Selective brain cooling and its vascular basis in diving seals Free

When seals undergo experimental dives they reduce their cardiac output to about 10% of the pre-dive value, primarily as a result of a profound bradycardia, to compensate for a selective peripheral arterial vasoconstriction. This ensures that the oxygen in the blood is delivered to hypoxia-sensitive tissues, like the brain, while the rest of the body has to subsist on local stores of oxygen and/or anaerobiosis (Ramirez et al., 2007). In this situation it is of obvious importance that brain metabolism, and hence brain oxygen requirements, are kept as low as possible to extend the animal's diving capability, and it follows that a reduction of brain temperature would meet this end.

Scholander and colleagues (Scholander et al., 1942) reported a drop in brain temperature of harbour seals (Phoca vitulina) during experimental diving, but their results were obtained by use of mercury thermometers and were generally regarded with scepticism and soon largely forgotten. Some 40 years later, however, Kooyman and colleagues (Kooyman et al., 1980) showed that the central arterial temperature of freely diving Weddell seals (Leptonychotes weddelli) decreased about 3°C during a 53 min dive, and Hill and coworkers (Hill et al., 1987) found that the aortic temperature of freely diving Weddell seals was reduced by about 2°C in periods of active diving. Finally, in preliminary reports we (Blix et al., 2002; Odden et al., 1999) confirmed the results of Scholander et al. (Scholander et al., 1942) showing that seals may indeed reduce their brain temperature, even during relatively short experimental dives.

Any reduction of brain temperature will normally lead to vigorous shivering in mammals (Simon et al., 1986), and if so this thermoregulatory response would compromise the brain cooling and in that context be rather counterproductive. However, Kvadsheim and colleagues (Kvadsheim et al., 2005) have previously shown that the normal shivering response to brain cooling is inhibited as part of the response package that is elicited upon diving. In the present study we add new insight about selective brain cooling in seals and endeavour to explain how it is achieved.

A total of 4 harp seals (Pagophilus groenlandicus, Erxleben 1777) and 9 hooded seals (Cystophora cristata, Erxleben 1777), aged from 1 to 3 years, of both sexes, were used in this study. Information on the diving behaviour of harp (Folkow et al., 2004) and hooded seals (Nordoy et al., 2008; Folkow and Blix, 1999) is available elsewhere. The animals used in the present study were originally caught as pups in the pack-ice of the Greenland Sea and raised in captivity at Tromsø in 5.5 m×5.5 m×1.2 m seawater pools with wooden ledges, where they were offered herring (Clupea harrengus) or capelin (Mallotus villosus) supplemented with a vitamin complex (Blix et al., 1973). The animals were taken under permits issued by The Royal Norwegian Ministry of Fisheries and The Royal Danish Ministry of Foreign Affairs, and the experiments were carried out under permit from the Norwegian Animal Research Authority. Animals used for morphological studies were killed for other purposes by overdoses of pentobarbital (Nembutal, 40 mg kg^–1) injected into the extradural intravertebral vein.

In preparation for the experiments the animal was placed on a specially designed restraining board of classic design and accustomed to experimental dives in water of 3–4°C over a period of several days. The duration of these preparatory dives was varied over the range of 10–20 min to avoid habituation. Before each experimental session, the animal was instrumented with electrodes for measurement of heart rate and thermocouples for measurement of brain, intra-aortic, musculus latissimus dorsi and rectal temperature. Before diving, the animal was allowed to equilibrate with its body immersed to the neck in the water for at least 1 h, whereafter it was submerged three times for periods of 10, 15 or 20 min separated by recovery periods of 40–60 min while still immersed to the neck in water.

Brain temperature was measured in 4 harp seals and 5 hooded seals as described previously (Odden et al., 1999): a 30 mm long and 1.5 mm thick blind-ended stainless steel tube was surgically implanted into the brain of the animals under full isoflurane anaesthesia. The steel tube was placed 10 mm lateral to the midline into the left cerebral hemisphere, near the third ventricle (as verified post mortem). Animals were not used in any experiments until at least 48 h after implantation of the tube. Before each experiment, a 0.5 mm isolated copper–constantan thermocouple was introduced into the tube under light sedation [0.6 mg kg^–1 i.m. injection of Zoletil forte vet (tiletamin–zolazepam); Virbac, Carros Cedex, France]. The thermocouple was connected through a thermocouple amplifier with internal temperature reference (AD 595 CD: Analog Devices, Norwood, MA, USA) to an A/D converter and data acquisition system that stored the data every 20 s (Lab-Acq Pro and Insta-Trend Pro; Dianachart, Oak Ridge, NJ, USA). After sedation, the animals rested in air for at least 1 h to allow complete recovery before the series of diving experiments commenced.

