Haemodynamic Responses to Haemorrhage in the Snake, Elaphe Obsoleta Obsoleta

The cardiovascular adaptations of snakes invite attention because of the long body shape and ecological specializations of these reptiles. Recent studies of terrestrial and arboreal species demonstrated that blood pressure is restored towards normal following disturbance by tilting (Seymour & Lillywhite, 1976; Lillywhite & Seymour, 1978; Lillywhite, 1980a). However, relatively little is known regarding cardiovascular regulatory mechanisms in these or other reptiles.

In this paper we report the cardiovascular responses of a terrestrial and partly arboreal snake to losses of blood volume. The observed compensatory responses are notable for their efficacy and invite comparison of the regulatory mechanisms with those of higher vertebrates.

Sixteen adult black ratsnakes, Elaphe obsoleta obsoleta (Say), were captured near Lawrence, Kansas, and maintained in the laboratory at 25–28 °C. Surgical procedures generally followed the methods developed by Seymour & Webster (1975) and Lillywhite & Seymour (1978). Snakes were allowed at least 36 h to recover from surger and were held within acrylic tubes having narrow longitudinal openings to permit free exit and movement of catheters. Only healthy snakes in positive water balance were used in these investigations.

Arterial pressures were measured by means of an occluding catheter (PE-50 polyethylene tubing) filled with heparinized saline and tied into the dorsal aorta at a level near the vent. Central venous pressures were measured from a non-occluding, beveltipped catheter (usually PE-10) inserted through a small needle puncture made in the inferior vena cava 10–20 cm posterior to the heart. The catheter tip was advanced to within a few cm of the heart and secured by external suture. Blood pressures were recorded from pressure transducers (P–1000 Linear Core) coupled to separate penchannels of a desk model DMP–4A physiograph (Narco-Biosystems, Inc., Houston, Texas).

The percentage of plasma trapped in the packed red cell column was determined in four separate tests (different snakes) where the red cell volume of a centrifuged blood sample was resuspended in 4 ml of 0-9 % saline and centrifuged a second time. The absorbance of a diluted sample of the supernatant was read from the spectrophotometer and compared with a standard volume dilution plot determined from dilutions of small volumes of plasma taken from the initially centrifuged blood sample. Subsequently, haematocrits were multiplied by 0·843 in calculations of blood volume using Evans blue dye, the percentage of trapped plasma taken as 15·7% (four determinations = 13, 16·5, 16·7, 16·7%).

Snakes were bled individually while lying at rest, unanaesthetized, in horizontal tubes. Fixed percentages (2–8 %) of the initially measured blood volume (IBV) were withdrawn at regular intervals into a clean syringe from the arterial catheter. Blood pressures and heart rate were recorded for 5 min between successive blood withdrawals. In this manner, graded haemorrhage was executed at roughly 6-8 min intervals (withdrawal times varied) until blood could no longer by obtained with reasonable ease. In some experiments the blood was stored at room temperature in heparinized containers and returned to the snake at the conclusion of haemorrhage. These snakes survived.

Experiments were conducted at temperatures of 25–28 °C. Arterial and venous pressures were recorded from nine snakes; only arterial pressure was measured in the remaining snakes. Heart rate was determined from the pulsatile arterial pressure trace or from ECG recordings signalled from two silver electrodes sutured to the skin near the heart. At regular intervals blood samples were used for determinations of haematocrit and plasma protein concentration. The latter was determined from a micro modification of the method of Bradford (1976) using Coomassie Blue G–250.

Initially measured haematocrits averaged 27-9% (s.e. = 1·9, N = 16). The mean concentration of plasma protein was 75-26 mg/ml (s.e. = 2·70, N = 9). Arterial pressure varied from 38 to 75 mmHg in different snakes and was significantly higher in males (mean = 54·45 + 3·65 s.e., N = 11) than in females (mean = 41·75+ 1·50 s.e. N = 4) (Mann Whitney U test, P < 0·05).

