Assessment of Multiple Cardiocentesis in Ball Pythons (Python Regius)

Assessment of Multiple Cardiocentesis in Ball Pythons (Python regius)

RAMIRO ISAZA, DVM, MS, DIPLOMATE, ACZM,1,* GORDON A. ANDREWS, DVM, PHD,2 DIPLOMATE, ACVP, ROB L. COKE, DVM,1 AND ROBERT P. HUNTER, MS, PHD3

This study evaluated the gross and microscopic effects of serial blood collection from six ball pythons (Python regius) by using cardiocentesis. We collected 39 blood samples from each snake over a 120-day period. Cardiocentesis was performed on manually restrained snakes, with each sample requiring ∼15 sec to collect. No clinically apparent complications were noted in any of the snakes after the cardiocentesis procedures, and all snakes survived until they were euthanized 73 days after the last blood sample. Minimal gross lesions were noted at necropsy; faint brown pigmentation of the pericardium was present in five of six snakes, and three snakes had ∼0.5 ml dark pigmented fluid in the pericardial space. One snake had a small, organized hematoma in the pericardial space. Microscopic findings were limited to moderate and regularly arranged collagen fibrosis and focal thickening of the epicardium. The pericardial sac in all snakes had a mild infiltrate of hemosiderin-laden macrophages and small numbers of heterophils. The results suggest that serial cardiocentesis is well tolerated in ball pythons.

During the evaluation of the protocol for a pharmacokinetic study in Materials and Methods snakes (1, 2), our Institutional Animal Care and Use Committee Animals. Six ball pythons (two male and four female; weight, 0.81 (IACUC) questioned the use of multiple cardiocentesis as a safe and to 0.85 kg) were used for this study. The snakes were housed indi- humane method of collection of multiple blood samples from con- vidually in 114-L aquaria with newspaper substrate and screen tops. scious snakes. When studies are proposed that include the collection of The snakes had ad libitum access to a hide-box and water bowl. The multiple cardiocentesis samples in mammals, legitimate questions of cages were housed in a thermostatically controlled room at 30°C cardiac damage, risks of the procedure, and stress and pain are often with a 12:12-h light:dark cycle. Physical examinations were performed raised. Similar concerns can be applied to experiments involving snakes, prior to the start of the study, after every cardiocentesis, and daily but to the authors knowlege no studies that assess the effects of mul- during the rest phase prior to euthanasia. This study was part of a tiple cardiocentesis in snakes had been published previously. Members pharmacokinetic and drug metabolism study approved by the IACUC of our IACUC suggested that after the conclusion of the studies, an of Kansas State University (1, 2). assessment of the effects of multiple cardiocentesis in snakes would help Cardiocentesis procedure. Over a 120-day period, we collected address these concerns and provide valuable information. 39 blood samples from each snake. Cardiocentesis was performed Superficial blood vessels in snakes are neither visibly apparent on manually restrained snakes, with each sample taking approxi- nor easy to palpate. This situation provides a challenge to the phle- mately 15 sec to collect. The location of the heart was estimated by botomist that must collect multiple blood samples from snakes in measuring 20% to 40% the length of the body from the tip of the an experimental study. Of the described methods of collecting per- rostrum. The exact location of the heart was then identified by ei- cutaneous blood samples from snakes, cardiocentesis is described as ther direct observation of beating through the ventral scutes, the method of choice in seven reptile medicine textbooks (3-9). In palpation of the heart within the ceolomic cavity, or detection of contrast, the authors of two articles have claimed that cardiocentesis blood flow by using Doppler ultrasonography. Prior to the start of sampling in snakes is stressful and dangerous (10, 11). None of the the study, the location of the heart was marked on each snake to references commented on the chronic effects of multiple help with subsequent blood collection. cardiocentesis. An assistant manually restrained the snakes in dorsal recumbency, A review of published pharmacokinetic drug studies in snakes found and the skin was aseptically cleaned with repeated applications of both that 9 of 11 used multiple cardiocentesis as the method of collecting 2% chlorhexidene solution and 10% isopropyl alcohol. Because the blood samples (12-22). In these studies, all cardiocentesis were per- heart is movable within the ceolomic cavity, the sample-taker placed formed on manually restrained, nonanesthetized, or sedated snakes, a thumb caudal to the heart and gently advanced it forward. The index and each snake underwent 4 to 14 (mean, 8) cardiocentesis blood finger of the same hand then was placed cranial to the heart so that the collections. None of these studies reported morbidity or mortality heart was further fixed in position. A 22 or 25 gauge needle attached to data from the blood collection technique, and no follow-up observa- a 3-ml syringe was placed between and under ventral scutes located a tions or histopathologic assessments of the hearts were reported. few millimeters cranial to the thumb and over the ventricle of the The purpose of the present study was to observe a group of ball heart. The needle was advanced cranially and dorsally until it en- pythons undergoing multiple cardiocentesis procedures during a ter- tered the ventral aspect of the ventricle. Blood usually entered the minal pharmacokinetic study and then to document the gross and syringe slowly, often only during diastole of the heart. Approximately microscopic long-term effects of multiple cardiocentesis in these animals. 0.75 ml of blood was collected for each sample. After the procedure the snakes were returned to their cages for observation. Department of Clinical Sciences,1 Department of Diagnostic Medicine and Pathobiology,2 Necropsy and histological preparations. The snakes were and Zoological Pharmacology Laboratory, Department of Anatomy and Physiology,3 Col- euthanized on day 120 of the study (73 days after the last lege of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506 cardiocentesis sample). All the snakes were submitted for complete *Corresponding author: Ramiro Isaza, DVM, MS, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, P.O. Box 100126, University of Florida, gross necropsy; three of the six snakes were unavailable for histo- Gainesville, Florida 32610-0126 pathologic evaluation. The heart, epicardium, and pericardial sac were

