NEW ORLEANS— "It was 100 years ago that the anatomy of the blood-brain barrier was first shown. But it is only today that we begin to understand the relevance the barrier has for medicine and neurological disorders," said Damir Janigro, PhD, at the 25th International Stroke Conference. Currently, researchers are taking a new look at the blood-brain barrier, interested in how they might find ways to cross this seemingly insuperable obstacle to deliver drugs to the brain or, conversely, to protect the integrity of the barrier in hopes of preventing diseases such as multiple sclerosis, AIDS, Alzheimer's disease, and Parkinson's disease.

DETECTING PATHOLOGIC CHANGES

The blood-brain barrier is an exclusive component of the endothelium of the over 400 miles of cerebral capillaries, where tight junctions prevent substances in the blood from crossing between cells and into the brain, said Dr. Janigro, Director of Cerebrovascular Research at the Cleveland Clinic Foundation in Cleveland. These undesirable substances include "not only molecules but also cells, primarily inflammatory cells," he said. While some agents do cross the barrier, including antidepressants and recreational drugs like cocaine and ethyl alcohol, a variety of desirable drugs, including antitumor and antiviral agents, growth factors, and gene delivery systems do not, he noted.

Conversely, it is becoming evident that the barrier itself may be subject to loss, perhaps underlying the initiation of chronic disease. In stroke, for example, there is a transient loss of blood-brain barrier function that happens within minutes or hours of the event, he said. In other settings, there appears instead to be a "leakage" across the barrier that may be protracted over time, leading to chronic diseases such as multiple sclerosis or Alzheimer's disease. "There's plenty of evidence … that if you lose blood-brain barrier function—even a low fraction of its function but for an extended period of time—you may allow entry into the brain, [of] molecules or cells that cause the development of pathology," he said.

Work is ongoing to develop simple blood tests or diagnostic imaging techniques that may allow evaluation of the integrity of the blood-brain barrier, he said. For example, in stroke, the loss of integrity of the barrier may herald adverse bleeding events after the administration of tissue plasminogen activator (t-PA). The ability to visualize this loss might allow those with existing breaches of the barrier to be excluded from treatment.

"How … [many] of these changes that are seen in pathological situations really relate to the loss of barrier function, and how early can we detect these changes in chronic neurological disease?" Dr. Janigro asked. The answers may make it possible to aggressively treat the blood-brain barrier disease, thereby preventing the cascade of events that lead to full-blown pathology.

ENDOTHELIAL RESPONSE TO ISCHEMIC INSULT

When an ischemic event occurs, whether thrombotic or embolic, there follows a sequence of events, including loss of cerebral blood flow, edema, and the breakdown of the blood-brain barrier, said Berislav Zlokovic, MD, PhD, Director of the Neurologic Surgery Lab at the University of Rochester, New York. This primary injury leads to a variety of secondary injuries at the microcirculation level that have led to the discovery of the role brain endothelium plays in the regulation of hemostasis in the brain, he said.

"When we began looking into these problems about seven years ago, we started with a very logical premise—that under physiologic conditions, clot-preventing pathways and clot-dissolving pathways are overruling clot-forming pathways," Dr. Zlokovic said. Except for the occurrence of intracranial bleeding, stroke or trauma result in this balance being upset, with procoagulant factors "prevailing" over anticoagulant and fibrinolytic factors.

Endothelial cells in the cerebral microcirculation have been found to actively regulate coagulation processes responsible for production and secretion of hemostatic factors, including fibrinolytic proteins such as t-PA and plasminogen-activator inhibitor-1 (PAI-1). "Another molecule that we cloned from brain microvessels and brain endothelium is thrombomodulin, which is a receptor for thrombin and activates protein C," he added. Activated protein C is a "very powerful player" because it inhibits procoagulant factors Va and VIIIa, as well as PAI-1.

"Local hemostatic pathways in brain microcirculation may critically influence development of brain thrombosis following an insult," Dr. Zlokovic said. In this setting, it appears that there occurs a downregulation of the endothelial t-PA/PAI-1 system, and there is a reduced capacity of the brain's microvessels to activate protein C, resulting in a "procoagulant transformation" of the blood-brain barrier, he said. In recent work, Dr. Zlokovic's group has shown that in Alzheimer's disease, endothelial cells from isolated capillaries lose their ability to activate protein C, so that thrombomodulin is also downregulated. "All this derangement, this procoagulant state, actually predisposes for larger microvascular obstruction following an ischemic insult," he said.