Graded haemorrhage elicited cardiovascular responses which acted to compensate for the reduction of blood volume. Arterial pressure was generally maintained until a ‘critical volume deficit’ was reached; following this, the pressure dropped precipitously in response to further blood loss (Figs. 1, 2). Critical volume deficits ranged from 63·3 to at least 121·5 % IBV (mean = 85·6 + 8·3 s.e., N = 6) and could not be determined in certain snakes whose behaviour (e.g. curling of the tail) prevented efficient withdrawal of blood even though arterial pressure remained high (e.g. Fig. 1).

Heart rate increased during haemorrhage and typically plateaued for variable periods before the critical volume was removed (Fig. 3). The mean increase in heart rate during haemorrhage was 119% (s.E. = 13·9, N = 12). Pulse pressures initially ranged from 2 to 13 mmHg (mean = 6·3, s.e. = 1·1, N = 12), but in all cases dimirnished by the conclusion of haemorrhage. Central venous pressures were consistent between 1 and 4 mmHg in quiescent animals and showed little net change during haemorrhage (Fig. 2).

Haematocrits progressively diminished during haemorrhage (mean total decrement = – 30·5% initial, s.e. = 4·1, N = 12). Plasma protein concentration generally declined, but transient increases sometimes attended movements of snakes and increases of arterial pressure (Fig. 2). Maximum changes in protein concentration varied from roughly o to – 25·6% (mean = – 10·0, N = 10).

The patterns of haematocrit and plasma protein imply a dilution of the circulating blood volume by extravascular fluids. The magnitude of haemodilution was quantified in six snakes by measuring the circulating blood volume at the conclusion of haemorrhage. This residual volume, when added to the known volume of blood removed from a snake during haemorrhage, should exceed the IBV by an amount equal to the volume of extravascular fluid entering the circulation during the haemorrhage period. Such measurements, using both Evans blue and washout methods, are given in Table 1 along with haemodilution volumes estimated from progressive changes of haematocrit in relation to known haemorrhage volumes and the IBV. Measurements derived from either procedure generally agreed.

A graphic illustration of the haemodilution in a snake which lost 100% of its IBV is shown in Fig. 3. Regression coefficients of similar time-dilution plots determined from haematocrit changes indicated that rates of haemodilution varied from 5·75 to 18·3% IBV·h ^{− 1} (mean = 11·4, s.e. = 1·64, N = 7). The percentage of blood loss that was replaced by haemodilution ranged from 20 to 70·8% (mean = 49’9, s.e. = 8-4, N = 7). There was a significant correlation between the cumulative (total haemodilution volume and the critical volume deficit (r = 0·78, P < 0·01). However neither variable bore a significant relationship to either the rate of blood withdrawal or duration of experiment (both P > 0·05).

Snakes typically were motionless during most of the haemorrhage period but became active at volume deficits ranging from 28 to 77 % IBV (mean = 54). Activity consisted of a snake slowly lifting its head and neck or moving the body more vigorously while crawling forward and backward in the tube. During periods of low arterial pressure snakes engaged in stereotyped ‘wiggling’ where lateral undulations were advanced sinusoidally along the body. These movements were nearly always propagated headward, usually from the rear of the body to the head but sometimes limited to the region near the heart or neck. Occasionally, the movements were vigorous, causing the body to ‘flap’ against the tube. The movements produced transient increments of venous and arterial pressures which persisted considerably longer than the behavioural acts themselves (Fig. 4).

The abilities of snakes to resist the disturbing effects of blood loss are probably the most potent reported in any vertebrate. Snakes maintained arterial pressure at IBV losses averaging 86 %, and one individual maintained pressure during deficits exceeding 100%. Studies of other snakes corroborate these data and demonstrate capabilities to recover from such treatment provided that blood is returned to the circulation or the animal is allowed to drink water (Lillywhite, unpublished data). The only comparable data for other reptiles are those of Hohnke (1975) who reported critical volume deficits ranging from 3·8 to 71 % (mean = 39·8) in Iguana iguana. It is generally accepted that among mammals blood losses in excess of 35 to roughly 50% result in irreversible shock (Kovach, Szasz & Pilmayer, 1969; Guyton, 1980). Only among avian species do reported haemorrhage tolerances approach that of snakes (Kovach & Szasz, 1968).

