Macrophages are eliminated from the injured peripheral nerve via local apoptosis and circulation to regional lymph nodes and the spleen

Abstract

The present study investigated the fate of macrophages in peripheral nerves undergoing Wallerian degeneration, especially their disappearance from the injured nerves after phagocytosis of axonal and myelin debris. Wallerian degeneration was induced in adult male C57Bl/6 mice by transecting the right sciatic nerve. Five days after transection, the male sciatic nerves were transplanted into female recipient mice by placing them exactly parallel to the host sciatic nerves. Nerves of the female recipient mice were also transected to induce breakdown of the blood-nerve barrier in the host animal. Apoptosis was assessed by morphological, immunohistochemical (activated caspase-3), and molecular (DNA fragmentation) methods in transplanted, recipient, and in control nerves. A subpopulation of macrophages within the degenerating nerves died locally by apoptosis in each experiment. The fate of the male macrophages within the transplanted nerves and the host organism was investigated by in situ hybridization with a Y-chromosome-specific DNA probe (145SC5). In situ hybridization specifically stained cells within the transplanted male nerve. Y-chromosome-positive cells were detected not only inside the transplanted nerve, but also inside the female host nerve, the perineurial tissue, the local perineurial blood vessels, draining lymph nodes and the spleen of the female host, suggesting hematogenous as well as lymphatic elimination of macrophages from the injured nerve. These data indicate that local apoptosis and systemic elimination via circulation to the local lymph nodes and the spleen are involved in the disappearance of macrophages from the injured peripheral nervous system.

Fig. 1.

Southern blot. Five micrograms of DNA extracted from female (a) and male (b) tissue were digested with EcoRI, electrophoresed in agarose gel (1%), blotted on a nylon membrane, and hybridized with the 145SC5 probe, which detected a strong signal of 1.5 kb in the male DNA. The 2.2 kb band detected by hybridization with a probe for the prion protein gene demonstrates similar amounts of DNA on each lane.

Fig. 2.

a,b, Numerous macrophages are present within the transplanted male sciatic nerve, as detected by immunocytochemistry for the Mac-3 (DPT 5) (a) or F4/80 (DPT 20) (b) antigens.c–h, Detection of apoptotic macrophages within the transplanted male donor nerve. c, Morphologically, cells with the typical hallmarks of apoptosis (arrows) such as chromatin condensation and margination and apoptotic bodies are present (H&E). d, Immunocytochemistry for Mac-3 shows an apoptotic macrophage (arrow) with a condensed and fragmented nucleus. e, Semithin section of sciatic nerve 10 d after transection. Myelin debris-containing macrophages with dark, condensed nuclei (arrows) typical for apoptosis are present. f, Detection of DNA fragmentation with thein situ tailing technique. Degenerating cells (arrows) are present within the transplanted nerve.g, Double staining for IST and F4/80 identifies a macrophage with DNA fragmentation (arrow).h, Immunocytochemistry for CM-1. CM-1-positive (arrow) and CM-1-negative (arrowhead) cells with the typical morphology of apoptosis. Scale bars, 25 μm.

Fig. 3.

Quantitative data of invading (a, b) and apoptotic (c, d) macrophages in transplant (a, c) and recipient nerves (b, d). The open square represents cell numbers in the degenerating control nerve. *Statistically significant (p < 0.05) versus the total number of endoneurial macrophages at DPT 2 and 5 and DPT 20 as well as the relative number of apoptotic macrophages at the same time points. **Statistically significant (p < 0.0005) versus the number of endoneurial macrophages at all other time points.

Fig. 4.

Double immunofluorescence for CM-1 (green) and Mac-3 (red). The double-labeled macrophage is clearly detected.

Fig. 5.

Detection of Y chromosome-positive cells. a, H&E stain showing the highly cellular transplanted nerve at the left margin and several fascicles of the host nerve at the right margin. b, The same nerve as shown ina stained for the Y-chromosome probe 145SC5. There are numerous positive cells within the transplanted nerve and the surrounding perineurial tissue. c, High magnification of the grafted nerve showing numerous large, round Y chromosome-positive cells with macrophage morphology. d, Y chromosome-positive cells are also present in the surrounding perineurial tissue of the transplanted nerve. e, Few Y chromosome-positive cells are detected in the female host nerve.f–h, Y chromosome-positive cells (f) closely attached to an endothelial cell at the luminal surface (arrow) of a perineurial blood vessel, (g) in a para-aortal lymph node and (h) in the spleen. Scale bars:a, b, 100 μm; c–h, 25 μm.