Volume 43, No. 6 / November 2004 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science 35 Figure 1. Photograph of a ball python heart after collection of 39 cardiocentesis blood samples over a 120-day period. P, pericardium; V, ventricle. placed into buffered formalin and submitted for routine histopatho- logical evaluation. The hearts from three ball pythons of similar size and age that had no cardiocentesis performed were examined as normal control tissues. Tissues submitted for microscopic examination were formalin-fixed (10% buffered neutral formalin), routinely processed, sectioned at 4µm, and stained with hematoxylin and eosin. Sections also were stained with Massons trichrome stain to evaluate the degree of fibrosis present in the myocardium, epicardium, and pericardial sac.

Results All snakes were normal upon physical examination prior to the beginning of the study, they remained clinically normal throughout the study, and all snakes survived to the end of the study. No clinically apparent complications were noted in any of the snakes after the cardiocentesis procedures. Figure 2. Photomicrographs of the heart from an unmanipulated control Gross lesions in the six snakes were confined to the heart. Abnor- (A) and an experimental (B) ball python after collection of 39 cardiocentesis malities included faint brown pigmentation of the pericardium in blood samples over a 120-day period. E, epicardium; M, myocardium. five of the snakes, and the presence of approximately 0.1 ml of dark Massons trichrome stain; magnification, 10×. pigmented fluid in the pericardial space in three of the snakes (Fig. 1). Focal adhesions of the pericardium to the ventricles were found in two of the snakes. A small, well-organized hematoma was found blood cells, and foci of crystallized hemoglobin pigment surrounded in the pericardial space of one snake. by multinucleated giant-cell macrophages suspended in fine fibrous Histologically, the pericardial sac in the control snakes was a thin connective tissue. membrane composed of parallel arrays of collagen with small num- In the experimental snakes the filamentous attachments between the bers of fibroblasts and lined by a single layer of mesothelial cells. The epicardium and myocardium were more pronounced, wider, and com- pericardial sac of the three experimental snakes contained a mild in- posed of more mature collagen than those in the control animals. filtrate of hemosiderin-laden macrophages and small numbers of Frequently fibrosis extending into the myocardium was present at the heterophils. No differences in thickness or amount of collagen in the point of attachment of these filaments. Neither the myocardium nor pericardial sacs were seen between the control and experimental snakes. endocardium differed histologically between the two groups of snakes. The epicardium in the control snakes was a membrane composed of a loose reticular network or meshwork of connective tissue (Fig. 2A). Discussion The epicardium contained occasional capillaries and nerve ganglia. This was an opportunistic, observational study to address IACUC The control epicardium varied markedly in thicknessfrom approxi- concerns about the long-term effects of serial cardiocentesis in snakes. mately 20 µm to as thick as 300 µmamong the sections and within A total of 39 serial cardiocentesis samples were collected from each an individual snake heart. The control epicardium almost always had of 6 snakes, and the gross and histological effects of repeated a loose reticulum and was attached to the myocardium by fine fila- cardiocentesis were examined 73 days after the last sampling. In light ments of collagen. of the physical examination and behavior of the snakes, the multiple The epicardium of the experimental snakes tended to be approxi- cardiocentesis appeared to have limited clinical consequences. After mately of the same thickness as those of the controls but composed each cardiocentesis the snakes had apparently normal heart function of more mature, densely arranged collagen and containing a diffuse as was evident by their return to normal behavior. Unfortunately, infiltrate of small numbers of hemosiderin-laden macrophages and subtle changes in heart function were not measured by ultrasonogra- heterophils (Fig. 2B). In the snake with the hematoma, the epicar- phy, radiography, or electrocardiography; these measurements would dium contained a focal thickening filled with hemosiderin-laden have been a supportive addition to this study. The gross and histo- macrophages, lymphocytes, a few plasma cells and heterophils, red logical findings were limited to evidence of mild chronic pericardial

