Alcoholic fixation over formalin fixation: A new,safer option for morphologic and molecular analysis of tissues

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.     

Laetispicine, an amide alkaloid from Piper laetispicum, presents antidepressant and antinociceptive effects in mice

In the present work, we studied the effect of laetispicine, an amide alkaloid isolated from the stems of Piper laetispicum (Piperaceae), in forced swimming, open field, acetic acid writhing and formalin tests in KM mice to assess antidepressant and antinociceptive effects. A significant and dose-dependent decrease in the immobility time, as evaluated by the forced swimming test, was observed after laetispicine administration (38.18, 39.79, 58.77 and 67.28% decreased at the doses of 5, 10, 20, 40 mg/kg, respectively), suggesting an antidepressant effect. Furthermore, in the open field test, laetispicine at the given doses did not alter the number of crossings and rearing, as compared to controls. Results from writhing and formalin tests showed that laetispicine reduced the number of writhing in mice in a dose-dependent manner, attenuated the licking and spiting time of the injected paw in the first phase of formalin test. The antinociceptive effect of laetispicine was not affected by pre-treatment (i.p.) with naloxone (2 mg/kg). In conclusion, we showed that laetispicine possessed significant antidepressant and antinociceptive properties, making this drug potentially useful in depression and pain.     

Infantile stage of rat development in behavioral parameters of depression-like state and pain response

In the 7–8- and the 10–11-day old male rat pups born to dams exposed to an immobilization stress for the last week of pregnancy and to the dams exposed to no stress (control), behavioral parameters were studied: the level of depression in the test of forced swimming (the Porsolt’s test) and 24 h after a long pain response during inflammation (the formalin test—a subcutaneous injection of 2.5% formalin into the hind leg plantar pad). In control pups, significant age-related changes in the forced swimming were revealed: the immobility time was longer in animals of the older age group, whereas no age differences were found in parameters of the persistent inflammatory pain and in flexing + shaking behavior. The prenatal stress produced an increase in the immobility time and the flexing + shaking behavior in the 7–8-day old, but not in the 10–11-day old rat pups. This resulted in elimination of the age differences in the immobility time in the prenatally stressed animals. Thus, use of different methodic approaches has allowed revealing peculiarities in the parameters of the degree of depression and duration of the pain response at inflammation in the 7–8- and 10–11-day old rat pups, which indicates heterogeneity of the infantile development stage that, according to literature data, includes in rats the period from the 5th to the 10th postnatal days.     

Combined morphological and molecular analysis of individual nematodes through short‐term preservation in formalin

Small metazoans such as marine nematodes are increasingly identified using both molecular and morphological techniques. Formalin is the preferred fixative for morphological analysis but specimens become unsuitable for molecular study due to formalin‐induced modification of DNA. Nematodes fixed in ethanol work well for molecular studies but become unsuitable for taxonomy due to shrinkage. Here we show for the first time that formalin can be used as a short‐term fixative (≤ 7 days) for marine nematodes, allowing both morphological and molecular work to be conducted on the same individual. No sequence ambiguities were detected in polymerase chain reaction (PCR) amplifications of 18S ribosomal DNA (rDNA) following short‐term formalin preservation.     

A comparative study on the effects of selected pesticides on hemato-biochemistry and tissue histology of freshwater fish Cirrhinus mrigala (Hamilton, 1822)

The aim of the present study was to investigate the comparative effects of pesticides Chlorfenapyr, Dimethoate and Acetamiprid on the health of Cirrhinus mrigala under long term exposure. Eighty C. mrigala were divided in four equal groups; one control and three treated groups. The blood was collected from both control and treated groups at intervals of 10th, 20th and 30th days for hemato-biochemistry and histopathological alterations. The result indicates significant difference (P < 0.05) in RBCs, Hb, PCV and MCHC whereas elevation in WBCs and Platelets counts were recorded. In 10th day sampling, MCV value of Dimethoate and Acetamiprid treatment had no difference in comparison with the control group, however it is significantly increased (P < 0.05) in rest of sampling. The MCH value of exposed fish showed significant increased (P < 0.05) after 20th and 30th days for Chlorfenapyr and after 30th days for Acetamiprid exposure while insignificantly increased for rest of sampling. It was also found that these pesticides significantly decrease (p < 0.05) the T3 and T4 levels while increase in the TSH, cortical, ALP, AST, ALT and LDH levels in the serum of the treated fishes in contrast to control group. Similarly, histopathological analysis of gills and liver showed significant alterations in all the treated groups. Toxicity trends of these pesticides was ranked as Chlorfenapyr > Acetamiprid > Dimethoate. It is concluded that indiscriminate use of such pesticides poses a noxious threat to non-target organisms, harm the ecosystems and jeopardizes human health.     

