Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11716-11720, December 1992 Neurobiology Insulin-like growth factor II stimulates motor nerve regeneration (somatomedin/sdatic nerve/axon/neurotropiuc/neurite) STEPHANIE L. NEAR*, L. RAYMOND WHALEN*, JAMES A. MILLERt, AND DOUGLAS N. ISHII0§ Departments of *Anatomy and Neurobiology, *Physiology, and §Biochemistry, Colorado State University, Fort Collins, CO 80523; and tAmgen Inc., Thousand Oaks, CA 91320 Communicated by Dale Purves, September 14, 1992 (receivedfor review, June 17, 1992)

ABSTRACT Injury to mammalian motor nerves can lead can increase the regeneration of motor axons in vivo and (ii) to paralysis, but relatively succul regeneration may occur endogenous IGFs contribute to the spontaneous regeneration when conditions are favorable. Elucidation of the mcanism of motor axons. A positive finding for hypothesis i is poten- upholding successful regeneration is of theoretical and clincal tially ofclinical significance, irrespective ofwhether hypoth- interest. In this study, the hypothesis that insulin-like growth esis ii is validated. factor H (IGF-ll) can stimulate motor nerve regeneration was tested. When IGF-H was infused continuously near a site of MATERIALS AND METHODS crush on the sciatic nerve, the distance of motor axon regen- eration was increased snlfcantly in rats. In contrast, spon- Materials. Recombinant human IGF-II (Amgen) was >97% taneous regeneration was inhibited when an anti-IGF-H anti- pure based on HPLC; only a single band was visible on serum was infused through a "window" in the epineurium. reducing SDS/PAGE. To produce antiserum, 0.3 mg of Thus, infused IGF-il can increase, and endogenous IGFs can keyhole limpet hemocyanin was added to 50 pg of IGF-II in support, the regeneration of motor axons in lesioned nerves. 50 mM sodium phosphate buffer (pH 6.8), glutaraldehyde was added to 1% (vol/vol), and the mixture was incubated for 30 min at room temperature. Rabbit polyclonal anti-hIGF-II Successful regeneration often is encountered following injury antiserum 489-42 (Amgen) had a titer of 1:30,000 in ELISAs. to the peripheral nervous system. Nevertheless, paralysis This antiserum cross-reacts 10%6 with IGF-I, but cross- can result from injury to motor axons in nerves, particularly reactivity with insulin was not observed. when lesions are in proximal nerve regions (closer to the IGF-II and its antiserum were each prepared in Roswell spinal cord). Such paralysis might be reduced in incidence Park Memorial Institute medium 1640 (RPMI), sterilized by someday with improved understanding of the mechanisms passage through 0.2-gum filters, and stored in aliquots at supporting successful regeneration. The consequences of -20'C. In addition, 30 jug of IGF-ll per ml and 30 Al of motor nerve injury continue to pose a serious medical, antiserum per ml in RPMI were incubated together at 37rC for economic, and societal problem. 3 hr, incubated at 40C for 30 min, and centrifuged at 15,000 The nerve distal to a site of injury contributes to sponta- x g for 30 min, and the supernatant fraction was stored at neous regeneration (1, 2). After transection, axons can cross -200C. a gap of several millimeters and enter the distal nerve stump, Surgical Procedures. Animal care and use procedures set indicating the presence of soluble neurotrophic substances. forth by the National Institutes of Health were followed. Supporting cells in the nerve distal to a lesion indeed produce Male Sprague-Dawley rats, 12-14 wk old, were housed in soluble factors, which attract and stimulate neurite growth plastic cages on a 12-hr light/dark cycle and randomly (3-5). Freezing the distal nerve greatly reduces the popula- assigned to treatment groups. Rats were fed four pellets per tion of Schwann, fibroblast, endothelial, and other cell types day of Prolab RMH 3000 Rat Chow prior to surgery, and, and impairs regeneration (6, 7). Motor (8) but not sensory (9) afterwards, were fed ad lib. Free access to water was axons regenerate at normal rates in Ola mice, indicating that provided. Rats were anesthetized with an initial i.p. dose of different factors may regulate regeneration in each neural 90 mg ofketamine per kg and 5 mg ofxylazine per kg and were system. A deeper understanding