar trend was observed for differences in the response of pancreatic A cells to both stimuli among various groups of LETO rats. There was no significant difference in ΣIRGs during both tests between OLETF and LETO rats with similar treatments, except during an insulin tolerance test (ITT) in saline-treated Px rats. The magnitude of the plasma glucagon response to both stimuli in the test animals was roughly parallel to the glucagon cotent in the pancreas. These findings suggest that differences in the proliferation and responsiveness of pancreatic A cells between OLETF and LETO rats after a 70% pancreatectomy are not nearly as significant as compared with B cells.

Effect of the pineal gland on circadian rhythmicity of colony forming units for granulocytes and macrophages (CFU-GM) from rat bone marrow cell cultures.

The present study provides evidence that the pineal gland has a physiological role in the proliferation of colony forming units for granulocytes and macrophages (CFU-GM). A biphasic circadian rhythm of CFU-GM proliferation in rat bone marrow cell cultures (BMC) from intact animals peaking at 0600 and 1800 was observed. Pinealectomy (Px) at 1600 obliterated the circadian rhythm patterns of CFU-GM. Afternoon injections of melatonin (1630, 20 micrograms/per day for 10 days) to Px animals restored the rhythmicity. When pinealectomy was done at 0800, the morning peak remained unaltered and the colony number at 1800 was higher than that found in the afternoon Px animals. In conclusion, the pineal gland or its main hormone melatonin seems to have a regulatory role in the proliferation of CFU-GM in rat BMC. Further, the expression of the activity of CFU-GM in rat BMC depends on the time when pinealectomy is done or melatonin is substituted.

The circadian activity rhythms of rats with mid- and parasagittal ‘split-SCN’ knife cuts and pinealectomy

To explore the multioscillator nature of the neurohumoral circuitry controlling the expression of circadian rhythmicity, rats' wheel running circadian activity rhythms were compared following sagittal knife cuts in the region of the suprachiasmatic nucleus (SSCN), following pinealectomy (PX) and following the combination of SSCN and PX. 25% of animals with knife cuts that passed through one SCN had disturbed running activity under constant illumination; rhythmic disturbances were seen neither in animals with sham knife cuts nor in rats with knife cuts on the midline or lateral to the SCN. Animals with both SSCN and PX were twice as likely to show severe rhythmic disruptions under free-running conditions as rats with SSCN and sham PX. Rats with PX and sham SSCN did not display disrupted activity rhythms. When animals with PX alone or SSCN alone were first observed under free-running conditions and then subjected to a second surgical procedure so that all animals underwent both PX and SSCN, all PX and most SSCN animals demonstrated coherent activity rhythms after the first operation, but 35% showed disruption in circadian activity patterns only following the second surgery. The activity rhythms of rats with knife cuts placed either on the midline or lateral to the SCN did not deteriorate when combined with PX. Rats with coherent rhythms following knife cuts damaging one SCN had rhythm disruptions after the addition of PX. The effects of pinealectomy may indicate that the pineal gland plays a role in maintaining the coupling relationships in the multioscillator system controlling circadian activity rhythms. The results of this study also suggest that neither the direct commissural connection of the SCNs nor the humoral output of the pineal gland is indispensable for the expression of coherent circadian activity rhythms in the rat.

Circadian analysis of mononuclear cells in the rat following pinealectomy and superior cervical ganglionectomy