The comparison of immobility time in experimental rat swimming models

Rat swimming models have been used in studies about stress and depression. However, there is no consensus about interpreting immobility (helplessness or adaptation) in the literature. In the present study, immobility time, glucose and glycogen mobilization, corticosterone and the effect of desipramine and diazepam were investigated in two different models: swimming stress and the forced swimming test. Immobility time was lower in swimming stress than in the forced swimming test. Both swimming models increased corticosterone levels in comparison with control animal levels. Moreover, swimming stress induced higher corticosterone levels than the forced swimming test did [F(2,14)=59.52; p<0.001]. Liver glycogen content values differed from one another (swimming stress相似文献   

Effects of Dominant/Subordinate Social Status on Formalin-Induced Pain and Changes in Serum Proinflammatory Cytokine Concentrations in Mice

Current investigations regarding social stress primarily focus on the health consequences of being in stressful social hierarchies. The repetitive nature of social conflicts seems to favor an induction of hyperalgesia or hypoalgesia, both in rodents and humans. Additionally, social conflicts may affect the immune system. In order to better establish the pain and immune responses to stress, the present study implemented a sensory contact model on 32 male BALB/c mice. Subsequent to establishing a dominance/submissive social relationship, each mouse was injected with formalin (20 μl, 2%) and their pain behavior was scored and serum concentrations of proinflammatory cytokines IL-1 and IL-6, and corticosterone were also measured. Test results revealed that subordinate mice were hypoalgesic during chronic phase of formalin test compared to control and dominant mice (P<0.05). On the other hand, subordinate mice were hyperalgesic compared to dominant mice during the whole acute phase of formalin test (P<0.05). Corticosterone, IL-1 and IL-6 concentrations were much higher in serum of dominant and subordinate mice than in the control group (p<0.05). The results indicated that, although both dominant and subordinate animals displayed an increase in serum corticosterone and proinflammatory cytokines during social interactions, their response to pain perception differently was affected with the social status.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

Neuroinflammatory reactions in sickness behavior induced by bacterial infection: Protective effect of minocycline

The neurological changes elicited by bacterial infection are called sickness behavior. Minocycline (MIN) is neuroprotective with a remarkable brain tissue penetration. MIN was orally administered at a dose 90 mg/kg for 3 days, whereas Escherichia coli was given as a single intraperitoneal injection (0.2 mL of 24 h growth) on the third day. After 24 h of bacterial infection, behavioral tests namely open field and forced swimming were carried out, then animals were decapitated. Rats infected with E. coli displayed reduced struggling time in forced swimming test, as well as, exploration and locomotion in open field test with reduction in neurotransmitters (norepinephrine, dopamine, and serotonin) versus elevation in the inflammatory (tumor necrosis factor‐alpha, interferon‐gamma) and oxidative stress (thiobarbituric acid reactive substance, reduced glutathione) biomarkers. Inflammatory infiltrates of nuclear cells were observed in brains of infected rats. MIN administration prevented the deleterious effects of E. coli infection, thus protects against sickness behavior possibly via defending from neuroinflammation.     

Attenuation of the neuronal stress responsiveness and corticotrophin releasing hormone synthesis after sexual activity in male rats

Beneficial effects of sexual activity and mating on the responsiveness to environmental stress can be observed in humans and other mammalian species alike, but the underlying neurobiological mechanisms are largely unknown. Sexual activity and mating with a receptive female has recently been shown to reduce the subsequent emotional stress response via activation of the brain oxytocin system. Therefore, we investigated the neuronal and hormonal responses to an acute stressor (forced swimming) after mating in male rats.Attenuation of the stress-induced increase of c-fos and CRH mRNA expression within the hypothalamic paraventricular nucleus 4 h after mating revealed that sexual activity reduced neuronal reactivity in this region. However, this effect was independent of oxytocin as oxytocin receptor blockade, by central administration of an oxytocin receptor antagonist, after mating did not prevent the reduced expression of c-fos mRNA in response to stressor exposure. Mating itself stimulated corticotrophin (ACTH) and corticosterone secretion, which was absent in males after contact with an unreceptive female (non-mated group). However, ACTH and corticosterone responses to forced swimming applied either 45 min or 4 h after female contact were similar between mated and non-mated males. These findings provide evidence for a stress-protective effect of sexual activity and mating in male rats and for dissociation between neuronal and neuroendocrine stress responses.     

