Gateway to the Brain

Winding through the human brain is a 400-mile network of capillaries. The cells that comprise its walls are so tightly packed that they form a barrier that efficiently protects delicate brain tissue from potentially harmful substances in the blood. If the walls of the capillaries were laid flat, the blood-brain barrier would cover an area 20 by 50 feet.

The capillaries here are far more discriminating than those elsewhere in the body. While allowing passage of oxygen and other essential chemicals, the capillariesí anatomical arrangement shields the brain from toxins in the circulatory system and from biochemical fluctuations that would be overwhelming if the brain had to continually respond to them. A steady chemical environment is especially important in this master organ, where specific levels of neurotransmitters are key to survival. However, the barrier can be a hindrance when it blocks therapeutic drugs from reaching the brain.

By dissecting the workings of the barrier, researchers are revealing details of cellular adhesion that provide this vital protection, and are developing new approaches to treating brain diseaseñwhich is on the rise.

Neurodegenerative diseases and those associated with advancing age (Alzheimerís and strokes, for instance) are becoming more prevalent as the population ages. Brain tumor incidence has increased in parallel to improved survival rates for patients with other cancers, which later can spread to the brain. According to the National Cancer Institute, each year 15,000 people are diagnosed with cancer that begins in the brain, and 150,000 others find that their cancer has spread to the brain.

Another source of increasing brain illness is AIDS; 30,000 people with the disease now have the brain infections cryptococcal meningitis (caused by a fungus) or toxoplasmosis encephalitis (caused by a protozoan). Inherited diseases such Huntingtonís disease and amyotrophic lateral sclerosis (ALS), for which genes have recently been discovered, someday also may be candidates for treatments based on blood-brain barrier research.

Physicians and biologists have been fascinated by the sanctity of the vertebrate brain for more than a century. In 1885, German bacteriologist Paul Ehrlich discovered that, when a dye called Evanís blue was injected into an animal, it appeared throughout the bloodstream but was excluded from the brain. For many years, investigators wondered just how the brain managed to seal itself off. Were the endothelial cells that form the walls of capillaries unpassable? Or was the seal created by astrocytes, the supportive neuroglial cells that wrap around capillaries in the brain?

In 1969, Thomas Reese and Milton W. Brightman, neurobiologists at the National Institutes of Health, visualized in an electron microscope precisely how the cells of brain capillary walls knit together so tightly: their cell membranes overlap to form a barrier of high-resistance tight junctions. Whereas the cells forming capillary walls elsewhere in the body are pocked with vesicles and windowlike portals called fenestrations, and sometimes tight junctions, those of the blood-brain barrier have few vesicles, no fenestrations, and continuous high-resistance tight junctions forming a tight sheet that curves to form the capillary tubule.

Regenerating capillaries in the brain also perpetuate the barrier. As new endothelial cells align, they wrap about each other, forming the characteristic tight fit.

What regulates the distinctive specialization of cells comprising the blood-brain barrier? Several investigators have shown that capillaries transplanted from outside the brain soon weld together, suggesting that an unidentified substance in the biochemical milieu of the brain controls establishment of the formidable barrier. The astrocytes embracing the capillaries of the barrier may be the source of the triggering substance.

The blood-brain barrier lets in some molecules, but not others. Whether or not a molecule is permitted entry depends on several factors, including its size, charge, and its solubility in lipids, the large, fatty molecules comprising much of a cell membrane, says Laurence Fritz, president of Athena Neurosciences, a biotechnology company in South San Francisco.

Because the endothelial cell membrane is lipid-rich, lipid-soluble molecules more easily cross the barrier. Many psychoactive drugs are lipid-soluble, Fritz says. Heroin, for example, traverses the barrier 100 times faster than its precursor morphine, which is only partially lipid soluble. Some psychoactive drugs, such as Valium, simply dissolve in the lipid of the membrane and cross it, without binding to a specific receptor, he adds.

Nicotine, cocaine, and alcohol easily travel from blood to brain, as users of these drugs can attest by their fast-appearing effects. On a healthier note, lipid-soluble oxygen also directly crosses through the endothelial cell membrane into the brain.

Water-soluble molecules take other routes across the blood-brain barrier. Insulin is engulfed into a vesicle by endocytosis on the blood side, travels through the cells, and is deposited on the brain side by exocytosis. Such vital chemicals as glucose, amino acids, and iron have their own carrier molecules. If the brain is starved of glucose for just a few seconds, unconsciousness results. Amino acids are constantly needed to build protein neurotransmitters, and iron is needed for metabolism.

A few brain areas lack the barrier. The vomiting center just above the brainstem is one such exception, and it makes evolutionary sense. An ingested toxin enters the brain here, triggering vomiting, so that the animal stems intake of the substance. Some toxins such as certain snake venoms, however, do make it past the blood-barrier into more fragile areas by literally dismantling capillaries.

Until recently, penetrating the bloodbrain barrier required brute force efforts. Surgeons drilled a hole in the skull and inserted a catheter to deliver a substance to a specific area. This approach made the patient vulnerable to infections.

