Simone Odília Fernandes Diniz

Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart.

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 × 106) were labeled with 99mTc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the 99mTc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90+, CD73+, CD54+, and CD45-. Five minutes after 99mTc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 ± 0.32 vs. 6.34 ± 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.

99mTechnetium labelled Escherichia coli.

Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. 99mTc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of 99mTc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with 99mTc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation.

Technetium-99m ceftizoxime kit preparation

The aim of this work was to prepare a kit of 99mTc-ceftizoxime (99mTc-CFT), with stability and biological activity preserved, able to identify a septic focus (E. coli) in the experimental infection model in rats. The preparation of the CFT kit involved the use of lyophilized solutions containing the antibiotic ceftizoxime and the sodium dithionite reducing agent (6.0 mg/mL). After lyophilization, the kit was reconstituted with 1.0 mL of sodium 99mTc-pertechnetate solution (Na99mTcO4-) with an activity of 370 MBq. The solution was boiled for 10 min and filtered through a cellulose ester filter. The labeling efficiency was on the order of 92%, remaining stable for six hours and the kit remained stable for two months. The biological activity of the 99mTc-CFT was evaluated by diffusion in agar impregnated with E.coli and S. aureus. Seven Wistar rats, weighing from 200 to 250 g, were used for the development of the septic focus. After 24 hours from the induction of the infectious site (E.coli), the animals were anesthetized and 0.1 mL of 99mTc-CFT (37 MBq) was injected into the tail veins of the animals. The images were obtained with a gamma camera one, two and six hours after injection and the regions of interest (ROIs) were calculated. The diameters of the inhibition halos for 99mTc-CFT were 27.16 ± 0.23 and 27.17 ± 0.20 for S.aureus and E.coli, respectively, while those for the unlabeled CFT were 30.4 ± 0.33 and 29.43 ± 0.26, respectively. The results for the biodistribution of 99mTc-CFT in infected animals furnished a ratio of 1.97 ± 0.31, 2.10 ± 0.42 and 2.01 ± 0.42 for cpm-target/cpm-no target for the one, two and six-hour periods, respectively. The images showed a clear uptake of labeled antibiotic (99mTc-CFT) by the infectious site during the experiment. The results attest to the viability of producing a kit with 99mtechnetium-labeled ceftizoxime for the investigation of infectious processes.

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 × 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 Ca(2+) channel blockers (ω-conotoxin GVIA, 10(-6) M) and sildenafil (3 × 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 Ca(2+) 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 Ca(2+) 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.

Scintigraphic imaging using technetium-99m-labeled ceftizoxime in an experimental model of acute osteomyelitis in rats.

Purpose To investigate inflammatory (zymosan) and infectious (Staphylococcus aureus) processes in experimental models in rats using technetium-99m-labeled ceftizoxime (CFT). Methods Male Wistar rats were used for the development of the inflammatory (zymosan) and infectious (S. aureus) processes in the medullary cavity of the left tibia. Sterile saline was used for the control group. At 48 h after induction of the processes, the animals were anesthetized and scintigraphic images were acquired at 1, 2, 4, and 6 h after intravenous injection of 0.1 ml of 99mTc–CFT (55 MBq). Quantitative analysis of the scintigraphic images was performed by counting the radioactivity in the regions of interest. Samples of tibia were taken for histopathological examination. Results The images showed that 99mTc–CFT presented higher tropism to infectious foci than with the inflammatory site. The average value of the target/nontarget ratio of the 99mTc–CFT was significantly higher in the infected (2.40±0.22) than in the inflamed tibia (1.50±0.05) and the control group (1.05±0.04) for all of the investigated times. The histological data showed a similar inflammatory response for both the S. aureus and zymosan groups. Conclusion The 99mTc–CFT presented a high tropism and retention for an infected region in this model of osteomyelitis, thereby constituting an interesting strategy to distinguish aseptic from septic sites.

Assessment of bacterial translocation in obstructive jaundice using Tc-99m Escherichia coli

ABSTRACT In obstructive jaundice, the passage of bacteria and endotoxins through the intestine wall to reach the systemiccirculation is associated with septicemia, renal failure and pulmonary dysfunction. The aim of this work was toinvestigate bacterial translocation utilizing 99m Tc-E.coli in an experimental model of obstructive jaundice. The levelsof bilirubin in rats that were subjected to ligature of the bile duct were significantly increased when compared tocontrol animals (p 0.05). Theevaluation of the intestinal permeability using “per os” administration of 99m Tc-DTPA showed 1.61 ± 0.57% and1.39 ± 0.72% of radioactivity in the urine of the control and jaundice animals, respectively. The histologicalanalysis of the distal wall of the ileum showed that thecovering epithelium and the crypt were morphologicallypreserved in both groups. The seven-day period after the ligature of the bile duct may not have been long enough topromote modifications in intestinal wall to occur soas to permit the passage of Tc-99m E.coli.Key words: Bacterial translocation, Technetium-99m E coli, obstructive jaundice

Biodistribution of free 99mTc-ovalbumin and 99mTc-ovalbumin encapsulated in liposomes

The oral administration of proteic antigens, like ovalbumin, may result in the induction of oral tolerance or immunization. The aim of this work was to label a protein antigen with 99mTechnetium, encapsulate it in liposomes and investigate its absorption and tissue distribution after oral administration in mice. Ovalbumin was labeled with 99mTechnetium and encapsulated in small unilamellar vesicles. 99mTc-OVA encapsulated or not in liposomes was administrated to mice that were sacrificed after different times. The radioactivity was measured in various organs of the animals. Differences concerning the biodistribution of 99mTc-OVA were noticed. The technique may represent alternatives for the induction of immunization or oral tolerance.

