Biomedical subjects

R F Potter

Publications and source records attributed to R F Potter.

At least 19 recordsLinked to original sources

PubMed2003

Cotransfection of heme oxygenase-1 prevents the acute inflammation elicited by a second adenovirus.

The acute inflammatory response elicited by adenovirus vectors results in loss of gene expression and tissue injury in the target organ. This acute inflammation is now believed to be the major limiting factor for the use of adenovirus vectors in gene therapy. While exploring the level of acute inflammation caused by the adenovirus encoding the gene for the anti-inflammatory enzyme heme oxygenase-1, we discovered that this adenovirus not only did not elicit acute inflammation, but could prevent the inflammation caused by a second adenovirus. Here we describe a new approach to gene therapy, which uses the encoding of the potent anti-inflammatory enzyme heme oxygenase-1 to prevent early host inflammatory responses normally associated with adenovirus vectors.

Acute Disease↗

PubMed2001

Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent.

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.

Animals↗

PubMed2001

Role of leukocyte accumulation and oxygen radicals in ischemia-reperfusion-induced injury in skeletal muscle.

The role of leukocytes and nonleukocyte-derived reactive oxygen metabolites (ROMs) in reperfusion-induced skeletal muscle injury was determined. Male rats received 2 h no-flow hindlimb ischemia-reperfusion (I/R, n = 6) or were rendered neutropenic via antineutrophil serum (ANS) before I/R (I/R + ANS, n = 5). Oxygen radicals in the absence of neutrophils were tested by administration of dimethylthiourea (DMTU) (I/R + ANS + DMTU, n = 5). Perfused capillaries (CD(per)) and rolling (L(r)), adherent (L(a)), and extravasated leukocytes (L(e)) in the extensor digitorum longus muscle were measured every 15 min during 90 min of reperfusion using intravital microscopy. The vital dyes bisbenzimide (BB) and ethidium bromide (EB) provided direct measures of tissue injury (EB/BB). CD(per) decreased immediately on reperfusion in the I/R and I/R + ANS groups. CD(per) in the I/R + ANS + DMTU group remained at baseline throughout reperfusion. L(a) increased in the I/R group; however, EB/BB was the same between I/R and I/R + ANS groups. Injury in the I/R + ANS + DMTU group did not differ from other groups > or =60 min, after which EB/BB became significantly lower. L(e) did not differ between groups and was highly correlated to tissue injury. The results suggest that L(e) lead to parenchymal injury, and ROMs lead to perfusion deficits during the early reperfusion period after ischemia.

Animals↗

PubMed1999

Initiation of remote hepatic injury in the rat: interactions between Kupffer cells, tumor necrosis factor-alpha, and microvascular perfusion.

Severe trauma may initiate a systemic inflammatory response, which in turn may result in remote organ injury. After limb ischemia/reperfusion (I/R), intravital fluorescence microscopy was applied to the livers of normotensive rats to investigate the initiation of remote injury to the liver. Additionally, we determined whether Kupffer cell activation and tumor necrosis factor-alpha (TNF-alpha) were involved, via perfusion deficits, in such injury. TNF-alpha, measured by immunoassay, peaked at 30 minutes of reperfusion, but returned to baseline within 60 minutes. Limb I/R resulted in significant increases to global hepatocellular injury measured by alanine transaminase (ALT) and lethal hepatocyte injury as seen with intravital fluorescence microscopy. Although the number of perfused sinusoids went unchanged, a significantly augmented perfusion heterogeneity was measured. After 1.5 hours of reperfusion, both TNF-alpha and Kupffer cells were shown to contribute to global hepatocellular injury (e.g., ALT). After 3 hours, TNF-alpha was no longer essential for this injury, suggesting that some other mechanism(s) activated Kupffer cells and initiated hepatocellular injury. Using propidium iodide and fluorescence microscopy, we found that both TNF-alpha and Kupffer cell activation were necessary to drive hepatocytes toward lethal injury. No additional benefits were observed with a combination of TNF-alpha inhibition and Kupffer cell suppression. These results not only implicate both Kupffer cells and TNF-alpha in the initiation of remote hepatic injury, but suggest that sinusoidal perfusion deficits are not essential for the initiation of such injury. Other mechanism(s) are likely involved in the pathogenesis of remote hepatic parenchymal injury.

