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Original Basic Science—General

Systemic Transplantation of Bone Marrow Mononuclear Cells Promotes Axonal Regeneration and Analgesia in a Model of Wallerian Degeneration

Usach, Vanina PhD1; Malet, Mariana MD2; López, Margarita3; Lavalle, Lucía MD1; Piñero, Gonzalo1; Saccoliti, María MD4; Cueto, Alicia MD5; Brumovsky, Pablo MD, PhD2; Brusco, Alicia PhD3; Setton-Avruj, Patricia PhD1

Author Information
doi: 10.1097/TP.0000000000001478
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Traumatic injury of peripheral nerves has a high incidence worldwide and is associated with partial or total functional loss of the affected limb and disabling neuropathic pain. Of relevance, reinnervation timing and the mechanisms underlying efficient regeneration become critical for favorable clinical evolution.1

Wallerian degeneration (WD) is a pathophysiological process characterized by loss of axon-Schwann cell (SC) contact followed by SC proliferation and myelin breakdown, essential steps for axonal regeneration and remyelination after nerve injury.2 Numerous experiments have shown that SC transplantation promotes remyelination and enhances nerve conduction,3,4 although drawbacks in its therapeutic application include the use of a healthy nerve and a significantly time-consuming culture procedure to obtain enough cells for transplantation. Currently, most efforts in stem cell-based therapies are directed toward characterizing a cell type which, in addition to driving remyelination, is multipotent and simple to obtain, can proliferate in vitro and is easily integrated in the host tissue. The cell types most commonly evaluated for the stimulation of nerve regeneration in different experimental models include bone marrow stromal cells (BMSC),5 olfactory ensheathing cells,6,7 endothelial precursors,8 adipocyte-derived stem cells,9-11 dental pulp–derived stem cells,12 and skin-derived stem cells.13

Recently, bone marrow mononuclear cells (BMMC)14,15 have emerged as a suitable option, as culture is not required and phenotypic rearrangements can be hence avoided.16 BMMC represent a heterogeneous population including BMSC, hematopoietic stem cells,17,18 hematopoietic,18 and endothelial cell precursors19 and produce numerous cytokines and trophic factors promoting recovery in various types of tissue injury.19-21 BMMC have been shown to differentiate into hepatocytes in animal models of hepatic damage,22 whereas, in response to heart infarct, they either become miocytes or contribute to a reduction in inflammation.23,24 BMMC also promote angiogenesis, neuroprotection and neuroregeneration, as observed in rodent models of central25-27 and peripheral28 nervous system injury.

We have previously shown that BMMC migrate, either spontaneously or after transplant, exclusively to the ipsilateral nerve in both irreversible (nerve ligation29) and reversible (sciatic nerve crush30) models of WD; in the latter, BMMC were also shown to colocalize with axonal and SC markers.30 In this context, the present work evaluated systemic transplant as a noninvasive method of BMMC administration and its impact on regeneration, nerve function recovery and pain modulation in rats with sciatic nerve crush.

MATERIALS AND METHODS

Animals

Adult Wistar rats were kept in standard conditions with food and water ad libitum on a 12-hour day/night cycle. Experimental procedures were performed following the guidelines of Comité de Bioética at Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, and in accordance with the NIH Guide for the Care and Use of Laboratory Animals and EU Directive 2010/63/EU for animal experiments of the European Commission. Pain behavior analysis was conducted following the Society for Neuroscience and the International Association for the Study of Pain guidelines for the use of animals in research, and approved by Comité de Ética at Instituto de Investigaciones en Medicina Traslacional.

For each experimental group and survival time, 5 animals were used for immunohistochemical and Western blot analyses, 3 animals for electron and optical microscopy analyses, 6 animals for electrophysiological studies and 11 animals for pain behavior analysis. A matching number of naive animals was used for methodological studies, with the exception of pain behavior analysis.

Sciatic Nerve Crush

Surgery was performed as previously described.30 Briefly, rats (70 days old, 270-300 g body weight) were anesthetized with ketamine (75 mg/kg intraperitoneal) and xylazine (10 mg/kg i.p.) and their right sciatic nerves were exposed and crushed for 8 seconds at mid-thigh level with jeweler’s forceps.

