Myocardial uptake of 99mTc-annexin-V and 111In-antimyosin-antibodies after ischemia-reperfusion in rats.

OBJECTIVES: Phosphatidylserin exposure on cell surfaces occurs early during apoptosis and is detected in vivo by using (99m)Tc-annexin-V (ANX). Cardiomyocyte membrane disruption is detected in vivo by using (111)In-antimyosin-antibodies (AM). We aimed to determine if ANX and AM allow evaluation of the time-course of these two distinct cell death events after myocardial ischemia-reperfusion. METHODS: Coronary tying (20 min) followed by reperfusion (IR) was performed in 31 rats. Twelve of the rats were injected with ANX, 11 with AM, and eight with both tracers. Myocardial uptake of tracers was studied 1-2 h, 4 h, or 24 h after IR by scintigraphy (ANX, n = 14) and autoradiography (all cases), and compared to histology and Apostain staining. RESULTS: Scintigraphy was positive in all rats 2 h after IR and in three of five rats at 24 h. On autoradiography, ANX activity was intense in myocardial lesions as early as 1 h post-IR, whereas AM activity was mild at 2 h then increased at 4 h post-IR. ANX and AM uptakes evolved from mid-myocardium to endocardial and epicardial regions from 2 h to 24 h post-IR. Apostain staining was significant in myocardial lesions (p < 10(6) compared to six sham-operated rats). On histology, myocardial lesion was characterized by interstitial oedema, myocytes necrosis, and dramatic thinning at 24 h. CONCLUSION: These data suggest that ANX and AM allow temporal and regional evaluations of PS exposure and membrane disruption, respectively, during myocytes death after 20-min myocardial ischemia followed by reperfusion. Also, (i) apoptosis starts very early in injured myocardium, (ii) myocyte necrosis occurs later (3-4 h post-reperfusion), and (iii) most dead cells are removed from mid-myocardium between 6 h and 24 h after reperfusion.

Mechanical ventilation and hemorrhagic shock-resuscitation interact to increase inflammatory cytokine release in rats.

OBJECTIVE: To determine whether hemorrhagic shock and resuscitation (HSR) and high lung stress during mechanical ventilation interact to augment lung and systemic inflammatory responses and whether their sequence affects these responses. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory. SUBJECTS: Fifty-six male Wistar rats. INTERVENTIONS: Controls were immediately killed after anesthesia. High lung stress was produced by mechanical ventilation with high tidal volume of 30 mL/kg and no positive end-expiratory pressure (HV) for 2 hrs. HSR consisted of lessening systemic arterial pressure to 30 mm Hg for 1 hr followed by reinjection of the withdrawn blood. Experimental groups consisted of HSR only and HSR preceded or followed by HV or conventional mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Interleukin-1beta, interleukin-6, and macrophage inhibitory protein 2 were determined in lung homogenate, bronchoalveolar lavage fluid, and plasma. HV ventilation alone did not increase plasma or lung cytokine content compared with controls. HSR significantly increased all mediators in lungs and plasma but not macrophage inhibitory protein 2 in plasma. Conventional ventilation, applied either before or after HSR, did not influence lung or systemic mediator release, whereas HV significantly increased mediator release when combined with HSR whatever the sequence of injuries. Lung mediator content was significantly higher in animals ventilated with HV before the HSR stress than in animals submitted to HSR and then ventilated with HV. Plasma macrophage inhibitory protein 2 concentrations followed the same pattern. CONCLUSIONS: This study shows that HSR and high lung tissue stress interact to increase lung and systemic release of inflammatory mediators in a way that depends on their sequence. Previous injury may sensitize lungs to inadequate mechanical ventilation, but inadequate mechanical ventilation may also sensitize lungs to postoperative complications.

Evaluation of 99mTc-UBI 29-41 scintigraphy for specific detection of experimental Staphylococcus aureus prosthetic joint infections.

