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Modification of microglia function protects from lesion-induced neuronal alterations and promotes sprouting in the hippocampus ¹

Institute of Anatomy, Department of Cell and Neurobiology, Humboldt University Hospital (Charité), 10098 Berlin, Germany

³Correspondence: Institute of Anatomy, Department of Cell and Neurobiology, Humboldt University Hospital (Charité), 10098 Berlin, Germany. E-mail: robert.nitsch{at}charite.de

SPECIFIC AIMS

Central nervous system (CNS) lesions, including neurodegeneration in the course of Alzheimer’s disease, multiple sclerosis, and stroke, regularly trigger rapid changes in microglial morphology and function. The effects of activated microglia with regard to secondary degeneration vs. regeneration and reorganization after lesion are still unclear. Therefore, our aim was to elucidate whether glial activation has any influence on presynaptic response to lesion (i.e., sprouting and reactive synaptogenesis) and on postsynaptic changes (i.e., the arborization of dentate neurons known to respond to lesion by loss of distal dendrites).

PRINCIPAL FINDINGS

1. TGF-ß1 treatment abrogates lesion-induced dendritic retraction and improves axonal sprouting in vivo
We tested the effects of intraventricularly injected TGF-ß1 on denervated dendrites of interneurons, which can be completely stained by parvalbumin after entorhinal cortex lesion (ECL). Thereafter, we calculated the dendritic length in relation to the entire area of the termination zone, i.e., the stratum radiatum lacunosum moleculare of CA1. The lesion plus TGF-ß1 treatment resulted in significantly enhanced dendritic arborization in contrast to the lesion alone. To verify this observed effect, we used heat-inactivated TGF-ß1 (TGF-ß^HI) and registered a total dendritic length similar to that of the lesioned group. Sprouting reaction was significantly reduced in the ECL group (24%) in contrast to the ECL group treated simultaneously with TGF-ß1 (42%). Groups treated with TGF-ß^HI showed AChE-positive areas similar to the untreated lesioned sections (27%). These data show that anti-inflammatory TGF-ß1 treatment after lesion rescues denervated dendrites from secondary retraction and enhances sprouting of the remaining afferents.

2. Modification of microglial function preserves dendritic length after lesion
To further analyze this in vivo effect, we used organotypic entorhino-hippocampal slice cultures (OEHSC). Assuming that rescue of denervated dendrites is triggered by glial cells, we manipulated glial activity with TGF-ß1 (immunomodulator), AG126 (tyrphostin, inhibitor of tyrosine kinase), LLME (lysosomotrophic, kills selectively mononuclear cells), AAA (glutamate-homologue, Na⁺-dependent uptake by astroglia), and Gal (complement-dependent, antibody-mediated cytotoxicity against oligodendroglia). The dendritic density after lesion in untreated slices was reduced sixfold compared with the control. TGF-ß1 (anti-inflammatory treatment), AG126 (anti-mitotic treatment), and LLME (kills microglia) protected dendrites to the point where their length per area was similar to the nonlesioned control slices. Treatment with AAA (kills astroglia) or Gal (kills oligodendroglia), by contrast, resulted in dendritic lengths comparable to the lesioned OEHSC group. Since dendritic retraction could be reduced to control levels by interfering with microglial functions, we continued to focus our analysis on these cells, registering their morphology and phagocytotic activity after treatment with the listed compounds (Table 1 ). Phagocytotic activity was measured using phagocytosis-dependent labeling. Entorhinal fibers were initially traced with fluorescent Mini Ruby, where incorporated and labeled debris demarcated phagocytosing cells (phagocytotic activity = phagocytosing microglia divided by all microglia in the denervated molecular layer). The status of microglial activity was correlated with microglial morphology (amoeboid vs. ramified) and calculated using the index of ramification.

3. The role of proinflammatory cytokines
These results strongly indicate that microglia are involved in dendritic damage after ECL. We speculated that cytokines produced by activated microglia are responsible for this secondary damage. Thus, we searched for changes induced by the tested compounds in the release of the inflammatory cytokines tumor necrosis factor (TNF-) and interleukin 1ß (IL-1ß). TNF- and IL-1ß were measured by ELISA in untreated/unlesioned, untreated/lesioned, and treated/lesioned slice cultures. ELISA for IL-1ß revealed that this inflammatory cytokine is up-regulated after lesion. Treatment with TGF-ß1 reduced this increase, but the effect was not visible until 4 days after treatment. Surprisingly, the concentration of TNF- was not affected.

4. Anti-inflammatory treatment does not affect neuronal morphology and survival
To prove that the observed effects are driven mainly by altered microglia function and not by enhancement of neuritic growth, we tested the direct effects of all three compounds on the neuritic length in neuronal single cell cultures. Whereas TGF-ß1 and LLME had no effect on neurons in vitro, AG126 decreased neuritic length. However, if relevant in the slice culture, this result is obviously overcome by its effects on microglia leading to enhanced dendritic survival.

CONCLUSIONS

This study was developed to estimate the final outcome of microglia activation on dendrites, which are known to be severely altered during the course of axonal degeneration, a fact that hinders proper reinnervation. Our study corresponds with the idea that microglia activation has detrimental effects on the survival of dendrites after deafferentiation. Using entorhinal cortex lesion of adult rats and organotypic entorhino-hippocampal slice cultures, we provide direct evidence that therapeutic inhibition of microglial immune maturation, proliferation, and survival rescues denervated dendrites from secondary retraction and enhances sprouting of remaining afferents. Selective abolishment of astrocytes or oligodendroglia, however, did not result in the protection of hippocampal dendrites. Moreover, using an enzyme-linked immunosorbent assay, we could show that primary damage to the CNS leads to an IL-1ß up-regulation attenuated by the immunomodulator TGF-ß1, indicating a key role for IL-1ß in secondary neuronal damage. However, since we found no evidence for any remarkable regulation of TNF--release, it is apparent that this proinflammatory cytokine does not act as a major contributor to transneuronal changes after lesion-induced reorganization.

The few in vivo studies of the impact of microglia/macrophages on axonal regeneration and sprouting in the CNS clearly show that interfering with their recruitment and activation strongly affects tissue repair. Depending on the lesion type and location, this impact may improve or worsen neurological deficits.

View larger version (34K): [in this window] [in a new window]   Figure 1. Summary schematic diagram: influence of glial cell function on postlesional neuronal reorganization.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0825fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0825fje

² These authors contributed equally.

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