Editor's Note: This is part two of a two-part series on stroke research. Part one ran in Tuesday's issue.

Sometimes it seems that nature has designed receptors with the express purpose of driving neuropharmacologists to distraction.

Take adenosine receptors. They come in four subtypes, exist in bone marrow, as well as in the brain, and research in the October 2004 issue of Nature Medicine suggests that it is the peripheral, rather than central, receptors that mediate at least some of the deleterious effects of stroke. Talk about adding insult to injury.

The counterintuitive findings are reported in a paper titled "Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury." It was published by scientists from Boston University and Harvard Medical School's Massachusetts General Hospital.

"Previous research had shown that both adenosine receptor agonists and antagonists showed a protective effect in ischemia," said Jiang-Fan Chen, associate professor of neurology at Boston University School of Medicine. "It was quite confusing.

"People, including our group, believed it was a neuronal mechanism, perhaps related to glutamate release," Chen told BioWorld Today. However, the protective effects of adenosine receptor antagonists had been demonstrated mainly in the periphery. Global adenosine A_2A (A2A) receptor knockout mice had been shown to be resistant against ischemia. But in the research in Nature Medicine, the goal was to separate the effects of brain and bone marrow A2A receptors on induced stroke.

The researchers used a combination of irradiation and bone marrow transplants in knockout mice to create mice that had A2A receptors in the bone marrow but not the brain.

Similar to work done by Cesario Borlongan and his colleagues in a study of the effects of cord blood cells on stroke, Chen and colleagues used middle cerebral artery occlusion to stop oxygen flow to a defined part of the brain and induce stroke, or ischemia. (See BioWorld Today, Oct. 19, 2004.)

The authors compared three groups of animals: wild-type animals with A2A receptors in the brain and bone marrow, global knockouts with no A2A receptors, and cross-transplanted mice with A2A receptors in the bone marrow but not the brain. In keeping with earlier findings, knockouts had less damage in the brain after stroke induction. However, the researchers found that cross-transplantation reversed that protective effect - suggesting it was the lack of bone marrow receptors, rather than those in the brain, that were responsible for the reduced vulnerability of knockout mice to stroke.

The researchers next investigated which mechanisms might be responsible for the peripheral influence on brain damage. In that set of experiments, mice again were cross-transplanted and two groups were compared: mice with a full set of A2A receptors and mice with A2A receptors only in the brain. Replicating previous experiments, mice lacking bone marrow A2A receptors showed reduced anatomical damage after induced stroke.

To determine the protective mechanism behind their findings, Chen and colleagues first used antibody staining to see whether there were inflammatory cells being activated by the bone marrow receptors and then crossing the blood-brain barrier to account for the neuroprotection.

"Under ischemic conditions, you can see cells crossing, because ischemia compromises the blood-brain barrier," Chen said. But the researchers found that lack of bone marrow A2A receptors did not alter the number of neutrophils or microglia, two major types of inflammatory cells, in the brain.

What they did find was that cross-transplanted mice had altered levels of inflammatory cytokines in response to induced stroke. Specifically, the expression of several inflammatory interleukins (IL-1, -6 and -12) was reduced in mice lacking bone marrow A2A receptors, whereas the anti-inflammatory cytokine IL-10 was increased.

In a final experiment, the researchers tested what effects bone marrow A2A receptors would have on peripheral ischemia. For that, they stopped blood flow to the liver. The effects were opposite to those seen in induced stroke; that is, mice lacking A2A bone marrow receptors had worse liver injuries than controls.

Chen said that the results of those studies have clinical relevance in several ways. New Jersey-based Kyowa Pharmaceutical Inc., a wholly owned subsidiary of Japanese company Kyowa Hakko Kogyo Co. Ltd, is testing A2A receptor antagonists in clinical trials for Parkinson's disease. The company said in April that it expected to initiate Phase III trials in the third quarter and potentially file for approval in 2006.

The research presented in Nature Medicine shows that A2A receptors might be more broadly used for neuroprotection. Chen, who is not affiliated with Kyowa, said that "based on these animal studies, we think there might also be a neuroprotective effect in stroke."

Also, "if there are opposite effects of A2A modulation in the brain and the periphery, this has important implications for clinical trials" in general, Chen said. Given the contrasting effects the researchers observed in the brain and the liver, he suggested it might be useful to prescreen those participating in trials, such as the Parkinson's trial, to make sure any peripheral conditions are not exacerbated.