Biomedical subjects

M W Julian

Publications and source records attributed to M W Julian.

14 recordsLinked to original sources

PubMed2000

Digital imaging analysis for the study of endotoxin-induced mitochondrial ultrastructure injury.

Primary defects in mitochondrial function have been implicated in over 100 diverse diseases. In situ, mitochondria possess unique and well-defined morphology in normal healthy cells, but diseases linked to defective mitochondrial function are characterized by the presence of morphologically abnormal and swollen mitochondria with distorted cristae. In situ study of mitochondrial morphology is established as an indicator of mitochondrial health but thus far assessments have been via subjective evaluations by trained observers using discontinuous scoring systems. Here we investigated the value of digital imaging analysis to provide for unbiased, reproducible, and convenient evaluations of mitochondrial ultrastructure. Electron photomicrographs of ileal mucosal mitochondria were investigated using a scoring system previously described by us, and also analyzed digitally by using six digital parameters which define size, shape, and electron density characteristics of over 700 individual mitochondria. Statistically significant changes in mitochondrial morphology were detected in LPS treated animals relative to vehicle control using both the subjective scoring system and digital imaging parameters (p < 0.05). However, the imaging approach provided convenient and high throughput capabilities and was easily automated to remove investigator influences. These results illustrate significant changes in ileal mucosal mitochondrial ultrastructure during sepsis and demonstrate the value of digital imaging technology for routine assessments in this setting.

Analog-Digital Conversion↗

PubMed2000

Endotoxin-induced ileal mucosal injury and nitric oxide dysregulation are temporally dissociated.

Despite recent investigations, the mechanisms responsible for intestinal epithelial injury during endotoxemia remain unclear. The present study tests the hypothesis that epithelial necrosis and/or apoptosis correlate with nitric oxide (NO) dysregulation in a nonischemic model of sepsis-induced ileal injury. To test this hypothesis, a well-established in situ, autoperfused, feline ileal preparation was employed. After endotoxin (lipopolysaccharide [LPS], 3 mg/ kg, intravenously; n = 9) or vehicle (control; n = 5) treatment, ileal segments were obtained at baseline, 2 and 4 h for simultaneous evaluations of cellular and mitochondrial ultrastructure, immunoprevalence of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (a stable biomarker of peroxynitrite), and histochemical evidence of apoptosis. Epithelial necrosis was prominent by 2 h post-LPS, despite unaltered global ileal tissue oxygen content, blood volume, and blood flow. Significant evidence of apoptosis and increases in the immunoprevalence of iNOS and 3-nitrotyrosine were not evident until 4 h post-LPS. These results suggest that the early ileal mucosal necrosis may be due to LPS-induced activation of inflammatory pathways and/or microcirculatory disturbances, whereas NO dysregulation may participate in later events, including protein nitration and epithelial apoptosis.

Animals↗

PubMed1999

Ileal VO(2)-O(2) alterations induced by endotoxin correlate with severity of mitochondrial injury.

Sepsis is usually associated with altered O(2) metabolism in systemic organs. Until recently, inadequate O(2) delivery was thought to be the putative mechanism underlying these metabolic alterations. However, current investigations suggest that impaired O(2) consumption due to disrupted O(2) use by mitochondria may be the culprit. Therefore, we hypothesized that endotoxin (LPS)-induced V O(2)- O(2) alterations would correlate with the severity of mitochondrial injury in a systemic organ (i.e., the ileum). Using an in situ autoperfused feline ileum preparation, we assessed V O(2)- O(2) relationships and mitochondrial ultrastructure after 2 h in LPS-treated (3 mg/kg, intravenous; n = 11) and time-matched control (n = 5) animals. Mitochondrial injury was graded in a blinded fashion on the basis of characteristics associated with established stages of cell injury. LPS-treated animals developed severe mitochondrial injury in the ileal mucosa despite unchanged regional tissue perfusion and ileal oxyhemoglobin levels compared with controls. Worsening of mitochondrial injury correlated with increases in the critical O(2) delivery (r = 0.85; p < 0.002) and decreases in the maximum O(2) extraction (r = -0.61; p < 0.02) in the ileum. These results suggest that mitochondrial injury, leading to impaired O(2) utilization, may be primarily responsible for altered V O(2)- O(2) relationships in systemic organs during sepsis.

