Glial cell line-derived neurotrophic factor added to a sciatic nerve fragment grafted in a spinal cord gap ameliorates motor impairments in rats and increases local axonal growth

Purpose: The aversive nature of regenerative milieu is the main problem related to the failure of neuronal restoration in the injured spinal cord which however might be addressed with an adequate repair intervention. We evaluated whether glial cell line-derived neurotrophic factor (GDNF) may increase the ability of sciatic nerve graft, placed in a gap promoted by complete transections of the spinal cord, to enhance motor recovery and local fiber growth. Methods: Rats received a 4 mm-long gap at low thoracic level and were repaired with a fragment of the sciatic nerve. GDNF was added (NERVE+GDNF) or not to the grafts (NERVE-GDNF). Motor behavior score (BBB) and sensorimotor tests-linked to the combined behavior score (CBS), which indicate the degree of the motor improvement and the percentage of functional deficit, respectively, and also the spontaneous motor behavior in an open field by means of an infrared motion sensor activity monitor were analyzed. At the end of the third month post surgery, the tissue composed by the graft and the adjacent regions of the spinal cord was removed and submitted to the immunohistochemistry of the neurofilament-200 (NF-200), growth associated protein-43 (GAP-43), microtubule associated protein-2 (MAP-2), 5-hidroxytryptamine (serotonin, 5-HT) and calcitonin gene related peptide (CGRP). The immunoreactive fibers were quantified at the epicenter of the graft by means of stereological procedures. Results: Higher BBB and lower CBS levels (p < 0.001) were found in NERVE+GDNF rats. GDNF added to the graft increased the levels of individual sensorimotor tests mainly at the third month. Analysis of the spontaneous motor behavior showed decreases in the time and number of small movement events by the third month without changes in time and number of large movement events in the NERVE+GDNF rats. Immunoreactive fibers were encountered inside the grafts and higher amounts of NF-200, GAP-43 and MAP-2 fibers were found in the epicenter of the graft when GDNF was added. A small amount of descending 5-HT fibers was seen reentering in the adjacent caudal levels of the spinal cords which were grafted in the presence of GDNF, event that has not occurred without the neurotrophic factor. GDNF in the graft also led to a large amount of MAP-2 perikarya and fibers in the caudal levels of the cord gray matter, as determined by the microdensitometric image analysis. Conclusions: GDNF added to the nerve graft favored the motor recovery, local neuronal fiber growth and neuroplasticity in the adjacent spinal cord.

Murine and rat cavernosal responses to endothelin-1 and urotensin-II Vasoactive Peptide Symposium

Endothelin-1 (ET-1) and urotensin-II (U-II) are the most potent constrictors of human vessels. Although the cavernosal tissue is highly responsive to ET-1, no information exists on the effects of U-II on cavernosal function. The aim of this study was to characterize ET-1 and U-II responses in corpora cavernosa from rats and mice. Male Wistar rats and C57/BL6 mice were used at 13 weeks. Cumulative concentration-response curves to ET-1, U-II, and IRL-1620, an ET(B) agonist, were performed. ET-1 increased force generation in cavernosal strips from mice and rats, but no response to U-II was observed in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME), or in strips prestimulated with 20 mM KCI. IRL-1620 did not induce cavernosal contraction even in presence of L-NAME, but induced a cavernosal relaxation that was greater in rats than mice. No relaxation responses to U-II were observed in cavernosal strips precontracted with phenylephrine. mRNA expression of ET-1, ET(A), ET(B), and U-II receptors, but not U-II was observed in cavernosal strips. ET-1, via ET(A) receptors activation, causes contractile responses in cavernosal strips from rats and mice, whereas ET(B) receptor activation produces relaxation. Although the cavernosal tissue expresses U-II receptors, U-II does not induce contractile responses in corpora cavernosa from mice or rats. J Am Soc Hypertens 2008;2(6): 439-447. Published by Elsevier Inc. on behalf of the American Society of Hypertension.

