Biomedical subjects

Qi Lu

Publications and source records attributed to Qi Lu.

At least 37 records · Page 2Linked to original sources

PubMed2004

A study of the biologic activity of trauma-hemorrhagic shock mesenteric lymph over time and the relative role of cytokines.

BACKGROUND: Gut-derived factors in intestinal lymph have been recently shown to cause lung injury, activate neutrophils, and injure endothelial cells in rats subjected to hemorrhagic shock (T/HS). However, the time course of the appearance and disappearance of these factors in intestinal lymph is unclear. Thus the goal of this study was to characterize the biologic activity of T/HS lymph collected at various times during and after shock. METHODS: Male rats subjected to trauma (laparotomy) plus hemorrhagic shock (mean arterial pressure, 90 mm Hg x 90 min) (T/HS) or trauma plus sham shock (T/SS) had their mesenteric lymph duct catheterized. Mesenteric lymph collected before shock, during shock, and hourly for 6 hours after shock was assayed for cytokine levels (tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, interleukin-1, and transforming growth factor-beta) as well as biologic activity on endothelial cells (cytotoxicity and permeability) and neutrophils (CD11b adhesion molecule expression and respiratory burst activity). RESULTS: T/HS, but not T/SS, lymph injured endothelial cells and activated neutrophils, although the cytokine levels did not differ between the T/HS and T/SS lymph samples. The biologic activity of T/HS lymph appeared during the shock (gut ischemic) period. The temporal pattern of activity varied on the basis of the biologic activity being tested, with the neutrophil-activating properties of the T/HS lymph persisting longest. CONCLUSIONS: These results suggest that gut ischemia itself is sufficient to induce the production of biologically active T/HS lymph and that the temporal pattern of biologic activity varies over time on the basis of the property being tested. Consequently, studies directed at identifying the active factors in T/HS lymph must take these temporal patterns of activity into account.

Animals↗

PubMed2004

Cloning and analysis of human Apg16L.

Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole. In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L. This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L. Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.

Alternative Splicing↗

PubMed2004

Female sex hormones protect red blood cells from damage after trauma-hemorrhagic shock.

BACKGROUND: Trauma/hemorrhagic shock (T/HS) is known to cause changes in red blood cell (RBC) deformability and resting shape. Our previous studies have shown that proestrus female rats are more protected from shock-induced RBC damage than diestrus females or males. However, it is unclear whether female or male sex hormones can influence the severity of these alterations. METHODS: Red blood cell deformability and shape were examined in proestrus female rats, and oophorectomized female rats, as well as in castrated and non-castrated male rats (5-10 animals per group) subjected to T/HS. Red blood cell deformability was measured by laser ektacytometry whereas erythrocyte shape was evaluated by scanning electron microscopy. RESULTS: Proestrus female rats subjected to T/HS did not show either significant RBC deformability changes (decrease in elongation index) or shape alterations (increase in the percentage of reversibly and irreversibly changed cells). Oophorectomized rats demonstrated more severe RBC changes than did non-oophorectomized rats. The degree of RBC damage was the same in castrated and non-castrated males, which was significantly worse than in proestrus females. CONCLUSIONS: Removal of female sex hormones increases the severity of T/HS-induced RBC changes, indicating that female sex hormones protect against RBC damage. In contrast, male sex hormones do not appear to modulate T/HS RBC dysfunction.

Analysis of Variance↗

PubMed2004

Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils.

