Article

In Vivo Imaging of Dorsal Root Regeneration: Rapid Immobilization and Presynaptic Differentiation at the CNS/PNS Border

Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 03/2011; 31(12):4569-82. DOI: 10.1523/JNEUROSCI.4638-10.2011
Source: PubMed

ABSTRACT

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

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Available from: Alessandro Di Maio
Development/Plasticity/Repair
In Vivo Imaging of Dorsal Root Regeneration: Rapid
Immobilization and Presynaptic Differentiation
at the CNS/PNS Border
Alessandro Di Maio,
1
Andrew Skuba,
1,5
B. Timothy Himes,
1,2
Srishiti L. Bhagat,
1
Jung Keun Hyun,
1,3
Alan Tessler,
1,2
Derron Bishop,
4
and Young-Jin Son
1,3,5
1
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129,
2
Department of Neurology
Department of Veterans Affairs Hospital, Philadelphia, Pennsylvania 19104,
3
WCU Nanobiomedical Science Research Center, Dankook University,
Cheonan 330-714, Korea,
4
Department of Medical Education, Indiana University School of Medicine, Muncie, Indiana 47306, and
5
Shriners Hospitals
Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS.
Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth
inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled,
large-diameter DR axons in thy1-YFPHmiceelongated through a DR crush site, but not a transection site, and grew along the root at 1.5
mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper
into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that
enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastruc-
tural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting
presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are
neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS
territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm
to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.
Introduction
Almost a century ago, Ramo´ n y Cajal labeled a subset of dorsal
root ganglion (DRG) axons with Golgi staining and showed that
regenerating dorsal root (DR) axons were redirected peripherally
or terminated at the dorsal root entry zone (DREZ), the transi-
tional zone between the CNS and PNS (Ramo´ n y Cajal, 1928).
This regeneration failure remains an important practical issue
because common spinal root injuries, such as brachial plexus or
cauda equina injuries, lack effective therapy (Hannila and Filbin,
2008; Havton and Carlstedt, 2009). The molecular and cellular
events that repel or arrest axons at the DREZ remain poorly un-
derstood, but this regeneration failure is generally attributed to
glia-associated growth-inhibitory molecules and lack of intrinsic
growth potential of DRG neurons (Ramer et al., 2001a).
Although spinal root injury evokes changes similar to those
induced by direct CNS injury, it does not cause an impassable
glial scar. Nevertheless, the axotomized DREZ prevents regener-
ation efficiently: Whereas peripheral conditioning lesions, which
enhance the growth potential of DRG neurons, promote intraspi-
nal regeneration of their central axons in the dorsal columns
(Neumann and Woolf, 1999; Cao et al., 2006), the same axons fail
to regenerate through the DREZ (Chong et al., 1999; Golding et
al., 1999; Zhang et al., 2007). Notably, repellent cues, including
oligodendrocyte-associated inhibitors (Nogo, MAG, and OMgp)
and astrocyte-associated chondroitin sulfate proteoglycans
(CSPGs), cause only brief growth cone collapse or retraction
(Snow et al., 1991; Li et al., 1996; Yiu and He, 2006). Moreover,
DRG axons grow despite growth cone collapse (Marsh and Le-
tourneau, 1984; Jones et al., 2006; Jin et al., 2009). These growth-
inhibitory molecules therefore seem to account for the turning
but not the arrest of DR axons at the DREZ. These considerations
led us to suspect that a novel mechanism plays a more decisive
role in preventing regeneration across the DREZ.
Previous studies relied heavily on conventional tracing tech-
niques and postmortem analyses. The spatial and temporal reso-
lutions of these studies were limited: one could only deduce
dynamic events associated with DR regeneration by comparing
Received Aug. 29, 2010; revised Jan. 25, 2011; accepted Feb. 3, 2011.
This work was supported by NS062320 (Y.-J.S.) and World Class University program (R31-2008-000-100069-0)
through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology
(Y.-J.S, J.K.H). We thank Rita Balice-Gordon, Wenbiao Gan, and Joshua Trachtenberg for advice on in vivo imaging;
Theresa Connors and Amy Kim for technical assistance; and Marion Murray, Mickey Selzer, and Veronica Tom for
comments on the manuscript.
*A.D.M., A.S., and B.T.H. contributed equally to this work.
Correspondence should be addressed to Dr. Young-Jin Son, Shriners Hospitals Pediatric Research Center, Depart-
ment of Anatomy and Cell Biology, Temple University School of Medicine, Medical Education Research Building, 6th
Floor, 3500 North Broad Street, Philadelphia, PA 19140. E-mail: yson@temple.edu.
DOI:10.1523/JNEUROSCI.4638-10.2011
Copyright © 2011 the authors 0270-6474/11/314569-14$15.00/0
The Journal of Neuroscience, March 23, 2011 31(12):4569 4582 4569
Page 1
static images. Fundamental questions therefore remained unan-
swered, including whether axons stop abruptly or attempt to turn
around at the DREZ and whether they remain immobile or regain
mobility over time. Axon turning would suggest that the DREZ
functions as a passive barrier that axons avoid. Brief collapse
without permanent immobilization would highlight the impor-
tance of repulsive growth inhibitors that continuously collapse
axons at the DREZ. On the other hand, rapid, permanent immo-
bilization would suggest a unique mechanism that arrests axons
by paralyzing or stabilizing them, as was speculated many years
ago but virtually forgotten (Carlstedt, 1985; Liuzzi and Lasek,
1987).
To address these questions, we applied in vivo imaging tech-
niques using fluorescent transgenic mice with a vital Golgi-like
stain, and monitored the regeneration of identified DR axons
repeatedly over weeks to months directly in living animals with
and without conditioning lesions. Combined with a targeted
ultrastructural analysis, we found that most axons were not
repelled, but were immobilized rapidly and chronically at the
DREZ, forming presynaptic terminal endings in its CNS
territory.
Materials and Methods
Mice. We used adult mice (2–4 months of age) of either sex from trans-
genic strains thy1-YFPH and thy1-YFP16, which express yellow fluores-
cent protein (YFP) under the control of the neuron-specific Thy-1
promoter (Feng et al., 2000). The original breeding pairs were purchased
from The Jackson Laboratory; subsequent stocks of mice used in these
experiments were reared in the animal facilities at Drexel University
College of Medicine (DUCOM). All experiments were performed in ac-
cordance with DUCOM’s Institutional Animal Care and Use Committee
and National Institutes of Health guidelines.
Surgical and postoperative procedures. Thy1-YFPH mice were anesthe-
tized with an intraperitoneal injection of xylazine (8 mg/kg) and ket-
amine (120 mg/kg). Supplements were given during the procedure as
needed. A 2- to 3-cm-long incision was made in the skin of the back; the
spinal musculature was reflected; and the L3–S1 spinal cord segments
were exposed by hemilaminectomies. The cavity made by the laminecto-
mies was perfused with warm sterile Ringer’s solution or artificial CSF. A
small incision was made in the dura overlying the L5 dorsal root near the
L3 DRG; a fine forceps (Dumont #5) was introduced subdurally and the
L5 dorsal root was crushed for 10 s. After images were collected (see
below), we attempted to minimize scar formation by tightly applying a
piece of thin synthetic matrix membrane (Biobrane, Bertek Pharmaceu-
ticals) over the exposed cord and dura, so that scarring accumulated on
the membrane rather than on the dura surface. The matrix membrane
was removed and replaced at each imaging session. This membrane was
stabilized with a layer of much thicker artificial dura (Gore Preclude
MVP Dura Substitute, W.L. Gore and Associates) that covered the lam-
inectomy site. The musculature was then closed with sterile 5-0 sutures,
and the skin with wound clips. Animals were given subcutaneous injec-
tions of lactated Ringer’s solution to prevent dehydration and kept on a
heating pad until fully recovered from anesthesia. Buprenorphine was
given as postoperative analgesia (0.05 mg/kg, s.c., every 12 h for 2 d). For
each imaging session, we reanesthetized and surgically reexposed the area
of interest and repeated the procedures. For conditioning lesions, the
sciatic nerve was crushed in the lateral thigh of the ipsilateral hind leg 10 d
before the root was crushed. Animals were anesthetized as described
above; the skin and superficial muscle layer of the midthigh were opened;
and the sciatic nerve was crushed for 10 s with fine forceps (Dumont #5).
