Brain Injury Reversed in Animal Model of AIDS

Damage from Other Neurological Disorders May Also Be Reversible

Image courtesy Woong-Ki Kim The brain’s comeback. Drugs that cannot breach the blood–brain barrier can reverse signs of neuroAIDS. Here, the brains of untreated animals show activated macrophages (top) clustered with simian immunodeficiency virus (second from top). In contrast, treated animals have minimal inflammatory macrophages (third) and few remaining traces of virus (bottom).

The brain may suffer a variety of insults, but damaged neurons share a common trait: a drop in levels of N-acetylaspartate (NAA), a molecule as small as glucose, more plentiful than glutamate, and easy to measure by noninvasive neuroimaging.

Depending on the circumstances, missing NAA may indicate Alzheimer’s disease, ischemic stroke, a brain tumor, or traumatic injury. And, as doctors soon learned with the AIDS epidemic, NAA levels drop in tandem with the neurological deterioration that further cripples people with HIV.

Yet Gilberto Gonzalez was unprepared for the precipitous fall and resurgence of this marker of neuronal injury in macaques with simian immunodeficiency virus (SIV) over the course of their AIDS-like disease, before and after they were treated with a potent antiretroviral cocktail.

“We expected a decline, but we saw a whopping decline,” said Gonzalez, HMS professor of radiology and chief of neuroradiology at Massachusetts General Hospital. “When we treated them, we expected the decline to stabilize, but the rebound was as stunning as the decline. I’ve never seen anything like it.”

The observations suggest that neuronal injury in AIDS—and perhaps other neurodegenerative diseases—may be reversible and that treating monocytes in the blood may ameliorate or prevent the brain damage, Gonzalez and his colleagues report in the September Journal of Clinical Investigation.

The decline and rebound of NAA most closely correlated with circulating levels of activated monocytes destined to mature as perivascular macrophages in the brain. In untreated monkeys, SIV-infected monocytes expanded in the blood and accumulated in the brain at the interface of blood vessels and nerves, where they presumably spewed destructive cytokines, cytotoxins, and chemokines. Within two weeks of treatment, monocytes calmed down in the blood and brain. Within months, the neurons appeared nearly fully restored in the treated macaques.

Repetitive-infection Model
The findings strengthen a popular hypothesis proposed several years ago by first author Kenneth Williams, a viral pathologist at Beth Israel Deaconess Medical Center, and Dartmouth colleague William Hickey that infected brain-bound monocytes drive AIDS-related neurological disease by continually reinfecting the brain with dysfunctional and destructive immune cells and new virus.

“Traditionally, because the virus enters the brain days after infection in monkeys and humans and because the emergence of dementia requires years, it was thought that it was a chronic progressive disease,” said Williams, HMS assistant professor of medicine. “But the virus does not get into neurons. And these drugs do not get into the brain. A cell outside the brain, a monocyte, is likely involved in driving the disease. We suggest that you need continual seeding of the virus and the activated macrophage for brain disease.”

Photo by Graham Ramsay Postdoctoral fellow Woong-Ki Kim (on left) and Kenneth Williams demonstrated that monocytes infected by simian immunodeficiency virus expanded in the blood and accumulated as macrophages in the brains of monkeys when unchecked by virus-controlling immune cells. Collaborators (below, clockwise from top left) Gilberto Gonzalez, Margaret Lentz, Sarah Pilkenton, and Eva Ratai showed that drugs shrank the number of circulating activated monocytes, reversing the signs of AIDS-related brain damage. Photo by Graham Ramsay

Born in the bone marrow, these monocytes circulate in the blood for about 48 hours as they begin to mature. They transform into macrophages when they arrive in the brain, gut, skin, or other tissue, where the bacteria-eating innate immune cells form the first line of defense against pathogens. Compared to the long-lasting resident brain macrophages called microglia, the transient perivascular macrophages that predominate in SIV- and HIV-infected brains move on in weeks or months.

The Measure of the Macrophage
Not surprisingly, much of AIDS research has focused on T cells, specifically the CD4+ T cells that are systematically infected and destroyed by the virus and carry about 95 percent of the viral load. Using the same cellular receptors, about 5 percent of the virus enters monocytes. There, it reproduces more slowly and lives longer, said Ronald Desrosiers, director of the New England Primate Research Center and HMS professor of microbiology and molecular genetics. About 10 years ago, Desrosiers found that the genetic sequences of SIV in the brain had subtle variations that made it more likely to infect macrophages.

Williams and Gonzalez do not know how the activated macrophages cause disease or even if they are a surrogate marker for another peripheral mechanism that controls disease in the central nervous system. But their paper demonstrates an approach that is fast and effective for testing the many plausible proposed mechanisms for neuronal injury associated with macrophages.

The researchers used a time-lapse macaque model of AIDS developed by Keith Reimann, HMS associate professor of medicine at BID, and Joern Schmitz, HMS assistant professor of medicine at BID. The monkeys are depleted of CD8+ T cells, which normally help fight the virus. Without CD8+ cells, the infection progresses directly to AIDS. The post-infection life span shrinks to three months, and the rate of encephalitis skyrockets from 30 to 100 percent. Activated monocytes greatly expand. And without CD8+ T cells, the amount of virus circulating in the blood remains high in spite of antiretroviral drugs.

Every two weeks, Gonzalez and his colleagues scanned the monkeys’ brains through magnetic resonance spectroscopy. The technique employs the magnetic resonance imaging unit normally used to see brain structure, but it is tuned to look at the brain chemistry, similar to the way biologists use the equipment to determine the atomic structure of molecules.

They tracked the changes in the brain during the normal course of the disease. Beginning at two weeks, the NAA of untreated monkeys dropped steadily. Traditional pathological markers showed extensive neuronal injury. Then they gave another group of monkeys antiretroviral drugs one month after infection. The steadily declining NAA levels began rebounding within two weeks. In two to three months, the animals recovered normal neuronal metabolism, as measured by NAA. Pathological exams showed only trace evidence of inflammation and virus in the brain.

Ultimately, the researchers want to identify ways to prevent or reverse the neurological damage in people. Within days of infection, HIV sneaks into the brain and can temporarily befuddle its host in the first flush of viremia as the body successfully represses the virus. Later, as the immune system fails, people can show symptoms as mild as forgetfulness or slow reaction times and, in late-stage AIDS, as severe as dementia. In children, the virus can interfere with normal brain development.

Since the late 1990s, the new and improving drug cocktails have reduced the incidence of neurological symptoms from about half to about one third of people with AIDS in this country. The antiretroviral drugs have halted and sometimes improved cognitive impairment in adults and helped infected children reach normal developmental milestones.

“The big clinical need is treatment,” said Ned Sacktor, a neurologist specializing in AIDS at the Johns Hopkins University School of Medicine. “We have highly active antiretroviral therapy, which has some benefit, but there is no treatment to specifically protect the brain against inflammatory mediators.”

Gonzalez wants to know how the neurons bounce back from the injury. “The ultimate injury in a variety of diseases probably has similar common pathways,” he said. “At a certain threshold, the recovery rate cannot keep up with the destruction rate. We’re showing that neurons can come back from disease if you do not exceed a threshold.”

With collaborators at the Tulane National Primate Research Center and elsewhere, Williams will be testing drugs that preferentially kill activated monocytes. “It shows we might not need to worry about the brain as much as a reservoir of disease [that evades the drug cocktails],” he said. “If you can target monocytes in the blood and get rid of the cellular reservoir, you can stop future infections and clear a significant viral reservoir.”

—Carol Cruzan Morton

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