OBJECTIVE: Our previous studies indicated that mesenteric lymph duct ligation prevented burn-induced lung injury. Thus, the goal of the present study was to begin to investigate potential mechanisms of this protective effect. DESIGN: Prospective animal study with concurrent control. SETTING: Small animal laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Mesenteric lymph and portal vein plasma were collected from male rats subjected to a 40% third-degree scald burn or sham burn. The biological effects of these lymph and plasma samples were tested for their ability to kill human umbilical vein endothelial cells (HUVECs), increase HUVEC monolayer permeability, and activate polymorphonuclear leukocytes (PMNs), as reflected in CD11b adhesion molecule expression and superoxide production. Additionally, ileal specimens were harvested at the end of the experiment (6 hrs postburn) for histologic analysis. MEASUREMENTS AND MAIN RESULTS: Postburn mesenteric lymph produced during the first 2 hrs after burn injury and tested at a 5% concentration, but not sham-burn lymph or portal plasma from burned rats, was toxic for HUVECs resulting in cell death after an 18-hr incubation period. Similarly, only postburn lymph increased HUVEC monolayer permeability. Postburn lymph activated both rat and human PMNs as reflected in increased CD11b expression and augmentation of the phorbol myristate acetate-induced superoxide response. Neither sham-burn lymph nor postburn portal vein plasma activated PMNs. Both the burn and sham-burn lymph samples were sterile, indicating that the effects of burn lymph on the HUVECs or PMNs were not due to translocating bacteria. Last, an association was found between burn-induced gut injury and the production of toxic burn lymph. CONCLUSIONS: Burn-induced gut injury results in the production of biologically active factors that are carried in the mesenteric lymph, but not the portal plasma, which injure endothelial cells and activate PMNs and thus could contribute to distant organ injury.

Animals↗

PubMed2004

Trauma-hemorrhagic shock-induced up-regulation of endothelial cell adhesion molecules is blunted by mesenteric lymph duct ligation.

OBJECTIVE: Previous studies have shown that mesenteric lymph duct ligation prevents trauma-hemorrhagic shock-induced lung injury and neutrophil activation. Since endothelial cells rapidly express adhesion molecules, such as P-selectin and intercellular adhesion molecule-1, after shock, and because trauma-hemorrhagic shock-induced lung injury appears to involve neutrophil-endothelial cell interactions, we tested the hypothesis that lymph duct ligation would diminish trauma-hemorrhagic shock-induced P-selectin and intercellular adhesion molecule-1 expression in the lung and other organs. DESIGN: Prospective animal study with concurrent control. SETTING: Small animal laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Four groups of male rats were studied: trauma (laparotomy) plus sham shock, trauma-sham shock plus lymph duct ligation, trauma-hemorrhagic shock (90 mins of shock at 30 mm Hg), and trauma-hemorrhagic shock plus lymph duct ligation. At 3 or 24 hrs after trauma-hemorrhagic shock or trauma-sham shock, lung, heart, liver, kidney, intestinal, and other visceral concentrations of P-selectin and intercellular adhesion molecule-1 expression were measured using the dual radiolabeled monoclonal antibody technique. MEASUREMENTS AND MAIN RESULTS: At 3 and 24 hrs, trauma-hemorrhagic shock increased endothelial cell P-selectin and intercellular adhesion molecule-1 adhesion molecule expression in the lung and liver. At 3 and 24 hrs after trauma-hemorrhagic shock, intercellular adhesion molecule-1 expression was increased in the heart, spleen, pancreas, intestine, and kidney, whereas at 24 hrs, but not 3 hrs, P-selectin expression also was increased in these organs. Lymph duct ligation prevented trauma-hemorrhagic shock-induced increased adhesion molecule expression in all of these organs with the exception of intestinal P-selectin expression. CONCLUSIONS: Trauma-hemorrhagic shock-induced increases in endothelial cell P-selectin and intercellular adhesion molecule-1 expression in the lung and liver as well as other tissues appear to be related to factors liberated from the ischemic gut and carried in intestinal lymph.

Analysis of Variance↗

PubMed2004

Hemorrhagic shock induces endothelial cell apoptosis, which is mediated by factors contained in mesenteric lymph.

