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Liuzzi&Lasek1987 - 15 Circumsporozoite...

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Unformatted text preview: 15 Circumsporozoite precipitation (CSP) tests 1} Van derberg, R Nussenzweig, H. Most, Milit. Med. 134,1183 (1969)] were performed as described (9). Twenty<five sporozoites were examined and the number with granular precipitates on the surface (2+ reaction) or with a long threadline filament at one end (4+ reaction) determined. The CSP score was the number of s oroz01tes with 2+ reactions multiplied by 2 plus t e number of sporozo1tes with 4+ reactions multiplied by 4. A score of 8 was considered positive. 16. Inhibition of sporozoite invasion of hepatoma cells (ISI) [M. R Hollingdale, E. H. Nardin, S Thara- vani), A L. Schwartz, R S Nussenzweig,] Imrnd 7101 )132, 909 (1984)] was performed .1s described (9). )Assays were performed in triplicate at a serum dilution of 1:20. The percent ISI— — [100][1 — (number of sporozoites that entered in the presence of test serum) + (number of sporozoites that en tered in the presence of control serum)]. The four sera with the highest 151 (Table 2) were serially diluted to determine the last dilution at which sporozoite invasion was inhibited 250% (ISIA50). The ISI»50 was >1 : 300 but <1:1000 in two sera, and <12300 in the other two. 17. Although monoclonal antibodies to CS protein re< peat epitopes react in the four assays the target antigens for the ELISA (purified protein from the repeat region of the CS protein), IFAT (air dried Egorozoites), )and I51 and CSP (live sporozoites) are iferent and these assays may measure antibodies to diflerent epitopes. In fact, when sera from all 83 individuals were compared there was a significant correlation only between the results of the IgG IFAT and IgG ELISA (r— v 0 45, 95% confidence interval: 0. 27—0. 61 P < 0.00001) and the IgG IFAT and ISI (r= 0,.30 95% confidence inter val: 0.11 to 0.,48 P— — 0.0028). 18. Concentrations of IgG subclass antibodies to R32tet32 were determined as described (9). Dilur tions of purified myeloma proteins of each of the four subclasses were used to establish standard curves. At the 1:50 serum dilution used in these assays, the minimum detectable concentrations of IgG1, IgG2, IgG3, and IgG4 antibodies to R32tet32 were 0.75, 0.95, 0 95, and 1 611 /ml, respectively (Myeloma dproteins and antiboi 1es to subclasses were provie d by R Wistar, Ir., and C. Cole.) 19. W. R. Ballou, in preparation. 20. M. F. Boyd, in Muluriolw, A Comprehemive Survey of All Axperts of This Group ofDiseuxex flow 11 Global Perspective, M. F. Boyd, Ed. (Saunders, Philadel» phia, 1949), pp. 638—641. 21. S. Cohen, I. A. McGregor, S. P. Carrington,Nuture (London) 192, 733 (1961). 22. R. S. Nussenzweig 21‘ 111., ibid. 216, 160 (1967). Astrocytes Block Axonal Regeneration in Mammals by Activating the Physiological Stop Pathway FRANCIS I. LIUZZI* AND RAYMOND I. LASEK Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells. N THE ADULT MAMMALIAN CENTRAL nervous system (CNS), regenerating ax- ons typically grow relatively short dis- tances—less than 1 mm. The restriction of axonal elongation in the adult CNS is not a limitation of the intrinsic capacity of axons to elongate (I, 2); but rather, cellular ele- ments of the CNS, particularly astrocytes, appear to restrict the elongation of axons (3, 4). The effects of mature mammalian astro- cytes on axonal elongation can be studied at the transitional zone between the dorsal root and the dorsal root entry zone of the spinal cord (1, 5—8). By transecting the axons in the dorsal root, it is possible to study their regeneration from the root into 64.2 the spinal cord without directly injuring the cord. Trauma to the cord results in the formation of a connective tissue scar that contains fibroblasts and a dense collagenous matrix (1, 4), which has been shown to prevent axonal elongation (1, 9). In the dorsal root regeneration model, there is no connective tissue scar and axonal growth in a purely astroglial environment can be stud- ied. Regenerating axons in the dorsal root are surrounded by a substrate that contains Schwann cells and their basal laminae. This milieu is similar to that in a peripheral nerve and supports the active elongation of regen— erating axons, which grow until they reach the dorsal root transitional zone. At this 23. D. F. Clyde, V C. McCarthy, R M Miller, R. B. Hornick, Am. ]. Med. Sci. 266, 398 (1973) 24. 