Ivan Hong Jun Koh

Microcirculatory evaluation in sepsis: a difficult task.

Microcirculatory dysfunction plays a pivotal role in the pathogenesis of severe sepsis and septic shock; hence, microcirculation blood flow monitoring has gained increasing attention. However, microcirculatory imaging is still investigational in human sepsis and has not yet been incorporated into routine clinical practice for several reasons, including the difficult interpretation of microcirculation imaging data, difficulty to draw a parallel between sublingual microcirculation imaging and organ microcirculation dysfunction, as well as the absence of microvessel dysfunction parameters defining sequential microcirculatory changes from the early to late stages of the disease, which could aid in the context of therapeutic approaches and of prognostic parameters. The purpose of this review was to bridge the experimental abdominal organ microvascular derangement kinetics and clinical aspects of microcirculatory findings in the early phase of severe sepsis/septic shock.

Injuries to the mesenteric microcirculation due to bacterial translocation.

CFU/g or mL of tissue. The recovery was 60% positive inblood and 100% positive in tissues. Despite the absence ofclinical symptoms or mortality at all time periods studied, amicrocirculatory injury was detected between 2 hour and 28days. At 2 hours after BT the microcirculatory injuriesincluded obstruction of distal capillaries with low flow, anda lesser degree of obstruction in small and medium venules.In addition multiple focal hemorrhages in capillaries andsmall venules were distributed throughout the entire intes-tinal mesentery. However, the majority of medium andlarge venules and arterioles were not affected; they main-tained a normal blood flow pattern. Also, an augmentedvolume of lymphatic flow was observed in association withan increased contractile frequency of lymphatic vessels(control 6 and BT 12 contractions/min). When com-pared to the normal microcirculation, these alterationswere progressive reaching maximal severity on day 3. At day7, the injuries were persistent albeit improving. At 14 dayspost-BT, the microvascular circulatory pattern was mostlyrestored; resolved at 28 days. However, the vast majority ofpreviously damaged vessels remained without restoration ofblood flow, suggesting that the recuperation of the mesen-teric microvascular damage was most likely due to a neo-vascularization process. Neither BT nor microcirculatorydamage were evident in the control group receiving vehicle.

Effect of pentoxifylline on lung inflammation and gas exchange in a sepsis-induced acute lung injury model

Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.

Accessing the biocompatibility of layered double hydroxide by intramuscular implantation: histological and microcirculation evaluation.

Biocompatibility of layered double hydroxides (LDHs), also known as hydrotalcite-like materials or double metal hydroxides, was investigated by in vivo assays via intramuscular tablets implantation in rat abdominal wall. The tablets were composed by chloride ions intercalated into LDH of magnesium/aluminum (Mg2Al-Cl) and zinc/aluminum (Zn2Al-Cl). The antigenicity and tissue integration capacity of LDHs were assessed histologically after 7 and 28 days post-implantation. No fibrous capsule nearby the LDH was noticed for both materials as well any sign of inflammatory reactions. Sidestream Dark Field imaging, used to monitor in real time the microcirculation in tissues, revealed overall integrity of the microcirculatory network neighboring the tablets, with no blood flow obstruction, bleeding and/or increasing of leukocyte endothelial adhesion. After 28 days Mg2Al-Cl promoted multiple collagen invaginations (mostly collagen type-I) among its fragments while Zn2Al-Cl induced predominantly collagen type–III. This work supports previous results in the literature about LDHs compatibility with living matter, endorsing them as functional materials for biomedical applications.

What is important for continent catheterizable stomas: angulations or extension?

