WO1988008728A1 - Device and method for perfusing an animal head - Google Patents

Abstract

This invention relates to a method for providing physical and biochemical support for an animal head which has been ''discorporated'' (i.e., severed from its body). This method uses processing equipment to supply a severed head with oxygenated fluid (such as blood) and nutrients, by means of tubes connected to arteries which pass through the neck. After circulating through the head, the deoxygenated fluid returns to the processing equipment by means of tubes connected to veins that emerge from the neck. A series of processing components including oxygenators (58) and at least one pump (52) will remove carbon dioxide and replenish the oxygen level in the fluid, and return the replenished fluid to the severed head via tubes attached to the arteries.

Description

DEVICE AND METHOD FOR PERFUSING AN ANIMAL HEAD

BACKGROUND OF THE INVENTION

This invention is in the fields of biochemistry, blood processing, and biomedical devices.

The following descriptions are intended to provide a basic introduction to the relevant prior art. For more extensive discussion, the reader should refer to the references listed, and to other references known to those skilled in the art. Complete citations for books and articles are provided in a list of references near the end of this patent, before the claims.

Terminology

It is often very difficult for people who do not specialize in medicine to understand medical textbooks and articles, because highly technical terms are used instead of common words. Even those who specialize in biochemistry or biology often have difficulty interpreting articles on medicine, especially surgery. Therefore, the following paragraphs contain a list of technical words used in this branch of medicine (followed by their plain language equivalents, in parentheses). These are not exact definitions, which can be found in medical dictionaries; instead, they are approximations, offered solely as a convenience for readers who aren't familiar with medical terms.

The medical terms that are relevant to this invention include prefixes such as 'cardio-' (involving the heart), 'neuro-' (involving nerves), and 'myo-' (involving muscle). Three different prefixes (hemo-, hema-, and hemato-) all refer to blood. Medical nouns include cephalon and encephalon (the head), cervix (the neck), thorax (the chest), cranium (the skull), and vasculature (a set of blood vessels which serve a certain organ or part of the body). Their corresponding adjectives, in the same order, are cephalic (involving the head), cervical (involving the neck), thoracic (involving the chest), cranial (involving the skull), and vascular (involving blood vessels). Other adjectives which refer to specific organs include cardiac (involving the heart), pulmonary (involving the lungs), hepatic (involving the liver), and renal (involving the kidneys).

Osseous and ossified refer to bones, or to something which has hardened such as a plastic which is injected in fluid form and which becomes hard. Cannula and catheter are sometimes used interchangeably, since both are tubes used for transporting fluid from or to the body; however, in proper use, a cannula (the plural form is cannulae) is inserted directly into a blood vessel, while a catheter is inserted into a body cavity (such as the bladder, to remove urine). An anastomosis is created when two tubes (such as blood vessels) are surgically connected to each other, allowing fluid to pass from one into the other. A ligation is a cut which severs something. For example, large blood vessels are ligated by clamping them to shut off the flow, suturing (stitching) them closed, cutting the vessel slightly beyond the stitches, and then releasing the clamp. Small blood vessels are usually ligated by cauterizing them (cutting them with an electric device which seals them shut).

Somatic refers to body; a cephalosomatic transplant refers to transplanting a cephalon (a head) onto a body. A transsection is a surgical cut which passes across the main dimension of something which is usually cylindrical in shape; for example, a cervical transsection is a cut through the neck, which separates the head from the body. A laminectomy is a surgical cut which passes through several layers of tissue.

In general, a 'prosthesis' is any device which substitutes for a missing part of the body. Although this usually refers to mechanical devices such as artificial limbs, it can also refer to devices which process blood either mechanically or chemically. Devices such as heart pacemakers and hemodialysis units (artificial kidneys) are usually referred to as biomedical devices rather than prostheses, since they assist diseased or injured organs but don't replace them. However, the term 'biomedical' is extremely broad and somewhat vague; any device with a medical or biological use (such as a hypodermic syringe) might be regarded as a biomedical device. Therefore, some people may refer to any device which either replaces or assists some part of the body (such as an artificial kidney) as a prosthesis.

As used herein, terms such as 'body,' 'patient,' and 'head' normally refer to research animals. However, it is possible that after this invention has been thoroughly tested on research animals, it might also be used on humans suffering from terminal illnesses or mortal injuries.

Cardio-pulmonary bypass devices

Cardio-pulmonary (heart-lung) bypass machines are commonly used during open heart surgery, since it is difficult for a surgeon to operate on a beating heart. In an operation involving a bypass machine, the heart is temporarily stopped by chilling it with a cold fluid, clamping the aorta, and administering potassium chloride. Cannulae (specialized tubes having rigid or semi-rigid ends, usually made of plastic) are inserted into the superior and inferior vena cavae (major blood veins which normally carry deoxygenated blood to the heart) through an opening that is created in the right atrial wall of the heart. These cannulae carry blood with low oxygen content and high carbon dioxide content from the body to the bypass machine.

The blood usually passes through a heat exchanger which cools the blood. During most bypass operations, the body temperature of the patient is lowered to about 28 to 30 degrees Celsius, to slow down metabolism and reduce oxygen consumption by the organs.

The cooled blood enters an oxygenation chamber. The two most commonly used types of oxygenation chambers are called 'bubble' oxygenators and 'membrane' oxygenators. In a bubble oxygenator, gas containing oxygen and a relatively small percentage of carbon dioxide enters the bottom of the chamber and flows upward through the circulating blood, creating bubbles. Contact between the bubbles and the blood adds oxygen to the blood and removes carbon dioxide. Upon leaving a bubble chamber, the blood passes through a defoaming device. By contrast, membrane oxygenators contain a membrane which separates the gas from the blood. Oxygen and carbon dioxide diffuse through the membrane, allowing oxygen to displace carbon dioxide in the blood. By eliminating the interface between blood and gaseous oxygen, membrane oxygenators tend to cause less denaturation of blood proteins, and less damage to blood cells, than bubble oxygenators. Membrane oxygenators are sold by several suppliers, including American Bentley, and Shiley Inc., both of Irvine, CA.

After the blood has been oxygenated, it flows to a pump, usually a 'peristaltic' pump (also called a 'roller' pump, described below). The blood is pumped to a suitable pressure, and it returns to the patient's body via a cannula which is inserted into the patient's aorta.

Several chemical parameters are usually measured during an operation involving a bypass machine, including dissolved oxygen content (expressed as pO), dissolved carbon dioxide content (expressed as pCθ2), acidity (expressed as pH), and potassium concentration (expressed as pK). If desired, other parameters (such as the concentration of glucose and other blood sugars) can also be measured. Each of those values can be increased or decreased by external manipulation if desired.

For additional information on cardio-pulmonary machines see, e.g., Utley 1982, Berger 1979, or Bregman 1977. For additional information on cardiac surgery see, e.g., Sabiston et al 1983, Cooley 1984, or Norman 1972.

Waste Products

As used herein, 'waste product' includes any molecule which is present at a concentration which causes a harmful effect on a patient, or which is accumulating in a manner which indicates that it should be removed to avoid a harmful effect on a patient. This category includes metabolites such as urea and lactate, proteins which have been degraded and have lost their desired activity, acids accumulating at levels which create acidosis symptoms, and nutrients which are present at excessive concentrations. It also includes dead cells.

A variety of devices have been developed which can be used to remove waste products from blood, including the following.

Hemodialysis Machines and Other Membrane Devices

In general, "dialysis" includes any process in which solute molecules are exchanged between two liquids that are separated by a membrane, in response to a difference in chemical potentials between the liquids. Hemodialysis is simply one type of dialysis, in which one of the liquids is blood.

Dialysis units use semi-permeable membranes which allow certain types of molecules to pass through, while retaining other molecules and blood cells. Hemodialysis machines, often called an 'artificial kidneys,' remove urea and several other waste products from the blood. They are used to treat people whose kidneys are injured or diseased, as follows.