PURPOSE: (99m)Tc-UBI 29-41 (UBI), an antimicrobial peptide, specifically targets bacteria. We tested the ability of UBI to discriminate between infected and uninfected prosthetic joints using a rabbit model previously validated. METHODS: Left knee arthroplasty was performed on 20 New Zealand rabbits, then 10(7) cfu of S. aureus (n = 12) or sterile saline (n = 8) was injected into the joint. On days 9 and 20 after surgery, planar UBI scintigraphy was performed in six infected and four uninfected rabbits, 1 h and 4 h p.i. (150 MBq), on a gamma camera. Operated-to-normal knee activity ratio (ONKR) was calculated on each scintigram. Then, after sacrifice, tissue samples of both knees were counted in a gamma counter. RESULTS: One rabbit injected with sterile saline had cutaneous infection at sacrifice and was excluded from analysis. ONKR was higher in infected than in uninfected animals 4 h p.i. 20 days after surgery: 1.75 +/- 0.48 vs 1.13 +/- 0.11, p = 0.04. From 1 h to 4 h p.i., ONKR increased in 9/12 infected and 0/7 uninfected animals. According to UBI uptake intensity and kinetics, scintigraphy was truly positive in all infected cases on day 9 and in four of six infected cases on day 20. It was truly negative in two of three sterile inflamed prosthetic knees on day 9, and in all cases on day 20. Biodistribution studies revealed increased UBI uptake in periprosthetic tissues in all animals 9 days after surgery, and only in infected animals on day 20. CONCLUSION: In this experimental study, (99m)Tc-UBI 29-41 scintigraphy permitted the early detection of acute prosthetic joint infection, and exclusion of infection in chronic sterile prosthetic joint inflammation.

99mTc-annexin-V functional imaging of luminal thrombus activity in abdominal aortic aneurysms.

BACKGROUND: The mural thrombus of abdominal aortic aneurysms (AAA) is involved in aneurysm progression via several interdependent biological processes including platelet activation. 99mTc-annexin V (ANX) is a scintigraphic tracer that binds to phosphatidylserine exposed on activated platelets and apoptotic cells. Here, we evaluated the potential of ANX imaging to assess mural thrombus biological activity in an experimental AAA model. The clinical applicability was further tested ex vivo on human samples of excised AAA thrombi. METHODS AND RESULTS: Experimental AAA was created by infusing elastase into infrarenal abdominal aorta in 17 rats, and 6 sham-operated rats were used as controls. Abdominal ANX scintigraphy was performed 2 weeks later followed by quantitative autoradiography and histological studies. Among the 13 rats which developed AAA, 11 displayed intense ANX uptake within AAA by scintigraphy. ANX uptake in the aneurysms on planar and single-photon emission computed tomography (SPECT) imaging was higher than that observed in infrarenal aorta of sham-operated controls (target/background ratio: 5.7+/-0.9 versus 1.33+/-0.21; P<0.005 for SPECT). Aneurysm-to-background activity ratios obtained by scintigraphy correlated with ANX activity in corresponding autoradiograms (R=0.69; P<0.02). This activity was located in the thrombus area where activated platelets and polymorphonuclear leukocytes accumulated. Similar patterns were also found in all of the 7 human AAA thrombi harvested during surgery. CONCLUSIONS: ANX imaging may assess mural thrombus renewal activity linked to permanent flowing blood interface.

(99m)Tc-annexin V and (111)In-antimyosin antibody uptake in experimental myocardial infarction in rats.