Role of complement C5a in mechanical inflammatory hypernociceptio: potential use of C5a receptor antagonists to control inflammatory pain

particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). Experimental approach: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. Key results: Local pretreatment of rats with PMX53 (60-180 mg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan- activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E-2 and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan- induced joint hypernociception in mice. Conclusions and implications: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

Genetic variability in Australian isolates of Puccinia coronata f. sp avenae assessed with molecular and pathogenicity markers

In a preliminary survey of genetic variability among 12 Australian isolates of Puccinia coronata f. sp. avenae Fraser and Led (Pca) collected from 1966 to 1993, two relatively diverse (

Giardia muris and Giardia duodenalis groups: ultrastructural differences between the trophozoites

Trophozoites of the Giardia muris group from hamsters, domestic rats and mice and of the Giardia duodenalis group from hamsters and domestic rats were examined by transmission electron microscopy. The basic ultrastructure of the trophozoites was similar. Differences were shown in the morphology of the ventrolateral flange of the trophozoites of Giardia muris and Giardia duodenalis groups. Marginal plates are less developed in the species of the Giardia duodenalis group. In this group, the distal extremity of the lateral flange is short and thick and the marginal plate does not penetrate into the distal extremity of the flange. In the Giardia muris group, the ventro-lateral flange is well developed and narrow and the marginal plate penetrates the distal extremity of the flange. The osmiophilic lamella, which accompanies the dorsal surface of the marginal plate is seen only in the Giardia muris group.

ISOLATION AND GENOTYPING OF Toxoplasma gondii IN SERONEGATIVE URBAN RATS AND PRESENCE OF ANTIBODIES IN COMMUNICATING DOGS IN BRAZIL

The role of rodents in the epidemiology of toxoplasmosis was investigated inLondrina, Paraná State, Brazil. One hundred and eighty-one Rattus rattus and one Mus musculus were caught in 37 places. Blood and tissues were collected and submitted to the indirect fluorescence antibody test (IFAT) and the bioassay. Serum samples from 61 contacting dogs were also collected. Sixteen rats (8.8%) were positive for Toxoplasma gondii, but just two of them were positive by serology and bioassay test. Antibodies were found in nine (4.9%) rats. Tissues of nine rats bioassayed were positive and four isolates were obtained. Restriction fragment length polymorphism (RFLP) analysis was performed using 12 markers (SAG1, SAG2, SAG2-alt, C22-8, C29-2, L358, PK1, BTUB, GRA6, SAG3, Apico, CS3). Genotyping revealed that the four strains isolated from this study have been isolated before in cats and chickens from Brazil. None of the isolates was identified like clonal archetypal T-types I, II, and III. The rats presented lower serologic Toxoplasma gondii prevalence (8.8%) compared to contacting dogs (70.5%).

GP38, P28-I and P28-II: candidates for a vaccine against Schistosomiasis

Three antigens protective against Schistosoma mansoni have been extensively characterized. The schistosomulum surface antigen GP38 possesses an immunodominant carbohydrate epitope of which the structure has been defined. Protection can be achieved via the transfer of monoclonal antibodies recognizing the epitope or by immunization with anti-idiotype monoclonal antibodies. The glycan epitope is shared with the intermediate host, Biomphalaria glabrata as well as being present on other molluscs, including the Keyhole Limpet. A group of molecules at 28 kDa were initially characterized in adult worms and shown to protect rats and mice against a challenge infection. One of these molecules, P28-I, was cloned and expressed in E. coli, yeast and vaccinia virus. The recombinant antigen significantly protected rats, hamsters and baboons against a challenge infection. P28-I is a glutathione-S-transferase and the recombinant antigen produced in yeast exhibits the enzyme activity and has been purified to homogeneity by affinity chromatography. A second P28 antigen, P28-II, has also been cloned, fully sequenced and expressed. This recombinant antigen also protects against S. mansoni infection.

