Talsma et al. Journal of Neuroinflammation(2024) 21:134
https://doi.org/10.1186/s12974-024-03132-5

Journal of Neuroinflammation

Open Access

RESEARCH

Neither injury induced macrophages within
the nerve, nor the environment createdby Wallerian degeneration is necessaryfor enhanced in vivo axon regeneration after
peripheral nerve injury
Aaron D. Talsma1, Jon P. Niemi1 and Richard E. Zigmond1*
Abstract
Background Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration
and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury,
macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia(DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident
macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites domacrophages enhance nerve regeneration, and is a particular population needed.
Methods Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using thesestrains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regenerationin vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition ofpharmacological macrophage depletion methods, tested the role of various macrophage populations in initiatingor sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, andmacrophages were quantified by immunofluorescent staining.
Results Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustainedfor 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in
dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained forthe pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent
in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs thancontrols due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited
Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramaticallyreduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a*Correspondence:
Richard E. Zigmond
rez@case.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Talsma et al. Journal of Neuroinflammation(2024) 21:134

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transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed,
yet CL-enhanced regeneration was not impaired.
Conclusions Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.
Keywords Arginase 1, Axotomy, CCR2, Clodronate, Conditioning lesion, Dorsal root, Macrophages, Neuroimmune,
Regeneration, Zymosan

Introduction
Peripheral nerves are well known for their ability toregenerate, often for weeks [1]. A peripheral nerve injury
also primes axotomized neurons to regenerate faster
after a subsequent injury in a phenomenon called theconditioning lesion (CL) response. Additionally, the CL
response permits growth through the restrictive CNS
environment, observed in the central branch of pseudounipolar sensory neurons conditioned by peripheralaxotomy [2–6]. Maximal regeneration enhancement
is achieved 8 days after the first nerve injury regardlessof the number of CLs given [7], implying that processes
induced by an injury and not the injury itself are responsible for the CL response. Injury induces a transcriptional
program in neurons, which is necessary for enhanced
regeneration [8–10]. A component of the CL response isneuron intrinsic as enhanced regeneration and regeneration-associated gene (RAG) expression can be induced
in sensory neuron monocultures [8]. However, this
response lasts less than 3 days, implying there are othercritical components in vivo [11]. Importantly, injuring
the central branch of sensory neurons does not induce
RAGs or enhance regeneration, indicating a peripheral
factor, such as inflammation, is necessary for the CL
response [4, 12–15].
A sciatic nerve injury creates 4 to 5 distinct compartments, depending on the type of injury, where inflammation and local signaling could influence axonal
regeneration. Those compartments include the dorsal
root ganglia (DRGs), the nerve segments proximal anddistal to the lesion, and the lesion site, which in a transection injury is split into proximal and distal nerve(DN) stumps. These macrophages are suggested to promote axon growth but an unresolved question is which
populations are required [16–18]. After a single injury,
macrophages have many roles in the repair and regeneration process. In the DN, macrophages assist with myelin
clearance [19, 20], growth factor secretion [21, 22],angiogenesis [21, 23, 24], and efferocytosis [17], which
may promote regeneration and the CL response [21, 25,26]. Retrograde signals in injured axons are necessary forenhancing regeneration [27, 28]. In particular, retrograde
transport of activated signal transducer and activator oftranscription 3 (STAT3) is necessary for accumulationof phospho-STAT3 in the nucleus and CL-enhanced
regeneration [29, 30]. Phosphorylation of STAT3 at twosites downstream of gp130 and neurotrophin signaling isnecessary for the maximal axonal outgrowth [31]. Macrophages can produce ligands for both receptor classes
suggesting a possible mechanism by which nerve macrophages enhance regeneration.
Both resident and injury-induced macrophages in thenerve and DRG were initially thought to be predominantly bone marrow derived (i.e., monocyte derived macrophages; MDMs) [26, 32]. Accordingly, loss of either
CCR2 or CCL2 was reported to dramatically impair DN
and DRG macrophage accumulation [18, 19, 33, 34] andCL-enhanced regeneration in vitro [18, 33], suggesting
MDMs and DRG macrophages are necessary for the CL
response. However, recent studies demonstrated that the
DRG macrophage response is dominated by proliferation of a true resident population [16, 17] independent of
CCR2 signaling [35]. The nerve response is dominatedby MDMs and thus is impaired in Ccr2 null animals [17,19, 36], although resident compensation results in only a
50% macrophage reduction [35]. Given these new results,
the impaired CL response reported in Ccr2 KOs may be
due to either the overall decrease in nerve macrophagesor an ineffective phenotype adopted by the residents dueto their distinct lineage. Indeed, the transition of injuryinduced nerve macrophages from a Ly6Chi to Ly6Clo phenotype may be necessary for CL enhanced regenerationin the dorsal columns [17].
In this study, we sought to examine the role of CCR2
signaling and injury-induced nerve macrophages in
CL-enhanced regeneration in vivo. We used Ccr2 KO
animals and Ccr2gfp knockin/knockout (KI/KO) animals [37], which express GFP under the control of theendogenous Ccr2 locus and can distinguish MDMs
from resident macrophages responding to nerve injury[35, 38], to examine both the role of MDMs in regeneration and the source and phenotype of nerve macrophages. Using a variety of CL assays, we determined thatmyelin removal, CCR2 signaling, DN MDMs, and the
DN environment were not necessary to initiate or sustain CL-enhanced regeneration in vivo. We find lesion
site macrophages are a distinct population that express
the pro-regenerative marker Arg1 independent of theirlineage and CCR2 signaling. However, when they are
depleted with the other injury-induced nerve macrophages, we observe no effect on CL-enhanced regeneration in vivo.

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Materials and methods
Mice

Eight- to 20 week-old age matched mice were used for all
studies. It has previously been shown that there is no sexual dimorphism in the CL response [35]; therefore, males
and females were used in approximately equal numbers
for all groups in all experiments. Mice were housed
under 12 h:12 h light: dark cycle with ad libitum access tofood (LabDiet; Prolab RHM 3000) and water. The strainsin this study were acquired from Jackson Laboratory (Bar
Harbor, ME, USA) and maintained in our animal facility.
C57BL/6J mice were used as WT animals (Jackson Laboratory; #000664) and as controls for Ccr2 KOs (JacksonLaboratory; #0004999). Ccr2gfp/gfp KI/KO animals (Jackson Laboratory; #027619) were bred with C57BL/6J tocreate heterozygous Ccr2gfp/+ controls or with themselvesto produce Ccr2gfp/gfp, which served as a second KO strain
for CCR2.
Surgeries

All surgical procedures were approved by the Case Western Reserve University Institutional Animal Care and
Use Committee. Surgeries were performed under isoflurane anesthesia. After all surgeries, animals were given
bupivacaine subcutaneously at the incision and carprofen
subcutaneously at the scruff. Wound clips were used toclose the wound and were removed 14 d after surgery, if
applicable.
Peripheral regeneration and the response to a CL. A
small skin incision was made in the mid-thigh on theright and left hind limb. On the right side, the sciatic
nerve was exposed and transected just before the trifurcation producing a CL, and an approximately 2 mmforceps-width of nerve was removed distally. With the
intrinsic retraction of the nerves, this created a criticalnerve gap (greater than 3 mm) which was not bridged
in our experiments [39]. On the left side, the trifurcation was exposed producing a sham control nerve. In
one experiment, rather than transecting the right sciatic nerve, it was crushed just before the trifurcation.
Wounds were closed with a wound clip, and the mice
were allowed to recover for the “conditioning period”
which was 7, 14 or 28 d as indicated. Then, the originalincisions were reopened, and the sciatic nerves were
exposed at the level of the greater trochanter of thefemur. The sciatic nerves were crushed bilaterally using
ultrafine hemostats (Fine Science Tools, Forest City, CA,
USA; 13006-12) for 45 s just distal to the greater trochanter producing the TL. The wounds were closed again withwound clips. The mice recovered for 2 or 5 d as indicatedand then were sacrificed by CO2 inhalation. The sciatic
nerves were removed, pinned in a 35 mm petri dish withdental wax on the bottom so the nerves were straight,
and fixed overnight in 4% paraformaldehyde in PBS.

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Growth of axons in the left sciatic nerve was taken as
control (unconditioned) growth.
CL and dorsal root regeneration. First, a unilateral sciatic nerve transection was performed as described above.
After a 7 d conditioning period, the TL was made on
the L4 dorsal roots. First, an expanded L4 laminectomy
was made. The L5 spinous process space is in line with
the iliac crest and was identified by that landmark [40].
An incision extending from ∼L6-L3 was made, and thespinal muscles were detached with sharp tipped 5 mm
micro scissors (Fine Science Tools (FST; 15003-08) fromthe L3, L4, and L5 vertebrae to completely expose the L4
DRG. Then, using fine-tipped rongeurs (FST; 16221-14),
the L5 spinous process, which projects slightly anteriorly,
was removed to expose the L4-L5 intervertebral space.
Next, we removed the L4 laminae, transverse processes,
and a portion of the L4 pedicles to expose the L4 nerveroots, the L5 superior articular processes to visualize the
L4 DRG, and a caudal portion of the L3 laminae to give
more access to the L4 dorsal root. Within this surgical
window, the L4 dorsal and ventral roots are the most lateral and can be seen entering the L4 DRG sitting at thecaudal border of the L4 vertebral body. Sterile Lactated
Ringer’s solution (VEDCO, St. Joseph, MO, USA; 50989883-17) was used to irrigate the surgical site as needed.
Using fine micro scissors (FST; 15000-08), the meninges
were carefully opened by making a midline sagittal incision followed by lateral incisions starting from the rostralend of the first incision. The meninges were folded backto expose the L4 dorsal roots. Each was crushed for 15 swith an angled suture tying forceps that we filed slightly
to ensure the faces contacted completely (FST; 1106307). The ventral root was usually crushed at the sametime since separating it from the dorsal root was likely tocause collateral tissue damage. The meningeal flaps werethen moved back into place, and the window was covered
with BIOBRANE (Smith + Nephew, Inc., Andover, MA,
USA). The remaining muscles were placed back near
their original position on top of the BIOBRANE, and thewound was closed. The mice were allowed 3 d to recover
before they were sacrificed, and the L4 DRGs with spinal
nerve and nerve roots were collected, pinned, and fixed
as with the sciatic nerves.
Zymosan conditioning of the sciatic nerve