Blood from the patient is carried to and from the dialysis machine via cannulae. The blood flows into a chamber where it contacts a membrane, usually made of a semi-permeable form of cellulose. On the other side of the membrane is a liquid called the 'dialysate'. Initially, this liquid does not contain those molecules which are to be removed from the blood (such as urea, uric acid, phosphates, and creatinine), all of which are relatively small molecules that can permeate through the membrane.

Waste molecules such as urea are present in a higher concentration in the blood than in the dialysate; this variation across the membrane is often referred to as an 'osmotic gradient or a 'concentration gradient'. Driven by that difference in concentration, the waste molecules permeate through the membrane into the dialysate, trying to establish an equilibrium concentration on both sides of the membrane. The dialysate is removed and discarded (thereby removing the urea and other metabolites) and replaced with fresh dialysate. This process of removal sustains a high osmotic gradient of waste molecules a cross the membrane, causing more urea and other undesired molecules to permeate into the dialysate. This removes the undesired molecules from the blood.

Proteins and blood cells do not pass through the membrane, since the pores in the membrane are too small. In addition, desirable nutrients and minerals such as potassium, glucose, sodium, and magnesium are added to the dialysate at approximately the same concentrations that are contained in normal blood. Since those molecules are in osmotic equilibrium on both sides of the membrane, they remain in the blood at the desired concentration.

After the undesired metabolites are removed, the blood is returned to the patient's body via a second catheter and a needle.

For additional information on hemodialysis machines, see Nissenson 1984, Cogan et al 1985, or Drukker et al 1983. For additional information on other types of membrane devices, see Buck 1982 and Leonard 1983.

As mentioned above, "dialysis" includes any process in which solute molecules are exchanged between two liquids that are separated by a membrane, in response to a difference in chemical potentials between the liquids. Osmotic gradients, described above, are one such chemical potential. Another involves ionic charge. For example, negatively charged ions such as lactate will be attracted by, and will permeate through, a positively charged membrane while negative or neutral molecules will either be repelled by the membrane or remain unaffected. Therefore, lactate can be removed from blood by using a dialysis unit having a positively charged membrane. The rate of lactate removal can be increased if a negative charge is applied to the blood and a positive charge is applied to the dialysate.

Affinity Columns

Another type of device which is often used to remove specific molecules from blood or other biological fluids is often referred to as an 'affinity column.' The term 'affinity binding' refers to binding reactions between molecules which cling to each other because of electrical charges (similar to a magnet and steel), but which do not form covalent bonds; see, e.g., Cuatrecasas et al 1971.

An important type of affinity binding involves antigens and antibodies. Scientists can generate antibodies which will bind to any type of protein or other large molecule; see, e.g., Schreier 1980. Briefly, this involves injecting the molecule (which is an antigen) into an animal, usually a mouse or rabbit. The animal's immune system will recognize the molecule as foreign. Certain types of blood cells in the animal (called lymphocytes) will secrete antibodies which bind to the foreign molecule; in the animal, these antibodies help the animal neutralize and then destroy the antigen.

The antibodies are present in high concentration in a body fluid called 'ascites fluid', which is removed from the animal. The antibodies are concentrated and attached to a 'support' substance (usually small particles or a permeable gel). The support with the antibodies is then immobilized inside a chamber, by packing the inlet and outlet of the chamber with filter material such as glass wool. The filter material allows fluid (such as blood) to pass through the chamber; however, it does not allow the support material, the antibodies bound to that support material, or the antigens bound to the antibodies, to leave the chamber.

When the antibodies in the chamber begin to get saturated with antigen molecules (as indicated by their passage through the column), the flow of fluid through the vessel is stopped, and the conditions inside the vessel are changed (usually by adding salt or acid). Under the salt or acid conditions, the antibodies release the affinity-bound antigen molecules, which are flushed out of the vessel and discarded. Then the salt or acid is removed and replaced with a buffer solution to stabilize the chamber. This regenerates the antibodies, which can be used many times.

When ascites fluid is removed from an animal, it contains 'polyclonal' antibodies (a mixture of different antibodies which bind to different antigenic molecules). If desired, 'monoclonal' antibodies can be created which bind to a single antigenic molecule, by a different process. Briefly, this process involves removing numerous lymphocyte cells from the animal. Each lymphocyte cell secretes a single type of antibody. The lymphocytes are joined to cancerous cells, by means of a chemical such as polyethylene glycol which causes cell walls to become soft. The softened cells fuse to each other, forming hybrid cells.

Each hybrid cell which survives the fusion process is called a 'hybridoma' cell (the suffix '-oma' refers to cancer). A hybridoma is a hybrid cell, composed of a cancer cell with an antibody-forming gene from a lymphocyte). Hybridomas have two characteristics that make them useful. First, each cell secretes a single type of antibody, due to the antibody-making gene it received from a lymphocyte cell. And second, each hybridoma will reproduce itself indefinitely in a flask if proper nutrients are supplied to it, because of the uncontrolled growth genes it received from a cancer cell. It's easy to make cancer cells reproduce in flasks and dishes, in any quantity desired; that's why they're used as one of the starting cell lines to make hybridomas.

Each hybridoma cell secretes one type of antibody, but the mixture of different hybridomas cells secretes a variety of antibodies, not all of which are desired. To find and isolate one particular cell line which secretes the desired antibody, the mixture of hybridomas is diluted, then it is divided among numerous culture chambers at a concentration which indicates that only one cell will be contained in each chamber. When a single cell which is isolated in its own culture chamber reproduces, it generates a 'clonal' colony (i.e., each colony was generated from a single parent cell, so all of the cells in that colony are genetically identical with each other). Each clonal colony secretes a single type of antibody. The different colonies are tested to see whether their antibodies bind to the specific antigen that was used at the very start of the procedure, and a clonal colony which generates the desired type of antibody is selected. The selected clonal hybridoma cell line generates 'monoclonal' antibodies, all of which are identical. Those antibodies can be immobilized on a support, loaded into an af¬finity columns, and used to remove one specific type of molecule from blood or other fluids.

Immobilized Enzyme Reaction Vessels

The third category of device which is used to remove undesired molecules from biological fluids involves enzymes. Most enzymes are proteins which catalyze chemical reactions; see, e.g., Lehninger 1983 or Stryer 1980. Most of the biochemical reactions in the body are controlled by enzymes, either directly or indirectly.

Many enzymes are highly specific; they will catalyze only one particular reaction, which may involve joining together two molecules, cleaving a certain molecule into two smaller molecules, etc. Some enzymes are less specific; for example, some oxidase enzymes in the liver can add oxygen atoms to a wide variety of molecules, in order to make those molecules more soluble in water. Molecule(s) that are transformed by enzymes are called 'substrate' molecules. Many enzymes are named by adding the suffix '-ase' to the name of a substrate or a reaction; for example, lipase enzymes cleave lipids; protease enzymes cleave proteins; RNA polymerase makes RNA; the enzyme glucose phosphotransferase adds phosphate groups to glucose molecules.

It is possible to treat blood outside of the body of an animal or human by passing the blood through a chamber (often called an 'extracorporeal' device) which contains enzymes that are immobilized yet active. The following example, involving heparin and heparinase, is derived from Langer et al 1982 and U.S. patent 4,373,023; also see Lavin et al 1985 for an example involving bilirubin.

Heparin is an anti-coagulant (a substance which prevents blood from clotting). It's a polymer (a very large molecule with a repeating structure; the building block of heparin is a ring structure with a sulfur atom). It's often used during open heart surgery to prevent blood from forming clots in a cardio-pulmonary bypass machine. However, heparin also inhibits the ability of the patient to form normal, healthy blood clots, which are mecessary to heal after the surgery is finished. In addition, heparin changes the viscosity (the thickness and ease of flow) of blood, which can cause hemorrhaging (internal bleeding).