The muscle and skin were then closed in layers and the animals were
allowed to recover on a heating pad until fully awake.
In vivo imaging and image acquisition. We used a Leica MZ16 fluores-
cent stereomicroscope or an Olympus BX51 microscope equipped with a
fast shutter and a highly sensitive cooled CCD camera (ORCA-Rx2,
Hamamatsu) controlled by MetaMorph software (Molecular Devices).
Body temperature was maintained by placing the animal on a thermo-
statically controlled heating pad. Warmed lactated Ringer’s solution was
used to superfuse the exposed portion of spinal cord. Images were ac-
quired either as single snapshots or as multiple streams of 10–20 frames
Figure1. YFP-labeledDRGneurons are large and neurofilamentpositive. A, Size distribution ofYFP L5 DRG neurons (green)inThy1-YFPH mice, superimposed on theentire population, labeled
with a fluorescent Nissl stain (red, inset). YFP neurons are large. B, Superficial layers of the dorsal horn of a Thy1-YFPH mouse, exhibiting little YFP fluorescence in lamina II, where CGRP and
IB4 innervation is abundant. Yellow dotted lines denote dorsal roots. C, Four-color immunolabeling of a DRG cross section illustrating expression of neurofilament (magenta) but not CGRP (blue)
or IB4 (red) in YFP DRG neurons (green) in Thy1-YFPH mice. Scale bars: A, 200
m; B, 200
m; C,50
m.
4570 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
Page 2
acquired within 30 40 ms exposure time. In-focus images were then
selected, and an overview montage was created using Photoshop (Adobe
Systems). High-resolution confocal images were obtained with a Leica
TCS 4D confocal microscope. Z stacks were obtained at 0.3
m step size
for 20 40
m depths. Leica TCS-NT acquisition software and Imaris
image software (Bitplane) were used to reconstruct z-series images into
maximum-intensity projections.
Immunohistochemistry of DREZ in whole mounts. Following in vivo
imaging, we harvested tissues and processed them in whole mounts to
immunolabel astrocytes, oligodendrocytes, or Schwann cells to locate the
CNS/PNS interface. The immunostaining procedure was standard
(Wright et al., 2009), except for the permeabilization steps in which
chilled MeOH and 1% sodium borohydride were also used. Mice were
perfused transcardially with 0.9% heparinized saline solution followed
by 4% paraformaldehyde in PBS. After 3 h in situ postfixation at 4°C, the
spinal cord segment (L3–L6) with attached dorsal roots was removed and
rinsed in PBS. The tissue was then washed for 30 min in a blocking
solution containing 0.1
M glycine and 2% bovine serum albumin (BSA)
in PBS and treated in cold MeOH for 10 min and then 1% sodium
borohydride for 5–10 min. After thorough and extensive rinsing in PBS,
the spinal cord was further permeabilized with 0.2% Triton X-100 with
2% BSA in PBS (TBP) for 1 h and then incubated with primary antibody
diluted in TBP overnight. The next day, the spinal cord was rinsed thor-
oughly in TBP and then incubated with appropriate fluorescently conju-
gated secondary antibodies diluted in the TBP for1hatroom
temperature. The tissue was then rinsed in PBS, and a thin sheet of dorsal
spinal cord was prepared from the DREZ and rootlet, mounted in
Vectashield (Vector Laboratories), and stored at 4°C.
Immunohistochemistry of DREZ on cryostat sections. To immunolabel
axons at the axotomized DREZ with synaptic vesicle markers, we used the
transgenic strain, thy1-YFP16, in which the entire population of large-
diameter axons expresses YFP (data not shown). To analyze more axons
than superficially located ones, we prepared cryostat sections, rather than
whole mounts, of the DREZ after crushing dorsal roots of cervical spinal
cord. Using the surgical procedures described earlier, C3–C5 roots were
crushed, and the animals were allowed to recover. At 20 d after injury, the
C3–C5 spinal cord and roots were harvested, postfixed overnight at 4°C,
cryoprotected in 30% sucrose in PBS, and rapidly frozen in Shandon M1
embedding matrix (Thermo Electron). Serial transverse sections were
cut on a cryostat at 10
m (CM3000, Leica) and collected on Superfrost
Plus slides (Fisher Scientific). For immunostaining, sections were post-
fixed in 4% paraformaldehyde in PBS for 20 min, rinsed in PBS, and
blocked for1hinTBP. The sections were then incubated overnight at
4°C in a cocktail of primary antibodies diluted in TBP. Sections were then
rinsed in PBS and incubated with secondary antibodies in TBP for1hat
room temperature and processed as described above.
Analysis of thy1-YFPH DRGs. L5 DRGs were dissected from unoper-
ated thy1-YFPH mice and processed to obtain serial cryostat sections
using the methods described above. Selected sections were stained with a
fluorescent Nissl stain (Neurotrace 530/615 red fluorescent Nissl stain;
Invitrogen) according to the manufacturer’s instructions, washed exten-
sively with PBS, and coverslipped using Vectashield (Vector Laborato-
Figure 2. Axons fail to elongate through a transection injury. A, Schematic illustration of a right-sided laminectomy (T12–L5) to partially expose the L5 root. B, Low-magnification fluorescence
view of the exposed spinal cord of a living Thy1-YFPH mouse. The laminectomy extends laterally to partially expose L3, L4, and L5 DRGs and medially to expose the midline dorsal vein. C,
High-magnification fluorescence view of the area where L5 DR axons enter the spinal cord. Superficially positioned YFP axons run parallel to the midline dorsal vein, curve perpendicularly to enter
the DREZ, and then bifurcate within the spinal cord. Highlighted box approximates DREZ. D, Repeated imaging of L5 DR axons for 7 d after root transection. Axons in L5 root were cut near the L3 DRG
(red scissors), 3 mm from the DREZ, and imaged on day 0, day 3 (not shown), and day 7. The area of the root transection (red boxes) is magnified in right panels (D1–D3). D1, Before the
transection. A few superficial YFP axons are visible. D2, After the transection. The medial portion of the L5 root was completely cut with spring scissors, and the proximal and distal ends were then
closely reapposed. D3, Seven days after the transection. The transection site is filled with 500
m collagenous scar tissue. No axons cross it. D4, A high-magnification confocal view of the area
prepared after sacrificing the mouse on day 7. YFP axons located deeper in the root than those imaged in vivo also failed to regenerate across the transection site (asterisks). Instead, they turned
around (arrows) and extended back along the proximal axons. Note that all the fluorescence in the stump beyond the cut (i.e., left of the asterisk) is due to fragments of degenerating axons that
retained YFP.
Di Maio et al. Dorsal Root Regeneration In Vivo J. Neurosci., March 23, 2011 31(12):4569 4582 4571
Page 3
ries). Neuron counts were made on five L5 DRGs from three animals. For
each DRG, at least three randomly selected sections were analyzed, taking
care not to use sections that were poorly mounted or stained. Each sec-
tion selected was at least 30
m away (three sections) from either of the
other selected sections. Using a Retiga EXi (Qimaging) digital camera,
the entire section was photographed in segments using the 20 objective
on a Leica DMRBE fluorescent microscope. The same section was pho-
tographed using both red (Nissl-stained cells) and green [YFP-positive
(YFP) cells] fluorescent filter cubes to identify neurons containing a
nucleus with a visible nucleolus and to determine whether such neurons
were YFP positive. Image segments were collected at 200 magnification
and combined to form a montage. Using ImageJ software (National In-
stitutes of Health), the cell area of all neurons containing a nucleus with
a visible nucleolus from each chosen section was measured. We counted
a minimum of 200 neurons per ganglion. If this number was not reached
in the three sections chosen, a fourth section was counted, also in its
entirety. However, because identification and counting continued even
after the minimum of 200 neurons were obtained, we always counted
200 Nissl-stained neurons per DRG (mean 218 6 Nissl cells mea-
sured/DRG, 4.3% of the measured were YFP). Histograms represent-
ing the cross-sectional area of all Nissl- and YFP-labeled DRG neurons
measured were compiled to compare the distribution of the YFP-labeled
cells with the total cell populations.