of the mechanisms behind placed on a 370C water blanket. Depth of anesthesia was motor axon regeneration in vertebrates is likely to emerge if monitored, and maintenance doses of ketamine (45 mg/kg) the identity of diffusible substances that support motor axon were administered at --30-min intervals, as needed. regeneration were known. Motor Axon Regeneration Assay. Sciatic nerves were The polypeptide known as insulin-like growth factor II crushed as described by De Koning et al. (26). The proce- (IGF-II) (10, 11) is one candidate diffusible substance. Brain dures for implantation of miniosmotic pumps containing and spinal cord comprise the most abundant tissue sources various test substances and measurement of motor axon for IGF-II mRNAs in adult rats (12-14). IGF-II is found in regeneration distances are described in the legend to Fig. 1. cerebrospinal fluid (15), and receptors for IGFs are distrib- The stimulus consisted of square wave pulses (10 mA, 25 ls, uted widely in neural tissues (16-18). IGF-II gene expression seven pulses per s) from a Grass S44 stimulator and Grass in muscle is correlated closely with the development and constant-current unit. Evoked potentials detected by the regeneration of neuromuscular synapses (19), and IGF-II recording electrodes were amplified 500,000 with a Grass P5 supports neurite growth in cultured neuroblastoma (20), 11K preamplifier. The amplified signal was split to a Tek- sensory, sympathetic (21), and motor (22) cells. Reviews are tronix 7854 oscilloscope for signal averaging and to a Compu- available providing additional detail on the actions ofIGFs in pro system 8/16 computer for storage and analysis. neural tissues (23-25). Two hundred fifty-six successive responses to nerve stim- Two independent, but closely interrelated, hypotheses ulation were averaged for analysis ofevoked potentials. Each were evaluated in this study: (i) locally administered IGF-II averaged action potential contained 1024 data points with a

The publication costs ofthis article were defrayed in part by page charge Abbreviation: IGF-II, insulin-like growth factor II (rat multiplication payment. This article must therefore be hereby marked "advertisement" stimulating activity). in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed at t. 11716 Downloaded by guest on October 1, 2021 Neurobiology: Near et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11717 sampling rate of 30 gs per point. The band pass was 3 Hz to electrophysiological procedure was devised to measure mo- 10 kHz (-3 decibels). tor axon regeneration distances (Fig. 1). The evoked com- Neurofilament Immunohsochstry. Nerve segments pound action potential in anonlesioned sciatic nerve is shown containing the front of regeneration (marked by suture) were in Fig. 2A. Several precautions ensured that these potentials frozen for 20 s in a mixture (80% propane/201% ethane) that were conducted actively to the recording site and were was cooled in liquid nitrogen. Longitudinal sections (10 !Lm) recorded from motor axons. A plastic shield was used to were fixed for 10 min in 4%o formalin/0.1 M sodium acetate, electrically isolate the nerve from muscle. Nerve crush or pH 6, and then for 10 min in 4% formalin/0.1 M sodium application of a local anesthetic (lidocaine) just proximal to borate, pH 11. Following exposure to 10 mg of collagenase the recording electrode abolished the evoked potentials. per ml (1540 units/mg; Sigma, type XI) in Hanks' salts at pH Because the transected dorsal roots were reflected away, it 7.4 for 30 min, the sections were incubated in 0.1% glacial was unlikely that sensory fibers were recruited inadvertently acetic acid in ethanol at -209C for 10 min and then in 3% by the stimulus. In addition, when intact dorsal root fibers horse serum in phosphate-buffered saline for 30 min. Mouse were stimulated, ascending evoked spinal cord potentials monoclonal anti-neurofilament antibody (SM132 from Stern- were detected in spinal cord segments cranial to the dorsal burger Monoclonals) was diluted 1:5000 in phosphate- root attachment zone (28). However, following transection of buffered saline and incubated with the sections overnight. dorsal roots, such potentials were not detected. Therefore, SMI32 binds to