The Brain and Microglial Recruitment—Think Local, Not Global

Microglia play an important, if controversial, role in Alzheimer disease (AD). While these phagocytic cells clear up deposits of amyloid-β (Aβ), they may also unleash a horde of cytokines harmful to neurons. On top of that, there is evidence that not all microglia are created equal. Those derived from the blood stream seem to be particularly adept at clearing Aβ from the brain (see ARF related news story), but there has been a nagging question about how much peripherally recruited cells contribute to the CNS pool of microglia. Not much, according to two papers published in the November 18 Nature Neuroscience online.

The problem with studies suggesting that brain microglia are derived from peripheral cells is that they commonly depend on detection of transplanted bone marrow-derived cells in the brain of irradiated recipient mice. That irradiation can lead to disruption of the blood-brain barrier, allowing cells normally restricted to the periphery to enter the brain, suggest researchers lead by Fabio Rossi at the University of British Columbia, Vancouver. That point is echoed by researchers in Germany led by Marco Prinz at the University of Gottingen. Both groups now show that in the absence of irradiation, none of the microglia found in the brain are recruited from the blood.

Rossi and colleagues circumvented the need for irradiation by using parabiosis, a technique where the circulatory systems of two genetically identical mice are shared. With one mouse expressing hematopoietic cells labeled with green fluorescent protein, the researchers were able to look for infiltrating GFP-positive microglia in the brain of the parasymbiotic partner under a variety of conditions.

First author Bahareh Ajami and colleagues found that in over 30 different sections taken from the brain, brainstem, and spinal cord of each mouse, not one GFP+ microglia was detected despite the presence of more than 2,000 cells that stained for the microglial marker Iba-1. The authors also failed to detect infiltration of peripheral microglia into the CNS in models of acute and chronic neurodegeneration and microgliosis. In one model, they severed the facial motor nerve after it emerges from the CNS. This typically results in acute local microgliosis, but when Ajami and colleagues carried out this procedure in one of the parasymbiotic pairs, they again failed to find infiltration of GFP+ microglia from the partner, suggesting that the gliosis is due to recruitment of local, CNS-based cells. For their chronic model, they coupled a normal mouse to an ALS mouse expressing mutant superoxide dismutase (mSOD). Again, despite rampant microgliosis in the spinal cord of the ALS parasymbiont, none of the cells detected were GFP-positive. As controls, the researchers used irradiated animals transplanted with hematopoietic bone marrow cells. In both the acute and chronic model, they found that the microglia surrounding the damaged neurons were derived from the transplant.

Taken together, these result indicated that the replacement of microglia by circulating precursors can be induced by the experimental manipulations associated with irradiation and transplantation, but this replacement does not take place under physiological conditions, write the authors. They tested this idea by irradiating the GFP+ symbiont partner and then carrying out the facial nerve axotomy 5 weeks later. In this case they found that 80 percent of the infiltrating microglia were GFP-positive, supporting the idea that irradiation, or perhaps some other type of damage to the BBB, is a prerequisite for peripheral microglial infiltration into the CNS.

Prinz and colleagues arrive at a similar conclusion but from a different direction. Because circulating monocytes have recently been shown to be heterogenous, first author Alexander Mildner and colleagues were interested in exactly which blood monocytes are the precursors of microglia. Monocytes fall into two types based on high or low expression of the cell-surface maker Ly-6C. The researchers found that both high expression of Ly-6C and expression of the chemokine receptor CCR2 are essential for infiltration of microglia into the brain following irradiation and bone marrow transplant (CCR2 was recently shown to be crucial for microglial clearance of Aβ in APP transgenic mice—see ARF related news story). But does this infiltration depend on a damaged blood-brain barrier

To answer that, Mildner and colleagues used a highly focused beam to protect the brain from irradiation, then, 2 weeks following bone marrow transplantation, they measured microglial infiltration. They found that the protected brains were devoid of Ly-6Chi/CCR2+/+ donor-derived microglia. To check if infiltration might require a trigger, such as CNS damage, the authors carried out a similar experiment but this time using the neurotoxic agent cuprizone to induce demyelination in the corpus callosum. Again, in the mice whose brains were protected from radiation, the scientists found no infiltration. In animals that had full body radiation, the CCR2+/+ microglia did infiltrate the corpus callosum, indicating that Ly-6Chi/CCR2+ monocytes were recruited to the site of damage. The data indicate that Ly-6Chi monocytes are only recruited to the demyelinating lesion when the brain was irradiated, write the authors. Similarly, using the same facial nerve axotomy protocol employed by Rossi’s group, Mildner and colleagues found no infiltration of microglia to the site of damage if the animals’ brains were initially protected from radiation damage.

Do these findings spell the death of the infiltrating microglia hypothesis Not if you ask Serge Rivest of Laval University, Quebec (see Simard et al., 2006). Rivest sounded a cautionary note about parabiosis in an e-mail to Alzforum. The technique employed may not allow adequate chimerization, and GFP cells are just not able to compete with resident myeloid cells,” he suggested in reference to the work from Rossi’s group. And even if microglial infiltration does depend on damage to the blood-brain barrier, peripheral microglia or their precursor could still be physiologically relevant to the brain. There is some evidence that the BBB is compromised in AD (see ARF related news story): stroke is a typical example of how circulating monocytes can gain entry into the CNS.

Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FMV. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nature Neuroscience. 2007 Nov 18, online. Abstract

Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch U-K, Mack M, Heikenwalder M, Bruck W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nature Neuroscience 2007 Nov 18, online. Abstract

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