Zymosan is a yeast cell wall extract, which can stimulate
inflammation in the nervous system [41–43]. We usedzymosan to induce inflammation in the sciatic nerve in
an attempt to condition the nerve. Zymosan A (Millipore Sigma. St. Louis, MO, USA; Z4250) was suspended
in sterile PBS at a concentration of 5 µg/µL for injection.
As zymosan does not dissolve, the mixture was agitated
before each injection to resuspend it. Animals were anesthetized, and the sciatic nerve was exposed bilaterally,

Talsma et al. Journal of Neuroinflammation(2024) 21:134

as described above. On the right side, a 10 µL Nanofil syringe (World Precision Instruments, Sarasota, FL,USA) with a 35 G beveled needle (WPI; NF35BV-2) was
inserted near the trifurcation and moved along the axisof the nerve until the needle tip was at least ∼5 mm into
the nerve. The nerves were slowly injected with 0.2 µLof either PBS or 5 µg/µL zymosan (1 µg total) while theneedle was pulled out to spread the injection along thelength of the nerve. Animals recovered for 7 d to allow
for inflammation and conditioning to occur. Then bilateral crush TLs were made, and 2 d later the animals weresacrificed. The nerves were treated as described for the
CL paradigm.
Conditioning lesion with CL site treatments

Treatment of CL macrophages with an M1 stimulation
mixture, an arginase inhibitor, or clodronate liposomes
was given by nerve injection. First, a unilateral sciatic
nerve transection was performed as described above
with a 7 d conditioning period. Then, on the day of thetransection and every day after until the animals weresacrificed, 1 µL of treatment was injected into the cut endof the nerve using a 10 µL Nanofil syringe (WPI) and a
35 G beveled needle (WPI). To ensure the treatment andany damage caused by the injection was restricted to the
CL area, the needle was inserted just past the end of thebevel, which is ∼250 μm. If the sciatic nerve had segregated into two fascicles before the trifurcation, each fascicle was injected with a portion of the 1 µL. After the
7 d conditioning period, proximal TLs were made, and 2
d were allowed for regeneration before the animals weresacrificed and nerves collected as described above.
M1 stimulation injections: Lipopolysaccharide (LPS)
stimulates the classic M1 macrophage phenotype in vitro[44] and stimulates an inflammatory environment in the
CNS [45, 46]. M1 and M2 polarization signals activate
antagonistic transcriptional programs, and many M2
signals function through signal transducer and activator of transcription 6 (STAT6) activation [47]. Thus, weused LPS (Sigma-Aldrich; O55:B5 E. coli, L5418-2ML)
and the STAT6 inhibitor AS1517499 (Millipore Sigma;
SML1906) to promote M1 polarization in vivo. In vitro,
M1 and M2 phenotypes fade in the absence of polarizing
signals [48] so we treated the CL sites every day. According to the manufacturer, the IC50 of AS1517499 is 21 nM
in vitro. We used a 50X concentration (10 µM) to maintain this dose locally. LPS was delivered at a concentration of 0.5 µg/µL or 0.5 µg per day. To prepare the M1
stimulation cocktail, AS1517499 was first prepared asa 10 mM stock solution in DMSO. An aliquot was serially diluted 1:10 and then 1:50 in sterile PBS to make a
20 µM solution. The LPS was received in a 1 µg/µL aqueous solution, which was mixed 1:1 with the 20 µM STAT6
inhibitor solution to make the 10 µM STAT6 inhibitor,

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0.5 µg/µL LPS cocktail for injection. Vehicle solution was
50 µL RNase free water, 0.1 µL DMSO and 49.9 µL sterile
PBS. A total of 9 injections were given during a standard
in vivo CL paradigm as described above.
Arginase inhibitor injections: The Arg1 inhibitor, alphaamino acid N(omega)-hydroxy-nor-l-arginine (norNOHA; Cayman Chemical, Ann Arbor, MI, USA;10,006,861), was dissolved in DMSO to create a 100 mM
stock solution (25 mg in 843 µL DMSO). The stock solution was diluted 1:10 in PBS to create a working solutionof 10 mM. PBS and DMSO were mixed 1:10 to create thesolution used for the vehicle controls. A total of 9 injections were given during a standard in vivo CL paradigm
as described above.
Clodronate injections: Clodronate acts intracellularly to
induce apoptosis, and encapsulating it in liposomes targets its uptake and toxicity to macrophages [49]. 1 µL of
clodronate liposomes (FormuMax, Sunnyvale, CA, USA;
Clophosome-A, F70101C-A) or control liposomes (FormuMax; F70101-A) were injected into the nerve at the
CL. For WT animals, the CL was injected daily for a totalof nine injections during a standard in vivo CL paradigm
as described above. For Ccr2gfpKOs, the CL was injected
the day of the transection, the day after, and then every
other day for a total of 5 injections.
Luxol fast blue (LFB) for myelin visualization

LFB staining was performed on 20 μm sections of the
distal sciatic nerve. Slides were rehydrated in water for
5 min, moved to 35% ethanol for 5 min, and then to 70%
ethanol for 5 min before being placed in sealed slide containers with filtered 0.1% LFB solution (Electron Microscopy Sciences; 26681-01). The slides were incubated at
60–65 °C overnight, rinsed briefly by dipping first in 95%
ethanol and then in distilled water, destained by incubating in 0.05% lithium carbonate (w/v) in ddH20 for 30 s,
and then immediately quenched by rinsing and incubating in 70% ethanol for 5 min. Slides were dehydrated byincubating in 95% ethanol and then 100% ethanol for
5 min each, soaked in xylenes for 5 min, and then coverslipped with VectaMount Mounting Medium (Vector
Laboratories, Newark, CA, USA; H-5000). Slides wereimaged at 20x with transmitted light on a Zeiss AxioImager M2 microscope with the investigator blinded to theexperimental groups.
Immunofluorescence and imaging

Pairs of sham and manipulated sciatic nerves from eachanimal were embedded in Tissue-Tek O.C.T. compound(Electron Microscopy Sciences), sectioned at 10, 20 and
40 μm using a cryostat (Leica Biosystems, Wetzlar, Germany), and direct mounted onto Superfrost Plus slides(Thermo Fisher; 12-550-15). Tissue was stained on theslides in humidified chambers, using a PAP pen ring

Talsma et al. Journal of Neuroinflammation(2024) 21:134

to contain the staining solutions. All tissue was rehydrated for 20 min in 0.25% PBS-Triton X100 (PBS-TX).
Blocking buffer was 10% normal donkey serum in either
PBS-TX or 0.25% PBS-Tween20 (PBS-T). Sections wereincubated overnight at 4 °C with primary antibodies andsubsequently incubated at room temperature in secondary antibodies for either 1 h (for 10 μm sections) or 2 h(for 20 and 40 μm sections). Tissues were washed with
PBS-TX or PBS-T 4 times after the primary and secondary antibody incubation steps for 10 min each for 10
and 20 μm sections and 15 min for 40 μm sections. The
last two washes after the secondary antibody step weredone with PBS. Primary antibodies combinations diluted
in PBS-TX blocking buffer were as follows: rabbit antiSCG10 (Novus Biologicals, Centennial, CO, USA; NBP149461) at 1:4000; rabbit anti-SCG10 at 1:4000 and rat
anti-CD68 (Bio-Rad Laboratories, Hercules, CA, USA;clone FA-11, MCA1957) at 1:400; rat anti-CD68 at 1:400
and chicken anti-Arg1 [50; Aves Labs; #1146] at 1:5000;
sheep anti-GFP (Bio-Rad; 4745 − 1051) at 1:500, rat antiCD68 at 1:400 and chicken anti-Arg1 at 1:5000; sheep
anti-GFP at 1:500 and rat anti-CD68 at 1:400. Rabbit
anti-GFP (ThermoFisher; A-11,122) at 1:800 and rat antiF4/80 at 1:1000; rat anti-CD68 at 1:400, Rabbit anti-iNOS(Sigma; N7782) at 1:500, and chicken anti-Arg1 at 1:5000
were diluted in PBS-T blocking buffer. When staining for
F4/80, tissue was incubated in preheated 10 mM sodium
citrate buffer, pH 6.0, for 20 min at 95 °C after rehydration for antigen retrieval. The following secondary antibodies were diluted 1:400 in the same blocking buffer as
the primary antibodies: AF488 donkey anti-rabbit (Jackson ImmunoResearch; 711-545-152), AF488 donkey Fab2
anti-rat (Jackson ImmunoResearch; 712-546-150), AF488
donkey Fab2 anti-chicken (Jackson ImmunoResearch;703-546-155), AF488 donkey Fab2 anti-sheep (Jackson
ImmunoResearch; 713-546-147), AF594 donkey anti-rat(Jackson ImmunoResearch; 712-585-153), AF594 donkey
anti-chicken (Jackson ImmunoResearch; 703-585-155),
AF647 donkey Fab2 anti-rabbit (Jackson ImmunoResearch; 711-606-152), AF647 donkey Fab2 anti-rat (Jackson ImmunoResearch; 712-606-150), AF647 donkey Fab2
anti-chicken (Jackson ImmunoResearch; 703-606-155).
4′,6-Diamidino-2-phenylindole (DAPI) 1:1000 in PBS(ThermoFisher; D1306) was used to label cell nuclei.
Vectamount PLUS (Vector Laboratories; H-1900) wasused to mount 40 μm sections and Fluoro-Gel (Electron
Microscopy Sciences; 17985-10) was used to mount 10
and 20 μm sections.
A Zeiss AxioImager M2 wide field microscope wasused to take 3-color images (DAPI, AF488 and AF647)
of 10 μm sections. Images to quantify nerve regenerationand macrophages in 40 μm sections, and 4-color images(DAPI, AF594, AF488 and AF647) were taken using a
Zeiss LSM 800. All slides stained in the same batch (i.e.,

Page 5 of 25

with the same blocking buffer and antibody solutions) foran experiment were imaged with constant settings (e.g.,laser powers, scan speed, resolution, PMT voltages, andexposure times). All confocal stacks were run througha 3 by 3 median filter to remove noise before they were
maximally projected for quantification. The investigator
was blinded to the groups before quantification. Controls
without primary antibody were used to guide selectionof minimum values for positive staining when doing cellcounts or measuring percent area stained. Images were
quantified using ImageJ software (1.53).
In vivo CL regeneration analyses