nonphosphorylated epitopes on heavy (220 the evoked potentials recorded in the sciatic nerve distal to kDa) neurofilament subunits. Using the procedure of Hsu et a crush site were actively conducted potentials in regener- al. (27), the sections were incubated subsequently with ating motor axons. biotinylated anti-mouse IgG antibodies and a complex of Two days after sciatic nerve crush, the ventral roots were avidin-biotinylated horseradish peroxidase, following recom- stimulated electrically, and motor axons had regenerated 4 mended procedures (Vectastain Elite ABC kit, Vector Lab- mm distal to the crush site. In all experiments, several passes oratories). 3,3'-Diaminobenzidine tetrahydrochloride was of the recording electrode were made to confirm the position used as substrate. of the most distal regenerating axons. The regeneration front was labeled with a suture through the epineurium. The recorded evoked potentials at sites 2 mm proximal to the RESULTS regeneration front, at the front, and 1 mm distal to the front Correspondence Between Electrophysiologic and Immuno- are shown in Fig. 2 B, C, and D, respectively. Evoked histochemical Detection of Sciatic Nerve Regeneration. An potentials were detected proximal, but not distal, to the A

Osrmotic u;rp B zzzz. Stmnnuius electrc-. Crush site suture CSut corsal rcot .V, s r-- Sciatic nerve I,';M$ site suture

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FIG. 1. (A) Method fordelivery oftest solutions to lesioned sciatic nerve. The right sciatic nerve was exposed by blunt dissection, and needle holders with 0.4-mm-wide jaws were tightly closed on the nerve for 20 s where the nerve emerged from under the middle gluteal muscle (26). The middle of the crusb site was labeled by placing an 8-0 Ethicon suture in the epineurium. In those experiments in which the effects of antiserum were tested, iridectomy scissors were used to clip a tiny "window" extending from to "15 mm distal to the crush site for the tibial and common peroneal branches of the nerve. Internal pressure caused the epineurium to spring open, exposing about half of the diameter of the nerve. Implanted Alzet 1007D (Alza) miniosmotic pumps delivered test solutions through a sterile silicone catheter (0.076 cm i.d.) to the site of nerve crush. The distal end of the catheter was perforated with a row of four or five holes, in order to bathe a 15-mm length of nerve, and was sutured to muscle underlying the nerve. If pumps did not deliver the expected volume of a solution, catheters were clogged with connective tissue, or inflammation was evident, rats were disenrolled from the study. (B) Evoked potential assay for motor axon regeneration distance. A dorsal laminectomy, at the third lumbar vertebra (L3), exposed the conus medullaris and cauda equina. The spinal cord and dorsal roots were transected, and the latter were reflected away. Ventral roots (levels L4-L5) that supply the sciatic nerve were left intact, and these roots ipsilateral to the crush site were placed over silver bipolar hook electrodes. Prior to electrical stimulation, 0.3 ml (2 mg/ml) ofpancuronum bromide was injected into the lateral tail vein, and respiration was controlled with an Edco animal ventilator. The regenerating sciatic nerve was exposed, and the pump and catheter were removed and carefully examined under a dissecting microscope. The possibility for volume conductance of evoked electrical activity was reduced by isolating the nerve. The nerve was transected just proximal to its bifurcation at the stifle, sutured with 8-0 Ethicon (nonconducting suture) to a thigh muscle that had been reflected, and a thin plastic shield was placed under the nerve. The sciatic nerve and ventral roots were bathed in warm mineral oil. The sciatic nerve was laid across silver chloride bipolar hook electrodes that were moved distally in about 1-mm increments to record evoked potentials. A ground electrode was placed s.c. at a surgical site on the rat's back. The most distal site at which an evoked potential could be detected (confirmed by repeat passes with the recording electrode) was marked with a suture ("regeneration front"). The regeneration distance was measured between this suture and the suture labeling the crush site. Downloaded by guest on October 1, 2021 11718 Neurobiology: Near et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A. Above Crush artifact conveniently marked the position of the suture used 400 to identify the front itself (arrow, Fig. 2G). Distal to the front 300 (within 1 mm), the neurofilament stain no longer revealed long, continuous axons (Fig. 2H). Rather, the dark staining 200 showed axonal fragments undergoing Wallerian degenera- 100 tion. These data showed a colocalization between the regen- eration front detected by electrophysiological and immuno- B. Proximal chemical methods. 