Analyzing a Predator-Prey Interaction: Muscular Performance in Boas (Boa Constrictor) and Cardiovascular Response in Rats During

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5-22-2011 Analyzing a Predator-Prey Interaction: Muscular Performance in Boas (Boa Constrictor) and Cardiovascular Response in Rats During Constriction Katelyn Josephine McCann Dickinson College

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Recommended Citation McCann, Katelyn Josephine, "Analyzing a Predator-Prey Interaction: Muscular Performance in Boas (Boa Constrictor) and Cardiovascular Response in Rats During Constriction" (2011). Dickinson College Honors Theses. Paper 123.

This Honors Thesis is brought to you for free and open access by Dickinson Scholar. It has been accepted for inclusion by an authorized administrator. For more information, please contact [email protected]. Analyzing a Predator-Prey Interaction: Muscular Performance in Boas (Boa

constrictor) and Cardiovascular Response in Rats during Constriction

Katelyn J. McCann

With the collaboration of Kevin Wood Pat McNeal and Emmett Blankenship, DVM

Submitted in partial fulfillment of Honors Requirements

for the Department of Biology

Dr. Scott Boback, Supervisor Dr. Charles Zwemer, Supervisor Dr. Carol Loeffler, Reader

May 19, 2011 Abstract:

Constricting snakes must balance the energetic cost of constriction with the potential danger in releasing their prey too early. Therefore, it would be advantageous for these snakes to possess a mechanism to determine the minimum pressure and duration required to ensure that a prey item has been subdued and is no longer capable of inflicting harm. We hypothesized that Boas (Boa constrictor) modulate their constriction based on endogenous cues from their prey such as a heartbeat. In previous work we demonstrated that Boas respond to a simulated heartbeat in a deceased rat model by constricting with greater pressure and duration than when constricting rats without a simulated heartbeat. We extended this work in the current study by testing how Boas respond to a more realistic model; a rat whose cardiovascular system fails during the constriction event. We presented snakes with rats with a simulated heartbeat that "failed" halfway into the constriction. Analysis of these data demonstrated that constriction events with a simulated heart which fails ten minutes into the constriction are of intermediate duration and total pressure when compared to constriction tests with no simulated heart and a continuously beating simulated heart. We have also conducted experiments to test the snake's response while constricting live, anesthetized rats.

This system allows us to observe the snake's response to an actual rat heartbeat while simultaneously monitoring cardiovascular function in the rat during the constriction.

Analyses of these data support our hypothesis that cardiac arrest, rather than suffocation, is the proximate cause of death in rats during constriction.

Introduction:

Predators have evolved prey capture techniques that balance success in capturing and incapacitating prey with the safety in doing so. Retaliation from prey is typical and predators

1 must have strong selective pressures to minimize risks associated with their prey capture and subduing strategies. Further, the energy expenditure associated with prey capture can be costly to the predator which may be left vulnerable to additional attacks while engaged with its prey. Therefore, the evolution of efficient and successful prey capture methods is of paramount importance to predators' fitness.

Snakes are notorious for their unique specializations used to subdue and swallow enormous prey which can exceed their own body mass (Greene, 1997). As limbless, elongate predators, snakes have evolved methods to restrain and incapacitate their bulky and often dangerous prey including envenomation, constriction, or a combination of the two (Shine,

1993). The method of subduing prey varies amongst species of snakes and often within a species depending on the type of prey being captured (Mehta and Burghardt, 2008). Relative to Caenophidians (superfamily of advanced snakes), most extant Henophidians (superfamily of basal snakes) show less variation in prey restraint behavior, employing constriction and coiling, regardless of prey type (Greene and Burghardt, 1978). Recent work has demonstrated that prey restraint behavior varies within some Henophidian groups while others exhibit little flexibility in feeding and prey restraint behavior (Mehta and Burghardt,

2008). Regardless, the consistency in behavioral pattern by those basal snakes was reported by Greene and Burghardt (1978) as evidence that early snakes utilized constriction to restrain and subdue prey and that this feature may have been a key innovation which determined the success of the snake radiation.

Boas (Boa constrictor), members of the Henophidia, are non-venomous constrictors feeding on a wide range of prey including lizards, birds and mammals (Greene,

1983). Constriction in Boas is initiated by a strike (typically at the anterior portion of the

2 prey: Mehta and Burghardt, 2008) whereby the snake rapidly propels the anterior portion of

its body forward contacting the prey prior to its maximum strike distance. The momentum of

the strike carries the snakes' head forward and facilitates the formation of the first coil with a

downward movement of the braincase (Cundall and Deufel, 1999). As the head moves

ventrally, the snakes body bends laterally creating what is referred to as a ventral-lateral coil. This type of coil orients the prey item horizontal to the substrate and .p laces the snake's ventral and lateral surfaces in contact with the prey, typically over the thorax (Mehta and

Burghardt, 2008).

Constriction is both energetically costly and potentially dangerous as the snake

remains intimately engaged with the prey throughout the constriction. During this time the

snake is vulnerable to retaliatory attacks from the prey itself (Erberle and Kappeler, 2008),

and it is defenseless to its own predators and even additional prey (e.g., altruistic aggression

from kin: Janzen, 1970) as its sensory apparati and means of defense are occupied by the

constriction. In addition, Canjani et al. (2003) examined the aerobic metabolism of Boa

constrictor amarali during constriction and found constriction times of up to sixteen minutes

and rates of oxygen consumption up to 0.325 ml 02 g-1 h-1, an eight-fold increase from

resting levels. Therefore, it would be advantageous for these snakes to have a mechanism to

determine the minimum pressure and duration of constriction required to ensure that a prey

item has been subdued and is no longer capable of inflicting harm.

