Spleen melanomacrophage centers response of Nile tilapia during Aeromanas hydrophila and Mycobacterium marinum infections.

In order to understand the pathophysiology of melanomcrophage centers (MMCs) formation during the tilapia defense response to bacterial infections, the present study evaluated the response, in terms of area, number and pigment constitution, of splenic MMCs of Oreochromis niloticus subjected to intraperitoneal (i.p.) infection with Aeromonas hydrophila and Mycobacterium marinum. Eighty-four fish (396.9 ±â€¯21.0 g) were randomly distributed into twelve plastic tanks (300 L), to constitute three treatments with 28 animals each: control group (inoculated with PBS); Infected with A. hydrophila (1 × 107 UFC mL-1); Infected with M. marinum (1 × 106 UFC mL-1). The spleen was collected in seven fish per treatment on the 3rd, 7th, 14th and 21st day post-infection (DPI). The results revealed the participation of MMCs in the defense response of tilapia during bacterial infection by A. hydrophila and M. marinum, since there was an increase in the number and size of these cell aggregates. Variation of pigment accumulation with significant increase of hemosiderin, in infected tilapias by A. hydrophila, bacteria responsible for causing hemolytic anemia in fish was also found. On the other hand, M. marinum-infected tilapia had high amount of melanin in MMCs. In general, mycobacterial infections are notoriously difficult to treat, being characterized as a chronic disease. These findings demonstrate different strategies of fish response during the evolution of these bacterial diseases.

CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates / Técnicas sorológicas e moleculares aplicadas na identificação de Plasmodium spp. em amostras de primatas não humanos

Abstract The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

Resumo Neste estudo objetivamos identificar Plasmodium spp. em amostras sangue de primatas não humanos (PNH) do estado do Maranhão, utilizando técnicas clássicas e alternativas para o exame da malária humana. Foram analisadas 161 amostras de sangue de PNH, sendo 141 de CETAS (cativeiro) e 20 de reserva particular (vida livre), utilizando microscopia, teste de diagnóstico rápido (RDT), imunofluorescência indireta (IFI) e técnicas moleculares (semi-nested PCR, PCR em tempo real quantitativo e LAMP). Dois métodos sorológicos (dot-ELISA e ELISA indireto) também foram padronizados com antígenos solúveis de roptrias de P. falciparum e P. berghei. Formas trofozoíticas de Plasmodium sp. foram identificadas em lâminas de cinco animais diferentes. Nenhuma amostra foi positiva em TDR e LAMP. Quatro amostras foram soropositivas para P. malariae na IFI. Os soros de PNH mostraram baixa reatividade pelo ELISA indireto. Plasmodium sp. foi detectado em 34,16% (55/161) das amostras utilizando a qPCR baseada no gene 18S rRNA. No sequenciamento, duas amostras mostraram identidade com P. malariae (100%), uma com Plasmodium sp. ZOOBH (97%) e uma com P. falciparum (99%). A PCR mostrou ser a técnica mais sensível para diagnósticos de Plasmodium em amostras de PNH.

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates.

The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

Identification of Plasmodium spp. in Neotropical primates of Maranhense Amazon in Northeast Brazil.

In the Brazilian Amazon region, malaria caused by Plasmodium malariae is considered to be a zoonosis because of cross-transfer of the parasite between humans and Neotropical primates. To contribute information on this issue, we investigated occurrences of natural infection with Plasmodium sp. among Neotropical primates in the Maranhense Amazon (Amazon region of the state of Maranhão), in the northeastern region of Brazil. Blood samples were collected from 161 Neotropical primates of six species that were caught in an environmental reserve (Sítio Aguahy) and from captive primates (CETAS-Wildlife Screening Center, municipality of São Luís), in Maranhão. Plasmodium sp. was diagnosed based on light microscopy, PCR, qPCR and LAMP for amplification of the 18S rRNA gene. Serum samples were also assayed by means of indirect immunofluorescence for IgG antibodies against P. malariae/P. brasilianum, P. falciparum and P. berghei. Parasites were detected through light microscopy on five slides from captive primates (four Sapajus spp. and one Callithrix jacchus). In the molecular tests, 34.16% (55/161) and 29.81% (48/161) of the animals sampled were positive in the qPCR and PCR assays, respectively. In the PCR, 47/48 animals were positive for P. malariae/P. brasilianum; of these, eight were free-living primates and 39 from CETAS, São Luís. One sample showed a band in the genus-specific reaction, but not in the second PCR reaction. Anti-P. malariae/P. brasilianum IgG antibodies were detected in four serum samples from Sapajus spp. in captivity. In this study, circulation of P. malariae/P. brasilianum in Neotropical primates was confirmed, with low levels of parasitemia and low levels of antibodies. The importance of these animals as reservoirs of human malaria in the region studied is still unknown. This scenario has an impact on control and elimination of malaria in this region.