Animals↗

PubMed1999

Cytokines contribute to early hepatic parenchymal injury and microvascular dysfunction after bilateral hindlimb ischemia.

PURPOSE: Hepatic dysfunction may contribute to death from multiple organ dysfunction after abdominal aortic surgery. Several factors are likely responsible, and the purpose of this study was to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are involved in initiating this remote hepatic injury. METHODS: In a normotensive rat model of 4-hour bilateral hindlimb ischemia/reperfusion (I/R), we measured systemic TNF-alpha and IL-1 levels throughout the I/R period. Rats were randomly assigned to either the 3-hour control group, the 3-hour I/R group, or the I/R group with administration of a polyclonal antibody (PAb) to TNF-alpha (I/R + TNF-alpha PAb). Direct evidence of lethal hepatocyte injury through the labeling of nuclei by propidium iodide (per 10(-1)mm(3)) and altered microvascular perfusion were assessed by using intravital microscopy. RESULTS: Systemic TNF-alpha peaked at 83.97 pg/mL (P <.05, n = 5) at 30 minutes of reperfusion and returned to baseline in 60 to 90 minutes. No significant change in systemic IL-1 was detected (P <.05, n = 4). Alanine aminotransferase increased 2.5-fold in the I/R group through 3 hours of reperfusion (P <.05, n = 4), and TNF-alpha PAb did not attenuate this alanine aminotransferase increase (P <.05, n = 6). Lethal hepatocyte injury increased by 8-fold in the I/R group compared with the control group (P <.05, n = 5), whereas TNF-alpha PAb significantly reduced this injury (P <.05, n = 4). No regional differences in injury were noted within the acinus. Total perfusion within the microvascular unit did not drop; however, significant flow heterogeneity was observed. The proportion of continuously perfused sinusoids declined in the I/R group after 3 hours of reperfusion in both periportal (62.0 +/- 2.2, P <.05) and, to a lesser, although significant, degree, in the pericentral regions (73. 2 +/- 1.73, P <.05). CONCLUSION: By scavenging extracellular TNF-alpha with a PAb, we provide direct evidence that TNF-alpha contributes to, but is not solely responsible for, early remote hepatocellular injury and microvascular dysfunction. The administration of TNF-alpha PAb reduced lethal hepatocyte injury in both regions of the acinus and also improved perfusion in the periportal region (76.8 +/- 5.41, P <.05), but not in the pericentral region. This suggests that TNF-alpha released during reperfusion mediates early remote hepatocellular injury and microvascular dysfunction after a remote ischemic insult.

Alanine Transaminase↗

PubMed1999

The effect of mannitol versus dimethyl thiourea at attenuating ischemia/reperfusion-induced injury to skeletal muscle.