The stability of arterial pressure and graded tachycardia observed during haemor-rbage (Fig. 2) suggest that baroreflexes (Hohnke, 1975 ; Seymour & Lillywhite, 1976,Lillywhite & Seymour, 1978; Berger, Evans & Smith, 1980; Millard & Moalli, 1980) are activated by the condition of blood loss. Adrenergic mechanisms probably play a dominant role in modulating cardiac performance (Lillywhite & Seymour, 1978; Berger et al. 1980) and may also be expected to enhance vascular tone (Lillywhite & Seymour, 1978). Possible regulation of venomotor tone can be inferred from the stability of central venous pressure (Fig. 2) which suggests that venous capacity is reduced in response to blood loss.

Movements of snakes clearly are important in elevating venous and arterial pressures, no doubt by improving the venous cardiac return. It appears also that snakes have evolved specific behaviours which function to control arterial pressure (Fig. 4) (Lillywhite, 1980b; unpublished).

Behavioural and reflexogenic mechanisms confer rapidity of response but are not suitable for long-term compensation of large blood losses. Hence, the restoration of blood volume is an important feature of homeostatic adjustment in snakes. Estimates of haemodilution based on haematocrit changes or derived more directly from measurements of intravascular volumes (Table 1) indicate that relatively large percentages of blood loss (20 – 71%) were replaced by the absorption of fluids from extravascular compartments. As expected, the degree of haemodilution was correlated with the severity of haemorrhage that was tolerated by a snake. We did not calculate haemodilution volumes based on observed changes in plasma protein because the latter were less consistent than were changes in red cell concentration. Transient increases in plasma protein concentration observed when snakes moved or increased arterial pressure suggest that filtration of plasma was temporarily enhanced in response to the improved haemodynamic state. However, we cannot exclude the possibility that protein entered the circulation from an extravascular source.

If the volume deficits tolerated by snakes are expressed in terms of the volume of blood withdrawn minus the volume of extravascular fluid added to the circulation during the same time period, the resulting net losses of vascular fluid are reduced in magnitude (Table 1) and appear more comparable to the critical deficits observed in mammals. The superior tolerance of snakes to blood loss is thus related, in part, to a high fluid transfer capacity as was shown for birds (Djojosugito, Folkow & Kovach, 1968). In birds, the efficient volume compensation was attributed to strong reflex vasoconstriction in skeletal muscle, resulting in increased pre-to postcapillary resistance ratio and attendant fall in capillary pressure. Similar responses seem likely to occur in snakes, since increased peripheral resistance is a dominant reflex in pressure adjustments to tilting (Lillywhite & Seymour, 1978). There appear to be no data indicating differences in the amount of fluid stores that is available for compensating blood loss in various vertebrates. Possibly snakes are capable of mobilizing part of the intracellular fluid which comprises a relatively larger space than in birds or mammals (Thorson, 1968).

The IBV of Elaphe obsoleta scales in direct proportion to the body mass, as was shown in a variety of mammals (Sjostrand, 1962). Since we used arterial haematocrits in calculations of IBV, a rather consistent discrepancy between Evans blue and washout estimates of the IBV (Table 1) is expected. Assuming that the ratio of whole body haematocrit to central haematocrit is somewhat less than unity (Gregerson & Rawson 1959; Lawson, 1962), correction of the present measurements for the distribution of ed cells would confer closer agreement since Evans blue calculations would be lowered somewhat and washout calculations similarly elevated. It suffices to conclude that the whole IBV of E. obsoleta is roughly 6 % of the body mass which is in agreement with data for other terrestrial snakes (Thorson, 1968; Smeller, Bush & Seal, 1978). Aquatic species may have larger blood volumes (Dunson, 1975; Feder, 1980).

This work was supported by NIH Grant HL 24640, Biomedical Sciences Support Grant RR07037, and University of Kansas general research award 3494 – 0038.