The role of the pineal gland and adrenocorticosteroids in circadian rhythmicity of immune function was investigated in Sprague-Dawley rats that either had been pinealectomized (PX) or had undergone superior cervical ganglionectomy (SCGX), which functionally denervates the pineal. For both experiments, conducted between August and October, rats were entrained to a 10-h light:14-h dark cycle and fed ad libitum. The circadian rhythm in monocytes (ANOVA, p < .01 for Control + Sham + PX samples) tended to parallel that of plasma corticosteroid levels (ANOVA, p < .01) with the peak during early dark and the nadir at early light. In comparison, suppressor/cytotoxic T cells and B cells tended to be more frequent during early light with minimum levels during early dark (ANOVA, p < .05 and .01, respectively, for Control + Sham + PX samples). Natural killer (NK) activity in control animals varied significantly (ANOVA, p < .05) over the light:dark cycle with peak activity during early dark in the SCGX experiment, but was only slightly elevated during late dark in the PX experiment. PX animals exhibited increased frequency of NK cells, but only in samples collected during the day ( t test, p < .05). This effect was not observed in sham-operated PX controls and in SCGX animals. Neither PX nor SCGX had a significant (ANOVA) effect on plasma corticosterone levels. These observations in the rat are consistent with findings in mice and humans that corticosteroids play an important general role in circadian rhythmicity of immune functions. In contrast, the present study suggests that pineal gland influence of immune function(s) is more specific and that pineal gland interaction with NK cells has a circadian component.

Gonadal Responses of the Male Syrian Hamster to Programmed Infusions of Melatonin are Sensitive to Signal Duration and Frequency but Not to Signal Phase Nor to Lesions of The Suprachiasmatic Nuclei1

This study investigated the roles of the melatonin signal and the circadian system in the induction of photoperiodic responses in the male Syrian hamster. Pinealectomized animals received programmed s.c. infusions of saline or melatonin. Saline infusions for 10 h or melatonin for 4 h during the night had no effect on the reproductive axis whereas nightly 10-h infusions of melatonin induced gonadal atrophy. Animals that received 10-h infusions of melatonin arranged such that consecutive daily signals were delivered alternately during the day and night also exhibited gonadal atrophy, whereas melatonin signals delivered every 48 h, exclusively during either the day or night, were without effect. These results demonstrate that the brain is able to read melatonin signals delivered at different phases of the circadian cycle and to use them in combination to generate an appropriate photoperiodic response. Melatonin signals lasting 10 h delivered to pinealectomized (PX) animals every 24 h induced gonadal regression. Melatonin delivered at periodicities of 20 h, 23 h, and 25 h also caused gonadal regression whereas infusions every 28 h were without effect, demonstrating that the systems responsive to melatonin are sensitive to signal frequency but do not need to receive the signal on a strictly circadian basis. These results are discussed in the context of the significance of the melatonin-free interval. PX animals that received sham or bilateral lesions of the suprachiasmatic nuclei (SCN) were infused nightly for 10 h with saline or melatonin. Melatonin infusions were equally effective at inducing gonadal atrophy and lowering serum testosterone levels in both sham- and SCN-lesioned animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Pinealectomy on the Mitotic Activity of Adrenomedullary Chromaffin Cells in Relation to Time of Day

The frequency of mitoses of adrenaline (A) cells and noradrenaline (N) cells in the adrenal medulla of nonoperated (NO), sham-operated (SPX), and pinealectomized (PX) male, 53-day-old Holtzman rats (n = 133) was investigated by means of light microscopy. Animals were killed at eight time points during a standardized 24-h light-dark (12:12) cycle 14 days after surgery. Mitotic indices (n/1,000) were determined in sections of adrenal medulla fixed with glutaraldehyde and OsO4. Overall frequency of mitoses was extremely low (mitotic index: 0.73 = 115/157,223). Daily mean mitotic index was maximum in A cells (0.83) and minimum in N cells (0.52) of PX group but did not show statistically significant differences between cell types or experimental groups. Neither cell type in NO animals showed 24-h changes in mitotic index, but cells in SPX animals did, with highest value in the late dark phase and lowest in the late light phase, when values of two cell types were combined (P less than 0.01-0.001). In PX animals, mitotic index followed a similar but more distinct 24-h change in A cells (P less than 0.009), but not in N cells, resulting in different time-of-day changes between two types of cells (P less than 0.01-0.05). The mitotic index was higher in PX than in control (NO and SPX) animals in the middark phase (P less than 0.05) and lower in operated (SPX and PX) than in nonoperated (NO) animals from late light to the early dark phase, suggesting that the latter was possibly due to a residual effect of the surgery. These results are consistent with the interpretation that the pineal has an inhibitory action on A cells and may coordinate the two types of cells in their mitotic activity, especially in the middark phase of the daily cycle.