In vivo regeneration analysis was performed using FIJI
and excel macros as previously described [35]. Briefly,
the center of the crush site was marked, and images
were blinded using the excel macro. In the FIJI macro, a
500 μm wide region of interest (ROI) was placed on the
crush site, a threshold for positive staining was set, andthe upper and lower bounds of the nerve were traced for
all nerves. The excel macro then calculated the fractionof regenerating axons at 100 μm intervals from the crush
site, and the average axon length in each nerve based on
the number of pixels at each distance and at the crushsite. The dorsal root crush sites violated assumption 2of the analysis from Talsma et al. [35], and thus the FIJI
macro was modified. A 300 μm wide ROI was placed on
the intact proximal portion of the crush and was used
to represent the maximum number of axons. The distal
crush was often notably disrupted, accounting in partfor reduced regeneration, and was defined by a second
ROI and excluded from the analysis. The threshold wasset, and the roots were traced as for the sciatic nerves.
The same formulas were used to calculate the fraction ofregenerating axons and average axon length.
Statistics and experimental design

Statistical tests were performed in Prism 10 (GraphPad,
San Diego, CA). All bar graphs show the mean +/- SEM.
Means were compared using a Two-Way ANOVA with
Sidak’s multiple comparisons post hoc tests unless otherwise noted, α = 0.05. Using this test, only comparisons
within genotypes or treatments and within injury conditions were made. Other comparisons are not meaningful
and were not performed (e.g. a sham WT group was notcompared to a conditioned KO group). For each experiment, approximately equal numbers of male and female
mice were used. At least 3 animals of each sex were slated
for an experimental group when planning, depending on the availability of the genotype, and 4 of each sex
was considered ideal in case a sample was lost. If a cageslated for an experiment contained extra mice those mice
were added to their corresponding experimental group.
Each animal was considered to be an experimental unit.

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Therefore, the minimum n for each experiment is 4 to 10,
and the n for each sex is 2 to 5.

Results
Ccr2 KO animals have normal enhancement of peripheral
regeneration after a peripheral CL

To measure CL-enhanced regeneration in vivo, we developed a peripheral CL assay, which generally is a unilateral CL of the sciatic nerve at the trifurcation (usually atransection) followed by a conditioning period (usually 7d), and then bilateral proximal crush lesions to test theregenerative ability of the nerves (i.e., test lesion, TL)
followed by a regeneration period (usually 2 d). Using
variants of this assay, we examined the effect of reducing injury-induced macrophage recruitment using both
the Ccr2 KO mouse [18, 19] and the Ccr2gfp/gfp mouse(referred to henceforth as Ccr2gfp KO). CCR2-dependent macrophage recruitment was hypothesized to berequired for CL-enhanced regeneration based on the
impairment in neurite outgrowth observed in cultured
ganglia from Ccr2 KO mice [18]. Since Ccr2 null animals are primarily deficient in both total macrophages(50% reduction) and MDMs specifically (80–90% reduction) in the DN [35], it suggests these macrophages arenecessary for CL-enhanced regeneration. This was firsttested by performing crush CLs in WT and Ccr2 KOs(Fig. 1A). Since crushed axons grow beyond the injury
within 2 days [35, 51], this paradigm allows the crushconditioned axons to interact with the DN environment during the 7 d conditioning period. The Ccr2 KOs
show normal regeneration enhancement indicating thatthe DN MDMs are not necessary for the CL response(Fig. 1B-G). To further test the necessity of DN macrophages, we performed a transection CL assay in which an
approximately 2 mm nerve segment distal to the transection is removed to prevent reattachment of the proximaland distal stumps [39] and thus any interaction of the
injured axons with the DN environment (Fig. 1H). This
paradigm was used to test regeneration enhancement
in both Ccr2 KOs compared to WT controls (Fig. 1I-N)
and in Ccr2gfp KO mice compared to Ccr2gfp/+ heterozygous controls (referred to henceforth as Ccr2gfp het;
Fig. S1 A-E). Surprisingly, CL-enhanced regeneration in
Ccr2 KOs and Ccr2gfp KOs was significantly increased
compared to baseline regeneration in sham conditioned
nerves and indistinguishable from CL-enhanced regeneration in their respective controls (Fig. 1I-N and Fig S1).
Importantly, there was no difference in CL-enhanced
regeneration between the crush and transection CL
groups in WT and Ccr2 KOs (Fig. 1O) showing that lossof the DN environment in the transection CL paradigm
does not impair CL-enhanced regeneration. Together,
this suggests that neither CCR2-recruited MDMs nor
the DN environment, including resident and recruited

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macrophages, are necessary for enhancing regeneration
over a 7 d conditioning period.
The CL response in DRG neuronal cultures fades within
3 d of plating [11], indicating that a key function of the invivo milieu, and reportedly the presence of activated macrophages [52], is to sustain the CL response. Thus, perhaps CCR2 signaling is necessary to recruit and replenish
macrophages to sustain the inflammatory response andmaintain CL-enhanced regeneration during the weeks
required for functional recovery. To test this, TLs were
performed 14 or 28 d after a transection CL in WT and
Ccr2 KOs (Fig. S2 A). Interestingly, CL-enhanced regeneration was maintained for 28 d, and the enhancement
was as robust after 14 or 28 d as after the 7 d CL in both
genotypes (Fig. S2 B-H). This indicates that CCR2 signaling, CCR2-dependent MDMs, and the DN environment
are not necessary for maintaining the CL response.
The most well studied role of macrophages in the
injured sciatic nerve is their clearance of myelin debris
during Wallerian degeneration (WD) [53, 54]. Incomplete
myelin clearance is thought to be inhibitory to regenerating axons [21, 55, 56], and significant myelin clearance distal to an injury site begins at 3 d after an injury[19, 57]. Thus, it was unlikely that myelin was cleared
during the 2 d regeneration period in the region immediately distal to the TLs, however faster myelin clearance from conditioned nerves could promote enhanced
regeneration. Luxol fast blue (LFB) staining was used tolabel myelin in cut-conditioned WT and Ccr2 KO nerves
given a 7, 14, or 28 d conditioning period and the contralateral sham nerves that only received a crush TL. The
region immediately distal to the TL was quantified inboth sham and conditioned nerves (Fig. S3 N). The presence of myelin, as measured by the percent area stained
by LFB, was unchanged by genotype or the presence ofa CL in the 7 and 14 d conditioning groups (Fig. S3 A-H,
M). In the 28 d conditioning group, there was a small but
significant decrease in the amount of myelin staining in
WT and Ccr2 KO CL groups compared to the contralateral nerves (Fig. S3 I-M). However, approximately 80%of the nerve area was still stained with LFB in all groups,
consistent with previously reported LFB values in naïve
nerves [19, 21], indicating a lack of degeneration. The
region quantified in Fig. S3 (Fig. S3 N) corresponds to
the regenerating segment quantified in Fig. 1I-N and S2,
demonstrating that cut-conditioned axons readily regenerate into an undegenerated nerve.
Recruited macrophages and CCR2 signaling are notrequired for enhancing regeneration in centrally
projecting axons

At baseline, the peripheral branch of sensory neurons has
a greater growth capacity than the central branch, butboth can be conditioned after a peripheral nerve injury

Talsma et al. Journal of Neuroinflammation(2024) 21:134

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Fig. 1 Sciatic nerves of Ccr2 KO animals show normal regeneration after a single crush injury and CL-enhanced regeneration. WT and Ccr2 KO animals
underwent an in vivo CL assay: a unilateral distal sciatic nerve injury, either a crush or transection, followed by a 7 d conditioning period, then bilateralproximal crush test lesions followed by 2 d for regeneration. A, H. Diagram showing the time course and relative positions of the CL and TL performedon sciatic nerves for the in vivo Crush CL (A) or Cut CL (H) assays, and the dotted box indicates the tissue area examined for the assays. B-E and I-L.
Representative images of regenerating nerves from WT and Ccr2 KO mice immunostained for SCG10, which labels regenerating sensory axons, in 40 μm
sections. F, M. Axon regeneration quantified at 100 μm intervals as the fraction of regenerating axons relative to the crush site for WT and Ccr2 KO mice
after a Crush CL (F) and a Cut CL (H). G, N, O. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating
axons for WT and Ccr2 KO mice after a Crush CL (G) and a Cut CL (N). O. Comparison of Crush and Cut conditioned regeneration distance. Unconditioned
regeneration (Sh; B, D, I, K), represents the baseline growth rate. Conditioned regeneration (CL; C, E, J, L) was the same between genotypes and type of CL,
and significantly increased compared to contralateral unconditioned nerves. The dotted line indicates the center of the crush site which was considered
to be 500 μm wide, and the solid line is 3000 μm from the crush. Scale bar = 500 μm. # indicates a significant (p < 0.05) difference between the Sh (unconditioned) and CL (conditioned) regeneration within a genotype. N = 24 WT Crush, 22 WT Cut, 15 Ccr2 KO Crush, 17 Ccr2 KO Cut[5, 58, 59]. Some early studies on the CL response suggested that the peripheral and central CL responses differ in that a peripheral nerve transection, but not a crush,enhances dorsal root and dorsal column regeneration[5, 60], whereas either lesion enhances peripheral nerve
regeneration [e.g., 3, 61]. In addition, one of the first
papers implicating macrophages in regeneration, foundthat injecting macrophages directly into the DRG only
enhanced dorsal root regeneration [62]. Thus, we hypothesized that MDMs may only be necessary for enhancing
regeneration of the central branch of sensory neurons.
This was tested in WT and Ccr2 KO mice using a transection CL paradigm with bilateral L4 dorsal root crush
TLs and a 3 d regeneration period (Fig. 2A). There was

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Fig. 2 (See legend on next page.)

