New Scientist vol 177 issue 2385 - 08 March 2003, page 46
A shark with an amazing party trick is teaching doctors how to protect the brains of stroke patients.
Douglas Fox made its acquaintance
IT IS 90 minutes since Lazarus last breathed oxygen, and he is finally slipping away. I reach into his aquarium, past the hoses that bubble pure nitrogen through his water, and flip him belly-up. No response. Just to make sure he's gone for real, Gillian Renshaw and I wait a while longer - for us a boring five minutes serenaded by the hum of fluorescent bulbs. But not for Lazarus. His oxygen-deprived brain must be drifting somewhere east of Jigangaland.
When I finally lift Lazarus from the water, he hangs limp in my hands like a slab of sashimi, and I worry we've waited too long. But then as I carry his flaccid frame over to another aquarium, it happens: like a blue bolt from heaven, a surge of slippery, writhing fishlife suddenly animates his body. Once again, Lazarus lives.
At 70 centimetres long, Lazarus is a typical epaulette shark, so called for the black patterns resembling military insignia on their "shoulders". In the wild, these fish are confined to the waters off eastern Australia and Papua New Guinea. But their reputation for surviving without oxygen is spreading far and wide. If we can understand how they do it, then we could vastly improve our treatment of medical conditions where organs are deprived of oxygen, such as stroke and heart attack. But it doesn't stop there. Somewhere within the shark's stoic capacity for life after breath may lie something even bigger: clues that could help us withstand many life-threatening conditions in which our cells commit suicide.
An animal living in the shallows around coral reef platforms might not seem an obvious candidate for becoming a Hercules of hypoxia. But the epaulettes' seemingly cosy home has a habit of turning nasty. The water, just centimetres deep, is isolated from the ocean at low tide, and although its oxygen is replenished during daylight hours by photosynthesising algae, this doesn't happen during low tides after dark. Instead, as reef inhabitants respire, oxygen levels plummet to as low as 20 per cent of normal - low enough for non-adapted fish to suffer permanent brain damage within a few minutes. But not epaulettes. They remain on the reef as oxygen levels fall, hunting shellfish and crabs.
Even more impressive, the sharks can survive being left high and dry. At extreme low tides you occasionally find them beached on the exposed reef, limp and comatose. But pick one up and it quickly reboots and wriggles for freedom. This amazing talent for resurrection is what first attracted Renshaw, a physiologist at Griffith University in Gold Coast, Australia. Five years on, she has found the epaulette's abilities go way beyond what it displays in the wild: in her lab Lazarus and his chums regularly survive three hours with nary a bubble of oxygen, and have even been known to recover after their tails begin to stiffen - an early sign of rigor mortis.
It's true that a few other animals can cope with oxygen deprivation for far longer - some fish and turtles pass the entire winter frozen, without oxygen, under ice (see "Masters of hypoxia"). But at low temperatures, cells tend to consume oxygen more slowly, delaying their death. What makes the epaulette so interesting is that it has evolved to survive oxygen starvation at up to 26 °C - tantalisingly close to human body temperature.
Unlike the shark, however, we are ill-equipped to deal with low oxygen. Nowhere is this more apparent than in the brain, where hypoxia caused by a stroke triggers a fatal chain reaction in just five minutes or so. That's because brain cells rely so heavily on aerobic respiration: unlike other cells such as muscle, they store almost no glucose that can be used for emergency energy production in the absence of oxygen. And the brain is the body's biggest energy guzzler, so supplies soon run low when the blood flow is cut off. Without energy, neurons become depolarised as potassium ions leak out and sodium and calcium ions rush in. Next, the pumps that usually pull neurotransmitters, especially glutamate, into the cell go into reverse. As glutamate flows out uncontrollably it triggers neighbouring neurons to fire, just when they should be conserving energy, and to release glutamate themselves, in turn exciting their own neighbours. And so the chain reaction goes on. All this conspires to push neurons over the brink - most will eventually opt for apoptosis, cell suicide (see Diagram).
Even cells that survive oxygen starvation aren't home free. In strokes, neurons continue to die for 48 hours after blood flow has been restored, and delayed cell death also occurs after heart attacks. The culprit is "reperfusion injury": during oxygen shortage, the mitochondria (the cell's energy-producing organs) mysteriously shift gear, producing free radicals from what oxygen they can get. But when blood flow returns, the mitochondria are still in this altered state, and convert the tide of life-giving oxygen into death-dealing radicals, damaging proteins and membranes, and sending more cells spiralling into apoptosis.