Animals were then divided into a nontreated (sciatic nerve crush plus saline solution injection) and a BMMC-treated (sciatic nerve crush plus BMMC injection) group and sacrificed at different days postinjury (DPI). For pain behavior analysis, a sham group (sciatic nerve exposed but left intact) was also included. At different DPI, the proximal, crush, and distal areas in the ipsilateral nerve and the contralateral nerve were carefully identified, dissected out and used for experiments.

BMMC Isolation

Femurs and tibias from adult Wistar rats weighing 300 to 400 g were dissected. Bone marrow was extruded with α-MEM + 10% fetal calf serum (Natocor, Argentina) and the aspirate centrifuged through a Ficoll-Paque Plus (1.077 g/mL) density gradient, as previously described.29,30 The interface containing the mononuclear cell fraction was used for further experiments.

BMMC Characterization

Flow Cytometry

Freshly isolated BMMC were washed in phosphate-buffered saline (PBS) supplemented with 1% fetal calf serum and then incubated with anti-CD34, a marker of progenitor cells (1/25, goat polyclonal IgG, Santa Cruz Biotechnology, Inc.), anti-CD90, a marker of multipotent cells and fibroblasts (1/25, mouse monoclonal IgG, clone OX-7, Chemicon Int), anti-CD45, a pan-hematopoietic marker (1/200, mouse monoclonal IgG, clone MRC OX-1 Chemicon Int), anti-CD11b, a myelinomonocytic marker (1/200, mouse monoclonal IgG, clone OX-42 Chemicon Int), FITC anti-CD3, a T lymphocyte marker (mouse monoclonal IgG clone G4.18, BD), anti-CD105, a MSC marker (1/25, rabbit polyclonal IgG, Bioss), anti-rat myelin basic protein (MBP) (generously donated by Dr. Campagnoni, UCLA Neuroscience Research Building Department of Psychiatry and Biobehavioral Sciences. LA, USA; 1/25, IgG rabbit polyclonal), anti-rat p75NTR, a nonmyelinated and immature SC marker (1/200, rabbit polyclonal IgG, Abcam), and anti-S100β, a SC marker (1/25, rabbit polyclonal IgG, Santa Cruz Biotechnology, Inc.) for 1 hour at room temperature. For macrophage lineage marker CD68 (1/50, mouse monoclonal IgG clone ED1, Serotec), before incubation with the antibody, cells were fixed in 0.01% paraformaldehyde for 10 minutes and then permeabilized with 0.5% PBS-Tween20 for 15 minutes. After several washes, cells were incubated with Cy3- or Cy2-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature in the dark. Cells were washed again and analyzed with a Becton-Dickinson FACScalibur flow cytometer (San Jose, CA). BMMC autofluorescence and nonspecific secondary antibody binding were used as controls.

RNA Preparation and RT-PCR

Total RNA was extracted from BMMC using RNeasy Mini Kit, according to the manufacturer’s instructions. Reverse transcription-polymerase chain reaction RT-PCR was performed as previously described.31 Primer sequences used are described in Table 1.

TABLE 1
TABLE 1:
Primer sequences

Injection of Freshly Isolated BMMC

Transplant of BMMC was done as previously described.29 Briefly, BMMC-treated rats were transplanted 15 × 106 cells through the caudal artery immediately after sciatic nerve crush. A group of animals were transplanted BMMC labeled with cell tracker orange CMTMR (5-(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). Nontreated animals were injected saline solution.

Preparation of Tissue Sections and Immunohistochemistry

After 7 or 14 DPI, animals were sacrificed and sciatic nerves prepared for immunohistochemistry as described previously.29,30 Anti-rat MBP (1/400, IgG rabbit polyclonal), conditioned medium from the RT-97 hybridoma cell line (against NF-h, axonal marker, 1:10 IgG mouse, a kind gift of Dr. P. Monje. The Miami Project to Cure Paralysis. Miami, USA), anti-rat P0 (myelin marker, 1:500, IgG rabbit polyclonal, a kind gift of Dr. O. Bizzozero. University of New Mexico Health Sciences Center, Albuquerque, NM) and antirat p75NTR (1/200, rabbit polyclonal IgG, Abcam) were used. Goat antirabbit Cy3 or donkey antimouse secondary antibodies (1/200) plus Höechst 32258 (2 μg/mL, Sigma Chem Co) were used accordingly. Analysis was performed using an Olympus FV1000 confocal microscope and an Olympus BX100 epifluorescence microscope.

