The study of insect blood-feeding behaviour: 1. Feeding equipment, physical and endogenous factors, dose effect analysis, and diet destination

Experimental techniques that we have found useful during our studies of insect blood-feeding behaviour are reviewed. Some of the principal findings resulting from these techniques are discussed. Where directly applicable, the work of others is included, but no complete review of the subject has been attempted.

Effect of endogenous hydrogen sulfide inhibition on structural and functional renal disturbances induced by gentamicin

Animal models of gentamicin nephrotoxicity present acute tubular necrosis associated with inflammation, which can contribute to intensify the renal damage. Hydrogen sulfide (H2S) is a signaling molecule involved in inflammation. We evaluated the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by gentamicin. Male Wistar rats (N = 8) were injected with 40 mg/kg gentamicin (im) twice a day for 9 days, some of them also received PAG (N = 8, 10 mg·kg-1·day-1, ip). Control rats (N = 6) were treated with saline or PAG only (N = 4). Twenty-four-hour urine samples were collected one day after the end of these treatments, blood samples were collected, the animals were sacrificed, and the kidneys were removed for quantification of H2S formation and histological and immunohistochemical studies. Gentamicin-treated rats presented higher sodium and potassium fractional excretion, increased plasma creatinine [4.06 (3.00; 5.87) mg%] and urea levels, a greater number of macrophages/monocytes, and a higher score for tubular interstitial lesions [3.50 (3.00; 4.00)] in the renal cortex. These changes were associated with increased H2S formation in the kidneys from gentamicin-treated rats (230.60 ± 38.62 µg·mg protein-1·h-1) compared to control (21.12 ± 1.63) and PAG (11.44 ± 3.08). Treatment with PAG reduced this increase (171.60 ± 18.34), the disturbances in plasma creatinine levels [2.20 (1.92; 4.60) mg%], macrophage infiltration, and score for tubular interstitial lesions [2.00 (2.00; 3.00)]. However, PAG did not interfere with the increase in fractional sodium excretion provoked by gentamicin. The protective effect of PAG on gentamicin nephrotoxicity was related, at least in part, to decreased H2S formation.

Endogenous and exogenously-induced immunomodulation of tumour-host responsiveness

In spite of the availability of multiple effector mechanisms of the immune system to combat tumour growth and metastases, their impairment frequently accompanies the appearance of cancer. Factors contributing to this impairment may be related to properties of the host and/or the tumour itself and may be with respect to their origin -endogenous or exogenour. Based on the unique biological behavior of prostate cancer (PCa), and its apparent escape from immune surveillance in the presence of tumour immuno genicity, continuing investigation of endogenous and exogenous factors thought to be relevant to its pathogenesis have been made. For this purpose further studies of the suggested role of human seminal plasma (SePl) and the synthetic oestrogen, diethylstiboestrol (DES), as representative endogenous and exogenous immunomodulatory factors (IMF) of tumour-host responsiveness, together with evaluation of human prostatic tissue extracts and leuprolide (the luteinizing-hormone-releasing-hormone proposed as an alternate to DES therapy) have been made by evaluating their effect on the lytic activity of natural killer (NK) cells. SePl and prostate extracts significantly suppressed NK cell lysis. Physicochemical studies suggest SePl and prostate IMF to be associated with high and low molecular weight macromolecules; and implicate the participation of transglutaminase and prostaglandins. Comparative study of therapeutic levels of DES vs. leuprolide on NK cell lysis demonstrated significant suppression by DES vs. a negligible effect of leuprolide. Metastases are highly prevalent in PCa, and contribute significantly to its morbidity and mortality. Further knowledge of the range of effects of endogenous and exogenous IMF on effector mechanisms of tumour-host responsiveness, to include suppression of NK cells, and elucidation of their nature, may contribute toward our understanding of the unique biological behavior of tumours of the prostate, in addition to improvement in their clinical management.

Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.

Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

A cell-free system from Plasmodium falciparum able to translate endogenous mRNA was used to determine the effect of artemisinin, chloroquine and primaquine on the protein synthesis mechanism of the parasite. The antimalarial drugs did not inhibit the incorporation of [³H] methionine into parasite proteins even at concentrations higher than the ones found to strongly inhibit the parasite growth. Results clearly indicate that these compounds do not have a direct effect on protein synthesis activity of P. falciparum coded by endogenous mRNA.

Alteration in the endogenous intestinal flora of swiss webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.