19 F. Clyde, V. C. McCarthy, R. M Miller, W. E Woodward, Am. ]. Tro. Med. Hyg. 24, 397(1973). 25. K H. Rieckmann, P E. Carson, R. L. Beaudoin, I. S. Cassells K. W. Sell, Tram. R. Sue. Trop. Med Hyg 68, 258 (1974) 26. K. H. Rieckmann, R. L Beaudoin,]. S. Cassells,K. W. Sell, 131111. WHO 57 (suppl. 1), 261 (1979). 27. R. S. Nussenzweig, I. P. Vanderberg, H. Most, C. Orton, Nature (London) 222, 488 (1969). 28. R. L. Beaudoin et 111., Exp. Puruxitol. 42, 1 (1977). 29. S. Aley et 111,]. Exp. Med. 164, 1915 (1986). 30. We thank the Saradidi Rural Health Project, D. O. Kaseje, I. D. Bales, R. Gore, 1. Williams, A. Adje- pong, C. Paul, R Mtahb, D. Achango, F Kilonzo, l9.A1nollo, R. Haj kowski, C. L. Diggs, C. R. Roberts, D Shanks, andR E Whitmire for support and encouragement, Smith, Kline, 8t French Labo» ratories for supplying R32tet32 and R32LR, and W. R. Ballou for manuscript review. S. L. H was sup» ported in part by the Naval Medical Research and Development Command, Work Unit No. 3M463750D808 AQ 061 and M.R.H. and G.RW. were supported by AID contract No. DPE70453-C7 00-3051-00. 2 April 1987; accepted 23 June 1987 interface between root and spinal cord, the regenerating axon tips encounter a substrate that consists almost entirely of reactive as- trocytic processes. Here, among these pro- cesses, many of the growth cones stop and form stationary axon terminals that remain in place for a year or more (6—8). These observations suggest that mature astrocytes can stop the intrinsic tendency of axons to elongate through their efiects on the axon tip. By what mechanisms do astrocytes stop axonal elongation? Normally, a physiologi- cal sequence is activated in the axon tip when it makes synaptic contact with an appropriate postsynaptic neuron in the CNS (10—12). One important part of this physio- logical pathway is the disassembly of cyto- skeletal polymers (neurofilaments and mi- crotubules) that are continuously transport- ed into the stationary axon tip. Specifically, proteases that degrade the neurofilaments are selectively activated in the presynaptic terminal (11~14); if these proteases are in- hibited by the protease inhibitor leupeptin, neurofilaments accumulate in the axon ter- minal (I5). Axons can also be stopped from elongat- ing without fiilly activating the physiologi- cal pathways that degrade the neurofila- ments. If axons are physically obstructed from elongating by being forced to grow into a cul-de-sac that is produced by tightly ligating a peripheral nerve, the trapped axon tips fill with neurofilaments and other axon— ally transported structures (13, 16). This Bio-architectonics Center, Medical School, Case Western Reserve University, Cleveland, OH 44106. *Present address: Department of Anatomy and Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23501. SCIENCE, VOL. 237 indicates that simply blocking the forward progress of the axon is not sufficient to activate the physiological pathway that nor- mally controls the transport of neurofila— ments and other transported structures into the axon terminal. Neurofilament accumulation in the axon tip is an index of the activity of the metabol- ic mechanisms that remove the cytoskeleton from a stationary axon terminal (13). To determine if adult astrocytes activate these metabolic mechanisms when they stop axo- nal growth or if they physically obstruct regenerating axonal elongation without acti- vating the metabolic mechanisms, we com- pared the axon tips of regenerating axons stopped by astrocytes at the dorsal root transitional zone with those that were physi- cally obstructed by a ligation neuroma, which is formed by tightly ligating a periph- eral nerve or dorsal root. We show that neurofilaments do not accumulate in the stationary axon terminals that contact reac- tive astrocytes in the dorsal root transitional zone. Adult rat lumbar (L5) dorsal root axons were completely transected 5 mm from the Flg. 1. (A) Growth cone (GC) of regenerating dorsal root axon observed 1n the vicinity of the root transitional zone. This axon tip has the typical features of a growth cone; it has a clear cytoplasm and a well- -developed system of tubulovesicular membranes. Note that the growth cone is in close apposition to both as- trocytic (Ast) and Schwann cell (Sc) processes. Scale bar, 1 0 pm. (B) Typical axon temiinal (Ter) 1n the CNS side of the root transitional zone This