Rutin and meloxicam attenuate paw inflammation in mice: Affecting sorbitol dehydrogenase activity

Rutin, naturally occurring flavonoid, has reported to cover interesting multiple pharmacological properties. This study evaluated rutin or/and meloxicam effects in paw inflammation induced by formalin in mice. Mice were divided into four groups: I‐Formalin group, II‐Rutin 60 mg/kg (p.o.), III‐Meloxicam 10 mg/kg (p.o.), plus IV‐Combined rutin and meloxicam. Therapies were administered once a day for 7 days. The curative effects were assessed on inflammatory, oxidative stress, and apoptosis. Both rutin and/or meloxicam induced marked improvement in paw licking time on the 1st day and by combined treatment only on the 3rd day as well reduction in paw edema% on the 3rd day. Moreover, noticeable progress in liver malondialdehyde content, superoxide dismutase, and sorbitol dehydrogenase activities as well decline in paw interleukin‐1β level and extent of apoptosis. The results spot light on the good influence of combined rutin and meloxicam in formalin‐induced mice paw inflammation to a better extent than either rutin or meloxicam lonely.     

The use of spontaneous behavior,swimming performances and metabolic rate to evaluate toxicity of PFOS on topmouth gudgeon Pseudorasbora parva

Perfluorooctane sulfonate (PFOS) is a ubiquitous environmental contaminant that has been found to pose various risks to fish health and the safety of aquatic ecosystem. Swimming performance is an integrated index of fitness in fish. However, little research has sought on the effects of PFOS on swimming performances of fish. Experiments were carried out to clarify the impacts of acute exposure to PFOS on behavior, swimming ability and metabolic rate in topmouth gudgeon (Pseudorasbora parva), to understand the underlying ecotoxicological effects of waterborne PFOS exposure on fish physiology and behavior. Fish were exposed to PFOS (0, 0.5, 2, 8 or 32 mg/L) for 96 h. Afterwards, the routine metabolic rate (RMR), spontaneous swimming behavior (SSB), fast-start swimming performance (FSP) and critical swimming speed (U_crit) of the topmouth gudgeon were examined. The results show reduced behavioral performance and increased physiological stress with increasing PFOS concentration. Both RMR, SSB and U_crit were significantly affected by PFOS exposure (p < 0.05). The lowest observed effect concentration (LOEC) is 2 mg/L for SSB. PFOS treatment resulted in increased RMR (p = 0.001) and decreased U_crit (p = 0.005), whereas FSP was not influenced by PFOS (p > 0.05). The results indicate that the anaerobic swimming capacity was conserved, but the metabolic level, SSB and aerobic swimming performance in topmouth gudgeon were susceptible to PFOS contamination, and hence might be useful as considerable potential biomarkers of pollution.     

In vivo and in vitro evaluation of pharmacological activities of Adenia trilobata (Roxb.)

Adenia trilobata, locally known as akandaphal in Bangladesh, has some traditional uses. Leaves and stems extracted with pure methanol (MEATL, MEATS) and fractioned by n-hexane (NFATL, NFATS), which was subjected to qualitative phytochemical analysis. The qualitative phytochemical analysis of four extracts showed the presence of secondary metabolites such as alkaloid, carbohydrate, glycosides, flavonoids, phenols, flavonol, and saponins. All four extracts of A. trilobata, exhibited a strong antioxidant activity while a moderately (MEATS = 328 μg/mL) to weakly toxic (NFATL = 616.85 μg/mL) LC₅₀ observed in brine shrimp lethality bioassay. In thrombolytic test, MEATL (18.54 ± 2.18%; P < 0.01) and MEATS (25.58 ± 4.76%; P < 0.0001) showed significant percentage of clot lysis in human blood. The in vivo analgesic activity carried by acetic acid test and formalin test, while the antidiarrheal activity assayed by two standard methods e.g., castor oil-induced diarrhea and castor oil-induced gastrointestinal motility. Both, in vivo model, showed an extremely significant (P < 0.0001) dose-dependent manner percentage of inhibition in comparison to the control group. Present results suggested, A. trilobata could be a potential source for antioxidative, cytotoxic, thrombolytic, analgesic, antidiarrheal agents which require further study to identify the mechanism of A. trilobata.     