So how do epaulette sharks avoid this catastrophic series of events? Renshaw has found that as oxygen levels drop, the shark first maintains its uptake by breathing more rapidly. Once levels fall below about 30 per cent of normal, however, it slows its breathing and heartbeat, and relaxes arteries so that it is easier to pump blood to the brain. Next, it powers down non-essential brain functions by releasing GABA - a depressive neurotransmitter that counteracts the excitement of glutamate - in brain areas that aren't needed. The shark becomes limp and paralysed, and possibly even blind. But its electroreception sense continues to run and, if it detects predators or prey, it can lurch back to life. As it shuts down brain centres, the epaulette also turns down its mitochondria. As well as saving energy, says Renshaw, this protects the mitochondria from reperfusion damage by minimising the production of free radicals once oxygen returns.
Of course, it's a gross oversimplification to compare a stroke victim to an oxygen-deprived epaulette shark, not least because a blocked artery doesn't just deprive cells of oxygen, but also glucose, which can serve as an anaerobic energy source. But Renshaw's studies highlight some promising approaches to stroke management. One obvious candidate is GABA, which is produced not only by epaulette sharks but also by other animals that can tolerate low oxygen levels. In the 1990s, researchers thought chemicals that mimic GABA or block the toxic effects of glutamate would become wonder drugs for treating stroke. But a decade and more than 30 failed clinical trials later, it is apparent that transferring lessons learned from animals that cope with low oxygen into humans isn't going to be so simple.
Part of the problem is that glutamate is not the only neurotransmitter that excites neurons to death: dopamine, aspartate and others also play a part. Although researchers tried to damp these down as well, most clinical trials tested only one drug at a time, and that may not have been enough to calm excited neurons. What's more, studies from hypoxia-tolerant turtles hint that small amounts of glutamate may somehow help - possibly by tuning neurons to be more sensitive to calming neurotransmitters such as GABA. If this is the case in humans, then blocking glutamate could do as much harm as good.
Another problem is that not all strokes are alike. Drugs that damp down glutamate should work best for patients in whom only a small bit of the brain has been starved of oxygen, and most of the damage is likely to be caused by the spreading wave of neurotransmitter hysteria. But these people can be difficult to distinguish from patients whose brain cells have mostly been killed directly by oxygen starvation.
While some clinicians still hold out hope for GABA and its ilk, Renshaw's studies have persuaded her to experiment with an alternative approach that has the potential to counteract hypoxia throughout the body, not just in the brain. She has found that by giving epaulettes several two-hour bouts of 5 per cent normal oxygen levels, she can increase their subsequent ability to survive without oxygen. Experiments with rats show that such "preconditioning" also works in animals that have not evolved to tolerate low oxygen. And there are some indications that humans are no exception. Patients who have experienced chronic chest pain (a sign their heart is short of oxygen) are more likely to survive a heart attack than those who haven't. And patients who suffer mini-strokes, are less likely to die when they have a major stroke.Surgeons performing a coronary bypass sometimes exploit the effect by clamping off arteries feeding the heart for several minutes before they halt circulation for longer while they attach a graft.
Preconditioning seems to evoke a wide range of defences, from inhibiting some types of glutamate receptors to producing enzymes that neutralise radicals. Exactly how it works is a bit of a mystery, but it looks like mitochondria are the key. Navdeep Chandel from Northwestern University in Chicago has found that when heart cells are stripped of their mitochondria, they lose the ability to turn on preconditioning in response to low oxygen, or undergo apoptosis. This supports the idea that mitochondria signal when oxygen is low - by emitting a trickle of oxygen radicals - and then give the cue to either turn on protective responses or commit cell suicide. The trick with preconditioning is to persuade them to choose life.
Low-oxygen preconditioning is already being tried on patients. In Russia, intermittent hypoxic therapy (IHT) has been used for years to treat all sorts of conditions from asthma to heart disease and chemotherapy toxicity. Elsewhere, a handful of doctors are trying it for diabetes and chronic fatigue. Patients are typically given IHT five minutes on, five off, for one hour a day, five days a week - starting near 90 per cent of the oxygen concentration at sea level and gradually decreasing to 50 per cent.
Critics doubt whether this is enough to induce the sort of preconditioning seen in rats given 50 per cent of normal oxygen levels for hours per day. Nonetheless, Renshaw is now testing whether humans given IHT have preconditioning-like responses, such as increases in levels of proteins that make blood vessels grow and heat shock proteins, which limit cell damage. If so, then regular IHT might work as prophylaxis for people at risk from heart attack and stroke.