Moon (2000) proposed a heartbeat, lung ventilations, and body movements as potential signals exploited by snakes during constriction to determine exactly when a prey

item has expired. Snakes have been shown to possess mechanoreceptors in their skin with high vibrational sensitivity (Proske, 1969), and therefore would likely have the ability to

3 detect changes in such endogenous signals from the prey item. However, in preliminary tests

Moon (2000) found that the presence of a heartbeat and ventilatory movements in a dead

mouse did not elicit increased constriction pressure or duration in a Caenophidian species,

the Kingsnake (Lampropeltis getula).

Despite the absence of a response to these endogenous signals in the Kingsnake, it

is possible that other species may have a response to such signals. A response may be

evident especially in Henophidian snakes because these species retain conservative

constriction behavior. Based on the mode of constriction and the wide range of prey items

consumed by Boas, it is possible that their constriction is modulated by physiologic cues

received from their prey during constriction rather than a pre-strike assessment on the basis

of prey size, temperature, or movement (Shine and Sun, 2003). In a previous study conducted

in our lab, it was determined that Boas constricting prey with a continuously beating,

simulated heart did so for longer and with greater total pressure as compared to snakes

constricting prey without a simulated heart (Hall et al., 2010, Figure 1 ). These findings

suggest that Boas have the ability to detect the presence of heart contractions in their prey and use this stimulus as feedback to determine the necessary duration and total pressure of constriction. However, these tests (heart beating continuously throughout the constriction event vs. no heart beating) do not precisely replicate the full heartbeat signal that would be present in a live prey item. When snakes constrict live prey, the prey would have a beating heart when first constricted, and then at some undetermined time the heart would likely fail.

The widely held belief (cited by at least 32 other herpetological books) that constricting snakes kill their prey by suffocation (Mushinsky, 1987) has recently been challenged in support of the theory that prey are killed via circulatory failure (Hardy, 1994;

4 Moon, 2000). To maximize efficiency of constriction, snakes would presumably have the ability to detect the proximate cause of death in their prey and use this as a cue to release. If the cause of death in prey is indeed circulatory failure then snakes likely possess some ability to determine when cardiac arrest has occurred. Therefore, to more fully understand the dynamic interaction between constricting snakes and their prey, it is important to determine how and when death occurs in prey.

In order to more accurately simulate a constriction event we adjusted our simulated heart treatment to stop the heart while the snake is constricting. We chose to turn the heart off at the midpoint of constriction when the heart was beating continuously. If snakes truly respond to the stimulus of a heartbeat in prey, we predicted that a change in this stimulus during constriction (i.e. cessation of a heartbeat) would elicit a responsive change in constriction pressure and/or duration.

These data are informative, and more fully establish that Boas respond to a beating heart. However, the simulated heart system only replicates one aspect of the cardiovascular system, an internal expansion and contraction of the heart (Hall et al., 2010). Other factors such as blood flow and pulse pressures cannot be simulated with this system. For instance the dynamic changes that would occur to a prey's heart during a constriction event such as changes in rate, rhythm, volume, and pressure cannot be simulated with the artificial heart.

Furthermore, a real heart under the pressure of constriction will likely behave differently than a simulated heart with its constant beating.

The physiologic cues generated by death in the prey may have influenced the evolution of snakes' ability to detect a heart beat in their prey. This study aimed to determine

5 the physiologic changes resulting in rat death during constriction, to determine the potential cues which snakes use to modulate constriction. Therefore we expanded our experimental system by using live, anesthetized rats to more accurately simulate the actual prey. The anesthetized rat model allows us to replicate tests done with a dead rat by eliminating skeletal muscle twitching and tonicity as a variable without inhibiting cardiovascular function. This model allows us to observe a snake's response to a real, functional rat cardiovascular system with few confounding variables.

This system was also designed to determine the proximate cause of death in rats during constriction. As snakes compress their prey during constriction (especially the prey's thoracic region), at least two critical homeostatic processes are probably affected: ventilation and circulation of blood. Thus prey are likely dying via circulatory arrest, suffocation, or a combination of both. For suffocation (asphyxia) to be the proximate cause of death, pressure from the snakes' coil must prevent expansion of the thoracic cavity (ventilation) while at the same time having no effect on the rat's cardiovascular system (Hardy, 1994). We distinguished circulatory arrest (defined as a cessation of cardiac output and effective circulation, Mosby, 2002) from suffocation (defined as an interruption in breathing with oxygen deprivation, Mosby, 2002) by monitoring a number of physiologic parameters in the anesthetized rats including blood pressures (mean arterial pressure [MAP] and central venous pressure [CVP]), the electrical activity of the heart via an electrocardiogram (ECG), and the relative amounts of oxygen and carbon dioxide in the blood.

Methods:

Part I

6 In the first part of this study, we used deceased rats (Rodentpro.com) to test whether snakes can perceive when a rat has expired by detecting the rat's heartbeat during the constriction event. This was accomplished by implanting a simulated heart inside a frozen• warmed rat. The simulated heart system was comprised of a water-filled endotracheal tube connected in series with plastic tubing and a rodent ventilator (Harvard Rodent Ventilator,

Model 683). The entire circuit was voided of air and filled with water to avoid complications involving the compressibility of air in the circuit during the constriction event. The bulb of the endotracheal tube was filled with 1 ml of water to approximate the volume of the heart of a rodent this size and the rate of the ventilator was set to 195 cycles per minute to simulate a normal rat heart rate (Japundzic, 1990).

Two additional water-filled endotracheal tubes connected in series with plastic tubing and a pressure transducer (Gould P.T.J. 4771) were used to detect the pressure of constriction in the rat. The first was placed in the thoracic cavity of the rat via the esophagus, and the second was placed in the abdominal region of the rat via the rectum. Prior to instrumentation, the rats were thawed overnight and their teeth were removed to avoid puncturing the bulbs of the endotracheal tubes. Rats were then instrumented and heated to normal rat body temperature, 38°C (Dilsaver et al., 1992), using a Harvard Homeothermic Blanket Control

Unit (Harvard, Edenbridge, KY). Their internal core body temperature was monitored using a temperature probe within the liver of the rat and recorded using a Physitemp BAT-10

(Physitemp, Clifton, NJ).