Antibodies. The primary, cell-type-specific antibodies included anti-glial
fibrillary acidic protein (GFAP, mouse monoclonal, 1:1000, Millipore
Bioscience Research Reagents, Millipore) to label astrocytes, anti-myelin
oligodendrocyte glycoprotein (MOG, goat polyclonal, 1:200, R&D Sys-
tems) for labeling oligodendrocytes and anti-SC/2E (mouse monoclonal,
1:1000, Cosmo Bio), or laminin-1 (rat monoclonal, 1:200, Abcam) to
label Schwann cells. Mouse monoclonal antibodies to a synaptic vesicle
protein, SV2 (1:10, Developmental Studies Hybridoma Bank), or to syn-
aptotagmin 2 (znp-1, 1:2000, Zebrafish International Resource Center)
were used to label synaptic vesicles. To learn more about the phenotype
of YFP DRG neurons, selected sections from L5 DRGs were labeled
with one or more of the following methods: Neurons containing phos-
phorylated epitopes of high-molecular-weight neurofilament were iden-
tified using the SMI 312 antibody (mouse monoclonal antibody, 1:1000
dilution, Covance). The population of small primary afferent neurons
that expresses the trkA neurotrophin receptor was labeled using an anti-
body to calcitonin gene-related polypeptide (CGRP, rabbit polyclonal
antibody to rat CGRP, 1:2000, Bachem). The population of small DRG
neurons that does not express the trkA neurotrophin receptor was
labeled using Griffonia simplicifolia IB4 lectin (biotin conjugate, 5
g/ml, Sigma-Aldrich). Secondary antibodies used were Alexa 647-
conjugated donkey anti-mouse 1:200, Invitrogen), Alexa-Fluor 568-
conjugated goat anti-mouse IgG
1
(1:200, Invitrogen), Alexa-Fluor
647-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), and
rhodamine-red-conjugated rabbit anti-goat IgG (Jackson Immu-
noResearch Laboratories).
Electron microscopy of the DREZ. The mice were perfused transcardi-
ally (with heparinized Tyrode’s solution followed by 2% paraformalde-
hyde and 2.5% glutaraldehyde in 0.1
M Na-cacodylate buffer. The spinal
cord segments L3–L6 were then removed as one piece and rinsed in 0.1
M
Na-cacodylate buffer, mounted on an agarose support, and placed in the
Figure 3. Axons elongate througha crush injury with little variability. A, Repeatedimaging of L5 DR axonsfor 7 d after root crush. The medialportion of the L5 rootwas crushed with a fine forceps
(red arrowhead) and imaged on days 0, 3, 5, and 7 after the crush. The area of the crush is magnified in the right panels (A1–A4 ). A1, Immediately after crush. Four injured YFP axons are shown
(colored arrows). A2, Three days after the crush. All four axons could be reidentified. Each axon extends a single neurite that crosses the site of crush. A3, A4, Five and seven days after crush. Neurites
remain stable and there is no additional growth from these or other proximal axons. Axon swellings are occasionally observed (e.g., A4; axon marked by blue arrows). A5, High-magnification
confocal view of the area prepared after sacrificing the mouse on day 7. YFP axons located deeper in the root also regenerate across the crush site. B, Confocal analysis of the regeneration after a
crush injury in another Thy1-YFPH mouse that did not receive in vivo imaging. The L5 root was crushed at the usual location (red arrowhead); 4 d later, the spinal cord was prepared in whole mounts
and analyzed by high-resolution confocal microscopy. The area near the crush injury (blue box) and the DREZ (yellow box) on day 4 are magnified in B1 and B2, respectively. B1, Consistent with in
vivo imaging observations, almost all YFP axons extended a single neurite that crossed the crush site and grew further without stopping. Blue arrows point to degenerating axon fragments. B2,
Almost all axons reached the DREZ and terminated at a location similar to that of single axons (arrows). Few axons turned around (e.g., arrowhead), further evidence of the consistency
of the axon response to crush injury. DC, Dorsal column; DR, dorsal root. Red stars indicate uninjured axons. Because their proximal portion was located laterally, they escaped crush of
the medial portion of the L5 root.
4572 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
Page 4
vibratome well containing chilled buffer. The most superficial longitudi-
nal slice containing the DREZ (250
m thickness) was cut and further
processed for electron microscopy. To target our electron micro-
scopic analysis to the area where axons had stalled, we applied fiducial
markers to the surface of the spinal cord slice. The spinal cord sections
were flattened with insect pins in Sylgard silicone elastomer-lined 35
mm Petri dishes. A 1.0% solution of 1,1-dioctadecyl-3,3,3,3-
tetramethylindodicarbocyanine-5,5-disulfonic acid (DiI, Invitrogen)
was dissolved in dichloromethylene and loaded into a micropipette (re-
sistance of 5–10 M). Crystals of DiI were iontophoretically applied to
the surface of the spinal cord slice in an area of the DREZ with bulb-
tipped axons (e.g., see Fig. 11 A). To render the DiI crystal electron dense,
we excited the DiI crystals near their excitation wavelength in the pres-
ence of 3,3-diaminobenzidine (DAB, 5.0 mg/ml, Sigma-Aldrich) until
the DiI crystal was replaced with a dark red/brown DAB precipitate (20
min). After photoconversion, spinal cord slices were trimmed to contain
the area of interest using the electron-dense fiducial markers as reference
points. Tissue blocks were stained with 1.0% osmium tetroxide reduced
in 1.5% potassium ferrocyanide for 45 min, then dehydrated in an as-
cending ethanol series, infiltrated with Araldite 502 Embed 812 resin, and
polymerized at 60°C for 48 h. Polymerized tissue blocks were sectioned
(0.5
m) with a glass knife on a Leica Ultracut R microtome (Leica) until
the fiducial markers were located. Serial ultrathin sections (60 –70 nm)
were cut and mounted on pioloform-coated slot grids. Sections were
counterstained with 2.0% aqueous uranyl acetate and Reynold’s lead
citrate. Sections were viewed at 75 kV on a Hitachi H-600 transmission
electron microscope. Serial electron micrographs were captured at
6000 and scanned at a resolution of 1000 dpi.
Results
YFP-labeled DRG neurons are large and
neurofilament positive
To monitor DR regeneration directly in living mice, we used the
H line of thy1-YFP mice (thy1-YFPH), which expresses high lev-
els of YFP in subsets of neurons, including sensory neurons in
DRGs (Feng et al., 2000). Because DRG neurons are heteroge-
neous, we first characterized YFP-labeled neurons in DRG of
thy1-YFPH mice. YFP neurons were large, with an average size
of 1244.7
m
2
(Fig. 1
A)(n 66, compared to the mean size of
594
m
2
of 1523 Nissl-stained neurons). As expected for large
DRG neurons (Ruscheweyh et al., 2007), YFP neurons were
neurofilament but CGRP-negative (CGRP) and IB4 (Fig.
1C). Additional analysis of superficial layers of the dorsal horn in
thy1-YFPH mice revealed little YFP fluorescence in lamina II,
Figure 4. Axons elongate 1.5 mm/d along preexisting endoneurial tubes. A, Repeated imaging of an identified axon for 4 d after root crush. On day 0, a Thy1-YFPH mouse with only a few
superficial YFP axons underwent crush of the most medial portion of the L5 root (red arrowheads) to minimize the number of damaged axons. A1, Magnified view of the crush site immediately
after injury. Two superficial axons are shown; the axon marked by a blue arrow (blue axon) was stretched but survived the injury, whereas the axon marked by a green arrow (green axon) was
damaged.On day 2, degeneration of both proximal and distal tips confirmed apparent damage of the green axon by the crush. A2,Magnified view of the crush site. Yellowarrow denotes dying-back
degeneration of the green axon. Note that no neurite has yet been formed by the green axon. On day 4, the green axon has extended 3 mm, and its tip has reached the DREZ. A3, Magnified view
of the green axon near the DREZ, illustrating its growth along the fluorescent fragments of degenerating distal axons (yellow arrowheads: i.e., endoneurial tube trajectory). B, Examples of newly
formed neurites on proximal stump axons imaged 2 d after injury. Day 0, Magnified view of the crush site immediately after injury showing two crushed axons (pink arrows). Day 2, Both axons
extendedshort neurites (green arrows) that have notyet reached thedistal stump axons (e.g., yellow arrowhead). Theseneurites have slendergrowing tips (green arrow,inset). Day 3,Both neurites
extended through the degenerating distal axons. Asterisks in A1 and A2 indicate a node of Ranvier on the blue axon that served as a landmark. Blood vessels served as a landmark in B. Red
arrowheads indicate the site of crush.