A less drastic strategy is to inject mannitol into the carotid artery. This substance temporarily dehydrates the barrierís endothelial cells, shrinking them so that tiny, transient openings appear between the cells. The mannitol injection provides a 15-second window when a drug given intravenously can sneak past the normally seamless brain capillary walls.

Neurosurgeon Edward Neuwelt, at the Oregon Health Science University in Portland, uses mannitol to deliver chemotherapy to brain tumors. Although Neuweltís approach has cured some advanced cancer patients, it is not widely used because it is nonspecific. As neurotransmitters, white blood cells, and hormones rush in along with the therapeutic drug, seizures and other deleterious neurological side effects can result.

Researchers at Brown University and CytoTherapeutics Corp., both in Providence, Rhode Island, are experimenting with an implant to deliver drugs to the brain, bypassing the circulatory system. The implants are tiny, hollow tubes about the length of a thumbnail. Contained in them are cells genetically engineered to secrete large amounts of therapeutic proteins. Nerve growth factor, which is used to halt degeneration of dopamine-secreting cells in Parkinsonís disease, and enkephalins, peptides that block pain, are among those being studied.

The cell implant is encased in a semipermeable polymer membrane that lets nutrients in and the therapeutic protein out. The device does not provoke the immune system. "We donít want the body to wall the implants off. Biocompatibility is the key," says Seth Rudnick, president of CytoTherapeutics.

Rudnick explains how an implant is being tested on an animal model of Parkinsonís disease. The researchers focus on the striatum, part of the basal ganglia, which contact the substantia nigra. It is these dopamine-producing neurons that degenerate in Parkinsonís disease, producing the characteristic tremors and rigidity. "A tether is implanted in the striatum, and we put dopamine-secreting cells in. We use monkeys trained to pick Froot Loops [a dry cereal] off a tray. When we damage the striatum [cutting off the dopamine supply], they canít do it. When we then implant cells, the monkeys can pick up Froot Loops at 80 to 90% of the baseline level."

Cells that secrete nerve growth factor (NGF) can also be successfully housed in implants. In the July 1993 issue of Experimental Neurology, the CytoTherapeutics-Brown University team describe delivering large quantities of recombinant NGF to the brain of an animal model. "NGF is one of a family of neurotrophic factors that previous studies have shown may play a critical role in the treatment of central nervous system disorders by preventing or reversing neuronal cell loss, which is characteristic of neurodegenerative disorders," says Patrick Aebischer, founding scientist of CytoTherapeutics. "Because we have been able to demonstrate that growth factors can be successfully delivered by encapsulating factor-producing cells and implanting them directly within the brain, we may significantly increase the potential applications of these growth factors in the treatment of numerous CNS diseases, such as Alzheimerís disease, ALS, and Parkinsonís disease." As early as this year, the scientists may begin to plan clinical trials.

Yet another route into the brain is via biochemicals that naturally open the barrier by binding to receptors on the endothelial cells that comprise the walls. Alkermes Inc. in Cambridge, Massachusetts, has developed "receptor-mediated permeabilizer" or RMP technology. It consists of a family of compounds based on bradykinin, a peptide that dilates blood vessels. "RMP loosens tight junctions enough to let small molecules through," says Richard Pops, president of the company. Preclinical trials showed that a compound called RMP-7 enhanced the entry of an analgesic called loperamide into brains of mice. Administering RMP-7 and loperamide enabled mice to withstand heat applied to their tails longer than could mice given loperamide without the RMP-7 boost.

Later this year, clinical trials are planned in which RMP-7 administration is to be followed by intravenous amphotericin-B. This drug is taken in high doses by AIDS patients to combat the infection cryptococcal meningitis.

Admission of amphotericin-B to the brain may make lower dosages as effective as higher ones given by conventional routes. A change in procedure might tame the severe nausea that earns the drug its nickname, "amphoterrible."

Similarly, standard chemotherapy agents may hitch rides with RMP-7 to treat brain tumors. "Chemotherapeutic drugs are water soluble and do not cross the blood-brain barrier. RMP-7 can get more chemo into the tumor," says Pops. Phase I clinical trials completed in 1993 showed RMP-7 to be safe and tolerable for this application, and trials are ongoing to test it with the standard chemotherapeutic drug carboplatin.

The routes into the blood-brain barrier mentioned so far are nonselective, allowing entry to whatever happens to be in the vicinity of the temporarily compromised barrier. Several research teams are pursuing a more targeted, carrier-mediated approach that uses the protein receptor for transferrin, the transport protein for iron.

Transferrin receptors are found on cells throughout the body, but they are especially abundant on brain endothelium, where they can number 100,000 per cell. Transferrin with iron attached binds the receptor, then is engulfed into the endothelial cell, migrates through the cytoplasm, and is extruded on the brain side, where the iron is released.

The brainís iron transport system was altered to make it carry drugs in the work of Philip M. Friden and co-workers at Alkermes, Barry J. Hoffer, professor of pharmacology at the University of Colorado School of Medicine in Denver, and collaborators at the Scripps Clinic and Research Foundation in La Jolla and at the University of Linkoping Health Science Center in Sweden. They produced a monoclonal antibody that binds to the rat transferrin receptor, and they linked the antibody to a drug. The first experiment, in 1991, piggybacked the cancer drug methotrexate into the rat brain.