OBJECTIVE: Mannitol is used as a treatment for skeletal muscle ischemia/reperfusion (I/R) injury in humans, despite the fact that its effectiveness in vivo is still disputed. The purpose of this study was to determine the efficacy of mannitol in attenuating I/R injury at the microcirculatory level. METHODS: The study was designed as an experimental study with male Wistar rats. The main outcome measures were intravital microscopy, which was used to measure capillary perfusion, capillary and venular red blood cell velocity (VRBC), and leukocyte-endothelial interactions in the extensor digitorum longus muscle of the rat hind limb before and after ischemia. In addition, tissue injury was assessed during reperfusion with the fluorescent vital dyes bisbenzimide and ethidium bromide. Dimethyl thiourea (DMTU), a highly effective therapeutic agent of experimental I/R injury, was used as a positive control. RESULTS: No-flow ischemia (2 hour) resulted in a 40% drop in capillary perfusion, a decline in capillary and venular VRBC, and increased leukocyte venular adherence and tissue infiltration. Tissue injury increased to a constant level during reperfusion. Mannitol attenuated capillary malperfusion during the first 60 minutes of reperfusion and prevented a decline in capillary VRBC. However, mannitol did not reduce tissue injury or leukocyte adherence and infiltration during reperfusion. By comparison, DMTU not only prevented the perfusion deficits and the increases in leukocyte venular adherence and tissue infiltration but significantly reduced the magnitude of tissue injury. CONCLUSION: Our findings suggest that mannitol may be of limited value for the prevention of early reperfusion-induced injury after no-flow ischemia in skeletal muscle. By comparison, DMTU was highly efficacious by not only reducing microvascular perfusion deficits but by also reducing leukocyte-endothelial cell interactions and the incidence of cellular injury.

Animals↗

PubMed1999

Microvascular perfusion deficits are not a prerequisite for mucosal injury in septic rats.

Our major objective was to investigate whether injury to the mucosa of the small intestine occurred in a normotensive model of sepsis and whether such injury was associated with microvascular perfusion deficits. Using fluorescence intravital microscopy, we show direct evidence of cell injury within the mucosa (pneumonia 12.4 +/- 2.6 cells/field, sham 2.2 +/- 0.7 cells/field), whereas use of 51Cr-labeled EDTA showed evidence of increased mucosal permeability (pneumonia 1.90 +/- 0.67 ml. min-1. 100 g-1; sham 0.24 +/- 0.04 ml. min-1. 100 g-1), 48 h following induction of pneumonia. Despite such injury the capillary density in the ileal mucosa and submucosa of pneumonic rats (1,027 +/- 77 and 1,717 +/- 86 mm2) was not significantly different compared with sham (998 +/- 63 and 1,812 +/- 101 mm2). However, a modest albeit significant decrease in capillary perfusion was measured in the muscularis layer of pneumonia (11.0 +/- 1.3 mm) compared with sham (13.9 +/- 0.63 mm) and appeared to be associated with leukocyte entrapment. Pretreatment using low doses of endotoxin to induce endotoxin tolerance not only increased muscularis capillary density but reduced the number of leukocytes trapped within the microvasculature, decreased myeloperoxidase activity within the ileum in pneumonic rats, and prevented mucosal injury. In conclusion, we have shown that pneumonia results in remote injury to the mucosa of the ileum and that such injury was not associated with concurrent mucosal perfusion deficits.

Animals↗

PubMed1999

Microcirculatory perfusion deficits are not essential for remote parenchymal injury within the liver.

A normotensive model of hindlimb ischemia-reperfusion in Wistar rats was used to test the hypothesis that microvascular perfusion deficits contribute to the initiation of remote hepatic injury during a systemic inflammatory response. Animals were randomly assigned to one of three groups: 4 h of ischemia with 6 h of reperfusion (I/R-6; n = 4), 4 h of ischemia with 3 h of reperfusion (I/R-3; n = 5), or no ischemia (naive; n = 5). With intravital fluorescence microscopy, propidium iodide (PI; 0.05 mg/100 g body wt) was injected for the in vivo labeling of lethally injured hepatocytes (number/10(-1) mm(3)). PI-positive hepatocytes increased progressively over the 6-h period (naive 32.9 +/- 7.8 vs. I/R-3 92.8 +/- 11.5 vs. I/R-6 232 +/- 39.2), with no difference between periportal and pericentral regions of the lobule. Additionally, a significant decrease in continuously perfused sinusoids (naive 70.0 +/- 1.5 vs. I/R-3 65.0 +/- 1.0 vs. I/R-6 48.8 +/- 0.9%) was measured. Regional sinusoidal perfusion differences were only observed after 3 h of limb reperfusion. Indirect measures of hepatocellular injury using alanine transaminase levels support the progressive nature of hepatic parenchymal injury (0 h 57.8 +/- 6.5 vs. 3 h 115.3 +/- 20.7 vs. 6 h 125.6 +/- 19.5 U/l). Evidence from this study suggests that remote hepatic parenchymal injury occurs early and progresses after the induction of a systemic inflammatory response and that microvascular perfusion deficits are not essential for the initiation of such injury.