Heparinase is an enzyme which inactivates heparin, by cleaving heparin into smaller molecules. Langer et al developed a device containing immobilized but active heparinase, as follows. Heparinase molecules were 'conjugated' (i.e., covalently bonded) to a 'support' material (small solid particles) in a way that did not prevent their enzymatic activity. The support material with the immobilized heparinase was loaded into a chamber and held in place by filters at the inlet and outlet of the vessel. Blood which contained heparin entered the chamber through the entrance filter, and contacted the immobilized heparinase. The heparin molecules in the blood were cleaved into innocuous molecules by the heparinase, thereby inactivating the heparin. The blood left the vessel through the exit filter, and flowed to a pump.

The exit filter would have become clogged by the support particles if not handled specially, so Langer et al developed a method of stirring the support material inside the chamber to keep it fluidized. This method involved setting up the chamber so the blood flowed in a downward direction. While it was running, the particulate support material that accumulated near the bottom of the chamber was continually suctioned into a tube and pumped back up to the top of the chamber.

The science of immobilizing enzymes is fairly well developed; see, e.g., US patent 4,331,767 (Nakajima et al 1982). By the proper selection of support material, activating or crosslinking agents, and reaction parameters, it is possible to immobilize most types of enzymes without destroying their enzymatic activity.

Enzymes have been identified and isolated which can degrade virtually any type of biological molecule. The standard nomenclature for enzymes, along with a numerical identification system which indicates what type of substrate they use and/or what type of reaction they perform, is contained in Enzyme Nomenclature 1984, which is updated and reissued every few years by the International Union of Biochemistry. In addition to listing enzymes according to their substrates and reactions, it contains citations to technical references which discuss each enzyme.

Nutrients

As used herein, the term 'nutrients' includes all molecules which help to maintain or restore metabolic activity when present at a proper concentration in the blood, or which are otherwise added to the blood of a patient in a therapeutic or research effort. This includes carbohydrates, vitamins, amino acids, nucleotides, proteins and peptides, hormones, substances involved in cyclic biochemical reactions, compounds added to the blood to sustain the proper pH, etc. As used herein, it also includes therapeutic or experimental drugs (there is no clear distinction between nutrients and drugs; for example, Vitamin C is a nutrient, but to someone suffering from scurvy, it is also a therapeutic drug). Nutrients does not include dissolved gases such as oxygen, nitrogen, or carbon dioxide.

Nutrients are discussed in numerous texts on biochemistry; for example, Stryer 1980 and Lehninger 1983 are good introductory texts, while Lehninger 1975 is more complex and comprehensive.

One particular type of sugar molecule, glucose, plays a key role in brain metabolism. In healthy animals, it is virtually the sole energy source for brain cells, which convert glucose into lactic acid, via an intermediate molecule called pyruvate. At normal physiological pH, a hydrogen proton dissociates from the lactic acid molecule; this forms the ionized molecule, lactate. After the lactate in the blood travels from the brain to the internal organs, it is recycled through the pyruvate intermediate by a set of enzymes, to form glucose again.

Devices for Adding Nutrients to Blood

A variety of devices and methods are known for adding any soluble substance to a fluid in a desired concentration. For example, a substance may be pumped directly into the fluid at a controlled rate; it may be allowed to seep out of a permeable device and diffuse into the fluid; or it may be allowed to pass through a membrane into the fluid. Various devices have been developed which allow sustained release of selected drugs into the blood stream at low concentrations for weeks or months after injection or implantation in an animal; see, e.g., Williams 1969 or Nixon 1976.

Methods are also known for monitoring the concentration of numerous substances in blood; see, e.g., Buck 1982, Richterich et al 1982, Whitby et al 1984, and Wolf et al 1972.

Blood-Compatible Materials

Various types of blood-compatible materials have been developed for catheters, prosthetics, and other medical uses. Such materials include various rigid or flexible plastics, and various metallic alloys. Blood compatible materials for any specific use must satisfy several requirements, which can vary depending upon the particular use. In general, a blood-compatible device must not cause clotting of the blood; it must withstand leaching, corrosion, or other degradation by body fluids; and it must have sufficient strength, flexibility, and durability for its intended use. For more information on blood-compatible materials, see, e.g., Gebelcin 1983 and Hench et al 1984.

Pumping

A variety of different types of pumps have been developed for medical use. 'Peristaltic pumps', also called 'roller pumps', are used most commonly to pump fluids containing living cells, because they inflict very little stress or damage on cells; see, e.g., Norman 1972. In a peristaltic pump, blood or some other fluid is contained inside a flexible plastic or rubber tube, and a pinch-roller is used to squeeze the tube in a manner such that the point of constriction moves along the length of the tube. The squeezing action forces fluid through the tube in the desired direction, and the fluid never touches anything except the inside of the tube. Peristaltic pumps cuse very little turbulence or shear force in the fluid being pumped, which means thai they inflict relatively little damage on blood cells when they are used to pump blood.

Multiple-finger pumps perform in a manner similar to roller pumps; a set of rods squeeze a flexible tube in a manner such that the point of constriction moves along the length of the tube, pushing the fluid along in front of the constriction.

If desired, peristaltic and other pumps can be used to create pulsatile flow which resembles the pulsing action of heartbeats; see, e.g., US 3,892,628 (Thorne et al, 1975). Another type of pulsatile blood pump, which involves side-by-side balloons inside a rigid casing, is described in US 4,116,589 (Rishton, 1078). Blood Vessels

The main arteries that supply blood to the head are the carotid arteries and the vertebral arteries. There is a left common carotid, which branches directly off the aorta (the main artery leading from the heart). There is also a right common carotid, which comes from the aorta via an intermediate artery, called the innominate artery or bracbiocephalic trunk. Both of the common carotids divide into internal and external carotids, up inside the neck.

In addition to the carotid arteries, there are two vertebral arteries (left and right), which branch off of subclavian arteries. They travel along the spine up to the base of the brain, where they join together in an artery called the basilar artery. An arterial structure supplied by several arteries at the base of the brain is called the Circle of Willis. If any of the arteries which supply the Circle of Willis are disrupted, the other arteries can supply sufficient blood to the brain (especially if the brain is chilled, as usually done during open heart surgery).

The major veins that take deoxygenated blood from the head back down to the heart are the jugular, cervical, and vertebral veins. There are four jugular veins; both the left and the right side have internal and external jugular veins. Each side also has a deep cervical vein and a vertebral vein, all of which come from a network of veins at the base of the brain called a cervical plexus. If one vein which drains a cervical plexus is disrupted, other veins can continue to drain the plexus.

Part of the tissue in the lower face and neck is supplied by numerous small blood vessels that pass through the neck, which are not in direct communication with the arteries and veins mentioned above.

The capillaries are the tiny blood vessels where oxygen and nutrients are transferred from the blood to the tissue, and where the carbon dioxide and waste products from the tissue are transferred to the blood for removal. In a sense, capillaries are where arteries turn into veins. Inside the brain, the capillary walls do not have the same type of structure and porosity as they have in the rest of the body. This is because the cells which form the walls of capillaries in the brain overlap each other in 'tight junctions,' while the cells that make capillary walls elsewhere in the body are separated by 'slit-pores.' The tight junctions in the brain capillaries form something referred to as the 'blood-brain barrier.' This barrier prevents certain types of molecules such as antibodies and neurotransmitters from diffusing into the brain; this is desirable, because antibodies would form tiny clumps in the brain, and neurotransmitters would cause brain cells to fire randomly, interfering with thought processes.

For information on vascular surgery and microsurgery, see Haimovici 1984 and Daniel et al 1977.

Cerebrospinal Fluid

The entire cavity which contains the brain and spinal cord of an adult human has a volume of about 1650 ml. About 150 ml is occupied by cerebrospinal fluid. About 800 ml of fluid is formed each day in the ventricles in the brain; it passes through the ventricles into the spinal cord, and is absorbed into the venous blood through the arachnoidal villi.