OBJECTIVE: Trauma-hemorrhagic shock is one of the leading causes of acute respiratory distress syndrome. This syndrome is associated with disruption of the alveolar barrier consisting of both epithelial and endothelial cells, which leads to a major increase in epithelial and microvascular permeability in the lungs. Although alveolar epithelial cell apoptosis has been documented as a contributing factor to this increase in permeability, it is unclear whether endothelial cell apoptosis occurs following trauma-hemorrhagic shock and, if so, the source of factors leading to this process. DESIGN: Prospective animal study with concurrent control. SETTING: Small-animal laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Trauma-hemorrhagic shock in rats was induced by laparotomy followed by blood withdrawal to achieve a mean arterial blood pressure of 30 mm Hg for 90 mins. At the end of the shock period, the rats were resuscitated, and 3 hrs later lungs were taken for histologic analysis. In other experiments, mesenteric lymph was collected from trauma-hemorrhagic shock and trauma-sham shock rats, and the biological activity of these lymph samples was tested for their ability to kill cultured endothelial cells or endothelial cells of isolated femoral veins. MEASUREMENTS AND MAIN RESULTS: Trauma-hemorrhagic shock triggered endothelial cell apoptosis in the lung as assessed using the Tunnel assay as well as by light and electron microscopic analysis. Since our previous studies have documented that mesenteric lymph is a major contributor to lung injury following shock, we also tested the hypothesis that factors in the mesenteric lymph were responsible for the endothelial cell apoptosis-inducing effect of shock. Preventing the mesenteric lymph from reaching the lung by mesenteric lymph duct ligation decreased endothelial cell apoptosis. Mesenteric lymph obtained from rats subjected to trauma-hemorrhagic shock elicited apoptosis in cultured endothelial cells and when placed into isolated femoral vein as well as increased endothelial cell monolayer permeability. CONCLUSIONS: Trauma-hemorrhagic shock induces endothelial as well as epithelial cell apoptosis in the lung via factors contained in the mesenteric lymph, thereby contributing to the pathophysiology of the acute respiratory distress syndrome.

Animals↗

PubMed2004

The role of lymph factors in lung injury, bone marrow suppression, and endothelial cell dysfunction in a primate model of trauma-hemorrhagic shock.

Studies in rodent models of trauma-hemorrhagic shock (T/HS) have shown that factors contained in the intestinal lymph are responsible for acute lung injury and bone marrow suppression, and that they contribute to a systemic inflammatory state. Because results observed in rodent T/HS models may not fully reflect the response of injured patients, it is necessary to determine if these results can be replicated in primates before the institution of invasive studies in humans. Thus, the three goals of this study were to determine if diversion of thoracic duct lymph reduced T/HS-induced lung injury; to compare the biologic activity of thoracic duct lymph from baboons subjected to T/HS or trauma sham-shock (T/SS); and to compare the biologic activity and composition of plasma from baboons subjected to T/SS, T/HS, and T/HS with thoracic duct lymph drainage. Three groups of baboons were studied: T/SS plus lymph diversion via a thoracic duct catheter, T/HS, and T/HS plus lymph diversion (T/HS-LD). The trauma component consisted of a neck dissection with resection of the proximal clavicle plus a laparotomy. HS was to a mean arterial pressure of 40 mmHg and was maintained at 40 mmHg until the base excess reached -5 mEq or the total shock period reached 3 h. Volume resuscitation was carried out by reinfusing the shed blood plus crystalloids. Before, during, and after the T/HS or T/SS period, blood and lymph samples were obtained for analysis, and lung samples were harvested for measurement of lung wet-to-dry ratio at 5 h after the end of the shock period. Diversion of thoracic duct lymph prevented T/HS-induced lung injury as reflected in lung wet-to-dry weight ratios (T/SS = 4.6 +/- 0.5; T/HS+LD = 4.8 +/- 0.7; T/HS = 5.4 +/- 0.6; P < 0.05). Lymph from the T/HS group collected during the early postshock period was cytotoxic for human endothelial cells (HUVECs; 16% vs. 100% survival in T/SS lymph) and increased HUVEC monolayer permeability almost 2-fold (P < 0.01). T/HS lymph and plasma also suppressed red blood cell (erythroid burst-forming unit) and white blood cell (granulocyte-monocyte colony-forming unit) progenitor cell growth of human bone marrow to approximately 50% of control, whereas T/SS lymph and plasma were not suppressive (P < 0.05). Plasma cytokine levels were increased to a similar degree in the two T/HS groups. Thus, in a primate model of T/HS, gut-derived factors carried in the lymph potentiates lung injury and endothelial dysfunction, and suppresses bone marrow progenitor cell growth.

