High performance liquid chromatography method for quantification of the N-phenylpiperazine derivative LASSBio-579 in rat plasma

A rapid HPLC analytical method was developed and validated for the determination of the N-phenylpiperazine derivative LASSBio-579in plasma rat. Analyses were performed using a C18 column and elution with 20 mM sodium dihydrogen phosphate monohydrate - methanol. The analyte was monitored using a photodiode array detector (257 nm). Calibration curves in spiked plasma were linear over the concentration range of 0.3-8 mg/mL with determination coefficient > 0.99. The lower limit of quantification was 0.3 mg/mL. The applicability of the HPLC method for pharmacokinetic studies was tested using plasma samples obtained after administration of LASSBio-579 to Wistar rats, showing the specificity of the method.

Sensitive method for determination of piplartine, an alkaloid amide from Piper species, in rat plasma samples by liquid chromatography-tandem mass spectrometry

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

The effects of maternal hyperglycemia on human umbilical vascular gene expression and neonatal rat lung development

Background: Maternal diabetes affects many fetal organ systems, including the vasculature and the lungs. The offspring of diabetic mothers have respiratory adaptation problems after birth. The mechanisms are multifactorial and the effects are prolonged during the postnatal period. An increasing incidence of diabetic pregnancies accentuates the importance of identifying the pathological mechanisms, which cause the metabolic and genetic changes that occur in offspring, born to diabetic mothers. Aims and methods: The aim of this thesis was to determine changes both in human umbilical cord exposed to maternal type 1 diabetes and in neonatal rat lungs after streptozotocin-induced maternal hyperglycemia, during pregnancy. Rat lungs were used as a model for the potential disease mechanisms. Gene expression alterations were determined in human umbilical cords at birth and in rat pup lungs at two week of age. During the first two postnatal weeks, rat lung development was studied morphologically and histologically. Further, the effect of postnatal hyperoxia on hyperglycemia-primed rat lungs was investigated at one week of age to mimic the clinical situation of supplemental oxygen treatment. Results: In the umbilical cord, maternal diabetes had a major negative effect on the expression of genes involved in blood vessel development. The genes regulating vascular tone were also affected. In neonatal rat lungs, intrauterine hyperglycemia had a prolonged effect on gene expression during late alveolarization. The most affected pathway was the upregulation of extracellular matrix proteins. Newborn rat lungs exposed to intrauterine hyperglycemia had thinner saccular walls without changes in airspace size, a smaller relative lung weight and lung total tissue area, and increased cellular apoptosis and proliferation compared to control lungs, possibly reflecting an aberrant maturational adaptation. At one and two weeks of age, cell proliferation and secondary crest formation were accelerated in hyperglycemia-exposed lungs. Postnatal hyperoxic exposure, alone caused arrested alveolarization with thin-walled and enlarged alveoli. In contrast, the dual exposure of intrauterine hyperglycemia and postnatal hyperoxia resulted in the phenotype of thick septa together with arrested alveolarization and decreased number of small pulmonary arteries. Conclusions: Maternal diabetic environment seems to alter the umbilical cord gene expression profile of the regulation of vascular development and function. Fetal hyperglycemia may additionally affect the genetic regulation of the postnatal lung development and may actually induce prolonged structural alterations in neonatal lungs together with a modifying effect on the deleterious pulmonary exposure of postnatal hyperoxia. This, combined with the novel human umbilical cord gene data could serve as stepping stones for future therapies to curb developmental aberrations.

Anomalous somatic embryos in Acca sellowiana (O. Berg) Burret (Myrtaceae)

Somatic embryogenesis represents a valuable tool for the studies on the basic aspects of plant embryo development. Today this process is used as a potencial technique for large-scale plant micropropagation although, so far, it has been applied to only a small number of species. However, when somatic embryos are malformed they are considered economically useless. In Acca sellowiana (O. Berg) Burret, an important fruit-producing crop, large amounts of anomalous somatic embryos (76.3%) were found just after 40 days of culture of explants in a 2,4-D containing medium. Among the anomalous forms found in the cotiledonary stage, 12.2% consisted of fused embryos, 40.4% displayed fused cotyledons, 13.0% presented supernumerary cotyledons, and 10.7% showed absence or poorly developed cotyledons, including those without the shoot apical meristem. Histological analyses indicated that the altered embryos were formed either directly from cotyledons, hypocotyl and radicle of the zygotic embryos used as explants, or indirectly from calli formed from these tissue parts. It is suggested that the formation of anomalous somatic embryos, as well as a low frequency of conversion into emblings reflect physiological and/or genetic disturbances triggered by the presence of 2,4-D in the medium. In vitro experimental alternative approaches are discussed in order to lessen the occurrence of malformed somatic embryos.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21% of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 µg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets

Circadian time-dependent effects of fencamfamine on inhibition of dopamine uptake and release in rat striatal slices