Excess fluid can build up inside the skull if the drainage ducts in one or more ventricles become plugged; this condition is called 'hydrocephalus.' It is often treated surgically by inserting a semi-permanent drainage tube into one of the ventricles, to drain the excess fluid (which is usually carried to the peritoneal cavity, where it is absorbed).

Tissue Culture Systems

As used herein, 'tissue culturing' refers to a process of growing and/or maintaining animal cells in a dish, flask, or other container outside of the body. Tissue culture can be done to study the cells, or to generate proteins or other products from the cells.

There are several important factors that affect tissue culture, including the following. First, animal cells are generally divided into two categories of cells. One category, known as 'suspension cells', includes cells which can reproduce while floating freely in liquid, unattached to any other cells or any solid surface. The most common suspension cells are white blood cells (red blood cells do not contain chromosomes, so they do not reproduce in culture). The second category is called 'anchorage dependent cells'. This reflects the fact that most cells which make up cohesive tissue such as muscle, skin, or organs will not reproduce unless they can attach to other cells or certain types of artificial surfaces. Anchorage dependent cells can be cultured on the insides of tiny hollow tubes (see, e.g., US 4,201,845, Feder et al, 1980) or on the surfaces of tiny particles which are floating in a liquid (see, e.g., US 4,293,654, Levine et al, 1981).

The second major factor which affects tissue culture relates to the number of generations of cells which can be grown outside the body. Usually, only a limited number of generations will grow from cells taken from non-cancerous tissue. The number of generations can vary depending on cell type, nutrients, and other factors, but it is not uncommon for non-cancerous mammalian cells to stop reproducing after about twenty generations. Such cells are often referred to as a 'strain' of cells. By contrast, cells taken from cancerous tissue will usually reproduce indefinitely if supplied with suitable nutrients. Such cells are often referred to as 'immortal' cells, or as a cell 'line'. Immortal cell lines have been established from virtually every type of organ. For example, scientists have isolated immortal cell lines with the basic characteristics of liver cells from liver tumors, and immortal lines of white blood cells from leukemia victims. In addition, most types of non-immortal mammalian cells can be converted into immortal lines by one of two processes. One process, which is usually called 'transformation' of the cells, involves the use of certain viruses such as the Epstein-Barr virus. The other process involves fusing non-immortal cells with cancerous cells, as was mentioned previously with hybridomas. Although all forms of transformation alter the genetic makeup of cells, it's usually possible to isolate the transformed cell lines into clonal colonies and screen each colony to identify a clonal line which has the desired characteristics, such as the expression of one or more enzymes.

An entirely different type of tissue culture is involved in U. S. patents 4,060,081 (Yannas et al, 1977) and 4,280,954 (Yannas et al, 1981). These patents (and various articles by I.V. Yannas and J.F. Burke) describe lattices made of collagen fibers. Collagen is the fibrous protein that holds cells together in animal tissue, including human skin and organs. The lattices developed by Yannas and Burke (and their assistants) are very porous structures, like a network of threads where the overall volume is about 2 to 5 percent threads and 95 to 98 percent open space. Yannas and Burke created these lattices in membrane form to provide a suitable synthetic covering for burn wounds. Cells from the periphery of the burn wound, or cells that were implanted in the lattice (see US 4,458,678, Burke et al 1984) will grow inside the pores in the lattice, creating new skin. This is comparable to tissue culture, except that it is in contact with a stillliving animal rather than in a flask or dish.

During the healing process, the collagen membrane is covered and protected by an outer layer of silicone rubber, which allows a controlled amount of moisture to evaporate (just like normal skin); this avoids the accumulation of excess fluid in or under the new skin (excess fluid is called edema). However, the pores in the silicone rubber which allow water molecules to pass through are too tiny to let in bacteria or viruses; therefore, the silicone layer prevents infection.

In healthy tissue, collagen goes through a cycle. The cells continuously secrete new strands of collagen, while a natural enzyme called collagenase chews up the old strands into amino acids, which permeate into the cells to be made into new collagen fibers. In this way, the collagen which holds tissue together is continually renewed, to maintain strength and flexibility. That process works out perfectly with the collagen lattice developed by Yannas and Burke; the collagenase secreted by the cells eventually chews up the synthetic collagen fibers (which are obtained from sources such as cowhide), and the cells replace the synthetic collagen with new, natural collagen. To complete the process, the layer of silicone rubber spontaneously separates from the new skin, since it can't attach to the new collagen fibers that are being secreted under wet conditions. The Yannas/Burke membrane is one of the most remarkable and humanitarian biotechnology inventions of this century; within the course of about three weeks, it's possible for someone whose body is almost completely covered with third degree burns to grow an entirely new skin which is less scarred, more flexible, and more comfortable than could be obtained using any previously available technique for treating severe burns.

Another branch of tissue culture involves cells which perform a desired function (usually involving one or more sepcific enzymes) but which are not actively reproducing. For example, Nose et al 1977 describes an artificial liver created by placing slices of liver in a perfusion chamber, and US patent 4,353,888 (Sefton 1982) describes a method of encapsulating living mammalian cells.

When discussing individual cells, isolated organs, or slices of tissue, the distinction between living cells (also called viable cells) and dead cells becomes hard to apply. One common test of Ufe or viability is whether a cell can reproduce. However, even when cells can no longer reproduce, their molecules often remain active and act in coordination for a substantial period of time, especially if they are provided with oxygen, glucose, and adenosine tri-phosphate. Indeed, many enzymes continue to function even after a cell membrane breaks and the cell spills its guts. If a cell is encapsulated, contained within an organ which has been isolated, or otherwise treated in some manner that will prevent it from reproducing, it can fall into a borderline area where words like "alive" need to be defined carefully before they can be applied, and the conclusion will probably depend on semantics and definitions rather than scientific facts. It's similar to arguing about whether a virus is alive or a fertilized egg cell is a human being; those are questions of semantics, not scientific facts, and people who say they're still hoping scientists will find some ultimate fact that will finally prove their political viewpoint to be correct are misleading the public.

The same comments about alive versus dead apply to perfused organs. They are in a borderline state; most of their cells remain active for some time, and some of their cells may retain the theoretical potential to reproduce for a while. But those cells, and the organs they comprise, gradually die as the perfusion period stretches on. There's no single minute that serves as a dividing line between living and dead.

Perfusion of Isolated Organs

Intact organs taken from lab animals can be studied outside the body, using a specialized type of tissue culture technique called 'perfusion'. This technology may seem new, but it's over fifty years old; the greatest milestone in the history of perfusion research occurred in 1938, with the publication of The Culture of Organs by Charles Iindbergh (the aviator who first crossed the Atlantic) and Alexis Carrel (a surgeon who won the Nobel Prize for medicine and physiology in 1912).

The perfusion of isolated organs usually involves killing a laboratory animal, removing the organ(s) of interest, and maintaining the intact organ in a condition which allows all or part of its metabolic activity to continue for a period of time (usually several hours; rarely more than three days). This is done by connecting the arteries and veins of the organ to tubes and a mechanical pumping system, which pump an oxygenated fluid with nutrients through the organ.

By isolating an organ, scientists make it accessible for direct observation and they eliminate the variables and interferences caused by other organs in an intact animal. Another type of perfusion involves the preservation of organs (such as kidneys) taken from people who have been killed in accidents until those organs can be transplanted into other people. Perfusion technology (in general, and on several specific organs) is described in Norman et al 1968 and Berger 1979, and in various patents classified in US class 435, subclass 283.

Usually, the perfusion period for an isolated organ does not last more than a couple of days. Scientists who try to keep isolated organs alive longer than that encounter difficulties such as edema, blood clotting, and infection.