Animals↗

PubMed2004

Persistent HIF-1alpha activation in gut ischemia/reperfusion injury: potential role of bacteria and lipopolysaccharide.

In both animal models of hemorrhagic shock and clinical settings, shock-induced gut ischemia has been implicated in the development of the systemic inflammatory response syndrome and distant organ injury, yet the factors transducing these events remain to be fully determined. Because hypoxia-inducible factor (HIF-1), a transcription factor composed of oxygen-labile HIF-1alpha and constitutive HIF-1beta subunits, regulates the physiologic/pathophysiologic response to hypoxia and ischemia, we examined the HIF-1 response in two rat models of gut ischemia-reperfusion. We found that ileal nuclear HIF-1alpha protein levels were induced in rats subjected to trauma (laparotomy) plus hemorrhagic shock for 90 min relative to their trauma sham-shock and naïve counterparts and that this trauma hemorrhagic shock-induced mucosal HIF-1alpha protein response persisted after 1 h and 3 h of reperfusion. Likewise, in a model of isolated gut ischemia-reperfusion injury, where the superior mesenteric artery was occluded for 45 min, nuclear HIF-1alpha were induced in the gut mucosa relative to their sham counterparts and persisted after 1 h and 3 h or reperfusion. Similar to the in vivo response, in vitro hypoxia induced HIF-alpha expression in three different enterocyte cell lines (rat IEC-6 and human Caco-2 and HT-29 cell lines). However, in contrast to the in vivo response, HIF-1 expression rapidly disappeared on subsequent reoxygenation. Because in vivo enterocytes are exposed to bacteria, we tested whether the in vitro HIF-1alpha response would persist on reoxygenation if the enterocytes were cocultured with bacteria. P. aeruginosa, an enteric bacterium, markedly induced enterocyte HIF-1alpha protein levels under normoxic conditions. Furthermore, the addition of P. aeruginosa during either the hypoxic or reoxygenation phase prevented the degradation of HIF-1alpha protein levels. Moreover, the observation that lipopolysaccharide induced HIF-1alpha expression in a time-dependent manner in IEC-6 cells indicated that the induction of HIF-1 by exposure to P. aeruginosa is not dependent on bacterial viability. In conclusion, these results suggest that HIF-1alpha activation is an early reperfusion-independent event in models of gut ischemia-reperfusion and that this HIF-1alpha response is potentiated by the presence of P. aeruginosa or lipopolysaccharide.

Animals↗

PubMed2004

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms.

OBJECTIVE: To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. SUMMARY BACKGROUND DATA: Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. METHODS: Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. RESULTS: T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. CONCLUSIONS: Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.

Animals↗

PubMed2004

Albumin protects against gut-induced lung injury in vitro and in vivo.