Aorta blood temperature was measured in 2 harp seals by exposing a small section of the brachial artery at the base of the fore-flipper under full isoflurane anaesthesia and inserting a 0.5 mm sterile copper–constantan thermocouple transmurally and advancing it retrograde to the level of the aorta. The temperature was recorded as described for brain temperature. Again the animals were not used in any experiments until at least 48 h after completion of the surgery.

Musculus lattissimus dorsi temperature was measured in 1 harp seal by inserting a hypodermic needle under local anaesthesia [subcutaneous injection of 2–3 ml Xylocaine (10 mg ml^–1); AstraZeneca, Södertälje, Sweden] and introducing a 0.5 mm copper–constantan thermocouple 2 cm into the lattissimus dorsi muscle through the hypodermic needle which was subsequently withdrawn. The temperature was recorded as described for brain temperature.

Rectal temperature was recorded in 4 harp seals and 5 hooded seals with a copper–constantan thermocouple probe that was inserted 20 cm into the rectum of the animal. The temperature was recorded as described for brain temperature.

All thermocouples were calibrated at 0 and 40°C, and the recorded temperatures were linearized according to Tøyen (Tøyen, 1992).

During all diving experiments, heart rate was recorded using two subcutaneous electrodes placed anterior and posterior to the heart along the dorsal mid-line, and a third reference electrode was placed laterally on the animal. The electrodes were connected to a monitoring unit (CM-4008; Medi-Stim, Oslo, Norway) where data were stored digitally, or recorded on a printer (TA 4000; Gould, Valley View, OH, USA).

The brachial arteries of 3 dead hooded seals were perfused first with saline and then with McDowell's fixative (McDowell and Trumph, 1976) immediately after death, whereupon tissue samples were excised from the ventral side of the fore-flippers, cut into smaller blocks and immersed in fresh fixative until further processing. In preparation for further processing all samples were rinsed in 2.5% glutaraldehyde in 0.1 mol l^–1 phosphate buffer adjusted to pH 7.2. Samples intended for light microscopic examination were subsequently dehydrated in graded series of ethanol up to 100% and embedded in Epon (Electron Microscopy Sciences, Hatfield, PA, USA), then cut on an Ultracut UCT microtome (Leica, Wetzlar, Germany) and examined using a Colour View Camera with accompanying software (Soft Imaging System, Münster, Germany). Samples intended for scanning electron microscopy were dehydrated in graded series of ethanol and critical point dried with carbon dioxide, then attached to metal stubs with silver paste, sputter coated with a 30 nm platinum layer and examined in a Phillips XL 30 ESEM (Eindhoven, The Netherlands).

Vascular injections of quick-setting polyester (Ashland, Porsgrunn, Norway) were made into the brachial artery of 4 dead hooded seals under 0.4–0.5 atm (∼40.4–50.5 kPa) pressure. Pressure was maintained for different periods in the different animals to obtain casts of the arterial part alone, and in combination with the venous part of the vasculature. After hardening of the cast the tissue was removed by digestion in concentrated HCl.

Photographs were taken of the arteries and veins in the fore-flippers of 2 dead hooded seals after rinsing of the vessels with saline and subsequent injection of radio-opaque fluid (Mixobar Colon, Astra Meditec, Göteborg, Sweden; 1 g ml^–1) into the brachial artery or the veins of the digits, using OEC 9600 (OEC Medical Systems Inc., Salt Lake City, UT, USA) X-ray equipment.

Temperature data were subjected to statistical analyses to test for significant differences in changes in temperature in connection with diving. The distribution of temperatures for repeated measurements in one animal was approximately symmetric, but with somewhat heavy tails. Therefore, the results are presented as Wilcoxon medians with corresponding 95% Wilcoxon's signed rank confidence intervals (Hollander and Wolfe, 1999). Different animals of both species showed very similar temperature responses to diving, as confirmed by ANOVA, where drop in temperature was the dependent variable and species and different animals and different dives were potential independent variables. Only a large random effect between different dives by the same animal was found. Dive series from different species and animals were therefore combined in one non-parametric presentation of the results. A P-value of <0.05 was taken to indicate statistically significant differences.

