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Address correspondence to Caroline Pot, Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Tel.: 41-79-520-8000. Fax: 41-1-635-3303. E-mail: caroline.pot{at}access.unizh.ch
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Abstract |
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Key Words: Nogo-A; growth-inhibitory protein; regeneration; peripheral nervous system; axonal repair
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Introduction |
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To investigate in vivo the inhibitory characteristics of Nogo-A, we generated transgenic mice expressing the rat nogo A gene under the inducible control of the Schwann cellspecific P0 promoter (unpublished data). P0 is the major structural protein of peripheral myelin, and previous studies demonstrated the usefulness and specificity of the P0 promoter for transgene expression in Schwann cells (Messing et al., 1992).
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Results and discussion |
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In the toe pinch reflex, before sciatic nerve crush, all mice showed equal sensitivity; a pinch of toe 3, 4, or 5 (tested separately) reliably induced a rapid retraction of the leg (digits 1 and 2 are innervated by the saphenous nerve and were therefore not tested). After the crush, the response was totally abolished in all animals. The time taken for the injured hindlimb to show any degree of a response to the stimulus was noted. Digit function recovery occurred in a medial to lateral direction in all mice (Fig. 4 B). The recovery was monitored over 30 d, and in many of the transgenic mice the sensitivity of the last two digits did not appear. The toe pinch reflex values from those animals were extrapolated from the recovery curve. The percentage of animals responding 30 d after the lesion was calculated (Fig. 4 C). For the digit 4 of control animals, 91% showed sensitivity (75% for digit 5) compared with 80% for Tg16 (60% for digit 5) and 25% for Tg11 (25% for digit 5) (Fig. 4 C). Thus, the Nogo-A transgenic mice showed a significant delay in the recovery of the toe pinch reflex.
Spinal motoneurons were retrogradely labeled with Fluorogold 7, 14, and 28 d after the crush from a site 7 mm distal to the lesion (Fig. 5, A and C). Fluorogold was chosen as a marker because it is a long-lasting and nondiffusible tracer that undergoes rapid retrograde axonal transport. Nonlesioned transgenic mice had the same number of motoneurons as wild-type mice. A time course with wild-type mice was established to determine the time at which the transgenic animals were to be traced after the lesion. By 5 d, no motoneuron axons had reached the injection site. Between 7 and 14 d, a gradual increase in the number of retrogradely labeled motoneurons was seen and the peak number of motoneurons was reached 17 d after the lesion. Therefore, 7 and 14 d after the lesion were chosen to analyze the motor axonal regeneration, and a later time point (28 d) to analyze axon numbers at a time when functional recovery in control animals was complete. 7 and 14 d after a sciatic nerve lesion, a significantly lower number of motor axons had grown 7 mm past the lesion in transgenic animals compared with control animals (Fig. 5 B), indicating a slower regeneration of the motoneuron axons. 28 d after the lesion, only a small difference was noted in both transgenic groups compared with the control group, indicating that almost all the axons had reached the injection site. The discrepancy between this last result and the behavioral data (Fig. 4) could be due to the fact that 28 d after the lesion, most of the motoneuron axons of the transgenic mice had reached the injection site 7 mm distal of the lesion but not the muscle target (15 mm away from the lesion), or not the appropriate target. Correct locomotion is also dependent on regeneration and correct targeting of sensory axons, which were not studied yet.
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Taken together, these data show that postnatal expression of Nogo-A in Schwann cells results in a significant delay in axon regeneration in the denervated adult mouse sciatic nerve. The selective postnatal expression prevented possible effects of Nogo-A on the development of Schwann cells and axons, or on the process of myelin formation. The doxycycline treatment cannot be responsible for the observed differences in regeneration because all the animals, controls and transgenics, were treated with the drug. It is also unlikely that the reporter lacZ had a negative effect because myelin and Schwann cell morphology was normal, and, previously, the reporter had been used in Schwann cells without any signs of toxicity (Arroyo et al., 1998).