OBJECTIVE: Since albumin has the ability to detoxify, we assessed whether low-dose albumin could protect against trauma/hemorrhagic shock (T/HS)-induced endothelial cell, lung, gut, and red blood cell (RBC) injury in vivo and endothelial cell injury in vitro. SUMMARY BACKGROUND DATA: T/HS cause ischemic insult to the gut, resulting in the release of biologically active factors into the mesenteric lymph, which then cause injury to multiple distant organs. METHODS: In vitro experiments tested the ability of albumin to reduce the cytotoxicity of mesenteric lymph from male rats subjected to T/HS (laparotomy + MAP 30 mm Hg for 90 minutes) for human umbilical vein endothelial cell (HUVEC). In subsequent in vivo experiments, the ability of albumin given as part of the resuscitation regimen to protect against T/HS-induced injury was tested by comparing the magnitude of injury in T/HS rats receiving human albumin (shed blood + 0.12, 0.24, or 0.36 g/kg) or lactated Ringer's solution (shed blood + 2 x volume of shed blood as LR) with that observed in rats subjected to trauma/sham shock. Rats were killed after a 3-hour recovery period and had lung permeability evaluated by bronchoalveolar lavage and myeloperoxidase assays, intestinal microvillous injury by histology, and RBC deformability using ektacytometry. RESULTS: Both bovine and human albumin prevented T/HS lymph-induced HUVEC cytotoxicity in vitro, even when added 30 minutes after the lymph (viability 15 +/- 4% to 88 +/- 3%, P < 0.01). In vivo RBC deformability was better preserved by blood plus albumin than blood plus lactated Ringer's solution (P < 0.01). Likewise, albumin administration reduced T/HS-induced lung permeability and neutrophil sequestration in a dose-dependent fashion, with 0.36 g/kg of albumin effecting total lung protection (P < 0.01). In contrast, albumin treatment did not prevent T/HS-induced gut injury. CONCLUSIONS: Low-dose albumin protects against gut lymph-induced lung, HUVEC, and RBC injury by neutralizing T/HS lymph toxicity.

Albumins↗

PubMed2004

Pancreatic duct ligation reduces lung injury following trauma and hemorrhagic shock.

OBJECTIVE: To determine whether pancreatic digestive enzymes released into the ischemic gut during an episode of T/HS are involved in the generation of distant organ injury. This hypothesis was tested by examining the effect of PDL on T/HS-induced intestinal injury, lung injury, and RBC deformability. SUMMARY BACKGROUND DATA: The effect of pancreatic duct ligation (PDL) on distant organ injury following trauma/hemorrhagic shock (T/HS) was examined. PDL before T/HS decreases lung and red blood cell (RBC) injury and exerts a limited protective effect on the gut. Pancreatic proteases in the ischemic gut appear to be involved in gut-induced lung and RBC injury. Based on recent work, it appears that proinflammatory and/or toxic factors, which are generated by the ischemic intestine, play an important role in the pathogenesis of multiple organ failure. The process by which these toxic factors are generated remains unknown. Previous experimental work has clearly documented that intraluminal inhibition of pancreatic proteases decreases the degree of T/HS-induced lung injury and neutrophil activation. One possible explanation for this observation is that the toxic factors present in intestinal lymph are byproducts of interactions between pancreatic proteases and the ischemic gut. METHODS: Male Sprague-Dawley rats were subjected to a laparotomy (trauma) and 90 minutes of sham (T/SS) or T/HS with or without PDL. At 3 and 24 hours following resuscitation, animals were killed and samples of gut, lung, and blood were collected for analysis. Lung permeability, pulmonary myeloperoxidase levels, and bronchoalveolar fluid protein content were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum (% villi injured). To assess RBC injury, RBC deformability was measured, as the RBC elongation index (RBC-EI), using a LORCA device. RESULTS: At 3 and 24 hours following resuscitation, PDL prevented shock-induced increases in lung permeability to both Evans blue dye and protein in addition to preventing an increase in pulmonary myeloperoxidase levels. T/HS-induced impairments in RBC deformability were significantly reduced at both time points in the PDL + T/HS group, but deformability did not return to T/SS levels. PDL did reduce the magnitude of ileal injury at 3 hours after T/HS, but the protective effect was lost at 24 hours after T/HS. CONCLUSIONS: PDL prior to T/HS decreases lung injury and improves RBC deformability but exerts a limited protective effect on the gut. Thus, the presence of pancreatic digestive enzymes in the ischemic gut appears to be involved in gut-induced lung and RBC injury.

Animals↗

PubMed2004

Intestinal bacterial overgrowth induces the production of biologically active intestinal lymph.