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Talsma et al. Journal of Neuroinflammation(2024) 21:134

Page 9 of 25(See figure on previous page.)
Fig. 2 The central process of sensory neurons shows enhanced regeneration after a peripheral CL in Ccr2 KO animals. A. Diagram showing the sciatic CL,
dorsal root TLs and the time course used for the in vivo dorsal root regeneration assay performed on WT and Ccr2 KO animals. B-E. Representative imagesof regenerating dorsal roots immunostained for SCG10 in 40 μm sections. Unconditioned regeneration (B, D) shows that the poor intrinsic regenerative
capacity of dorsal root axons is the same in both genotypes. Conditioned regeneration (CL; C, F) was also the same between genotypes and significantly
increased compared to contralateral unconditioned roots. The dotted line indicates the center of the crush site which was considered to be 500 μm wide,
and the solid line is 3000 μm from the crush. Scale bar = 500 μm. F. Axon regeneration quantified at 100 μm intervals as the fraction of regenerating axons
relative to the axons in the 300 μm proximal to the crush site. The fraction of axons at each distance from the crush was calculated from ratios of SCG10
immunofluorescence. G. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating axons. # indicates asignificant (p < 0.05) difference between the Sh (unconditioned) and CL (conditioned) regeneration within a genotype. N = 7–8 per group

dramatic regeneration enhancement in conditioned dorsal root axons in Ccr2 KOs which was indistinguishable
from that in WT mice (Fig. 2B-G), demonstrating CCR2
signaling, CCR2-dependent MDMs, and the DN environment are dispensable for enhancing central regeneration.
Macrophages at lesion sites are a spatially restricted
population of arginase 1+ cells

Our transection CL paradigm combined with the use of
CCR2 null animals suggests that MDMs and the environment distal to the transection (i.e., the distal stumpand DN) do not play a role in CL-enhanced regeneration. Thus, we examined the macrophages that remainedin our transection CL paradigm, which are primarily
lesion site macrophages found in the TL and CL. Additionally, crush lesions (i.e., our TLs) cause a unique WDindependent inflammation and macrophage recruitment[63], and recent work has indicated that there are spatialdifferences in gene and protein expression between the
immune cells responding to a nerve injury site and those
responding to the DN to participate in WD [64]. It is notknown if other types of lesions also induce a distinct macrophage phenotype, the source of those macrophages,
or if they play a role in regeneration enhancement. We
sought to investigate these questions.
Macrophages can take on a spectrum of activation
states that include the classically activated pro-inflammatory phenotype (M1) or the alternatively activated antiinflammatory phenotype (M2) [65, 66]. M2 macrophages
have been posited to be beneficial for axonal regeneration and can be labeled by staining for arginase 1 (Arg1)[67, 68]. To examine lesion site macrophages without thelesion site complexity present after a transection injury,
an experiment was performed using crush CLs and TLs
because they create spatially restricted lesion sites thatare visually apparent in the tissue, and whose boundaries can be defined by the instrument used to create them.
Animals received a unilateral crush CL followed by a 7
d conditioning period and then bilateral crush TLs witheither a 2 d or a 5 d regeneration period (Fig. 3A). Macrophages were labeled by CD68 staining, which strongly
labels all macrophages but can be expressed at lower levels in other myeloid populations [69–71]. Nerves were
stained for DAPI, Arg1, and CD68 and were quantified
using 600 μm wide regions of interest because the lesions

were made using a hemostat with a nominal width of
600 μm. Arg1+ CD68+ macrophages were present specifically in the crushed areas under all injury conditions andas early as 2 d after a single TL (Fig. 3B-E). These lesion
site macrophages are distinct from the Arg1− CD68+
macrophages located distal to all injury sites which presumably participate in WD, evidenced by the consistently
low Arg1 expression in the nerve immediately distal to
the lesions (Fig. 3G, I) regardless of the presence of macrophages (Fig. 3F, H). Thus, the lesion-associated macrophages are distinct from those in other areas of the
injured nerve, which could indicate that they have a special function after injury, perhaps in axon regeneration.
Origins of Lesion Site macrophages in Ccr2gfp mice

The Ccr2gfp animals were next used to examine thesource and phenotype of macrophages found at the TL
and CL sites in control and Ccr2 null backgrounds. We
first analyzed the TLs of the nerves from the transection
CL paradigm in Figure S1 (Fig. 4A). In the Ccr2gfp hets,
macrophages were recruited to the TL sites in approximately equal numbers in unconditioned and conditioned
nerves; however, in the Ccr2gfp KOs, macrophages were
nearly completely absent from both TL sites (Fig. 4B-I).
Approximately 95% of macrophages at the TL site in theheterozygous controls were GFP+ indicating that they
are recruited MDMs (Fig. 4D). In the DN of Ccr2gfp KOs,
resident macrophages proliferate, compensating partiallyfor the lack of MDMs [35], but the scarcity of TL macrophages indicates residents are not able to compensate
in a lesion 2 d post injury. In the Ccr2gfp hets, 90% of TL
macrophages express Arg1 by 2 d post injury (Fig. 4E).
Macrophages are also greatly reduced in the TL sitesof all Ccr2 KO nerves (Fig. 4J-M). However, the macrophage deficiency in the KOs decreases markedly withtime, as demonstrated by comparing WT and Ccr2 KO
nerves in a crush CL paradigm with a 7 d conditioning and 5 d regeneration period. Macrophages are only
slightly reduced in the Ccr2 KOs at 5 d post-TL and areequivalent in the 12 d crush CL as measured by CD68%
area stained in 40 μm sections (Fig. 4M). Together, thesedata indicate CCR2 is required for rapid recruitment ofmacrophages to nerve lesion sites, but alternative sourcesof macrophages, either proliferating residents or nonCCR2 recruited MDMs, can eventually compensate inTalsma et al. Journal of Neuroinflammation(2024) 21:134

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Fig. 3 Arginase 1 expression is primarily associated with macrophages within crush sites. A. Diagram showing WT animals given a unilateral 7 d crush CL,
followed by crush TLs with a 2 d or 5 d regeneration period. The dotted box indicates the area examined in the assays. The “Sham” contralateral nerve only
received the TL. B-C. Representative sciatic nerves from animals given a crush CL and a 2 d TL, immunostained for the macrophage marker CD68 and theproregenerative macrophage marker arginase 1 (Arg1). D-E. Representative sciatic nerves from animals given a crush CL and a 5 d TL, immunostained forCD68 and Arg1. For B-E, the dotted line indicates the center of the crush TL which was nominally 600 μm wide, and the box is 600 μm wide and placedat the crush CL. F. Macrophages at and immediately distal to the TL site represented by CD68 percent area stained. CD68 was measured for all groups
by placing a 600 μm wide rectangular ROI centered over the TL site (Lesion Site) and then a second 600 μm wide ROI adjacent to the distal edge of thefirst ROI (Distal Nerve). ROIs were adjusted for the height of the nerve. G. M2 phenotype in macrophages at and immediately distal to the TL site represented by Arg1 percent area stained. Arg1 was measured for all groups as in F. H. Macrophages at and immediately distal to the CL site represented by
CD68 percent area stained. CD68 was measured for all groups as in F except the first ROI was placed over the CL site. I. M2 phenotype in macrophages atand immediately distal to the CL site represented by Arg1 percent area stained. Arg1 was measured for all groups as in H. Scale bar = 500 μm. * p < 0.05;** p < 0.01; *** p < 0.001. N = 4–5 per group

Talsma et al. Journal of Neuroinflammation(2024) 21:134

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Fig. 4 TL site macrophages are primarily CCR2+ MDMs and are rapidly recruited to control but not Ccr2 null TL sites. A. Diagram showing the lesion
paradigm with the dotted boxes indicating the location quantified in B-I. B-E. Quantification of the macrophages responding to the Ccr2gfp het and
KO TLs shown in F-I. Ten micron sections were immunostained with CD68 to label all macrophages, GFP to label infiltrating MDMs, and Arg1 to label
proregenerative macrophages and cells were counted in a 600 µm wide by 300 µm tall area centered on the TL. B. The total macrophages in the TL sites
is represented by the number of CD68+ cells per 0.01 mm2 and is significantly higher in both Ccr2gfp het TL sites compared to the KOs. C. Resident macrophages quantified as CD68+GFP− cells per 0.01 mm2. D. Recruited macrophages quantified by percentage of CD68+GFP+ cells per 0.01 mm2. Nearly all
TL site macrophages are derived from CCR2+ infiltrating monocytes and are absent in the 2 d TL of Ccr2gfp KOs. E. The percentage of CD68+ macrophages
per 0.01 mm2 that express Arg1 are significantly decreased in Ccr2gfp KOs. F-I. Representative images of macrophages in the TL sites of Ccr2gfp het and KO
animals from a CL paradigm. F’-I’. Insets of Arg1 staining in images in A-D. J-M. Ccr2 KOs also have greatly diminished numbers of macrophages in theirTLs compared to WT, quantified by the percent area stained of CD68 in the TL site of 40 μm sections. Macrophages are significantly reduced from Ccr2
KO TLs in both the 14 d CL paradigm (I) and the 28 d CL paradigm (J) as well as dorsal root TL 3 d after injury (K). L. Quantification of TL sites in the 7 d CLwith a 5 d TL injury paradigm shows that the macrophage deficiency is largely temporary. Scale bar = 600 μm. ** p < 0.01; *** p < 0.001. N = 7–10 per groupthe absence of CCR2. Notably, this compensation also
occurs more rapidly than in the DN [19, 35], suggesting that additional or more effective signals stimulating
proliferation or CCR2-independent recruitment are activated at the lesion site. Indeed, in nerve crush injuries
on mice with mutations preventing axon degeneration,
macrophage recruitment to the DN, but not the crush
site, is impaired [63, 64]. These data also demonstratethat TL macrophages are neither necessary nor sufficientfor CL-enhanced regeneration since TL macrophages arepresent in the WT sham CL nerves, which do not haveenhanced regeneration, and absent in the CCR2 nullconditioned nerves, which have enhanced regeneration(Fig. 4B-I J-K, and 4 L correspond to nerves in Fig. S1, S2,
and 2 respectively).
Having excluded a role for TL macrophages in enhancing regeneration, we turned to the CL macrophages
in our transection paradigm (Fig. 1H). Because thisTalsma et al. Journal of Neuroinflammation(2024) 21:134