Western Blot of BMMC and Tissue Proteins

For tissue protein extraction, the proximal and distal areas and the contralateral nerve were dissected at different DPI and tissue was homogenized in 0.32 M sucrose containing protease inhibitors. Protein quantification was done through Bradford method.32

Proteins were electrophoresed on 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinyl difluoride membranes (PVDF) for immunoblotting. Nonspecific protein binding sites were blocked with 5% nonfat dry milk in 0.1% PBS-Tween20. Anti-rat MBP (1/3000, IgG rabbit polyclonal) was used and peroxidase-conjugated goat anti-rabbit (1/10 000) antibody was used accordingly (Jackson Immuno Research). Immunocomplexes were revealed using diaminobenzidine.

Electron and Optical Microscopy Analysis

Three, 7, 14, and 21 DPI, animals were anesthetized and perfused with 4% paraformaldehyde plus 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4. The proximal, crush, and distal area of the ipsilateral nerve and the contralateral sciatic nerve were removed and tissue prepared as previously described.33

Freshly isolated BMMC were processed in the same way for morphological analyses.

Semithin tissue sections were mounted onto glass slides and dyed with 0.5% toluidine blue in sodium carbonate 2.5% (w/v).

Ultrathin sections were collected on 300 mesh copper grids, dyed with 2% (w/v) aqueous uranyl, stained with Reynolds solution and later analyzed in a Zeiss EM 109 T electron microscope. Photographs were obtained using Kodak 5301 films and an Epson Perfection V500 Photo scanner or a GAETA digital camera.

Electrophysiological Studies

Electrophysiological studies were performed in both experimental groups 3, 7, 14, 21, and 60 DPI. In rats under anesthesia, and maintaining body temperature at 37°C, nerve measurements were practiced by means of a portable electromyography instrument (Cadwell Wedge Sierra II, Cadwell Labs, Inc., Kennewick, WA). Recording and ground electrodes were placed in the soleus muscle and the tail, respectively. The ipsilateral sciatic nerve was exposed and the proximal and distal areas were electrically stimulated. The same procedure was carried out in contralateral and naïve nerves. Stimulus intensity was 30 mA and a slight twitching of the limb was observed in all rats. The distal latency and the amplitude of the compound muscle action potential (CMAP) were recorded.

Mechanical Withdrawal Threshold

Hindpaw mechanical threshold was evaluated by using the Von Frey method as previously described.34,35 The withdrawal threshold was taken as the force needed to induce paw withdrawal in at least 2 out of 3 consecutive stimuli. A paw withdrawal reflex obtained with 6 g force or less was considered allodynia (pain-like response to a stimulus that does not normally evoke pain, International Association for the Study of Pain).

Quantitative Analysis

All data were analyzed and quantified by experimenters who were blind to the experimental design. Briefly, microscopy images were analyzed using Image Pro Plus software (version 5.1). For semithin sections, the total number of axons/100 μm2 was calculated in 30 randomly selected fields. In ultrathin sections, the number of myelin sheaths/100 nm was calculated, in addition to g-ratio (axon diameter/axon diameter including myelin sheath). The quantitative analysis of Western blot studies was performed using Gel Pro Analyzer. For each protein, the ratio of integrated optical density band was calculated to total protein content in each line, as measured through Coomassie blue.

Statistical Analysis

Parametric statistics studies were performed between experimental groups by applying analysis of variance (ANOVA), followed either by Dunnett or Tukey test.

Nonparametric statistics was used to analyze effects on pain behavior. Comparison between contralateral and ipsilateral hind-paw withdrawal thresholds was carried out by applying the Wilcoxon matched pairs test. Treatment effects at each DPI were analyzed using the Kruskal-Wallis rank sum test. When appropriate, comparisons among groups were conducted with Dunn multiple comparison post hoc test.

In all cases significance was stated at P less than 0.05.

