At the end [of a lecture on astronomy by Bertrand Russell] a little old lady at the back of the room got up and said: `What you have told us is rubbish. The world is really a flat plate supported on the back of a giant tortoise.' The scientist gave a superior smile before replying, `What is the tortoise standing on?' `You're very clever, young man, very clever,' said the old lady, `but it's turtles all the way down!'

Steven Hawking (1988)

A Brief History of Time

5.1 Animal Selection

In order to demonstrate the utility of simultaneous optical and NMR spectroscopy, we chose to investigate the cardiac physiology of the western painted turtle Chrysemys picta bellii, specifically the response to prolonged anoxia. It has been shown previously that the cardiomyocytes and whole isolated hearts of these turtles exhibit remarkable tolerance to hypoxic conditions maintaining calcium homeostasis even as oxidative energy stores are depleted [18]. In this chapter, the preparation and instrumentation of animal subjects as well as data collection methods are detailed. Animal subjects were western painted turtles (Chrysemys picta bellii) weighing approximately 300 g and small enough to fit inside of the 180 mm diameter gradient set of the NMR system.

5.2 Gross Preparation

Animals were anesthetized by intramuscular injections of ketamine (40 mg/kg) and diazepam (0.4 mg/kg) and were prepared by cutting the bridges on either side of the plastron (lower shell) and dissecting away the skin surrounding the limb pockets allowing for removal of the entire plastron. This preparation is similar to that described in [46]. Figure 5.1 below shows the anatomical relationship of the heart to the body and a diagram of a heart similar to that of Chrysemys picta bellii.

Figure 5.1: Relative Anatomy of the Turtle Chrysemys scripta. (Reprinted from [46] with permission of The Company of Biologists, Ltd.)

5.3 Pericardial Loading of fluo-3/AM

Loading of the fluorescent calcium indicator fluo-3/AM was accomplished by bathing the heart in a solution of the indicator made from TES Ringer's electrolyte solution (The composition of TES Ringer's solution is included in APPENDIX C.) The solution was prepared by dissolving 50 mg of fluo-3/AM in 20 mL of dry dimethyl sulfoxide (DMSO). This was then mixed with a 20 mL of a 20% (weight/volume) solution of F-127 pluronic dispersing agent in DMSO. The resulting 40 mL of fluo-3/DMSO solution was then added to 3 mL of TES Ringer's solution and kept in the dark until it was to be used. Bathing of the heart was accomplished by taking advantage of the fairly unique anatomy of the turtle in which the heart floats in a large loose pericardial sac. Using a small gauge needle on a syringe the pericardial fluid (approximately 1.5 mL) surrounding the heart was withdrawn while leaving the pericardial membrane in tact. This fluid was replaced with half (1.5 mL) of the fluo-3/AM solution, and bathing ensued for 45 minutes during which time the animal was tidally ventilated through the tracheostomy tube. After an initial 45 minute period, the fluo-3/AM solution was withdrawn from the pericardium and replaced with the other unused 1.5 mL. Bathing continued for another 45 minutes during which time further preparation was performed on the animal. At the end of the second bathing period, the pericardium was resected (cut away) and the heart was bathed thoroughly with clean TES Ringer's solution to remove any unabsorbed fluorophore.

In order to ensure that sufficient AM loading was taking place via this loading protocol, we conducted experiments to monitor fluorescence on a SPEX fluorolog-2 spectrafluorometer. Results of these studies are described in Chapter VI.

5.4 NMR Coil and Optical Fiber Placement

After the pericardial sac surrounding the heart was resected, the ligamentous aponeurosis (frenulum) at the apex of the heart was clipped and used to pull the ventricle of the heart up into the solenoidal coil where it was fixed with a small amount of tissue cement. After fixation of the heart in the coil, the animal was placed into a 4 inch diameter plexiglass tube with an access port cut into the top. Foam pads were wedged between the sides of the shell and the walls of the tube to hold the animal in place. A fiber-optic probe was positioned for sensing of optical fluorescence in the ventricle and fixed into place via a specially designed plastic guide. The fiber was brought within 1 mm of contact with the ventral surface of the ventricle of the exposed heart in the coil. After placement of the fiber-optic, the cables, tubing, and fiber are taped into place on the tube. The resonator was pretuned for resonances at 34.63 and 85.56 MHz, so only the feed coils need be adjusted. Both ³¹P and ¹H channels were matched to 50 W using an HP 4195A network analyzer in the S₁₁ mode. The tube containing the animal was then placed into the bore of the superconducting magnet. Figure 5.2 below is a photograph showing the in situ coil and optical fiber.

Figure 5.2: Dual Spectroscopy Preparation. Animal subject has been fitted with coil and fiber-optic and sits inside the specially designed plastic sample holder.

5.5 Unidirectional Ventilation Technique

Though they do not have a diaphragm, per se , terrestrial turtles do breathe via generation of a negative pressure inside of the thorax in much the same way mammals do [23]. Because the preparation used here leaves the thorax cavity open and exposed to air, the animal has essentially suffered an induced pneumothorax and is unable to breathe of its own volition. Therefore, the animal must be ventilated to maintain blood oxygenation.

In preliminary experiments, a tracheostomy was performed and manual tidal inflation and deflation of the lungs was used to oxygenate the animal. The results of this technique proved unsatisfactory, however, since tidal inflation of the lungs causes movement of the NMR coil relative to the heart and also a change in the magnetic field shim in the region of interest inside the magnet.

Instead, ventilation of the animal was effected by blowing moisturized gas through a tracheostomy tube in the trachea and out through a cannula inserted and tied into the apex of the left lung. The ventilation gases passed through the bronchi, the lung, and out through the cannula. Figure 5.3 is a schematic of the breathing setup employed. It was found that lungs of the animal inflated bilaterally and uniformly to a fixed and constant volume roughly determined by the gas flow rate.

In order to explore the efficacy of this novel ventilation technique, analysis of arterial blood samples from animal subjects ventilated both tidally and unidirectionally was performed. The results of these experiments and a discussion of the findings are presented in Chapter VI.