Fencamfamine (FCF) is a central stimulant that facilitates central dopaminergic transmission through inhibition of dopamine uptake and enhanced release of the transmitter. We evaluated the changes in the inhibition of uptake and the release of striatal [3H]-dopamine at 9:00 and 21:00 h, times corresponding to maximal and minimal behavioral responses to FCF, respectively. Adult male Wistar rats (200-250 g) maintained on a 12-h light/12-h dark cycle (lights on at 7:00 h) were used. In the behavioral experiments the rats (N = 8 for each group) received FCF (3.5 mg/kg, ip) or saline at 9:00 or 21:00 h. Fifteen minutes after treatment the duration of activity (sniffing, rearing and locomotion) was recorded for 120 min. The basal motor activity was higher (28.6 ± 4.2 vs 8.4 ± 3.5 s) after saline administration at 21:00 h than at 9:00 h. FCF at a single dose significantly enhanced the basal motor activity (38.3 ± 4.5 vs 8.4 ± 3.5 s) and increased the duration of exploratory activity (38.3 ± 4.5 vs 32.1 ± 4.6 s) during the light, but not the dark phase. Two other groups of rats (N = 6 for each group) were decapitated at 9:00 and 21:00 h and striata were dissected for dopamine uptake and release assays. The inhibition of uptake and release of [3H]-dopamine were higher at 9:00 than at 21:00 h, suggesting that uptake inhibition and the release properties of FCF undergo daily variation. These data suggest that the circadian time-dependent effects of FCF might be related to a higher susceptibility of dopamine presynaptic terminals to the action of FCF during the light phase which corresponds to the rats' resting period

Rapid eye movement (REM) sleep deprivation reduces rat frontal cortex acetylcholinesterase (EC 3.1.1.7) activity

Rapid eye movement (REM) sleep deprivation induces several behavioral changes. Among these, a decrease in yawning behavior produced by low doses of cholinergic agonists is observed which indicates a change in brain cholinergic neurotransmission after REM sleep deprivation. Acetylcholinesterase (Achase) controls acetylcholine (Ach) availability in the synaptic cleft. Therefore, altered Achase activity may lead to a change in Ach availability at the receptor level which, in turn, may result in modification of cholinergic neurotransmission. To determine if REM sleep deprivation would change the activity of Achase, male Wistar rats, 3 months old, weighing 250-300 g, were deprived of REM sleep for 96 h by the flower-pot technique (N = 12). Two additional groups, a home-cage control (N = 6) and a large platform control (N = 6), were also used. Achase was measured in the frontal cortex using two different methods to obtain the enzyme activity. One method consisted of the obtention of total (900 g supernatant), membrane-bound (100,000 g pellet) and soluble (100,000 g supernatant) Achase, and the other method consisted of the obtention of a fraction (40,000 g pellet) enriched in synaptic membrane-bound enzyme. In both preparations, REM sleep deprivation induced a significant decrease in rat frontal cortex Achase activity when compared to both home-cage and large platform controls. REM sleep deprivation induced a significant decrease of 16% in the membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) in the 100,000 g pellet enzyme preparation (home-cage group 152.1 ± 5.7, large platform group 152.7 ± 24.9 and REM sleep-deprived group 127.9 ± 13.8). There was no difference in the soluble enzyme activity. REM sleep deprivation also induced a significant decrease of 20% in the enriched synaptic membrane-bound Achase activity (home-cage group 126.4 ± 21.5, large platform group 127.8 ± 20.4, REM sleep-deprived group 102.8 ± 14.2). Our results suggest that REM sleep deprivation changes Ach availability at the level of its receptors through a decrease in Achase activity

Role of endothelium in angiotensin II formation by the rat aorta and mesenteric arterial bed

We investigated the angiotensin II (Ang II)-generating system by analyzing the vasoconstrictor effect of Ang II, angiotensin I (Ang I), and tetradecapeptide (TDP) renin substrate in the absence and presence of inhibitors of the renin-angiotensin system in isolated rat aortic rings and mesenteric arterial beds with and without functional endothelium. Ang II, Ang I, and TDP elicited a dose-dependent vasoconstrictor effect in both vascular preparations that was completely blocked by the Ang II receptor antagonist saralasin (50 nM). The angiotensin converting enzyme (ACE) inhibitor captopril (36 µM) completely inhibited the vasoconstrictor effect elicited by Ang I and TDP in aortic rings without affecting that of Ang II. In contrast, captopril (36 µM) significantly reduced (80-90%) the response to bolus injection of Ang I, without affecting those to Ang II and TDP in mesenteric arteries. Mechanical removal of the endothelium greatly potentiated (70-95%) the vasoconstrictor response to Ang II, Ang I, and TDP in aortic rings while these responses were unaffected by the removal of the endothelium of mesenteric arteries with sodium deoxycholate infusion. In addition, endothelium disruption did not change the pattern of response elicited by these peptides in the presence of captopril. These findings indicate that the endothelium may not be essential for Ang II formation in rat mesenteric arteries and aorta, but it may modulate the response to Ang II. Although Ang II formation from Ang I is essentially dependent on ACE in both vessels, our results suggest the existence of an alternative pathway in the mesenteric arterial bed that may play an important role in Ang II generation from TDP in resistance but not in large vessels during ACE inhibition

Effect of protein malnutrition on the glycolytic and glutaminolytic enzyme activity of rat thymus and mesenteric lymph nodes

The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of Wistar rats submitted to protein malnutrition (6% protein in the diet rather than 20%) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87% (from 0.30 ± 0.05 to 0.04 ± 0.01) and 75% (0.40 ± 0.04 to 0.10 ± 0.02), respectively. The protein content was reduced only in the thymus from 102.3 ± 4.4 (control rats) to 72.6 ± 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by half in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.