27 September 1985 Discarded cannulae used for hormone research

Using discarded cannulae - fine tubes for drawing blood or fluids from the body or for injecting medicines - and 10 cents worth of local anaesthetic, a Lincoln College veterinary scientist has developed a technique for taking blood from the brain of a live horse that has implications for existing neuro-endocrinological research.

Profeesor Cliff Irvine, Professor of Veterinary Science at the College, set out to found the way ovulation in horses is controlled by the brain and pituitary gland.

He wanted to sample blood from the brain to measure the hormones present.

Medical researchers have for years been trying to get enough hormonal substance from blood in the cranial cavity to identify and synthesise,

Two scientists shared the Nobel Prize for identifying one of the hormones, after millions of dollars had been spent.

Getting a minute amount of the substance has been more expensive than getting one kilogram of moon rock.

And the process has meant killing millions of pigs and sheep under rigid conditions.

Professor Irvine's technique, using secondhand cannulae, is used on live horses in the Equine Research Unit at the College.

Although he has been working on the technique for little more than a year and a half, he has already attracted a lot of interest from medical scientists in New Zealand and overseas. 

Local medical researchers have been working with him since September last year, processing blood from his horses.

Getting a minute amount of the substance has been more expensive than getting one kilogram of moon rock.

And the process has meant killing millions of pigs and sheep under rigid conditions.

Professor Irvine's technique, using secondhand cannulae, is used on live horses in the Equine Research Unit at the College.

Although he has been working on the technique for little more than a year and a half, he has already attracted a lot of interest from medical scientists in New Zealand and overseas.

Local medical researchers have been working with him since September last year, processing blond from his horses.

Earlier he had tried reaching the "pituitary gland of a dead sheep, by boring a hole in the front of the nose and inserting a needle.

"This was ·a difficult and troublesome technique, but I got this working well at Davis," he said.

Then, instead of drilling a hole, he thought he would try to insert a tube up the jugular vein through which blood from the cranial cavity drains.

"I couldn't do it in the sheep ; I tried and tried with dead sheep's heads, but didn't have a show."

"Then I thought I might be able to do it in a horse, so I went down to the pathology room, where lots of horses·were being post-mortemed every day. 

"I tried it on a horse head ; I put a nick in the vein there and I put the cannula along, and I threaded and twisted.

"I had all the tissues off so I could see where it was going ; I could see where all the bends, T-junctions and Y-junctions were.

"I found if I twisted the cannula in the right way I could get right into the brain."

"So that is the way it developed, and it really has turned out remarkably easy."

Professor Irvine said he could now insert the cannula in a couple of minutes.

"I had fiddled around over the years thinking· that blood gets out of the brain, therefore we must be able to get in somehow.

"The blood in these very fine passages that we are trying to get access to, that blood has to get out to the surface somewhere."

Professor Irvine said the blood had to get out along the jugular vein eventually, because all blood from the head drains into that vein.

So he decided to try to get into the jugular vein, using 50-cm-long  teflon cannula.

"There is quite a lot of fiddling, and twisting and turning to do, so you have to know what you are at," he said.

Professor Irvine said that in the last two weeks before he left Davis he had plenty of horse heads to work on.
"Over the Christmas-New Year holidays no horses had been post-mortemed, so there were a lot of horses· in the freezer," he said.

"When they thawed out the horse heads I went in with  my tubes .

"Helped by an eager American student Bob Huun, we fiddled around day and night until finally we got into the jugular vein just beneath the cheek muscle.''

Professor Irvine said he tried the technique on a live horse. about two days before leaving the United States.

"This was successful, so when I got back to New Zealand tried the technique again, and again it worked. "

That was in March last year, but he did very little on the research in the winter,. but started again last September.

After being inserted, the cannula goes along the vein under the jawbone, alongside the face and up into the cranial cavity.

A small lifebelt-type balloon near the tip of the cannula is used to direct the tube along the vein .

The balloon is inflated to prevent the cannula being diverted into small blood vessels, and deflated when the tube is going in the right direction.

Two small metal bands hold the balloon in place near the end of the cannula, and air is blown in through a narrow passage in the teflon cover of the tube.

At first Professor Irvine used x-rays to see which way the cannula was going, but he has become so expert he now inserts by feel.

He usually takes about five minutes to insert the cannula, but ''good ones I can get in in one minute," he said.

Once inserted, the cannula can be plaited into the mane of a horse so samples of blood can be taken easily at any time.

Blood can he taken by syringe, or withdrawn by en infusion pump strapped to the headstall of the horse, just under the jaw.

Professor Irvine said the importance in developing the technique was not in finding things out about horses.

"This is about finding out things important in human medicine," he said.

"In the past it had not been possible to get access to the part of the brain through which the signals travel along blood vessels to other parts of the brain. 

"Now I can get a cannula right into the brain cavity of a horse to sample the blood, so that the hormones can be measured .

"The same system applies to humans ; the brain and pituitary are connected by the same blood vessels in humans, and the same hormones occur in humans.