Di Maio et al. Dorsal Root Regeneration In Vivo J. Neurosci., March 23, 2011 31(12):4569 4582 4573
Page 5
where CGRP and IB4 innervation is
abundant (Fig. 1B). We also observed that
CGRP axons were not labeled in the line
16 thy1-YFP (thy1-YFP16) mice, which
were thought to express YFPs in nearly all
neurons (data not shown) (cf. Fig. 10).
These results are consistent with an earlier
characterization of the M line thy1-YFP mice
(thy1-YFPM), in which fewer neurons are
labeled than in the H line (Kerschensteiner
et al., 2005). Large, neurofilament neurons
are myelinated and their central axon pro-
cesses are thought to regenerate more
poorly than those of small-diameter, non-
myelinated neurons (Tessler et al., 1988;
Guseva and Chelyshev, 2006). Thus, thy1-
YFPH mice provide a unique opportunity
to study in vivo the axon regeneration of
sensory neurons whose regeneration po-
tential may be particularly weak.
Axons turn around and fail to elongate
through a transection injury
While optimizing the in vivo imaging tech-
niques for DR regeneration, we learned that
regeneration of individual DR axons is
better imaged in lumbar than in cervical
spinal cord of thy1-YFPH mice (A. Skuba,
T. Mimes, and Y. J. Son, unpublished ob-
servations). In our typical preparation, we
performed a right-sided laminectomy
(Fig. 2 A) (T12–L5) to partially expose the
L5 root and the DREZ where these pro-
cesses enter the spinal cord (Fig. 2B, C;
highlighted box in Fig. 2C approximates
the DREZ). Superficially positioned
YFP axons in the medial portion of the
L5 root (3–10 axons) run parallel to the
midline dorsal vein, curve perpendicu-
larly to enter the DREZ, and then bifur-
cate within the spinal cord, with one
branch that enters the dorsal column
(DC) and another that enters the gray
matter (Fig. 2C). This stereotypical trajec-
tory helped us to follow regeneration of
several identified axons simultaneously
and repeatedly over time in vivo. It also
permitted us to identify reliably the loca-
tion of the DREZ in living spinal cord.
We first monitored DR axons after complete transection. The
medial portion of the L5 root was cut near the L3 DRG, 3mm
from the DREZ, using a fine-spring scissors (Fig. 2D2). After
lifting the proximal stump to confirm that the DR had been com-
pletely transected, we closely apposed the cut ends of the proximal
and distal stumps and then examined axons on both sides of the
transection 3 and 7 d after injury. Even 7 d after the cut, no YFP
axons from the proximal stump extended across the transection site
(Fig. 2 D3)(n 4 mice). The two ends were separated by 500
m
of collagenous scar tissue, which seemed to prevent proximal axons
from penetrating this region. After the mice were killed, we used
confocal imaging to confirm that YFP axons located deeper in the
root than those that we imaged in vivo also failed to regenerate across
the injury site (Fig. 2 D4). Importantly, however, few if any neurites
formed bulblike endings or terminated abruptly. Instead, they
turned around at the transection site and extended back along the
proximal axons (e.g., arrows in Fig. 2D4 )(n 95 axons, 3 mice).
Axons elongate through a crush injury with little variability
Next, we monitored YFP axons every 24 48 h for 7 d after a
crush injury (Fig. 3). We crushed the medial portion of the L5
root with a fine forceps (Fig. 3A1). The next day we observed
dying-back degeneration of proximal stump axons (data not
shown, e.g., Fig. 4 A2) and fragmentation/degeneration of the
same axons distal to the crush (e.g., yellow arrowheads in Fig.
4A3) (cf. Kerschensteiner et al., 2005), which confirmed that
axons had been appropriately damaged. We applied several ad-
ditional criteria for unambiguously distinguishing regenerating
axons from axons that had been spared or recovered from the
injury. These included the following: (1) in regenerating axons,
Figure5. Regeneratingaxonsare rapidly immobilized at theDREZ.Repeated imaging of axon tipsatthe DREZ 6 –13d after root
crush. On day 0 (data not shown), the L5 root was crushed at the usual site, 3 mm away from the DREZ. On day 6, tips of five
regenerating axons are observed at the DREZ (colored arrows). DC, Dorsal column; DR, dorsal root. Magenta asterisks denote
fluorescent debris of degenerating old axons that served as landmarks. Note that the location of an axon tip relative to other axon
tips and landmarks remained unchanged in subsequent imaging sessions on days 8, 10, and 13, indicating that the axons were
immobilized after entering the DREZ. A1–A4, Magnified view of the area outlined by magenta boxes showing tips of four axons.
They did not grow, retract, or turn around and remained in the same location in subsequent imaging sessions. Their appearance
also was unchanged except for swellings that developed on the tips or shafts of some axons (e.g., white arrows in A2–A4 ).
4574 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
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there was expansion of the nonfluorescent portion of the YFP
axon at the crush site due to proximal and distal degeneration (in
contrast to narrowing of the unlabeled gap due to fluorescent
cytoplasm refilling the crush site if axons survived the injury); (2)
regenerating axons were much thinner, less brightly fluorescent,
and more undulating than axons that survived the injury; (3)
regenerating neurites were thinner and more dimly fluorescent
than the degenerating fluorescent fragments of axons through
which they extended; (4) in contrast to surviving or spared axons,
regenerating axons stopped at the DREZ; and (5) in contrast to
surviving or spared axons, regenerating axons did not exhibit
nodes of Ranvier.
In contrast to the transection injury, almost all crushed YFP
axons extended a single neurite that grew across the site of injury
by 3 d (Fig. 3A2)(n 25 axons, 5 mice) without forming addi-
tional branches (Fig. 3A3,A4). Confocal analysis 7 d after injury
confirmed that almost all YFP axons located deep in the DR
also grew through the crush site (Fig. 3A5).
To exclude the possibility that these observations were due to
imaging-associated artifacts, we examined mice 4 d after crush
injury that we had not previously imaged in vivo (Fig. 3B). Con-
sistent with in vivo imaging observations, almost all YFP axons
extended a single neurite through the crush site and grew until
reaching the DREZ (Fig. 3B1)(n 85 axons, 2 mice). Notably, by
4 d, almost all of the axons had already arrived at the DREZ, 3
mm beyond the injury site, and terminated at a similar location
(Fig. 3B2, arrows). We observed few axons turning around, fur-
ther evidence of the consistency of the axon response to crush
injury. Thus, most, and perhaps all, YFP sensory axons, which
have previously been thought to have a relatively weak regenera-
tive capacity, can nevertheless regenerate
across a crush site, extend within the PNS,
and arrive at the DREZ.
Axons grow >1.5 mm/d
We next investigated in vivo how quickly
YFP DR axons regenerated along the
root (Fig. 4). This process was difficult to
accomplish, however, because fragments
of degenerating YFP axons (e.g., yellow
arrowheads in Fig. 4A3) retained fluores-
cence for 7–10 d and obscured the leading
tips of regenerating YFP axons. To cir-
cumvent the problem, we minimized the
number of damaged YFP axons by
crushing only the most medial portion of
the L5 root (Fig. 4 A1). This strategy en-
abled us to crush only one or two YFP
axons and follow their regeneration with
little residual YFP fluorescence. The crush
was made at the usual location, 3mm
away from the DREZ, and the crushed
axons were imaged daily. Typically, the
proximal tips of the crushed axons degen-
erated for 2 d after injury (Fig. 4 A2) (e.g.,
an axon marked by green arrow) and then
extended a short neurite, which, by day 4,
had grown 3 mm along the fluorescent
fragments of degenerating axons (i.e., en-
doneurial tubes marked by yellow arrow-
heads; Fig. 4A3) and arrived at the DREZ
(Fig. 4A3). We also observed short neu-
rites extending from some of the proximal
axons imaged 2 d after injury (Fig. 4B, green arrows). Thus,
YFP axons elongated at a rate of 1.5 mm/d along their origi-
nal endoneurial tube trajectory. These observations are consis-
tent with confocal analysis of mice that we did not image in vivo
(cf. Fig. 3B), and demonstrate that large-diameter axons grow
well along the dorsal root, with little variability.