Animals↗

PubMed1998

Ischemic tolerance in skeletal muscle: role of nitric oxide.

We tested the hypothesis that ischemic preconditioning (PC) of skeletal muscle provided tolerance to a subsequent ischemic event 24 h later, and that such protection was due to nitric oxide (NO). Male Wistar rats, anesthetized with halothane, were randomly assigned to groups: ischemic (no PC; n = 11), PC (n = 11), PC + N-nitro-L-arginine methyl ester (L-NAME; 100 micromol/l; n = 5), PC + N-nitro-D-arginine methyl ester (100 micromol/l; n= 4), PC + aminoguanidine (AMG; 100 micromol/l; n = 4), ischemic + L-NAME (n= 4), or ischemic + AMG (n = 4). PC consisted of 5x 10 min of ischemia and reperfusion, and, 24 h later, 2 h of ischemia were induced by a tourniquet applied to the limb. With the use of intravital microscopy, the number of perfused capillaries (Npc) in the extensor digitorum longus (EDL) muscle was measured over a 90-min reperfusion period. The ratio of ethidium bromide- to bisbenzimide-labeled nuclei was used to estimate tissue injury. PC preserved Npc (23.6 +/- 2.5) following 2 h of ischemia compared with sham muscles (11.5 +/- 5.1), significantly elevating inducible NO synthase (iNOS) activity (81% increase), but did not afford protection to the parenchyma. L-NAME and AMG prevented ischemia-reperfusion-induced reduction in Npc in muscles without PC. However, after 90 min of reperfusion, L-NAME (Npc = 15.0 +/- 1.7), but not AMG (Npc = 22.8 +/- 3.1), significantly reduced the microvascular protection afforded by PC. We conclude that PC of the EDL muscle resulted, 24 h later, in protection to microvascular perfusion only, and that such protection was due to NO from sources other than iNOS.

Animals↗

PubMed1998

Leukocyte activation and flow behavior in rat skeletal muscle in sepsis.

In animal models of endotoxemia, sepsis is associated with the accumulation of leukocytes and altered microvascular perfusion. In order to test the hypothesis that bacterial sepsis upregulates leukocyte-endothelial adhesion, we used intravital microscopy to examine the flow behavior of leukocytes in the postcapillary venules (PCV) of rats made septic by cecal ligation and perforation (CLP). Animals were randomized to CLP or sham study groups and studied 6 h, 24 h, or 48 h later. In postcapillary venules of the extensor digitorum longus muscle, we found that: (1) over the course of the study, leukocyte adhesion and extravasation increased in both experimental groups (analysis of variance [ANOVA], significant time effect: adhesion, p < 0.001; extravasation, p < 0.05); (2) leukocyte adhesion was decreased by CLP treatment (ANOVA, sepsis effect, p = 0.05), particularly after 24 to 48 h of sepsis (ANOVA, sepsis x time interaction, p < 0.05); and (3) the reduction in leukocyte adhesion in CLP animals was associated with a decrease in leukocyte extravasation (ANOVA, sepsis effect, p < 0.01). After correction for the reduction in systemic leukocyte count associated with CLP, the effect of sepsis on leukocyte adhesion and extravasation no longer reached statistical significance. These findings suggest that chronic (6 to 48 h) bacterial sepsis does not upregulate leukocyte adhesion in a manner similar to that seen in models of acute endotoxemia. These data suggest that the increased microcirculatory flow heterogeneity seen in this and other models of bacterial sepsis may not be explained by leukocyte entrapment in postcapillary venules.