OBJECTIVE: We have previously documented that gut-derived lymph from rats subjected to trauma plus hemorrhagic shock (T/HS) is injurious to vascular endothelial cells and activates neutrophils (PMNs), two key events in postshock organ injury. Because T/HS leads to gut injury, intestinal bacterial overgrowth, and the loss of gut barrier function, the relative role of gut injury as opposed to intestinal bacterial overgrowth per se in the pathogenesis of biologically active intestinal lymph is unclear. We therefore studied whether mesenteric lymph can injure endothelial cells and/or active PMNs in an intestinal bacterial overgrowth model where there is no gut injury (monoassociation). METHODS: Bacterial overgrowth was established in male rats by treating the animals with 4 days of oral antibiotics followed by administration of a nonpathogenic, streptomycin-resistant strain of Escherichia coli C25. Mesenteric lymph was then collected from rats with normal flora and from E. coli C25 monoassociated rats. Its effects were tested on human umbilical vein endothelial cells (HUVECs) and human PMNs. As an additional control, lymph was collected from antibiotic-decontaminated rats that received antibiotics but were not colonized with E. coli C25. RESULTS: As compared with medium, normal flora intestinal lymph, antibiotic-decontaminated lymph, or portal plasma from the monoassociated rats, mesenteric lymph from the monoassociated rats killed HUVECs and increased the permeability of a HUVEC monolayer. In contrast to the effects on HUVECs, lymph from the monoassociated rats did not increase PMN CD11b expression or prime PMNs for an augmented respiratory burst, as compared with lymph from the rats with normal flora or from antibiotic-decontaminated rats. The effects of lymph from the monoassociated rats was not caused by bacteria, because these lymph samples were sterile. CONCLUSION: These results indicate that disruption of the normal intestinal microflora resulting in bacterial overgrowth with enteric bacilli may participate in the production of mesenteric lymph that is injurious to endothelial cells in shock, but this mechanism does not appear to be significantly involved in the activation of PMNs.

Animals↗

PubMed2003

Burn-induced red blood cell deformability and shape changes are modulated by sex hormones.

BACKGROUND: Burns are known to cause changes in red blood cell (RBC) deformability and resting shape. However, it is unclear whether sex and sex hormones can influence the severity of these alterations. METHODS: Red blood cell deformability and shape were examined in proestrus and diestrus female rats, ovariectomized female rats, as well as castrated and non-castrated male rats (6 animals per group) subjected to scald burn. Red blood cell deformability was measured by laser ektacytometry and erythrocyte shape was evaluated by scanning electron microscopy. RESULTS: Burn-induced RBC deformability changes (decrease in elongation index) and shape alterations (increase in the percentage of reversibly and irreversibly changed cells) were less severe in proestrus females than in diestrus females or males. Ovariectomized rats demonstrated more severe RBC changes than non-ovariectomized ones. The degree of RBC damage was the same in castrated and non-castrated males. CONCLUSIONS: Removal of female sex hormones increases the severity of burn-induced RBC, indicating that female sex hormones protect against burn-induced RBC dysfunction. In contrast, male sex hormones do not appear to modulate burn-induced RBC dysfunction.

Animals↗

PubMed2003

Relocalization of neuronal nitric oxide synthase (nNOS) as a marker for complete restoration of the dystrophin associated protein complex in skeletal muscle.

A lack of effective treatments for Duchenne muscular dystrophy, a fatal X-linked myopathy, has focused attention on the possibility of gene therapy. The aim of the gene therapy approach is the restoration of the dystrophin associated complex of proteins, one member of which is neuronal nitric oxide synthase, an important enzyme in signal transduction. Transgenic mdx mice and plasmid gene transfer of both human and murine recombinant dystrophins was used to assess whether nNOS could be restored to the sarcolemma following dystrophin gene transfer at a variety of levels of expression. Murine revertant fibres and human patients with different dystrophin deletions were used to assess the relationship between exon deletion and loss of neuronal nitric oxide synthase localization to the sarcolemma. We demonstrate that the domain encoded by exons 45-48 is required for localization of neuronal nitric oxide synthase to the sarcolemma. On the basis of these observations we suggest that neuronal nitric oxide synthase is a useful marker for complete restoration of the dystrophin associated complex and should be used as one of the criteria for selecting the recombinant molecule to be used for gene therapy in Duchenne muscular dystrophy.