Myxobolus sp. and Henneguya sp. (Cnidaria: Myxobolidae) natural co-infection in the kidney of Piaractus mesopotamicus (Characiformes: Serrasalmidae).

This study evaluated the myxozoan infection and histopathology of the kidney of freshwater fish Piaractus mesopotamicus from intensive fish farming in Brazil. A total of 55 fish were examined for myxozoan infection. Infected organs were processed by usual histology and stained with hematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). From the total of 55 fish analyzed, 47 (85.45%) presented myxospores, being 9.09% (5/55) only with Myxobolus sp., 5.45% (3/55) only with Henneguya sp., and 70.91% (39/55) presenting both parasites. The presence of myxospores was associated with histological alterations in both stromal and renal parenchyma. Myxospores were found mostly in the peritubular interstitial tissue and in low intensity in the glomerulus which caused nuclear hypertrophy and loss of Bowman space. An increase in the glomerular tuft and a reduction in the lumen of the collector tubules were also observed, besides the high number of melanomacrophage cells in the glomerulus. This study reports for the first time detection of myxozoan mixed infection in one organ of pacu and discuss the possible transportation of myxospores in the circulating blood.

Metazoan parasites of Plagioscion squamosissimus, an invasive species in the Tietê River, São Paulo, Brazil.

This study focused on the characterization and analysis of communities and infra-communities of metazoan parasites of Plagioscion squamosissimus caught in Promissão Reservoir in the Tietê River in Borborema (21°39'58"S, 49°8'49"W), state of São Paulo, Brazil. Fifty adult specimens caught by professional fishermen in March 2015 were necropsied. The fish presented an average standard length of 25.2 ± 2.2 cm and average weight of 328.82 ± 89.03 g. A total of 5,227 specimens of metazoan parasites were collected: 2,880 (55.1%) adult Diplectanum piscinarius (Monogenoidea: Diplectanidae) and 2,347 (44.9%) Austrodiplostomum compactum metacercariae (Digenea, Diplostomidae), both with 100% prevalence and mean abundance of 57.6 and 46.9, respectively. Parasite diversity was low (species richness = 2), with a Simpson index (D) equal to 0.505, and low values of Brillouin (HB = 0.687) and Margalef diversity (I = 0.117) indices. Berger-Parker's index of dominance (p = 0.551) indicated a slight dominance of the monogenean parasite D. piscinarius. There was a positive correlation, assessed by Pearson coefficient between parasite abundance of D. piscinarius and standard length (r = 0.43) and weight (r = 0.51) of hosts.

Influence of dexamethasone and levamisole on macrophage recruitment, giant cell formation and blood parameters in the tropical fish Piaractus mesopotamicus / Influência da dexametasona e levamisol no recrutamento de macrófagos, formação de células gigantes e parâmetros hematológicos no peixe tropical Piaractus mesopotamicus

