RESEARCH ARTICLE

ErbB2 Blockade with Herceptin (Trastuzumab) Enhances Peripheral Regeneration after Repair of Acute or Chronic Peripheral

J. Michael Hendry, MD,1,2,3 M. Cecilia Alvarez-Veronesi, MASc,1,4 Eva Placheta, MD,5 Jennifer J. Zhang, MD,1 Tessa Gordon, PhD,1 and Gregory H. Borschel, MD1,2,3,4

Objective: Attenuation of the growth supportive environment within the distal nerve stump after delayed peripheral nerve repair profoundly limits nerve regeneration. Levels of the potent mitogen neuregulin and its receptor ErbB2 decline during this period, but the regenerative impact of this change is not completely understood. Herein, the ErbB2 receptor pathway is inhibited with the selective monoclonal antibody Herceptin (trastuzumab) to determine its significance in regulating acute and chronic regeneration in a rat hindlimb. Methods: The common peroneal nerve of Sprague–Dawley rats was transected and repaired immediately or after 4 months of chronic denervation, followed by administration of Herceptin or saline solution. Regenerated motor and sensory neurons were counted using a retrograde tracer 1, 2, or 4, weeks after repair. Distal myelinated out- growth after 4 weeks was quantified using histomorphometry. Immunofluorescent imaging was used to evaluate Schwann cell proliferation and epidermal growth factor receptor (EGFR) activation in the regenerating . Results: Herceptin administration increased the rate of motor and sensory neuron regeneration and the number of proliferating Schwann cells in the distal stump after the first week. Herceptin also increased the number of - ated that regenerated 4 weeks after immediate and delayed repair. Reduced EGFR activation was observed using immunofluorescent imaging. Interpretation: Inhibition of the ErbB2 receptor with Herceptin unexpectedly enhances nerve regeneration after acute and delayed nerve repair. This finding raises the possibility of using targeted molecular therapies to improve outcomes of peripheral nerve injuries. The mechanism may involve a novel inhibitory association between ErbB2 and EGFR. ANN NEUROL 2016;00:000–000

hronic denervation of Schwann cells during nerve viral delivery systems8 can restore the regenerative Cregeneration over distance and time is one of the capacity of chronically denervated nerves. However, these major contributors to poor functional outcomes follow- effects are dose dependent and optimizing their delivery ing peripheral nerve injuries.1 The transient expression of is being actively researched.7–10 Alternative strategies have growth-associated genes by denervated Schwann cells is involved the endogenous support or “protection” of the basis of the decay in the regenerative environment denervated Schwann cells in the distal nerve stump using within the chronically denervated distal nerve stump.2–6 axons from an uninjured donor nerve. We demonstrated Exogenous supplementation of this neurotrophic environ- that multiple nerve isografts, termed “side-to-side ment through microsphere delivery systems7 and adeno- bridges,” allowed axons to regenerate through epineurial

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24688

Received Dec 28, 2015, and in revised form Apr 11, 2016. Accepted for publication May 1, 2016.

Address correspondence to Dr Hendry, Division of Plastic and Reconstructive Surgery, Hospital for Sick Children, 555 University Ave, Roy C. Hill Wing, Rm 5408, Toronto, ON, M5G 1X8, Canada. E-mail: [email protected]

From the 1Division of Plastic and Reconstructive Surgery, Hospital for Sick Children, Toronto, Ontario, Canada; 2Department of Surgery, University of Toronto, Toronto, Ontario, Canada; 3Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 4Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; and 5Division of Plastic and Reconstructive Surgery, Medical University of Vienna, Vienna, Austria

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TABLE . Summary of the Experimental Groups Used in This Study

Cohort Group Sample Size, No. Immediate repair 1 week 16 2 weeks 20 4 weeks 24 Sham 16 Delayed repair Acutely axotomized nerve 2 weeksa 12 repaired to chronically denervated stump (TIB–CP cross suture) 4 weeksb 13 Chronically axotomized nerve 2 weeks 10 repaired to chronically denervated stump (CP–CP suture) The sample size was the number of nerves used in each experimental group. Bilateral surgeries were performed in each rat in all cases. aRetrograde labeling only. bHistomorphometry data only. CP 5 common peroneal; TIB 5 tibial. windows from the side of an intact donor nerve into the examining the effects of Herceptin’s blockade of the side of an injured recipient nerve.11–13 Their opposition ErbB2 receptor on peripheral nerve regeneration in a rat of chronic denervation led to complete reinnervation of model of acute and chronic nerve injury. We report that the denervated muscles by increasing motor and sensory in both models, Herceptin administration has the unex- axonal regeneration 2- to 3-fold, thereby restoring muscle pected effect of accelerating early axon regeneration of weight and contractile force.11,12 However, the molecular both motor and sensory neurons whether or not the dis- mechanisms of this effect are poorly understood and tal nerve stump was chronically denervated, the enhanced therefore investigations were undertaken to implicate the axon outgrowth stimulating Schwann cell proliferation. potent neurotrophic factor neuregulin (NRG) in media- Herceptin was however ineffective if the neurons were ting these effects. chronically axotomized. Findings that Herceptin reduced NRG signals through the ErbB2–ErbB3 hetero- the activation of epidermal growth factor receptors dimer combination and has many well-established roles (EGFRs) provide the basis for a novel role of Herceptin in Schwann cell mitogenesis, dedifferentiation after in peripheral nerve regeneration, because the binding peripheral nerve injury, and redifferentiation after axonal likely blocks the inhibitory heterodimerization of ErbB2 contact with regenerating nerves.14–17 Despite these with EGFR, independent from the NRG pathway. known roles, we reported the paradoxical finding that blockade of the NRG receptor, ErbB2, with the chemo- Materials and Methods therapeutic monoclonal antibody Herceptin (trastuzu- Experimental Design mab)18,19 increased axonal regeneration after immediate We aimed to implicate the NRG receptor, ErbB2, as a key repair. Furthermore, these effects likely occurred inde- regulator of axon regeneration following acute nerve transec- pendently of NRG signaling, as there was no change in tion. To this end, the common peroneal (CP) nerve of thy-1 the levels of ErbB2 activation in the treatment group.20 green fluorescent protein (GFP)-positive rats whose axons flu- 22 Other inconsistencies in the literature include the decline oresce green were transected and repaired either immediately or after a 3-month delay (Fig 1). In the immediate repair in Schwann cell proliferation 18 days postinjury despite cohort, outcomes were analyzed at 1, 2, and 4-week end- persistently elevated levels of soluble NRG14 and the points. In the delayed repair cohort, the relative contributions observation that selective knockout of the ErbB2 receptor of chronic axotomy and chronic denervation to this model on Schwann cells in a regenerating nerve has no impact were isolated by performing 2 types of repair. In every case, 21 on their rate of proliferation 4 and 12 days postinjury. the chronically denervated distal CP nerve stumps was mobi- The purpose of this study was to clarify the role of lized followed by repair to either an acutely axotomized (Sub- ErbB2 signaling in peripheral nerve regeneration by re- group 1) tibial (TIB) nerve or the chronically axotomized