Rats were then be presented to the snakes head first, using forceps, as the snakes cue on this region for strike placement (Kardong, 1992). The rat was presented with the simulated heart turned on. After l 0 minutes of constriction the simulated heart was turned

7 off. The snake was allowed to continue its constriction until it disengaged its mouth from the

rat at which point data collection was stopped and the instrumented prey was removed from

the snake. The endotracheal tubes were removed and the rat was placed back into the snake's

cage to allow it to consume the uninstrumented rat.

Part II

For this part of the study we used live, anesthetized rats to analyze snakes'

constriction behavior. Ten Wistar rats (300g - 400g) were purchased from Charles River

Laboratories Inc. for use in constriction tests. Rats were anesthetized using Inactin (a

thiobutabarbital sodium salt hydrate) solubilized in saline immediately prior to each use.

Inactin was chosen as the optimal anesthetic for our purposes because it is a long-acting,

injectable anesthetic that minimized pain and discomfort of the rat and is effective for at least

3-4 hours. For instance, Inactin has been used with predictable success for highly invasive,

long-duration, abdominal procedures in rats (Buelke-Sam et al., 1978; and Vasthare et al.,

1988; Phillipson, M. et al., 2009). Like other anesthetics, Inactin inhibits voluntary skeletal

muscle; however it also has little effect on the cardiovascular system (Polakowski et al.,

2004). These combined effects allowed us to evaluate cardiovascular response to constriction without the confounding variable of skeletal muscle tonicity or twitching.

Inhalational anesthesia with isoflurane was used initially by placing a saturated cotton

ball in a desiccator to sedate rats before injection with Inactin. Once rats lost their righting reflex they were removed from the desiccator and injected intraperitoneally with an induction dose (120mg/kg) oflnactin. Rats were monitored and maintained at Stage III, Plane 1 of

surgical anesthesia as judged by loss of palpebral reflex, insensitivity to deep periosteal pain,

8 and continued and obvious spontaneous ventilation. When necessary, additional quarter doses of Inactin were administered intraperitoneally to maintain this level of anesthesia.

Surgical Procedure:

Constriction Pressure Probe:

We constructed a pressure probe to measure the pressure of constriction exerted by the snake on the rat. Typically this was done by filling a tube with tap water which was connected to a Gould pressure transducer. Each time we ensured that no air was within the tube. Initially, we attempted to measure the pressure of constriction intrathoracically by inserting a saline filled Foley Catheter down the esophagus of the rat (intraorally) about 10 cm to the level of the heart. This method asphyxiated the rats by preventing the epiglottis from opening to allow air into the lumen of the trachea. As an alternative we tried to externalize the esophagus and insert the Foley Catheter through an incision in the lumen of the esophagus. Although this allowed for successful insertion of the Foley Catheter, when the blub at the distal end was filled with water it appeared to inhibit venous return and caused death in the rat. Therefore we chose to measure constriction pressure using a subcutaneous pressure probe placed along the entire lateral aspect of the rat, posterior to the limbs. The pressure probe was constructed using 21 cm of tygon tubing (1/4 inches OD X 1116 inches

ID, Sams Inc., 3M, Ann Arbor, MI) connected by a stop cock to a length of IV tubing

(Figure 2). The entire system was filled with saline and all air bubbles removed. Three stab wounds were made (posterior to the right hind limb, dorsolaterally parallel to the diaphragm, and at the base of the skull posterior to the right ear) and a subcutaneous tunnel was made using hemostats, through which the pressure probe was inserted. The pressure probe was then sutured in place at the cranial and caudal wounds and the midline wound was closed with

9 suture. The IV tubing of the pressure probe was then connected to a pressure transducer

(Gould/Statham P23Xl) and constriction pressure was recorded using Acknowledge software.

We used two different sized pressure probes to measure constriction pressure. Each of the pressures probes used was validated by applying a known pressure to the probe in a recently deceased rat using a sphygmomanometer. The known pressures applied were then compared to the pressure recorded by Acknowledge. We examined the relationship between the known pressures and the Acknowledge recorded pressures and found the relationship to be linear in each case. We fitted each with a best-fit line and adjusted our constriction pressure data according to the equation to estimate the actual pressure.

ECG Placement:

Electrodes (Grass Ag [silver cup], Grass Technologies, West Warwick, RI) to monitor cardiac electrical function were placed subcutaneously via stab wounds. Three electrodes were placed in an equilateral triangle (ventrolateral right coracoid, ventrolateral left coracoid and ventral median sagital/epigastric) in the thorax. Electrode cups were affixed with suture, the wound was closed with interrupted suture, and leads were externalized via a common exit wound located in the right upper abdominal quadrant. The three ECG electrodes were attached to an ECG cable and cardiac electrical function was recorded using

Acknowledge software at a rate of 2000 Hz.

Blood Sampling and Vascular Catheters:

Two vascular catheters (2.5 cm of PE-50 affixed to 1 m of surgical tygon tubing) were used to monitor both central venous pressure (CVP) and mean arterial pressure (MAP).