OBJECTIVE We developed an experimental ex-vivo model to define factors that may influence continence of catheterizable channels by urinary and colonic stomas based on the principle of imbrication of the outlet tube. MATERIALS AND METHODS From 20 pigs, colon specimens with 25 cm length were obtained and a transverse flap with 3.0 cm length x 1.5 cm width in the average point of the intestine was tubulated to create an efferent tube. With the tube configured, it was embedded by 3 seromuscular stitches far 0.5 cm each other. A pressure study of both intra-luminal surface and channel was then conducted during the filling of the submerse piece with environmental air in a water container, to define the efferent channel continence. The study was repeated after the progressive release of suture stitches until only one stitch remains. RESULTS Channel continence analyzed in each segment in three different valve length situations, making a total of 20 segments, revealed that with 3 stitches (1.5 cm valve) the maximum average pressure prior to overflow was 54 cm H2O; 53.65 cm H2O with 2 stitches (1.0 cm of valve), and 55.45 cm H2O with only one stitch (0.5 cm of valve), which are the same values. The record at the segment explosion pressure was 67.87 cm H20. CONCLUSION The study showed that angulation of channel with colon, maintained by only one stitch (0.5 cm imbrication) was more important than a larger extension of the valve, represented by 3 suture stitches (1.5 cm imbrication) in order to allow continence to the efferent channel.

Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites

Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.

Typical and atypical enteropathogenic Escherichia coli bacterial translocation associated with tissue hypoperfusion in rats

Although enteropathogenic Escherichia coli (EPEC) are well-recognized diarrheal agents, their ability to translocate and cause extraintestinal alterations is not known. We investigated whether a typical EPEC (tEPEC) and an atypical EPEC (aEPEC) strain translocate and cause microcirculation injury under conditions of intestinal bacterial overgrowth. Bacterial translocation (BT) was induced in female Wistar-EPM rats (200-250 g) by oroduodenal catheterization and inoculation of 10 mL 10(10) colony forming unit (CFU)/mL, with the bacteria being confined between the duodenum and ileum with ligatures. After 2 h, mesenteric lymph nodes (MLN), liver and spleen were cultured for translocated bacteria and BT-related microcirculation changes were monitored in mesenteric and abdominal organs by intravital microscopy and laser Doppler flow, respectively. tEPEC (N = 11) and aEPEC (N = 11) were recovered from MLN (100%), spleen (36.4 and 45.5%), and liver (45.5 and 72.7%) of the animals, respectively. Recovery of the positive control E. coli R-6 (N = 6) was 100% for all compartments. Bacteria were not recovered from extraintestinal sites of controls inoculated with non-pathogenic E. coli strains HB101 (N = 6) and HS (N = 10), or saline. Mesenteric microcirculation injuries were detected with both EPEC strains, but only aEPEC was similar to E. coli R-6 with regard to systemic tissue hypoperfusion. In conclusion, overgrowth of certain aEPEC strains may lead to BT and impairment of the microcirculation in systemic organs.