Vitamin C and endogenous cortisol in foreign-body inflammatory response in pacus

The objective of this work was to evaluate the effect of food supplementation with vitamin C on macrophage and multinucleated giant cell (MGC) activities of pacus at two stocking densities. The experiment was carried out in a 2x2x3 split-plot factorial arrangement with: 0 and 500 mg kg-1 vitamin C; 5 and 20 kg m-3 stocking densities; and evaluation times at 3, 6, and 12 days after the subcutaneous implantation of glass coverslips (DPI). The number of macrophages and MGC, as well as cortisol and glucose plasma levels were determined. The number of macrophages and MGC with two to five nuclei was significantly greater in fish supplemented with vitamin C at 5 kg m-3 stocking density at 3 DPI in comparison to nonsupplemented ones. The macrophage and MGC counts were lower in fish with high-plasma cortisol concentration. Supplementation with 500 mg vitamin C benefits macrophage activity on foreign-body inflammation, and high-cortisol concentration has suppressive effects on this response.

Effect of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts

There is increasing evidence that angiotensin-(1-7) (Ang-(1-7)) is an endogenous biologically active component of the renin-angiotensin system (RAS). In the present study, we investigated the effects of Ang-(1-7) on reperfusion arrhythmias in isolated rat hearts. Isolated rat hearts were perfused with two different media, i.e., Krebs-Ringer (2.52 mM CaCl2) and low-Ca2+ Krebs-Ringer (1.12 mM CaCl2). In hearts perfused with Krebs-Ringer, Ang-(1-7) produced a concentration-dependent (27-210 nM) reduction in coronary flow (25% reduction at highest concentration), while only slight and variable changes in contraction force and heart rate were observed. Under the same conditions, angiotensin II (Ang II; 27 and 70 nM) produced a significant reduction in coronary flow (39% and 48%, respectively) associated with a significant increase in force. A decrease in heart rate was also observed. In low-Ca2+ Krebs-Ringer solution, perfusion with Ang-(1-7) or Ang II at 27 nM concentration produced similar changes in coronary flow, contraction force and heart rate. In isolated hearts perfused with normal Krebs-Ringer, Ang-(1-7) produced a significant enhancement of reperfusion arrhythmias revealed by an increase in the incidence and duration of ventricular tachycardia and ventricular fibrillation (more than 30-min duration). The facilitation of reperfusion arrhythmias by Ang-(1-7) was associated with an increase in the magnitude of the decreased force usually observed during the post-ischemic period. The effects of Ang-(1-7) were abolished in isolated rat hearts perfused with low-Ca2+ Krebs-Ringer. The effect of Ang II (27 nM) was similar but less pronounced than that of Ang-(1-7) at the same concentration. These results indicate that the heart is a site of action for Ang-(1-7) and suggest that this heptapeptide may be involved in the mediation of the cardiac effects of the RAS

Use of nucleotides in weanling rats with diarrhea induced by a lactose overload: effect on the evolution of diarrhea and weight and on the histopathology of intestine, liver and spleen

Until recently, dietary sources of nucleotides were thought not to be essential for good nutrition. Certain states with higher metabolic demands may require larger amounts that cannot be provided by endogenous production. The objective of the present study was to determine the action of nucleotides on the recovery from lactose-induced diarrhea in weaned rats. Thirty-six weanling Fisher rats were divided into two groups. Group 1 received a standard diet and group 2 received a diet containing lactose in place of starch. On the 10th day, six animals per group were sacrificed for histopathological evaluation. The remaining animals were divided into two other subgroups, each with 6 animals, receiving a control diet, a control diet with nucleotides (0.05% adenosine monophosphate, 0.05% guanosine monophosphate, 0.05% cytidine monophosphate, 0.05% uridine monophosphate and 0.05% inosine monophosphate), a diet with lactose, and a diet with lactose and nucleotides. On the 32nd day of the experiment all animals were sacrificed. Animals with diarrhea weighed less than animals without diarrhea. The introduction of nucleotides did not lead to weight gain. Mean diet consumption was lower in the group that continued to ingest lactose, with the group receiving lactose plus nucleotides showing a lower mean consumption. Animals receiving lactose had inflammatory reaction and deposits of periodic acid-Schiff-positive material in intestinal, hepatic and splenic tissues. The introduction of nucleotides led to an improvement of the intestinal inflammatory reaction. In lactose-induced diarrhea, when the stimulus is maintained - lactose overload - the nucleotides have a limited action on the weight gain and on recovery of intestinal morphology, although they have a protective effect on hepatic injury and improve the inflammatory response.