Major stroke risk factors including diabetes, smoking, and hypertension all predispose to this coagulant transformation of the vessels, he added. Microvascular obstruction and fibrin deposition positively correlate with the extent of neuronal injury, said Dr. Zlokovic. He and his colleagues have shown that the deposition of fibrin and platelets at the blood-brain barrier is associated with the expression of leukocyte adhesion molecules that "invite" macrophages and monocytes into the brain. "Migration of leukocytes across the blood-brain barrier correlates with the extent of neuronal injury, so if you prevent leukocytes coming, you prevent also, to a certain extent, neuronal injury," Dr. Zlokovic said.

TRANSPORTERS—MORE THAN MEETS THE EYE

The disruption of the blood-brain barrier as a result of stroke presents potential advantages and disadvantages, according to Fiona Parkinson, PhD, Associate Professor in the Department of Pharmacology and Therapeutics at the University of Manitoba, Winnipeg. The main advantage is that of permeability; drugs that do not generally cross the blood-brain barrier might be facilitated in reaching the tissue of interest by this disruption. "However, there are a number of disadvantages to the increased blood-brain barrier permeability, including the loss of ionic homeostasis and loss of neurotransmitter homeostasis," she said. Immune cells and toxic compounds would also be free to enter the brain during that period, providing an added neurotoxic insult, she added.

While a number of studies have documented this increased permeability after stroke, it has also been shown that the increase is not consistently progressive but that changes can occur over time. An opening might occur, then resolve, then recur several hours later, she said. Regional variations also exist, with some areas of the brain experiencing greater permeability than others. Finally, the degree of opening varies with the severity of the ischemic insult. "It appears that just utilizing the increased permeability of the blood-brain barrier for drug delivery is not very reliable," she said.

Other strategies for gaining drug access to the brain after stroke have been examined, including the transcellular lipophilic pathway, which in some cases has allowed small, lipophilic compounds to cross the blood-brain barrier. A second pathway is "receptor-mediated endocytosis." Endothelial cells express a number of different receptors, she explained. Ligands for those receptors can bind, be engulfed, and "transcytose" across the endothelial cell layer. Some experimental work has shown that a monoclonal antibody for the transferrin receptor, coupled with brain-derived neurotrophin factor, which is neuroprotective but cannot cross the barrier itself, can both cross the barrier and exert neuroprotective effects. "The monoclonal antibody binds to the transferrin receptor in the endothelial cells, goes across the blood-brain barrier by receptor-mediated endocytosis, and pulls the conjugated brain-derived neurotrophic factor across the barrier as well," she explained.

Finally, she explained, endothelial cells of the blood-brain barrier also express a number of transport proteins, including transporters for glucose, amino acids, nucleosides, and other compounds. These transporters might be approached by two strategies: one is to design compounds that would gain access to the brain by going through these transport processes; the other would be to block these processes, in that way bolstering brain levels of endogenous permeant.

Adenosine levels increase in the brain up to 100-fold following a stroke, and this adenosine can reduce stroke-induced neuronal injury. Bound to A1 receptors, it acts by inhibiting calcium influx, decreasing glutamate release, and by increasing potassium reflux to decrease the excitability of postsynaptic neurons. Adenosine also binds to A2A receptors, present in the vasculature and some neuronal populations, again producing positive effects such as vasodilation and inhibiting inflammation.

Based on these findings, Dr. Parkinson's group has been working to enhance endogenous levels of adenosine, or at least to reduce the amounts of adenosine washed out of the brain and into the vasculature after an ischemic episode. One method they have been exploring is using nucleoside transport inhibitors, which block the transport processes utilized by adenosine, in that way elevating extracellular adenosine levels. At least in an in vitro model, they have been able to block the ENT-1 and ENT-2 transporters using dipyridimole, which blocks both types of transporters. "It did appear that in this situation, the nucleoside-transport inhibitors were able to inhibit the penetration of adenosine across the in vitro blood-brain barrier and in this way may be able to protect brain adenosine levels," she concluded.