Reconstituição da válvula ileocecal em cães

PURPOSE The importance of keeping the ileocecal valve in the intestinal ressections has been reported by several authors. When preserved, the ileocecal valve was related to a longer survival and prevention of the short bowel syndrome, due to its ability to block the colonic content reflux into the ileum and to avoid the rapid empting of the ileal content into the cecum. It was assessed a tecnique of ileocecal valve reconstitution, based on vesicoureteral anti-reflux tecniques. METHODS Fourteen beagles were operated. Seven underwent ileocecal valve reconstitution following the tecnique proposed and in the other seven a simple end-to-end anastomosis was performed. To assess the new valve, it was done the clinical follow up, the microbiologic analysis and the manometric study. RESULTS Clinically, during 45 days of follow up, there was no difference between the dogs with and without ileocecal reconstitution. In the aerobic bacteria analysis, the predominant bacterium was Escherichia coli. Quantitatively, the cultures grew in an irregular way, so that it was not able to compare the bacterial growth between the groups with or without ileocecal valve. The new valve had a colo-ileal reflux pressure similar to that of the physiological valve (P > 0.05). However, when compared to the non valve group, the reflux pressures of the physiological valve and new valve were significantly higher, with P < 0.05 and P < 0.001, respectively. CONCLUSION In this study, the reconstituted ileocecal valve served as a barrier to the colo-ileal reflux just as the physiological valve does.PURPOSE: The importance of keeping the ileocecal valve in the intestinal ressections has been reported by several authors. When preserved, the ileocecal valve was related to a longer survival and prevention of the short bowel syndrome, due to its ability to block the colonic content reflux into the ileum and to avoid the rapid empting of the ileal content into the cecum. It was assessed a tecnique of ileocecal valve reconstitution, based on vesicoureteral anti-reflux tecniques. METHODS: Fourteen beagles were operated. Seven underwent ileocecal valve reconstitution following the tecnique proposed and in the other seven a simple end-to-end anastomosis was performed. To assess the new valve, it was done the clinical follow up, the microbiologic analysis and the manometric study. RESULTS: Clinically, during 45 days of follow up, there was no difference between the dogs with and without ileocecal reconstitution. In the aerobic bacteria analysis, the predominant bacterium was Escherichia coli. Quantitatively, the cultures grew in an irregular way, so that it was not able to compare the bacterial growth between the groups with or without ileocecal valve. The new valve had a colo-ileal reflux pressure similar to that of the physiological valve (P>0.05). However, when compared to the non valve group, the reflux pressures of the physiological valve and new valve were significantly higher, with P<0.05 and P<0.001, respectively. CONCLUSION: In this study, the reconstituted ileocecal valve served as a barrier to the colo-ileal reflux just as the physiological valve does.

Vascular adventitia is a suitable compartment to transplant transduced vascular smooth muscle cells for ex vivo gene expression.

Vascular smooth muscle cells (VSMC) are ideal for systemic gene therapy because of their proximity to blood vessels and they have demonstrated long-term exogenous gene expression in vivo. However, the procedure generally followed to seed the transduced VSMC onto arteries denuded of endothelial cells usually induces stenosis and thrombosis, with a consequent high risk for use in humans. We demonstrate here that the vascular adventitia is a suitable place to introduce transduced VSMC and to secrete therapeutic proteins into the blood stream by a simple procedure, avoiding postoperative vascular complications. Transduced VSMC, with the retroviral vectors carrying the human growth hormone gene (hGH), were seeded into the adventitia of the rat abdominal aorta by single injection of a cell suspension. Based on the hGH and anti-hGH production in serum and on histological analysis of the removed aortas, we demonstrated hGH production over the 2-month experimental period. None of the animals used in the experiment showed stenosis, thrombosis, or other vascular or visible physiological abnormalities.

Analysis of the Virulence of an Atypical Enteropathogenic Escherichia coli Strain In Vitro and In Vivo and the Influence of Type Three Secretion System

Atypical enteropathogenic Escherichia coli (aEPEC) inject various effectors into intestinal cells through a type three secretion system (T3SS), causing attaching and effacing (A/E) lesions. We investigated the role of T3SS in the ability of the aEPEC 1711-4 strain to interact with enterocytes in vitro (Caco-2 cells) and in vivo (rabbit ileal loops) and to translocate the rat intestinal mucosa in vivo. A T3SS isogenic mutant strain was constructed, which showed marked reduction in the ability to associate and invade but not to persist inside Caco-2 cells. After rabbit infection, only aEPEC 1711-4 was detected inside enterocytes at 8 and 24 hours pointing to a T3SS-dependent invasive potential in vivo. In contrast to aEPEC 1711-4, the T3SS-deficient strain no longer produced A/E lesions or induced macrophage infiltration. We also demonstrated that the ability of aEPEC 1711-4 to translocate through mesenteric lymph nodes to spleen and liver in a rat model depends on a functional T3SS, since a decreased number of T3SS mutant bacteria were recovered from extraintestinal sites. These findings indicate that the full virulence potential of aEPEC 1711-4 depends on a functional T3SS, which contributes to efficient adhesion/invasion in vitro and in vivo and to bacterial translocation to extraintestinal sites.