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FIGURE 1: Surgical methods for the immediate repair and chronic denervation experiments. (A) The rat hindlimb was opened, and the common peroneal (CP) and the tibial (TIB) nerves were exposed. The CP nerve was sharply divided and repaired in the same surgery. (B) The 2 cut ends of the CP nerve were sutured with two 9-0 epineurial sutures. This repair was incorporated within a silicone tube as shown in the panel that demonstrates the repaired CP nerve and the TIB nerve. (C) To distinguish between the effects of chronic denervation and chronic axotomy, 2 methods of delayed repair were performed and are pre- sented schematically. The first surgery involved transection of the CP nerve and suturing proximal and distal stumps into adja- cent muscle in opposite directions. After 15.5 weeks, the distal CP nerve stump was either cross-sutured to a freshly cut TIB proximal nerve stump (Subgroup 1) or sutured to the chronically axotomized proximal CP nerve stump (Subgroup 2). (D) The TIB cross-suturing technique involved mobilizing the distal CP nerve stump from its attachment to the adjacent muscle (i, ii) and repairing it to an acutely transected TIB nerve in a tension-free manner (iii, iv).

(Subgroup 2) original proximal CP nerve stump. In each of In all cases, transected nerves regenerated their axons in these cohorts, rats weighed between 250 and 300g. See the the presence or absence of Herceptin, an inhibitory monoclonal Table for a summary of the 6 experimental groups and their antibody specific to the 4D5 epitope of the ErbB2 receptor sample sizes. (Roche Pharmaceuticals, Hoffmann-La Roche, Basel,

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Switzerland). Rats in the experimental group received 2 intra- were positioned prone and the bilateral, repaired CP nerve peritoneal Herceptin doses of 8mg/kg to ensure therapeutic lev- stumps were exposed beyond the knee joint. The CP nerve in els prior to CP nerve transection.23–25 Thereafter, rats in the all cases was sharply transected 10mm beyond the repair site Herceptin group received 8mg/kg of Herceptin diluted to a vol- and the transected tip of the proximal nerve stump was sub- ume of 0.3ml with sterile saline every 3 days. Rats in the con- merged in a Vaseline well containing 10ll of 4% FG. After 60 trol group received 0.3ml of saline injected intraperitoneally minutes, the well was removed and the wound was then irri- every 3 days. gated and closed in layers. All protocols used in this study were approved by the One week following the retrograde labeling procedure, Hospital for Sick Children’s Laboratory Animal Services Com- rats were euthanized with an intraperitoneal injection of sodium mittee (Toronto, Ontario, Canada), and adhered strictly to the pentobarbital (240mg/kg). The rats underwent immediate Canadian Council on Animal Care guidelines. transcardial perfusion with 240ml of ice-cold saline followed by 120ml of 4% paraformaldehyde (PFA). The lumbar enlarge- Surgical Techniques ment containing the sciatic nerve motoneurons and the L4/L5 DRG were harvested and fixed in a solution of 4% PFA with IMMEDIATE REPAIR COHORT. All surgical procedures were 30% sucrose for 5 days. All tissue was embedded in frozen carried out under isoflurane inhalation anesthesia and per- OCT medium (Tissue-Tek; Andwin Scientific, Woodland Hills, formed in an aseptic manner using standard microsurgical tech- CA) and mounted on cryostat blocks for sagittal sectioning of niques with an operating microscope. Both hindlimbs were the spinal cords and cross-sectioning of the DRG. shaved in all the rats and a lateral skin incision was made. The and DRG at 50lm and 20lm thickness, respectively, were intermuscular septum between the biceps femoris and the vastus mounted on Fisher Scientific (Pittsburgh, PA) Superior glass lateralis muscles was divided and the sural, TIB, and CP nerves slides. Motoneurons were counted on every spinal cord section were identified (see Fig 1A). under a fluorescent microscope, the experimenter blinded to The CP nerve was identified and separated from the TIB the group from which the spinal cords were removed for analy- nerve using blunt dissection as far proximally as possible without sis. Counts for sensory neurons involved counting every fifth compromising the . The dissected CP nerve was section and multiplying the raw total by a factor of 5. Final transected sharply with iris scissors 5mm distal to the bifurcation, raw counts were multiplied by a correction coefficient as after which the proximal and distal nerve stumps were opposed described by Abercrombie.27 for immediate repair (see Fig 1A). The nerve was repaired with 2 epineurial 9-0 nylon microsutures that apposed proximal and dis- Histomorphometry tal nerve stumps within a silicone cuff that maximizes epineurial In the immediate repair 4 week cohort and a subset of rats alignment, as described previously,26 and is shown in the exam- from Subgroup 1 from the delayed repair cohort, a 2mm seg- ple of surgery in Figure 1B. The wound was closed in layers. ment of CP nerve was harvested immediately distal to the site Routine postoperative monitoring and analgesia with meloxicam of retrograde labeling. This segment was fixed in 2% glutaralde- (0.5mg/kg) were provided during recovery. hyde, postfixed with 1% osmium tetroxide, ethanol dehydrated, DELAYED REPAIR COHORT. In the first surgery on the and embedded in Araldite 502 (Polyscience, Warrington, PA). delayed repair cohort, the surgical exposure was the same as for Thin (0.6lm) sections were cut using a LKB II Ultramicrotome the immediate nerve repair cohort. During this denervation (LKB-Produckter, Stockholm, Sweden) and then stained with procedure, the proximal and distal stumps of the transected CP 1% toluidine blue for examination by light microscopy. At nerve were sutured in opposite directions to the innervated 3100 overall magnification, the entire nerve cross section was muscle to prevent reinnervation. The second repair was carried captured using Image-Pro Analyzer v9.0 (Media Cybernetics, out after a mean duration of 15.5 weeks of chronic injury. The Rockville, MD) and the number, size and myelination of the repairs were performed in one of two ways as depicted in Fig- axons evaluated using a custom designed program in MATLAB ure 1C and D. The chronically denervated distal CP nerve (MathWorks, Natick, MA). The sections were evaluated for stump was sutured to the chronically axotomized proximal CP overall nerve architecture and quality of the regenerated fibers. nerve stump (Subgroup 2) or cross-sutured to the acutely trans- The number of myelinated axons was counted and the nerve ected proximal stump of the adjacent TIB nerve (Subgroup 1). fiber size and myelination thickness were measured from the entire imaged nerve cross section. Retrograde Labeling In a final surgery, the retrograde fluorescent neurotracer Fluoro- Immunohistochemistry gold (FG; hydroxystilbamidine bis[methanesulfonate]; Sigma, St Louis, MO; #39286; 4% [wt/vol]) was applied to the cut sur- SCHWANN CELL PROLIFERATION ASSAY. Rats from both face of the distal CP nerve stump 10mm from the site of surgi- the Herceptin and saline control groups that had undergone cal repair. Thereby the cell bodies of motor and sensory CP nerve transection with immediate repair 1 week earlier (see neurons within the spinal cord and dorsal root ganglia (DRG) Table) were administered a sterile solution of 100mg/kg bromo- that had regenerated their axons 10mm distal to the repair sites deoxyuridine (Sigma) through intraperitoneal injection. Two were back-labeled with the FG dye. To accomplish this, the rats hours later, the remaining CP nerve distal to the site of