36 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science Volume 43, No. 6 / November 2004 hemorrhage and moderate epicardial fibrosis. Serial cardiocentesis in snakes is associated with several potential problems. The use of poor technique requiring multiple attempts to collect the blood sample has been suggested as a cause of trauma to the heart and associated structures (3-9). Laceration of vascular, pul- monary, or gastrointestinal structures would be expected to cause increased morbidity or mortality. We recommend the techniques used in the present study, which were performed by persons experienced in cardiocentesis in snakes, in order to make blood collection as safe and atraumatic as possible. 1) The hearts position should be determined definitively prior to introduction of the needle. 2) The heart should be immobilized between the fingers prior to introducing the needle. 3) The snake must be firmly restrained. 4) Once the heart is entered, the phlebotomist should be patient and allow for the relatively slow collection of blood. Often blood is collected only during diastole, and there is a temptation to needlessly redirect the needle. Physical restraint has been the accepted and is the preferred method of performing cardiocentesis in snakes (3-9). The effects of short pe- riods of physical restraint in ball pythons has been determined to produce minimal stress (23). The stress of cardiocentesis in combination with physical restraint has not been studied in snakes, but a single cardiocentesis may be similar to manually restrained mammals subjected to manual restraint for venipuncture. The momentary stress and pain caused by cardiocentesis appears to cease when the snake returns to the cage. Snakes react to noxious cutaneous stimulation, such as that caused by a needle entering the skin (24), and therefore likely are perceiving pain during any phlebotomy including cardiocentesis. It is currently unknown if this perception of pain differs from that of phlebotomy techniques used in mammals. General anesthesia may be effective Figure 3. Schematic drawing illustrating of the path of a cardiocentesis needle for a single blood collection but is of limited use for serial cardiocentesis entering the heart of a snake (A) and a mammal (B). Note that in the snake the needle passes through fewer structures before entering the heart. sampling over an extended period of time, as is needed for pharmacokinetic studies. The use of local analgesia prior to cardiocentesis has not been described, but local anesthetics such as lidocaine in Snakes lack a diaphragm and a true pleural cavity (24, 30). Expan- snakes have been mentioned for surgical procedures (24). Unfortu- sion of the lung is produced by a combination of movement of nately the effectiveness, duration of action, and systemic effects of intercostal muscles and smooth muscle within the lungs (24). On local analgesia have not been well documented in snakes. the ventral midline, snakes lack a sternum and ribs, and have a rela- Pre-placement of a catheter has been described and may be an tively thin, muscular body wall (30). During cardiocentesis the needle alternative to cardiocentesis for collecting multiple blood samples in passes through skin, a thin body wall, pericardium, and directly into snakes (25). This procedure requires general anesthesia, invasive sur- the ventricle of the heart. The needle does not pass through pleura or gery, post-surgical convalescence, and associated surgical a pleural space, and presumably snakes have a decreased propensity complications such as sepsis and possible catheter failure. Of the 11 for developing a pneumothorax. Because of these anatomical differ- pharmacokinetic studies published involving snakes, two utilized pre- ences, percutaneous entry into the heart of a snake is more analogous placed catheters (12, 22). The risks and pain associated with surgical to venipuncture of a large mammalian vein (such as the jugular vein implantation of a catheter must be considered and balanced against in a dog) than cardiocentesis in a mammal. the use of multiple cardiocentesis. The postoperative pain and dis- Obtaining serial blood samples from snakes, as is needed for phar- comfort associated with a catheter and bandages are consistent macologic studies, often presents a technical challenge. Cardiocentesis throughout the study period, while not sparing the stress associated has been described as the preferred method of collecting routine clini- with handling and restraint. cal blood samples and has been used for serial blood sampling in When evaluating the use of cardiocentesis in a snake study, it is snake pharmacokinetic studies (3-22). However, investigators and important to consider the marked anatomical differences between IACUC committees have a responsibility to address issues of safety, snakes and mammals (Fig. 3). The snake heart is three-chambered, acute pain, stress, and chronic effects of experimental procedures of with two atria and a single ventricle (26, 27). In contrast to the typi- any species. Valid concerns were raised at the beginning of the phar- cal mammalian ventricle, the interior chamber of the snake ventricle macokinetic study (1, 2), and this observational study attempted to is filled with a network of myocardial muscles that divide it into address these concerns. All the snakes in this study survived and were three subchambers (26, 27). The snake ventricle has a compact myo- apparently healthy at the conclusion of the study. The gross and cardial layer surrounding the spongy medullary myocardium, which histological changes were minimal and probably of limited clinical is relatively thick and often trabeculated (27, 28). The arterial blood significance in light of the observations of this study. The acute pain pressures generated by the snake ventricle are generally lower than and stress associated with serial cardiocentesis of snakes was not di- those in mammals (29). The combination of a thick ventricular wall rectly assessed, but may be, from an anatomical perspective, more and low blood pressure may help decrease the amount of blood leak- similar to pain from multiple percutaneous blood collections than to age from the ventricular wall after cardiocentesis. cardiocentesis of mammals.

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