Efficient and successful regeneration in peripheral nerve is influenced by Schwann cellreleased neurotrophic factors, extracellular matrix molecules, and basement membrane components. The expression of Nogo-A slows down the rate of axon growth compared with that seen in a normal denervated sciatic nerve. The difference in the regenerative potential observed between the two nogo Aexpressing mouse lines (Fig. 4) is probably related to their difference in expression levels of nogo A (Fig. 2 B). P0 promoter activity is also known to be temporarily decreased distal to a lesion in the context of Schwann cell dedifferentiation (Gupta et al., 1988), thus possibly creating a window of increased opportunity for regenerating axons, although significant levels of Nogo-A protein may still be present on myelin debris. P0 promoter is known to be strongly induced starting 7 d after peripheral nerve lesion when the remyelination takes place (Gupta et al., 1988), and thus could represent an impediment for late growing axons. All these data strongly indicate that Nogo-A is a potent neurite growth inhibitor that can override multiple strong regeneration-enhancing factors known to be present in lesioned peripheral nerves.
Nogo-A, constitutively present in CNS myelin, biases the balance of growth-promoting and -inhibitory factors toward inhibition of regeneration, leading to a restriction of plasticity and functional recovery. The inhibitory property of Nogo-A is further demonstrated by enhanced regeneration, compensatory sprouting, and functional recovery of lesioned CNS tracts resulting from in vivo application of the monoclonal antibody IN-1 (Schnell and Schwab, 1990; Thallmair et al., 1998; Merkler et al., 2001; Papadopoulos et al., 2002). Similar results were obtained with autoantibodies against myelin (Huang et al., 1999) and antibodies against specific regions of the Nogo-A molecule (unpublished data). All these results suggest that blockade of Nogo-A signaling by antibodies, receptor-blocking reagents (GrandPre et al., 2002), or drugs acting at the postreceptor level represent exciting experimental approaches for therapies of CNS injuries, including spinal cord or brain trauma and stroke.
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Materials and methods |
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Controlling the doxycycline-regulatable expression of nogo A in HeLa cells
HeLa cells, stably expressing the tTA construct, were transfected with pBI-3-nogoA construct using FuGENE 6 transfection reagent (Roche). 3 µg/ml doxycycline solution was added to the culture medium for 24 h. The cells were fixed for 15 min in 4% paraformaldehyde (PFA) and immunostained for myc and Nogo-A, and the activity of ß-galactosidase was assessed. For ß-galactosidase staining, the cells were incubated for 1 h at 37°C in a solution containing 1 mg/ml X-Gal (Roche), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 0.1% Triton X-100, and 2 mM MgCl2 in PBS. For immunofluorescence, the cells were permeabilized with 0.1% Triton X-100 in PBS and blocked with 10% FCS. Mouse anti-myc antibodies (clone 9E10; Sigma-Aldrich) were incubated simultaneously with the rabbit antiNogo-A antiserum 472 (Chen et al., 2000) for 30 min at RT. Rabbit antibodies were visualized by antirabbit FITC-conjugated secondary antibody, and mouse antibodies by antimouse TRITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories).
Mouse breeding and genotyping
All transgenic lines were created using the hybrid strain B6D2F1. For both constructs, founder lines were crossed for two to six generations into the inbred strain C57BL/6. Double transgenic mice were obtained from crosses between mice heterozygous for each transgene. The line with the strongest expression of the transactivator (Tg-[P0Cx-rtTA2] 693 Zbz) and of two independent reporter lines (Tg-[Nogo/LacZ] 728 and 732 Zbz) were crossed, yielding the double transgenic lines Tg11 and Tg16. The genotypes of the mice were determined by PCR analysis of genomic DNA isolated from mouse tails. Primers 5'-CACGGCGGACAGAGCGTACAG-3' and 5'-CCGAATTCACCATGTCTAGACTGG-3' were used to amplify a 600-bp fragment from the rtTA2 construct, and primers 5'-CCTGCTGCATCTGAGCCTGTG-3' (first exon of nogo A) and 5'-ACAGGTGCTACTACTGACATCTG-3' (second exon of nogo A) to amplify a 548-bp fragment from the cDNA for the tetOnogo A construct. Transgene expression was induced at birth by replacing normal drinking water with 5% sucrose containing doxycycline (2 mg/ml). The mice were kept under doxycycline until the end of the experiments.