Central venous pressure was measured in the right atrium via the left external jugular vein. A medial incision was made from just below the mandible to the manubrium and the left

10 external jugular vein was then exposed using blunt dissection with curved mosquito forceps and isolated with two strands of suture. Once the vein was isolated, an incision was made

(approximately 50% of the way through the vessel) to gain access to the vessel lumen. The vein was then cannulated by lifting the lifting the distal end of the opening using a 23-guage needle (Tyco Kendall monoject hypodermic needle) with the beveled end bent to a 90° angle, as an introducer. Once the lumen was exposed the catheter was inserted and advanced approximately 2.5 cm to the right atrium. The catheter was then secured to the vessel, using a cuff on the catheter, with 4-0 black braided silk ligature (Ethicon, Somerville, NJ). Once secured the jugular catheter was looped around and sutured into a subcutaneous pocket and externalized via the same common exit wound as the electrodes. The tygon tubing of the catheter was then connected via a blunt 18-guage needle to a length of IV tubing (saline• filled) (Figure 3C, 3D). The IV tubing was then connected to a pressure transducer

(Gould/Statham P23Xl) and pressure was recorded using Acknowledge software.

To measure MAP we initially catheterized the right carotid artery. We found that this may have caused death in several animals due to minimized cranial perfusion with only one functioning carotid artery; therefore we modified our procedure to catheterize the femoral artery instead. A medial longitudinal incision from the mid-inguinal ligament to a point just proximal to the medial femoral condyle was made and the femoral triad (femoral nerve, artery and vein) were visualized. The femoral artery was then exposed using blunt dissection with curved mosquito forceps and isolated with two strands of suture. An arterial blood sample was taken using a 24-guage IV catheter and needle (Angiocath. Abbott Laboratories,

Abbott Park, IL). About 0.25 cubic centimeters (cc) of arterial blood was withdrawn from the catheter using a 1 cc syringe and 23 gauge needle and a pre-constriction blood gas analysis

11 (IRMA blood gas analyzer series 2000, Edison, NJ) was performed immediately. Once the blood sample had been taken the catheter was secured to the femoral artery and an infusion plug (intermittent infusion plug 0.1 ml Tyco Healthcare, Covidien, Mansfield, MA) was secured to the end of the catheter. A 19-guage catheter needle (winged infusion set with 30 cm tubing, Terumo Surflo, Somerset, NJ) was inserted through the septum of the infusion plug. The catheter needle was secured to the indwelling catheter in the artery and connected to a length ofIV tubing (all filled with saline) (Figure 3A, 3B). The IV tubing was then connected to a pressure transducer (Gould/Statham P23Xl) and pressure was recorded using

Acknowledge software.

Constriction:

Instrumented rats were presented to snakes as described above for dead rats (Methods

Part I). Once a strike occurred the snakes were allowed to coil on the rats and proceed to the constriction phase until the snake disengaged its head and began to release its coil, at which point the rat was removed. Rats were inspected immediately after constriction via necropsy to assure proper placement of catheters and electrodes. An arterial blood sample was taken from the left ventricle, the aortic arch, or the abdominal aorta and a post-constriction blood gas analysis was performed immediately. Snakes were then fed with a different dead rat.

Analvsis:

The mean venous and arterial pressures were compared at several time points throughout the constriction event using an analysis of variance (ANO VA). Heart rate and duration of QRS complex were also measured at several time points during constriction and compared using an ANOV A. Heart rate was calculated from our recorded ECG trace by counting the number of full cardiac cycles in a five second time interval. The duration of the

12 QRS complex was then measured using Acknowledge Software. The time interval from the beginning of the QRS wave to the end (Q-S interval) was measured and the average length of five QRS waves at each time point was calculated. Pre and post-constriction blood parameters were compared using paired T-tests.

Results:

Part I

An analysis of these data in which the simulated heart was turned off after ten minutes of constriction suggests that there is a difference in total constriction pressure and duration as well as rate of decay of pressure, when the heart is "beating" and when the heart is turned off (Figure 1 ).

The results from the mixed model ANOV A indicated that our heart treatment had a significant effect on duration (F = 6.08, DF = 2, 52, P = 0.0043). The mean duration of constrictions with the no heart treatment was the shortest (763.16±137.7 seconds), the mean duration of constrictions with the continuous heart treatment was the longest

(1397.60±141.01 seconds) while the mean duration of constrictions with the 10-minute heart treatment was intermediate (1052.91±160.41 seconds). A post-hoc test of the least squares means with a Tukey-Kramer adjustment for multiple comparisons showed a significant difference between the no heart treatment and the continuous heart treatment (P = 0.0029).

There was no significant difference between the 10-minute heart treatment and either the no heart treatment (P = 0.3220) or continuous heart treatment (P = 0.2103) (Figure 4).

Results from a second mixed model ANOV A indicated that our heart treatment had a significant effect on the total pressure of constriction [integral of pressure (mmHg) vs. time

(sec)] (F = 8.17, DF = 2, 52, P = 0.0008). The mean total pressure of constrictions with the

13 no heart treatment was 40788±11346 mmHg·seconds. The mean total pressure of constrictions with the 10-minute heart treatment was 70990±13259 mmHg·seconds. And the mean total pressure of constriction with the continuous heart treatment was 103975±11595 mmHg·seconds. A post-hoc test of the least squares means with a Tukey-Kramer adjustment for multiple comparisons showed a significant difference between the no heart treatment and the continuous heart treatment (P = 0.0005). There was no significant difference between the

10-minute heart treatment and the no heart treatment (P = 0.1871) or the 10-minute heart treatment and the continuous heart treatment (P = 0.1419) (Figure 5).

Finally, a third mixed model ANOV A indicated that our heart treatment had a significant effect on the proportion of the integral (total pressure) within the first 10 minutes of the constriction. (F = 6.90, DF = 2, 52, P = 0.0022). The no heart treatment had 85.58% of the total pressure within the first 10 minutes of the constriction. The 10-minute heart treatment had 71.14% of the total pressure within the first 10 minutes of the constriction, and the continuous heart treatment had 62.10% of the total pressure within the first 10 minutes of the constriction. A post-hoc test of the least squares means with a Tukey-Kramer adjustment for multiple comparisons showed a significant difference between the no heart treatment and the continuous heart treatment (P = 0.0016). There was no significant difference between the

10-minute heart treatment and the no heart treatment (P = 0.1086) or continuous heart treatment (P = 0.4147) (Figure 6).

