Anesthesiology 2001; 94:489 –95 © 2001 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Technique for Using Video Microscopy and Indicator Dilution for Repeated Measurements of Cardiac Output in Small Animals Richard J. Rivers, M.D., Ph.D.,* Judy B. Beckman, B.S.,† Mary D. S. Frame, Ph.D.‡ Background: The authors developed an indicator dilution technique for small animals to repeatedly determine cardiac output and blood volume without cardiac instrumentation or blood sampling. Methods: Observations were made in the hamster (N  32, 70 mg/kg pentobarbital) cremaster using in vivo fluorescence videomicroscopy. Fluorescein isothiocyanate– conjugated bo- vine serum albumin (10 mg/ml) was injected as a bolus dose (right jugular) while video recording the light intensity in a 20-m arteriole (intensified charge-coupled device [CCD] cam- era at fixed gain). The intensity signal was analyzed over time (background subtracted) and calibrated to the dye concentra- tion. The ex vivo calibration was performed using a constant optical path length (20 m) and a range of dye and hematocrit concentrations. In vivo tube hematocrit was determined using standard methods with fluorescently labeled erythrocytes. Thus, quenching of the fluorescence signal by hemoglobin was corrected for the calibration, and the plasma space in the arte- riole was determined. The steady state dye concentration mea- sured by the light intensity at 2 min was not different from the dye concentration found by direct spectrophotometric analysis of the plasma. Results: Cardiac index was calculated as milliliters of blood per minute per kilogram body weight. The calculated cardiac index was 359  18 ml · min 1 · kg 1 , which is not different from the reported values for hamsters. Cardiac output was increased twofold when enough intravenous nitroprusside or nitroglycerine was injected to decrease mean arterial pressure from 90 to 70 mmHg. Cardiac output was elevated during do- butamine infusion (16 g · kg 1 · min 1 ) and decreased during esmolol infusion (50, 75 · kg 1 · min 1 ). Blood volume deter- mined from the steady state dye concentrations was 6.2  0.5 ml/100 g body weight, within the normal range for hamsters. Conclusions: Fluorescent dye dilution and video microscopy can be used to repeatedly determine cardiac output or blood volume in small animals. MEASUREMENT of cardiac output (CO) is technically challenging using the usual methods of thermodilu- tion, 1–3 microspheres, 4,5 or classical indicator dilution. 6 In small laboratory animals such as hamsters or mice, the technical difficulty is greatly amplified. There are realis- tic concerns with the extensive cardiac instrumentation required using thermodilution because the catheters cannot be made small enough to work properly while having no direct effect on cardiac function. 3 On the other hand, microspheres, or indicator dilution tech- niques, require whole-blood sampling of 0.5 ml or more per measurement, which amounts to 10 –20% of the blood volume in a 100-g animal. 5 Thus, repeat measure- ments in the same animal involves significant volume loss. There is an additional concern with repeat measure- ments using large microspheres ( 5 m) that signifi- cant microcirculatory changes occur between measure- ments because of vessel occlusion by the large particles. 4 With an increasing move toward the small animal mod- els for reasons of both cost and genomic knowledge, and to interpret peripheral changes in conjunction with sys- temic state, we developed an indicator dilution method to determine CO and blood volume in small laboratory animals. This allows direct observation of an indicator passing through a 20-m arteriole in an in vivo micro- vascular preparation. This modification is relatively non- invasive, does not require cardiac instrumentation, does not require blood sampling for the measurement, and can be used for repeated measurements in the same animals. Here we outline the method and its verification. Materials and Methods After obtaining approval from the University of Roch- ester School of Medicine and Dentistry, adult male Golden hamsters (HSD:Syr; age, 78  2 days; weight, 122  9 g [mean  SD]; N = 32) were anesthetized with pentobarbital sodium (70 mg/kg intraperitoneally) and tracheostomized. Body temperature was maintained be- tween 37 and 38°C. The following catheters were placed: a right jugular catheter (PE50, drawn to a 200-m tip) for injection of fluorescently labeled eryth- rocytes or fluorescein isothiocyanate conjugated– bovine serum albumin (FITC-BSA; dye for CO), left carotid cath- eter for injection of microspheres, left femoral arterial catheter for withdrawal of the microspheres, and left femoral venous catheter for intravenous administration of nitroprusside, nitroglycerine, dobutamine, or esmolol (dosages given in Results; catheters each PE50, drawn to approximately 100-m tip). The carotid artery catheters were placed only in studies with microspheres. The right cremaster (n = 16 animals) or left cheek pouch (n = 16 animals) was prepared for in vivo microcircu- latory observations. 7 The preparation was continuously *Associate Professor, Departments of Anesthesiology, Pharmacology and Phys- iology, and Biomedical Engineering, † Technical Associate, Department of Anes- thesiology, ‡ Assistant Professor, Departments of Anesthesiology and Biomedical Engineering. Received from the Departments of Anesthesiology, Pharmacology and Physi- ology, and Biomedical Engineering, University of Rochester School of Medicine and Dentistry, Rochester, New York. Submitted for publication September 13, 1999. Accepted for publication October 13, 2000. Supported by grant No. 55492 from the National Institutes of Health, Bethesda, Maryland (to Dr. Frame). Presented at the annual meeting of Experimental Biology, Washington, DC, April 16 –21, 1999. Address reprint requests to Dr. Frame: Department of Anesthesiology, Univer- sity of Rochester, 601 Elmwood Avenue, Box 604, Rochester, New York 14642. Address electronic mail to: molly_frame@urmc.rochester.edu. Individual article re- prints may be purchased through the Journal Web site, www.anesthesiology.org. Anesthesiology, V 94, No 3, Mar 2001 489 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/94/3/489/402801/0000542-200103000-00021.pdf by guest on 16 July 2023