paradigm prevents reattachment of the proximal anddistal stumps, axons injured by the CL reside in theproximal stump and nascent nerve bridge with macrophages throughout the 7 d conditioning period. Indeed,
CD68+ CL macrophages can be seen in close proximity to
SCG10 labeled regenerating axons in the proximal stumpof transected Ccr2gfp het and Ccr2gfp KO nerves 4 and 7 dafter injury (Fig. 5A-D). Thus, we examined Arg1+ CL site
macrophages 9 d post injury in the transection CL paradigm (Fig. 5E) by staining for Arg1 and CD68 to identify
lesion site macrophages, and for GFP to identify CCR2+derived MDMs (Fig. 5F-S). In Ccr2gfp hets, most CL site
macrophages are MDMs (i.e., are GFP+) and express
Arg1 (Fig. 5F-J, P-S); however, in Ccr2gfp KOs, Arg1+
macrophages are still abundant but there are very few
MDMs (Fig. 5K-O, P-T). While there is an overall reduction in CD68+ macrophages in the CL site of Ccr2gfp KOs
compared to hets (Fig. 5P), an increase in the number of
GFP−CD68+ macrophages (Fig. 5Q) partially compensate
for the loss of MDMs in the KO mice. The relative M2
macrophage number is also maintained (Fig. 5S) due toincreased Arg1 expression among residents (Fig. 5T),
suggesting they are adopting the appropriate phenotype.
These observations are consistent with a model suggested by the TL site data (Fig. 4) that MDMs are preferentially recruited to lesion sites, but residents eventually
migrate to and proliferate at a lesion site compensating
for impaired recruitment.
To further evaluate the time course of compensation in CCR2 nulls and ascertain the longevity of Arg1+
CL macrophages, the CL site in WT and Ccr2 KO mice
9, 16, and 30 d post-injury were stained for CD68 andArg1 (Fig. 6A-G). At every time point, the number of
CD68+ macrophages in Ccr2 KO CLs was 75–80% of WT(Fig. 6H), although the number is maximal at 9 days anddeclines at a similar rate in both genotypes. This suggests that an equilibrium was reached in both genotypes
rather than inefficient recruitment mechanisms causing
a delayed inflammatory response in Ccr2 KOs. Notably,
the Arg1+ percentage of macrophages was equivalent
between genotypes at all time points (Fig. 6I) despite
being derived from different sources, which implies thelesion environment and not the macrophage lineage
determines the phenotype. Macrophages in the Ccr2
KO CLs also occupy less relative area than in WT CLs(Fig. 6J). However, the difference in macrophage area
between genotypes diminishes over time (Fig. 6J) implying that the Ccr2 KO macrophages are slightly larger on
average which may be another compensatory mechanism. The CL site macrophages persist to some extent for
at least 30 d (Fig. 6F-H) along with CL-enhanced regeneration (Fig. 1 and S2) in both WT and Ccr2 KO animals. The macrophage response and Arg1 expression arelessening at the 30 d time point (Fig. 6F-K), raising thePage 12 of 25

possibility that the CL response may also fade at a latertime point, possibly due to resolution of the macrophage
response.
Activated macrophages in the sciatic nerve do not enhanceregeneration

Lesion site macrophages are a spatially unique population of immune cells responding to nerve injury, in thatthey are preferentially monocyte-derived, have a potentially pro-regenerative Arg1+ phenotype, and are present
in every nerve that demonstrated enhanced regeneration;
all characteristics of a population that could induce CLenhanced regeneration. Therefore, we hypothesized that
recruiting MDMs into an uninjured sciatic nerve could
increase axon regeneration after a subsequent crush
injury. To accomplish that we used zymosan, a yeast cellwall extract, which recruits and activates macrophages
within nervous tissues [72], and increases the speed offunctional recovery when injected into an injured sciatic
nerve [41]. Additionally, zymosan-activated macrophages
can enhance regeneration of sensory neurons in vitro andin vivo over long distances in the spinal cord [67, 72, 73].
To test if zymosan can induce a CL-like effect, again our
CL paradigm was modified. Instead of a transection CL,
the sciatic nerve was injected with either 1 µg zymosan(at 5 µg/µl) or vehicle, with a 7 d conditioning period
before performing crush TLs with a 2 d regeneration
period (Fig. 7A). The zymosan injection induced a robust
inflammatory response throughout the segment distal to
the TL (Fig. 7D-I) but did not enhance axonal regeneration (Fig. 7B, C, E, F). Interestingly, approximately 95%
of zymosan-recruited macrophages, 1 mm distal to thecrush site, express Arg1 (Fig. 7G-I). Since zymosan-activated macrophages can enhance regeneration in sensory
neurons [67, 72, 73], these results indicate that inducing macrophage infiltration into the uninjured sciatic
nerve is not sufficient to induce a CL-like regeneration
enhancement in uninjured axons.
CL-enhanced regeneration is resistant to alterations in CL
macrophage phenotype

Lesion site macrophages express Arg1, a marker of antiinflammatory M2 macrophages [74]. Arg1, along withinducible nitric oxide synthase (iNOS), are two of themost well documented macrophage polarization markers for anti-inflammatory/M2 and pro-inflammatory/
M1 macrophages, respectively [44]. These markers arewidely used because they are enzymes in competing
pathways that utilize arginine as a substrate to produce
either trophic polyamines via Arg1 or the inflammatory
radical nitric oxide via iNOS [75]. The Arg1 inhibitor,
nor-NOHA, can produce pro-inflammatory macrophage
polarization in vivo [76]. Since M2 macrophages havebeen characterized as pro-regenerative for neurons [67],Talsma et al. Journal of Neuroinflammation(2024) 21:134

Fig. 5 (See legend on next page.)

Page 13 of 25

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Page 14 of 25(See figure on previous page.)
Fig. 5 Cut axons reside in the CL with macrophages during the conditioning period, and Arg1+ CL site macrophages can be derived from either CCR2+
recruited macrophages or CCR2− resident macrophages. A-D. Representative images of the proximal CL 4 and 7 days post-injury (DPI) of Ccr2gfp hets
and KOs, showing axons residing near CL macrophages during the conditioning period. Ten micron sections were immunostained for SCG10 labelingcut and regenerating axons, CD68 labeling all macrophages, and GFP labeling recruited macrophages. Dotted box indicates the area displayed in A’-D’.
Scale bar = 250 μm. A’-D’. Enlarged 500 by 600 μm images from A-D. E. Diagram showing the lesion paradigm with the dotted boxes indicating the location examined in F-S. F-O. Representative images of Ccr2gfp het and KO proximal CLs after the in vivo regeneration paradigm. F, K. Ten micron sections
immunostained for GFP, labeling recruited CCR2+ macrophages shown in magenta, CD68, labeling all macrophages shown in red, and Arg1, labelingproregenerative macrophages shown in green. With these colors, recruited Arg1+CD68+GFP+ macrophages appear white and resident Arg1+CD68+GFP−
macrophages appear yellow. Arg1− resident (CD68+ GFP−) and recruited (CD68+ GFP+) appear red and magenta, respectively. Dotted boxes indicate
the magnified images in G-H and L-O. Scale bar = 500 μm. G-J. Enlarged portion of the Ccr2gfp het CL site in F illustrates that most macrophages are
Arg1+CCR2+ recruited macrophages although a few are resident derived. H-J Show single grayscale channels of G. L-O. Enlarged portion of the Ccr2gfp KO
CL site in K illustrates macrophages are mostly resident derived (CD68+CCR2− cells) but are still Arg1+. M-O Show single channel grayscale images from
L. P-S. Quantification of the macrophages in the CL sites shown in F-O by counting cells in an approximately 250 μm square centered on the inflammation at the cut nerve end in CLs or 3 mm distal to the TL in Sh. P. Total macrophages in the CL site, quantified as the number of CD68+ cells per 0.01 mm2,
is significantly higher in Ccr2gfp hets compared to KOs. Q. Resident macrophages, quantified as the number of GFP−CD68+ cells per 0.01 mm2, shows asignificant increase in residents in Ccr2gfp KO CLs compared to Ccr2gfp hets. R. Recruited macrophages quantified as the GFP+ percentage of CD68+ cells
per 0.01 mm2. Most CL macrophages are GFP+ in Ccr2gfp hets, indicating they are derived from CCR2+ infiltrating monocytes, and are reduced but notabsent in Ccr2gfp KOs. S. M2 macrophages, quantified as the Arg1+ percentage of CD68+ cells per 0.01 mm2, demonstrates similar proportions of M2 CL
macrophages in both genotypes. T. Proportion of M2 macrophages derived from residents, quantified as the GFP− percentage of Arg1+CD68+ cells per
0.01 mm2, reveals residents expand and adopt the appropriate phenotype when MDMs are deficient in Ccr2gfp KOs. ** p < 0.01. *** p < 0.001. # indicates asignificant (p < 0.05) difference between the Sh (unconditioned) and CL (conditioned) groups within a genotype. N = 6–7 per groupit was of interest to test if shifting the macrophages at the
CL site to an inflammatory M1 phenotype would resultin loss of regenerative support from macrophages and areduction in CL-enhanced regeneration.
One of two treatments were injected daily into the
CL site to alter the M2 phenotype (Fig. 8A). First norNOHA, an arginase inhibitor, was used. It did notalter the number of CD68+ macrophages at the CL site
(Fig. 8B-C) but did result in a small decrease in the percentage of Arg1+ macrophages (Fig. 8E, B-C cyan orwhite cells) and a substantial increase in the percentage
of iNOS-expressing macrophages (Fig. 8F, B-C magenta
or white cells). However, treated mice displayed a CL
response that was unaltered compared to vehicle treatedcontrols (Fig. 8G-K). The second treatment was an “M1stimulation cocktail” containing LPS, which is sufficient
to induce the classical M1 phenotype in culture [44,77], and an inhibitor of STAT6, which is a transcription
factor that helps produce the M2 phenotype and inhibits the LPS response [47]. The M1 stimulation cocktail
did not alter the number of CL macrophages or Arg1
expression but it did significantly increase CD68+ macrophage expression of iNOS (Fig. 8L-P, G-K magenta
or white cells) perhaps to a slightly greater extent than
did nor-NOHA. The preserved Arg1 expression withan increase in iNOS expression consequently manifests
as an increase in triple-positive (white) cells. Interestingly, there was a small decrease in CL-enhanced regeneration in the M1 stimulated nerves compared to vehicle(Fig. 8Q-U); however, there was also still significantly
enhanced regeneration compared to sham. These twotreatments, which were both intended to inhibit an M2
and promote an M1 phenotype, altered the CL macrophage phenotypes in slightly different ways and to slightly
different degrees but did not change Arg1+ macrophages

into iNOS+ macrophages, demonstrating that macrophage phenotypes in vivo are more complex than an
M1/M2 dichotomy. The modest inhibition of regeneration from the M1 stimulation suggests macrophages can
impair regeneration, but it is unclear whether this is due
to an impaired CL response or if the macrophages are
creating an environment hostile to axons.
Inducing apoptosis to deplete CL macrophages does not
disrupt CL-enhanced regeneration