VECTORS AND TRANSGENES

The daunting task of crossing the blood-brain barrier with viral vectors, for the delivery of potentially neuroprotective transgenes, was discussed by E. Antonio Chiocca, MD, PhD, Associate Professor of Surgery and Director of Neuro-Oncology at the Massachusetts General Hospital Neurosurgical Service and Brain Tumor Center in Boston.

Gene transfer into the brain might be useful to address a variety of neurodegenerative disorders, but many of these would require permanent expression of the therapeutic gene, he said. However, in certain states, such as ischemic hypoxic injury, even transient expression of the gene may be sufficient to partially reverse, or at least limit, injury to the tissue.

According to Dr. Chiocca, there are "three ingredients" to gene transfer approaches to neuroprotection: the neuroprotective gene itself, the vector that transfers the gene across cellular membranes and into the nucleus where expression of the gene will take place, and the route of delivery for the vector-gene combination.

During ischemia, a variety of changes occur, including decreased energy availability, excitotoxic amino acid production, and reactive oxygen species formation, that lead to apoptotic and necrotic death of the cells. Potentially, the transfer of genes such as those for glucose transporters, superoxide dismutase, or heat shock protein, could be used to target and limit these events, he said. "However, a major limitation of the entire gene therapy field is the efficient delivery of these genes to large anatomic extent or area of brain," he added.

Vectors that have been investigated include viral vectors, such as adenovirus and herpes simplex, and nonviral vectors, such as liposomes, naked DNA, or protein transporters. In their laboratory, he said, they have been experimenting with a herpes simplex virus, which he described as "an exquisitely neurotropic virus" that "loves to infect neurons." The virus will carry up to 15 copies of the therapeutic gene, but none of the viral genes are expressed once inside the cell.

Limiting the utility of these vectors, however, is the fact that components of the immune system, such as complement, antibodies, and cytokines, may recognize the vector and neutralize it, decreasing the effective concentration of the vector. The tight junctions of the barrier can also limit the passage of these larger molecules—adenovirus measures 70 nanometers in diameter, herpesvirus about 150 nanometers. Even with disruption of the barrier, the maximum opening of these junctions is estimated to be 20 nanometers, he said.

To improve vector passage across the blood-brain barrier, a variety of approaches have been investigated: One approach would be to improve vector survival in circulation by using immunosuppressive measures to limit the action of complement or immunoglobulin action against the virus, he said. "The second would be to increase vector concentration in cerebral vasculature by trying intra-arterial routes of delivery, rather than just generalized systemic routes," he added. Another approach would be to try to increase vector infection of endothelial or astrocytic cells by altering vector surface ligands. Finally, it may be possible to increase vector transit simply by disrupting the barrier.

Most initial work has combined intra-arterial routes of injection with barrier disruption, Dr. Chiocca noted. In their own laboratory, they have disrupted the barrier in animal models both by pharmacologic means and by the introduction of tumors. "Tumors are known to have a leaky blood-brain barrier, and this may also apply to disrupted blood-brain barrier in ischemic events," he said. "We do think there are ways that we can get around the issue of the blood-brain barrier," he concluded.

—Susan Jeffrey

Suggested Reading
Janigro D. Blood-brain barrier, ion homeostasis and epilepsy: possible implications towards the understanding of ketogenic diet mechanisms. Epilepsy Res. 1999;37:223-232.
Stanness KA, Neumaier JF, Sexton TJ, et al. A new model of the blood-brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions. Neuroreport.1999;10:3725-3731.
Preston E, Foster DO. Evidence for pore-like opening of the blood-brain barrier following forebrain ischemia in rats. Brain Res. 1997;761:4-10.
Preston E, Webster J, Palmer GC. Lack of evidence for direct involvement of NMDA receptors or polyamines in blood brain barrier injury after cerebral ischemia in rats. Brain Res. 1998;813:191-194.
Wu D, Pardridge WM. Neuroprotection with noninvasive neurotrophin delivery to the brain. Proc Natl Acad Sci. 1999;96:254-259.

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