Effects of Lidocaine Infusion during Experimental Endotoxemia in Horses

Background The clinical efficacy of IV infusion of lidocaine for treatment of equine endotoxemia has not been studied. Hypothesis Lidocaine infusion after exposure to lipopolysaccharide (LPS) will inhibit the inflammatory response and have inhibitory effects on the hemodynamic and cytokine responses to endotoxemia. Animals Twelve horses. Methods Two equal groups (n = 6): saline (GI) and lidocaine (GII). In all animals, endotoxin (500 ng/kg body weight [BW]) was injected intraperitoneally over 5 minutes. Twenty minutes later, animals received a bolus of GI or GII (1.3 mg/kg BW) over 5 minutes, followed by a 6-hour continuous rate infusion of GI or GII (0.05 mg/kg BW/min). Treatment efficacy was judged from change in arterial blood pressure, peripheral blood and peritoneal fluid (PF) variables (total and differential cell counts, enzyme activities, and cytokine concentrations), and clinical scores (CS) for behavioral evidence of abdominal pain or discomfort during the study. Results Compared with the control group, horses treated with lidocaine had significantly lower CS and serum and PF tumor necrosis factor-alpha (TNF-alpha) activity. At several time points in both groups, total and differential cell counts, glucose, total protein and fibrinogen concentrations, and alkaline phosphatase, creatine kinase, and TNF-alpha activities were significantly different from baseline values both in peripheral blood and in PF. Conclusions and Clinical Importance Lidocaine significantly decreased severity of CS and inhibited TNF-alpha activity in PF.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.

Histological analysis of cobalt-chromium stents with and without Camouflage (R) polymer coating: experimental porcine carotid artery model

This study evaluated the arterial response to cobalt-chromium stents with and without polymer coating (Camouflage (R), Hemoteq AG, Wuerselen, Germany) implanted in pigs. Cobalt-chromium balloon-expandable stents (4 x 16 mm) were implanted in the common carotid arteries of nine pigs. Histological analysis of endothelialization, inflammation and injury was performed one month later. All stents were successfully deployed, and all but one animal survived the 30 study days. All arteries were patent. Endothelialization was nearly complete in most sections of all carotid stents in both groups. There were mild inflammatory infiltrate and mild-to-moderate injury, which were associated with the stent shafts and not significantly different between groups. Our findings suggest that, in porcine carotid arteries, the histological response to balloon-expandable cobalt-chromium stents coated with polymer (Camouflage (R), Hemoteq AG) is similar to the response to non-coated cobalt-chromium stents.

Hemodynamic effects of PEEP in a porcine model of HCl-induced mild acute lung injury

Positive end-expiratory pressure (PEEP) and sustained inspiratory insufflations (SI) during acute lung injury (ALI) are suggested to improve oxygenation and respiratory mechanics. We aimed to investigate the hemodynamic effects of PEEP with and without alveolar recruiting maneuver in a mild ALI model induced by inhalation of hydrochloric acid. Thirty-two pigs were randomly allocated into four groups (Control-PEEP, Control-SI, ALI-PEEP and ALI-SI). ALI was induced by intratracheal instillation of hydrochloric acid. PEEP values were progressively increased and decreased from 5, 10, 15 and 20 cmH(2)O in all groups. Three SIs maneuvers of 30 cmH(2)O for 20 s were applied to the assignable groups between each PEEP level. Transesophageal echocardiography (TEE), global hemodynamics, oxygenation indexes and gastric tonometry were measured 5 min after the maneuvers had been concluded and at each established value of PEEP (5, 10, 15 and 20 cmH(2)O). The cardiac index, ejection fraction and end-diastolic volume of right ventricle were significantly (P < 0.001) decreased with PEEP in both Control and ALI groups. Left ventricle echocardiography showed a significant decrease in end-diastolic volume at 20 cmH(2)O of PEEP (P < 0.001). SIs did not exert any significant hemodynamic effects either early (after 5 min) or late (after 3 h). In a mild ALI model induced by inhalation of hydrochloric acid, significant hemodynamic impairment characterized by cardiac function deterioration occurred during PEEP increment, but SI, probably due to low applied values (30 cmH(2)O), did not exert further negative hemodynamic effects. PEEP should be used cautiously in ALI caused by acid gastric content inhalation.

Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2)

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil. (C) 2007 Elsevier Ltd. All rights reserved.

