COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis. Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05). Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam. Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Influence of Antiplatelet Drugs in the Pathogenesis of Experimental Periodontitis and Periodontal Repair in Rats

Background: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. Methods: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. Results: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-alpha and II-6 (P < 0.05), but only Asp decreased thromboxane A(2) (P < 0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P < 0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. Conclusion: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed. J Periodontol 2011;82:767-777.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Effects of neonatal handling on central noradrenergic and nitric oxidergic systems and reproductive parameters in female rats

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NO x), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats. Copyright (c) 2007 S. Karger AG, Basel.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

Involvement of the heme oxygenase-carbon monoxide-cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase-carbon monoxide-cGMP (HO-CO-cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO-CO-cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5-7 days) were subjected to the measures of baseline tail flick test Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na(2)CO(3)), heme-lysinate (substrate overload) or vehicle (L-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO-CO-cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain. (C) 2011 Elsevier B.V. All rights reserved.

Prolactin differentially modulates the macrophage activity of lactating rats: possible role of reproductive experience

Reproductive experience (i.e., pregnancy and lactation) induces physiological changes in mammals. We recently showed that a previous reproductive experience can modulate the activity of dopaminergic hypothalamic systems while decreasing serum prolactin (PRL) levels and oxidative burst activity in peritoneal macrophages. Dopamine receptor antagonists increase serum PRL levels, and both PRL and dopamine receptors might be involved in the modulation of macrophage activity, providing a means of communication between the nervous and immune systems. The present study evaluated the in vitro effects of PRL and the dopamine receptor 02 antagonist domperidone (DOMP) on the peritoneal activity of macrophages from primiparous and multiparous female rats during lactation. Oxidative bursts and phagocytosis in peritoneal macrophages were evaluated by flow cytometry. Primiparous and multiparous Wistar rats, during the period of lactation (i.e., days 5-7 after parturition) were used. Samples of peritoneal fluid from these rats were first incubated with PRL (10 and 100 nM) for different periods of time. The same procedure was repeated to evaluate the effects of DOMP (10 and 100 nM). Our results showed that macrophages from multiparous rats respond more effectively to in vitro incubation with PRL, especially with regard to oxidative bursts and the percentage of phagocytosis. Additionally, these effects were more pronounced after 30 min of incubation. These data suggest that reproductive experience is associated with a reduction in serum PRL levels, and cells in experienced female animals, including their macrophages, become more sensitive to the effects of PRL (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Nitric oxide synthase inhibitors improve prepulse inhibition responses of Wistar rats

Introduction: Cognitive and attentional deficits in schizophrenia include impairment of the sensorimotor filter as measured by prepulse inhibition (PPI). In this way, the study of animals that naturally present low PPI responses could be a useful approach for screening new antipsychotic drugs. Several pieces of evidence suggest that dopamine and nitric oxide (NO) can modulate PPI but their role in those animals is unknown. Objectives: The aim of this study was to investigate the role of dopamine and NO in Wistar rats with naturally low PPI response. Methods: Male Wistar rats with low PPI responses received an i.p. injection of the antipsychotics haloperidol (0.1, 0.3 or 1 mg/kg) or clozapine (0.5, 1.5 or 5 mg/kg), the anxiolytic diazepam (1 or 3 mg/kg) or the NO synthase (NOS) inhibitors, N(G)- nitro-L-arginine (L-NOARG; 40 mg/kg, acutely or sub-chronically) or 7-Nitroindazole (7-NI; 3, 10 or 30 mg/kg). All animals were submitted to the PPI test 1 h after injection. Striatal and cortical dopamine, DOPAC, and noradrenaline levels of rats with low PPI responses were compared to rats with normal PPI responses. Results: We found increased levels of catecholamines on the striatum and prefrontal cortex of Wistar rats with low PPI. In these animals, both antipsychotics, typical and atypical, and NOS inhibitors significantly increased PPI. Conclusion: Taken together, our findings suggest that the low PPI phenotype may be driven by an over-active catecholamine system. Additionally, our results corroborate the hypothesis of dopamine and NO interaction on PPI modulation and suggest that Wistar rats with low PPI may represent an interesting non-pharmacological model to evaluate new potential antipsychotics. (C) 2010 Elsevier B.V. All rights reserved.

