Rapid response of identified resident endoneurial macrophages to nerve injury

Abstract

Macrophages play a central role in the pathogenesis of peripheral neuropathy but the role of resident endoneurial macrophages is undefined because no discriminating markers exist to distinguish them from infiltrating hematogenous macrophages. We identified and characterized resident endoneurial macrophages during Wallerian degeneration in radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into irradiated TK-tsa transgenic Lewis rats. In such animals, resident cells carry the transgene, whereas hematogenous cells do not. As early as 2 days after sciatic nerve crush and before the influx of hematogenous macrophages, resident transgene-positive endoneurial macrophages underwent morphological and immunophenotypic signs of activation. At the same time, resident macrophages phagocytosing myelin were found, and proliferation was detected by bromodeoxyuridine incorporation. Continuous bromodeoxyuridine feeding revealed that resident endoneurial macrophages sequentially retracted their processes, proliferated, and expressed the ED1 antigen, rendering them morphologically indistinguishable from hematogenous macrophages. Resident endoneurial macrophages thus play an early and active role in the cellular events after nerve lesion before hematogenous macrophages enter the nerve. They may thus be critically involved in the pathogenesis of peripheral neuropathy particularly at early stages of the disease and may act as sensors of pathology much like their central nervous system counterparts, the microglial cells.

Figure 1.

Resident endoneurial macrophages in normal nerves and early changes during Wallerian degeneration. In normal sciatic nerve (A,C, D and E), immunocytochemistry using the polyclonal macrophage antibody against Iba1 (A, C, and D; red color) reveals low numbers of resident endoneurial macrophages. They frequently are small, have a triangular shape, and carry tiny slim processes. They are positioned around blood vessels or within the endoneurium between myelin sheaths as revealed by phase contrast microscopy (A and D) or double-labeling immunocytochemistry for myelin basic protein (C, green color). D and E: Co-localization with the TK-tsa transgene on serial semithin sections revealed that some of them carry the transgene (red spot in E), whereas others had undergone physiological turnover from the blood and are TK-tsa-negative. Three days after sciatic nerve crush, there is also a marked increase in number of endoneurial macrophages (B). As early as 2 days after sciatic nerve crush (F–J) and more pronounced after 3 days (B), Iba1-positive endoneurial macrophages begin retracting their processes, become slightly thicker (F), and sometimes already take up a rounded appearance (H). Co-localization of Iba1 (F) with the TK-tsa transgene (red spot in G) on adjacent serial sections revealed that many of these activated macrophages are resident. I and J: Also as early as 2 days after lesion, resident endoneurial macrophages, identified by in situ hybridization for the TK-tsa transgene (red spots), were found that newly express the ED1 antigen (green color) present on activated macrophages. These cells exhibit an activated phenotype and are large and round. Cross-sections from sciatic nerve 7-mm distal from a crush lesion. Blue nuclear counterstain with DAPI. Scale bars: 30 μm (A and B); 10 μm (C–J).

Figure 2.

Phagocytosis of myelin by resident endoneurial macrophages. A and B: Adjacent semithin serial sections double-stained for the TK-tsa transgene (red spots) and either ED1 (A) or myelin basic protein (B). A representative ED1-positive resident macrophage is shown that has phagocytosed myelin debris. The specificity of the stain is documented by semithin serial sections through another resident macrophage (C–E, asterisk) stained for ED1 (C), the TK-tsa transgene (D, red spot), and myelin basic protein (E). This resident macrophage does not contain myelin debris. Two (A and B) and 3 (C–E) days after crush lesion. A and B show additional phase contrast to delineate the morphology. Blue nuclear counterstain with DAPI. Scale bars, 10 μm (A and B); 15 μm (C).

Figure 3.

Proliferation of resident endoneurial macrophages. A and B: Adjacent serial semithin sections stained with ED1 (A) and BrdU to mark proliferating cells (B) reveal no proliferation of ED1-positive macrophages 3 days after crush injury. Note that proliferating cells (green color in B) are always ED1-negative (nuclei marked with asterisks in A). C: Co-localization of Iba1 (red) and BrdU (green) reveals many proliferating macrophages. D and E: Co-localization of Iba1 (red) and BrdU (green) on one semithin section (D) and the TK-tsa transgene on another adjacent semithin section (E, red spot) reveals that identified resident endoneurial macrophages proliferate 3 days after crush injury. Blue nuclear counterstain with DAPI. Scale bars, 30 μm (A and B); 20 μm (C); 10 μm (D and E).

Figure 4.

Evolution of cellular changes in macrophages during Wallerian degeneration. Cross-sections through sciatic nerve 7-mm distal to a crush lesion. A–F: Co-localization of ED1 (green) and Iba1 (red) in a normal nerve (A) and 2 (B), 3 (C), 4 (D), 7 (E), and 28 days (F) after crush lesion in representative macrophages. There is a progressive expression of the ED1 antigen concomitant with a change of morphology toward large phagocytic macrophages. Blue nuclear counterstain with DAPI. Scale bar, 5 μm.

Figure 5.

Evolution of cellular changes in macrophages during Wallerian degeneration. A–D: Co-localization of the TK-tsa transgene (A, red spot), Iba1 (B), ED1 (C), BrdU (D, green), and S-100 (D, red) on adjacent serial sections from an animal fed BrdU continuously for 7 days after crush lesion. The cell marked with an asterisk is positive for the TK-tsa transgene, Iba1, ED1, and BrdU, but negative for S-100, indicating its identity as a resident endoneurial macrophage that has proliferated within the 7 days between lesioning and sacrifice. As ED1-positive macrophages do not proliferate, the cell must first have proliferated and subsequently expressed the ED1 antigen. Blue nuclear counterstain with DAPI. Scale bar, 5 μm.