Since the CL macrophage phenotype does not seem to benecessary for CL-enhanced regeneration, we attempted
to directly test their necessity by specifically ablatingthese macrophages with clodronate liposomes. Clodronate is a small molecule that acts intracellularly to induce
apoptosis. By encapsulating it in liposomes, its toxic
activity can be restricted to phagocytic cells, predominantly macrophages [49, 78], and clodronate liposomes
have been used to deplete infiltrating macrophages innerve injury models [79–81]. To restrict the activityof the clodronate further, clodronate or control liposomes were injected directly into the proximal CL site
in a transection CL paradigm to target CL macrophages.
The injections were given daily from the time of the CL
until the animals were sacrificed at the end of our transection CL paradigm. Surprisingly, the CL sites seemed
minimally affected and CD68+ cells were still abundantin the clodronate treated group. The cells appeared more
numerous, but smaller, and rounder, likely explaining theslight decrease in CD68 area (Fig. S4 F-H) and suggesting that they were monocytes or neutrophils infiltrating in response to clodronate induced apoptosis of CL
macrophages. CL-enhanced regeneration was also notinhibited in clodronate treated CLs (Fig. S4 A-E). However, since clodronate seemed to be causing localized CL

Talsma et al. Journal of Neuroinflammation(2024) 21:134

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Fig. 6 Arg1+ macrophages are still observed in the CL sites of both WT and Ccr2 KO animals at 30 d after the CL. A. Diagram showing the lesion paradigms
used with the dotted box indicating the location quantified in B-K. B-G’. Representative images of Arg1+ macrophages in WT and Ccr2 KO CL sites from
7 d CL (B-C’), 14 d CL (D-E’), and 28 d CL (F-G’) paradigms displayed as CD68 and Arg1 colabeling (B-G) and Arg1 alone (B’-G’) in 10 μm sections. H. CL
site macrophages were quantified by counting CD68 labeled cells within two 300 μm diameter circles centered on the inflammation at the nerve end
and expressed as CD68+ cells per 0.01 mm2. I. Arg1+ macrophages were quantified by counting cells with immunofluorescence staining that colocalizedwith CD68+ cells and expressed as the percentage of Arg1+ CD68+ cells. J. Macrophages quantified by measuring the percent area of the CL labeled by
CD68 which represents relative macrophage area. I. Total Arg1 expression quantified by the percent area of the CL labeled by Arg1. Scale bar = 500 μm.
* p < 0.05. ** p < 0.01. N = 4–8 per group

macrophage apoptosis, we were able to use it to create
another paradigm that nearly completely depleted CL
macrophages.
We have shown that lesion site macrophages are predominantly MDMs but that residents can compensate
relatively quickly and completely in the absence of CCR2dependent recruitment (Figs. 4, 5 and 6). Importantly,

MDM recruitment is also almost completely inhibitedin Ccr2gfp KOs [Figs. 5 and 35]. However, some CCR2independent recruitment mechanisms may remain [35].
We also previously found that resident macrophages area major chemokine source to recruit MDMs in the DN[35], and so they may also be the source of CCR2 independent recruitment signals. Thus, we hypothesizedTalsma et al. Journal of Neuroinflammation(2024) 21:134

Page 16 of 25

Fig. 7 Zymosan induced recruitment of macrophages into the sciatic nerve does not condition axons. A. Diagram of the zymosan conditioning paradigm in which WT sciatic nerves received a unilateral conditioning injection of zymosan (Zym) or vehicle (Veh) 7 d before the nerves were given bilateralcrush TLs and assayed after a 2 d regeneration period. B. Axon regeneration quantified at 100 μm intervals as the fraction of regenerating axons relative
to the crush site using SCG10 staining. C. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating
axons. D. Macrophages in the 3 mm immediately distal to the TL measured by the percent area immunostained for CD68. E-F. Representative images ofregenerating nerves immunostained for regenerating axons with SCG10 and macrophages with CD68 in 40 μm sections. The dotted line indicates thecenter of the crush site which was considered to be 500 μm wide, and the solid line is 3000 μm from the crush. Scale bar = 500 μm. Forty-micron sections
were used in B-E. G-H. Vehicle and zymosan injected nerves were stained for CD68, Arg1, and DAPI. I. Arg1 expressing macrophages were quantified by
the percentage of CD68+ cells that express Arg1 per 0.01 mm2. Scale bar = 100 μm. # indicates a significant (p < 0.05) difference between the Sh (unconditioned) and Injected (conditioned) regeneration within treatment groups (vehicle or zymosan). *** p < 0.001. N = 4–8 per group

Talsma et al. Journal of Neuroinflammation(2024) 21:134

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Fig. 8 Effect of driving CL-macrophages toward an M1 phenotype on CL-enhanced regeneration. A. Diagram showing the CL paradigm performed
on WT animals with daily CL injections to pharmacologically inhibit an M2 phenotype and promote an M1 phenotype at the CL. The arginase inhibitor,
nor-NOHA (left, B-K), or an M1 stimulation cocktail, containing LPS and a STAT6 inhibitor (right, L-U), was injected daily into the CL site to polarize the macrophages from M2 to M1. A-B, L-M. Representative images for vehicle and arginase inhibitor (A-B), or M1 stimulation cocktail (L-M) treated mice showing
CD68, Arg1, and iNOS, immunostaining. CD68 is shown in blue so that macrophages co-labeled with Arg1 will appear Cyan and those co-labeled withiNOS will appear magenta. Triple labeled cells will appear white. Note the increase in magenta cells and decrease in cyan cells in the nor-NOHA treated
CL (C), compared to the increase in white cells with few magenta cells in the M1 stim CL (M). Scale bar = 100 μm in A, B, L, M. D, N. Macrophage number, displayed as cells per 0.01 mm2, was not altered by the treatment. E, O. The percentage of CD68+ macrophages expressing Arg1 after the arginase
inhibitor (E) or M1 stimulation cocktail (O) treatment compared to vehicle controls. F, P. The percentage of CD68+ macrophages expressing iNOS after
arginase inhibitor (F) or M1 stimulation cocktail (P) treatment compared to vehicle. G, Q. Axon regeneration quantified at 100 μm intervals as the fractionof regenerating axons relative to the crush site for arginase inhibitor (G) or M1 stimulation (Q) treated nerves. H, R. Mean regeneration distance calculated
by integrating SCG10 immunofluorescent staining of regenerating axons for arginase inhibitor (H) or M1 stimulation (R) treated nerves. I-K, S-U. Representative images of regenerating nerves treated with arginase inhibitor (I-K) or M1 stimulation cocktail (S-U) immunostained for regenerating axons with
SCG10 in 40 μm sections. Unconditioned regeneration (I, S) was the same for both treatment groups. Conditioned regeneration (J-K, T-U) was also thesame between treatments and significantly increased compared to contralateral uninjected nerves. The dotted line indicates the center of the crush sitewhich was considered to be 500 μm wide, and the solid line is 3000 μm from the crush. Scale bar = 500 μm in I-K, S-U. * p < 0.05. *** p < 0.001. # p < 0.05
between injury groups within the same treatment group. N = 4–11 per group

Talsma et al. Journal of Neuroinflammation(2024) 21:134

that removing residents and CCR2 signaling in the DN
would completely abolish the macrophage response.
To test this, a single dose of control or clodronate liposomes was injected into the 2 mm of nerve distal to a sciatic nerve transection in Ccr2gfp Het and KO mice, andthe DNs were examined 7 d after injury (Fig. S5 A-D).
Clodronate liposome injected Ccr2gfp het mice showeda small but significant decrease in macrophages distal tothe injury site compared to vehicle controls (Fig. S5 E).
However, ∼80% of macrophages in the nerve were GFP+,
suggesting that macrophages were still being recruited to
the nerve (Fig. S5 G). Strikingly, in clodronate liposome
treated Ccr2gfp KO mice, both GFP+ and GFP− macrophages were still nearly completely absent 7 d after injury
and significantly reduced compared to all other groups(Fig. S5 F-G), indicating that ablating residents prevents
the DN macrophage response in Ccr2 null animals. Mostof the remaining macrophages in the clodronate treated
Ccr2gfp KO mice were GFP− (Fig. S5 F), possibly residents
migrating from beyond the injected area.
These data support a model in which macrophages
responding to a nerve injury are recruited by the fastest
available mechanism. Under normal circumstances thisis recruiting MDMs via CCR2 which can still maintainan inflammatory response even when macrophages arebeing constantly depleted (Fig. S4). When the rapid CCR2
dependent mechanism is lost, resident macrophages
and alternate recruitment mechanisms are able to compensate eventually. Additionally, lesion site macrophage
accumulation seems to have more robust compensatory
mechanisms than the DN (Figs. 4, 5 and 6, S5). Thus, todeplete CL macrophages, residents need to be removed
by injecting clodronate, and MDM recruitment needs
to be prevented by using Ccr2 null animals. To test this,Ccr2gfp KO mice underwent a transection CL paradigm
with injection of either clodronate or vehicle liposomes
into the proximal CL on days 0 (with the CL), 1, 3, 5, and7 (with the TLs) before sacrificing on day 9 (Fig. 9A). As
hypothesized, macrophages in the CL site were at least
80–90% depleted after clodronate treatment as measured
by GFP, F4/80, and CD68 staining (Fig. 9H-L; Fig. S6 A, C,
E). F4/80 staining is poorly colocalized with GFP staining(Fig. 9J) and CD68 staining in the adjacent section (Fig.
S6 C, arrowheads) raising the novel possibility that there
is a population of F4/80 expressing cells in the nerve thatare not from a myeloid lineage. Despite the substantial
macrophage depletion, there was minimal change in the
total cell number measured by DAPI staining (Fig. 9M).
Indeed, there was an increase in Ly6G staining at the CL
site, indicating an increase in neutrophils (Fig. S6 B, D, F)
which may be compensating for the decrease in macrophages and which colocalized with some of the remaining CD68 signal (Fig. S6 C, D arrowheads). Since these
are Ccr2gfp KO animals, TL macrophages are also nearly

Page 18 of 25

completely depleted (Fig. 4 and illustrated in Fig. 9C, dotted line). Surprisingly, however, CL-enhanced regeneration was not impaired in the clodronate injected nerves(Fig. 9B-G) demonstrating that lesion site macrophages
are not necessary for CL-enhanced regeneration. Further, this was again our transection CL paradigm which
prevents reattachment of the proximal and distal stumps.
Additionally, in this paradigm, the proximal CL was visualized and mobilized every other day for injection ensuring that the severed axons in the proximal stump and thedegenerating DN were always separated by several millimeters. Thus, there was no opportunity for the DN topromote regeneration through providing a supportive
substrate or through juxtacrine or paracrine signaling.