4 Volume 00, No. 00 Hendry et al: Herceptin and Regeneration histomorphometry sample collection was removed for assay of data in the immediate repair cohort used a 2-tailed t test, and cellular proliferation. The dissected CP nerve was straightened those of delayed repair cohort used a 1-tailed t test. An alpha and fixed in 4% PFA with 30% sucrose, embedded in frozen of 0.05 was used in all cases and significance was determined if media, and sectioned at 30lm thickness. Antigen retrieval using p < 0.05. Means and standard error of the mean (SEM) were a brief 5-minute enzymatic digestion with 0.1M proteinase K reported. was performed. Slides were then incubated in 2M HCl for 30 minutes at room temperature to denature the double-stranded Results DNA of all nuclei, followed by neutralization in 0.1M borate Animal Survival buffer (pH 8.3) for 5 minutes. The sections were then blocked All surgeries and intraperitoneal injections of Herceptin with a 5% solution of bovine serum albumin and incubated or saline were tolerated well with 1 exception. One rat in overnight with anti-bromodeoxyuridine (BrdU) monoclonal pri- the Herceptin group died intraoperatively from anesthetic mary antibodies (1:50; Sigma #B8434). The remainder of the complications while undergoing retrograde labeling. The immunostaining procedure followed the general immunohisto- labeling could not be completed, but the CP nerve tissue chemistry protocol outlined below. Double immunostaining distal to the surgical repair site was harvested for immu- with immunoglobulin G antibodies to S100 b-chain (1:50; nohistochemistry and histomorphometry. Santa Cruz Biotechnology, Santa Cruz, CA; sc-7852) and ED1 (1:100; Abcam, Cambridge, MA; ab31630) was performed to Administration of Herceptin Enhances Early label Schwann cells and macrophages, respectively. Proliferating 2 Motor and Sensory Neuron Regeneration after cell density estimates (cells/100lm ) were generated by count- Immediate Repair ing within a measured area of the proximal transected tip of Fifty-two CP nerves from 26 rats were successfully trans- the distal nerve segment that was 12mm distal to the original ected and repaired immediately within a silicone tube repair site. This analysis was carried out using Image-Pro Pre- followed by retrograde labeling and blinded analysis (see mier software (Media Cybernetics). Table, immediate repair cohort). After 1 week, but not 2 EGFR IMMUNOHISTOCHEMISTRY. Six CP nerves from thy-1 and 4 weeks of nerve regeneration, there was a significant GFP transgenic rats underwent transection with immediate increase (p < 0.05) in the number of motor and sensory repair to be analyzed for activated phospho-EGFR expression neurons that regenerated axons 10mm beyond the repair levels after 1 week of regeneration (see Table). Two additional site in the rats that received intraperitoneal injections of nerves, 1 from each of the Herceptin and saline treatment Herceptin as compared to rats that received saline injec- group, were intended for analysis but damaged during tissue tions of the same volume (Fig 2). removal and excluded. The CP nerves were straightened, fixed in 4% PFA with 30% sucrose, and sectioned at 20lm thickness The overall extent of regeneration observed in these on a cryostat. Specimens were postfixed with ice-cold 100% experiments was excellent when compared to the pool of ethanol, rinsed with phosphate-buffered saline (PBS; pH 7.6), uninjured motor and sensory neurons. After 4 weeks of and washed with PBS with 0.1% Triton X-100 (PBST; Sigma). regeneration, motoneuron counts reached 84% of unin- Nonspecific staining was blocked using PBST with 1% bovine jured controls (see Fig 2, dotted line). This was attrib- serum albumin (Fisher Scientific) and 10% donkey serum uted to meticulous 2-suture epineurial repair, (Sigma). Polyclonal anti–phospho-EGFR primary antibodies incorporating the repair site within a silicone cuff, and (1:200; Abcam ab5652) were applied and incubated overnight rigid application of the criteria for counting the neurons at 48C, followed by the fluorescent secondary antibodies (Alexa in the spinal cord and the DRGs (see Fig 1). The tech- Fluor series; Invitrogen, Carlsbad, CA; 1:200). The CP nerve nique of performing the repair within a cuff is believed was imaged longitudinally on either side of the repair site using to limit the extent of extraneural, misguided regeneration a standard fluorescence microscope (DM2000; Leica Biosys- into the extraneural space and was demonstrated to be tems, Wetzlar, Germany). The region of interest proximal to superior to repair without a cuff in a rat model.28 the repair site was delineated clearly by the strong GFP signal of intact axons that had not undergone distal Wallerian degen- Herceptin Administration Increases the Number eration. Within this region, EGFR fluorescence intensities were of Myelinated Axons Regenerating into the normalized relative to the GFP fluorescence intensity of the Acutely Denervated CP Distal Nerve Stump same section using ImageJ software (National Institute of The CP nerve was harvested 10mm distal to the site of Health, Bethesda, MD). A mean relative fluorescence intensity transection and surgical repair for histomorphometric level was derived for nerve sections from Herceptin-treated ani- analysis 2 and 4 weeks after repair. There was no visible mals and saline-treated animals (n 5 3 per treatment group). difference between the nerves of uninjured, sham- Statistical Analysis operated animals from either Herceptin or saline groups Normalcy of the data sets was compared using the Kolmo- (Fig 3). Histomorphometry sections after 2 weeks of gorov–Smirnov goodness-of-fit test (p > 0.05). Comparisons of nerve regeneration were not cut because of the limited

