Inflammaging impairs peripheral nerve maintenance and regeneration

Abstract The regenerative capacity of peripheral nerves declines during aging, contributing to the development of neuropathies, limiting organism function. Changes in Schwann cells prompt failures in instructing maintenance and regeneration of aging nerves; molecular mechanisms of which have yet to be delineated. Here, we identified an altered inflammatory environment leading to a defective Schwann cell response, as an underlying mechanism of impaired nerve regeneration during aging. Chronic inflammation was detected in intact uninjured old nerves, characterized by increased macrophage infiltration and raised levels of monocyte chemoattractant protein 1 (MCP1) and CC chemokine ligand 11 (CCL11). Schwann cells in the old nerves appeared partially dedifferentiated, accompanied by an activated repair program independent of injury. Upon sciatic nerve injury, an initial delayed immune response was followed by a persistent hyperinflammatory state accompanied by a diminished repair process. As a contributing factor to nerve aging, we showed that CCL11 interfered with Schwann cell differentiation in vitro and in vivo. Our results indicate that increased infiltration of macrophages and inflammatory signals diminish regenerative capacity of aging nerves by altering Schwann cell behavior. The study identifies CCL11 as a promising target for anti‐inflammatory therapies aiming to improve nerve regeneration in old age.


Supporting Information Inventory
Quantifications of axon density (A), average axon diameter (B), myelin thickness (C) and g-ratio (D) of sciatic nerves 4 weeks after crush injury (lesion side) and intact contralateral nerves (control side) from mature adult (3-6 months) and old mice (18-20 months). n = 4 mature adult mice and n = 5 old mice, between 156 and 448 axons plus myelin sheath were measured per sciatic nerve. *** p < 0.001 by Wilcoxon rank sum test (U-test).

Figure S2
(A) Dot blots of cytokine detection array for comparison of mature adult and old mice sciatic nerve lysates without crush and 3 days or 8 weeks after crush. Assay overlay is shown as provided by the manufacturer, hyperlink to assay layout is indicated as well. (B) Toe-spread test for motor reinnervation of 'old' mice treated with ASA (10 mg/kg body weight) or vehicle (PBS) following unilateral sciatic nerve crush. n = 6 mice per cohort, mean ± SEM. * p < 0.05 by two-way ANOVA with Holm-Sidăk multiple comparisons test.
(C) Cytokine detection array dot blots for comparison of sciatic nerve lysates from uninjured or injured old mice 4 weeks after crush injury with or without ASA treatment. (D) Quantification of cell density, overall macrophage number, M1 and M2 macrophage populations in the crush area of sciatic nerves of 'old' mice 4 weeks after crush treated with ASA or vehicle; mean ± SD. n = 3 biological replicates. * p < 0.05, ** p < 0.01 in unpaired, two-tailed ttest.

Supporting experimental procedures Experimental animals
All animal procedures were approved by the local authorities (Thüringer Landesamt für Verbraucherschutz, Germany) and conformed to international guidelines on ethical use of animals. Animals had free access to food and water and were housed under constant temperature and humidity conditions on a 12/12-h light/dark cycle. Old mice (20 months) as well as most of the young and mature adult mice (3-6 months) were purchased from Janvier Lab (Le Genest-Saint-Isle, France), remaining mice were bred in-house. All animals were on a C57BL/6J background.

Sciatic nerve crush injury
For unilateral injuries of sciatic nerves mice were anaesthetized with isoflurane in oxygen, fur was removed with an electric razor (Aesculap ISIS, B. Braun AG, Melsungen Germany) and skin incised minimally. The biceps femoris muscle was separated to reveal the sciatic nerve. Using a smooth hemostatic forceps (Fine Science Tools GmbH, Heidelberg, Germany) GmbH, Heidelberg, Germany) the nerve was crushed mid-thigh. Muscle tissue was sutured using non-absorbable surgical suture material (Catgut GmbH, Markneukirchen, Germany) and skin was closed by suturing or with an AutoClip System (FST).