Rapid immobilization of axons entering the DREZ
To determine the behavior of axons at the PNS/CNS border, we
continued imaging axons beyond 4 d after crush, when they ar-
rive at the DREZ. Because more degenerating fluorescent axon
fragments (i.e., residual YFP fluorescence) disappeared over the
next few days, we resumed imaging 6 or 7 d after injury when the
growth tips of regenerating axons were fairly easy to recognize. In
several mice with L5 root crush, we were able to identify a number
of axon tips arriving at the DREZ on day 6 or 7 (n ⫽⬎40 axons,
6 mice) and relocate them again in subsequent imaging sessions
over 2 weeks after crush (Fig. 5). Unexpectedly, the leading tips of
these axons did not continue to grow forward, retract, or turn
around but were completely immobile; they remained in the
same location in subsequent imaging sessions (i.e., as identified
by the relative location with respect to adjacent axons or land-
marks such as blood vessels and fluorescent debris). Notably,
their appearance also did not change except that swellings formed
on the tips or shafts of some axons (Fig. 5, white arrows).
We occasionally observed slowly growing and retracting ax-
ons (Fig. 6). Close observation, however, indicated that this
growth was short neuritic sprouting extending from (Fig. 6 A5),
and then being reabsorbed by (Fig. 6A6 ), axon tips that remained
stationary and developed swollen endings over time (Fig. 6A4
Figure 6. Immobilized axon endings at the DREZ extend and retract a sprout. Shown is continued imaging of the mouse shown
in Figure 4, 7–11 d after L5 root crush. On day 7, the tip of the green axon (magenta arrows) is at the same location as on day 4 (cf.
Fig. 4). The blue axon that was stretched on day 0 has degenerated. Red asterisks indicate axon debris, a landmark, that remained
stable during the imaging sessions. Note that the relative distance between the tip of the green axon and the debris remained the
same on subsequent imaging sessions on days 9 and 11. A4, Magnified view of the axon tip on day 7. A fine neurite extends from
the slightly swollen tip (magenta arrow). A5, Two days later on day 9, the neurite has elongated 50
m; the axon tip remains
at the same location and is more swollen. A6, Two days later on day 11, the neurite appears to have absorbed into the tip, which
remains immobilized at the same location. Thus, the apparent mobility of this axon at the DREZ was due to fruitless sprouting of a
stabilized axon tip.
Di Maio et al. Dorsal Root Regeneration In Vivo J. Neurosci., March 23, 2011 31(12):4569 4582 4575
Page 7
A6, pink arrows). On the last day of imaging, we killed the mouse,
analyzed the same axon endings with high-resolution confocal
microscopy, and confirmed that no growth tip structures had
been missed due to poor resolution of our live imaging setup
(data not shown) (compare Fig. 8, day 20). The apparent mobility
of these axons at the DREZ was therefore due to fruitless sprout-
ing of immobilized axon tips. Collectively, considering that axons
arrive at the DREZ as early as 4 d after crush injury (compare Fig.
4), these observations demonstrate that axons are immobilized
surprisingly quickly after arriving at the DREZ. It is also notable
that, in contrast to axons at the site of a transection injury (Fig.
2D), axons arriving at the DREZ after crush injury (Fig. 4) rarely
turned around along the dorsal root even though this pathway
contains growth-promoting Schwann cells.
Chronic immobilization of axon endings at the DREZ
We also observed the long-term response of DR axons stopped at
the DREZ in mice whose L5 root had been crushed 4 months
previously (Fig. 7) (n 15 axons, 2 mice). When we initiated
imaging at 128 d after the crush, we observed several superficially
positioned YFP axons and their tips at the DREZ (Fig. 7A,
colored arrows). In subsequent imaging sessions at days 131 and
140 after crush, these axons were not motile but remained in the
same place and looked unchanged, demonstrating chronic im-
mobilization or stability. We also analyzed a mouse whose L5
root had been crushed 4 months previously but had not been
studied with in vivo imaging (Fig. 7B). Confocal analysis revealed
axon profiles at the DREZ that appeared similar to those observed
within the first week after crush (compare Fig. 3B2): almost none
of the YFP axons turned around; instead, almost all of them
terminated as single processes at a similar location at the DREZ.
Axon swellings, similar in appearance to synaptic varicosities, were
also frequently observed on axon shafts (Fig. 7B, yellow arrow-
heads). These observations show that axons quickly immobilized on
entering the DREZ remained completely immobile and stable for
long periods despite the absence of target innervation.
Rapid immobilization of axons at the DREZ even after a
conditioning lesion
Next, we asked whether axons became motile at the DREZ fol-
lowing a conditioning lesion of peripheral processes that en-
hances the growth potential of DRG neurons (Chong et al.,
1999). To this end, we crushed sciatic nerves in the ipsilateral leg
10 d before crushing the L5 root at the usual location (Fig. 8) (n
6 mice). We then imaged these mice in vivo every 2 or 3 d over 3
weeks, and monitored regeneration of identified axons at the
crush site, along the root, and at the DREZ. We found that most
YFP axons had already extended through the crush site 2 d after
crush (Fig. 8, day 2) (95%, n 15), 1 d faster than noncondi-
tioned axons. In addition, axons frequently extended more than
one neurite, further illustrating the enhanced growth within the
root due to a conditioning lesion. However, we observed no ax-
ons that had regenerated through the DREZ at 4 d after crush
(Fig. 8, day 4) (n 85 axon tips located at the DREZ). Instead,
like nonconditioned axons at 4 d after crush (compare Figs. 3B,
4), these conditioned axons did not turn around upon arriving at
the DREZ but terminated as single processes at a similar location
at the DREZ. In subsequent imaging sessions over the next 3
weeks, they did not grow forward or retract but remained immo-
bile (Fig. 8). The only noticeable change was the swelling of the
tips and shafts of some axons (Fig. 8, yellow arrowheads). Thus,
even conditioned axons with enhanced peripheral growth were
quickly immobilized or stabilized at the DREZ.
Figure 7. Axons are chronically immobilized at the DREZ. A, Repeated imaging of axons crushed 4 months previously. L5 root of a Thy1-YFPH mouse was crushed (red arrow) and imaged on days
1, 128, 131, and 140 following injury. On day 1, the root crush is indicated by degeneration of proximal and distal axon stumps at the injury site. On day 128, several axons are located at the DREZ
outlined by a magenta box. A1, Magnified view of the DREZ area showing at least five axons and their tips (colored arrows). A2, Magnified view of the yellow boxed area in A1, showing an axon and
its tip marked by red arrows. The location and appearance of these axons and their tips remained unchanged on subsequent imaging sessions on days 131 and 140. A3, Magnified view of the axon
in A2 on day 131, illustrating its chronic stability. The day 140 image is a confocal view of the area prepared in a fixed whole mount. B, Confocal analysis of a different mouse that was not imaged
in vivo. Top, L5 root was crushed at the usual location (red arrowhead) and, 4 months later, the spinal cord was analyzed in a whole-mount preparation. White dotted line, Location of axon tips at
the DREZ. Asterisks, Intact axons that were uninjured because of their location lateral to the crush site. Bottom panel, Magnified view of the magenta-boxed area. Almost no axons turned around,
and their tips (white arrows) were found at a location similar to those in mice imaged in vivo on day 140 (cf. Fig. 7A) and on day 4 (cf. Fig. 3B2) after root crush, further evidence of the chronic stability
of axons at the DREZ. Yellow arrowheads, Swellings formed on axon shafts.