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FIGURE 2: Administration of Herceptin-enhanced motor and sensory neuron regeneration 1 week after immediate repair. Retro- grade tracer was applied to the cut end of the common peroneal nerve at 1, 2, and 4 weeks following immediate repair. (A) Sig- nificantly more motoneurons had regenerated their axons into the distal stump at 1 week after repair in the rats that received intraperitoneal Herceptin compared to saline. (B, C) There was no difference in the number of motoneurons that regenerated at either 2 (B) or 4 (C) weeks in rats that had received saline or Herceptin. (D) Similarly, there were significantly more sensory neu- rons counted in the dorsal root ganglia that regenerated their axons after 1 week in the rats that were administered Herceptin as compared to saline. (E, F) The maximal number of regenerated neurons was observed at 2 (E) and (F) 4 weeks after repair. The error bars represent 1standard error of the mean, and significance is denoted by an asterisk (p < 0.05). [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.] myelination at this time point and excessive debris of the Quantitatively, there were significantly more ongoing Wallerian degeneration. In the 4-week group, myelinated axons counted in distal nerve cross sections 4 CP nerve cross sections from the rats treated with Her- weeks after the CP nerve repair from rats treated with ceptin demonstrated increased density of regenerated, Herceptin (2,752 6 180) as compared with saline- myelinated fibers in the CP nerves compared with the treated animals (2,071 6 220; p < 0.05; Fig 4). There densities in the CP nerves of rats treated with saline. were no significant differences in the fiber and axon

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FIGURE 3: Herceptin increased the number of axons in the distal nerve stump after immediate repair. Representative histomor- phometric cross sections are shown of the common peroneal (CP) nerve 10mm distal to the repair site 4 weeks postoperatively (original magnification, 3100 under light microscopy). (A–D) Representative images of sham-injured (Uninjured) CP nerves treated with Herceptin injections (A) or saline injections (B), 4 weeks after cut-and-repair treated with Herceptin (C), and 4 weeks after cut-and-repair treated with saline (D). (E, F) Magnifications of C and D are shown in E and F to better visualize myelinated axons. Note that regenerated axons in Herceptin-treated rats are dispersed throughout the cross section of the nerve as opposed to clustering within endoneurial channels. diameters, myelin thicknesses, or g-ratio in the Herceptin- ected acutely at the time of delayed repair, or to the chroni- treated animals compared to controls after 4 weeks of cally axotomized proximal CP nerve stump (see Fig 1; regeneration. The frequency distribution of fiber diameters Table, delayed repair cohort). When compared after 2 weeks between these two groups was significantly different (p < of regeneration, Herceptin was found to significantly 0.001) using Kolmogorov–Smirnov test. increase the regeneration of acutely axotomized TIB moto- neurons regenerating into the chronically denervated CP Herceptin Enhances Nerve Regeneration when nerve (Herceptin, 282 6 31; saline, 210 6 24; p < 0.05; Acutely but Not Chronically Axotomized Fig 5, Top). In contrast, no effect of the Herceptin was Motoneurons Regenerate Their Axons through observed on the regeneration of chronically axotomized a Chronically Denervated Nerve Stump motoneurons with the same number of motoneurons regen- The distal stumps of 22 CP nerves were denervated for 15.5 erating into the distal denervated CP nerve in animals weeks and repaired to either an adjacent TIB nerve trans- treated with Herceptin or saline injections (Herceptin, 84 6

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FIGURE 4: Herceptin increased the number of axons in the distal nerve stump after immediate nerve repair. The common peroneal nerve stump >10mm distal to the repair site was harvested 4 weeks postoperatively for histomorphometric analysis of nerve cross sections. (A–E) Herceptin treatment significantly increased the numbers of myelinated axons (A) but did not affect other histomor- phometric indices (B–E). (F) Histograms present distributions of fiber diameters in regenerating nerve between saline and Herceptin treatment groups. The error bars represent 1standard error of the mean, and significance is denoted by an asterisk (p < 0.05). [Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.annalsofneurology.org.]