The aim of this study was to evaluate the effect of parenteral administration of dexamethasone and levamisole on the accumulation of macrophages and formation of giant cells in chronic inflammation by foreign body and blood parameters in pacu (Piaractus mesopotamicus). It was used 50 mg/kg of levamisole and 2.0 mg/kg of dexamethasone and the combination of both drugs. Coverslips were implanted under the skin. After 2, 7, and 15 days postimplantation (DPI), fish were anesthetized for removal of coverslips and the number of macrophages and giant cells were count. It was also collected blood from the caudal vessel to evaluate: red blood cell count, hematocrit, hemoglobin concentration, leukocytes count, thrombocytes count, MCV, and MCHC. We observed that dexamethasone affects negatively the formation of giant cells in the chronic inflammation for foreign body. Levamisole, despite being immunostimulatory in several species, showed limited action. However, it was enough to counteract the effect of dexamethasone; the association of the drugs did not interfere significantly in erythrocyte and leukocyte number in most of the treatments and times studied. In dexamethasone group there was a reduction in the number of erythrocytes and hemoglobin associated with increased mean corpuscular volume, suggesting slight macrocytic anemia. At 15 DPI, most groups showed the recovery of hematologic response. As in mammals, dexamethasone affects negatively the inflammatory response. Levamisole showed little effect by itself. However, in some parameters the association of both drugs causes similar response to control and naïve groups, showing the antagonistic effect of these drugs.

O objetivo deste estudo foi avaliar o efeito da administração parenteral de dexametasona e levamisol na acumulação de macrófagos e formação de células gigantes na inflamação crônica por corpo estranho e avaliação de parâmetros sanguíneos em pacu (Piaractus mesopotamicus). Utilizou-se 50 mg/kg de levamisol e 2,0 mg / kg de dexametasona e a combinação de ambos fármacos. As lamínulas foram implantadas sob a pele. Depois de 2, 7 e 15 dias pós-implantação (DPI), os peixes foram anestesiados para a remoção das lamínulas e contagem do número de macrófagos e células gigantes. Foi coletado sangue da veia caudal para realizar contagem de células vermelhas, leucócitos, trombócitos, concentração de hemoglobina, MCV e MCHC. Observou-se que a dexametasona afeta negativamente a formação de células gigantes na inflamação crónica por corpo estranho. O levamisol, apesar de ser considerado imunoestimulante em várias espécies, mostrou ação limitada. No entanto, foi suficiente para neutralizar o efeito da dexametasona; a associação das drogas não interferiu significativamente no número de eritrócitos e de leucócitos na maioria dos tratamentos e períodos estudados. No grupo da dexametasona, houve redução do número de eritrócitos e concentração de hemoglobina associado ao aumento do volume corpuscular médio sugerindo leve anemia macrocítica. Aos 15 DPI, a maioria dos grupos mostrou recuperação na resposta hematológica. Como em mamíferos, a dexametasona afeta negativamente a resposta inflamatória. O levamisol mostrou pouco efeito por si só. No entanto, em alguns parâmetros a associação das duas drogas provoca resposta similar ao grupo controle e naive, mostrando o efeito antagonista de estas drogas.

Metazoan parasites of Plagioscion squamosissimus, an invasive species in the Tietê River, São Paulo, Brazil / Parasitas metazoários de Plagioscion squamosissimus, uma espécie invasora no Rio Tietê, São Paulo

Abstract This study focused on the characterization and analysis of communities and infra-communities of metazoan parasites of Plagioscion squamosissimus caught in Promissão Reservoir in the Tietê River in Borborema (21°39′58"S, 49°8′49"W), state of São Paulo, Brazil. Fifty adult specimens caught by professional fishermen in March 2015 were necropsied. The fish presented an average standard length of 25.2 ± 2.2 cm and average weight of 328.82 ± 89.03 g. A total of 5,227 specimens of metazoan parasites were collected: 2,880 (55.1%) adult Diplectanum piscinarius (Monogenoidea: Diplectanidae) and 2,347 (44.9%) Austrodiplostomum compactum metacercariae (Digenea, Diplostomidae), both with 100% prevalence and mean abundance of 57.6 and 46.9, respectively. Parasite diversity was low (species richness = 2), with a Simpson index (D) equal to 0.505, and low values of Brillouin (HB = 0.687) and Margalef diversity (I = 0.117) indices. Berger-Parker's index of dominance (p = 0.551) indicated a slight dominance of the monogenean parasite D. piscinarius. There was a positive correlation, assessed by Pearson coefficient between parasite abundance of D. piscinarius and standard length (r = 0.43) and weight (r = 0.51) of hosts.