Acute Disease↗

PubMed1997

Transfusing red blood cells stored in citrate phosphate dextrose adenine-1 for 28 days fails to improve tissue oxygenation in rats.

OBJECTIVE: To determine whether the time that red blood cells are stored in citrate phosphate dextrose adenine-1 solution before transfusion alters the ability to improve tissue oxygenation. DESIGN: Prospective, randomized, controlled study. SETTING: University research institute laboratory. SUBJECTS: Male Sprague-Dawley rats (350 to 450 g). INTERVENTIONS: Twenty-four hours after randomization to sham laparotomy (n = 21) or cecal ligation and perforation (n = 16)1 supply-dependency of systemic oxygen uptake (VO2) was induced in rats by isovolemic hemodilution. Rats were then re-randomized to receive either rat red blood cells stored in citrate phosphate dextrose adenine-1 for 3 days ("fresh" n = 17) or rat red blood cells stored in citrate phosphate dextrose adenine-1 for 28 days ("old" n = 20). MEASUREMENTS AND MAIN RESULTS: Changes in systemic VO2 were measured for 90 mins to determine the efficiacy of the treatment. Statistical analysis included a fully factorial repeated-measures, generalized linear model. No significant interaction was found between cecal ligation and perforation or sham animals and transfusion with fresh or old red blood cells. However, comparing the combined groups of animals receiving either fresh or old red blood cells, we found that after the transfusion of old red blood cells, systemic VO2 was not significantly improved (after hemodilution 1.68 +/- 0.27 mL/100 g/min, after transfusion 1.86 +/- 0.17 mL/100 g/min; p > .05). In contrast, transfusion with fresh red blood cells acutely increased systemic VO2 (after hemodilution 1.62 +/- 0.06 mL/100 g/min, after transfusion 2.10 +/- 0.09 mL/100 g/min; p = .049). CONCLUSION: Storage of rat red blood cells for 28 days in citrate phosphate dextrose adenine-1 impaired their ability to improve tissue oxygenation when transfused into either control or septic rats placed into supply dependency of systemic VO2.

Adenine↗

PubMed1997

Animal models for blood transfusion: a model for sterile blood sampling in the rat.

Rat blood is frequently used for experimental transfusion. However, no data are available concerning the quality of the blood used, although bacterial contamination could severely alter results. To obtain large quantities of sterile rat blood for a transfusion study, we tested carotid artery cannulation, known as a standard procedure. Blood cultures from the collected blood showed polymicrobial overgrowth even after sterility measures were improved. In contrast, the puncture of the abdominal aorta proved to be a simple and reliable method for the collection of sterile blood. We conclude that studies using blood collected from donor rats should be controlled and the quality of such blood be tested before transfusion.

Animals↗

PubMed1996

Leukocyte activity and tissue injury following ischemia-reperfusion in skeletal muscle.

Few studies have correlated the occurrence of leukocytes with the time course of ischemia-reperfusion (I-R)-induced tissue injury in skeletal muscle. The goal of this study was to test the hypothesis that leukocytes were responsible for not only the onset, but progression of parenchymal cell injury within skeletal muscle following no-flow ischemia. Thirteen male Wistar rats (150-250 g) were randomly allocated to either a control (no I-R; n = 3), I-R (n = 5), or a leukopenic I-R group (n = 5). Under halothane anesthesia, the extensor digitorum longus muscle was prepared for intravital microscopy to allow video recording of microvascular perfusion and leukocyte flow behavior following 3 hr no-flow ischemia of the hindlimb. Tissue injury was assessed as the ratio of ethidium bromide (impermeant dye)-labeled nuclei to bisbenzimide (permeant dye)-labeled nuclei (E/B). During reperfusion, the I-R group showed a progressive decline in the number of perfused capillaries (N(C)) (from 19.37 +/- 0.04 to 3.34 +/- 1.18), while leukopenic and control rats were not significantly different. In the I-R group, the number of rolling leukocytes increased from 4.05 +/- 1.93 to 14.77 +/- 1.33 at the onset of reperfusion and remained stable throughout the reperfusion period. The number of stuck leukocytes, in the I-R group, progressively increased from 1.41 +/- 0.01 prior to ischemia to 4.66 +/- 0.01 at the onset of reperfusion to 11.96 +/- 0.01 after 90 min. The index of tissue injury (EIB) increased asymptotically from 0.60 +/- 0.02 to 0.95 +/- 0.01 after 90 min of reperfusion in the I-R group, while leukopenia significantly reduced both the magnitude of tissue injury (i.e., 35% reduction from untreated I-R group) and the onset of such injury. In spite of the benefit afforded by leukopenia, evidence of tissue injury persisted (20% above control baseline level). We conclude that although leukocytes were responsible for the onset of parenchymal injury in skeletal muscle following 3 hr no-flow ischemia they are not the sole mediators of such injury.