Animals↗

PubMed1998

Dithiothreitol improves recovery from in vitro diaphragm fatigue.

There is increasing evidence that reactive oxygen species are produced during strenuous skeletal muscle work and that they contribute to the development of muscle fatigue. Although the precise cellular mechanisms underlying such a phenomenon remain obscure, it has been hypothesized that endogenously produced reactive oxygen species may down-regulate force production during fatigue by oxidizing critical sulfhydryl groups on important contractile proteins. To test this hypothesis, we fatigued rat diaphragm strips in vitro for 4 min at 20 Hz stimulation and a duty cycle of 0.33. Following fatigue, the tissue baths were drained and randomly replaced with either physiologic saline or physiologic saline containing the disulfide reducing agent, dithiothreitol (DTT) at varying doses (0.1-5.0 mM). Force-frequency characteristics were then measured over a 90-min recovery period. At the 0.5 and 1.0 mM doses, DTT treatment was associated with significantly greater force production in the recovery period. DTT's effects were observed at most frequencies tested, but appeared more prominent at the higher frequencies. The beneficial effects of DTT were not evident at the 0.1 or 5.0 mM doses and appeared to be specific for fatigued muscle. These recovery-enhancing effects of a potent disulfide reducing agent suggest that important contractile proteins may be oxidized during fatigue; such changes may be readily reversible.

Analysis of Variance↗

PubMed1997

Endotoxin-induced ileal Vo2-Do2 alterations do not correlate with the severity of ileal injury.

PURPOSE: Altered Vo2-Do2 relationships are most often noted to occur in the setting of sepsis or endotoxin (LPS)-induced systemic organ microvascular injury and are generally thought to be causally linked to that injury. However, we have recently shown that ileal microvascular injury is not associated with altered ileal Vo2-Do2, relationships. Thus, we hypothesized that the severity of LPS-induced systemic organ microvascular injury would not correlate with the development of systemic organ Vo2-Do2 alterations. MATERIALS AND METHODS: To test this hypothesis, we used the in situ autoperfused feline ileal preparation to simultaneously examine microvascular permeability, reflected as the ileal lymph to plasma protein concentration ratio (CL/CP), and ileal Vo2-Do2 relationships 2 hours after intravenous LPS (0.75-2.0 mg/kg; n = 9) and in matching controls (n = 5). RESULTS: As expected, all LPS-treated animals were found to have extensive ileal histological damage and marked increases in the CL/CP compared with controls (0.308 +/- 0.019 v 0.097 +/- 0.009; P < .001). In addition, although the critical Do2 (Do2c) was elevated in the LPS-treated animals relative to controls (34.2 +/- 5.0 v 16.7 +/- 1.4 mL/min/kg; P < .03), there was no correlation between the Do2c and the CL/CP in the LPS-treated animals. Finally, ileal wet to dry weight ratios after LPS did not differ from controls. CONCLUSION: Taken together, these data suggest that factors other than organ injury, as assessed by morphological and permeability alterations, are important in the pathogenesis of altered systemic organ Vo2-Do2 relationships after LPS.

Animals↗

PubMed1997

Ischemia/reperfusion injury to the ileum does not account for the ileal VO2-DO2 alterations induced by endotoxin.

PURPOSE: Endotoxin (lipopolysaccharide [LPS])-induced systemic organ injury leads to disruption of normal systemic organ metabolic processes, which are manifest clinically by signs of accelerated anaerobic metabolism (e.g., tissue acidosis and hyperlactatemia) and altered VO2-DO2 relationships. The association of increased anaerobic metabolism with VO2-DO2 alterations has led to the notion that ischemia/ reperfusion (I/R) injury may be a prerequisite for the development of VO2-DO2 alterations during endotoxemia. However, in contrast to sepsis, in which oxygen consumption is often increased, oxygen consumption is severely decreased after I/R injury. Based on these observations, we hypothesized that I/R injury would result in systemic organ VO2-DO2 alterations, which are distinct from those that occur in sepsis. MATERIALS AND METHODS: We used the in situ autoperfused feline ileal preparation to simultaneously examine microvascular permeability, reflected as the ileal lymph to plasma protein concentration ratio (CL/CP), and ileal VO2-DO2 relationships after either intravenous LPS (2.0 mg/kg; n = 5) or I/R injury (n = 5), and in matching controls (n = 5). RESULTS: As expected, all LPS-treated and I/R-injured animals were found to have extensive ileal histological damage and marked increases in the CL/CP compared with controls (0.315 +/- 0.009 and 0.329 +/- 0.034, respectively, v 0.097 +/- 0.009; P < .001, both comparisons). In addition, the critical DO2 (DO2c) was elevated, and the critical oxygen extraction was decreased in both the I/R and LPS groups relative to controls. However, as initially hypothesized, the VO2 at the critical DO2 was markedly decreased in the I/R group compared with that of the LPS group. CONCLUSIONS: These data indicate that I/R injury is insufficient to account for the systemic organ VO2-DO2 alterations that occur with LPS injury.