Discussion
Signals derived from the injured nerve and the macrophages found there [17, 21] have been proposed toenhance regeneration and the CL effect. This is the firststudy to investigate the various macrophage subtypes
found in the injured nerve and their effect on in vivo
regeneration. Using Ccr2 null models, we interrogated
the role of MDMs and found that the WD-independent
inflammation at a lesion site relies on CCR2 signaling butthat resident macrophages and CCR2-independent MDM
recruitment are rapidly able to compensate. Further, weshow the lesion site macrophages are a long-lasting population with a unique M2-like phenotype that is independent of their lineage. To test their effect on regeneration,
WD-dependent and -independent macrophages in thenerve were systematically depleted by combining Ccr2
null genetic models with surgical and pharmacologic
techniques. MDMs, and TL macrophages in particular,
were depleted with Ccr2 null animals, the DN environment with all WD-dependent inflammation was physically prevented from interacting with injured axons usingtransection CLs, and WD-independent macrophages at
the CL were depleted using clodronate injections into
the CL of Ccr2gfp KO animals. There was no differencein CL-enhanced regeneration between Ccr2 KO transection CL nerves and WT crush CL nerves showing that
the CL response does not require MDMs or the interaction of axons with the WD environment in the DN. Further, even when injured axons do not interact with any
populations of injury-induced nerve macrophages, both
unconditioned and CL-enhanced regeneration are unimpaired indicating these macrophages are not necessary
for the CL effect or axon regeneration.
Ccr2 KOs and removal of Wallerian degenerationdependent inflammation does not impair regeneration
enhancement

Previous publications utilizing the Wallerian degeneration slow (Wlds) mouse or the Sarm1 KO mouse, which

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Page 19 of 25

Fig. 9 Clodronate liposomes injected into the CL site of Ccr2gfp KO mice significantly reduces macrophages but does not prevent the peripheral CL
response. A. Diagram depicting a CL paradigm performed on Ccr2gfp KO animals in which CL macrophages were targeted for ablation by injecting clodronate liposomes into the proximal CL site on the day of the CL (day 0) the next day (day 1) and every other day thereafter. Control animals received vehicle
liposome injections instead. B-E. Representative images of regenerating nerves immunostained for regenerating axons with SCG10. Unconditioned
regeneration (B, D) was the same for both treatment groups. Conditioned regeneration (C, E) was also the same between treatments and significantly
increased compared to contralateral uninjected nerves. The dotted line indicates the center of the crush site which was considered to be 500 µm wide,
and the solid line is 3000 µm from the crush. Scale bar is 500 µm. F. Axon regeneration quantified at 100 µm intervals as the fraction of regenerating
axons relative to the crush site. G. Mean regeneration distance calculated by integrating SCG10 immunofluorescent staining of regenerating axons. H.
Macrophages quantified by percent CD68 positive area in a circle placed on the CL with a diameter equal to the largest width of the uninjured portionof the nerve. Forty-micron sections were used in B-H. I-J. Representative images of a PBS (vehicle) liposome-treated (I) and clodronate liposome-treated(J) Ccr2gfp KO CL site immunostained for macrophages with F4/80, proregenerative macrophages with Arg1, recruited CCR2+ macrophages with GFP,
and DAPI. F4/80 is a membrane marker, shown in green, while Arg1, shown in red, and GFP, shown in cyan, are both intracellular. These colors cause
proregenerative F4/80+, Arg1+ macrophages to appear yellow or red ringed by yellow, recruited proregenerative GFP+, Arg1+ monocyte/macrophages toappear cyan and white, and triple positive cells to appear white surrounded by yellow-green. I’-J’’’’. Individual channel images from I and J. Percent area
stained was quantified by outlining the tissue of the entire CL site. K. GFP staining was significantly reduced within the CL site of clodronate liposome
treated Ccr2gfp KO mice compared to vehicle treated mice. L. F4/80 macrophage staining was also significantly reduced in clodronate liposome versus
PBS liposome treated mice. M. Despite the significant reduction in macrophages in the CL site of clodronate liposome treated mice, DAPI staining was
not altered in the CL site. Scale bar is 500 μm. N = 18–20 per group for B-H and N = 5 per group for I-M. ** p < 0.01; *** p < 0.001. # p < 0.05 between injury
conditions within the same treatment group

Talsma et al. Journal of Neuroinflammation(2024) 21:134

both display significantly delayed WD, still show a robust
immune response including neutrophils, MDMs, andresident macrophages at the site of injury [63, 64]. In theSarm1 KO mouse, two waves of inflammation involving
macrophages have been demonstrated in the injured sciatic nerve [64]. The first is a response at the site of nerve
injury, which is maximal by 3 to 5 d post injury, restricted
to the injury site, and independent of WD. The second
wave is maximal at 7 to 9 d post injury and is localized tothe entirety of the DN [64].
MDMs recruited by CCR2 are the major component of
WD-dependent inflammation [35, 82] and were thought
to be necessary to enhance regeneration [33]. To determine if WD-dependent inflammation played a direct orindirect role in the CL response, a crush CL was used
to leave the epineurium and other ECM components of
the nerve intact. Axons grow into the nerve distal to thecrush by 2 d post-injury, evidenced by the sham nerve
growth in all our experiments, and thus interact with the
DN environment during the conditioning period. Neither
reducing DN macrophages, by performing a crush CL
in Ccr2 KOs, nor completely removing the DN environment, by performing transection CLs in WT and Ccr2
KOs, decreased CL-enhanced regeneration comparedto the WT crush CL. Finally, we showed that myelin
removal, also thought to promote enhanced regeneration, was not necessary for regeneration enhancement,
although it likely still has a small benefit for conditioned
axons [7]. Further, the minimal myelin removal in theregenerating segment during the 2 d regeneration period
suggests WD had not yet begun. However, any changes
that did occur were not sufficient to enhance regeneration, evidenced by the comparatively slow regenerationin the unconditioned sham nerves. If WD-dependent
inflammation served as a necessary source for retrograde
injury signals required for regeneration [83], or influenced the regenerative response through alternative
mechanisms, a change in the CL response elicited by acrush CL versus a cut CL would be apparent.
MDMs and the DN environment also were not necessary for maintenance of CL-enhanced regeneration in
the peripheral or central branch. We extended the conditioning period from the normal 7 d to 28 d to test if
WD-dependent inflammation and MDMs were needed
to maintain the CL response. A transection CL maintained CL-enhanced regeneration in the sciatic nerve
for 4 weeks indicating that neither the DN environment
nor MDMs and CCR2 signaling are needed to enhanceregeneration peripherally. We also tested the necessity ofthese macrophage subsets in supporting CL-enhanced
regeneration of injured dorsal root axons. The possibility existed that different signaling and/or non-neuronal
support were necessary for CL-enhanced regenerationof normally regeneration-deficient dorsal root. Again,

Page 20 of 25

WT and Ccr2 KO mice displayed equivalent levels of CLinduced regeneration of the dorsal root 3 d post-crush
injury.
The Ccr2 KO mouse, which has an approximately 50%
reduction of macrophages in the sciatic nerve after injury[18, 19, 82] displayed no difference in axonal regenerationcompared to WT after either a 2 d crush injury or a CL.
It was surprising that CCR2 signaling was not needed forenhanced regeneration as such a dependence was previously reported in culture experiments [18, 33], and it isunclear what important differences exist between the invitro and in vivo assays, as both were performed in the sameway up until the TLs. The quantification methods for axonal growth may contribute to the discrepancy. For in vivo
regeneration, we have derived a method to quantify thetheoretical average axon length without measuring individual axons [35]. In vitro, DRG explants were quantified bycounting the 20 longest neurites, and dissociated cultures
were quantified by measuring the longest axons of neurons
that had grown a certain minimum distance [18]. These latter two methods quantify a subset of neurons with the mostrobust growth and thus will tend to represent the growthof an outlier population. That population may be representative of a true biologic subset of neurons that respond tocues induced by CCR2 signaling but which do not represent the whole population of conditioned neurons.
To examine the effect of macrophages on the initialperiod of peripheral nerve regeneration and the importance of the local environment for the CL response, sciatic nerves were conditioned with zymosan 7 d prior toa crush lesion. Zymosan, when injected into the DRG,
has been shown to increase axonal regeneration of thecentral branch of unconditioned sensory neurons acrossthe dorsal root entry zone and into the spinal cord [72].
Additionally, injection of zymosan into the intraocularspace of the eye promotes significant regeneration of the
injured optic nerve [84]. Despite achieving substantial
recruitment of Arg1+ zymosan-activated macrophages in
the nerve, there was no regeneration enhancement aftera sciatic nerve crush injury compared to vehicle-treatedcontrols, demonstrating that these macrophages can neither interact with uninjured axons to stimulate a CL-like
effect nor alter the nerve environment to improve basal
regeneration. Endogenous Arg1+ TL macrophages arealso incapable of increasing regeneration of newly injured
axons demonstrated by comparing WT and Ccr2 nullsham nerves. Throughout the CL paradigm, sham WT
nerves are only ever exposed to TL macrophages whilesham Ccr2 null nerves never encounter macrophages,
and yet the axons show equivalent growth. Thus, any
benefits regenerating axons derive from nerve macrophages [21, 41] may come at later time points, possibly
requiring induction of the CL response, or from othermacrophage populations.