Resumo O objetivo deste trabalho foi caracterizar e analisar as comunidades e infracomunidades de metazoários parasitos de corvinas capturadas no Reservatório de Promissão, Rio Tietê, município de Borborema (21° 67′S, 49° 14′O), Estado de São Paulo. Foram examinados 50 espécimes, capturados por pescadores profissionais no mês de março de 2015, e os parasitas coletados foram quantificados, preparados e montados para identificação taxonômica e análise das comunidades de parasitos. Os peixes analisados no estudo apresentaram comprimento padrão médio de 25,2 ± 2,2 cm e peso médio de 328,82 ± 89,03 g. Foram coletados 5227 espécimes de parasitas metazoários, sendo 2880 (55,1%) Diplectanum piscinarius (Monogenoidea: Diplectanidae) e 2347 (44,9%) metacercárias de Austrodiplostomum compactum (Digenea, Diplostomidae), ambos com prevalência de 100% e abundância parasitária de 57,6 e 46,9, respectivamente. Foi encontrada baixa diversidade parasitária (riqueza de espécies=2), com índice de Simpson (D) igual a 0,505 e baixos valores dos índices de Shannon (H'=0,688) e de diversidade de Margalef (I=0,177). O índice de dominância de Berger-Parker (d=0,551) indicou uma leve dominância do monogenético D. piscinarius. Houve correlação positiva intermediária, avaliada pelo coeficiente de Pearson, entre a abundância parasitária de D. piscinarius e comprimento padrão (r=0,43) e peso (r=0,51) dos hospedeiros.

Molecular identification of Plasmodium spp. and blood meal sources of anophelines in environmental reserves on São Luís Island, state of Maranhão, Brazil.

BACKGROUND: Considering the diversity of feeding habits that females of some species of anophelines present, it is important to understand which vertebrates are part of blood food sources and how important is the role of each in the ecoepidemiology of malaria. There are many vector species for Plasmodium spp. in the State of Maranhão, Brazil. In São Luís Island, Anopheles aquasalis is the main vector for human malaria; this species is abundant in areas with primates that are positive for Plasmodium. Anopheles aquasalis has natural exophilic and zoophilic feeding behavior, but in cases of high density and absence of animals, presents quite varied behavior, and feeds on human blood. In this context, the objective of the present study was to identify Plasmodium spp. and the blood meal sources of anophelines in two environmental reserves on São Luís Island, state of Maranhão, using molecular methods. METHODS: Between June and July 2013, female anophelines were collected in the Sítio Aguahy Private Reserve, in the municipality of São José de Ribamar, and in the Sítio Mangalho Reserve, located within the Maracanã Environmental Protection Area, in the municipality of São Luís. CDC-type light traps, Shannon traps and protected human bait were used during three consecutive hours in peridomestic and wooded areas. Pools of anophelines were formed using mosquitoes of the same species that had been caught at the same site on the same date. A genus-specific amplification protocol based on the 18S rRNA gene was used for qPCR and cPCR. RESULTS: A total of 416 anophelines were collected, of the following species: An. aquasalis (399), An. mediopunctatus (3), An. shannoni (1), An. nuneztovari (sensu lato) (1), An. goeldii (1), An. evansae (2) and An. (Nyssorhynchus) sp. (9), comprising 54 pools. Two pools were positive for Plasmodium (2/54) based on the 18S rRNA gene. In the phylogenetic analysis using the maximum likelihood method, based on a 240 bp fragment of the 18S rRNA gene, it was found that the sequences of Plasmodium sp. amplified from pools of An. aquasalis (pool 2) and An. nuneztovari (s.l.) (pool 10) were phylogenetically related to a clade of P. falciparum isolates from India, and to a clade of Plasmodium sp. isolates from psittacines in Brazil, respectively. Cat, dog and human DNA were identified in the blood meals of the anophelines sampled. CONCLUSION: The species An. aquasalis was the most abundant anopheline species in São Luís Island. Plasmodium spp. DNA was detected, thus confirming the importance of this species as the main vector on São Luís Island, Brazil. In addition, the presence of An. nuneztovari (s.l.) with DNA positive for Plasmodium spp. confirms its importance as a secondary vector.