stopped axon tip is characterized by small (35 to 40 nm in diameter) agranular vesicles, occasional dense- cored vesicles, and numerous, small, normal-appearing mitochondria. Notably, the cytoplasm of these terminals contains few, if any, neurofilaments and microtubules. These terminals were completely encapsulated by astrocytic processes, which were easily identified by their numerous intermediate filaments. Scale bar, 1.0 pm. (C) A large dorsal root axonal terminal within the dorsal root away from the astrocytes of the root transitional zone. These stopped axonal tips were packed with neurofilaments and membranous or- ganelles and were surrounded by Schwann cells and the connective tissue matrix of the root endoneurium. Moreover, these swollen terminals were the same as those that formed at a cul-de-sac in ligated peripheral nerves and dorsal roots. Scale bar, 5.0 pm. (D) High magnification of a part of the temiinal in (C). Neurofilament bundles cut in every plane of section are the predominant cytological feature of these terminals. Scale bar, 1.0 pm. 7 AUGUST 1987 REPORTS 64.3 spinal cord by crushing the root with Du- mont no. 5 forceps twice in the same place for 10 seconds. After 3 weeks to 3 months, the treated animals were anesthetized and perfused intracardially with a fixative. Liga- tion neuromas were formed in hypoglossal and saphenous nerves and in dorsal roots by tightly ligating the nerve with 6.0 suture. After 3 to 6 weeks, the animals were anes- thetized and perfused with the same fixative. The perfusion solution consisted of 5.0% paraformaldehyde, 2.5% glutaraldehyde, and 7.0% sucrose in 0.1M phosphate buffer (pH 7.4). The spinal cords were processed by removing the dorsal quadrant of the cord, containing the root entry zone of the crushed L5 root, which was cut into 1-mm- thick slabs. Tissues were then fixed by im- mersion for 2 hours in the same fixative, washed for 0.5 hour in bulfer, and “post— fixed” in 2.0% OsO4 for 1 hour. After washing in distilled water, the tissue was stained en bloc in 0.5% aqueous uranyl acetate for 3 hours, dehydrated in a graded series of ethanols, and embedded in Mara- glas. In I-um-thick sections stained with toluidine blue, the dorsal root transitional zone was identified, and thin sections of this 1pm Growing tip Axo-glial ending region were cut, placed on ZOO—mesh hexag- onal grids, and routinely stained. The grids were examined and photographed on a IEOL IOOCX transmission electron microscope. Light microscopic analyses of horseradish peroxidase—filled dorsal root axons con- firmed studies showing that the axons re- generate through the dorsal root into the transitional zone of the spinal cord (1 , 5—7, 16). At the transitional zone, axons showed a number of reproducible grOWth patterns: some axons turned around and grew back into the dorsal root. A small number of axons grew through this zone and continued for a few hundred micrometers before stop- ping in the dorsal horn. Many of the axons stopped at the transitional zone; these axons formed a discrete row of terminals at the junction between the dorsal root and the spinal cord, the root—cord interface. At 3 weeks after transection, some growth cones were observed in the region of the dorsal root where astrocytic processes pro- ject from the spinal cord into the root (Fig. 1A). These growth cones often were ap- posed to glial processes but were not com- pletely surrounded by astrocytic processes. Axon tips that had penetrated farther into Neuromitic ending Fig. 2. Summary drawing depicting the two mechanisms by which regenerating axons (AX) can be stopped. In one case, the axo—glial ending, the growth cone is stopped by activating the physiological stop pathway within the axon. This pathway operates during the development of axon terminals on target cells, and we propose that astrocytes can also stop axonal elongation through this pathway. Axonal elongation can also be stopped by physically obstructing axonal elongation. In this case, the neuromitic ending, connective tissue elements block the forward progression of the growth cone, and it is converted to a neurofilament-swollen terminal packed with membranous organelles. 