Effects of the povidone-iodine (PVPI) in treatment of bacterial peritonitis induced in rats

PURPOSE To evaluate the effectiveness of the use of the povidone-iodine (PVI) added to the liquid of wash of the peritoneal cavity in the reduction of bacterial absorption and in the remainder non-phagocyted bacteria in the circulating blood of rat. METHODS Thirty four Wistar females rats were used, distributed in the following groups: A (n=10), non-treated; B (n=9), wash of the peritoneal cavity with solution of PVI to 1% in saline solution; C (n=15), wash of the cavity with saline solution. After anesthesia, it was made intraperitoneal infusion of solution of Escherichia coli labeled with 99mTc containing 10(8) CFU/ml. After 40 minutes, it was made the treatment, in the group A, manipulation of the viscera; in the group B, irrigation of the peritoneal cavity with warm solution of 1% PVPI to 37,5 degrees C, and in the group C irrigation with warm saline (37,5 degrees C). After 15 minutes of the treatment, blood samples and fragments of liver, spleen and lung was obtained for count of the radioactivity, and animals killed by abdominal aorta section. There were determined the bacterial absorption index and the remainder index in the bloodstream. RESULTS Of the total of bacteria infused in the peritoneum, there was absorption of 0,92% (0,14% to 2,13%) in the animals of the group A (controls), 0,49% (0,18% to 0,71%) after use of topical PVPI (group B) and 0,80% (0,04% to 3,8%) after wash with saline solution (group C). There was significant reduction of the absorption when compared the treated animals with PVPI and the controls (p=0,003). Of the total of bacteria absorbed for the circulatory current, the percentile amount of bacteria non-phagocyted in the outlying blood was of 2,9% (1,1% to 17,7%) in the control group, 15,2% (8,3% to 21,4%) in those treated with PVPI (group B) and 6,9% (0,8% to 29,7%) after wash with saline solution (group C), with difference among controls and treated with PVPI (p=0,01). CONCLUSION The wash of the cavity peritoneal of mice with solution containing PVPI showed to be capable to reduce the absorption of bacteria by peritoneum of rat; however it seems to interfere with the function of the phagocytic cells for the observation of the increase of viable bacteria in the outlying blood of those animals.

Scintigraphic imaging with technetium-99M-labelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats

PURPOSE To evaluate whether scintigraphy with technetium-99m-labeled ceftizoxime ((99m)Tc-CFT) can differentiate mediastinitis from aseptic inflammation associated with sternotomy. METHODS Twenty female Wistar rats were randomly distributed into four groups: S (control) -partial upper median sternotomy with no treatment; SW (control) - sternotomy and treatment of sternal wounds with bone wax; SB - sternotomy and infection with Staphylococcus aureus; SWB - sternotomy with bone wax treatment and bacterial infection. Scintigraphy with (99m)Tc-CFT was performed eight days after surgery and images were collected 210 and 360 min after infusion of the radiopharmaceutical. RESULTS No animals exhibited clinical signs of wound infection at the end of the experiment, although histological data verified acute inflammatory response in those experimentally infected with bacteria. Scintigraphic images revealed that tropism of (99m)Tc-CFT to infected sternums was greater than to their non-infected counterparts. Mean counts of radioactivity in bacteria-infected sternal regions (SB and SWB) were significantly higher (p = 0.0007) than those of the respective controls (S and SW). CONCLUSION Scintigraphy with technetium-99m-labeled ceftizoxime is a method that can potentially detect infection post sternotomy and differentiate from aseptic inflammation in animals experimentally inoculated with S. aureus.

Influência das operações sobre o baço na distribuição da escherichia coli no sistema mononuclear fagocitário

OBJETIVOS: Para se evitar o estado asplenico, muitas medidas preservadoras do baco tem sido propostas na literatura, como a esplenorrafia, a esplenectomia parcial com preservacao dos vasos hilares e o auto-implante de tecido esplenico. A esplenectomia subtotal, com conservacao do polo superior do baco, nutrido apenas pelos vasos esplenogastricos e uma alternativa quando o pediculo esplenico precisa ser ligado. O objetivo deste estudo foi avaliar a influencia das esplenectomias parcial, subtotal e total na distribuicao da Escherichia coli no sistema mononuclear fagocitario. METODO: Foram estudados 32 ratos divididos em 4 grupos: operacao simulada (mantendo todo o baco), esplenectomia parcial, esplenectomia subtotal e esplenectomia total. Apos cinco semanas da operacao, uma aliquota de Escherichia coli marcada com 99m-tecnecio foi injetada por via venosa. Apos 20 minutos, os animais foram mortos, e o baco, os pulmoes e o figado foram retirados para se verificar a distribuicao das bacterias marcadas. RESULTADOS: A quantidade de Escherichia coli no tecido esplenico foi maior no grupo com o baco integro em comparacao com os grupos esplenectomia parcial e subtotal. A distribuicao da bacteria marcada pelo baco nao diferiu nos grupos com esplenectomia parcial ou subtotal. A quantidade de bacterias no pulmao foi maior no grupo esplenectomia parcial do que a do grupo com esplenectomia subtotal. Apos esplenectomia subtotal, a distribuicao da bacteria marcada foi maior no figado em comparacao com a captacao desse orgao nos demais grupos. CONCLUSOES: O polo superior do baco, suprido apenas pelos vasos esplenogastricos, tem capacidade de remover da circulacao bacterias vivas, mostrando que, mesmo sem a vascularizacao pelo pediculo esplenico, ha uma eficiente depuracao sanguinea. A distribuicao da Escherichia coli pelo sistema mononuclear fagocitario apresenta comportamentos diferentes, dependendo do tipo de esplenectomia a que o animal e submetido.