Adult↗

PubMed2003

Sexual dimorphism in the activation of neutrophils by shock mesenteric lymph.

BACKGROUND: After trauma/hemorrhagic shock (T/HS), inflammatory products exit the gut via mesenteric lymph. These products can prime neutrophils (PMN) and predispose to lung injury. Female gender and the proestrous state (PE) may confer protection against lung injury after T/HS. We therefore studied the dependence of T/HS-induced PMN priming on gender and stage of estrous. MATERIALS AND METHODS: T/HS was induced in male (M) and proestrous female (F) rats by laparotomy plus hemorrhagic shock (30 mm Hg, 90 min) followed by reinfusion of shed blood. Six hours later rats were sacrificed and plasma was obtained. Control male rat PMN were primed 5 min in buffer or in the plasma of M-T/HS or F-T/HS rats (n = 4-6/group). PMN were then assayed using DHR for respiratory burst (RB) initiated by sequential MIP-2 and PAF stimulation (MIP/PAF). Because MIP and PAF mobilize cell calcium ([Ca(2+)](i)) in a step crucial for RB initiation, we also assayed PMN [Ca(2+)](i) responses to MIP/PAF. RESULTS: M-T/HS plasma primed PMN RB (208 +/- 8 [SEM] U/sec versus buffer 51 +/- 12 U/sec, p < 0.01, ANOVA/Tukey's). F-T/HS plasma did not (87 +/- 20 U/sec, NS). PMN basal [Ca(2+)](i) was increased by pre-incubation in both M-T/HS and F-T/HS plasma (183 +/- 26 and 225 +/- 20 nM, p < 0.02, p < 0.01 compared to buffer [80 +/- 3 nM]). Peak PMN [Ca(2+)](i) response to MIP/PAF was 159 +/- 2 nM without priming. Priming PMN in either M-T/HS or F-T/HS plasma increased peak [Ca(2+)](i) responses to MIP/PAF to 274 +/- 35 (p < 0.04) and 330 +/- 24 nM (p < 0.02), but the effects of M-T/HS and F-T/HS plasma on [Ca(2+)](i) mobilization were indistinguishable. CONCLUSION: Plasma from male rats subjected to T/HS primes PMN respiratory burst, but plasma from proestrous females subjected to T/HS does not. In contrast, the male and proestrous female plasma primed PMN [Ca(2+)](i) mobilization by MIP/PAF equally. The decrease in pathologic PMN activation seen after T/HS in proestrous female rats depends on soluble mediators present in plasma. The decreased PMN RB seen after T/HS in proestrous females is mediated by calcium-independent pathways.

Analysis of Variance↗

PubMed2003

Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury.

The objective of this work was to test the hypothesis that Intraluminal serine proteases are involved in trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury. Male Sprague-Dawley rats were administrated the serine protease inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, Nafamostat) either intraluminally into the gut or intravenously after a laparotomy (trauma) and then subjected to 90 min of hemorrhagic shock (T/HS) or sham shock (T/SS). Intestinal and lung injury was assessed at 3 h after resuscitation with Ringer's lactate solution. In a second set of experiments, mesenteric lymph was collected from the groups of rats subjected to T/HS or T/SS and its ability to activate normal neutrophils was tested. Lung permeability, pulmonary myeloperoxidase levels, and the bronchoalveolar lavage fluid protein to plasma protein ratio were increased after T/HS but were significantly decreased in the T/HS rats receiving intraluminal (P < 0.05), but not intravenous, nafamostat. Likewise, T/HS-induced intestinal villus injury was less in the nafamostat-treated shock rats (P < 0.05). Last, the ability of T/HS mesenteric lymph to increase PMN CD11b expression or prime neutrophils for an augmented respiratory burst was significantly reduced by the intraluminal administration of nafamostat. Because intraluminal nafamostat reduced T/HS-induced gut and lung injury as well as the neutrophil activating ability of intestinal T/HS lymph, the presence of serine proteases in the ischemic gut may play an important role in T/HS-induced gut and hence lung injury.