Impact of mycogenic zinc nanoparticles on performance,behavior, immune response,and microbial load in Oreochromis niloticus

This work aims to evaluate the antibacterial activity of biological zinc nanoparticles (BIO-ZnONPs) against pathogenic fish bacteria and assess the effect of BIO-ZnONPs on the performance, behavior, and immune response in Nile tilapia (Oreochromis niloticus) as compared to chemical zinc nanoparticles (CH- ZnONPs). Aspergillus niger TS16 fabricated the BIO-ZnONPs were spherical shape with the average size of 45 nm and net charge of ?27.23 mV. Generally, the results indicate that BIO-ZnONPs were more effective than CH- ZnONPs in enhancing the performance properties of Nile tilapia. Five experimental groups of Nile tilapia (initial body weight of 20.2 g) were treated with two concentrations of 0.5 and 1 mg L^?1 from biological and chemical ZnONPs, while the fifth group was served as a control. After ten weeks of treated water with ZnONPs, the performance, feed efficiency parameters, feeding, and swimming behaviors significantly improved in BIO-ZnONPs treated groups (P < 0.05). The liver function, LYZ activity, and NBT values were significantly enhanced in the 0.5 mg L^?1 BIO-ZnONPS group compared to CH- ZnONPs group and control (P < 0.05). Furthermore, the lowest cortisol and the highest testosterone and growth hormone levels were recorded in 1 mg L^?1 BIO-ZnONPs group. Regarding the antibacterial effects, BIO-ZnONPs displayed the lower total bacterial loads in water and fish tissues (intestine, gills, skin, and muscle) and the maximum antibacterial properties against pathogenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Aeromonas hydrophila). Our study exemplifies novel findings of BIO-ZnONPs in the promotion of fish health and production and its antibacterial properties in Nile tilapia.     

Alterations in luteal production of androstenedione,testosterone, and estrone,but not estradiol,during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.     

Effects of semi-purified diet on depressive behaviors in aged mice

Diet is a key modifiable factor influencing the composition of gut microbiota. There are two types of commercially available diets for experimental animals: non-purified and semi-purified diets. Non-purified diets are composed of complex ingredients from multiple sources, while semi-purified diets are formulated with refined ingredients. Accumulating evidence has demonstrated a link between the gut microbiota and depression, and feed ingredients may influence depressive physiology and behaviors. To test this hypothesis, we examined how chronic non-purified (CRF-1) and semi-purified (AIN-93G) diets affected phenotypes, including depressive behaviors, plasma corticosterone levels, and small-intestine microbiota in young (2 months old) and aged (22 months old) inbred C57BL/JJcl mice. In young mice, similar phenotypes were associated with non-purified and semi-purified diets. However, in aged mice, semi-purified diets increased depressive behaviors in the tail suspension (P < 0.05) and forced swimming tests (P < 0.01). The corticosterone levels were similar between the two diets under normal rearing conditions. However, immediately after exposure to the stressful conditions of the forced swimming test, the corticosterone levels in the aged mice fed the semi-purified diet were higher than those of mice fed the non-purified diet (P < 0.05). There were fewer Lactobacillales in the small intestines of aged mice fed the semi-purified diet compared to those fed the non-purified diet (P < 0.01). Further, α-diversity was lower in aged mice fed the semi-purified versus non-purified diet (P < 0.01). Our results indicate that host physiology and gut microbiota differed according to whether the aged mice were fed a non-purified or semi-purified diet. Specifically, those fed the semi-purified diet were more vulnerable to stress than age-matched mice fed the non-purified diet. Our findings indicate that researchers should consider the effects of feed ingredients on depressive physiology and behaviors, and select diets that are appropriate for their particular research design. Further, identification of the ingredients in non-purified diets could facilitate examination of the mechanisms by which gut microbiota composition might increase resistance to stress and depression.     

Honey as a substitute for formalin?

Formalin has long been the standard fixative for clinical routines worldwide. After the Formaldehyde Standard became law in the US in 1987, as a result of increasing concerns about the potential carcinogenicity of formaldehyde, attempts have been made to find safer alternatives. Alcoholic formalin is a useful fixative, because in addition to fixation, dehydration also is begun. For centuries, honey has been known to be an antibacterial agent with the potential to preserve compounds without harmful effects on its users. We compared the effects of honey fixation with other routine fixatives using conventional histochemical and immunohistochemical staining methods. Our results demonstrated that tissues fixed in either honey or alcoholic formalin and 10% neutral buffered formalin (NBF) have similar histomorphology. Honey fixation showed minor histomorphological differences among the various tissues; however, it did not influence affect correct diagnostic conclusions. Our results suggested that honey can be used as a safe alternative to formalin in histopathology.     