Animals↗

PubMed1996

Intestinal mucosal permeability to 51Cr-ethylenediaminetetraacetic acid is increased after bilateral lower extremity ischemia-reperfusion in the rat.

BACKGROUND: Despite successful revascularization of ischemic extremities, multiorgan dysfunction syndrome develops in some patients. Mechanisms responsible for this are not known; however, the gastrointestinal tract has been implicated as a possible mediator. Our objective was to demonstrate increased intestinal mucosal permeability after bilateral hindlimb ischemia-reperfusion (I-R) in a rodent model. METHODS: Sixteen male Wistar rats were randomized either to 4 hours of bilateral hindlimb tourniquet ischemia and 24 hours of reperfusion (n = 8) or control groups (n = 8). The animals received 10 MuCi 51Cr-ethylenediaminetetraacetic acid (EDTA) by gavage, and excretion was measured in urine collected every 8 hours in 16 animals and every 4 hours in 8 animals. Arterial blood pressure was monitored continuously. Intravenous normal saline solution (3 ml/hr) with fentanyl (2 microgram/100 gm/hr) was continuously administered. Immediately before death complete blood count and levels of arterial lactate, creatinine, and urea were obtained. Mesenteric lymph nodes were harvested from the ileocecal region and cultured. Distal small bowel and proximal colon were preserved for histologic analysis. An additional 11 rats, six experimental and five control, were evaluated for mesenteric lymph node cultures only. RESULTS: Urinary excretion of 51Cr-EDTA was significantly greater in the I-R group between 0 and 8 hours (p < 0.02) and 8 to 16 hours (p < 0.0002) of reperfusion. This increase occurred as early as 4 to 8 hours of reperfusion (p < 0.0001). Urine volume in the I-R group was significantly reduced during 0 to 4 hours of reperfusion (p < 0.002). Hemoglobin and lactate level were significantly different in the I-R group. Leukocyte and platelet counts, levels of creatinine and urea, and colony counts from mesenteric lymph nodes were similar in I-R and control groups. Blinded histologic analysis of bowel segments did not reveal morphologic differences. CONCLUSIONS: Bilateral hindlimb I-R produces remote intestinal mucosal injury shown by significantly increased permeability to 51Cr-EDTA. Such increased mucosal permeability may be important in the development of multiorgan dysfunction syndrome in patients who sustain lower extremity I-R injury.

Animals↗

PubMed1996

Microcirculatory changes in rat skeletal muscle in sepsis.