PURPOSE: (99m)Tc-annexin V (ANX) allows scintigraphic detection of apoptotic cells via specific binding to exposed phosphatidylserine. In myocardial infarction, apoptosis of myocytes is variable and depends especially on the presence or absence of coronary reperfusion. In this study, ANX uptake in non-reperfused experimental myocardial infarcts was compared with uptake of a marker of myocyte necrosis ((111)In-antimyosin antibodies, AM) and an immunohistochemical marker of apoptosis (Apostain). METHODS: The left anterior coronary artery was ligated in 47 Wistar rats, which were then injected with ANX (n=20), AM (n=21) or both (n=6). Myocardial uptake of ANX and AM was determined at 2 h (n=14), 4 h (n=14) and 24 h (n=19) after coronary ligation (CL), by quantitative autoradiography with (n=23) or without (n=24) gamma imaging. Heart-to-lung ratios (HLRs) and infarct-to-remote myocardium activity ratios (INRs) were calculated on the scintigrams and autoradiograms respectively. Cardiac sections were stained with haematoxylin-eosin and Apostain. The above studies were repeated in 12 normal rats. RESULTS: All rats with CL showed increased ANX and AM uptake in cardiac areas on scintigrams 24 h after CL, with HLRs higher than in controls: 3.1+/-0.6 versus 1.5+/-0.3 (p=0.001) for ANX and 1.99+/-0.44 versus 1.01+/-0.05 (p<0.0005) for AM. Autoradiography showed intense ANX and AM uptake in infarcts, with comparable topography and INRs at 2 h, 4 h and 24 h after CL (4.6+/-0.9 versus 5.0+/-1.8 at 24 h), while Apostain staining was very low (0.06+/-0.06% of cells). CONCLUSION: In this model of persistent CL, we observed increased ANX uptake in injured myocardium, comparable in intensity, topography and kinetics to that of AM. There was only minimal Apostain staining in the same areas.

Noninvasive evaluation of acute capillary permeability changes during high-volume ventilation in rats with and without hypercapnic acidosis.

OBJECTIVE: To evaluate whether hypercapnic acidosis attenuates acute alterations of pulmonary capillary permeability due to high lung stretch in rats using a simple, noninvasive, scintigraphic method. DESIGN: Prospective, randomized, controlled animal study. SETTING: University research laboratory. SUBJECTS: Male adult Wistar rats weighing 291 +/- 7.5 g. INTERVENTIONS: Three groups of rats were studied: controls ventilated with a low (6 mL/kg body weight) tidal volume and rats ventilated with a high (38 mL/kg body weight) tidal volume under normocapnic (Paco(2) = 35.2 +/- 1.65 mm Hg) or hypercapnic (Paco(2) = 102.5 +/- 5.63 mm Hg) conditions. MEASUREMENTS AND MAIN RESULTS: Pulmonary capillary permeability alterations were assessed by monitoring the rate of (111)In-transferrin accumulation in lung tissue. Respiratory system pressure-volume curves were registered and analyzed. High tidal volume ventilation increased In-transferrin plasma to lung flux in such a way that I(111)In-transferrin behaved like a marker of water. The rate of initial (first 30 mins of high tidal volume ventilation) lung transferrin accumulation measured by scintigraphy (standardized lung/heart ratio) was steady, correlated with the percent decrease in respiratory system compliance (a marker of edema progression), and did not differ between normocapnic and hypercapnic groups (18.9 +/- 3.97 vs. 14.2 +/- 2.89%/hr, not significant). However, lung In-tranferrin accumulation rate was highly scattered due to variable interindividual mechanical properties of the respiratory system. This rate was correlated with initial values of volume of the upper inflection point of the pressure-volume curve (r = -.53, p < .001) and end-inspiratory pressure (r = .54, p < .001). Mechanical properties were similar in normocapnic and hypercapnic rats. There was no difference between In-transferrin accumulation rates in these rats when a stringent selection was made based on end-inspiratory pressure (28-32 cm H(2)O) or body weight (330-360 g). CONCLUSIONS: Hypercapnic acidosis does not influence in vivo the acute increase in pulmonary capillary permeability due to high-volume ventilation.

Ventilation strategy affects cytokine release after mesenteric ischemia-reperfusion in rats.