The antidepressant effect of an antiulcer pentadecapeptide BPC 157 in Porsolt's test and chronic unpredictable stress in rats. A comparison with antidepressants

Various antidepressants have antiulcer activity. Likewise, the models currently used in ulcers and depression disorders research have a considerable degree of similarity. Therefore, the possibility that depression disorders could be effectively influenced by a primary antiulcer agent with a cyto/organoprotective activity, such as the novel stomach pentadecapeptide BPC 157, was investigated in two rat depression assays. First, a forced swimming test (a Porsolt's procedure) was used. As a more severe procedure, chronic unpredictable stress (after 5 d of unpredictable stress protocol, once daily drug application during stress procedure, open field-immobility test assessment at fourth or sixth day of medication) was used. In a forced swimming test, a reduction of the immobility time in BPC 157 (10 μg, 10 ng.kg^–1 i.p.) treated rats corresponds to the activity of the 15 mg or 40 mg (i.p.) of conventional antidepressants, imipramine or nialamide, respectively, given according to the original Porsolt's protocol. In chronic unpredictable stress procedure, particular aggravation of experimental conditions markedly affected the conventional antidepressant activity, whereas BPC 157 effectiveness was continuously present. The effect of daily imipramine (30 mg) medication could be seen only after a more prolonged period, but not after a shorter period (i.e., 4-d protocol). In these conditions, no delay in the effectiveness was noted in BPC 157 medication and a reduction of the immobility of chronically stressed rats was noted after both 4 and 6 d of BPC 157 (10 μg, 10 ng) medication.     

Effect of GnRH analogs in postnatal domestic cats

The aim of this study was to reproductively assess the clinical and hormonal effects of a GnRH agonist (AG) and an antagonist (AN) administered during the postnatal period in domestic cats. Forty-eight male and female postnatal kittens were randomly assigned to deslorelin acetate 1.6 mg subcutaneous (AG; n = 16), acyline 33 μg/100 g subcutaneous weekly for 3 months (AN; n = 16), or control (CO; n = 16) which remained untreated. The cats were followed up (behavioral observation, physical examination, fecal sexual steroid determinations, mating test, and pregnancy diagnosis) up to puberty. Puberty was delayed (weeks) in the AG animals (62.9 ± 3.5; P < 0.01) but not in the AN (15.5 ± 1.7; P > 0.05) when they were compared with CO kittens (13.4 ± 0.4). Fifteen (15/16) of the AN and CO animals, and only 11 of 16 cats of the AG group were fertile (P > 0.1). No differences were found in body weight (P > 0.1) and measurements (P > 0.1), libido (P > 0.1) and in the appearance of side effects (P > 0.1; except a pyometra in an AG female) among groups. In both AG- and AN-treated males (testosterone; P < 0.01) and females (estradiol-17β; P < 0.01) fecal hormone concentrations were lower than in CO group during the first five postnatal weeks but not later. It is concluded that the neonatal administration of these AG and AN decreased fecal sexual steroids during the first postnatal weeks causing, the agonists but not the antagonist, a significant, reversible delay in puberty appearance.     

Acute administration of levetiracetam in tonic pain model modulates gene expression of 5HT1A and 5HT7 receptors in the thalamus of rats (Rattus norvergicus)

The nociceptive effect of Levetiracetam (LEV) on the expression of 5-HT_1A and 5-HT₇ receptors found in the thalamus was evaluated. Thirty-six male rats (Wistar) were randomized into six groups: in the Control group without treatment; LEV50 group LEV was administered in a single dose of 50 mg/kg i.g.; in the LEV300 group LEV dose of 300 mg/kg i.g.; in the FORMALIN group the formalin test was performed; in the LEV50/FORMALIN group LEV dose of 50 mg/kg i.g and the formalin test was performed; in the LEV300/FORMALIN group LEV dose of 300 mg/kg i.g and the formalin test was performed, subsequently the thalamus was dissected in all groups. In the formalin tests LEV exhibited an antinociceptive effect in the LEV300/FORMALIN group (p?<?0.05) and a pronociceptive effect in the LEV50/FORMALIN group (p?<?0.001). The results obtained by Real-time PCR confirmed the expression of the 5-HT_1A and 5-HT₇ receptors in the thalamus, 5-HT_1A receptors increased significantly in the FORMALIN group and the LEV300/FORMALIN group (p?<?0.05). 5-HT7 receptors are only over expressed at a dose of 300 mg/Kg of LEV with formalin (p?<?0.05). This suggests that LEV modulates the sensation of pain by controlling the expression of 5-HT_1A and 5-HT₇ in a tonic pain model, and that changes in the expression of 5-HT_1A and 5-HT₇ receptors are associated with the sensation of pain, furthermore its possibility to be used in clinical treatments for pain.