The aim of this study was to confirm that microvascular perfusion was abnormal during the early phases of normotensive sepsis and to determine whether these changes were due to the development of tissue edema. Skeletal muscle red blood cell (RBC) flow was studied in rats made septic by cecal ligation and perforation (CLP). After anesthesia with halothane, arterial and venous cannulae were inserted and, in the treatment group, a CLP performed. At 6, 24, and 48 h after entry into the study, the incidence of microcirculatory absence of flow in the extensor digitorum longus muscle (EDL) was examined with intravital microscopy. The number of capillaries containing RBCs were counted over a 60-s interval, and the flow status of each capillary was recorded. A significant increase in the number of stopped-flow capillaries was observed in the CLP group (p < 0.01) as compared with time-matched controls. In both groups the number of capillaries with stopped flow was greater than in naive animals. The severity of absence of flow was negatively correlated with the systemic hemoglobin concentration. These changes were not associated with an increase in tissue wet/dry weight ratio or albumin flux. This study shows that sepsis was associated with increased RBC flow heterogeneity. These changes, which occur within 24 h of the septic insult, are a persistent feature of the evolving septic process in the absence of tissue edema. These observations support the view that extrinsic compression of the microcirculation by tissue edema is not the primary cause of alterations in microcirculatory flow in sepsis.

Animals↗

PubMed1995

Measurement of tissue viability using intravital microscopy and fluorescent nuclear dyes.

Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injury following disease processes. However, this assumes that prolonged exposure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (EDL) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbital (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbenzimide (5 micrograms/ml; labels nuclei of all cells) and ethidium bromide (5 micrograms/ml; labels nuclei of injured cells) or had dyes topically applied 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. Noxious stimuli (i.e., hypoxia:FiO2 of 8-10% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were used to test the ability of ethidium bromide, when used in conjunction with intravital microscopy, to differentiate injured tissue. Video recordings at the surface of the EDL muscle were made every 30 min for 5 hr from which the number of perfused capillaries was counted (NCper). The numbers of bisbenzimide- and ethidium bromide-labeled nuclei were counted at the surface of the muscle and at two to three additional locations within the muscle (to a maximum depth of approximately 120-160 microns). The average NCper (19.05 +/- 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 +/- 125.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Skeletal muscle injury induced by ischemia-reperfusion.

OBJECTIVE: To study the temporal progression of injury in skeletal muscle after ischemia-reperfusion insult by means of intravital videomicroscopy and nuclear fluorescent dyes. DESIGN: A controlled study in an animal model. SETTING: A vascular research laboratory at a university-affiliated hospital. SUBJECTS: Eight male Wistar rats, for each of which the extensor digitorum longus muscle of the hind limb was exposed and prepared. INTERVENTIONS: Two hours of complete no-flow ischemia followed by 90 minutes of reperfusion in five of the rats; the other three rats acted as controls and underwent the same surgical procedure but not ischemia. During the reperfusion period the fluorescent vital dyes bisbenzimide, which permeates all cells, and ethidium bromide, which permeates cells with damaged membranes, were applied. Recordings to videotape were made with the intravital microscope very 15 minutes during the reperfusion period. MAIN OUTCOME MEASURES: The number of perfused capillaries crossing three straight lines on the video monitor were counted as a measure of microvascular dysfunction. An index of tissue injury was calculated as the ratio of the number of nuclei stained by ethidium bromide to the number stained by bisbenzimide (E/B). The number of stuck and rolling leukocytes and the velocity of the rolling leukocytes were determined in postcapillary venules. RESULTS: The mean number of perfused capillaries (and standard error of the mean) fell from 20.71 (1.64)/mm before ischemia to 11.69 (1.18)/mm during reperfusion in the experimental group but remained constant in the control group. In the experimental group E/B progressed from 0.43 (0.05) at the onset of reperfusion to 0.87 (0.03) at the end of reperfusion, the number of rolling leukocytes increased from a preischemia mean of 4.00 (1.90) to 14.80 (1.30)/1000 microns2, and the number of stuck leukocytes increased from 1.42 (0.20) to 9.20 (0.70)/1000 microns2. The velocity of the rolling leukocytes did not differ between the control and the experimental groups. CONCLUSIONS: Although microvascular perfusion decreased quickly to a constant level after 2 hours of noflow ischemia plus reperfusion, a progressive increase in tissue injury occurred, which may correlate with the number of stuck leukocytes.

Animals↗

PubMed1993

Ischemia-reperfusion induced microvascular dysfunction in skeletal muscle: application of intravital video microscopy.

Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.

Animals↗