OBJECTIVE: To evaluate the impact of different ventilation modalities on lung and plasma concentrations of cytokines in a model of secondary lung inflammation, mesenteric ischemia-reperfusion, in rats. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory of a university. SUBJECTS: Sixty-four male adult Wistar rats weighing 320-380 g. INTERVENTIONS: Eight groups were studied. Two groups underwent no surgical procedure: They were either not ventilated or ventilated with an injurious modality consisting of 30 mL/kg tidal volume (Vt) without positive-end expiratory pressure (PEEP). Animals of the other groups underwent laparotomy with or without 2-hr mesenteric ischemia followed by 4 hrs of reperfusion during which the rats were mechanically ventilated. Ventilation modalities were conventional (tidal volume 10 mL/kg, PEEP 3 cm H2O), protective (6 mL/kg, 6 cm H(2)O), or injurious (tidal volume 30 mL/kg and no PEEP). Rats were killed by exsanguination, and their lungs were excised and homogenized in buffer. Supernatants of lung homogenates and plasmas were stored at -80 degrees C for subsequent measurements. MEASUREMENTS AND MAIN RESULTS: Tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, macrophage inhibitory protein 2, and interleukin-10 were determined in lung supernatants and plasmas with a rat-specific enzyme-linked immunosorbent assay. Lung and plasma cytokine concentrations were not significantly different between rats ventilated with the injurious modality only and nonventilated rats. Lung and plasma cytokine concentrations were higher in rats that had undergone mesenteric ischemia-reperfusion than in rats with laparotomy only, whatever ventilation modality. Lung and plasma cytokine concentrations were higher in these rats after the injurious ventilation modality than after the other modalities. CONCLUSION: This study shows that an injurious ventilation does not produce significant in vivo release of cytokines in intact animals but promotes the release of pro- and anti-inflammatory cytokines in an inflammatory context.

Imaging apoptosis with (99m)Tc-annexin-V in experimental subacute myocarditis.

UNLABELLED: 99mTc-Annexin-V (ANX), which allows in vivo detection of apoptotic cells, is potentially a promising noninvasive tool to diagnose myocarditis. To test this assumption, we compared the myocardial uptake of ANX (imaging and quantitative autoradiography) in experimental subacute myocarditis (Wistar Bonn/Kobori rats [WBN/Kob]) and in normal Wistar rats. WBN/Kob is an inbred strain of Wistar rat in which myocardial injury mimicking subacute catecholamine-induced myocarditis spontaneously develops (course duration, 18 mo). The apoptotic myocardial rates were determined by immunohistochemical studies. METHODS: Fourteen WBN/Kob rats (8-10 mo old) and 12 control rats were injected with ANX (7.4 MBq/100 g). Ten-minute anterior planar thoracic images (matrix, 128 x 128) were obtained using a pinhole collimator, 1 and 4 h after injection. Heart-to-lung activity ratios were calculated on the scintigrams. Four hours after ANX injection, quantitative autoradiography of myocardial slices was performed, as well as histologic studies with hematoxylin-eosin and with a staining assay specific for apoptotic cells. RESULTS: Heart-to-lung activity ratios were higher in WBN/Kob rats than in control rats on 4-h images (2.07 +/- 0.07 vs. 1.66 +/- 0.06, P = 0.0007). Autoradiographic studies showed moderate diffuse, homogeneous myocardial ANX uptake significantly higher in WBN/Kob rats than in control rats: 54 +/- 4 versus 37 +/- 3 counts/mm(2) (P < 0.007). The apoptotic rate, evaluated with an apoptotic cell-staining assay, was 0.51% +/- 0.14% of cells in WBN/Kob rats versus 0.0042% +/- 0.0008% in control rats (P < 0.008). CONCLUSION: Compared with control rats, rats with subacute myocarditis mimicking catecholamine-induced myocarditis showed increased ANX myocardial uptake. This suggests a potential role for ANX imaging in the diagnosis of myocarditis.