Acidosis↗

PubMed1996

Acid aspiration results in ileal injury without altering ileal V(O2)-D(O2) relationships.

Systemic organ endothelial injury and V(O2)-D(O2) relationship alterations occur frequently in the setting of acute lung injury, and they are believed to play an important role in the pathogenesis of the multiple organ dysfunction syndrome (MODS). However, the relationship, if any, between systemic organ endothelial injury and V(O2)-D(O2) relationship alterations remains unknown. We hypothesized that microvascular endothelial injury and attendant interstitial edema would result in increased diffusion distances for oxygen and altered systemic organ V(O2)-D(O2) relationships. To test this hypothesis, we utilized the in situ autoperfused feline ileal preparation to evaluate ileal V(O2)-D(O2) relationships in control animals (n = 5) and in animals with HCl-induced acute lung and systemic organ injury (n = 5). As expected, ileal endothelial protein permeability (CL/CP) was increased in HCl-injured animals compared to control animals (0.187 +/- 0.024 versus 0.097 +/- 0.009; p < 0.01). However, contrary to our original hypothesis and despite marked morphologic and endothelial protein permeability alterations, ileal V(O2)-D(O2) relationships were not altered in the HCl-injured animals. Moreover, V(O2)-D(O2) relationships in the ileum remained unchanged even when ileal venous pressures were increased to 15 mm Hg. Taken together, these findings do not support an important role for oxygen diffusion limitation in the pathogenesis of altered systemic organ V(O2)-D(O2) relationships.

Animals↗

PubMed1995

Anti-CD18 antibody does not block ileal injury induced by phorbol myristate acetate.

Acute lung injury (ALI) is frequently complicated by systemic microvascular injury. In previous studies, ALI from acid aspiration resulted in ileal microvascular injury. This was due to systemic inflammation and was prevented by MoAb 60.3, an antibody against the CD18 antigen that mediates leukocyte adherence. The present study was designed to test the hypothesis that ileal microvascular injury during phorbol myristate acetate (PMA)-induced systemic inflammation is also blocked by MoAB 60.3. We assessed the injury by measuring the concentration ratios of lymph protein to plasma protein (i.e., CL/CP) at steady-state lymph flows in autoperfused cat ileum preparations. As expected, the CL/CP increased in the ilea of animals given PMA (15 micrograms/kg; n = 5) compared with the ilea of control animals (n = 5) (0.202 +/- 0.024 versus 0.106 +/- 0.010; p = 0.006) and was accompanied by widespread morphologic alterations. Intravenously administering MoAb 60.3 (2 mg/kg) to animals before the PMA infusion (n = 5) yielded a CL/CP value indistinguishable from that of the PMA group (0.222 +/- 0.024 versus 0.202 +/- 0.024; p = NS). These results suggest that CD18-mediated leukocyte adherence is not important in the mechanism of PMA-induced ileal microvascular injury.

Animals↗

PubMed1994

Loss of diaphragm glutathione is associated with respiratory failure induced by resistive breathing.