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro-fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 mu g/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX-blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Efeito da jabuticaba (Myrciaria caulifora), do fruto da palmeira juçara (Euterpe edulis Martius) e do jambolão (Syzugium cumini) sobre o perfil lipídico, a glicemia e a endotoxemia em camundongos submetidos à dieta de cafeteria

O excesso de gordura corporal induz a um quadro inflamatório associado à endotoxemia metabólica e aumento da resistência à insulina, bem como altera o perfil lipídico que resulta em prejuízos a função hepática e renal. Estudos sugerem que a ingestão de alimentos antioxidantes, como os polifenóis, proporcionam efeitos benéficos sobre os metabolismos glicídico e lipídico. O objetivo deste estudo foi investigar o efeito da casca de jabuticaba (Myrciaria cauliflora), da polpa do açaí juçara (Euterpe edulis Martius) e do jambolão (Syzygium cumini) sobre o perfil lipídico, a glicemia e a endotoxemia em camundongos Swiss submetidos à dieta de cafeteria. Inicialmente, os frutos foram liofilizados e submetidos à avaliação da composição centesimal. O ensaio biológico contou com 50 camundongos machos adultos da raça Swiss distribuídos em 5 grupos (n=10/grupo), a saber: grupo tratado com dieta comercial padrão (controle negativo), grupo tratado com dieta de cafeteria (controle positivo) e grupos teste que receberam por 14 semanas a dieta de cafeteria suplementada com 2% de casca de jabuticaba, ou polpa do jambolão ou polpa do açaí juçara liofilizados. Na 13ª e 14ª semana foram determinadas a tolerância à insulina e à glicose dos animais. Ao final do período experimental, avaliaram-se o ganho de peso, os parâmetros bioquímicos sanguíneos, histopatológicos e endotoxemia. Os parâmetros bioquímicos avaliados foram: colesterol total (CT) e as frações HDL-c, LDL-c, triacilgliceróis (TAG), bem como proteína C reativa (PCR), aspartato aminotransferase (AST) e alanina aminotransferase (ALT). Na histopatologia foram avaliados os efeitos da dieta hipercalórica sobre a área dos adipócitos, esteatose hepática e função renal a partir do número e área dos glomérulos. A endotoxemia foi avaliada pela concentração de lipopolissacarídeos (LPS) no soro dos animais. Aplicou-se o teste t para comparação dos resultados entre os grupos controle e ANOVA, complementada com teste de Tukey (α=5%), para comparação dos grupos suplementados com os frutos e o controle positivo. A suplementação com 2% de jambolão à dieta de cafeteria resultou em redução significativa (p<0,05) do conteúdo de CT, LDL-c, TAG, da razão CT/HDL, bem como diminuição da área dos adipócitos dos animais tratados com os frutos. A suplementação com açaí juçara também foi capaz de reduzir o conteúdo de CT, TAG e a área dos adipócitos, além de elevar a tolerância à glicose. Por outro lado, a jabuticaba não foi eficaz na melhoria dos parâmetros relacionados ao metabolismo lipídico, ao metabolismo da glicose e dos aspectos histopatológicos. A suplementação com 2% dos frutos liofilizados não promoveu efeitos positivos na redução do ganho de peso, resistência à insulina e endotoxemia provocada pela ingestão da dieta de cafeteria. Além disso, os frutos também não foram eficientes na preservação da histologia renal e infiltração lipídica no fígado. Conclui-se que a inclusão do jambolão e do açaí juçara na dieta pode apresentar efeitos positivos sobre danos causados por dietas hiperlipídicas, especialmente no que se refere à dislipidemia, à tolerância à glicose e à hipertrofia dos adipócitos.

Three-dimensional technology facilitates surgical performance of novice laparoscopy surgeons: a quantitative assessment on a porcine kidney model

Objective To determine whether the use of 3-dimensional (3D) imaging translates into a better surgical performance of naïve urologic laparoscopic surgeons during pyeloplasty (PY) and partial nephrectomy (PN) procedures. Materials and Methods Eighteen surgeons without any previous laparoscopic experience were randomly assigned to perform PY and PN in a porcine model using initially 2-dimensional (2D) and 3D laparoscopy. A surgical performance score was rated by an "expert" tutor through a modified 5-item global rating scale contemplating operative field view, bimanual dexterity, efficiency, tissue handling, and autonomy. Overall surgical time, complications, subjective perception of participating surgeons, and inconveniences related to the 3D vision were recorded. Results No difference in terms if operative time was found between 2D or 3D laparoscopy for both the PY (P =.51) and the PN (P =.28) procedures. A better rate in terms of surgical performance score was noted by the tutors when the study participants were using 3D vs 2D, for both PY (3.6 [0.8] vs 3.0 [0.4]; P =.034) and PN (3.6 [0.51] vs 3.15 [0.63]; P =.001). No complications occurred in any of the procedures. Most (77.2%) of the participating na??ve laparoscopic surgeons had the perception that 3D laparoscopy was overall easier than 2D. Headache (18.1%), nausea (18.1%), and visual disturbance (18.1%) were the most common issues reported by the surgeons during 3D procedures. Conclusion Despite the absence of translation in a shorter operative time, the use of 3D technology seems to facilitate the surgical performance of naive surgeons during laparoscopic kidney procedures on a porcine model.