25; saline 87 6 32; see Fig 5, Bottom). As acutely and Herceptin Administration Increases Axonal chronically axotomized motoneurons both regenerated into Outgrowth of Acutely Axotomized similar chronically denervated distal CP nerve stumps, these Motoneurons into the Distal Denervated CP findings indicate that the neurons and not the distal nerve Nerve Stump stump are the likely site of the enhanced regeneration of the Histomorphometry was performed after 4 weeks of nerve acutely axotomized motoneurons by Herceptin. regeneration following delayed repair of the acutely

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acutely but not chronically axotomized motoneurons that regenerated their axons into the distal nerve stump observed after 2 weeks, irrespective of chronic denervation of the stumps. There was no other difference in structural features of the regenerated nerves between these groups, including axon diameter, g-ratio, and mean fiber diameter. The frequency distribution of fiber diameters between these two groups was significantly different (p < 0.001) using Kolmogorov–Smirnov test.

Herceptin Administration Increases Cellular Proliferation in the Nerve Distal to the Repair Site after 1 Week A possible reason for the Herceptin-induced enhance- ment of axon regeneration by both motor and sensory neurons may be due to enhanced proliferation of Schwann cells in the distal nerve stumps. Using intraperi- toneal injection of a thymidine analogue, BrdU (100mg/ kg), 2 hours prior to retrograde labeling of the motor and sensory neurons, we examined the proliferation of cells in the distal nerve stumps of GFP transgenic rats (Fig 7). The number of proliferating cells was signifi- cantly greater in rats treated with Herceptin (2.3 6 0.32 cells/100lm2, mean 6 SEM) compared with rats treated with saline (0.9 6 0.18 cells/100lm2, mean 6 SEM) 1 week after immediate repair. The proliferating cells were predominantly Schwann cells and macrophages as deter- mined by morphological criteria described by Asbury29 in addition to colabeling with both S100 and ED1. In comparison with the background of DAPI-stained nuclei of all cells within the distal nerve stumps, a clear increase FIGURE 5: Herceptin increased the number of acutely but not of mitotically active BrdU-stained cells is seen amidst the chronically axotomized motoneurons that regenerated into actively degenerating GFP fluorescent axonal debris. chronically denervated distal common peroneal (CP) stumps. Top: The number of acutely axotomized motoneurons that Herceptin Administration Decreases EGFR regenerated axons 2 weeks after repair into a 15.5-week chroni- cally denervated distal CP stump. Bottom: The number of Phosphorylation in the Proximal Stump of the chronically axotomized CP motoneurons that regenerated Regenerating CP Nerve axons 2 weeks after repair into a 15.5-week chronically dener- An alternative mechanism was explored whereby Hercep- vated distal CP stump. The error bars represent 1standard error tin alters the interaction of ErbB2 with EGFR, a receptor of the mean, and significance is denoted by an asterisk (p < 0.05). [Color figure can be viewed in the online issue, which is pathway known to inhibit axonal regeneration. The available at www.annalsofneurology.org.] chronic denervation experiments described above indi- cated that Herceptin might exert its influence on neurons axotomized TIB proximal nerve stump to the distal CP proximal to the repair site. Therefore, comparison of nerve stump (see Table, delayed repair cohort, TIB-CP phosphorylated EGFR activity proximal to the repair site cross suture group). Visualization of nerve cross sections was undertaken between rats treated with Herceptin or taken beyond 10mm from the repair site from rats treated saline 1 week following immediate repair. Interestingly, with Herceptin indicated an increased density of myelin- there was a significant decrease in EGFR immunoreactiv- ated axons compared with sections from rats treated with ity on nerves proximal to the repair sites when compar- saline (Fig 6). Consistent with this, significantly greater ing Herceptin-treated and saline-treated animals (Fig numbers of myelinated axons 10mm beyond the repair 8A). A comparison of EGFR fluorescence intensity, nor- site were counted in Herceptin-treated animals (1,714 6 malized to nerve GFP intensity from the same section, 156) compared with saline-treated animals (1,178 6 262; was consistent with these observations. There was a sig- p < 0.05). This correlates with the increased number of nificant reduction in EGFR fluorescence intensity noted

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FIGURE 6: Herceptin significantly increased the number of acutely axotomized myelinated fibers regenerating into a 15.5-week chronically denervated common peroneal (CP) nerve stump. Nerve tissue 10mm distal to the repair site was harvested 4 weeks after delayed repair of acutely axotomized tibial nerves to chronically denervated CP nerves. Herceptin treatment as compared to saline treatment significantly increased the numbers of myelinated axons (A–C) but did not affect axon size (D), g-ratio (E), or mean fiber diameter (F). The frequency distribution of fiber diameters between saline and Herceptin treatment groups was significantly different when compared using the Kolmogorov–Smirnov test (F). The error bars represent 1standard error of the mean, and significance is denoted by an asterisk (p < 0.05). NS 5 not significant. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.] proximal to the repair site in animals treated with Her- sory neurons after peripheral nerve transection and surgi- ceptin (p < 0.05; see Fig 8B), a finding that suggests a cal repair in rats (see Figs 2 and 5). Back-labeling novel inhibitory action of ErbB2 on axonal regeneration experiments demonstrated more neurons regenerating acting through the EGFR. axons after 1 week and more axons regenerating in distal nerve stumps after 4 weeks of regeneration in rats treated Discussion with Herceptin (see Figs 2–6). The enhanced regenera- This study demonstrated that Herceptin, a systemically tion was isolated to acutely and not to chronically administered chemotherapeutic monoclonal antibody injured neurons, the acutely injured neurons regenerating against the ErbB2 receptor for breast cancer, significantly their axons as well through freshly denervated nerve accelerated early axonal regeneration of motor and sen- stumps as through chronically denervated distal nerve