It has been suggested that oxidant stress may contribute to dysfunction of respiratory muscles undergoing severe work loads. We examined changes in glutathione content and redox status in the diaphragm and intercostal muscles of anesthetized Sprague-Dawley rats exposed to prolonged inspiratory resistive loading while breathing 70% O2. These results were compared with those from control groups breathing air or 70% O2. Changes in liver glutathione were also examined. Freeze-clamping and an enzymatic recycling assay were used. Results show that 1) in controls, glutathione content was higher in the diaphragm than in the intercostals, 2) severe hypercapnic acidosis without hypoxemia was present with loading, 3) total diaphragm glutathione decreased approximately 35% with no increase in glutathione oxidation with resistive breathing, whereas intercostal and liver glutathione remained unchanged, and 4) the drop in diaphragm glutathione correlated significantly with the drop in minute ventilation and the increase in arterial PCO2, whereas it was not directly related to intensity of respiratory muscle activity. In conclusion, although diaphragm susceptibility to oxidant stress may be increased with resistive breathing, it is unlikely that the modest decrease in total glutathione contributed significantly to respiratory failure in this model.

Airway Resistance↗

PubMed1994

Paraaminosalicylate blocks that ileal injury induced by phorbol myristate acetate.

Activation of neutrophils with the release of oxidant radicals has been implicated in the pathogenesis of gut injury in inflammatory bowel disease (IBD). The pathogenesis of gut injury in the multiple organ dysfunction syndrome associated with acute lung injury, although less focal, appears to be similar. Paraaminosalicylate (PAS) has been shown to be effective in treating IBD, most likely because of its ability to scavenge oxidant radicals. The present study was therefore designed to test the hypothesis that PAS attenuates the gut injury typically seen during systemic neutrophil activation by phorbol myristate acetate (PMA). We assessed gut injury by measuring the concentration ratio of lymph to plasma protein (CL/CP) at steady-state lymph flows in autoperfused cat ileum preparations. As expected, the CL/CP increased in animals given PMA (15 micrograms/kb; n = 6) compared with control animals (n = 5) (0.205 +/- 0.033 versus 0.118 +/- 0.004; p = 0.04) 0.04) and were accompanied by morphologic alterations. In contrast, the intravenous administration of PAS (100 mg/kg) to animals prior to PMA infusion (n = 5) yielded a CL/CP value indistinguishable from that in control animals (0.113 +/- 0.017 versus 0.118 +/- 0.004). Additional in vitro studies suggested that the protective effects of PAS were not the result of altered neutrophil margination, chemotaxis, or oxidant burst. Although PAS appeared to protect the ileum from PMA-induced microvascular injury, it had no protective effects on the lungs.

Acute Disease↗

PubMed1993

Preservation of sustainable inspiratory muscle pressure at increased end-expiratory lung volume.

Previous studies in isolated muscles have shown that decreases in muscle length reduce the loss of force resulting from fatigue in response to repeated maximal stimulations. However, increases in end-expiratory lung volume (EEV), which presumably decrease the length of the inspiratory muscles, appear to make the inspiratory muscles more susceptible to fatigue. To address this paradox, we studied the influence of changes in EEV on inspiratory muscle fatigue resulting from repeated maximal voluntary inspirations for 15 min in normal humans. Tidal volume and breath timing were constant between runs. Fatigue runs were compared with atmospheric, positive or negative pressures applied to the mouth, sufficient to change EEV by approximately +30% or -20% of inspiratory capacity. Although the maximal initial pressure-time product for the inspiratory muscles (PTmus) was reduced by increased EEV, sustainable PTmus was not significantly affected. In contrast, both initial and sustainable pressure-time products for the diaphragm were reduced at elevated EEV. The rate at which the fatigue process developed was also reduced at increased EEV. There were no significant effects of decreased EEV on any measured pressures. We conclude that when EEV is elevated, within a moderate range, sustainable inspiratory muscle pressure is preserved. However, the contribution of the diaphragm to inspiratory pressure development during fatigue may be uniquely compromised by increased EEV.

Adult↗

PubMed1993

Tumor necrosis factor and endotoxin do not directly affect in vitro diaphragm function.