Ultrastructural description of Myxobolus cuneus (Myxosporea) in the skeletal muscle and kidney of tropical farmed fish Piaractus mesopotamicus (Characiformes: Characidae).

This study characterizes by transmission electron microscopy (TEM) and morphometric features the myxozoan Myxobolus cuneus (Myxosporea) in Piaractus mesopotamicus and reports the skeletal muscle and kidney as site of infection. The register was based in 21 young fish from intensive fish farming in Southeast Brazil and the spores were analyzed in fresh-mounted slides of the infected organs stained with Toluidine blue and processed as usual for TEM. It differs from Myxobolus cunhai from the fish host and different polar capsule size, and from Myxobolus serrasalmi on the pyriform spore shape and an oval macrospore, differently to that reported in this study. Morphometric characteristics and TEM study confirmed the present material as M. cuneus.

Uncaria tomentosa increases growth and immune activity in Oreochromis niloticus challenged with Streptococcus agalactiae.

Cat's claw (Uncaria tomentosa) is an Amazon herb using in native cultures in Peru. In mammals, it has been described several effects of this herb. However, this is the first report of its use on the diet of fish. The aim of this study was to determinate the effect of this plant on the growth and immune activity in Oreochromis niloticus. Nile tilapia (81.3 ± 4.5 g) were distributed into 5 groups and supplemented with 0 (non-supplement fish), 75, 150, 300, and 450 mg of U. tomentosa.kg(-1) of diet for a period of 28 days. Fish were inoculated in the swim bladder with inactivated Streptococcus agalactiae and samples were taken at 6, 24, and 48 h post inoculation (HPI). Dose dependent increases were noted in some of the evaluated times of thrombocytes and white blood cells counts (WBC) in blood and exudate, burst respiratory activity, lysozyme activity, melanomacrophage centers count (MMCs), villi length, IgM by immunohistochemistry in splenic tissue, and unexpectedly on growth parameters. However, dietary supplementation of this herb did not affect red blood cells count (RBC), hemoglobin, and there were no observed histological lesions in gills, intestine, spleen, and liver. The current results demonstrate for the first time that U. tomentosa can stimulate fish immunity and improve growth performance in Nile tilapia.

Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9.

PURPOSES: To assess the effects of 0.5% ketorolac tromethamine without preservatives on the expression of iNOS and MMP-9 in alkali burn ulcers. METHODS: Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were collected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry. RESULTS: Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS. CONCLUSIONS: Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9.

First report of Myxobolus sp. infection in the skeletal muscle of Neotropical freshwater fish Piaractus mesopotamicus.

Although there are several reports of Myxobolus species infecting the somatic muscle of teleost fish, species of this genus have not been described parasitizing the muscle tissue of pacus, Piaractus mesopotamicus. This study presented the first report of natural infection by Myxobolus sp. in the skeletal muscle of pacus reared in intensive system. Twenty-one fish (±142.2 g; ±23.1 cm) were captured (April 2013) from an intensive fish farm in São Paulo State, southeast Brazil. Spores of Myxobolus sp. were contained within plasmodia and showed oval morphology with the apical portion slightly pyriform and two polar capsules pyriform retaining the same ratio to each other. In the histopathological study, the skeletal muscle do not present signs of inflammation. The integrity of myofibrils within the infected fibers showed some degree of degeneration, with partial loss of myofibrillar details and striations. Spores were found infecting the skeletal muscle of 18 fish (85.7%). Finally, the high prevalence of Myxobolus sp. infection in the skeletal muscle of P. mesopotamicus and the absence of macroscopic lesions in the muscle tissue indicate the necessity of more meticulous examinations during the health management in the rearing system and at slaughter of pacus to ensure best quality of meat.

Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9 / Colírio de cetorolaco reduz inflamação e atrasa a epitelização em resposta a queimadura por álcali em coelhos, sem afetar iNOS ou MMP-9

Purposes: To assess the effects of 0.5% ketorolac tromethamine without preservatives on the expression of iNOS and MMP-9 in alkali burn ulcers. Methods: Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were collected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry. Results: Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS. Conclusions: Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9. .

Objetivos: Avaliarem-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a expressão da iNOS e da MMP-9, em córneas com úlceras químicas. Métodos: Doze olhos de coelhos machos, 120 dias de idade, foram tratados (GT ), a cada 6 horas, com o cetorolaco de trometamina 0,5% e outros 12 com solução salina (GC), imediatamente à ocorrência de úlceras por hidróxido de sódio (NaOH) 1 mol/L. A reepitelização foi monitorada por fluresceína a cada seis horas. Decorridas 24 horas, seis córneas (n=6) de cada grupo foram colhidas (primeiro momento). As demais (n=6) o foram após a sua reepitelização (segundo momento). Em ambos os momentos, avaliaram-se o infiltrado inflamatório e as condições do epitélio neoformado (HE). Por imuno-histoquímica, avaliou-se a imunomarcação de iNOS e de MMP-9. Resultados: A média do tempo de epitelização no GT foi de 55 ± 0,84 horas. No GC, ela foi de 44 ± 1,06 horas (p=0,001). Às 24 horas, as córneas do GT apresentaram menor exsudação inflamatória (p<0,01). No segundo momento, o GT mostrou discreta exsudação inflamatória (p>0,05) e menor número de camadas epiteliais comparativamente ao GC. A média de imunomarcação de iNOS em células do estroma não diferiu do GT, em ambos os momentos (p>0,05). No segundo momento, a região central da córnea expressou mais iNOS, comparativamente à periférica, em ambos os grupos. Não se observaram diferenças significativas nos escores de imunomarcação epitelial de iNOS entre os grupos e os momentos (p=0,69). Os escores de imunomarcação epitelial para MMP-9 não diferiram entre os grupos (p=0,69). A média de imunomarcação da MMP-9 em células do estroma não exibiram diferenças entre os grupos e momentos da avaliação (p=0,32). Não houve correlação entre a imunomarcação de iNOS e de MMP-9, assim como quanto ao quantitativo de células inflamatórias e à imunomarcação de iNOS. Conclusões: Cetorolaco 0,5% reduziu a inflamação e atrasou a epitelização na queimadura corneal ...

Expression of cellular components in granulomatous inflammatory response in Piaractus mesopotamicus model.

The present study aimed to describe and characterize the cellular components during the evolution of chronic granulomatous inflammation in the teleost fish pacus (P. mesopotamicus) induced by Bacillus Calmette-Guerin (BCG), using S-100, iNOS and cytokeratin antibodies. 50 fish (120±5.0 g) were anesthetized and 45 inoculated with 20 µL (40 mg/mL) (2.0 x 10(6) CFU/mg) and five inoculated with saline (0,65%) into muscle tissue in the laterodorsal region. To evaluate the inflammatory process, nine fish inoculated with BCG and one control were sampled in five periods: 3rd, 7th, 14th, 21st and 33rd days post-inoculation (DPI). Immunohistochemical examination showed that the marking with anti-S-100 protein and anti-iNOS antibodies was weak, with a diffuse pattern, between the third and seventh DPI. From the 14th to the 33rd day, the marking became stronger and marked the cytoplasm of the macrophages. Positivity for cytokeratin was initially observed in the 14th DPI, and the stronger immunostaining in the 33rd day, period in which the epithelioid cells were more evident and the granuloma was fully formed. Also after the 14th day, a certain degree of cellular organization was observed, due to the arrangement of the macrophages around the inoculated material, with little evidence of edema. The arrangement of the macrophages around the inoculum, the fibroblasts, the lymphocytes and, in most cases, the presence of melanomacrophages formed the granuloma and kept the inoculum isolated in the 33rd DPI. The present study suggested that the granulomatous experimental model using teleost fish P. mesopotamicus presented a similar response to those observed in mammals, confirming its importance for studies of chronic inflammatory reaction.