Drug treatment
Application of ASA (Sigma-Aldrich, St

Single frame motion analysis (SFMA)
To evaluate locomotor function by SFMA mice were accustomed to beamwalking before walking voluntarily from end to end on a horizontal beam.
Rear views of walking trials were captured by a high definition video camera once prior and at indicated time-points after surgery. Video sequences were examined with VirtualDub 1.10.4 (GNU General Public License) and footbase angles measured in selected frames with ImageJ v1.47t to serve as marker for reinnervation.

Toe-spread test
To test motor reinnervation, mice were placed on a transparent elevated walking beam. Once accustomed, mice walked voluntarily, and spreading of toes was recorded by a high definition video camera. Selected frames were analyzed in VirtualDub 1.10.4 (GNU General Public License) and scored according to Fig. 1d.

Semmes-Weinstein monofilament test
To investigate the sensory threshold of perception, animals were placed on a grid, accustomed and examined with Baseline ® Tactile Semmes-Weinstein monofilaments (Fabrication Enterprises Inc., White Plains, NY, USA). Scoring was based on the threshold force of the most flexible filament still evoking a lifting of the paw: score 0 -no response; 1 -300 g; 2 -4 g; 3 -2 g; 4 -0.4 g; 5 -0.07 g.

Electrophysiology
Mice were anaesthetized using isoflurane/O 2 inhalation. Hind limb fur was removed; needle electrodes were used to stimulate the left intact and right lesioned sciatic nerve in-situ, at a proximal and distal stimulation site separated by 12 mm. The neuromuscular response from the gastrocnemius muscle was recorded using a sensing ring electrode. A reference electrode was placed further distal. Nerve conduction velocity (NCV) and compound motor action potential (CMAP) were quantified from averaged response curves.

Immunohistochemistry
Paraffin-embedded sections of sciatic nerves were deparaffinized, rehydrated, boiled in 10 mM sodium citrate buffer for epitope retrieval and permeabilized with 0.5% Triton X-100. Afterwards, sections were incubated for at least 2 h in blocking buffer (0.2% fish skin gelatin, 1% goat serum and 1% donkey serum in PBS), followed by overnight incubation at 4 °C with primary antibody in blocking buffer. After multiple washing steps sections were incubated with secondary antibody at room temperature for 2 h. The contrast and axons and myelin were encircled using the hand tool. Axon and myelin were circumscribed manually using the freehand selection tool. Based on the measured areas, thicknesses of axons, thicknesses of myelin sheaths and g-ratios were calculated. At least 150 axons were evaluated per section.
Statistics and graphs were made in R statistics.

Immunoblotting
Following SDS-PAGE, proteins were transferred to Roti ® -NC nitrocellulose membranes (Carl Roth GmbH, Karlsruhe, Germany), which were incubated in blocking buffer containing 5% dried milk (Saliter, Obergünzburg, Germany) and 10% Roti-Block (Carl Roth) at room temperature for at least 1 h, followed by incubation with primary antibodies in blocking buffer overnight at 4 °C.
Subsequently, membranes were washed three times with TBS-T buffer and incubated with secondary antibody in blocking buffer at room temperature for 1 h. The used primary and secondary antibodies are listed below.
Membranes were washed three times with TBS-T, followed by development and visualization with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Europe GmbH, Freiburg, Germany).
List of primary and secondary antibodies for Immunoblot

Statistical analysis
Statistical analysis was performed using Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). Dependent on the data (i) unpaired, two-tailed t-test, (ii) oneway ANOVA with Tukey post-hoc test, (iii) two-way ANOVA with Holm-Sidăk or Tukey's post-hoc test, (iv) linear mixed models with Tukey's post-hoc test or (v) Wilcoxon rank sum test (U-test) were used for analysis. A confidence interval of 95 % was applied in all cases and the p value calculated.