8 Effect of Locally Infused IGF-iH on Motor Axon Regenera- .. k tion. Infused IGF-II increased the regeneration distance of 6,. motor axons. Implantation of a miniosmotic pump that re- 4 leased RPMI vehicle near the site of nerve crush had no w 2. detectable effect on the distance of regeneration (Fig. 3). The I- regeneration distance, however, was increased significantly in rats treated with 0.3 Ag of IGF-II per ml relative to those C. Front treated with RPMI vehicle (P < 0.0002). Insulin (molecular 8 G -j mass, 6 kDa) at a 3-fold higher concentration was inactive, 0 6 showing that increased regeneration was not the result of 4 IGF-II (molecular mass, 7.5 kDa) activation of insulin recep- 2 1- tors, assuming both ligands had equal access to sites of action. The activity of IGF-II was not entirely surprising, because proteins can penetrate the epineurial and blood- D. Distal nerve barrier after crush injury to nerves (29); also, one may 8 not exclude the possibility of an IGF transport mechanism. 6 Concentrations of locally infused IGF-II, at or above 50 ng/ml, were found to increase significantly the regeneration 4 distance (Fig. 4). The half-maximally effective dose (ED50) 2 was between 30 and 50 ng/ml. The IGF-II dose-response curve was much steeper than expected, indicating that the 4 8 12 response may have been influenced by a local gradient of TIME (ms) IGF-II. A gradient could result from retardation of IGF-II by the epineurium and perineurium as well as from formation of FIG. 2. Electrophysiological and immunocytochemical detection complexes with IGF binding proteins (30, 31) and dilution due of regenerating axons. Two days after sciatic nerve crush, evoked to diffusion and tissue perfusion. The that low motor axon potentials were measured about 1 cm proximal to the possibility crush site (A). The nerve had regenerated 4 mm (front), and evoked concentrations of IGF-II might adsorb significantly to os- potentials were additionally measured 2 mm proximal to the regen- motic pumps or catheters was not evaluated. The overall eration front (B), at the front (C), and about 1 mm distal to the front dilution of IGF-II at the active site appeared to be S50-fold, (D). Note the difference in voltage scale in A. (E) Neurofilament or less, because infused IGF-II at 50 ng/ml was active (Fig. immunohistochemistry in a nonlesioned nerve. (x20.) Two days 4) and 1 ng/ml can readily induce neurite growth in culture postcrush, the motor axon regeneration front located by electro- (20, 21). These data showed that locally infused, exogenous physiology was labeled with a suture. The nerve was prepared for IGF-II can increase motor axon regeneration. immunohistochemical detection of neurofilaments, and a series of Effect of Locally Infused Anti-IGF Antiserum on Motor photographs is shown from one longitudinal section of the nerve: Axon Regeneration. To determine whether endogenous IGFs within 1 mm proximal (F, x20), 0 mm (G, x10); and within 1 mm distal (H, x 20) to the regeneration front. The arrow shows a staining contribute to spontaneous regeneration, an anti-IGF-II anti- artifact created by the suture used to mark the regeneration front (G serum (30 1.l/ml) was infused. Negative results were ob- and H). Note that G was taken at a lower magnification to encompass tained. This was not entirely unexpected, because large the entire regeneration front. As a result, the neurofilament staining antibody molecules (150 kDa) are more apt than small IGF-II in G is somewhat less discernible than in other panels. (H) A higher magnification of the distal half of G (the same point near the suture 12 * * site is marked by the arrow in each panel). (Bars = 18 Aum.)