RESULTS

BMMC Characterization Previous to Transplant

Three different phenotypes were identified by microscopic observation, revealing a heterogeneous population (Figure 1A). RT-PCR analyses showed isolated BMMC to express CD34, CD90, and S100β mRNAs (Figure 1B), but not p75NTR, GFAP, MBP or P0 mRNAs (data not shown). Flow cytometry analyses using FSC/SSC and CD45/SSC gate revealed that purification was effective in enriching the percentage of mononuclear cells (Figure 1C1, regions C, D, and E) and reducing the number of other cell types such as granulocytes (Figure 1C1, region A) and erythroid precursors (Figure 1C1, region B)36 (Figure 1C1). Two subpopulations were found in the BMMC fraction, 1 with low and 1 with high CD45 expression (Figure 1C2). Analysis of different cell markers revealed the presence of a small proportion of CD45low/CD34+ cells (2.3% ± 0.78%, Figures 1C3 and C4), a small proportion of CD45low/CD105+ cells (2.75% ± 0.16%, Figure 1C3 and C4), and a large proportion of CD45+/CD90+ cells (34.1% ± 2.2%, Figures 1C3 and C4). Some T lymphocytes were also found (CD45low/CD3+; 9.52%), and a higher percentage of CD45high cells was observed to express CD68 and CD11b with similar pattern and levels (Figures 1C3 and C4). In terms of SC markers, barely detectable levels of p75NTR, S100β and MBP were measured in BMMC (Figures 1C3 and C4). As expected, the erythroid precursor region was negative for all the markers analyzed, whereas remaining granulocytes were all CD11b+ and weakly CD68+ (data not shown).

FIGURE 1
FIGURE 1:
Morphological (n = 3) and biochemical (n = 6) characterization of BMMC. A1-3, Electron microscopy photomicrographs of freshly isolated BMMC (12 000×; scale bar, 0.4 μm). B, RT-PCR products run in 1% agarose gel for detection of multipotent cell markers CD34 (B1), CD90 (B2) and SC marker S100β (B3), visualized with Sybr Safe. Reactions without template cDNA were run as negative controls. In all cases, from left to right: marker, negative control and independent BMMC samples. C, Flow cytometry analysis of BMMC; dot plot showing different subpopulations and their proportions, A: granulocytes, B: erythroid precursors, C: blasts, D: lymphocytes, E: monocytes (C1); histogram of CD45 expression (purple) and autofluorescence of cells (light blue) (C2); histogram of multipotent markers CD34 and CD90 and mesenchymal stem cell marker CD105; SC markers S100β, p75NTR and MBP (C3); dot plot of CD3, CD68 and CD11b expression (C4). Quantification of cells positive for each marker using FlowJ software (C5). D, Cells dyed with cell tracker orange CMTMR (red) and Hoechst (blue) (D1); analysis of BMMC-CMTMR migration in a naïve nerve and the distal area of the ipsilateral nerve of nontreated and BMMC-treated animals (D2). E, Semithin section images of a naïve nerve and the distal area of the ipsilateral nerve of nontreated and BMMC-treated rats 3 DPI. Asterisks indicate myelin and axon debris, arrowhead indicates a BMM-like cell (40×; scale bar, 20 μm).

BMMC dyed with CMTMR cell tracker (Figure 1D1) were used to confirm migration exclusively to the ipsilateral nerve (Figure 1D2) as previously described.30 Microscopic analysis of semithin sections was used to evaluate nerve morphology. Naive animals exhibited intact axons, SC and fibroblasts (Figure 1E). In contrast, distal areas showed irregular axons with myelin and axon debris (Figure 1E, asterisk) and cells resembling those described in Figure 1A (Figure 1E, arrowheads), which lack the phenotype of constituent tissue cells.

Effects of BMMC on Injured Nerve Morphology

Morphological evaluation was conducted on semithin (light microscopy, Figure 2) and ultrathin (electron microscopy, Figure 3) sections of all tested nerves. Because no significant differences were observed across contralateral nerves, proximal areas, and naive nerves at any of the DPI analyzed (Figures 2A, 3A), assays focused on the crush and distal stumps (Figures 2B, C; 3B, C).

FIGURE 2
FIGURE 2:
Analysis of semithin sections of axonal bundles in nontreated and BMMC-treated animals at different survival times (n = 3, 40×). Semithin sections of A, naive and contralateral nerves and proximal stump of the ipsilateral nerve. B, Crush area of nontreated and BMMC-treated animals. C, Distal area of nontreated and BMMC-treated animals. Scale bar, 20 μm. Arrows indicate intact axons; arrowheads indicate irregular axons; asterisks indicate myelin and axon debris.
FIGURE 3
FIGURE 3:
Ultrastructural analysis of sciatic nerves from nontreated and BMMC-treated animals at different survival times (n = 3, 3000×). Electron microscopy images of A, naive and contralateral nerves and proximal stump of the ipsilateral nerve. B, Crush area of nontreated and BMMC-treated animals. C, Distal area of nontreated and BMMC-treated animals. Scale bar, 2 μm. Arrows indicate intact axons; arrowheads indicate irregular axons; asterisks indicate myelin and axon debris.

