TNF-alpha is involved in the regulation of normal tissue homeostasis affecting cell proliferation, differentiation, and death. We previously reported that TNF-alpha reduces anterior pituitary cell proliferation and PRL release in an estrogen-dependent manner. In the present project we studied the induction of apoptosis by TNF-alpha in anterior pituitary cells from female rats. TNF-alpha (50 ng/ml) decreased the viability of anterior pituitary cells. Incubation with TNF-alpha for 24 h increased the percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive cells. TNF-alpha increased the percentage of somatotropes and lactotropes with apoptotic nuclear morphology without affecting the proportion of apoptotic corticotropes or gonadotropes. TNF-alpha increased the percentage of apoptotic lactotropes in cultured cells from rats killed in proestrus and estrus, but not in diestrus. This effect was significantly higher in cells from rats in proestrus than in estrus. In anterior pituitary cells from ovariectomized rats, TNF-alpha significantly increased the percentage of apoptotic lactotropes only when the cells were incubated in the presence of 17beta-estradiol. These results indicate that TNF-alpha induces apoptosis in somatotropes and lactotropes from female rats. The apoptotic effect of TNF-alpha on lactotropes is dependent on estrogens and could be involved in the regulation of anterior pituitary cell renewal during the estrous cycle.

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25 mg/kg). One day or 3 wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 wk of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.

Because ⌬-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10 ؊9 M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.AM251 ͉ CB1 receptor ͉ medial basal hypothalamus I t is well known that ⌬-9-tetrahydrocannabinol (THC), the active principle isolated from Cannabis sativa, alters many reproductive parameters in both male and female laboratory animals and humans (1). It has been reported that THC decreases secretion of hormones that control reproduction in male rats (2) but on the other hand stimulates sexual behavior in female rats (3). Also, a marked depression within 1 h in luteinizing hormone (LH) secretion after THC administration has been reported in male rats (2, 4). The molecular target for the action of this plant-derived cannabinol is the endocannabinoid system. This endogenous system consists of two types of GTP-binding protein-coupled receptors, cannabinoid receptors type 1 (CB1-r) and cannabinoid receptors type 2, and their endogenous ligands named anandamide (AEA) and 2-arachidonyl glycerol (5). This endocannabinoid system plays modulatory roles in many processes of the brain, such as the regulation of appetite, temperature, memory, and sexual and motor behavior (6). The activation of the endocannabinoid system acts in series with changes in the activity of several neurotransmitters including ␥-aminobutyric acid (GABA), dopamine, and glutamate (7).We had previously demon...

Objective: Periodontitis is an infectious disease leading to inflammation and destruction of tissue surrounding and supporting the tooth. The progress of the inflammatory response depends on the host’s immune system and risk factors such as stress. The aim of the present study was to investigate the role of the endocannabinoid anandamide (AEA) in experimental periodontitis with restraint stress, since the endocannabinoid system is known to modulate the hypothalamo-pituitary-adrenal axis as well as immune functions and has been found in human gingival tissues. Methods: Experimental periodontitis was induced by ligature around first inferior molars and immobilization stress for 2 h twice daily for 7 days in a rat model. Results: Corticosterone plasma levels, locomotor activity, adrenal gland weight and bone loss were increased in periodontitis and stress groups, and there was also less weight gain. The inflammatory parameters such as prostaglandin E₂ (radioimmunoassay), nitric oxide (radioconversion of ¹⁴C-arginine), tumor necrosis factor (TNF)-α (ELISA) and interleukin (IL)-1β (Western blot) measured in the gingival tissue were significantly increased in the periodontitis groups compared to the control group. Local injection of AEA (10^–8M, 30 µl) decreased corticosterone plasma levels and the content of the cytokines TNF-α and IL-1β in gingival tissue in periodontitis-stress groups. These AEA-induced inhibitions were mediated by CB₁ and CB₂ cannabinoid receptors since the injection of both antagonists together, AM251 (10^–6M) and AM630 (10^–6M) in 30 µl, prevented these effects. Conclusion: The endocannabinoid AEA diminishes the inflammatory response in periodontitis even during a stressful situation.

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because N-nitro-L-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by Ϸ40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE 2, which in turn releases corticosterone stored in microvesicles and other organelles.cyclooxygenase ͉ indomethacin ͉ nitric oxide synthase ͉ N-nitro-L-arginine methyl ester

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Optic neuritis (ON) is an inflammatory, demyelinating, and neurodegenerative condition of the optic nerve, which might induce permanent vision loss. Currently, there are no effective therapies for this disorder. We have developed an experimental model of primary ON in rats through a single microinjection of 4.5 μg of bacterial lipopolysaccharide (LPS) into the optic nerve. Since melatonin acts as a pleiotropic therapeutic agent in various neurodegenerative diseases, we analyzed the effect of melatonin on LPS-induced ON. For this purpose, LPS or vehicle were injected into the optic nerve from adult male Wistar rats. One group of animals received a subcutaneous pellet of 20 mg melatonin at 24 hr before vehicle or LPS injection, and another group was submitted to a sham procedure. Melatonin completely prevented the decrease in visual evoked potentials (VEPs), and pupil light reflex (PLR), and preserved anterograde transport of cholera toxin β-subunit from the retina to the superior colliculus. Moreover, melatonin prevented microglial reactivity (ED1-immunoreactivity, P < 0.01), astrocytosis (glial fibrillary acid protein-immunostaining, P < 0.05), demyelination (luxol fast blue staining, P < 0.01), and axon (toluidine blue staining, P < 0.01) and retinal ganglion cell (Brn3a-immunoreactivity, P < 0.01) loss, induced by LPS. Melatonin completely prevented the increase in nitric oxide synthase 2, cyclooxygenase-2 levels (Western blot) and TNFα levels, and partly prevented lipid peroxidation induced by experimental ON. When the pellet of melatonin was implanted at 4 days postinjection of LPS, it completely reversed the decrease in VEPs and PLR. These data suggest that melatonin could be a promising candidate for ON treatment.

TNF-alpha plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-alpha from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-alpha. The presence of lipopolysaccharide (LPS, 0.1 microg/mL) in the culture medium significantly increased TNF-alpha release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 beta-estradiol in the culture medium also increased TNF-alpha release. LPS significantly stimulated TNF-alpha release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-alpha on prolactin release. The presence of TNF-alpha (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-alpha and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-alpha could affect pituitary hormone release during endotoxemia.