Macrophage presence is essential for the regeneration of ascending afferent fibres following a conditioning sciatic nerve lesion in adult rats

Abstract

Background: Injury to the peripheral branch of dorsal root ganglia (DRG) neurons prior to injury to the central nervous system (CNS) DRG branch results in the regeneration of the central branch. The exact mechanism mediating this regenerative trigger is not fully understood. It has been proposed that following peripheral injury, the intraganglionic inflammatory response by macrophage cells plays an important role in the pre-conditioning of injured CNS neurons to regenerate. In this study, we investigated whether the presence of macrophage cells is crucial for this type of regeneration to occur. We used a clodronate liposome technique to selectively and temporarily deplete these cells during the conditioning phase of DRG neurons.

Results: Retrograde and anterograde tracing results indicated that in macrophage-depleted animals, the regenerative trigger characteristic of pre-conditioned DRG neurons was abolished as compared to injury matched-control animals. In addition, depletion of macrophage cells led to: (i) a reduction in macrophage infiltration into the CNS compartment even after cellular repopulation, (ii) astrocyte up-regulation at rostral regions and down-regulation in brain derived neurotrophic factor (BDNF) concentration in the serum.

Conclusion: Activation of macrophage cells in response to the peripheral nerve injury is essential for the enhanced regeneration of ascending sensory neurons.

Figures

Figure 1

Confirmation of Macrophage Cell Depletion Examined in the Spleens of Liposome-Treated Animals. A-B) 1 week after liposome administration no macrophage cells were present in the spleens of treated animals, as confirmed by the absence of CD68 (macrophage marker) immunoreactivity (***P < 0.0001; I). C-D) By 2 week after liposome treatment completion, macrophage cells (CD68+) had already began to repopulate in the spleen of treated animals (*P < 0.05; I). E-F) At the end of the experimental period (day 35) macrophage numbers in the spleen of treated animals had returned to normal levels, as compared to untreated animals (G-H). I) Quantification of immunoreactive CD68+ cells as shown in panels A-H, demonstrated a significant cellular reduction during liposome administration and during repopulation 2 weeks after treatment completion. No differences in macrophage cells were found at the end of the study. Columns represent an averaged mean (5 sections per animal, n = 5) and error bars indicate error of mean (+/- S.E.). Scale bars A, C, E, G 500 μm, enlarged views B, D, F, H 200 μm.

Figure 2

No Regenerated FB+ DRG Neurons Found in Liposome-Treated Animals. A-B) In the ipsilateral DRG, only saline-treated control animals showed retrograde labelled FB+ neuronal cell bodies (white arrows), as compared to the contralateral DRG (C). D-E) No FB labelled neurons were found in any of the liposome-treated animals, in the ipsilateral (red arrows) or contralateral DRG (F). G) Quantification of FB+ cell bodies in ipsilateral (black bar) and contralateral (white bar) DRGs revealed significantly more labelled neurons in saline-treated animals, as compared to unlabelled DRG neurons in liposome-treated (***P < 0.0001). Note that the contralateral DRG was used from the uninjured side as a control. Columns represent an averaged mean (5 sections per animal, n = 10) and error bars indicate error of mean (+/- S.E.). FB = fast blue tracer, DRG = dorsal root ganglia, CL = contralateral, IL = ipsilateral. Scale bars A and D 500 μm, C and F 300 enlarged views 200 μm.

Figure 3

Anterograde BDA-Labelled Fibres in the Spinal Cord of Saline-Treated Animals. A) Montage of SCI epicentre (black asterisk) demonstrating extensive anterograde labelling of ascending fibres in both proximal and distal stumps of the injured spinal cord (black arrows). B-G) Higher magnification images confirming the presence of BDA-labelled fibres extending across both stumps (black arrows), consistent with previous retrograde labelled results described in Figure 2. H) Quantification of percentage of BDA-labelled fibres revealed the presence of regenerated ascending fibres found 1 mm rostral and at the injury epicentre (0 mm) in control animals only (***P < 0.0001). Similar number of labelled fibres was found 1 mm caudal in the spinal cord of control and liposome-treated animals. BDA = biotinylated dextran amine, DCC = dorsal column cut. Directional key: D = dorsal, V = ventral, C = caudal, R = rostral. Scale bars montage 800 μm (horizontal bar) and 1 mm (vertical bar), B -E 200 μm, F-G enlarged views 100 μm.

Figure 4

Anterograde BDA-Labelled Fibres in the Spinal Cord of Liposome-Treated Animals. A) Histology-processed section through the SCI epicentre (black asterisk) revealed extent of lesioning through the dorsal columns (dashed line; shown as insert in panel B). B) Montage of lesion site (black asterisk) demonstrating anterograde BDA-labelled fibres present only in the distal stump of the spinal cord (D), with no visible fibres found in the proximal stump (C). E-G) Closer observation distally revealed extensive neuronal collapse and retraction of BDA-labelled fibres evident by their bulb-like structure (black arrows). BDA = biotinylated dextran amine, DCC = dorsal column cut. Directional key: D = dorsal, V = ventral, C = caudal, R = rostral. Scale bars, A (horizontal bar) 500 μm and (vertical bar) 1 mm, montage B 800 μm, C-E 200 μm, F-G enlarged views 100 μm.

Figure 5

Macrophage and Astrocyte Quantification in the Injured Spinal Cord. A) Statistical analysis of macrophage numbers in the spinal cord of both treatment groups indicated a greater macrophage presence in control animals than in liposome-treated. Specifically, in control animals these numbers were significantly higher 1-2 mm both rostral and caudal from the SCI epicentre, as compared to liposome-treated animals (*P < 0.05, **P < 0.01, respectively), while no differences were found at the SCI epicentre. B) Astrocyte quantification 3 mm rostral from the lesion epicentre revealed a greater reduction in astrocyte activation in control animals (*P < 0.05). No differences were observed between groups caudally. Columns represent an averaged mean (n = 10) and error bars indicate error of mean (+/- S.E.).

Figure 6

Higher BDNF Concentration Serum Levels in Saline-Treated Controls. Analysis of serum trophic levels at the end of the experimental period revealed an increased BDNF serum concentration in saline-treated controls, as compared to liposome-treated animals (*P < 0.05). Columns represent an averaged mean (n = 10) and error bars indicate error of mean (+/- S.E.). BDNF = brain derived neurotrophic factor.

Figure 7

Proposed Mechanistic Model for the Regeneration of Ascending Fibres in Pre-Conditioned DRG Neurons. Regenerative competence of pre-conditioned DRG neurons is described as a multi-faceted cascade of events: (1-2) SNI results in DRG and macrophage activation, upregulation of BDNF, cAMP and regeneration-associated genes (RAGs); all contributing to the beneficial phenotypic 'priming' of macrophage cells. (3) Facilitated entry of primed macrophage cells into the CNS compartment prior to CNS lesion. (4) SCI resulting in axonal injury, axon-myelin breakdown associated with the process of Wallerian degeneration. (5-6) Efficient removal of axonal and myelin debris by BDNF expressing macrophages beneficially activated with enhanced phagocytic capabilities, (7) consequently leading to axonal regeneration of the injured CNS-DRG branch. BNB = blood nerve barrier, RAGs = regeneration-associated genes, BBB = brain-blood barrier.