Animals↗

PubMed2003

The role of oxidant-mediated pathways in the cytotoxicity of endothelial cells exposed to mesenteric lymph from rats subjected to trauma-hemorrhagic shock.

Because gut-derived factors carried in mesenteric lymph are implicated in multiple organ dysfunction syndrome and have been shown to injure endothelial cells, we investigated several cellular pathways by which this process could occur. To accomplish this, mesenteric lymph (5%, v/v) collected at 1 to 3 h postshock from male rats undergoing trauma (5-cm laparotomy) and hemorrhagic shock (90 min of mean arterial pressure [MAP] of 30 mmHg; T/HS) was tested for endothelial cell cytotoxicity on human umbilical vein endothelial cells (HUVECs). Over 30 pharmacologic agents that had been reported to inhibit endothelial cell death were tested for their ability to prevent T/HS lymph-induced HUVEC cell death. These included agents documented to protect against oxidant-mediated, calcium-mediated, and arachidonic acid pathway-mediated endothelial cell injury and death. These pharmacologic inhibitors were preincubated with HUVECs for 1 h or were added to the HUVECs simultaneously with lymph, and were then incubated for 18 h. Controls were lymph alone, inhibitor alone, or medium alone. Mitochondrial tetrazolium (MTT) and LDH release assays were used to determine cell viability. The inhibitors that significantly protected HUVECs from the cytotoxicity of T/HS lymph (P < 0.001) included the antioxidant combination of vitamins C and E and the antioxidant-lipooxygenase inhibitor nordihydroguaretic acid (NDGA). These agents were equally effective when added simultaneously with lymph or preincubated with the HUVECs, suggesting an extracellular or membrane-bound process. In summary, the inhibitors that provided protection from toxic lymph appear to work at the membrane and are involved in limiting membrane peroxidation. Based on this study, it appears that an oxidant pathway is involved in T/HS lymph-induced endothelial cell injury and death.

Animals↗

PubMed2003

Gut injury and gut-induced lung injury after trauma hemorrhagic shock is gender and estrus cycle specific in the rat.

BACKGROUND: The purpose of this study was to test the hypothesis that trauma-hemorrhagic shock (T/HS)-induced gut and lung injury is modulated by gender and the stage of the estrus cycle at the time of injury. METHODS: We compared the incidence and magnitude of gut and lung injury in male and female rats subjected to a laparotomy (trauma) followed by 90 minutes of shock (mean arterial pressure, 30 mm Hg) (T/HS) or sham shock. RESULTS: Lung injury and pulmonary neutrophil sequestration as well as gut injury were increased after T/HS in the diestrus female and the male rats, but not in the estrus or proestrus female rats. Although T/HS caused gut and lung injury in the male and the female diestrus rats, the magnitude of injury was less in the female diestrus than in the male rats (p < 0.05). A strong correlation was found between intestinal villous injury and lung injury as well as between gut injury and pulmonary leukosequestration in the female rats subjected to T/HS (p < 0.0001). Plasma nitric oxide levels were approximately two- to threefold higher in the male and the diestrus female rats subjected to T/HS than in other groups (p < 0.05), and a high degree of correlation (r2 = 0.68, p < 0.0001) was found between villous injury and plasma nitric oxide levels. Ileal constitutive nitric oxide synthase (NOS) and inducible NOS (iNOS) activity was measured. Ileal constitutive NOS activity was similar between the groups, but iNOS activity was three- to fourfold higher in the T/HS male rats than in the sham shock or the T/HS proestrus groups (p < 0.01). CONCLUSION: Gender and estrus cycle stage influence susceptibility to T/HS-induced gut and lung injury. This difference in susceptibility to organ injury was associated with increased plasma nitric oxide levels and increased ileal iNOS activity.

Animals↗