Limiting oxygen may not be an option for really sick patients, but there are other ways to get a similar response. In rats, preconditioning has been induced by low doses of poisons such as cyanide, by a synthetic version of a bacterial endotoxin, by electrical stimulation of a brain region called the cerebellar fastigial nucleus, and even by mild cooling of 5 °C for 20 minutes. Cooling might be an especially benign way to precondition, says Kevin Lee, a neuroscientist at the University of Virginia in Charlottesville. But since the protective effect takes six hours to mount and lasts no more than 10 days, cooling would only be useful if you could somehow anticipate oxygen deprivation.
Many of the protective effects of preconditioning require cells to turn up or down certain genes, which takes hours, but some preconditioning effects kick in within minutes. Researchers are now looking for precise ways to activate these. For example, certain pores in mitochondrial membranes open up and allow potassium ions to flow in during low-oxygen preconditioning. And studies in rabbits show that drugs that promote opening prevent apoptosis and cause cells to respond as if they have already been preconditioned.
Another short cut exploits the finding that two days after preconditioning, brain cells increase production of chemicals called EETs, which reduce inflammation and cause brain capillaries to widen. By injecting EETs into rats 20 minutes before clamping off a brain artery, Nabil Alkayed from Johns Hopkins University School of Medicine in Baltimore reduced the volume of dead brain tissue by 30 per cent. He speculates that a drug that had this effect could provide rapid protection to patients who have just suffered a stroke.
But the word is that preconditioning drugs could turn out to have much wider uses than simply treating oxygen starvation. Targeting mitochondria gives you the potential to treat a whole variety of stresses throughout the body. Mitochondria stand at the crossroads, determining whether a cell dies by apoptosis or survives by preconditioning, so a drug that nudged them in the right direction could protect against damage following head trauma and seizure, spinal cord injuries, heat stroke, severe haemorrhage, the toxic effects of chemotherapy, and on and on. Experiments have already shown that hypoxic preconditioning protects rats against several of these insults.
"If it doesn't kill you," concludes Renshaw, "it makes you stronger. What we're interested in is why." She and others in the field are well on their way to finding out - and what better ally to have than a fish that's perfectly happy floating belly-up?
Douglas Fox is a freelance science writer living in northern California
"Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia" by Matthew Routley, Goran Nilsson and Gillian Renshaw, Comparative
Biochemistry and Physiology Part A, vol 131, p 313 (2002)
"Surviving anoxia with the brain turned on" by Goran Nilsson, News in Physiological Sciences, vol 16, p 217 (2001)
Masters of hypoxia
GOLDFISH : In its natural habitat can spend winter months sealed, with no oxygen, under pond ice. It depresses its metabolism by 90 per cent, swimming slowly. Like a runner, it uses glucose as an anaerobic energy source. Unlike a runner, it gets rid of the toxic waste - lactic acid - by converting it to alcohol and peeing it away. Low-oxygen tolerance made goldfish the world's first aquarium fish a thousand years ago.
RED-EARED SLIDER TURTLE : Also passes winter under ice. It depresses its metabolism by 95 per cent, becoming comatose, and uses glucose as an energy source. The turtle detoxifies lactic acid waste by neutralising it with carbonate that it leaches from its shell.
WOOD FROG : During winter, its breathing and heartbeat stop and 65 per cent of the water in its body freezes. It uses glucose as a cryoprotectant and energy source. European explorers described finding frozen frogs in Canada for the first time. "A remarkable Experiment," wrote Captain Francis Smith in 1747, "is to take the Earth in which the Frog is froze, and break it in Pieces without thawing it, the Frog will break like Glass. But thaw that Earth by the Fire, the Frog will recover and leap."
BAR-HEADED GOOSE : Migrates from India to Tibet, flying across the Himalayas at altitudes of up to 11,000 metres. There, oxygen levels are 20 per cent those at sea level - low enough to kill a human, but not the goose. While flying, it still has enough spunk left to honk, or so say delirious mountaineers who claim to have heard it.
LOÌC LEFERME, FREEDIVER : Last year he descended to a world-record depth of 162 metres on a single breath of air. When freedivers like Leferme hit the water, their hearts slow by 50 per cent, and their circulation shifts, lavishing most of their blood on the heart and brain.