4576 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
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Axons do not stop upon encountering astrocytes
At the DREZ, both astrocytes and oligodendrocytes are juxta-
posed to PNS Schwann cells. Following DR injury, astrocytes
proliferate and hypertrophy, occupy the DREZ, and are the first
cells encountered by axons regenerating from the periphery (Big-
nami et al., 1984; Fraher et al., 2002). Stalled axons at the DREZ
were observed to contact astrocytes (Carlstedt, 1985; Fraher,
2000), and reactive astrocytes were thought to form a primary
regenerative barrier at the DREZ. We were intrigued by the un-
expectedly rapid and long-lasting immobilization or stabilization
Figure 8. Axons are rapidly immobilized at the DREZ even after conditioning lesion. Repeated imaging of conditioning lesioned DR axons over 20 d after L5 root crush. Day 10, A schematic
drawing illustrating a conditioning lesion of the ipsilateral sciatic nerve 10 d before DR crush. Day 0, Immediately after the root crush, 10 d after conditioning lesion. L5 root was crushed at the usual
location (red arrowhead). Three crushed axons are shown in the large yellow inset box that magnifies the superficial site of crush (small yellow box). Day 2, All three axons have already extended
neurites across the crush site, illustrating enhanced growth within the root due to a conditioning lesion. Magenta box, An area of the DREZ where several axons were monitored in subsequent
imaging sessions, presented in magnified views in the right panels. Day 4, No axons regenerated through the DREZ (data not shown). The tips of these axons remain in the same location and have
a similar appearance in subsequent imaging sessions on days 7, 9, 13, 15, and 20. Positions of an axon tip relative to other axon tips and landmarks were used to determine axon motility between
imaging sessions. Yellow arrowhead denotes the tip of an axon with a particularly large increase in size over time. These axons were found again after the mouse was killed, and high-resolution
confocal microscopy confirmed the location and appearance of the axon tips [day 20 (fixed)].
Di Maio et al. Dorsal Root Regeneration In Vivo J. Neurosci., March 23, 2011 31(12):4569 4582 4577
Page 9
of axons arriving at the DREZ and won-
dered whether axons become immobi-
lized as they encounter astrocytes. To this
end, we developed a unique immuno-
staining protocol that permitted antibod-
ies to penetrate deep into the surface of
spinal cords prepared in whole mount
(see Materials and Methods). This
method allowed us not only to identify
axons that we imaged in vivo but also to
label simultaneously oligodendrocytes,
astrocytes, and Schwann cells that were
near or directly associated with axons at
DREZ (Fig. 9A, B).
We crushed L5 roots (n 2 mice),
monitored them for 2 weeks in vivo, and
confirmed that most axons in these mice
terminated in the usual location at the
DREZ 4 d after crush, and then remained
immobile (data not shown). Subse-
quently, oligodendrocytes (Fig. 9B) and
astrocytes (data not shown) were immu-
nolabeled in whole mounts and their rela-
tionships with the axons imaged in vivo
were analyzed with high-resolution con-
focal microscopy. As expected (Fraher, 1999; Fraher et al., 2002),
astrocytic processes extended further into the periphery than the
oligodendrocytes, even in the intact, noninjured DREZ (Fig. 9A–
A) and invaded the PNS even more extensively after injury (data
not shown) (compare Fig. 10 A,B). We found that axons did not
stop when they encountered astrocytic processes at the astrocyte-
PNS interface but extended along them and terminated deeper in
the CNS territory containing degenerating oligodendrocytes
(Fig. 9B)(95%, n 46 axon, 2 mice).
Presynaptic differentiation of axons at the DREZ
The rapid immobilization and subsequent swellings often
formed on axon shafts and tips resembled the synaptogenic pro-
cess and prompted us to test whether DR axons form synapses as
they enter the CNS territory of the DREZ. We first immunola-
beled cryostat sections of the DREZ where many stalled axons
were observed (Fig. 10 B–B⬙⬙), with synapse markers such as SV2
or synaptotagmin, together with a GFAP antibody to mark CNS
territory. Whereas no SV2 or synaptotagmin immunoreactivity
existed at the DREZ of uninjured mice (Fig. 10A), numerous,
intense immunopositive profiles were observed in the CNS terri-
tory of the DREZ of injured mice (Fig. 10 B, asterisk), and they
colocalized with tips or adjacent shafts of stalled YFP axons
(Fig. 10B, inset).
We next performed an ultrastructural analysis of axons that
had stopped at the DREZ (Fig. 11). To target our analysis to the
CNS territory of the DREZ where axons had stopped (Fig. 11B,
yellow arrowheads), we placed DiI crystals (Fig. 11A, white ar-
rows) at the completion of in vivo imaging at 13 d after the L5 root
crush. DiI crystals were then photoconverted and used as land-
marks to relocate the area with electron microscopy. This strategy
allowed us to observe abundant axonal profiles at the DREZ,
embedded in non-neuronal profiles such as Schwann cells and
astrocytes (Fig. 11C). These axonal profiles were filled with mi-
tochondria and 40 nm vesicles but lacked the vacuoles and
disorganized microtubules that are typical of dystrophic endings
(Fig. 11 D,E) (Ertu¨rk et al., 2007). Moreover, vesicles and mito-
chondria were differentially distributed within the nerve-
terminal-like profiles so that vesicles were highly clustered onto
one side of electron-dense membranes that resembled active
zones, and mitochondria were distributed toward the other side
(Fig. 11D,E). Although features of presynaptic differentiation
were apparent in these axonal profiles, no indications of differ-
entiation such as postsynaptic densities were observed postsyn-
aptically (Fig. 11 D1,E1, red-pseudocolored), excluding the
possibility that postsynaptic cells are adjacent axons. Together,
these observations indicate that axons became immobilized at the
DREZ by forming presynaptic endings on non-neuronal cellular
elements as they entered the CNS territory of the DREZ.
Discussion
The generally accepted explanation for the regeneration failure at
the DREZ is the combination of growth-inhibitory molecules
and limited intrinsic growth potential. However, growth inhibi-
tors that transiently collapse growth cones cannot account for the
unexpectedly rapid and long-lasting immobilization of YFP
axons that we observed in vivo even after a conditioning lesion.
Furthermore, they rarely turned around at the DREZ and exhib-
ited obvious features of presynaptic terminal endings. We pro-
pose that regeneration fails at the DREZ at least in part because
axons are rapidly and chronically stabilized by induction of pre-
synaptic differentiation (Fig. 12).
Dorsal root regeneration in vivo
The present study is the first to apply in vivo imaging to monitor
DR regeneration directly in living animals. Because we were con-
cerned about the potential confounding effects of artifacts due to
phototoxicity and multiple invasive surgical/anesthetic proce-
dures, we performed extensive control experiments with mice
that did not receive repetitive imaging or surgery. Several obser-
vations convinced us of the reliability of our techniques. First, our
estimated axon elongation rate (1.5 mm/d) was not slower
(rather, it was slightly faster) than previous estimates based on
conventional methods (Ramer et al., 2001a) (1 mm/d). Second,
consistent with an earlier study (Ylera et al., 2009), we observed
that conditioning lesions caused the injured dorsal roots to begin
Figure 9. Axons terminate in CNS territory containing oligodendrocytes. A–A, Low-magnification confocal view of the glial
interface at the DREZ in an intact Thy1-YFPH animal. Oligodendrocytes (A, red) and astrocytes (A, blue) were labeled with MOG
and GFAP antibodies, respectively, in a whole-mount preparation. Note that astrocytes extend further into the periphery than
oligodendrocytes even in intact, noninjured DREZ (A). Axons (green) were omitted. B–B, Confocal view of the glial interface at
the injured DREZ. The L5 root was crushed 2 weeks previously. Oligodendrocytes are degenerating (B, red), whereas astrocytes
invade further into the PNS (data not shown) (cf. Fig. 10A,B). Axons (B, YFP) do not stop when they encounter astrocytic
processes in the interface (data not shown) but terminate deeper in CNS territory containing oligodendrocytes (B, white arrows).
4578 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
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to grow earlier and to extend more neurites than after injury
alone. Third, we observed little variability among YFP axons in
initiation of growth, elongation along the root, or behavior at the
DREZ.
Our observation that YFP axons fail to regenerate across the
DREZ following a conditioning lesion is consistent with results
from previous studies in rats, in which only minor effects were
reported (Chong et al., 1999; Golding et al., 1999; Zhang et al.,
2007). It conflicts, however, with a recent report of significant
regeneration in mice, which the authors attributed to species
differences (Quaglia et al., 2008). It is possible that the capacity
for growth varies greatly among different populations of DRG
neurons in mice and that the latter study emphasized a different
subpopulation, possibly small-diameter axons, which were not
examined in the present in vivo imaging studies. Incomplete le-
sions or labeling artifacts may also be responsible (cf. Steward et
al., 2007). A clear advantage of in vivo imaging was that it permit-
ted us to evaluate immediately the extent of damage and to ascer-
tain whether the lesion was complete or incomplete.