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FIGURE 7: Herceptin increased cellular proliferation within the distal stump of the common peroneal nerve 1 week following immediate repair. (A, B) Longitudinal sections of common peroneal nerve 12mm distal to the repair site. More mitotically active cells stained for bromodeoxyuridine (BrdU; red) were seen in nerve sections from rats that received Herceptin injections (A) com- pared to rats that received saline injections (B). Inset panels depict only BrdU-labeled proliferating cells for clarity at a 1:1 scale with their adjacent sections. Cell nuclei are counterstained with DAPI (blue) seen among the actively degenerating axonal debris (green). (C) A colabeled Schwann cell (S100) with its sheath encasing a core of axonal debris (green; original magnification, 3100). (D) The proliferating macrophages double-staining with ED1 (blue) and BrdU (red; original magnification, 3100). (E) The significant increase in mean number (1standard error of the mean) of BrdU-labeled proliferating nuclei in Herceptin-treated ani- mals after 1 week is shown in the bar graph. Scale bars in panels C and D represent 10lm. GFP 5 green fluorescent protein. stumps (see Figs 5 and 6). Hence, Herceptin’s efficacy of Given the apparent lack of altered NRG signaling, promoting nerve regeneration overcomes the less permis- we explored alternative mechanisms to explain the effi- sive growth environment of chronically denervated distal cacy of Herceptin in enhancing early nerve regeneration. nerve stumps. An alternative mechanism that we examined was whether Herceptin appears to operate through a mechanism Herceptin blocked the normally strong binding and acti- separate from the NRG pathway, as our previous study vation of the EGFR (also known as ErbB1), a recently had indicated, where ErbB2 receptor activation was not identified inhibitor of axon regeneration.30–33 This possi- affected by its administration despite demonstrations of bility was verified by the significant effect of Herceptin normal pharmacological activity.20 Furthermore, axon- in reducing the EGFR phosphorylation on neurons prox- derived NRG also functions as a critical myelination sig- imal to the repair site in rats treated with Herceptin (see nal postinjury.17 Our finding that myelin thickness was Fig 8). Blockade of this transactivation of inhibitory preserved in the presence of Herceptin (see Figs 4 and EGFR by ErbB2 provides an explanation for the para- 6), the average (6 standard error) g-ratio being 0.52 6 doxical finding that Herceptin enhances peripheral nerve 0.02 as compared to >0.85 in NRG knockout models,17 regeneration independent of NRG signaling. further indicates that Herceptin does not impact on The ErbB receptor family is comprised of 4 highly NRG signal transduction. homologous transmembrane receptors (ErbB1, ErbB2,

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FIGURE 8: Herceptin administration is associated with a reduction in epidermal growth factor receptor (EGFR) activation. (A) Longitudinal sections of common peroneal nerves at the repair site. Note the presence of microsutures. The region proximal to the repair site (outlined, indicating intact proximal axons) demonstrates reduced levels of phospho-EGFR (p-egfr) immunore- activity in rats treated with Herceptin compared with those treated with saline. (B) Quantitation of the relative phospho-EGFR fluorescence intensity on the axons proximal to the repair site is compared for Herceptin-treated and saline-treated rats. There was a significant reduction in the mean intensity (1standard error of the mean) on the axons proximal to the repair site in the presence of Herceptin. Significance is denoted by an asterisk (p < 0.05).

12 Volume 00, No. 00 Hendry et al: Herceptin and Regeneration

ErbB3, and ErbB4) that are versatile because they are rats treated with Herceptin (see Fig 7). Given the capable of interacting and dimerizing in different combi- observed acceleration in axon regeneration and prior nations to produce a breadth of signals for complex sig- observation of unaltered NRG signaling, we hypothesize naling regulation.34–38 The extracellular region of these that this effect relates to earlier axonal contact with receptors has 4 discrete domains.39 Herceptin binds to denervated Schwann cells. This in turn induces a subse- the 4D5 epitope on the extracellular juxtamembrane quent earlier second wave of the axon-induced Schwann domain of the ErbB2 receptor.18,19 cell proliferation first described by Pellegrino and ErbB2 is the preferred binding partner of the other Spencer.46 This indirect influence on Schwann cell prolif- 3 ErbB transmembrane receptors. Interestingly, ErbB2 eration is also consistent with some reports in the litera- has no endogenous ligand of its own, but rather adopts a ture that NRG signaling through Schwann cell–derived constitutively active conformation that forms the strong- ErbB2 is dispensable for proliferation in response to est signaling transducer pairings with the other ErbB nerve injury.14,21 family members.30,34,37,38 These pairings result from the The ErbB family of receptors are known to regulate projection of a binding loop that forms strong hydrogen axon regeneration through soluble and axon-bound NRG bonds with the core of its adjacent binding partner. The acting through the ErbB2 receptor in addition to inhibi- close extracellular pairing aligns the intracellular domains tory regulation by activated Erb1 (EGFR).14,17,21,32,33 of the receptors where the catalytic intracellular domain We now demonstrate that the ErbB2 receptor may nega- of one receptor can transactivate (phosphorylate) the tively regulate axonal outgrowth through its potential 40 other and propagate downstream signals. Importantly, inhibitory association with the EGFR. This expanded this receptor interaction is stabilized by hydrophobic con- role of ErbB2 emphasizes its function of integrating mul- tacts between adjacent receptor juxtamembrane regions tiple signals for regeneration and remyelination and now that are allosterically blocked by binding of the 4D5 epi- possibly as a way to limit vigorous, inefficient sprouting 34,35,41 tope on ErbB2 by Herceptin. at the repair site through the EGFR pathway. One may The hypothesis that EGFR/ErbB2 dimer formation speculate that it could also serve as a form of maturation has an inhibitory effect on peripheral nerve regeneration signal that directs axons to stop elongating and Schwann is supported by the finding that activated EGFR is an cells to start myelinating. established inhibitor of axon regeneration in the central Targeted molecular therapies are increasingly used nervous system. Activated EGFR acts through mecha- to treat a wide range of disease states. From a transla- nisms involving transactivation by an unknown fac- tional perspective, Herceptin is already US Food and 32,33,42 tor. Of note, transactivation of EGFR by ErbB2 Drug Administration–approved and is an established elicits a strong and unique signal that can be blocked by therapy for treating HER2/ErbB2-overexpressing breast in vitro application of antibodies directed at the ErbB2 cancer.18,48,49 Based on the evidence presented in this 30,31,37,43 receptor. Last, EGFR and ErbB2 are both study and the evidence suggesting the inhibitory function expressed on the surface of peripheral motor and sensory of neuronal EGFR activation on peripheral nerve regen- 44,45 neurons as well as Schwann cells. eration,32,33 it would be important to examine how other The findings in this study may also add to our ErbB2 and EGFR inhibitors perform in similar animal understanding of how ErbB2 participates in Schwann and clinical models of peripheral nerve injury. Of note, cell proliferation. Typically, the soluble NRG isoform, dual EGFR and ErbB2 small molecule inhibitors such as known as glial growth factor (NRG type II), is upregu- afatinib and lapatinib are now available and can inhibit lated in the proximal nerve stump of an injured periph- the activation of both receptors in an EGFR–ErbB2 het- eral nerve within 3 days of injury, and its expression erodimer.50 Future work must clarify the functional and coincides with a surge in Schwann cell proliferation sensory outcomes following the administration of Her- 14,46,47 within the distal nerve stump. Thereafter, a second ceptin as an adjunct to the surgical treatment of periph- wave of proliferation proceeds as the front of regenerat- eral nerve injuries. ing axons progresses distally through the nerve stump.46 This second wave of proliferation is triggered by Conclusion Schwann cell contact with the axon-bound NRG I type Unexpectedly, Herceptin promotes nerve regeneration III isoform known as sensory and motoneuron-derived through a mechanism that blocks ErbB2’s transactivation factor.15 Herceptin appeared to have the additional effect of a negative regulator of axon regeneration, the EGFR. of increasing cellular proliferation, with both proliferat- This hypothesis was validated by demonstrating that ing Schwann cells and macrophages identified in greater EGFR is activated in the presence of ErbB2 and that numbers 12mm distal to the repair site after 1 week in EGFR and ErbB2 colocalize on injured neurons. This is