Part II

Constriction tests with anesthetized rats typically showed a drop in mean arterial pressure and an increase in central venous pressure upon formation of the coil. Figure 7 displays a representative trace from one constriction event. Our snakes generally achieved a

14 maximum constriction pressure shortly after the initial formation of the coil, which averaged

302.98 mmHg. The application of this constriction pressure to the thoracic cavity had numerous effects on the cardiovascular system in rats.

The results from a one-way repeated measures ANOVA indicated that there was a significant decrease in mean arterial pressure (measured in the femoral artery) during constriction (F = 1.187, DF = 2, P = 0.0268). Mean arterial pressure during constriction was measured 60 seconds after the strike (25.5 mmHg) and 60 seconds before the end of the constriction (19.5 mmHg) in two trials. Both of these values were significantly lower than baseline measurements (60 seconds prior to the strike, 72.0 mmHg, P < 0.05). A post-hoc test for multiple comparisons (Tukey) showed a significant difference between baseline mean arterial pressures and mean arterial pressure at two time points during constriction.

The same test was applied to measurements of central venous pressure (measured in the right atrium). There was no significant difference between baseline central venous pressure and central venous pressure during constriction, measured in two trials (F = 3 .111,

DF = 2, P = 0.2433). Although central venous pressures 60 seconds after the strike (35.0 mmHg) and 60 seconds prior to the end of constriction (29.0 mmHg) were greater than baseline central venous pressure (-7.5 mmHg), a post-hoc test for multiple comparisons

(Tukey) did not show a significant difference in central venous pressures (P > 0.05) (Figure

8).

The results of a Welch's ANOVA indicated that there was a significant effect of constriction on heart rate (F = 42.32, DF = 3, P < 0.0001). Mean heart rate values (bpm) from ten trials were evaluated at four time points: baseline, 60 seconds after the strike, 60 seconds prior to the end of constriction and 30 seconds prior to the end of constriction. A

15 post-hoc test of the least squares means showed a significant difference in heart rate at each time point when compared to the baseline heart rate (P = 0.0002, P <0.0001, P <0.0001)

(Figure 9).

The results of a second Welch's ANOV A indicated that the length of the QRS complex (ms) increased significantly during constriction (F= 21.98, DF= 3, P < 0.0001).

The length of the QRS complex was measured at four time points: baseline, 60 seconds after the strike, 60 seconds prior to the end of constriction and 30 seconds prior to the end of constriction. The mean lengths of the QRS complexes at each time point were compared for ten trials. A post-hoc test of the least squares means showed a significant difference in QRS complex length at each time point when compared to the baseline QRS complex length (P =

0.052, P <0.0001, P <0.0001) (Figure 10).

From baseline (post-surgery, pre-constriction) and post-constriction blood samples we were able to measure partial pressures of oxygen and carbon dioxide (mmHg), as well as blood pH and potassium levels (mM). The results of a paired t-test indicated that there was a significant increase in the partial pressure of C02 (mmHg) (T = -2.51, DF = 8, Ptone-tail) =

0.018, n = 9) and no significant change in the partial pressure of 02 (mmHg) during constriction (T= 1.25, DF = 9, Ptone-tail) = 0.124, n = 10) (Figure 11).

A paired t-test comparing mean blood pH levels before constriction (7.36) and after constriction (6.98) showed that there was a significant decrease in blood pH (T = -12.68, DF

= 16, P < 0.0001, n = 9) (Figure 12). The same test was applied to mean pre-constriction

( 4.827 mM) and post-constriction (9.348 mM) blood potassium levels. The results of the paired t-test indicated that there was a significant increase in blood potassium during constriction (T= 5.63, DF = 18, P < 0.0001, n = 10) (Figure 13).

16 Discussion:

Part I

The results from our constriction tests with a simulated heart establish that Boas have

the ability to detect a heartbeat in their prey and alter the duration and total pressure of constriction accordingly. The 10-minute heart treatment further establishes that not only do

Boas respond to the presence or absence of the heartbeat but they are also capable of responding to changes in this stimulus during constriction. The evolution of this ability to detect and respond to the heartbeat in a prey item would be evolutionarily advantageous if cessation of a heartbeat was indicative of death in the prey item. The pressure of constriction has the potential to cause cardiac arrest in rats and this cessation of a heartbeat in prey may act as a cue to the snake, indicating that it is safe to remove constriction pressure.

Part II

We observed a statistically significant decrease in femoral mean arterial pressure upon the initial strike and coil formation, which was maintained throughout the constriction event. In two trials in which femoral mean arterial pressure was measured, the pressure dropped and average of 75% from baseline, to below 25 mmHg (Mohrman and Heller,

2003). Peripheral mean arterial pressures below 25mmHg are indicative of severe hypoperfusion and likely complete circulatory arrest.

In addition to low mean arterial pressures we also observed increases in central venous pressure (CVP) measured in the right atrium (via the jugular vein). The mean

increase in central venous pressure was not statistically significant, however this may explained by a low sample size (n = 2). The central venous pressure measurements during constriction were also highly variable (SEM = 24.0 [ 60 seconds post strike]). The central

17 venous pressure is a direct measure of intrathoracic pressure which is, in turn directly affected by the constriction pressure. Because constriction pressures vary amongst snakes the intrathoracic pressures during constriction, and therefore the central venous pressures, vary accordingly. However, the mean central venous pressure 60 seconds after the strike was 35.0 mmHg (compared to a mean baseline CVP of -7.5mmHg). Central venous pressures above 7 mmHg result in the absence of venous return (Mohrman and Heller, 2003). Therefore, with a

CVP of 35.0 mmHg we can assume that there is no venous return, therefore insufficient pre loading, or filling of the ventricles during diastole.