Whole mount sciatic nerve preparation and immunofluorescence microscopy
Sciatic nerves of control and nogo A transgenic animals were dissected and fixed by immersion in 4% PFA for 20 min on ice. For ß-galactosidase staining, the whole sciatic nerve was incubated overnight at 37°C in X-Gal solution. After staining and photographing, the tissue was washed in PBS, postfixed for 1 h in 4% PFA, and then frozen in the same block. Cryostat sections (20 µm) were cut. For immunofluorescence, the sections from wild-type and transgenic animals were processed on the same slide by permeabilization with 0.030.3% Triton X-100 in PBS and blocked by 1% BSA or 2% rat serum. Cy3-conjugated mouse anti-myc antibodies (clone 9E10; Sigma-Aldrich) were incubated simultaneously with a rabbit antiNogo-A antiserum 472 or a rabbit anti-S100 antibody (Dako). The sections were analyzed using a confocal ZEISS LSM 410 microscope or a ZEISS Axiophot microscope equipped for epifluorescence.
Ultrastructural analysis
Mice were deeply anesthetized with pentobarbital and transcardially perfused by Ringer solution followed by 2% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer. All fixative and buffer solutions were supplemented with 2 mM CaCl2. Sciatic nerves were removed, placed in fresh fixative for 3 h, and then placed in cacodylate buffer overnight at 4°C. Tissues were postfixed in 2% OsO4 for 2 h, serially dehydrated, and embedded in Epon. Semi-thin sections were stained with toluidine blue and viewed under an Olympus microscope. Ultrathin sections (90 nm) were analyzed using a ZEISS EM 902.
Surgery
Mice (1626 g) were deeply anesthetized by intraperitoneal injection of fentanyl citrate (0.0189 mg/100 mg), fluanisone (0.6 mg/100 mg; Hypnorm; Jansen Biochemica), and midazolam (0.6 mg/100 mg; Dormicum; Hoffmann-La Roche). The sciatic nerve was exposed in the upper thigh and freeze crushed with watch-maker forceps that had been previously cooled in liquid nitrogen for 30 s. The epineurium remained intact. The crush site was marked with charcoal powder. The experiments were performed in conformation with the Swiss animal protection laws and were approved by the Cantonal Veterinary Department of Zurich.
Behavioral tests
Rotarod.
Animals were tested for two consecutive days on a rotating rod; the first day for the acceleration test and the second day for the fatigue test. The acceleration test was performed by placing a mouse on the revolving rod. Once balanced, the rod was accelerated from 4 to 40 rpm over a 300-s period. The fatigue test was performed at a fixed speed of 40 rpm for 300 s. For both tests, the latency for the mouse to fall off the rod was determined with a cut-off of 300 s. The mice were given five trials with a 20-min rest interval between each trial. The average time on the rod for each mouse was used for analysis.
SFI.
After first pressing their hind paws onto an ink pad, the animals were tested along a confined 60-cm-long walkway lined with plain white paper. The tracks were analyzed according to the empirical equation determined by de Medinaceli et al. (1982).
Toe pinch reflex.
Recovery of pain sensitivity was tested on awake mice by lightly pinching the most distal portion of the last three digits of the lesioned hind limb with forceps. The first day after the lesion at which foot withdrawal was restored was recorded.
Motoneuron tracing and histological analysis
Motoneurons tracing.
7, 14, and 28 d after surgery, crystals of Fluorogold (Molecular Probes) were applied 7 mm distal to the crush lesion onto the cut nerve. 48 h later, mice were deeply anesthetized with pentobarbital and perfused with 4% PFA. Spinal cords were removed and processed for cryosectioning as described by Sagot et al. (1998). Cryostat serial sections (30 µm) were viewed under fluorescence illumination, and Fluorogold-labeled motoneurons, identified by size, shape, and location in the ventral horn, were counted on every section.
Regenerating axons.
7 d after surgery, sciatic nerves were removed, fixed as described above, and cut transversally 4 mm distal to the lesion. After permeabilization with ethanol/acetic acid (95:5; 15 min), the sections (15 µm) were stained with a rabbit antiserum against GAP-43 (Chemicon) or with a mice neurofilament antibody, SMI-32 (Sternberger Monoclonals Inc.). Areas of 10 µm2 were randomly photographed from the two or three fascicles of each nerve at 630x, and all labeled axons were counted by two different, blinded observers.
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the Swiss National Science Foundation (No. 31-63633.00 to M.E. Schwab and No. 31-55525.98 to P. Berger and U. Suter), the National Center of Competence in Research "Neural plasticity and repair," and the Spinal Cord Consortium of the Christopher Reeve Paralysis Foundation.
Submitted: 17 June 2002
Revised: 22 July 2002
Accepted: 30 July 2002
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References |
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