Tecnología de igy anti-escherichia coli aplicada mediante nanotubos sobre cultivos de enterocitos porcinos. Anti- escherichia coli igy technology applied through nanotubes on porcine enterocyte cultures.

La diarrea neonatal representa uno de los problemas sanitarios de mayor relevancia en las primeras semanas de vida del cerdo. Provoca importantes pérdidas económicas por morbilidad y mortalidad. El cultivo de enterocitos primarios representa una herramienta valiosa para el estudio de patologías causadas por agentes infecciosos que afectan la integridad del epitelio intestinal. La producción de anticuerpos extraídos a partir de la yema de huevo de gallinas inmunizadas (IgY), es una tecnología innovadora, que ha mostrado ser protectiva contra diarreas causadas por agentes víricos y bacterianos. La nanotecnología permite mejorar la eficiencia en la administración de distintas drogas. Los nanotubos de carbono han ganado una enorme popularidad por sus propiedades y aplicaciones únicas. La investigación sobre los aspectos toxicológicos de estas nanopartículas es escasa. Una vez dentro de la célula, las nanopartículas pueden inducir estrés oxidativo intracelular por perturbar el equilibrio oxidativo. Las hipótesis de trabajo es: La administración de IgY anti-Escherichia coli a través de nanotubos protegerá in vitro e in vivo a los enterocitos de una infección por E. coli previniendo la diarrea neonatal porcina. Los objetivos del trabajo son: Evaluar la protección por un anticuerpo aviario IgY anti-E. coli aplicado mediante nanotubos de carbono a cultivo de enterocitos porcinos primarios sometidos a una post-infección con E. coli; Analizar los efectos secundarios de los nanotubos con IgY anti-E coli en la citotoxicidad, el balance oxidativo y la apoptosis de los enterocitos porcinos cultivados in vitro y Evaluar la acción terapeútica de la IgY anti-E coli aplicada a porcinos y efectos secundarios de la administración con nanotubos. Se implementará un diseño experimental in vitro con diferentes grupos de cultivos con nanotubos, con IgY anti-E. coli e inespecifica y con exposición a E. coli. Se realizará cultivo de enterocitos porcinos primarios con una técnica de disgregación enzimática con colagenasa según protocolo de Bader et al. (2000). Se evaluará la viabilidad por la prueba de azul tripan. Para la obtención del anticuerpo anti-E. coli aviario se aplicarán un total de 3 dosis de E. coli (109 UFC/ml de adyuvante) a gallinas Legorhn en condiciones fisiológicas. Se recolectarán los huevos diariamente. Se purificará la IgY según método de Polson et al. (1985) utilizando PEG 6000. La concentración de IgY se medirá por ELISA de alta sensibilidad. La IgY será incorporada a nanotubos según protocolo de Acevedo et al. 2006. Para analizar los posibles efectos secundarios de los nanotubos se evaluará: 1. Citotoxicidad por técnica de MTT 2. Estrés oxidativo por técnica de TBARS y 3. Apoptosis por técnica de TUNEL.Además, se implementará un diseño experimental in vivo para probar la acción terapeútica de este nutraceútico aplicados a lechones destetados y los efectos secundarios de la administración con nanotubos. Se realizará un cultivo de enterocitos de lechones que previamente fueron tratados con la IgY anti-E. coli administrada mediante nanotubos y efectuarán las técnicas descriptas anteriormente. Los resultados esperados son: Elaboración de un Ac aviario IgY anti-E. coli para prevenir infección de enterocitos, Profundización en el conocimiento acerca de los efectos citotóxicos de los nanotubos de carbono multilamelares, Generación de tratamiento alternativo para enfermedades entéricas porcinas.