"This has great relevance to the way the human brain controls the body ; the hormone mechanisms are identical to those of the horse."

The pituitary gland is in a separate compartment in the centre of the cranial cavity under the brain, and separated by a very tough diaphragm.

The brain produces hormones which are carried in the blood to the pituitary, and the pituitary produces hormones that travel around and act on parts of the body.

The hormones in the blood Professor Irvine is sampling are those directing the function of the pituitary.

Professor Irvine started off with his research because he was interested in reproductive failure in horses.

But the technique can also be used for studying the response of the body to stress.

That is why the medical scientists visit the college when Professor Irvine cannulates the horses to collect blood samples for processing.

"'The hormones they are looking at are hormones that are released when the body is subjected to stresses, emotional, physical, heat, exercise all 'forms of stresses.

"The body responds to these by sending a signal to the brain, and the brain processes those signals and makes an appropriate response.

"The brain releases chemicals which travel to other parts of the brain, and these trigger certain events and those events send signals around the body."

Professor Irvine said New Zealand medical scientists were looking at the signals initiated within the brain itself in response to emotional,
physical, chemical and other forms of stress.

Medical  scientists and researchers from Christchurch Hospital Princess Margaret Hospital in suburban Christchurch
and the University of Otago Clinical School have been involved with the technique for about a year. 

And they became involved through a chance conversation Professor Irvine had with a former colleague, Dr Margaret Evans, working at the Princess Margaret Hospital Clinical School.

He told her about the development of his work, and she told some of her colleagues.

"I think they first became interested about last September, and they came out the following month to collect blood samples," he said.

"A  lot of the substances being measured are absolutely new hormones that no one has ever been able to get to before."

Collaborating with Professor Irvine is American endocrinologist Dr Susan Alexander, at the college especially to work on the project, and Mrs Carolyn Redekopp and 'Professor R.A. Donald, of the Department of Endocrinology at Princess Margaret Hospital.

Professor Irvine said the blood taken from the horses had to be processed very quickly.

"The blood is out of the horse and into a refrigerated centrifuge.

"The cells in the plasma are separated, and the plasma is frozen immediately, or chemials are used to preserve the substances .

"These are very delicate hormone, not like the hormones that go right around the body.

"These are very delicate, very short lived, and easily damaged, so the blood has to be processed quickly that is why a team of scientists is needed."

Professor Irvine said the horses used in the research were quite used to being handled, and there was no stress involved in taking blood.

"They eat ; they drink. They are loose in a big shed, and wander around, eat hay and fight with each other.

"Their normal activities are not interrupted, and we just go up and take samples.

"They are not stressed in any way, nor are their functions disturbed."

Professor Irvine said a horse was quite unaware that blood was being taken; whether by collection pump or syringe.

Horses were turned out into the"paddock with infusion pumps attached, and when the horses were not being bled the line was kept clear with anti-coagulant.

Another method of bleeding was to use a withdrawal pump and a long coil.

Blood was drawn into the coil of fine tubing, attached to the side of the horse's head, and kept cool with a freezer pack.

"But we can only do this if the hormone we are measuring is very stable some are not and have to be processed immediately after coming out of the horse.

"Other hormones are quite stable and last for hours ," he said.

"The coil just gradually fills up, but has to be emptied every two or three hours because capacity is limited.

"Then we just gently pump the blood out into separate tubes for measuring the hormones."

Blood can be collected from the coil while the horse is in the paddock.

Medical scientists use a syringe to collect blood samples from humans because the blood has to be processed immediately.

The blood is then put into a centrifuge, chilled or preserved.

Professor Irvine said he was more confident of results when processing samples immediately after blood was taken.

The only reason the coil system was used because the scientists wanted to get sleep ; bleeding went on over 24 hours, he said.

In 24 hours 4 mls of blood was taken every five minutes, 48 mls an hour and 1.15 litres in the period.

Blood volume of a horses is about 30 litres, so the blood taken is only about three per cent of blood volume.

The horse makes up this amount of blood as .fast as the blood is taken out.

Sometimes larger samples are taken, because occasionally a number of different hormones have to be measured in the same sample for interaction.

Professor Irvine said that then eight to 10 mls might be taken, but this was usually done over an hour or so, never over a long period.

Cannulae are usually left in place  for five or six days, because this usually is the time taken for experiments.

"There is no reason why we couldn't leave the cannula in place longer but we have no reason to do this."

And the cost of the materials for using the technique ?

Professor Irvine said the cost was relatively low "these are actually discarded cannulae which have been used for getting into people's hearts."

The cannulae could not be used again for humans, because these could not be sterilized satisfactorily.

However, the cannulae could be sterilized well enough for his purpose.

"When I insert the cannula I put in a little local anaesthetic - about half a mill costing 10 cents that's all.

"The horses were given to us years ago, and some of the horses are mine," Professor Irvine said .

Further information from Professor Cliff Irvine, Department of Veterinary Science, Lincoln College