Histological and Ultrastructural Analyses

Three DPI, the crush area of both nontreated and BMMC-treated rats showed fewer axons with an irregular structure, some of them smaller in size (Figure 2B), indicating the beginning of a WD process with sharp axonal degeneration, fewer myelinated axons and the appearance of myelin debris (Figure 3B).

Seven DPI, nontreated animals presented typical WD morphology, with large amounts of myelin and axon debris, while BMMC-treated animals showed a partial recovery in axon morphology and a significantly larger number of axons (10.53 ± 1.55 vs 37.48 ± 3.79, P < 0.001). Fourteen DPI, nontreated rats still exhibited significantly fewer axons as compared to the BMMC-treated group (58.86 ± 3.21 vs 81.55 ± 6.5, P < 0.01), the latter also presenting a higher degree of myelination. Finally, 21 DPI, the nontreated group showed the beginning of spontaneous remyelination, with the presence of small irregular axons, whereas BMMC-treated animals presented a remarkable recovery both in axon morphology and number (65.82 ± 4.23 vs 114.32 ± 6.44, P < 0.001) (Figures 2B, 3B).

Distal to the crush, both nontreated and BMMC-treated rats exhibited similar alterations in axon number and structure 3 DPI. Seven DPI, nontreated animals presented typical WD morphology, with large amounts of myelin and axon debris, while BMMC-treated rats showed a slight recovery in axon morphology. Fourteen DPI, nontreated animals also showed less recovery in axon morphology and number than the BMMC-treated group (36.2 ± 4.78 vs 82.3 ± 7.14, P < 0.001). Also, 21 DPI, the nontreated group showed incipient remyelination with the appearance of scarcely myelinated axons, while BMMC-treated rats presented a substantial recovery in axon morphology and number (74.29 ± 4.97 vs 117.02 ± 10.47, P < 0.001) (Figures 2C, 3C).

Regarding the evolution of axonal degeneration-regeneration within each experimental group, the number of axons in the crush area of nontreated rats fell sharply until 7 DPI and began to recover afterwards, though still failing to reach naïve values as late as 21 DPI. In contrast, BMMC-treated rats showed a milder reduction in the number of axons, with recovery beginning as early as 7 DPI and full recovery at 21 DPI (Figure 4A, left panel). When comparing the distal area of nontreated and BMMC-treated rats, an abrupt decrease in axon number was observed in both groups until 7 DPI. However, compared to nontreated rats, BMMC-treated rats presented a much sharper recovery starting at 14 DPI and full recovery at 21 DPI (Figure 4A, right panel).

FIGURE 4
FIGURE 4:
Axon quantification in nontreated and BMMC-treated animals at different survival times (n = 3). A, Percentage of axons in naïve nerves and the crush (left panel) and distal (right panel) areas in both experimental groups as a function of survival time. B, Percentage of highly myelinated axons in naïve nerves and in the crush (left panel) and distal (right panel) areas in both experimental groups as a function of survival time. C, g-ratio of naïve nerves and crush and distal areas of the ipsilateral nerve of nontreated and BMMC-treated animals. Values expressed as the mean ± SEM. Statistical analysis performed through ANOVA followed by Dunnett test or by t-test analysis; in all cases *P < 0.05, **P < 0.01, ***P < 0.001 (each experimental group vs. naïve rats) and #P < 0.05, ##P < 0.01, ###P < 0.001 (comparison between experimental groups). ANOVA, analysis of variance.

A more specific analysis of highly myelinated axons (Figure 4B) in the crush area rendered a smaller proportion in nontreated rats than BMMC-treated rats starting at 7 DPI (47.22 ± 13.1 vs 82.59 ± 3.84) and onwards. In distal areas, a profound decrease in the percentage of highly myelinated axons was observed in both experimental groups, although slower in BMMC-treated rats and evident as from 7 DPI (7.037 ± 4.05 vs 48.53 ± 8.28). g-ratio analysis confirmed that the axons found were myelinated (Figure 4C).