Edema is fluid accumulation, which swells the organ and renders it difficult to pump the perfusate through it. This can be avoided or eliminated by routing the circulating blood through the body of a support animal; however, this technique generates a large number of other problems involving the support animal.

Blood clotting can be avoided by the use of anti-coagulants such as heparin, but that creates other problems as discussed above. More commonly, if a support animal is not involved, blood clotting is avoided by using a perfusion fluid other than blood, such as buffered saline solution (which can carry some oxygen, but not much) or certain types of fluorocarbons (which can carry as much oxygen as blood, but which are expensive).

Infection can be held in check for several days by using systemic (circulating) antibiotics such as penicillin, and topical (surface-acting) antibiotics such as neosporin.

Brain Perfusion

Research on perfused monkey brains is described in White et al 1963, 1964, and 1968A. As mentioned in White 1968A, there was a substantial drop in electroencephalic activity within three hours after the brain was removed from the skull, if no effort was made to remove metabolites from the circulating blood or add fresh blood fractions to the blood. If the blood circulating through the brain was also circulated through the body of a second monkey, the period of 'viability' could be extended to more than 3 days; see White et al 1971 A and White 1968. A retrospective summary of this line of work which appeared years later were White 1972 and White 1981. More recently, the technique has been extended to tests involving rat brains; sec Takaoka et al 1985.

White et al 1968B described work which involved isolating the brain of a donor monkey and placing that brain in the skull of a recipient monkey in order to assess how long the brain would survive. There was no attempt to connect the brain to the sensory organs or spinal cord of the recipient monkey. The transplanted brains generated electrical signals for more than 3 days.

Transplanted Heads

White 1971A describes two distinct surgical projects. In the first, a section of the neck of a rhesus monkey was removed, through a process called 'cervical transection'. This operation completely severed the spinal cord and all nerves and tissue except for the carotid arteries and jugular veins, which were the only remaining links between the head and the body. After that operation, each of the six monkeys operated on awakened and remained conscious during the period of study (eight hours). At the end of the study period, each monkey was painlessly killed for further analysis.

In the second type of operation described in White 1971A, three primate heads were completely severed from their bodies and grafted onto the sides of the necks of other monkeys, which remained intact. All of the transplanted heads regained consciousness for the study period (24 hours).

White 1971B describes a third type of operation. Four monkeys were cervically transected to remove their heads (intact) from their bodies. Each head was transplanted onto the body of a monkey which had been decapitated. All four transplanted heads regained consciousness, which lasted up to 36 hours. Hemorrhaging problems were encountered, attributed to chronic heparinization.

Spinal Cord Injuries

In general, a person or animal can survive total loss of spinal cord functioning, so long as the injury occurs below the intersection where the nerves that control respiration join the spinal cord.

If a nerve bundle in the spinal cord is 'divided' (severed by cutting, as is often done during neurosurgery to relieve severe chronic pain), the portion of the nerve bundle which remains connected to the brain does not carry any impulses to the brain. This leads to a feeling of numbness in the part of the body which was previously serviced by that nerve bundle. This procedure can be done to eliminate "phantom pain", which amputees often suffer. Phantom pain is generated when severed nerve endings that formerly served an amputated limb are contacted by activating substances inside the stump of the amputated limb.

It is possible to permanently inactivate (i.e., deaden) nerve cells in the spinal cord by injecting them with certain substances (such as ethyl alcohol), or to temporarily alleviate pain by means of analgesic drugs such as morphine.

For information on spinal cord functioning, injuries, and research, and on neurosurgery, see Youmans 1982 and Wilkins 1985.

SUMMARY OF THE INVENTION

This invention relates to a method for providing physical and biochemical support for an animal head which has been 'discorporated' (Le., severed from its body). This method involves processing equipment which supplies a severed head with oxygenated fluid (such as blood) and nutrients, by means of tubes connected to arteries which pass through the neck. After circulating through the head, the deoxygenated fluid returns to the processing equipment by means of tubes connected to veins that emerge from the neck. A series of processing components including oxygenators and at least one pump will remove carbon dioxide and replenish the oxygen level in the fluid, and return the replenished fluid to the discorped head via tubes attached to arteries. If desired, waste products and other metabolites may be removed from the fluid by chemical processing; nutrients, therapeutic or experimental drugs, and other substances may be added to the fluid; and the fluid may be routed through the body of a second animal.

The device of this invention will provide physical support for the head, by means of a collar around the neck, pins or other devices attached to one or more vertebrae, or similar mechanical means. If desired, the spine may be left attached to the discorped head.

The fluid processing and pumping steps will sustain various metabolic activities in the head after it has been severed from the body. This will allow analyses to be performed on the head (including pharmaceutical, toxicological, hormonal, and neurological analyses) without being affected by metabolites generated by digestive and other internal organs when such analyses are performed on intact animals.

The severed head preferably should retain all of the sensory organs, and the vocal cords if desired. Depending on the surgical procedures used to sever the head from the body and the type of fluid processing and drugs used during and after the operation, the discorped head might experience a period of consciousness after it has been severed from the body. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a cross-sectional depiction of the neck showing the major arteries and veins passing through the neck.

Figure 2 depicts an arrangement for transferring blood flow from the heart to the device of this invention.

Figure 3 is a schematic diagram depicting various pumping and treatment components used in this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a device for maintaining metabolic activity in an animal head which has been severed from its body. The components of this device comprise a unit referred to herein as a cabinet. The cabinet includes various types of blood processing and monitoring equipment. It may be attached to an external computerized control panel or other accessory devices if desired.

The head of a laboratory animal such as a dog or monkey may be severed from the body and coupled to the cabinet described herein, using means known to experimental surgeons. After this invention has been thoroughly tested on lab animals, it might also be possible to use this invention on terminally ill persons, subject to various government approvals and other legal requirements.

The major arteries and veins that pass through the neck of a primate are shown in Figure 1. That figure also shows, for orientation, thyroid gland 2, trachea 4, esophagus 6, vertebral body 8, spinal cord 10, and spinous process 12. The four major arteries are the left common carotid, the left vertebral, the right common carotid, and the right vertebral. There are also eight major veins; each side has internal jugular, vertebral, external jugular, and deep cervical veins.

Each blood vessel may be severed and attached to a cannula. The location of each cut may be varied along the length of the blood vessel. The attachment may be made by ordinary techniques; for example, a slit may be made in a blood vessel, a cannula (with a slightly enlarged end, if desired) can be inserted into the slit, and the blood vessel can be sutured or constricted around the end of the cannula. Alternately, if the blood vessel is large enough, it can be severed and the cannula may be inserted into the severed end and sutured directly to the cannula, or sutured in a manner which constricts the artery around an enlarged ring near the end of the cannula. Alternately, blood vessels may be attached to cannulae by means of temporary or semi-permanent coupling devices which do not require suturing.

When an operation has been completed, the arterial cannulae will carry oxygenated blood from the cabinet to the arteries. The arteries will carry the blood into the head. After the blood circulates through the head, it will return to the cabinet, via severed veins attached to veinous cannulae.

The equipment should be primed (filled with a suitable fluid, such as blood, plasma, or Ringer's lactate) before any connections are made between the head and the cabinet, to avoid circulating any air bubbles through the head. As used herein, the term 'blood' is used broadly to include blood substitutes (such as fluorocarbon solutions), blood fractions such as serum or plasma, and any other fluid which is used as a perfusate.

Any arterial or veinous cannula may pass through a manifold, an adjustable constriction, or a comparable flow control system to ensure that the rate and the pressure of blood flowing through that cannula (and through the connected artery or vein) remains at approximately physiological levels. However, the different internal diameters of the blood vessels will tend to serve that function even in the absence of a mechanical flow control system.