Heart rate always fell promptly from about 90 beats per minute (b.p.m.) to 8–10 b.p.m. upon submergence, stayed at this level throughout the dive, except for occasional brief episodes of muscle movement, and rose promptly to about 100 b.p.m. upon emergence (Fig. 1).

Brain temperature (T_brain) always fell significantly upon submergence (Fig. 2), in some cases as much as 2.5°C, but that was unusual (Tables 1 and 2). The drop in T_brain was not linear throughout the dive. It was slow during the first few minutes, but dropped quite linearly, and significantly more (P>0.001), after about 5 min of diving (Figs 2 and 3; Table 1). In some cases we noted that T_brain fell progressively during series of three consecutive dives (Fig. 4), but this was not always the case. On a few occasions we also noted that T_brain started to fall prior to the commencement of the dive. The lowest T_brain ever recorded was 34.2°C.

Intra-aortic temperature (T_aorta) fell by and large in parallel with T_brain, but was always significantly (about 0.5°C, P=0.002) lower than T_brain, and the difference tended to decrease throughout the dive (Fig. 5; Table 2).

Musculus lattissimus dorsi temperature (T_m) was fairly stable throughout the dives (Fig. 5; Table 2).

Rectal temperature (T_r) dropped very significantly (Table 2) during dives. In fact, in some cases it dropped even more than T_brain (Fig. 5), but overall significantly less (P=0.025; Table 1).

Angiography (Fig. 6) as well as the plastic casts revealed that the brachial artery of the hooded seal consists of one prominent vessel with few significant side branches until it splits up to the five digits of the fore-flipper. However, at the base of the flipper a conspicuous arterial rete is shunted in parallel with the brachial artery. The light and scanning electron microscopical examination of tissue from this area (Fig. 7) revealed that the blood in the arterial rete runs in parallel with blood in an even more conspicuous venous rete, creating what appears to be an ideal counter-current heat exchanger.

Angiography (Fig. 6) also revealed a system of prominent superficial veins which may lead the venous effluent from the flippers outside the heat exchanger described above and thereby provide cold blood to the heart.

This study has shown that seals respond to experimental diving not only with the well known host of cardiovascular responses (Ramirez et al., 2007), as reflected in a profound bradycardia (Fig. 1), but also with a controlled cooling of their brain (Figs 2, 3, 4, 5). The median cooling recorded in this study was of the order of 1°C over 15 min, but brain cooling of 2.5°C was recorded in single experiments, confirming the old and defamed results of Scholander and colleagues (Scholander et al., 1942). Moreover, our finding of a blockade of the thermoregulatory responses of hooded seals during diving (Kvadsheim et al., 2005) strongly supports the notion that the brain cooling is indeed intended and under physiological control. This also includes reduction of the thermoregulatory ‘set-point’ for body temperature, as part of the diving response package. This is basically a medullary reflex package, but in expert divers it may be profoundly affected by cortico-hypothalamic input, such as the controlled blockade of the thermoregulatory responses to hypothermia. It is by now also quite clear that expert divers may decide to what extent they will activate their (diving) defence responses depending on the challenge of the dive to come (Blix and Folkow, 1983; Folkow and Blix, 2010).

Without the recorded cooling, brain temperature is likely to increase during diving because of increased insulation of the head caused by vasoconstriction in skin and blubber and a marked reduction in cerebral blood flow (Blix et al., 1983a), which reduces the removal of heat from the brain during the early part of the dive. This is further supported by the fact that the difference between brain and aorta blood temperature was gradually reduced towards the end of the dive (Fig. 5), when cerebral blood flow is supernormal (Blix et al., 1983a). Moreover, assuming that Q₁₀ is the same in seals as in piglets (Busija and Leffler, 1987; Laptook et al., 1995), a reduction in brain temperature of 2.5°C in the seal should lead to a reduction in brain oxygen demand of about 25%. In cases of prolonged diving, when the brain is pretty much the sole consumer of haemoglobin-bound oxygen in seals, this obviously represents a very significant expansion of diving capacity. Hindell and colleagues (Hindell et al., 1992), and others, have pondered over the fact that several species of seals, notably the southern elephant seal, repeatedly exceed their calculated aerobic dive limit, and brain cooling may well, at least in part, also be the explanation for this.

In any case, all this begs the question, will the brain work adequately at such reduced temperatures? This is of course anybody's guess in seals, but Moser and Andersen (Moser and Andersen, 1994) found that spatial learning in rats is unimpaired at brain temperatures as low as 30°C, and we therefore boldly suggest that a cool head is a great advantage in diving seals.