The researchers describe in the 15 January 1993 issue of Science how they have transported NGF into the rat brain. In the experiment, pieces of fetal brain tissue were transplanted into the anterior chamber of the eye of adult rats, where the tissues could be directly observed through a stereomicroscope. Not only did the transplanted brain tissueís capillaries form a blood-brain barrier in the eye, but NGF conjugated to antitransferrin antibody injected into the peripheral circulation appeared in the ratsí eye chambers.

Degenerating neurons are just one mechanism of brain disease. Equally damaging is inflammation, the immune systemís nonspecific response whereby fluid and white blood cells go to the site of injury or infection. (The immune systemís specific response is to produce antibodies.) "Normally the blood-brain barrier keeps immune cells in the blood from entering the brain. In certain diseases, thereís a large influx of leukocytes across the bloodbrain barrier into the brain," says Fritz.

Leukocytesí entry into the brain is the culmination of a complex interplay between the blood cells and various cell-adhesion molecules on the inner capillary wall. First, a leukocyte rolls along the lumen of the capillary, guided by receptors on the capillary. Then the white blood cell adheres to the endothelial wall, where it is held in place by adhesion molecules. Finally, in the transmigration step, the white blood cell contorts itself by ameboid motion and squeezes through the narrow interface between adjacent endothelia.

Athena Neurosciences is using an in vitro model of the blood-brain barrier to dissect the steps of this leukocyte trafficking. The model consists of a monolayer each of blood cells, endothelium, and human neural tissue, forming what Fritz calls "a complex biological system that we can reproduce in a laboratory dish." If the researchers add astrocytes to the medium, along with cyclic adenosine monophosphate (cAMP), the endothelium forms tight junctions. (cAMP is a key component of signal transduction pathways, by which cells receive extracellular messages.)

Athenaís approach is to identify the steps of each moleculeís role in leukocyte trafficking by using monoclonal antibodies to block the passage of blood cells across the model barrier. For example, a monoclonal antibody raised against either of the two subunits of an adhesion molecule called alpha-4 beta-1 integrin prevents lymphocytes and monocytes from binding to brain endothelium. These are the types of white blood cells implicated in multiple sclerosis, in which the immune system attacks the fatty myelin coating around neurons.

To investigate multiple sclerosis further, Fritz and co-workers tested antibodies to this adhesion molecule in a rat version of the disorder, called experimental autoimmune encephalomyelitis. An antibody against either integrin subunit indeed halts leukocyte trafficking and relieves the symptoms of paralysis in the rats. This work is still in preclinical stages.

Understanding the molecular underpinnings of the blood-brain barrier can have rapid clinical consequences. Recent improvements in treatment for meningitis is a direct result of unravelling how components of the immune system, alerted by infection, invade the normally off-limits brain.

Meningitis is an inflammation of the brain that occurs when Streptococcus pneumoniae, Neisseria meningitidis, or Hemophilus influenzae bacteria cross the barrier. There are 50,000 cases of this malady per year in the United States. An attack is rapid, beginning with fatigue, fever, and irritability, followed by a stiff neck, seizures, coma, and death. Physicians typically administer several antibiotics, hoping that enough of an effective one would cross the blood-brain barrier to halt the infection. Until 1991, this approach worked only 70% of the time, with half of the survivors left deaf or paralyzed.

The symptoms of meningitis are not caused by bacteria but by the immune systemís reaction, inflammation. Alexander Tomasz of the Ciba-Geigy Research Laboratory in Basel, Switzerland, made this discovery in 1984, when he found that rabbits accidentally infected with dead pneumococci nevertheless fell ill. After additional experiments, he concluded that a component of the disintegrating cell walls of the microbes triggered the immune response. Pieces of the cell walls signal white blood cells to secrete the cytokines interleukin-1 and tumor necrosis factor, which separate the tightly spaced cells of the blood-brain barrier. White blood cells cross, where they produce even more cytokines, which open the barrier further. Some previous treatments for meningitis, such as penicillin-based antibiotics, may actually have accelerated the process. These antibiotics are well known to act by destroying the cell walls of certain bacteria.

Once researchers recognized the role of inflammation in meningitis, they realized that giving an anti-inflammatory steroid drug along with an antibiotic can help. Doing so, they lowered the death rate from 30% to 5%, with a more rapid resolution of symptoms and fewer lasting effects.

Elaine Tuomanen, head of the laboratory of molecular infectious diseases at Rockefeller University in New York City, and her group are working with monoclonal antibodies that latch on to bacterial cell wall fragments, thereby preventing them from triggering the immune response. They are trying to isolate the precise part of the cell wall that breaches the blood-brain barrier, and they are collaborating with Alkermes scientists to use that information to design carriers to take drugs into the brain. And so understanding the role of the blood-brain barrier in one diseaseñmeningitisñmay lead to ways to treat others, such as the infectious and degenerative neurological diseases now on the rise.

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