ARTICLE IN BRIEF
Investigators were able to show blood-brain barrier disruption using an in vivo imaging technique, demonstrating fibrinogen as a novel molecular signal that triggers rapid perivascular microglial responses, and contributes to axonal damage in neuroinflammatory diseases such as multiple sclerosis.
Researchers using a powerful in vivoimaging technique have documented for the first time how leakage of the blood protein fibrinogen into the central nervoussystem triggers an immune attack on nerves in the preclinical stages of multiple sclerosis in mice.
Scientists at the Gladstone Institute forNeurological Disease at the University ofCalifornia, San Francisco, used two-photon microscopy to view the activity of fibrinogen infiltration across the blood-brain barrier in mice and thesubsequent inflammatory response of microglia — the immune system's principal agents in the brain and spinal chord.
After leaking across the blood-brain barrier, fibrinogen binds to a microglial surface receptor called CD11b/CD18. This causes microglia to release reactive oxygen molecules that damage neurons, they reported in the Nov. 27, 2012, online edition of Nature Communications.
The National Institute of Neurological Disorders and Stroke (NINDS) and the National Multiple Sclerosis Society funded the research.
“This is the first study in living animals to show how leakage of fibrinogen activates microglia and induces rapid clustering of microglia around blood vessels, which contributes to axon damage and neuroinflammation,” said lead author Katerina Akassoglou, PhD, professor of neurology at UCSF, and an associate investigator at Gladstone Institute.
“Our study showed that vascular changes may instigate neurotoxicity and, until now, the initial trigger(s) that cause these changes in MS and their role in axonal damage have been unknown,” she told Neurology Today in a telephone interview.
ACTIVATION PRECEDES DAMAGE
In animal models, suppressing activation of microglia reduces inflammatory demyelination and neurological deficits in neuroinflammatory disease, and while evidence indicates that such activation is one of the earliest events in the pathology of these diseases, it has been unclear what triggers microglial activation or how these early events relate to lesion formation and localized axonal damage.
Dr. Akassoglou said the team's findings suggest disruption of the blood-brain barrier is needed for the mobilization of the microglia around the CNS vasculature, which then triggers and amplifies inflammatory demyelination and axonal damage. The activated microglia showed patterns of movement specifically toward animals' vasculature, which preceded the onset of any neurological symptoms or formation of lesions.
“We have demonstrated that microglia form perivascular clusters well before there is a loss of myelin, nerve damage, or the onset of paralysis. Of blood plasma proteins, we found that fibrinogen specifically triggers rapid and sustained microglial responses that in turn contribute to the development of axonal damage,” Dr. Akassoglou said.
The researchers were also able to inhibit the binding of fibrinogen to microglia, and the subsequent cascade of neurodegenerative events, using both an anticoagulant and by genetically modifying fibrinogen in the mice to eliminate its binding to receptors on microglia.
Current MS therapies are designed to treat later immune processes, Dr.Akassoglou noted, adding that the ability to inhibit fibrogen's activation of microglia could be an “upstream” target that could prevent or reduce neuroinflammation and nerve damage. However, because fibrinogen plays a major role in blood clotting throughout the body, any pharmacological approach would have to inhibit CNS activity without interfering with fibrinogen's action elsewhere, she said.
Dr. Akassoglou and her colleagues are now trying to translate the data in order to develop potential strategies for treating MS in humans, she toldNeurology Today.
“This study fills in some gaps,” said Richard Ransohoff, MD, director of Cleveland Clinic's Neuroinflammation Research Center, who was not involved with the study.
“It has been known for years that plasma proteins from blood can leak into the CNS when the blood-brain barrier is not working properly.The great contribution of this group is to clarify mechanisms by which plasma proteins activate microglia, and specifically to demonstrate fibrinogen's role. Remember, there are untold numbers of proteins in plasma so they really found a needle in a haystack.”
Two-photon microscopic imaging of the spinal cord is very difficult, he noted, and what the researchers have accomplished is very cutting edge, he said in a telephone interview withNeurology Today.
“The pictures in this article are spectacular. That they have been able to show little areas of leakage well before the onset of any clinical signs is important, as is the fact that the clusters of microglia they show occur around blood vessels near where damage occurs.”
However, in terms of potential therapeutics, much work remains to be done, Dr. Ransohoff cautioned.
“Showing fibrinogen's binding site on microglia is notable, but whether or not this binding is a choke point to stop brain damage in MS remains to be seen. Many questions need to be answered before taking this in a therapeutic direction, but knowing that fibrinogen crosses the blood-brain barrier and attaches to microglia is a very important insight, and might be useful to understand diverse other conditions beyond MS.”
NINDS: FINDINGS ARE ‘UNCLEAR’
Bibiana Bielekova, MD, chief of the Neuroimmunological Diseases Unit at the NINDS, said she had a number of questions about the conclusions the researchers reached and is not fullyconvinced by their findings.
“I think that neurotoxicity of fibrinogen-activated microglia in neurological diseases is a highly interesting concept, but further studies are needed before this concept can be fully accepted,” she told Neurology Today in a telephone interview. “At this stage, the significance for MS remains unclear. These individual researchers have been pursuing this concept for several years, but I have reservations about several parts of the current study.”
While there is little doubt that leakage of plasma, and fibrinogen in particular, occurs in neuroinflammatory diseases like MS and its animal model experimental autoimmune encephalitis (EAE), and that this leads to microglial activation, whether or not microglial activation causesirreversible axonal damage, especially in humans, remains speculative, she said.
“I am also not sure that I understand the mechanism behind the described phenomenon: on one hand, anticoagulant therapy inhibits axonal damage, but it would not be expected to abrogate binding of leaked fibrinogen to CD11b/CD18 on microglia. So is it possible that anticoagulant therapy also does something to the adaptive immune responses, which the authors did not measure?”
Moreover, she said, the effect ofanticoagulation on axonal and myelin damage was only measured at the peak of EAE, when T cells were also present in the affected tissue. Limiting binding of fibrinogen to CD11b/CD18 on microglia by genetic manipulation decreased microglial cluster formation, but the data about its effect on the course of EAE were not provided.
Dr. Bielekova also said that she was disappointed that the study did not provide more clarity with regard to specific research methods, such as randomization, blinding, and data handling, as was recommended in an Oct. 11, 2012, NINDS report published in Nature.
“Because the study relied heavily on microscopy, which is highly susceptible to selection bias, these technical details are essential for interpretation of the results. Although the authors repeatedly pointed to the presence of microglia clusters in pre-symptomatic EAE, I have not observed statistical significance between these pre-onset EAE data and control data in any of the presented figures.”
She concluded: “Hopefully, this research team will continue to pursue this line of research and provide definite evidence in future studies. Perhaps other fields, such as stroke, where large human studies utilizing immediate anticoagulation were performed, may add to the discussion. It would be expected that if neurotoxicity of fibrinogen-activated microglia exists, it should not represent an MS-specific phenomenon.”