We have documented that Boas are capable of exerting a remarkable pressure during constriction. Constriction pressures greater than 300 mmHg not only prevent venous return but also impose a tremendous afterload pressure (pressure in the arterial system which must be overcome by the pressure generated within the ventricles in order to eject blood). Arteries in the thoracic cavity, specifically the aorta, are directly affected by the high intrathoracic pressure generated by the constriction. Cardiac muscle fibers must generate a contractile force greater than the force imposed by the afterload pressure to cause the aortic valve to open and allow blood to be ejected. It is unlikely that the myocardial contractile force generated could overcome the opposing force on the aortic valve resulting from the constriction pressure. Rossen et al. (1943) showed that loss of consciousness occurs within ten seconds when cerebral circulation is arrested. Therefore, the application of extremely high thoracic pressure for a short period of time may be sufficient to cause loss of consciousness in rats during constriction. Following loss of consciousness, the threat of retaliation by prey is eliminated and sustained lower pressure may be sufficient to cause cardiac arrest in the rat.

18 This concurrent combination of minimal preloading and excessive afterload pressure results in no cardiac output and circulatory arrest. Peripheral mean arterial pressures below capillary closing pressure support the theory that circulatory arrest is occurring (cardiac output = 0 liters/minute). With circulatory arrest, blood flow to all tissues including the myocardium is inhibited. Myocardial ischemia significantly impairs the pumping capability of the heart, initially causing bradycardia, uncoordinated depolarization, and abnormal contraction, eventually resulting in arrhythmia.

Analysis of the electrocardiogram throughout the constriction revealed degeneration of the normal rhythm to atrial flutter followed by atrial fibrillation, coupled with severe bradycardia and widening of the QRS complex. These rhythms occasionally led to ventricular fibrillation or asystole. These arrhythmias are likely to result in full cardiac arrest and may be caused by myocardial ischemia due to circulatory arrest.

There is also the possibility that despite the appearance of electrical signals on the electrocardiogram, the electrical signal being conducted throughout the heart does not result in ventricular contraction, therefore no blood is ejected. This condition is known as electrical mechanical dissociation (EMD) and is defined by the presence of electrocardiographic complexes in the absence of cardiac output (Hardy, 1994). The non-perfusing rhythm resulting from EMD causes circulatory arrest due to functional cardiac arrest. EMD is likely to occur with high after load pressures, as the contractile cells of the myocardium are unable to generate a force great enough to produce isotonic contraction, despite normal depolarization.

Circulatory arrest could be caused by either asystole, ventricular fibrillation, EMD, or a combination of these. Nevertheless, the functional effect is the same: blood is not

19 circulated, therefore tissues become ischemic. Myocardial ischemia leads to further deterioration in heart function while peripheral tissue ischemia results in accumulation of metabolic waste products in the blood.

Arterial blood samples taken immediately before and after the constriction showed evidence of ischemia such as low pH, high potassium levels (hyperkalemia) and high partial pressures of C02. There was a significant decrease in blood pH during constriction (P <

0.0001). Changes in blood pH have profound physiologic effects on many metabolic processes. The normal blood pH for live (un-anaesthetized) rats is 7.371±0.010 (Hagerdal et al., 1975), similar to the mean baseline blood pH we observed in our anesthetized rats (7.36).

Blood pH below 7.35 indicates acidosis in rats (Kurpinsak and Skrzypczak, 2010). We observed mean post-constriction blood pH values of 6.98, indicating significant acidosis.

This acute acidosis is likely metabolic and may be the result of the accumulation of aerobic and anaerobic metabolic waste products such as C02 and lactic acid due to tissue ischemia.

Respiratory acidosis is not likely the cause of this low blood pH level because, despite the increase in partial pressures of C02, there was no significant change in partial pressures of

02.

Low intracellular pH due to ischemia has been shown to be closely related to membrane potential and extracellular pH (Hagberg, 1985). As intracellular hydronium ion concentration increases, potassium ions are released to maintain a normal intracellular ion balance and membrane potential. Therefore, hyperkalemia is associated with low pH

(Lindinger, 1995). Our post-constriction blood samples showed a significant increase in serum potassium (mM), which could be attributed to the drop in blood pH. Post-constriction hyperkalemia could also be attributed to tissue damage as a result of the constriction

20 (analogous to a crush injury), or a combination of both tissue damage and ion imbalances

(DeFronzo et al., 1982). In either case, hyperkalemia has been shown to increase susceptibility to heart arrhythmias (Gerst et al., 1966). Gerst et al. (1966) showed that metabolic acidosis, and associated hyperkalemia, result in an unstable resting membrane potential and lower threshold for depolarization in conducting cardiac myocytes, making them more easily excitable and therefore susceptible to arrhythmia. We observed post• constriction serum potassium levels of 9.348 mM in addition to blood pH levels of 6.98.

These levels of hyperkalemia and acidosis would likely cause fatal arrhythmia upon removal of constriction pressure if the heart was still beating. Thus, if myocardial ischemia did not induce cardiac arrest, hyperkalemia and acidosis would. Hyperkalemia is known to produce characteristic electrocardiographic changes at specific blood potassium levels. Blood potassium concentrations of about 6 mM cause a decrease in the amplitude of the P wave which was observed in our ECG recordings early in the constriction event. As potassium concentrations increase to 10 mM, lengthening of the duration of the QRS complex occurs, which was evident throughout the constriction event in our ECG recordings (Bisogno et al.,

1994; Figure 10)

The pressure of constriction has profound detrimental effects on the cardiovascular system. Concomitantly, constriction pressure on the thoracic cavity could completely inhibit ventilation by exerting a pressure which cannot be overcome by the respiratory musculature resulting in ventilatory failure. Cessation of ventilation will eventually lead to suffocation; defined as an interruption in breathing with oxygen deprivation (Mosby's, 2002). However,

Hardy (1994) suggested that it is unlikely that suffocation is the proximate cause of death during constriction, because suffocation also requires maintained cardiac output and

21 circulation (Hardy, 1994). Therefore without the depletion of oxygen from circulating blood, death by suffocation (the circulation of deoxygenated blood) is impossible.