regeneration front. Potentials measured at sites of regener- 1-1 8 *F ation (Fig. 2B) were considerably smaller in amplitude and V showed greater temporal dispersion relative to potentials measured 1 cm proximal to the crush site (Fig. 2A). z 4 The evoked potential fell off precipitously at recording sites distal to the regeneration front. The evoked potential shown at the regeneration front (Fig. 2C) was completely lost at a site 1 mm distal to the front (Fig. 2D), irrespective of signal amplification. Untreated RPMI IGF-II Insulin Axons were examined by immunohistochemistry. In a FIG. 3. IGF-II increases motor axon regeneration distance fol- nonlesioned sciatic nerve (Fig. 2E), darkly immunostained lowing nerve crush. Rats were randomly assorted into treatment neurofilaments revealed long, continuous axons in a longi- groups, and the sciatic nerve was crushed. Miniosmotic pumps tudinal section. Under lower power microscopy, many of released either vehicle (RPMI), 0.3 jug of IGF-II per ml, or 1 jig of these darkly stained axons could be followed for several insulin per ml. Pumps were not implanted in the untreated group. without Regeneration distances were measured 3 days postcrush. Values are millimeters interruption. Two days after nerve crush, means + SD; n = number ofrats. All data in this study were analyzed the motor axon regeneration front was located by electro- together using the Neuman-Keuhls posthoc test to detect interac- physiology. Immunostaining for neurofilaments revealed that tions between groups. P values indicate differences between group long, continuous axons were present in a section proximal means. *, Not significant, RPMI vs. untreated or insulin-treated (within 1 mm) to the regeneration front (Fig. 2F). A staining groups. **, P < 0.0002, RPMI vs. IGF-II-treated groups. Downloaded by guest on October 1, 2021 Neurobiology: Near et aL Proc. Natl. Acad. Sci. USA 89 (1992) 11719

12 Æ' < 0.0002) spontaneous regeneration, indicating that the basis for inhibition was antibody recognition of IGFs within the ~12 nerve. The same dilution of preimmune serum was not inhibitory. The following experiment was done to study further the inhibitory capacity of the anti-IGF-I1 antiserum. IGF-I (30 .~ ~*/ ,ug/ml) was incubated together with anti-IGF-II antiserum (30 jL/ml) at 3TC for 3 hr and at 4TC for 30 min. The 15,000 x g supernatant fraction was prepared and, when infused, did not inhibit motor regeneration at 3 days (Fig. SB). In contrast, 6 anti-IGF-I! antiserum alone did inhibit motor axon regener- ation in the presence of epineurial windows. These results show that the inhibitory capacity ofthe anti-IGF-II antiserum 0 0.01 0.1 1 10 was overturned by adding IGF-II to neutralize the anti-IGF IGF-II DOSE (gg/ml) antibodies. It, therefore, seemed improbable that the inhibi- tion was due to components in serum other than anti-IGF FIG. 4. Effect of IGF-II dose on motor axon regeneration dis- antibodies. These antibodies recognize IGF-I as well as tance. The procedure was the same as that described in the legend IGF-II. Consequently, endogenous IGF-I, IGF-II, or both to Fig. 3, except that miniosmotic pumps released either RPM! or support spontaneous regeneration of motor axons. various concentrations of IGF-II as shown. Regeneration distances were measured 3 days postcrush. Values are means + SD; n = 3 or 4. *, P < 0.0002, RPM! vs. indicated IGF-II-treated group. DISCUSSION molecules (7.5 kDa) to have difficulty in crossing the epineu- These results show that locally infused IGF-II increased the rium and To overcome this a window regeneration distance of motor axons. IGF-II acted, most perineurium. barrier, likely, on rather was cut the and regeneration was stud- IGF than insulin receptors (Figs. 3 and 4). through epineurium, IGF receptors are distributed in the region