Ventilatory pump failure can occur in the setting of severe infection. Recent in vivo studies have shown a significant decrease in diaphragm force production in rats with pneumococcal sepsis and sepsis secondary to Escherichia coli endotoxin. We hypothesized that diaphragm impairment during sepsis may be mediated by a direct effect of tumor necrosis factor-alpha (TNF) or endotoxin. To test this hypothesis we studied the mechanical characteristics of isolated rat diaphragm strips in tissue baths containing rTNF-alpha or endotoxin and compared the results with control strips. The strips were stimulated to contract isometrically in the tissue baths that were aerated with 95% O2-5% CO2. Baseline force-frequency determinations were made at 60 min. Following this, the strips were fatigued over a 4-min period (20 Hz, 0.33-s trains, 1 train/s) and force-frequency relationships determined 30 s, 10 min, and 60 min post-fatigue. There were no significant differences found between control and experimental strips in any aspect of contractile function tested, including force-frequency characteristics, fatiguability, and recovery from fatigue. Using an isolated cell line assay (L929), we found evidence of attenuated cytotoxicity of TNF at 26 degrees C compared with 37 degrees C. Therefore, we repeated the experiments studying the effects of TNF on in vitro muscle at 37 degrees C. We once again found no effect of TNF on contractile function. We conclude that the impairment of diaphragm function during sepsis is not mediated by a direct effect of TNF or endotoxin.

Animals↗

PubMed1991

Muscle shortening increases sensitivity of fatigue to severe hypoxia in canine diaphragm.

The effects of inspired O2 on diaphragm tension development during fatigue were assessed using isovelocity (n = 6) and isometric (n = 6) muscle contractions performed during a series of exposures to moderate hypoxia [fraction of inspired O2 (FIO2) = 0.13], hyperoxia (FIO2 = 1), and severe hypoxia (FIO2 = 0.09). Muscle strips were created in situ from the canine diaphragm, attached to a linear ergometer, and electrically stimulated (30 Hz) to contract (contraction = 1.5 s/relaxation = 2 s) from optimal muscle length (Lo = 8.9 cm). Isovelocity contractions shortened to 0.70 Lo, resulting in a mean power output of 210 mW/cm2. Fatigue trials of 35 min duration were performed while inspired O2 was sequentially changed between the experimental mixtures and normoxia (FIO2 = 0.21) for 5-min periods. In this series, severe hypoxia consistently decreased isovelocity tension development by an average of 0.1 kg/cm2 (P less than 0.05), which was followed by a recovery of tension (P less than 0.05) on return to normoxia. These responses were not consistently observed in isometric trials. Neither isovelocity nor isometric tension development was influenced by moderate hypoxia or hyperoxia. These results demonstrate that the in situ diaphragm is relatively insensitive to rapid changes in O2 supply over a broad range and that the tension development of the shortening diaphragm appears to be more susceptible to severe hypoxia during fatigue. This may reflect a difference in either the metabolic or blood flow characteristics of shortening contractions of the diaphragm.

Animals↗

PubMed1990

Sustainable inspiratory pressures over varying flows, volumes, and duty cycles.

This study identifies the influence of flow (0.5-2.0 l/s), duty cycle (0.29-0.57), and tidal volume (1.08-2.16 liters) on sustainable inspiratory muscle pressure (Pmus) and transdiaphragmatic pressure (Pdi) development. Six normal humans performed endurance tests using an isoflow method, which allowed for measurements of maximum dynamic Pmus and Pdi, with controlled lung inflation. The subjects repeated maximum dynamic voluntary inspirations for 10 min. Pressures dropped exponentially from initial measurements at rest (Pmusi or Pdi) to sustainable values (Pmus or Pdis). As flow and tidal volume increased, maximum initial and sustainable pressures decreased significantly. However, at a constant duty cycle, the sustainable dynamic pressures remained predictable fractions of initial dynamic pressures (i.e., Pmuss/Pmusi or Pdis/Pdii), regardless of changes in flow and tidal volume. In contrast, as duty cycle increased, the sustainable fractions significantly decreased for both Pdi and Pmus. For example, at a duty cycle of 0.29, Pmuss/Pmusi was approximately 0.71, and at a duty cycle of 0.57, Pmuss/Pmusi was approximately 0.62. Calculated sustainable pressure-time indexes varied significantly between 0.16 to 0.32 for Pmus and 0.11 to 0.22 for Pdi over the breathing patterns studied. We conclude that 1) the maximum dynamic pressure that can be sustained at a given duty cycle is a predictable fraction of the maximum dynamic pressure that can be generated at rest when measured under the same conditions of inspiration and 2) the sustainable fraction of initial dynamic pressure significantly decreases with increasing duty cycle.

Abdomen↗