Axons are immobilized, rather than repelled, at the DREZ
Our observation that a conditioning lesion did not promote
growth into the spinal cord agrees with the notion that the failure
is likely due to growth-inhibitory molecules (Golding et al., 1999;
Ramer et al., 2001b), such as CSPGs synthesized by astrocytes
(Rhodes and Fawcett, 2004; Silver and Miller, 2004), Nogo,
MAG, and OMgp present in myelin debris (Oertle and Schwab,
2003; Yiu and He, 2006), and semaphorins expressed by fibro-
blasts (Fawcett, 2006). However, these inhibitors act as repulsive
cues that cause brief growth cone or filopodial collapse and allow
axons to turn and grow away without a significant pause or long-
term immobilization (Raper and Kapfhammer, 1990; Snow et al.,
1990; Drescher et al., 1995; Li et al., 1996). Moreover, DRG axons
extend despite growth cone collapse (Marsh and Letourneau,
1984; Jones et al., 2006; Jin et al., 2009). Some growing axons
appeared to be trapped, forming dystrophic endings when ex-
posed to a gradient of proteoglycans, but these axon endings
remained extremely motile (Tom et al., 2004). Last, axons enter-
ing the DREZ in vivo are accompanied by Schwann cells, which
would provide an alternative growth pathway (cf. Adcock et al.,
2004) or constrain migration of axons into CNS territory
(Grimpe et al., 2005). We therefore anticipated that axons enter-
ing the DREZ would be mobile and dynamic, frequently turning
back to the PNS.
However, we found that YFP DR axons rarely turned
around at the DREZ but terminated abruptly and remained com-
pletely immobile. In our injury paradigm, axons arrived at the
DREZ 4 d after root crush. We often resumed imaging 6 or 7 d
after crush due to residual fluorescence that obscured leading
axon tips. It is unlikely, however, that we failed to observe local
motility of axons that might be particularly substantial soon after
arrival (i.e., 4 –7 d after crush). Conditioning lesions led to earlier
clearing of residual fluorescence (Skuba et al., unpublished ob-
servation), which permitted us to monitor axon tips 4 –7 d after
injury. These axon tips remained immobile at the same location
during this period, without turning, retracting, or growing for-
ward (Fig. 8). The large-diameter DR axons that we monitored in
vivo are, therefore, unexpectedly and quickly immobilized as they
enter the DREZ. Consistent with our in vivo results, neurites
cultured on cryosections of the DREZ rarely turned around and
did not collapse or retract, but became permanently immobile
within 20 min (Golding et al., 1999).
Presynaptic differentiation at the DREZ
In vivo imaging enabled us to target our ultrastructural analysis
specifically to the region of the DREZ where recently arrived axon
tips were abundant. We were able to identify numerous axonal
profiles within a few sections. They often looked “synapse-like”
(data not shown), exhibiting abundant mitochondria and mem-
branous vesicles, and resembled the structures called “synap-
toids” (Carlstedt, 1985; Liuzzi and Lasek, 1987). However, these
previously described “synapse-like” profiles lacked the character-
istic features of synaptic differentiation. It is also known that
Figure 10. Intense immunoreactivity of synapse markers associated with axon tips and shafts at the DREZ. Cross sections of the cervical roots of intact (A–Aⴖⴖ) and crushed (B–Bⴖⴖ) Thy1-YFP16
mice were immunolabeled with antibodies against SV2 (red) and GFAP (blue). The cervical roots (C3–C5) of the injured mouse were crushed peripherally 20 d previously. A, In the intact Thy1-YFP16
mouse, all of the large-diameter DR axons were labeled. DR, Dorsal root; DH, dorsal horn. A, SV2 immunoreactivity is not observed along the DR or at the DREZ. A, GFAP-labeled astrocytes denote
the glial interface at the DREZ (arrow). No SV2 (A) or synaptotagmin (data not shown) immunoreactivity is present in the CNS territory of the DREZ (Aⴖⴖ–Aⴖⴖ). Insets magnify an area of the DREZ.
B, In the injured mouse, numerous regenerating axons stop at the DREZ. The inset is a magnified view of an area of the DREZ showing two axons and their tips. Yellow arrowhead indicates debris of
degeneratingYFP axons. B, Intense SV2immunoreactivity is present at theDREZ where axons terminate. Theinset illustrates intense SV2immunoreactivityassociated with the twoaxon tips and
shafts. B, Astrocytes invade further into the periphery in the injured mouse. Note that SV2 immunoreactivity associated with the axon tips and shafts is present in the CNS territory as marked by
GFAP-labeled astrocytes (Bⴖⴖ, Bⴖⴖ).
Di Maio et al. Dorsal Root Regeneration In Vivo J. Neurosci., March 23, 2011 31(12):4569 4582 4579
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mitochondria and vesicles are abundant
even in nonsynaptic, dystrophic endings
(Ertu¨rk et al., 2007).
By contrast, the “synaptic” profiles that
we encountered exhibited obvious features
of presynaptic differentiation, including ac-
tive zones, differential distribution of mito-
chondria and synaptic vesicles, and absence
of microtubules and vacuoles. Moreover,
they were intensely immunolabeled by syn-
apse markers. These synaptic profiles were
relatively small and did not seem to belong
to the large, club-shaped bulbous endings
that developed in some axons at the DREZ
(e.g., Fig. 8, yellow arrowheads). They more
likely represent those axon endings that did
not increase in size or varicosities that we
often observed on axon shafts near the tips
(e.g., Fig. 9B).
Earlier ultrastructural studies were per-
formed relatively long after dorsal root in-
jury: 6 –9 months (Carlstedt, 1985) and 3
weeks to 3 months (Liuzzi and Lasek, 1987).
These studies therefore could not determine
whether axon endings became “synapse-
like” as they entered the DREZ or whether
they occasionally exhibited a synapse-like
appearance after chronic remodeling. Thus,
our studies provide novel and compelling
evidence that axons become differentiated
into presynaptic terminal endings immedi-
ately after entering the DREZ.
What causes presynaptic differentiation?
Synaptoids were speculated to be trig-
gered by a physiological stop signal asso-
ciated with reactive astrocytes (Liuzzi and
Lasek, 1987). We also observed that por-
tions of the axons exhibiting presynaptic
profiles were in contact with filament-rich
astrocytes (cf. Fig. 11D, E). Importantly,
however, we did not observe abundant in-
termediate filaments in the postsynaptic
cells that were in direct apposition to pre-
synaptic profiles. This observation raises
the possibility that nonastrocytic cell types,
such as NG2 cells, may provide the sta-
bilizing activity or induce the presynaptic
differentiation of regenerating axons at
the DREZ. In support of this notion, (1) the postsynaptic cells did
not exhibit postsynaptic densities, excluding that these profiles
are axon–axon synapses (Bernstein and Bernstein, 1971); (2) ax-
ons did not stop when they encountered astrocytes at the CNS–
PNS border but terminated deeper in the CNS territory; and (3)
presynaptic active zones were thinner than at neuron–neuron
synapses and resembled those reported at neuron–NG2 cell
synapses (Bergles et al., 2000; Lin and Bergles, 2004). Impor-
tantly, NG2 cells are present at the DREZ (Zhang et al., 2001;
Beggah et al., 2005) and stabilize dorsal column axons that mac-
rophages cause to retract at the lesion site (Busch et al., 2010).
The role of NG2 cells is speculative, however, and it is pos-
sible that reactive astrocytes are involved in the process. Recent
studies revealed that astrocytes are capable of inducing and pro-
moting synapse formation, presumably by releasing thrombos-
pondins (Ullian et al., 2004; Wang and Bordey, 2008; Allen and
Barres, 2009; Eroglu et al., 2009). An intriguing possibility, which
we are currently testing, is that astrocytes prevent regeneration at
the DREZ by mediating presynaptic differentiation between ax-
ons and NG2 cells. Heparin sulfate proteoglycans may also be
involved, since they trigger presynaptic assembly in the absence
of specific target recognition (Lucido et al., 2009).