of growth cones ied 5 The distance of days postcrush (Fig. 5A). spontaneous as well as along the axon shaft of motor neurons (22). regeneration was no different whether a window was present Occupancy of IGF receptors is correlated with increased or not (RPMI vs. RPMI-W groups). However, when a neurite growth (32) and increased transcripts that encode window was present, the antiserum significantly inhibited (P structural proteins ofaxons, such as a- and 3-tubulin mRNAs in vitro (33). Tubulin mRNAs are elevated following nerve 20 r A lesion (34, 35), albeit the factors responsible for their eleva- tion have yet to be identified. With respect to additional 15E aspects ofthe cellular mechanism, emerging data suggest that * * neurite outgrowth directed by IGFs, insulin, and nerve w10 growth factor receptors is stimulated through activation of tyrosine kinases. Reviews may be consulted for further FU4 discussion on transmembrane signaling, putative role of second messengers such as cAMP, G proteins, and protein kinase C, and a hypothesis of a common biochemical path- RPMI RPMI-W Serum Ab way regulating neurite growth (36, 37). An anti-IGF-II antiserum inhibited spontaneous motor 12 B axon regeneration (Fig. 5). Regeneration was inhibited by 2.2 mm (32% inhibition) on day 3 (Fig. SB) and 3.6 mm (26%

E inhibition) on day 5 of treatment (Fig. SA). This increase in 8 inhibition from 2.2 to 3.6 mm is an indication that the antibody probably inhibited the rate of spontaneous regen- eration. This inference is complemented by the observation 4 that infused IGF-II increased, whereas the anti-IGF-II anti- serum inhibited significantly, the rate of sensory axon regen- eration for at least a week (life span of miniosmotic pump used) following crush injury in sciatic nerves (38). Sensory RPMI Ab Ab + IGF-H IGF-I1 regeneration was measured by a pinch test in which a reflex contraction revealed the regeneration front (39). Although FIG. 5. (A) Effect of epineurial window, preimmune serum, and IGF-II was found to increase regeneration of motor and anti-IGF-1I antiserum on spontaneous regeneration of motor axons. sensory axons, using two different assay procedures, further The procedure was the same as that described in the legend to Fig. is needed to establish whether complete regeneration 4 for the vehicle (RPMI)-treated group. Other rats had windows cut study to in the epineurium, and pumps released vehicle (RPMI-W), 30 Al of target tissues would be enhanced by IGF-II. antiserum per ml (Ab), or 30 pl of preimmune serum per ml (serum). After sciatic nerve crush, IGF-I immunoreactivity (40) and Regeneration was measured 5 days postcrush. Values are means + IGF-II mRNA content (G. W. Glazner and D.N.I., unpub- SD; n = number of rats. *, Not significant, RPMI-W vs. RPMI or lished data) are increased in distal nerves. The properties of serum-treated groups. **, P < 0.0002, RPMI-W vs. Ab-treated the antiserum used in this study, however, were such that the groups. (B) IGF-1! overturns the inhibitory capacity of anti-IGF-1I relative contribution of endogenous IGF-I, IGF-II, or both, antiserum. Rats had windows cut in the epineurium. Pumps released to motor regeneration remains to be established. Neverthe- vehicle (RPMI), 30 Al of anti-IGF-II antiserum per ml (Ab), or 30 Al less, at long last, these data reveal the identity of soluble of anti-IGF-11 antiserum per ml (Ab) plus 30 Ag of IGF-1! per ml (see factors, endogenous to nerve, that contribute to text). For comparison the effect of 30 ,&g of IGF-1I per ml without spontaneous windows is shown. Motor regeneration was measured 3 days post- motor axon regeneration. The cellular source of endogenous crush. *, P < 0.0005, RPMI vs. Ab or IGF-!!