Our results are consistent with Hardy's idea that suffocation is unlikely as the proximate cause of death in constriction. Suffocation would be evident if the post• constriction, blood gas analysis of arterial blood revealed a significantly increased partial pressure of carbon dioxide as well as a significantly decreased partial pressure of oxygen.

Our data show that while there is a significant increase in the partial pressure of C02, there is no significant depletion of 02. In fact, partial pressures of 02 show no significant change during constriction. Marked decreases in MAP and heart rate as well as increases in CVP all point to circulatory arrest. In the case of cardiac arrest partial pressures of blood gases post• mortem should remain unchanged (Mitchoefer et al., 1967). Therefore, it is likely that some combination of suffocation and cardiac arrest is occurring as a result of the constriction; however it is the circulatory failure which inflicts the most imminent, life threatening, physiologic effects.

Acknowledgements:

I would like to thank my honors committee, Professor Scott Boback, Professor

Charles Zwemer and Professor Carol Loeffler for all of their help throughout this project. I would especially like to thank Professor Boback for his dedication and continuous support throughout this project. I would like to acknowledge my collaborators Kevin Wood and Pat

McNeal, without whom this project would not have been possible. Dr. Emmett Blankenship, from Canton Georgia, assisted with protocol development and surgical procedures. Emmett's suggestions helped refine our surgical procedures and ultimately provided the protocol we adopted. I would also like to thank Lily Bieber-Ham and Allison Hall for all of

22 their help and support throughout this project. Thank you to Professor Forrester for his help with statistics. Finally, thank you to Mike Potthoff and Jim Kuenzie for their technical support.

References

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25 Figure 1.

A. :iOO 250 Continuous Heart 200

1:..0

100

!;0

,-... 0 bi) B. 0 5-00 1000 1500 2000 2500 3000 ~ 300 250 § No Heart Thoracic <:» 200 (!) !-< Heart ;::s 150 tr: ifJ (!) 100 !-< ~ so) 0 ' C. 0 500 1000 1500 2000 2500 3000 300

2!>0 10-Minute Heart 200

150

100

0 0 500 1000 15-00 2000 2500 3000 Time (sec)

Figure 1. Representative constriction pressure vs. time traces with each of the three heart treatments. A. Continuous heart treatment in which the simulated heart was beating at 195 bmp throughout the constriction event. B. No heart treatment in which the simulated heart was not beating during constriction. C. 10-Minute heart treatment in which the heart was beating at 195 bpm for the first 10 minutes during the constriction event and then turned off for the remainder of the event.

26 Figure 2.

Figure 2. Probe used to measure constriction pressure constructed from 21 cm of tygon tubing (1/4 inches OD X 1116 inches ID, Sams Inc., 3M, Ann Arbor, Ml) connected by a stop cock to a length of IV tubing. The entire length of tubing was filled with water and all air bubbles were removed. It was then connected to a pressure transducer to record constriction pressure. The probe was placed subcutaneously in the rat, positioned on the dorsolateral aspect of the rat just posterior to the left hind and forelimbs.

27 Figure 3.

A c

B D

Figure 3. A. 24-gauge IV catheter and needle (Angiocath. Abbott Laboratories, Abbott Park, IL) was used to insert the catheter into the femoral artery. B. Once inside the artery, the needle was removed and the catheter was left in the artery. The catheter was then attached to an infusion plug (intermittent infusion plug 0.1 ml Tyco Healthcare, Covidien, Mansfield, MA) and secured to the end of the catheter. A 19-guage catheter needle (winged infusion set with 30 cm tubing, Terumo Surflo, Somerset, NJ) was inserted through the septum of the infusion plug. The catheter needle was secured to the indwelling catheter in the artery and connected to a length ofIV tubing (all filled with saline). CID. Jugular and carotid artery catheters were constructed using a lm length of tygon tubing. A small length of PESO tubing ( 4.0cm for jugular and 2.5 cm for carotid) was inserted into the end of the tygon tubing and a small ring of tygon was used as a cuff with which the catheter was secured once inside the blood vessel.

28 Figure 4.

25 - - -.. en ...... Q) 20 - -- ::J c ...._E 15 - -- c -- 0 ...... ro I- 10 - ::J 0 5 -

0 I None 10-Minute Continuous

Figure 4. Histogram displaying the mean(± SE) duration of constriction tests with each of the three heart treatments. The mean duration of the no heart treatment (i.e., None) was 763.16±137.7 seconds, 10-minute heart treatment (i.e., 10-Minute) was 1052.91±160.41 seconds and continuous heart treatment (i.e., Continuous) was 1397.60±141.01 seconds.

29 Figure 5.

140000

120000

100000 - cu ""- 80000 0) ...... Q) c 60000 11 40000 - 20000

0 None 10-Minute Continuous

Figure 5. Histogram displaying the mean (± SE) total pressure (Integral of pressure vs. time) of constriction tests with each of the three heart treatments. The mean total pressure of constriction tests with the no heart treatment was 40788± 11346 mmHg·seconds, 10-minute heart treatment was 70990±13259 rnrnHg·seconds, and continuous heart treatment was 103975±11595 rnrnHg·seconds.