Detección de circovirus pocino tipo II en cerdos que presentan diarrea en criaderos intensivos. Detection of porcine circovirus type II in pigs that present diarrhea in intensive hatcheries.

Se propone detectar la presencia del agente etiológico circovirus porcino (CVP) en cerdos con diarrea. El cual se considera un patógeno emergente importante asociado a diferentes sindromes y enfermedades porcinas. Se comunicó a nivel internacional en 1997 y en Argentina fue descripto en el 2003 en animales de cria extensiva, aunque se encuentra en los diferentes sistemas de producción. Su presencia causa grandes pérdidas económicas, ya que muchas veces no tiene una sintomatología clínica, sino que se manifiesta subclinicamente. En la provincia de Córdoba no se han realizado estudios sistemáticos para determinar la presencia de circovirus. Además, en animales que presentan diarrea no se ha realizado un relevamiento de la presencia del CVP y su asociación con el cuadro clínico. Diversos investigadores estan viendo la necesidad de incluir al CVP a la hora de pensar en los diagnósticos etiológicos, en aquellos que no solo presentan índices productivos ineficientes sino también que no responden a la terapia antimicrobiana. Es agente causal del Sindrome de Desmedro Posdestete y podria favorecer la acción de otros agentes patógenos que actuarían tanto en Sistema Respirastorio como en Sistema Digestivo. La complejidad del trabajo radica en demostrar lo antes mencionado a traves de las lesiones a observar en los tejidos y la determinación de la presencia del agente. La ejecución de este estudio sería un importante aporte a la producción porcina, en donde este agente produce importantes pérdidas económicas. La metodología, así como los resultados obtenidos serán útiles en la formación de recursos humanos aplicados a la investigación y transferidos directamente a la docencia. Hipotesis: Establecer la presencia del CVP en cerdos con signos clinicos de enteropatías Objetivos: Determinar en cortes histopatológicos de intestino y ganglio mesentério, lesiones compatibles con CVP; Determinar por inmunohistoquímica la presencia del antígeno CVP en las muestras. Identificar las células en las que se encuentre el antígeno. Materiales y método: Muestreo a campo y toma de muestra. En diferentes establecimientos porcinos, de aquellos animales que presenten el cuadro clínico de diarrea, serán elegidos 3 animales aleatoriamente para sacrificio y realización de la técnica de necropsia. Se tomarán muestras de intestino delgado y ganglio mesentérico que serán fijadas en formol; la técnica histopatológica y su lectura tendrán fin de encontrar lesiones compatibles con circovirus porcino tipo II; Realización de la técnica de inmunohistoquímica con anticuerpo monoclonal, para la detección del CVP en los tejidos; Interpretación de resultados; Publicación de los resultados.

Acute endotoxemia inhibits microvascular nitric oxide-dependent vasodilation in humans.

Nitric oxide (NO) is crucial for the microvascular homeostasis, but its role played in the microvascular alterations during sepsis remains controversial. We investigated NO-dependent vasodilation in the skin microcirculation and plasma levels of asymmetric dimethylarginine (ADMA), a potent endogenous inhibitor of the NO synthases, in a human model of sepsis. In this double-blind, randomized, crossover study, microvascular NO-dependent (local thermal hyperemia) and NO-independent vasodilation (post-occlusive reactive hyperemia) assessed by laser Doppler imaging, plasma levels of ADMA, and l-arginine were measured in seven healthy obese volunteers, immediately before and 4 h after either a i.v. bolus injection of Escherichia coli endotoxin (LPS; 2 ng/kg) or normal saline (placebo) on two different visits at least 2 weeks apart. LPS caused the expected systemic effects, including increases in heart rate (+43%, P < 0.001), cardiac output (+16%, P < 0.01), and rectal temperature (+1.4°C, P < 0.001), without change in arterial blood pressure. LPS affected neither baseline skin blood flow nor post-occlusive reactive hyperemia but decreased the NO-dependent local thermal hyperemia response, l-arginine, and, to a lesser extent, ADMA plasma levels. The changes in NO-dependent vasodilation were not correlated with the corresponding changes in the plasma levels of ADMA, l-arginine, or the l-arginine/ADMA ratio. Our results show for the first time that experimental endotoxemia in humans causes a specific decrease in endothelial NO-dependent vasodilation in the microcirculation, which cannot be explained by a change in ADMA levels. Microvascular NO deficiency might be responsible for the heterogeneity of tissue perfusion observed in sepsis and could be a therapeutic target.

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.