-treated groups. **, P nerve IGF-II, however, remains unidentified. Schwann cells, < 0.0002, Ab vs. Ab plus IGF-II-treated groups. Not significant, known to proliferate after nerve lesion, might be a primary RPMI vs. Ab plus IGF-II-treated groups. source of increased IGF-I1 mRNAs. The potential contribu- Downloaded by guest on October 1, 2021 11720 Neurobiology: Near et al. Proc. Natl. Acad. Sci. USA 89 (1992) tion of endothelial cells, fibroblasts, and migratory macro- 15. Haselbacher, G. K. & Humbel, R. E. (1982) Endocrinology phages may not be excluded. 110, 1822-1824. to sensory as well as motor axon regen- 16. Bohannon, N. J., Corp, E. S., Wilcox, B. J., Figlewicz, D. P., IGFs contribute Dorsa, D. M. & Baskin, D. G. (1988) Brain Res. 444, 205-213. eration distances. IGF-I infused at concentrations above 17. Smith, M., Clemens, J., Kerchner, G. A. & Mendelsohn, L. G. 50-100 Ag/ml increases sensory axon regeneration distances (1988) Brain Res. 445, 241-246. in freeze-injured (41) or crushed (42) sciatic nerves, and in 18. Lesniak, M. A., Hill, J. M., Kiess, W., Rojeski, M., Pert, C. B. silicone chambers (43). Cycloheximide inhibits regeneration, & Roth, J. (1988) Endocrinology 123, 2089-2099. and IGF-I can overcome this inhibition (44). Moreover, 19. Ishii, D. N. (1989) Proc. Natl. Acad. Sci. USA 86, 2898-2902. IGFs support a constant rate of sensory regen- 20. Recio-Pinto, E. & Ishii, D. N. (1984) Brain Res. 302, 323-334. endogenous 21. Recio-Pinto, E., Rechler, M. M. & Ishii, D. N. (1986) J. eration (38). Neurosci. 6, 1211-1219. The specificity of regenerated nerve cell connections has 22. Caroni, P. & Grandes, P. (1990) J. Cell Biol. 110, 1307-1317. been studied intensively (reviewed in ref. 45). Following 23. Recio-Pinto, E. & Ishii, D. N. (1988) Neurochem. Int. 12, nerve transection, regenerating motor axons send collaterals 397-414. in a random manner to both sensory and motor branches of 24. Sara, V. R. & Carlsson-Skwirut, C. (1988) Prog. Brain Res. 73, distal nerve, but inappropriate collaterals are pruned later, 87-99. leading to improved specificity (46). However, following 25. Ishii, D. N. (1992) in Neurotrophic Factors, eds. Loughlin, S. E. & Fallon, J. H. (Academic, New York), pp. 415-442. nerve crush, there would be much less disruption ofSchwann 26. De Koning, P., Brakkee, J. H. & Gispen, W. H. (1986) J. cell columns which serve to guide axons correctly back to Neurol. Sci. 74, 237-246. targets. Thus, the role of endogenous IGFs in regenerating 27. Hsu, S. M., Raine, L. & Fanger, H. (1981) J. Histochem. axons should be considered in light of the type of nerve Cytochem. 29, 577-580. injury. As regenerating motor axons approach peripheral 28. Carsten, R. E., Whalen, L. R. & Ishii, D. N. (1989) Diabetes targets, the up regulated IGF-II gene expression in dener- 38, 730-736. vated muscle may help further to restore connectivity (19). 29. Seitz, R. J., Reiners, K., Himmelmann, F., Heininger, K., Hartung, H.-P. & Toyka, K. V. (1989) Muscle Nerve 12, 627-635. We thank James R. Bamburg and Marilyn J. Anderson for helpful 30. Hardouin, S., Hossenlopp, P., Segovia, B., Seurin, D., Porto- comments on this manuscript, James Russell for assistance in lan, G., Lassarre, C. & Binoux, M. (1987) Eur. J. Biochem. 170, technical illustration, and Amgen for generously providing IGF-II 121-132. and anti-IGF-II antiserum 489-42. This work was supported by Grant 31. Hossenloppi P., Seurin, D., Segovia-Quinson, B. & Binoux, M. RO1 NS24787 from the National Institute of Neurological Disorders (1986) FEBS Lett. 208, 439 444. and Stroke and Grant IB2-9201 from the American Paralysis Asso- 32. Recio-Pinto, E. & Ishii, D. N. (1988) J. Neurosci. Res. 19, ciation. 312-320. 33. Mill, J. F., Chao, M. V. & Ishii, D. N. (1985) Proc. Natl. Acad. 1. Ramon y Cajal, S. (1928) Degeneration andRegeneration ofthe Sci. USA 82, 7126-7130. Nervous System (Oxford Univ. 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