One method of transferring the blood vessels to the cabinet is shown in Figure 2. In this figure, the internal jugular vein is clamped at two locations, then severed between the clamps. The cephalic end 20 (i.e., the end which is attached to the head) is coupled to cannula 22, and the thoracic end 24 (the end which remains attached to the body) is coupled to cannula 26, Cannulae 22 and 26 are attached to valve 28, which is shaped like a T (or a Y). A third cannula, 30, which is attached to the third arm of the T-valve, leads to the blood processing equipment of this invention. In other words, T- valve 28 and two of its cannulae are inserted into the internal jugular vein. Similarly, T-valve 40 is inserted into the common carotid artery 42.

After each T-valve (or Y-valve) is inserted, the clamps on both sides of it are opened. The flow-directing device inside each valve is initially oriented in a manner that allows blood to flow through the vein or the artery, travelling between the chest and the head. After both valves have been inserted, the flow-directing devices inside both of them are turned, to redirect the blood to the processing equipment. At the same time, valve 44 is opened; it serves as a shunt which allows the heart to continue pumping normal volumes of blood. This arrangement allows the heart to provide the remainder of the head with a continuous supply of blood while the remaining vessels are being transferred to cannulae. This arrangement also provides approximately steady-state blood flow through the processing equipment. This procedure can be used as soon as valves have been inserted into one common carotid and one internal jugular, or after all four such valves have been inserted. If desired, the blood of the animal may be cooled during the operation to reduce oxygen consumption in the brain, as is commonly done during open heart surgery.

In addition to the major veins and arteries shown in Figure 1, a number of smaller blood vessels carry blood to and from the face and neck. Any such blood vessel may be severed and attached to a cannula or to a manifold which leads to or from the cabinet. Capillaries and other very small vessels may be cauterized, or transferred to the cabinet through microsurgery techniques. If some region of tissue does not have sufficient blood flow after the operation, it can be treated with angiogenesis factor (a hormone which causes the growth of new blood vessels).

When deoxygenated blood enters the cabinet through the veinous cannulae, it passes through various pumps and treatment chambers as described below and indicated in Figure 3. Each fluid-handling component is connected to fluid conduits in a manner such that when a severed head is coupled to the device by means of the veinous and arterial cannula, the entire system is capable of circulating blood through each component and through the head.

It is anticipated that any waste products which are to be removed from the blood preferably should be removed before any nutrients are added; however, the exact sequence of treatment and pumping steps is not critical, and may be varied and optimized using routine experimentation.

The cabinet may be equipped with pumps at various locations, such as pumps 52 and 54 shown in Figure 3. Such pumping should be performed by devices which cause relatively low levels of damage to blood cells and other blood components, such as peristaltic pumps (also called roller pumps). Proper sizing of a pump is a matter of routine calculation or experimentation. If desired, the pumps may be used to supply pulsatile flow which emulates the surges created by heartbeats. Exact location of the pumps is variable, so long as sufficient pressure is provided to cause the blood to flow through all treatment devices and through the head at satisfactory flow rates.

If desired, the cabinet may be equipped with one or more preliminary treatment chambers, such as chamber 56 shown in Figure 3. In such chambers, the blood may be treated in various ways. For example, heparin may be added to the blood to prevent clotting; the blood may be heated or cooled in order to raise or lower the solubility of desired or undesired components; and buffering compounds may be added to prevent swings in acidity or other conditions which might damage cells or sensitive molecules.

In oxygenation chamber 58, the blood is contacted with a gas that contains molecular oxygen, such as purified oxygen or air. If desired, the gas may be filtered or otherwise treated to reduce the risk that it will contain viruses or bacteria. This chamber can incorporate any of the components (including heat exchangers, anti-foaming devices, sensors, etc.) that are used by cardiopulmonary bypass machines during open heart surgery. Preferably, the oxygenation chamber should use a membrane device, rather than a bubble or rotating disk device, to minimize exposure of the blood to free gas interfaces.

Since the head does not contain any digestive organs, it generates waste products at a relatively low level compared to an intact animal body. This is highly advantageous with respect to the present invention. If desired, certain types of neurological, pharmaceutical, or other metabolic activity in a discorped head may be studied for a substantial period of time without suffering from high levels of interference by undesired metabolites. However, for long term study of the head, it is possible to reduce the level of any specific waste product or other metabolite in the blood by adding fresh blood to the system, or by treating the circulating blood to remove metabolites from it.

Waste products generated by metabolism inside the head (or created as a byproduct of any step in the blood processing) can be removed in one or more waste removal chambers, such as chamber 60 shown in Figure 3. Depending on the type of molecule(s) being removed, such chambers can use any suitable selected mechanism. For example, small molecules such as urea and lactate can be removed by dialysis. Proteins and other large molecules can be removed by affinity binding, using antibodies. Ionic substances may be removed by dialysis or ion exchange devices.

If desired, the waste removal system may contain one or more pretreatment chambers, which may contain active cells. For example, liver cells contain a complex mixture of enzymes which conjugate foreign molecules (often called xenobiotic molecules) to electronegative molecules such as oxygen or glutathione. Such conjugation reactions usually increase the solubility of the undesired molecule in water; this allows the undesired compounds to be removed by other organs, primarily the kidneys. To simulate this natural process, a reaction chamber containing active liver cells (growing in culture or obtained from cadavers) can be placed upstream of a dialysis unit. Alternately, a mixture of liver enzymes can be immobilized in a reaction chamber which is placed upstream of a dialysis unit.

Chamber 62 is a nutrient and/or drug addition unit This unit can use devices such as metered pumping units, sustained release devices which work by diffusion, or any other device capable of adding a soluble substance to the circulating blood at a desired rate.

If desired, the circulatihg blood may be separated into fractions (e.g., plasma, serum, or red blood cells) by means such as centrifugation, or into volumetric portions by means of valve arrangements. If desired, a selected fraction or portion of the blood may be treated by any of the methods described herein and circulated through the cabinet and/or the head, without treating the remainder of the blood. For example, all of the blood can be sent through the oxygenation chamber, while only a fraction or portion of the blood might be sent to a device such as affinity column 62 by using a parallel flow system.

An outlet port 66 and an inlet port 68 may be provided to route all or part of the blood to an additional treatment device, or to the body of a second animal. This can be accomplished by injecting the blood into a vein of the support animal and collecting it from an artery. The intact animal body will process the blood in its internal organs, removing waste products and adding nutrients, hormones, and other molecules. Such treatment will convert lactic acid (and its ion, lactate) into glucose, a nutrient.

Certain types of undesired molecules such as heparin can be degraded by enzymes that are immobilized in chambers such as chamber 70. It is possible to immobilize a variety of different enzymes independently on supporting material, and then combine the immobilized enzymes in a single chamber. Alternately, if two enzymes do not function satisfactorily in the same chamber, they can be placed in separate chambers.

For the purposes of this invention, a substance is regarded as removed from the blood if it is converted into a different substance; for example, heparin is removed from the blood when it is cleaved into smaller molecules that don't function as anticoagulants, regardless of whether those smaller molecules remain within the blood. If heparin is added to the blood, it preferably should be removed before the blood is returned to the head, if long-term support of the head is desired. Heparin removal chamber 70 can be similar to the device described in Langer 1982. If a support animal is used, a heparin removal unit can be used to remove heparin from the blood before the blood is routed through the body of the second animal.

The cabinet may be equipped with various devices to monitor chemical conditions in the blood at any desired location. Devices and methods for measuring the status of any condition that is of interest are known to those skilled in the art of blood chemistry. Certain types of monitors (such as thermometers, pressure gauges, and electrodes which measure such things as acidity and potassium concentration) function continuously while remaining in contact with the blood. Such monitors do not add any undesired chemicals to the blood. However, some types of chemical or biological analyses require samples of blood to be removed from the system and treated with chemicals which should not be circulated through the head. To allow for such chemical and biological analyses, sampling ports may be provided at any desired location to allow quantities of blood to be removed from the cabinet and analyzed.