Central NOS inhibition differentially affects vasopressin gene expression in hypothalamic nuclei in septic rats

Our aim was to investigate the effect of central NOS inhibition on hypothalamic arginine vasopressin (AVP) gene expression, hormone release and on the cardiovascular response during experimental sepsis. Male Wistar rats were intracerebroventricularly injected with the non-selective NO synthase (NOS) inhibitor (L-NAME) or aminoguanidine, a selective inhibitor of the inducible isoform (iNOS). After 30 min. sepsis was induced by cecal ligation and puncture (CLP) causing an increase in heart rate (HR), as well as a reduction in median arterial pressure (MAP) and AVP expression ratio (AVP(R)), mainly in the supraoptic nucleus. AVP plasma levels (AVP(P)) increased in the early but not in the late phase of sepsis. L-NAME pretreatment increased MAP but did not change HR. It also resulted in an increase in AVP(P) at all time points, except 24 h, when it returned to basal levels. AVP(R), however remained reduced in both nuclei. Aminoguanidine pretreatment resulted in increased MAP in the early phase and higher AVP(R) in the supraoptic, but not in the paraventricular nucleus, while AVP(P) remained elevated at all time points. We suggest that increased central NO production, mainly inducible NOS-derived, reduces AVP gene expression differentially in supraoptic and paraventricular nuclei, and that this may contribute to low AVP plasma levels and hypotension in the late phase of sepsis. (c) 2010 Elsevier B.V. All rights reserved.

Reduced stress fever is accompanied by increased glucocorticoids and reduced PGE(2) in adult rats exposed to endotoxin as neonates

Immune challenges during neonatal period may permanently program immune responses later in life, including endotoxin fever. We tested the hypothesis that neonatal endotoxin exposure affects stress fever in adult rats. In control rats (treated with saline as neonates; nSal) body temperature peaked similar to 1.5 degrees C during open-field stress, whereas in rats exposed to endotoxin (lipopolysaccharide, LPS) as neonates (nLPS) stress fever was significantly attenuated. Following stress, plasma corticosterone levels significantly increased from 74.29 +/- 7.05 ng ml(-1) to 226.29 +/- 9.87 ng ml(-1) in nSal rats, and from 83.43 +/- 10.31 ng ml(-1) to 324.7 +/- 36.87 ng ml(-1) in nLPS rats. Animals treated with LPS as neonates and adrenalectomized one week before experimentation no longer displayed the attenuated febrile response to stress. This attenuated stress fever caused by an increased corticosterone secretion is likely to be linked to an inhibitory effect of glucocorticoids on cyclooxygenase activity/PGE(2) production in preoptic/anteroventral third ventricular region (AV3V) since stress failed to cause a significant increase in PGE(2) in nLPS rats, and this effect was reverted by adrenalectomy. Altogether, the present results indicate that endogenous glucocorticoids are key modulators of the attenuated stress fever in adult rats treated with LPS as neonates, and they act downregulating PGE(2) production in AV3V. Moreover, our findings also support the notion that neonatal immune stimulus affects programming of stress responses during adulthood, despite the fact that inflammation and stress are two distinct processes mediated largely by different neurobiological mechanisms. (C) 2010 Elsevier B.V. All rights reserved.

Reproductive development and function of female rats exposed to di-eta-butyl-phthalate (DBP) in utero and during lactation

Phthalates are environmental contaminants used in the production of plastics, cosmetics and medical devices. Studies on the effects of phthalates on female reproductive health are particularly sparse and mostly restricted to high-dose exposure in rats. In the present study, pregnant rats were treated with 100 mg/kg-d of di-eta-butyl-phthalate (DBP) or only the vehicle (control group), from GD 12 to GD 20 for evaluation of reproductive outcomes and fetal gonads analysis (F0), and from GD 12 to PND 21 to evaluate reproductive development and function on F1 female offspring. Results showed that all parameters were comparable between groups, although there was a significant increase in the fetal weight after DBP exposure. However, the body weight at birth was normal. Based on these data we can conclude that, in these experimental conditions, DBP did not disturb the reproductive development or function of female rats. (C) 2009 Elsevier Inc. All rights reserved.

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100 mu g/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4,6 or 24 h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24 h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24 h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6 h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24 h after surgery, and in both hypothalamic nuclei only at 6 h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood. (C) 2011 Elsevier B.V. All rights reserved.