30 Figure 6.

20 -

0 I None 10-Minute Continuous

Figure 6. Histogram displaying the mean(± SE) percentage of the integral in the first 10 minutes of the constriction event for each of the three heart treatments. The mean percentage of the integral in the first 10 minutes of constriction for the no heart treatment was 85.58%, 10-minute heart treatment was 71.14%, and continuous heart treatment was 62.10%.

31 Figure 7.

500 Constriction Pressure Femoral 400 Jugular 25mmHg -0) :r: 300 E ..._E 200 ~ ::J (/) (/) ,_Q) 100 o, ------0

-100 3 4 5 6 7 8 9 Time (min)

Figure 7. Representative pressure recording from one trial in which constriction pressure, femoral mean arterial pressure, and right atrial central venous pressure (via the jugular vein) were recorded throughout the constriction. The dotted line indicates capillary closing pressure (25 mmHg).

32 Figure 8.

100 !Femoral MAPI I Right Atrial CVPj

~- 80 ! E 60 E - * * 20 • • 0 ·························································

-20---..-

Figure 8. Scatter plot displaying the mean(± SE) femoral mean arterial pressure (MAP) and mean(± SE) right atrial central venous pressure (CVP) at baseline (60 seconds prior to strike) and two time points during the constriction event (60 seconds post strike and 60 seconds prior to the end of constriction) (n = 2). The red rectangle indicates the time during which constriction was occurring. The asterisks indicate significant differences from baseline. The mean femoral MAP at (a) baseline was 72.0±11.31 mmHg, (b) 60 seconds after the strike was 25.5±3.54 mmHg, and (c) 60 seconds prior to the end of constriction was 19.5±2.12mmHg. The mean right atrial CVP at (a) baseline was -7.5±0.71mmHg, (b) 60 seconds after the strike was 35.0±33.94mmHg, and (c) 60 seconds prior to the end of constriction was 29±5.66mmHg.

33 Figure 9.

400 - ..-.. o, p = 0.0002 E .._..a 300 - * ...... (1) co ! p < 0.0001 n:: t 200 - * p < 0.0001 co (1) I * 100 - ! !

0 I T Baseline Strike +60s -60s to End -30s to End

Figure 9. Scatter plot displaying the mean heart rate (beats per minute± SE) at baseline and three time points during constriction (60 seconds after the strike, 60 seconds prior to the end of constriction and 30 seconds prior to the end of constriction). The mean heart rate at (a) baseline was 428±0. 70bpm, (b) 60 seconds after the strike was 267±0.85bpm, ( c) 60 seconds prior to the end of constriction was 142±1.07bpm, and (d) 30 seconds prior to the end of constriction was 99±0.53 bpm. Asterisks indicate a significant difference from baseline (n = 10).

34 Figure 10.

80 p <0.0001 70 - *

...--. p <0.0001 (/) 60 ! ..._E * ro 50 - c: p = 0.052 ...... (1) f c 40 * (/) 0:: 0 30 ! 20 !

10 I I I Baseline Strike +60s -60s to End -30s to End

Figure 10. Scatter plot displaying the mean duration(± SE) of the QRS complex (milliseconds) at baseline and three time points during constriction (60 seconds after the strike, 60 seconds prior to the end of constriction and 30 seconds prior to the end of constriction). The mean duration of the QRS complex at (a) baseline was 18.7±0.28ms, (b) 60 seconds after the strike was 3 l .35±0.40ms, ( c) 60 seconds prior to the end of constriction was 48.75±0.52ms, and (d) 30 seconds prior to the end of constriction was 63.5±0.53ms. Asterisks indicate a significant difference from baseline (n = 10).

35 Figure 11.

160 • Ventilatory Arrest 140 • Cardiac Arrest Pre-constriction 120 • Post-constriction ...--... • • 0) • I 100 .,. • E ,,. • ! ..._E 80 •• N • 0 • ._. o 60 • a... • ••• 40 ~· •••• ~ 20 • •• • •• 0 0 20 40 60 80 100 120 140 160 p02 (mmHg)

Figure 11. Scatter plot displaying arterial blood gas data (p02 versus pC02). Black points are estimated from a previous study on dogs that were sacrificed by ventilatory arrest (black circles) or cardiac arrest (black diamonds; Mitchoefer et al., 1967). Colored points represent values from rats used in constriction tests in the present study. The blue diamond represents the mean(± SE) of the pre-constriction blood gas values. The red circle represents the mean (±SE) of the post-constriction blood gas values. Values for p02 did not differ before and after constriction whereas there was a significant increase in pC02 (P = 0.018, n = 9).

36 Figure 12.

7.6

7.4

7.2 p < 0.0001 * 7.0

I o, 6.8

6.6

6.4

6.2

6.0 Normal Pre-constriction Post-constriction

Figure 12. Histogram displaying normal rat blood pH and mean(± SE) pre and post constriction blood pH values. Normal rat blood pH is 7.37 l(Hagerdal, 1975). The mean blood pH in rats prior to constriction was 7.406±0.020, and the mean blood pH in rats immediately after constriction was 6.982±0.027. Asterisks indicate a significant difference from pre-constriction potassium level (P < 0.0001, n = 9).

37 Figure 13.

p < 0.0001

10 *

-:iE 8 E -E ·;;;:J 6 sI/) 0 a.. 4

o~----'---r--~- Normal Pre-constriction Post-constriction

Figure 13. Histogram displaying normal rat blood potassium concentration (mM) and mean (± SE) pre and post constriction blood potassium concentrations. Normal rat blood potassium level is approximately 5.3 mM (ranging from 4.6 - 6.0 mM [Runglar and Dabich, 1979]). The mean blood potassium level in rats prior to constriction was 4.827±0.299mM, and the mean blood potassium level in rats immediately after constriction was 9.348±0.745mM. Asterisks indicate a significant difference from pre-constriction potassium level (P < 0.000 l, n = l 0).