If desired, the monitoring devices and the devices which control the blood processing units may be coupled to each other to provide for automated control of one or more blood parameters. For example, a pH monitor which generates an electronic signal may be placed in contact with the blood. This signal may be directed to a visual display, and to an electronic circuit which allows the signal to control the output of an acidity control device. If the signal indicates that the acidity of the blood is approaching an undesirably high level, the control device can activate a pump which will add an alkaline or buffering substance to the blood. It is possible to operate numerous automated control systems simultaneously. The cabinet is equipped with a device for mounting the severed head in a position such that the veins and arteries which emerge from the head can be connected to the veinous and arterial cannulae. This can be accomplished by means such as inserting one or more surgical pins into the vertebrae in the neck, or by immobilizing the neck with an inflatable or padded collar. Preferably, the collar should be securely fitted around the neck to reduce the chance of infection at the exposed subcutaneous area at the base of the neck. If desired, the cabinet may be equipped to allow the head to be inclined in any direction, for ease of access during or after surgery. If desired, the exposed subcutaneous tissue at the base of the neck may be covered by one or more flaps of skin which remain attached to the neck during the operation, or by a synthetic membrane, to control moisture flux and reduce the chance of infection. For example, a bilayer membrane comprising a collagen-glycosaminoglycan lattice and a layer of silicone rubber, as described in US 4,060,081 (Yannas et al, 1977) may be seeded with skin cells taken from the same patient. The membrane may be cut to fit the exposed base of the neck with holes allowing arteries and veins (or cannulae) to pass through it, and sutured onto the exposed base of the neck near the end of the operation. The skin cells seeded into the membrane will grow to confluence, creating a new layer of skin, and the silicone layer will spontaneously detach from the new layer of skin.

The nerve signals entering the brain may be terminated or reduced by severing the nerves in the spinal cord and surrounding them with a suitable fluid containing no nerve-activating substances. Alternately, the nerves in the spinal cord or elsewhere can be killed with a substance such as ethyl alcohol, or treated with analgesic (pain-reducing) drugs. If desired, the spine may remain attached to the head during the discorp operation, and enclosed in a sheath device which should contain a suitable fluid.

If desired, the surgical cuts may be made in such a manner that the larynx (which contains the vocal cords) remains attached to the head. The severed end of the trachea (vand pipe) may be connected to a tube carrying slightly compressed, humidified air, so that the primate or human head may use its vocal cords if it is conscious. The compressor may be controlled by a switch mounted below the chin, so the severed head may turn the compressor on or off by opening its mouth.

In addition, the head may be severed from the body in such a manner that various organs contained in the neck or upper chest may remain attached to the head. Such organs may include the thymus gland (which, in young animals, is involved in generating antibodies to help fight infections), the thyroid gland (which is involved in iodine metabolism and metabolic rate regulation), and the parathyroid gland (which is involved in regulating calcium in the blood).

A drainage tube may be placed in one or more ventricles in the brain to drain ex cess cerebrospinal fluid.

INDUSTRIAL APPLICABILITY

The device of this invention can be manufactured and sold, and it may be used by industrial firms such as pharmaceutical companies for various purposes such as analyzing commercial drugs which are difficult to study in intact animals, to determine the fate of such drugs in the brain.

EQUIVALENTS

Those skilled in the art will recognize, or may ascertain using routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are within the scope of the claims.

REFERENCES

Berger, E.C., The Physiology of Adequate Perfusion (C.V. Mosby, St. Louis, 1979)

Bregman, D., Mechanical Support for the Failing Heart and Lungs (Appleton- Centuiy-Crofts, Norwalk, CT, 1977)

Buck, R.P., "Ion-selective Membranes and Electrodes," pρ.338-342 in Volume 7, McGraw-Hill Encyclopedia of Science and Technology, Fifth ed., 1982

Cogan, M.G. and Garovoy, M.R., Introduction to Dialysis (Churchill-Livingstone, New York, 1985)

Cooley, D., Techniques of Cardiac Surgery (Williams and Wilkins, Baltimore, 1984)

Cuatrecasas, P. and Anfinsen, C.B., "Affinity Chromatography," Ann. Rev. Biochem. 40: 259-278, 1971

Daniel, R.K. and Terzis, J.K, Reconstructive Microsurgery, (Little Brown and Co., Boston, 1977)

Drukker, W. et al, Replacement of Renal Function by Dialysis, 2nd ed. (Martinus Nijhoff Publ., Boston, 1983)

Enzyme Nomenclature 1984, issued by the International Union of Biochemistry (Academic Press, Orlando, Florida, 1984).

Gebelein, C.G., "Prosthetic and Biomedical Devices," pp. 275-313 in Vol. 19 of

Kirk-Othmer Chemical Encyclopedia, 3rd ed. 1983

Guyton, A.C., Textbook of Medical Physiology, 6th ed. (Saunders and Co., Philadelphia, 1981)

Haimovici, H., Vascular Surgery, 2nd ed. (Appleton-Century-Crofts, Norwalk, CT, 1984)

Hench, L.L. and Wilson, J., "Surface Active Biomaterials," Science 226: 630-636 (1984)

Kenedi, R.M. et al, Artificial Organs (University Park Press, Baltimore, MD, 1977)

Langer, R.S. et al, "An Enzymatic System for Removing Heparin in Extracorporeal Therapy," Science 217: 261-263, 1982. Also see U.S. Patent 4,373,023 (Langer et al 1983)

Lavin, A. et al, "Enzymatic Removal of Bilirubin from Blood: A Potential Treatment for Neonatal Jaundice", Science 230: 543-545, 1985

Lehninger, A.L., Biochemistry, 2nd ed. (Worth Publ., New York, 1975)

Lehninger, A.L., Principles of Biochemistry, (Worth Publ., New York, 1983)

Leonard, E.F., "Dialysis," pp. 564-579 in Vol. 7 of Kirk- Othmer Chemical Encyclopedia, 3rd. ed. 1983

Mann, R.W., "Cybernetic Limb Prosthesis", Annals of Biomedical

Engineering 9: 1-43, 1981

Nisseman, A.R. et al., Clinical Dialysis (Appleton-Century-Crofts, Norwalk, Connecticut, 1984)

Nixon, J.R., Microencapsulation (Marcel Dekker, New York, 1976)

Norman, J.C., et al, ed., Organ Perfusion and Preservation (Appleton-Century-Crofts, Norwalk, CT, 1968)

Norman, J.C., Cardiac Surgery, 2nd ed. (Appleton-Century-Crofts, Norwalk, CT, 1972)

Nose, Y. et al, "Hepatic Assist 1: Use of Liver Tissue in an Extracorporeal Device,", p. 372-377 in Kenedi 1977

Richterich, R. and Colombo, J.P., Clinical Chemistry (John Wiley and Sons, New York, 1981)

Sabiston and Spencer (ed.), Gibbons' Surgery of the Chest, (Saunders Publ., Philadelphia, 1983)

Schreier, M. et al, Hybridoma Techniques (Cold Spring Harbor Labs, New York, 1980)

Stryer, L., Biochemistry (Freeman and Co., San Francisco, 1981)

Takaoka, Y. et al, "A High Performance Isolated Rat Brain Preparation," Parts I and II, Resuscitation 12: 253-264, 1985

Unger, F. (ed.) Assisted Circulation (Springer-Verlag New York, 1979)

Utley, J.R., Pathophysiology and Techniques of Cardiopulmonary Bypass (Williams and Wilkins. Baltimore, 1982)

Whitby, L.G. et al, Lecture Notes on Clinical Chemistry, 3rd ed, (Blackwell Scl,

Publ., Boston, 1984)

White, R.J. et al, "Isolation of the Monkey Brain: In Vitro Preparation and Main tenance," Science 141: 1060 (1963)

White, R J. et al, "Preservation of viability in the isolated monkey brain utilizing a mechanical extracorporeal circulation," Nature 202: 1082 (1964).