Talsma et al. Journal of Neuroinflammation(2024) 21:134

Page 21 of 25

Wallerian degeneration-independent inflammation

following a single injection, Ccr2gfp KO mice treatedwith clodronate liposomes displayed a 79% reduction
in CD68 staining compared to PBS liposome treated
Ccr2gfp KO mice. In CCR2-competent mice, it is likelythat the continual apoptosis caused by the local clodronate treatment indirectly leads to recruitment of
MDMs to the site of injury. Indeed, this is supportedby the fact that 78% of macrophages in the clodronate
treated Ccr2gfp heterozygous mice express GFP, defining them as MDMs. In the absence of CCR2, this major
recruitment mechanism is unavailable, explaining the
79% depletion at 7 days in Ccr2gfp KO mice treated withclodronate liposomes compared to PBS liposomes.
Thus, local clodronate administration can effectively
eliminate macrophages in the Ccr2 null sciatic nerve.
Given that MDMs are the primary source of macrophages at lesion sites in WT mice and that macrophages in the nerve are a major source of CCL2, theprimary CCR2 ligand [35], we hypothesized that clodronate administration into the CL site of Ccr2 nullmice would yield a significant reduction in macrophages. When clodronate liposomes were injected into
the CL site of Ccr2gfp KO mice, lesion site macrophages
were reduced by 80–90% in the CL site compared to
PBS liposome controls as measured by either CD68 or
F4/80 staining. Macrophages were also almost completely absent at the TL site in Ccr2gfp KO mice, as discussed earlier, and since a transection CL was used,injured axons were prevented from interacting with the
DN. Surprisingly, the CL effect in clodronate treated
Ccr2gfp KO mice was indistinguishable from the PBS
liposome-treated mice and was essentially equivalentto CL-enhanced regeneration observed in all otherexperiments indicating that the DN environment andlesion site macrophages are not necessary for the CL
response. This also implies that any nerve-derived signals required for the CL response must come from theproximal CL or proximal nerve and the cells foundthere.
The depletion of nerve macrophages was nearlycomplete in this CL paradigm with the only remaining macrophages consisting of at most 10–20% of CL
macrophages. Our primary macrophage marker, CD68,
can label other myeloid cells such as neutrophils andmonocytes [69–71]. Indeed, depletion of CL macrophages led to a large increase in neutrophils measured by Ly6G staining which accounts for some of
the remaining CD68 staining. Additionally, our staining revealed many F4/80+ cells which did not express
GFP or CD68, indicating they were not derived from
CCR2+ monocytes and likely not any myeloid lineage.
They are primarily located in the nerve segment adjacent to the CL, and we have observed similar F4/80
profiles in the DN 2 and 3 d post-injury [35]. Their

The peripheral transection CL paradigm utilized for mostof this paper primarily tests the role of WD-independent
inflammation. The nerve segment where regeneration ismeasured is proximal to the CL site and distal to the 2d old crush TL. WD-dependent inflammation begins tooccur 72 h [35] post injury and is maximal at 7 d [19, 32,85], and thus is not present in the regenerating segment.
Indeed, we confirm no myelin degeneration has begun in
the regenerating segment we examine. Whereas, we show
WD-independent inflammation at the lesion sites occurs
more rapidly. Thus, macrophage accumulation and infiltration is mainly localized to the injury sites of WT micein this nerve segment, while CCR2 null animals haveabsent TL macrophages and reduced CL macrophages.
We confirm here that the lesion site is a unique environment, which shapes the macrophages found there
differently from those located in the DN compartment[17, 64]. Most macrophages in the CL site of both WT
and Ccr2 null animals express Arg1 which is both an M2/
anti-inflammatory macrophage marker [86] and enzymatically promotes healing by metabolizing the nitrogenin arginine to produce polyamines and shunting nitrogen away from nitric oxide production [87]. The proportion of Arg1+ CL site macrophages is similar in WT
and Ccr2 nulls despite a significant reduction in MDMs
in the Ccr2 nulls, suggesting they share a phenotype andpossibly a function despite being derived from different populations. This is a novel finding as macrophages
derived from different sources often respond distinctly
to the same set of signals [88] and suggests that the signals present in the lesion sites are potent drivers of a particular macrophage phenotype. Interestingly, lesion site
macrophages have been shown to express high levels ofefferocytotic receptors suggesting that clearance of dying
cells may be the primary driver of their unique expression profile [17, 64].
Sciatic nerve macrophages are not required for CL-induced
peripheral axon regeneration

Clodronate liposomes are a well-used method to systemically and locally deplete monocytes and macrophages [89]. In WT mice, daily administration of
clodronate liposomes to the CL only reduced CD68
staining at the CL site by ∼20% compared to PBSliposome treated mice, indicating recruitment mechanisms are sufficient to overcome apoptosis. Further
characterization of clodronate liposome treatment was
carried out by performing a single injection of clodronate liposomes into the sciatic nerve 2 mm distal to atransection injury in Ccr2gfp het and KO mice. Ccr2gfp
het mice treated with clodronate liposomes displayed
a 41% reduction in CD68+ staining 7 d post-injury
compared to PBS-liposome treated mice. Conversely,Talsma et al. Journal of Neuroinflammation(2024) 21:134

elongated morphology and presence in the nerve bothproximal and distal to a lesion suggests that they are
Schwann cells. Thus, while it is possible that the fraction of remaining nerve macrophages are inducing
CL-enhanced regeneration, it is far more likely that
another cell population, possibly Schwann cells, neutrophils, or macrophages in another compartment, areeither compensating for the loss of nerve macrophagesor are responsible for CL-enhanced regeneration.
Alternative sources of stimuli for CL-enhanced regeneration

Barrette et al. [21] demonstrated that depletion of
myeloid cells in the lesioned sciatic nerve dramatically impairs regeneration in response to a single injury.
While this paper is sometimes referred to as showing
the importance of the nerve macrophage response to
injury, the authors emphasize that they have depleted
both macrophages and granulocytes. It is noteworthy,
however, that neutrophil depletion by itself did not affectnerve regeneration from a single lesion [90]. The latter
result together with our findings that depletion of nerve
macrophages does not inhibit the CL response raises animportant question about the interplay between neutrophils and macrophages within the injured nerve environment. Notably, a population of novel pro-regenerative
neutrophils has been identified, which unlike other neutrophils are not depleted by antibodies to Ly6G and can
induce a CL response in the dorsal columns after being
injected into the sciatic nerve [91]. Thus, the compensatory increase in neutrophils we observe when CL macrophages are depleted could be involved in initiating or
sustaining the CL response.
In the absence of resident and MDMs in the nerve
parenchyma, other populations of macrophages could
be compensating for their loss and support the CL
response. Significant macrophage accumulation isobserved in L (lumbar) 3–5 DRGs following a sciatic
nerve injury. Proliferating resident macrophages anda small influx of MDMs lead to a 2-fold increase in
macrophages by 3 to 7 d post-injury [16, 17, 34, 35].
A recent paper used a CSF1R inhibitor (PLX73086)
to deplete macrophages in the DRG after injury, while
maintaining a normal macrophage response in the sciatic nerve, and found that axonal regeneration after
a single nerve crush injury was significantly reduced[16]. Additionally, single cell RNA-sequencing hasrevealed that epineurial macrophages are a distinct
Relmα+Mgl1+ population of macrophages in the sciatic nerve that respond differently to injury than macrophages located within the nerve parenchyma [36].
Another defect revealed by Barrette et al. [21] wasthat angiogenesis in response to nerve injury, muchof which occurs in the epineurium, was absent when
myeloid cells were depleted. Macrophages within the

Page 22 of 25

DRG and/or epineurial macrophages in the sciatic
nerve could play a significant role in the CL response
through direct or indirect mechanisms.
Role of M1 macrophages and the nerve environment

We have shown that induction of CL-enhanced regeneration occurs with or without the M2-like macrophages found in the CL and further cannot be inducedby the recruitment of M2-like macrophages using
Zymosan. This implies that M2 signals (and macrophage derived signals in general) in the nerve are notenhancing regeneration. However, we were able toimpair CL-enhanced regeneration with our M1 stimulation cocktail containing a STAT6 inhibitor and LPS.
This cocktail increased pro-inflammatory marker
expression in CL macrophages without affecting theexpression of M2 markers. Thus, this CL contained its
own unique signaling environment with macrophagederived M1 and M2 signals as well as the injected
LPS and STAT6 inhibitor. This could have led to themodestly reduced regeneration through incomplete
induction of the CL response, inhibition of the CL
response, or creation of substances capable of inhibiting CL-enhanced axon growth. Since CL-enhanced
regeneration is maximally induced in the absenceof macrophages, it is likely that the impaired CLenhanced regeneration is either due to the inhibitory
nature of the environment or inhibitory signals. This
could be further elucidated in the future by examining
regeneration in vitro to remove any inhibition due tothe environment, and by sorting and sequencing M1
stimulated macrophages to characterize their phenotype in detail.

Conclusions
Here we provide clear evidence that both DN nerve
and lesion site macrophages, defining features of WDdependent and -independent inflammation, respectively, are not necessary for CL-enhanced regenerationin the sciatic nerve. These data have many implications for the future study of the CL response, suchas the source(s) of specific retrograde injury signals
including growth factors and gp130 cytokines, thefunctional significance of Arg1 expressing macrophages at the nerve lesion site, and the role of macrophages in other nervous system compartments inaxonal regeneration.
Abbreviations
Arg1	arginase 1
CCR2	C-C motif chemokine receptor 2CL	conditioning lesion
CNS	central nervous system
DAPI	4′,6-diamidino-2-phenylindole
DRGs	dorsal root ganglia
iNOS	inducible nitric oxide synthase

Talsma et al. Journal of Neuroinflammation(2024) 21:134

KO	knock out
DN	distal nerveKI	knock in
DPI	days post-injury
GFP	green fluorescent protein
LFB	luxol fast blue
LPS	lipopolysaccharide
MDM	monocyte-derived macrophage
nor-NOHA	alpha-amino acid N(omega)-hydroxy-nor-l-arginine
PNS	peripheral nervous system
PBS	phosphate buffered saline
RAG	regeneration associated gene
ROI	region of interest
SARM1	sterile alpha and TIR motif containing 1
SCG10	superior cervical ganglion 10 or stathmin 2
STAT6	signal transducer and activator of transcription 6
TL	test lesion
WD	Wallerian degeneration
WT	wild type

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03132-5.
Supplementary Material 1
Acknowledgements
We thank Brian Balog for helpful comments on the manuscript.
Author contributions
ADT, JPN, and REZ designed research; ADT and JPN performed research; ADT
and JPN analyzed data; ADT, JPN, and REZ wrote the paper. All authors readand approved the final manuscript.
Funding
This research was funded by NIH Grant NS095017 and NS114891. Genotypingof mice was performed by the Case Western Reserve University Visual Sciences
Molecular.
Biology and Genotyping Core (EY11373). ADT is supported in part by an MSTP
grant (T32 GM007250) and a neurodegeneration grant (T32 NS077888).
Data availability
No datasets were generated or analysed during the current study.

Declarations
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
The authors have no declarations.
Ethical approval and consent to participate
All animal care, experimental, and surgical procedures were approved by theCase Western Reserve University Institutional Animal Care and Use Committee.
(Approval Number: 2015-0009) and were conducted in accordance with theguidelines of the National Institutes of Health on the care and use of animals.
Author details
1
Department of Neurosciences, Case Western Reserve University, 10900
Euclid Avenue, Cleveland, OH 44106-4975, USA
Received: 23 April 2024 / Accepted: 13 May 2024

Page 23 of 25

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