Month 2016 13 ANNALS of Neurology a newly identified pathway that regulates peripheral nerve 11. Ladak A, Schembri P, Olson J, et al. Side-to-side nerve grafts sus- tain chronically denervated peripheral nerve pathways during regeneration. These data also suggest that ErbB2 partici- axon regeneration and result in improved functional reinnervation. pates in Schwann cell proliferation indirectly by increas- Neurosurgery 2011;68:1654–1666. ing axonal contact due to the accelerated outgrowth seen 12. Gordon T, Hendry JM, Lafontaine CA, et al. Nerve cross-bridging with Herceptin administration. to enhance nerve regeneration in a rat model of delayed nerve repair. PLoS One 2015;10:e0127397.

13. Hendry JM, Alvarez-Veronesi MC, Snyder-Warwick A, et al. Side- Acknowledgment to-side nerve bridges support donor axon regeneration into chronically denervated nerves and are associated with characteris- This work was funded by the Canadian Institute for tic changes in Schwann cell phenotype. Neurosurgery 2015;77: Health Research (MOP-114950) and support provided 803–813. through the Hospital for Sick Children, Toronto, 14. Carroll SL, Miller ML, Frohnert PW, et al. Expression of neuregu- lins and their putative receptors, ErbB2 and ErbB3, is induced Ontario. during Wallerian degeneration. J Neurosci 1997;17:1642–1659.

15. Morrissey TK, Levi AD, Nuijens A, et al. Axon-induced mito- Author Contributions genesis of human Schwann cells involves heregulin and The study conception and design were developed by p185erbB2. Proc Natl Acad Sci U. S. A 1995;92:1431–1435. J.M.H., T.G., and G.H.B. Data acquisition and analysis 16. Michailov GV, Sereda MW, Brinkmann BG, et al. Axonal were carried out by J.M.H., M.C.A.-V., E.P., and J.J.Z. neuregulin-1 regulates myelin sheath thickness. Science 2004;304: 700–703. Drafting of text and preparation of figures were per- 17. Fricker FR, Lago N, Balarajah S, et al. Axonally derived formed by J.M.H., T.G., and G.H.B. neuregulin-1 is required for remyelination and regeneration after nerve injury in adulthood. J Neurosci 2011;31:3225–3233.

Potential Conflicts of Interest 18. Carter P, Presta L, Gorman CM, et al. Humanization of an anti- Nothing to report. p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 1992;89:4285–4289.

19. Fendly BM, Winget M, Hudziak RM, et al. Characterization of murine monoclonal antibodies reactive to either the human epi- dermal growth factor receptor or HER2/neu gene product. Cancer References Res 1990;50:1550–1558. 1. Sulaiman OAR, Midha R, Gordon T. Pathophysiology of surgical 20. Placheta E, Hendry JM, Wood MD, et al. The ErbB2 inhibitor Her- nerve disorders. In: Winn HR, ed. Youmans neurological surgery. ceptin (trastuzumab) promotes axonal outgrowth four weeks after Philadelphia, PA: Saunders/Elsevier, 2011:2368–2379. acute nerve transection and repair. Neurosci Lett 2014;582:81–86.