White, R.J. et al, "The Isolated Monkey Brain: Operative Preparation and Design of Support Systems," J. Neurosurg. 27: 216, 1967

White, R.J., "Experimental Transplantation of the Brain," (1968A) in Human Transplantation, Raraport F.T. and Dausset, J., eds. (Grune and Stratton, New York, 1968).

White, R.J. and Albin, M.S., "Mechanical Circulatory Support of the Isolated Brain," (1968B) pp. 747-757 in Norman 1968

White, R.J. et al, "Primate Cephalic Transplantation: Neurogenic Separation, Vascular Association," Transplantation Proceedings 3: 602-604, 1971A

White, R.J. et al, "Cephalic Exchange Transplantation in the Monkey," Surgery 70: 135-139, 1971B

White, R.J., "Preparation and Mechanical Perfusion of the Isolated Monkey Brain", Acta Endocrinologica (Suppl.) 158: 200-216 (1972)

White, RJ., "Brain", p. 655-674 in Organ Preservation for Transplantation, 2nd edition, A.M. Karow and D.E. Pegg, eds. (Marcel Dekker, New York, 1981).

Wilkins, R.H., and Rengachaiy, S.S., Neurosurgery. McGraw-Hill, NY, 1985

Williams, A., Sustained Release Pharmaceuticals (Noyes Development Corp., Park Ridge, NJ, 1969)

Wolf, P.L. et al, Methods and Techniques in Clinical Chemistry (Wiley-Interscience, New York, 1972)

Youmans, J.R., Neurological Surgery, 2nd ed. (W.B. Saunders, Philadelphia, 1982)

Claims

1. A device for maintaining metabolic activity in a mammalian head which has been severed from its body, comprising the following components: a. veinous cannulae which are capable of being attached to veins which pass through the neck and receiving fluid from the veins; b. arterial cannulae which are capable of being attached to arteries which pass through the neck and transporting fluid into the arteries; c. an oxygenation device which is in fluid communication with the veinous and arterial cannulae, and which is capable of displacing carbon dioxide contained in the fluid with oxygen; d. one or more pumps; e. fluid conduits which are attached to each of the components listed above in a manner such that the components, when coupled to the veins and arteries of a severed head by means of the cannulae of parts (a) and (b), will form a system capable of circulating fluid through the oxygenation device and through the head after the head has been severed from the body; and, fc means for securely mounting the head upon the device after the head has been severed from the body, in a position such that the veins and arteries which emerge from the head can be connected to the veinous and arterial cannulae.

2. A device of Claim 1 comprising at least one device or inlet port suited for allowing the addition of a nutrient, drag, anti-coagulant, or other desired substance to the fluid at a desired concentration.

3. A device of Claim 1 comprising at least one monitoring device or sampling port which allows the measurement of selected physical or chemical characteristics of the fluid at a selected location in the device.

4. A device of Claim 1 comprising one or more components which can remove one or more waste products from the fluid.

5. A method of Claim 4 wherein the component which can remove waste products from the fluid is selected from the group consisting of dialysis devices, affinity binding devices, ion exchange devices, immobilized enzyme chambers, and immobilized cell chambers.

6. A device of Claim 1, wherein the oxygenation device comprises a membrane oxygenator.

7. A device of Claim 1 which comprises a component designed for removing an anti-coagulant from the fluid.

8. A device of Claim 1 wherein the pumps are selected from the group consisting of peristaltic pumps and multiple finger pumps.

9. A device of Claim 1 which is equipped with one or more heat exchangers.

10. A device of Claim 1 which is equipped with monitors for measuring fluid temperature, fluid pressure, oxygen content, carbon dioxide content, and acidity.

11. A device of Claim 1 comprising a control system containing: a. a monitor designed for evaluating one or more fluid parameters, which generates a variable electronic signal which depends on the value of the parameter being measured; b. a control device which is capable of generating an output which can affect the value of the parameter being measured; and, c. an electronic circuit which allows the electronic signal from the monitor to control the output of the control device.

12. A device of Claim 11 comprising a multiplicity of feedback control systems capable of simultaneously regulating a multiplicity of different fluid parameters.

13. A device of Claim 1 comprising a flow control system designed for regulating the rate of fluid flowing into each of the arterial cannulae.

14. A device of Claim 1 which is equipped with an outlet port suitable for directing fluid which is passing through the device, or a portion of such fluid, to a second fluid processing device or to the body of a second animal, and an inlet port designed for receiving fluid from the second fluid processing device or from the body of the second animal.

15. A device of Claim 1 which is equipped with a compressor suitable for compressing air, and a conduit which can be coupled to a trachea.

16. A device of Claim 15 which contains means for humidifying the air.

17. A device for maintaining metabolic activity in a mammalian head which has been severed from its body, comprising the following components: a. veinous cannulae which are capable of being attached to veins which pass through the neck and receiving fluid from the veins; b. arterial cannulae which are capable of being attached to arteries which pass through the neck and transporting fluid into the arteries; c. an oxygenation device which is in fluid communication with the veinous and arterial cannulae, and which is capable of displacing carbon dioxide contained in the fluid with oxygen; d. one or more pumps; e. one or more components which can remove one or more waste products from the fluid; f. at least one device or inlet port suited for allowing the addition of a nutrient, drug, anti-coagulant, or other desired substance to the fluid at a desired concentration; and, g. fluid conduits which are attached to each of the components listed above in a manner such that the components, when coupled to the veins and arteries of a severed head by means of the cannulae of parts (a) and (b), will form a system capable of circulating fluid through the oxygenation device and through the head after the head has been severed from the body; h. at least one monitoring device or sampling port which allows the measurement of selected physical or chemical characteristics of the fluid at a selected location in the device; i. means for securely mounting the head upon the device after the head has been severed from the body, in a position such that the veins and arteries which emerge from the head can be connected to the veinous and arterial cannulae.

18. A device of Claim 17 which comprises a component designed for removing an anti-coagulant from the fluid.

19. A device of Claim 17 which is equipped with an an outlet port designed for directing fluid which is passing through the device, or a portion of such fluid, to a second fluid processing device or to the body of a second animal, and an inlet port designed for receiving fluid from the second fluid processing device or from the body of the second animal.

20. A device of Claim 17 comprising an automated control system containing: a. a monitor designed for evaluating one or more fluid parameters, which generates a variable electronic signal which depends on the value of the parameter being measured; b. a control device which is capable of generating an output which can affect the value of the parameter being measured; and, c. an electronic circuit which allows the electronic signal from the monitor to control the output of the control device.

21. A method of sustaining metabolic activity in an intact mammalian head which has been severed from its body, comprising the following steps: a. transporting fluid which has circulated through the head to a fluid processing device, via cannulae which are attached to veins which pass through the neck of the head; b. contacting the fluid with gaseous or dissolved oxygen in a manner which displaces carbon dioxide in the fluid with oxygen; c. pumping the fluid into cannulae attached to arteries which pass through the neck.

22. A method of Claim 21 wherein one or more nutrients are added to the fluid by the fluid processing device.

23. A method of Claim 21 wherein one or more metabolites are removed from the fluid by the fluid processing device.

24. A method of Claim 21 wherein all or a portion of the fluid is routed through the body of a second animal.

25. A method of sustaining metabolic activity in an intact mammalian head which has been severed from its body, comprising the following steps: a. transporting fluid which has circulated through the head to a fluid processing device, via cannulae which are attached to veins which pass through the neck of the head; b. contacting the fluid with gaseous or dissolved oxygen in a manner which displaces carbon dioxide in the fluid with oxygen; c. adding one or more nutrients or drugs to the fluid; d. removing one or more metabolites from the fluid; e. pumping the fluid into cannulae attached to arteries which pass through the neck.

26. A method of Claim 25 wherein all or a portion of the fluid is routed through the body of a second animal.