Useful for enhancing regeneration?
Efforts to overcome regeneration failure at the DREZ have in-
cluded enhancing the regeneration capacity of sensory axons with
neurotrophic factors and neutralizing growth inhibitors (Ramer
et al., 2002; Steinmetz et al., 2005; Cafferty et al., 2007, 2010;
Figure 11. Ultrastructural analysis of DR axons stopped at the DREZ, revealing presynaptic differentiation. The L5 root of a
Thy1-YFPH mouse was crushed 13 d previously. A, Low-magnification, transmitted-light view, superimposed on fluorescence
image, of the DREZ in a vibratome slice. Photoconverted DiI crystals (white arrows) were placed in an area where axons were
stopped, and the area between the two crystals was examined in the transmission electron microscope. Yellow arrowheads point
to two axon tips. The asterisk indicates axon debris and marks the same spot in B. B, Magnified fluorescent view of the boxed area
in A. Axons stopped at the usual location in the DREZ, showing axonal debris (asterisk) and stalled axon tips (yellow arrowheads).
C, An electron micrograph of the targeted area of the DREZ showing axonal profiles (pseudocolored green) embedded within
non-neuronal cellular processes. D, Magnified view of a boxed area in C, revealing a nerve-terminal-like profile in contact with a
non-neuronal cellular process (pseudocolored red). Note that the presynaptic profile is filled with differentially distributed mito-
chondria and abundant 40 nm vesicles but lacks vacuoles and disorganized microtubules. D1, An enlarged area of synaptic
contact in D. Vesicles are highly clustered and docked at an electron-dense membrane that resembles an active zone (white
arrows). No postsynaptic densities are present on the non-neuronal cell process. E, Magnified view of another area in C, revealing
a nerve-terminal-like profile of an adjacent axon. E1, An enlarged area of synaptic contact in E. Note that this axon also shows
presynaptic differentiation in contact with a fine non-neuronal cellular process (pseudocolored red) that does not exhibit postsyn-
aptic densities. SC, Schwann cell; As, astrocyte. Scale bars: A, 250
m; B, 100
m; C,1
m; D, 250 nm; E, 200 nm.
4580 J. Neurosci., March 23, 2011 31(12):4569 4582 Di Maio et al. Dorsal Root Regeneration In Vivo
Page 12
Wang et al., 2008; Andrews et al., 2009; Harvey et al., 2009, 2010;
Ma et al., 2010). The effectiveness of these efforts may have been
limited because they did not treat the stabilizing activity at the
DREZ. Conversely, even if axons are prevented from being stabi-
lized at the DREZ, growth inhibitors might nevertheless inhibit
regeneration into and across the DREZ. It is therefore reasonable
to expect the best outcome from combinatorial strategies target-
ing both stabilizing and inhibitory activities. It is, however, worth
pointing out that axons can regenerate along degenerating white
matter (Davies et al., 1999; Kerschensteiner et al., 2005) and that
simultaneous elimination of multiple inhibitory molecules alone
did not promote intraspinal regeneration (Lee et al., 2010a,b)
(but see Cafferty et al., 2010). It is tempting to speculate, there-
fore, that preventing presynaptic differentiation alone, particu-
larly during the early stage of injury before full deposition of
nonpermissive cues (Ramer et al., 2001b), might lead to signifi-
cant regeneration after spinal root injury. It will also be important
to determine whether such stabilizing activity restricts regenera-
tion and/or anatomical plasticity elsewhere in the injured CNS.
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  • Source
    • "The idea that synaptic-like connections form between regenerating axons and reactive glia, and may serve to curtail axonal regrowth after injury, had been suggested many years ago (Carlstedt 1985 ), although the importance of this phenomenon in regeneration failure had largely been abandoned. After a dorsal root crush, even following a peripheral conditioning lesion, injured sensory axons can regenerate rapidly within the proximal root until they reach the dorsal root entry zone (DREZ), a transitional region between the peripheral nervous system (PNS) and the CNS, where they abruptly halt their forward progress and remain (Carlstedt 1985; Liuzzi and Lasek 1987; Di Maio et al. 2011 ). Early studies suggested that, as peripheral axons regenerate toward the CNS, they contact reactive astrocytes, which initiate the early stages of so-called synaptoid formations in close association with the astrocyte surface. "
    [Show abstract] [Hide abstract] ABSTRACT: Animal studies are now showing the exciting potential to achieve significant functional recovery following central nervous system (CNS) injury by manipulating both the inefficient intracellular growth machinery in neurons, as well as the extracellular barriers, which further limit their regenerative potential. In this review, we have focused on the three major glial cell types: oligodendrocytes, astrocytes, and microglia/macrophages, in addition to some of their precursors, which form major extrinsic barriers to regrowth in the injured CNS. Although axotomized neurons in the CNS have, at best, a limited capacity to regenerate or sprout, there is accumulating evidence that even in the adult and, especially after boosting their growth motor, neurons possess the capacity for considerable circuit reorganization and even lengthy regeneration when these glial obstacles to neuronal regrowth are modified, eliminated, or overcome. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.
    Preview · Article · Dec 2014 · Cold Spring Harbor perspectives in biology
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    • "Proteoglycans produced by astrocytes in response to dorsal root injury are generally viewed as factors that prevent injured sensory axons from re-entering the adult spinal cord [26]. More recent studies have provided evidence that growing sensory axons are not repelled when they reach the DRTZ, but cease to grow after making synapse-like contacts with astrocytes within the spinal cord [27]. The GFAP-positive cells in the tubes thus appear to lack factors that induce stable contacts between growing sensory axons and bNCSC associated astrocytes. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The boundary cap is a transient group of neural crest-derived cells located at the presumptive dorsal root transitional zone (DRTZ) when sensory axons enter the spinal cord during development. Later, these cells migrate to dorsal root ganglia and differentiate into subtypes of sensory neurons and glia. After birth when the DRTZ is established, sensory axons are no longer able to enter the spinal cord. Here we explored the fate of mouse boundary cap neural crest stem cells (bNCSCs) implanted to the injured DRTZ after dorsal root avulsion for their potential to assist sensory axon regeneration. Results Grafted cells showed extensive survival and differentiation after transplantation to the avulsed DRTZ. Transplanted cells located outside the spinal cord organized elongated tubes of Sox2/GFAP expressing cells closely associated with regenerating sensory axons or appeared as small clusters on the surface of the spinal cord. Other cells, migrating into the host spinal cord as single cells, differentiated to spinal cord neurons with different neurotransmitter characteristics, extensive fiber organization, and in some cases surrounded by glutamatergic terminal-like profiles. Conclusions These findings demonstrate that bNCSCs implanted at the site of dorsal root avulsion injury display remarkable differentiation plasticity inside the spinal cord and in the peripheral compartment where they organize tubes associated with regenerating sensory fibers. These properties offer a basis for exploring the ability of bNCSCs to assist regeneration of sensory axons into the spinal cord and replace lost neurons in the injured spinal cord.
    Full-text · Article · May 2014 · BMC Neuroscience
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    • "These observations are consistent with the hypothesis that regenerating axons form synapse-like terminals with reactive glia, which was originally advanced by Carlstedt (1985). Advances in imaging have allowed live in vivo studies of acute and chronically injured axons in the lesion environment (Di Maio et al., 2011; Evans et al., 2014; Farrar et al., 2012; Kerschensteiner et al., 2005; Ylera et al., 2009). Ylera et al. (2009) recently used in vivo imaging to demonstrate that chronically injured axons can, indeed, be aroused into a robust regenerative state. "
    [Show abstract] [Hide abstract] ABSTRACT: Astrocytes react to CNS injury by building a dense wall of filamentous processes around the lesion. Stromal cells quickly take up residence in the lesion core and synthesize connective tissue elements that contribute to fibrosis. Oligodendrocyte precursor cells proliferate within the lesion and help to entrap dystrophic axon tips. Here we review evidence that this aggregate scar acts as the major barrier to regeneration of axons after injury. We also consider several exciting new interventions that allow axons to regenerate beyond the glial scar, and discuss the implications of this work for the future of regeneration biology.
    Full-text · Article · Jan 2014 · Experimental Neurology
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