Effect of repeated stress on novelty-induced antinociception in rats

There is extensive evidence that acute stress induces an analgesic response in rats. On the other hand, repeatedly stressed animals may present the opposite effect, i.e., hyperalgesia. Furthermore, exposure to novelty is known to induce antinociception. The effects of repeated restraint stress on nociception after exposure to novelty, as measured by the tail-flick latency (TFL), were studied in adult male rats. The animals were stressed by restraint 1 h daily, 5 days a week for 40 days. The control group was not submitted to restraint. Nociception was assessed with a tail-flick apparatus. After being familiarized with the TFL apparatus, each group was subdivided into two other groups, i.e., with or without novelty. Animals were subjected to the TFL measurement twice. For the animals exposed to novelty, the first TFL measurement was made immediately before, and the second 2 min after a 2-min exposure to a new environment. While the control group presented an increased TFL after exposure to a novel environment, chronically stressed animals did not show this effect. These results suggest that repeated restraint stress induces an alteration in the nociceptive response, perhaps as a result of an alteration in endogenous opioids in these animals.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 ± 6 and 2 ± 1 in phases 1 and 2) and lickings (6 ± 2 and 1 ± 1 in phases 1 and 2) in the formalin test when compared with control (75 ± 13 and 22 ± 6 flinches in phases 1 and 2; 28 ± 7 and 17 ± 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 ± 0.1 and 0.53 ± 0.13, respectively) when compared with control (-0.10 ± 0.08 at 30 min and -0.09 ± 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses.

Do endogenous opioids and nitric oxide participate in the anticonvulsant action of dipyrone?

It was previously reported that systemic administration of dipyrone inhibited the tonic component of generalized tonic-clonic seizures in both the electroshock and the audiogenic seizure models. The aim of the present study was to investigate the mechanisms involved in the anticonvulsant action of dipyrone by assessing the role of nitric oxide and opioids in the electroshock (female 60- to 90-day-old Wistar rats, N = 5-11) and audiogenic seizure (female 60- to 90-day-old Wistar audiogenic rats, N = 5-11) models of epilepsy. Naloxone (5 mg/kg, sc) significantly reversed the anticonvulsant effect of dipyrone in rats submitted to the induction of audiogenic seizures (ANOVA/Bonferroni's test), suggesting the involvement of opioid peptides in this action. In the electroshock model no reversal of the anticonvulsant effect of dipyrone by naloxone (5 mg/kg, sc) was demonstrable. The acute (120 mg/kg, ip) and chronic (25 mg/kg, ip, twice a day/4 days) administration of L-NOARG did not reverse the anticonvulsant action of dipyrone in the audiogenic seizure model, suggesting that the nitric oxide pathway does not participate in such effect. Indomethacin (10, 20 and 30 mg/kg, ip) used for comparison had no anticonvulsant effect in the audiogenic seizure model. In conclusion, opioid peptides but not nitric oxide seem to be involved in the anticonvulsant action of dipyrone in audiogenic seizures.

Effect of (-)-∆9-tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered ∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: ∆9-THC (N = 10), treated with 10 mg/kg body weight ∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Δ9-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Δ9-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Δ9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

Participation of endogenous opioids in the antinociception induced by resistance exercise in rats

Exercise is a low-cost intervention that promotes health and contributes to the maintenance of the quality of life. The present study was designed to investigate the influence of different resistance exercise protocols on the nociceptive threshold of rats. Female Wistar rats were used to perform exercises in a weight-lifting exercise model. The following groups were examined (N = 6 per group): untrained rats (control group); an acute protocol group consisting of rats submitted to 15 sets of 15 repetitions of resistance exercise (acute group); rats exercised with 3 sets of 10 repetitions, three times per week for 12 weeks (trained group), and a group consisting of trained rats that were further submitted to the acute protocol (trained-acute group). The nociceptive threshold was measured by the paw-withdrawal test, in which the withdrawal threshold (escape reaction) was measured by an apparatus applying force to the plantar surface of the animal paw. The opioid antagonist naloxone (2 mg/kg) was administered subcutaneously 10 min before the exercise protocols. The trained group demonstrated antinociception only up to day 45 of the 12-week training period. A significant increase (37%, P < 0.05) in the nociceptive threshold was produced immediately after exercise, decreasing to 15% after 15 min, when the acute exercise protocol was used. Naloxone reversed this effect. These data show that the acute resistance exercise protocol was effective in producing antinociception for 15 min. This antinociceptive effect is mediated by the activation of opioid receptors.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.