Immunohistochemical Analysis

MBP—the most sensitive protein in this experimental model30—was used to show the evolution of the myelination process after injury. Comparisons between nontreated and BMMC-treated rats were made 7 and 14 DPI, 2 time points of clear degeneration. Immunohistochemical analyses revealed continuous MBP immunoreactivity (IR) in the contralateral nerves of both experimental groups. Seven DPI, loss of continuity and MBP reorganization in clusters in the proximal and distal areas were observed in nontreated rats, in contrast to a more continuous MBP-IR detected in BMMC-treated rats (Figure 5A). Fourteen DPI, nontreated rats were still in the demyelination period but exhibited fewer clusters; in contrast, BMMC-treated rats showed barely any clusters and further improvement in MBP-IR along the ipsilateral nerve (Figure 5B).

FIGURE 5
FIGURE 5:
Immunohistochemical and Western blot detection of MBP in nontreated and BMMC-treated animals at different survival times (n = 5, 60×). A, B, Fluorescence photomicrographs of sections of Contralateral nerves, and proximal and distal areas of ipsilateral nerves after incubation with MBP antiserum (in red) and Höechst (in blue, for detection of nuclei) at 7 and 14 DPI. Scale bar, 30 μm. C, Western blot analysis (magnified for image clarity), Coomassie blue-colored PVDF membrane and quantification of MBP 7 DPI (left panel) and 14 DPI (right panel). MW, molecular weight markers; C, contralateral nerve; P, proximal area; D, distal area. Values expressed as the mean ± SEM. Statistical analysis performed through ANOVA followed by Tukey test; *P < 0.05; **P < 0.01, *** P < 0.001. PVDF, polyvinyl difluoride.

In support of the immunohistochemical results described above, Western blot analyses revealed a decrease in MBP in the ipsilateral nerve of nontreated rats as compared with their contralateral nerve 7 DPI. In contrast, MBP levels in the ipsilateral and the contralateral nerve of BMMC-treated rats were virtually unaffected. Fourteen DPI, nontreated rats only showed a decrease in MBP content in the distal area of the ipsilateral nerve, while BMMC-treated rats again presented similar MBP levels both in the contralateral and the ipsilateral nerves (Figure 5C).

Further support for BMMC effects on regeneration was obtained from immunohistochemical analyses using NF-h, P0, and p75NTR (Figure 6).

FIGURE 6
FIGURE 6:
Immunohistochemical detection of NF-h, P0 and p75NTR in nontreated and BMMC-treated animals at different survival times (n = 5, 60×). A, B and C, Fluorescence photomicrographs of sections of contralateral and distal areas of ipsilateral nerves after incubation with conditioned media for NF-h, antibodies anti-P0 and anti-p75NTR (red) and Höechst (blue, for detection of nuclei) at 7 and 14 DPI. Scale bar, 30 μm.

Effects of BMMC on Injured Nerve Functionality

Electrophysiological analyses were conducted to determine whether histological improvements correlated with functional recovery in BMMC-treated animals. Because no significant differences were observed between naive and contralateral nerves at any of the survival times and groups analyzed, ipsilateral nerves were compared to naive nerves. Figure 7 shows an increase in distal latency between 3 and 21 DPI in both nontreated and BMMC-treated rats, although complete recovery was only reached in the BMMC-treated group 60 DPI (Figure 7A). On the other hand, a sharp decrease in CMAP amplitude was observed between 3 and 21 DPI in nontreated rats and between 3 and 7 DPI in BMMC-treated animals. However, BMMC-treated rats showed a substantial recovery in CMAP amplitude at 14 DPI, and both experimental groups exhibited almost complete recovery at 60 DPI (Figure 7B).

FIGURE 7
FIGURE 7:
Analysis of the distal latency and amplitude of the CMAP in naïve, nontreated and BMMC-treated rats at different survival times (n = 6). A, Distal latency of naive and ipsilateral nerves in nontreated and BMMC-treated animals. B, Amplitude of CMAP of naive and ipsilateral nerves in nontreated and BMMC-treated animals. Values expressed as the mean ± SEM. Statistical analysis performed through ANOVA followed by Dunnett test or byt test analysis; in all cases *P < 0.05, **P < 0.01, ***P < 0.001 (each experimental group vs naive rats) and #P < 0.05, ##P < 0.01, ###P < 0.001 (comparison between experimental groups).