This study suggests that when brachial arterial blood flow to the fore-flippers is directed to the capillaries of the tissues for nutritional purposes, the blood may return in the deep central veins through a sophisticated counter-current vascular heat exchanger at the base of the flippers (Figs 6, 7, 8). This heat exchanger contains elements of an arterial and a venous rete which are reminiscent of structures described from the tail fluke and dorsal fin of the porpoise (Lagenorhynchus acutus) by Scholander and Schevill (Scholander and Schevill, 1955). This structure was later erroneously represented as a venous rete around the brachial artery, as the sole elements of (fore-) flipper circulation by Schmidt-Nelsen (Schmidt-Nelsen, 1975), and because of the prominence of his book and probably because of the simplicity of the concept, it unfortunately has become the general conception of marine mammal fluke and flipper circulation. However, at least in hooded seals, the arterial rete, which runs in parallel with the venous rete, is shunted in parallel with the prominent brachial artery, in much the same way as described in mallard ducks (Anas platyrhynchos) by Midtgård (Midtgård, 1980), and this difference is not trivial from a brain cooling point of view, as elaborated upon below. In any case, the counter-current vascular heat exchanger ensures that any capillary blood in the flippers may return to the body core at a temperature very close to core temperature and thereby avoid any body cooling in circumstances when that is undesirable. In that case the numerous cutaneous arterio-venous (A–V) shunts in the skin of the flippers (Bryden, 1978) will be closed and the entire venous drainage routed through the heat exchanger. The A–V shunts are under the control of the thermoregulatory ‘centre’ in the hypothalamus. They are not activated as part of the medullary controlled cardio-vascular diving responses if the animal is in positive thermal balance, as first shown by Djojosugito and colleagues (Djojosugito et al., 1969) in the duck, and later by Hammel and coworkers (Hammel et al., 1977) in the harbour seal, and A–V shunts are not affected by chemoreceptor activation later in the dive (Chalmers and Korner, 1966; Löfving, 1961).

When, on the other hand, the A–V shunts are opened, venous drainage occurs through the huge superficial veins (Figs 6 and 8) and thereby bypasses the vascular heat exchanger at the base of the flippers and provides cold blood from the cold flipper skin (Irving and Hart, 1957) to the heart. It follows that the cold blood will henceforth be delivered selectively to those tissues that are circulated, notably the brain, and the cooling rate of the brain will be determined by the ratio of carotid to brachial arterial blood flow, and of course water temperature, during diving. We (Blix et al., 1983a) have shown that A–V shunts are indeed open during experimental dives. This is not only because of the need for brain cooling but also because of the need for some A–V shunt flow in the hind flippers to allow the considerable oxygen-containing venous depots (Hol et al., 1975) to reach the heart in a situation when most organs are shut off from the circulation. The latter is clearly supported by the finding of maintained saphenous venous flow during experimental diving in the harp seal (Hol et al., 1975). This will inevitably bring some cold blood from the hind-flipper venous plexuses (Tarasoff and Fisher, 1970) to the venous plexuses of the pelvic region (Blix et al., 1983b). This is most likely the reason for the decrease in the temperature recorded in the rectum (Fig. 5), but the effect of this cooling on brain temperature will be minor, since this blood will be heated by its passage through the central venous blood pool. The muscles, on the other hand, are not circulated during experimental dives (e.g. Blix et al., 1983a) and the temperature of the lattissimus muscle was stable or tended to rise during the dive in the present study (Fig. 5; Table 2). This is in accordance with Ponganis and coworkers (Ponganis et al., 1993) who found stable muscle temperatures in the Weddell seal diving voluntarily in the Antarctic.

Then why is the degree of brain cooling variable from dive to dive in the same animal? This is quite certainly due to the fact that the phenomenon depends on the control of the cutaneous A–V shunts and that emotional stimuli exert profound effects on the vascular tone in these shunts. Thus, any transient or permanent apprehension on one side, and any degree of habituation on the part of the animal on the other, are bound to cause less cooling than would have been the case under natural conditions at high sea, when the animal is in control.

We conclude that the brain temperature may be actively down-regulated by at least as much as 2.5°C by the use of cold blood from the fore-flippers in the diving seal. By the Q₁₀ effect (e.g. Schmidt-Nielsen, 1975) alone cerebral oxygen requirements may as a result be reduced by as much as 25% and diving capacity thereby substantially extended. This may explain, at least in part, how seals diving voluntarily repeatedly exceed their calculated aerobic dive limit.