Modulatory role of locus coeruleus and estradiol on the stress response of female rats

The activity of the hypothalamic-pituitary-adrenal axis is modulated by the norepinephrinergic system and, in females, also by the ovarian hormones. We investigated the role of ovarian steroids and the locus coeruleus (LC) on stress-induced corticosterone secretion in female rats. Ovariectomized rats without hormonal replacement (OVX) or treated with estradiol (OVE) or estradiol plus progesterone (OVEP) were subjected to jugular cannulation. Immediately after that, each hormonal treatment group was subjected to LC lesion or sham surgery or no brain surgery. After 24 h, blood samples of all 9 groups were collected before and after ether inhalation. Other four groups (OVX control, sham and lesioned, and OVE) were perfused for glucocorticoid receptor (GR) immunocytochemistry in hippocampal CA1 neurons and paraventricular nucleus (PVN). Estradiol replacement decreased while LC lesions increased stress-induced corticosterone secretion. The effect of LC lesion was potentiated with the removal of ovarian steroids. Since GR expression of lesioned animals decreased in the hippocampus, but not in PVN, we suggest that the effect of LC lesion on corticosterone secretion could be due to a reduction in the efficiency of the negative feedback system in the CA1 neurons. However, this mechanism is not involved in the estradiol modulation on corticosteroid secretion, as no change in GR expression was observed in estradiol-treated animals.

Persistent Impairment of Testicular Histology and Sperm Motility in Adult Rats Treated with Cisplatin at Peri-Puberty

Cisplatin is one of the most widely used and effective chemotherapeutic agents for the treatment of several human malignancies. This study evaluated the effects of peri-pubertal cisplatin administration on several reproductive end-points and the reversibility of these effects in adulthood. Peri-pubertal Wistar male rats (45 days old) were divided into two groups: control (saline 0.9%) and cisplatin (1 mg/kg/day, 5 days/week, for 3 weeks, i.p.). The study was conducted in two steps and evaluations were performed at ages of 66 (post-pubertal age) and 140 (adult age) days on: (i) organ weights, serum gonadotropins and testosterone levels, sperm counts, motility and morphology, testicular histomorphometry, spermatogenesis kinetics, Sertoli cell number and in situ detection of apoptotic germ cells and (ii) sexual behaviour, fertility and intratesticular testosterone. At the end of cisplatin therapy, rats showed reductions in sperm production and reserves, sperm with progressive movement, tubular diameter, intratesticular testosterone and fertility potential, but increased numbers of TUNEL-positive seminiferous tubules, immotile sperm and pre-implantation losses compared with control. Moreover, cisplatin-treated post-pubertal rats displayed impaired testicular histopathology and sexual behaviour. Serum gonadotropins and testosterone levels, sperm morphology, spermatogenesis kinetics and Sertoli cell number were comparable between experimental groups at both ages. Alterations found in post-puberty were recovered at adulthood, except for sperm motility and damage to testicular histology. The persistence of these cisplatin effects, despite the unaltered fertility after natural mating in rats, may have implications for reproductive function of young boys undergoing cancer therapy, given the lower reproductive efficiency in human beings compared with rats.

Neonatal handling reduces the number of cells in the medial preoptic area of female rats

Early-life events may induce alterations in neuronal function in adulthood. A crucial aspect in studying long-lasting effects induced by environmental interventions imposed to the animal several weeks before is finding a stable change that could be causally related to the phenotype observed in adulthood. In order to explain an adult trait, it seems necessary to look back to early life and establish a temporal line between events. The neonatal handling procedure is an experimental tool to analyze the long-lasting impact of early-life events. Aside from the neuroendocrine response to stress, neonatal handling also alters the functionality of the hypothalamus-pituitary-gonad (HPG) axis. Reductions in ovulation and surge of the luteinizing hormone (LH) on the proestrous day were shown in female rats. Considering the importance of the medial preoptic area (MPA) for the control of ovulation, the present study aimed to verify the effects of neonatal handling on the numerical density and cell size in the MPA in 11-day-old and 90-day-old female rats. Cellular proliferation was also assessed using BrdU (5-bromo-2`-deoxyuridine) in 11-day-old pups. Results showed that neonatal handling induces a stable reduction in the number of cells and in the size of the cell soma, which were lower in handled females than in nonhandled ones at both ages. Cellular proliferation in the MPA was also reduced 24 h after the last manipulation. The repeated mother-infant disruption imposed by the handling procedure ""lesioned"" the MPA. The dysfunction in the ovulation mechanisms induced by the handling procedure could be related to that neuronal loss. The study also illustrates the impact of an environmental intervention on the development of the brain. (C) 2008 Elsevier B.V. All rights reserved