Effects of BMMC on Pain Behavior After Nerve Injury

Nontreated rats showed a considerable decrease in ipsilateral mechanical withdrawal threshold beginning 7 DPI and becoming most evident between 14 and 21 DPI. A progressive recovery was observed starting at 28 DPI, with mechanical withdrawal threshold reaching sham levels 35, 42 and 49 DPI (Figure 8A). In contrast, mechanical withdrawal threshold in BMMC-treated rats virtually never changed, remaining comparable to sham rats throughout survival times analyzed (Figure 8A). Neither sham nor nontreated or BMMC-treated rats exhibited changes in contralateral withdrawal threshold throughout the different survival times (Figure 8B).

FIGURE 8
FIGURE 8:
Analysis of mechanical withdrawal threshold in sham, nontreated and BMMC-treated rats at different survival times (n = 11). A, Values are expressed as median ± median absolute deviation. Statistical analysis performed through Kruskal-Wallis rank sum test followed by Dunn multiple comparison post hoc test; *P < 0.05; ** P < 0.01 (nontreated rats vs sham) and ## P < 0.01 (nontreated vs BMMC-treated rats). B, Table showing the comparison between contralateral and ipsilateral paws of sham, nontreated or BMMC-treated rats between 1 and 49 DPI. Values expressed as the mean ± SEM. Statistical analysis performed using the Wilcoxon matched pairs test; significant differences indicated in bold * P < 0.05; ** P < 0.01.

Comparisons between nontreated and BMMC-treated rats showed statistically significant differences 14 and 21 DPI. Comparisons between sham and BMMC-treated rats only rendered statistically significant differences 14 DPI (Figures 8A, B).

DISCUSSION

Within a model of WD, the present study proves the beneficial effects of systemically transplanted BMMC on regeneration and thus provides insights into a potentially therapeutic approach. Our experimental model encompasses 3 relevant features that may be important for functional nerve recovery and may supersede previous studies using either the mononuclear14,37 or MSC38 fraction of bone marrow cells: first, immediate cell availability and acute intervention minimize time-dependent damage and even allow for autologous transplant (only 60 minutes between bone marrow isolation and BMMC injection)39,40; second, transplant of BMMC using the middle caudal artery constitutes a noninvasive method41; and, last but not least, the spontaneous migration of transplanted cells helps functional recovery. Such an approach also has the potential to lower costs and risk of contamination and reduce the need for Good Manufacturing Practice facilities,42 thus opening doors for new therapeutic strategies.

BMMC have recently become the focus of attention, not only because they are easily obtained, but also because they do not require culturing for expansion and/or differentiation. The heterogeneous population of BMMC is in line with the detectable configurations of cell memory disc fluidity.43 Positive aspects of BMMC transplantation also include cell death delay through transient trophic effects44,45 or effects associated to cell memory disc theory.43 Moreover, a synergic effect has been postulated for the stromal and nonstromal components of BMMC on regeneration, which indicates that BMMC may supersede the BMSC population in their regenerating ability.46 Furthermore, our studies prove the potential of adult BMMC isolated right after injury, which prevents long term maintenance47 and the resulting uncertain effects, such as malignant transformation,48 and enables autologous transplant, thus reducing collateral effects associated to allotransplant.39

Posttransplant images showing the presence of cells lacking a SC phenotype in the distal area confirm the arrival of transplanted cells. This selective migration to the injured nerve30 reinforces our hypothesis of an endogenous recruitment mechanism triggered by nerve injury and suggests the existence of a cross-talk between the injured nerve and the transplanted cells.

Flow cytometry analysis of living cells through CD45/SSC-H gate showed the classical 2 populations in the mononuclear fraction, CD45low associated with blast cells and CD45high related to lymphocytes and monocytes. The presence of contaminating granulocytes was singled out through CD45high expression and increased cellular complexity, whereas the erythroid precursors were identified through CD45 expression and less cellular complexity. Under 20% BMMC expressed pan-macrophage marker CD68 and myeloid lineage marker CD11b. The other population showing CD45+/CD68/CD11b/CD3+ and low granularity proved to reflect lymphoid lineage cells. A group of CD45+/CD105+ cells with middle granularity was identified in the blast region, corresponding to the small proportion of MSC present in the BMMC fraction. Worth pointing out, Wistar rats have a different myeloid/lymphoid proportion (10:90) from other species like humans in peripheral blood,49 which can be extrapolated to bone marrow or BMMC composition. This can explain the low expression levels observed in leukocyte markers CD68 and CD11b and the high abundance of lymphoid lineage cells.