The water extract of Coleus barbatus Benth decreases gastric secretion in rats

Coleus barbatus (Labiatae) Benth is popularly used in Brazil "for the healing of liver and stomach diseases". The water extract (WE 1 to 10 g/Kg, p.o.) of stem and leaves given to rats and mice did not induce signs of intoxication. Preveious treatment of mice with WE (1 g/kg, p.o.) shortened the sleeping time induced by pentobarbital (50 mg/Kg, i.p.) by 37 por cento, althoyugh the extract alone did not increase the spontaneous activity nor did it induce hyperexcitability. In mice WE (2 g/Kg, p.o.) increased the intestinal transit of charcoal by 30 por cento, while reduced gastric secretions ion rats treated with WE (2g/Kg intraduodenal) 3,9 ± 1.0 to 0.5 ± 0.2 ml/4h, respectively). The treatment also reduced the total acid secretion from 34.4 ± 11.0 to 2.7 ± 0.5 mEq/l and raisedgastric pH from 2.2 ± 0.3 to 6.5 ± 0.8. Treatment with WE (2g/Kg, p.o.) protected against gastric ulcers induced by stress (5.3 ± 1.6 and 1.5 ± 0.5 ulcers/cm²), but did nor protect against indonethacin induced ulcers. The results show that the water extract of C barbatus Benth produces mild stimulation of thecentral nervous system and increases intestinal movements. The extract also reduces gastric secretion indicating an antidyspeptic activity, and protects against gastric ulcers induced by stress.

Pharmacological screening of Ageratum conyzoides L. (mentrasto)

The pharmacological activities of a water extract (WE) of Ageratum conyzoides L, a plant populary known for its analgesic and anti-inflamatory properties, were studied in vivo and in vitro preparations. Oral administration (p.o.) of the water extract (WE, 0.1 to 5 g/Kg) to rats and mice induced quietness and reduced the spontaneous motility. the sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) in mice was not altered by previous treatment with We (2 g/Kg, p.o.). The same treatment did not influence the paw edema induced by carrageenan or dextran, nor did it reduce the chronic paw edema induced by complete Freund's adjuvant or formaldehyde in rats. The tail flick response in immersion test and writhings induced by 0.8%acetic acid in mice were not altered by WE either. In isolated guinea-pig ilea WE (0.4 to 4 mg/ml) did not alter the EC50 values of histamine or acetylcholine, but reduced the maximal response to the agonists by 20 to 50%. We (0.01 to 10 mg/ml) produced tonic contractions of the ileal smooth muscle proportional to the doses, reaching a maximum of 75% relatively to the maximum obtained with histamine. Those contractions were blocked by diphenhydramine (10 nM) and reduced by 32% in presence of atropine (10 nM). The results indicated that oral treatment of rodents with A. conyzoides L neither reduced the inflammatory edema nor did it decrease the reaction to pain stimuli. In vitro the extract presented an unexpected histamine-like activity characteristic of a partial agonist. The results did not confirm the popular medicinal indications of the plant.

Analgesic activity of a triterpene isolated from Scoparia dulcis L. (vassourinha)

Analgesic and anti-inflammatory activities of water (WE) and ethanolic (EE) extracts of Scoparia dulcis L. were investigated in rats and mice, and compared to the effects induced by Glutinol, a triterpene isolated by purification of EE. Oral adminsitration (p.o.) of either WE or EE (up to 2 g/Kg) did not alter the normal spontaneous activity of mice and rats. The sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) was prolonged by 2 fold in mice pretreated with 0.5 g/Kg EE, p.o. Neither extract altered the tail flick response of mice in immersion test, but previous administration of EE (0.5 g/Kg, p.o.) reduced writhings induced by 0.8% acetic acid (0.1 ml/10 g, i.p.) in mice by 47% EE (0.5 and 1 g/Kg, p.o.) inhibited the paw edema induced by carrageenan in rats by respectively 46% and 58% after 2 h, being ineffective on the paw edema induced by dextran. No significant analgesic or anti-edema effects were detected in animals pretreated with WE (1 g/Kg, p.o.). Administration of Glutinol (30 mg/Kg, p.o.) reduced writhing induced by acetic acid in mice by 40% and the carrageenan induced paw edema in rats by 73%. The results indicate that the analgesic activity of S dulcis L. may be explained by explained by an anti-inflammatory activity probably related to the triterpene Glutinol.