2. You S, Petrov T, Chung PH, Gordon T. The expression of the low 21. Atanasoski S, Scherer SS, Sirkowski E, et al. ErbB2 signaling in affinity nerve growth factor receptor in long-term denervated Schwann cells is mostly dispensable for maintenance of myelin- Schwann cells. Glia 1997;20:87–100. ated peripheral nerves and proliferation of adult Schwann cells after injury. J Neurosci 2006;26:2124–2131. 3. Hoke€ A, Gordon T, Zochodne DW, Sulaiman OAR. A decline in glial cell-line-derived neurotrophic factor expression is associated 22. Magill CK, Moore AM, Borschel GH, Mackinnon SE. A new model with impaired regeneration after long-term Schwann cell denerva- for facial nerve research. Arch Facial Plast Surg 2010;12:315–320. tion. Exp Neurol 2002;173:77–85. 23. Pegram M, Ngo D. Application and potential limitations of animal 4. Hoke€ A, Redett R, Hameed H, et al. Schwann cells express motor models utilized in the development of trastuzumab (Herceptin): a and sensory phenotypes that regulate axon regeneration. case study. Adv Drug Deliv Rev 2006;58:723–734. J Neurosci 2006;26:9646–9655. 24. Guler€ M, Yilmaz T, Ozercan I, Elkiran T. The inhibitory effects of 5. Boyd J, Gordon T. Glial cell line-derived neurotrophic factor and trastuzumab on corneal neovascularization. Am J Ophthalmol brain-derived neurotrophic factor sustain the axonal regeneration 2009;147:703.e2–708.e2. of chronically axotomized motoneurons in vivo. Exp Neurol 2003; 183:610–619. 25. Frank RT, Edmiston M, Kendall SE, et al. Neural stem cells as a novel platform for tumor-specific delivery of therapeutic antibod- 6. Brushart TM, Aspalter M, Griffin JW, et al. Schwann cell pheno- ies. PLoS One 2009;4:e8314. type is regulated by axon modality and central-peripheral loca- tion, and persists in vitro. Exp Neurol 2013;247:272–281. 26. Furey MJ, Midha R, Xu Q-G, et al. Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate. Neu- 7. Wood MD, Kim H, Bilbily A, et al. GDNF released from micro- rosurgery 2007;60:723–732; discussion 732–733. spheres enhances nerve regeneration after delayed repair. Muscle Nerve 2012;46:122–124. 27. Abercrombie M. Estimation of nuclear population from microtome sections. Anat Rec 1946;94:239–247. 8. Blits B, Carlstedt TP, Ruitenberg MJ, et al. Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation 28. Sulaiman OAR, Gordon T. Transforming growth factor-beta and combined with intraspinal adeno-associated viral vector-mediated forskolin attenuate the adverse effects of long-term Schwann cell expression of glial cell line-derived neurotrophic factor or brain- denervation on peripheral nerve regeneration in vivo. Glia 2002; derived neurotrophic factor. Exp Neurol 2004;189:303–316. 37:206–218.

9. Gordon T. The role of neurotrophic factors in nerve regeneration. 29. Asbury AK. Schwann cell proliferation in developing mouse sciatic Neurosurg Focus 2009;26:E3. nerve: a radioautographic study. J Cell Biol 1967;34:735–743.

10. Boyd JG, Gordon T. A dose-dependent facilitation and inhibition 30. Klapper LN, Vaisman N, Hurwitz E, et al. A subclass of tumor- of peripheral nerve regeneration by brain-derived neurotrophic inhibitory monoclonal antibodies to ErbB-2/HER2 blocks crosstalk factor. Eur J Neurosci 2002;15:613–626. with growth factor receptors. Oncogene 1997;14:2099–2109.

14 Volume 00, No. 00 Hendry et al: Herceptin and Regeneration

31. Pinkas-Kramarski R, Soussan L, Waterman H, et al. Diversification of 41. Cho H, Leahy DJ. Structure of the extracellular region of HER3 Neu differentiation factor and epidermal growth factor signaling by reveals an interdomain tether. Science 2002;297:1330–1333. combinatorial receptor interactions. EMBO J 1996;15:2452–2467. 42. Douglas MR, Morrison KC, Jacques SJ, et al. Off-target effects of 32. Koprivica V, Cho K-S, Park JB, et al. EGFR activation mediates epidermal growth factor receptor antagonists mediate retinal gan- inhibition of axon regeneration by myelin and chondroitin sulfate glion cell disinhibited axon growth. Brain 2009;132(pt 11):3102– proteoglycans. Science 2005;310:106–110. 3121.

33. Leinster VHL, Joy MT, Vuononvirta RE, et al. ErbB1 epidermal growth 43. Olayioye MA, Graus-Porta D, Beerli RR, et al. ErbB-1 and ErbB-2 factor receptor is a valid target for reducing the effects of multiple acquire distinct signaling properties dependent upon their dimeri- inhibitors of axonal regeneration. Exp Neurol 2013;239:82–90. zation partner. Mol Cell Biol 1998;18:5042–5051.

34. Garrett TPJ, Mckern NM, Lou M, et al. The crystal structure of a 44. Audisio C, Nicolino S, Scevola A, et al. ErbB receptors modulation truncated ErbB2 ectodomain reveals an active conformation, in different types of peripheral nerve regeneration. Neuroreport poised to interact with other ErbB receptors. Mol Cell 2003;11: 2008;19:1605–1609. 495–505. 45. Pearson RJ, Carroll SL. ErbB transmembrane tyrosine kinase 35. Garrett TPJ, McKern NM, Lou M, et al. Crystal structure of a trun- receptors are expressed by sensory and motor neurons cated epidermal growth factor receptor extracellular domain bound projecting into sciatic nerve. J Histochem Cytochem 2004;52: to transforming growth factor alpha. Cell 2002;110:763–773. 1299–1311.

36. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling 46. Pellegrino RG, Spencer PS. Schwann cell mitosis in response to network: receptor heterodimerization in development and cancer. regenerating peripheral axons in vivo. Brain Res 1985;341:16–25. EMBO J 2000;19:3159–3167. 47. Stoll G, Muller€ HW. Nerve injury, axonal degeneration and neural 37. Karunagaran D, Tzahar E, Beerli RR, et al. ErbB-2 is a common regeneration: basic insights. Brain Pathol 1999;9:313–325. auxiliary subunit of NDF and EGF receptors: implications for 48. Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of breast cancer. EMBO J 1996;15:254–264. weekly intravenous recombinant humanized anti-p185HER2 mono- 38. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. clonal antibody in patients with HER2/neu-overexpressing meta- Nat Rev Mol Cell Biol 2001;2:127–137. static breast cancer. J Clin Oncol 1996;14:737–744.

39. Citri A, Skaria KB, Yarden Y. The deaf and the dumb: the biology 49. Agus DB, Akita RW, Fox WD, et al. Targeting ligand-activated of ErbB-2 and ErbB-3. Exp Cell Res 2003;284:54–65. ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2002;2:127–137. 40. Macdonald-Obermann JL, Piwnica-Worms D, Pike LJ. Mechanics of EGF receptor/ErbB2 kinase activation revealed by luciferase 50. Geuna E, Montemurro F, Aglietta M, Valabrega G. Potential of fragment complementation imaging. Proc Natl Acad Sci U S A afatinib in the treatment of patients with HER2-positive breast 2012;109:137–142. cancer. Breast Cancer Targets Ther 2012;4:131–137.

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