After sciatic nerve crush, neutrophils are the first immune cells to infiltrate the distal area within 8 hours, but their presence is short-lived.50 Within days, circulating monocytes are recruited and become dominant and responsible for complete degeneration.51 Considering the composition of the transplanted cells (9% granulocytes, 8% erythroid precursors and more than 60% mononuclear cells) and their regenerating ability after WD, it could be hypothesized that the small amount of remaining transplanted granulocytes will leave the lesion area within the first hours much like endogenous ones, which might thus confirm that the beneficial effect observed on sciatic nerve regeneration is explained by the mononuclear fraction.

As regard BMMC effect on nerve repair, this article shows that BMMC systemic transplant accelerates the onset of regeneration improving CMAP amplitude, which hints at the presence of regenerated axons. Besides, results from the present work show that a single systemic injection of BMMC completely prevents the decrease in mechanical withdrawal threshold compatible with mechanical hypersensitivity typically observed in rats with sciatic nerve crush.52,53 In fact, BMMC-treated rats remained analgesic throughout the whole tested period, as compared with nontreated rats. Studies analyzing the analgesic role of BMMC are scarce; one of them, however, has shown that BMMC unilateral injections into the hindlimb skeletal muscle of rats with diabetic neuropathy ameliorate mechanical and cold hyperalgesia and improve vascular flow, motor and sensory conduction velocities of the affected nerves.54,55

Our results are also in line with a number of studies using BMSC to control chronic neuropathic pain induced by various injury models. Systemic injection of BMSC has been shown to reduce thermal and mechanical allodynia in rodents subjected to several types of nerve injury.52,56,57 The ipsilateral intramuscular, intranganglionic, and intrathecal administration of BMSC have also been proven effective,56,58,59 with effects appearing to be long-lasting and equally efficient when BMSC are injected either at early or late stages of chronic constriction injury-induced neuropathic pain, at least with the intrathecal approach.57 Finally, BMSC have also succeeded in reducing pain in patients with lumbar disc degeneration60 or osteoarthritis.61

The mechanisms involved in the analgesic action of systemic BMMC remain to be established. However, if evidence on BMSC homing in the lumbar spinal cord, neighboring lumbar dorsal root ganglion and the prefrontal cortex56,57,62 could be extrapolated to BMMC, paracrine actions at the site of nerve injury may be expected.30 These actions against neuropathic pain may involve a reduction in the activation and proliferation of microglia and astrocytes, the suppression of excitatory synaptic transmission and an increase in action potential frequency.57 To sum up, the prevention of neuropathic pain in BMMC-treated animals throughout periods of major behavioral challenge induced by nerve crush suggests that these cells are continually acting as analgesic agents.

A number of mechanisms have been proposed to explain BMMC beneficial effects once they have been recruited. Transdifferentiation to SC,63 acceleration of debris removal,64 and/or release of trophic and growth factors contributing to axonal growth and neuroprotection27,65 have all been implicated. However, neuroimmunomodulation has more recently been postulated as a prevailing mechanism.30,66,67 On the basis of recent evidence on the actions of BMSC, BMMC could be speculated to exert anti-inflammatory effects including the switching of pro-inflammatory to anti-inflammatory macrophages, a reduction in proinflammatory interleukins and the secretion of anti-inflammatory cytokines IL-1055 and TGF-β1,58 all currently under study in our laboratory.

To conclude, the beneficial role of BMMC in nerve injury exposed in the present work, through mechanisms yet to be established, suggests that these cells could be useful adjuvants to anti-inflammatory/analgesic drug treatments in preventing their severe contralateral effects.

ACKNOWLEDGMENTS

The authors thank Dr. María Victoria Rosato-Siri, Dr. Roberto Diez and Ms. María Marta Rancez for their assistance in the elaboration of this article; Marianella Vence for animal care assistance; Biochem Plácida Baz and Biochem Ariel Billordo for flow cytometry assistance; LANAIS-MLE for electron microscopy images; and Mr. Guillermo Gastón for technical assistance.

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