644 the dorsal root transitional zone were com- pletely enveloped by astrocytic processes (Fig. 1B). These axo-glial terminals were present in the dorsal root transitional zone at 3 weeks and at 3 months, when regenera- tion is effectively complete. These results support the proposal that regenerating sen- sory axons form stable terminals on astro- cytes at the dorsal root entry zone (6—8). All of the axo—glial terminals were small (1.0 to 2.0 mm in diameter) and had rela- tively smooth contours, which suggests that they had few, if any, filopodia. The axo-glial terminals contained small agranular vesicles, a few dense-cored vesicles, tubulovesicular profiles, and normal mitochondria. The cy- toplasm between these membranous organ- elles was relatively unstructured and had few, if any, neurofilaments or microtubules. These results indicate that neurofilaments did not accumulate in the axo-glial termi— nals. In contrast with the axo-glial terminals at the transitional zone, a few (less than 1.0%) large swollen axon terminals were found in the dorsal root among the collagenous con- nective tissue elements (Fig. I, C and D). These terminals, which were 10 to 15 pm in diameter, were packed with neurofilaments and a variety of membranous organelles including dense bodies. Furthermore, these abnormal terminals were similar to those that formed when axons regenerated into a cul~de—sac produced by tightly ligating a peripheral nerve. Both in the dorsal root and in the cul-de-sac, the large swollen terminals were enwrapped by Schwann cells that were, in turn, surrounded by the collagenous ma- trix of the endoneurium. Thus, neurofila- ments will accumulate in the terminals of regenerating dorsal root axons that have been stopped from elongating by a connec— tive tissue obstacle. Our results indicate that astrocytes stop axons that are regenerating through physio- logical pathways within axons. During elon- gation, axons extend filopodia from their tips. When an axon tip contacts a target cell, filopodial extension is reduced and eventual- ly ceases. In this way, the target cell stops the exploratory behavior of the axon tip. Just as with stationary axon terminals on target cells, the axo—glial terminals that contacted astrocytes had few, if any, filopodia. Appar- ently, adult mammalian astrocytes can inhib- it the extension of filopodia from elongating axon tips. Our results also indicate that after the astrocytes have stopped the axon tip from advancing, they activate the intrinsic axonal mechanisms that prevent slowly transported cytoskeletal polymers from ac- cumulating in the stationary axon terminal. On this basis, we propose that astrocytes stop axonal elongation through their effects SCIENCE, VOL. 237 on the intrinsic physiological pathways that normally Operate when axons form synaptic contacts with target neurons or peripheral receptors. These observations and others indicate that regenerating axons can be stopped by two different mechanisms—(i) by activating the physiological stop pathway that is built into the axon and (ii) by physically obstruct- ing the advance of the axon tip (Fig. 2). In the adult mammalian spinal cord, both tar— get neurons and astrocytes have the poten- tial to stop axonal growth because of their capacity to activate the physiological stop pathway. Iust as with target cells, astrocytes may contribute to the formation of neural connections by controlling the intrinsic ten— dency of axons to elongate. In the mature CNS, astrocytic processes envelop a sub- stantial part of the surface of synaptic termi- nals; one of their roles may be to prevent , axon terminals from wandering away from their synaptic contacts (17). For effective regeneration in the CNS, axons must reconnect with their targets. The first and essential step in the sequence of reconnection is elongation of the axons. To promote axonal elongation within die sub- stratum of the CNS, two different kinds of environmental factors must be considered. If the spinal cord of an adult mammal is transected, a connective tissue scar forms in the damaged region, and swollen axon ter- minals that are often filled with neurofila- ments have been observed in contact with the scar (9). In this case, preventing or removing the connective tissue obstacle is clearly essential for axonal regeneration. This, however, may not be suflicient to promote axonal regeneration. In addition, the tendency of the mammalian astrocytes to limit the motility of the axon tip through the physiological stop pathway must also be considered. REFERENCES AND NOTES 1. S. Ramén y Cajal, Degeneration and Regeneration of the Nervous System (Oxford Univ. Press, London, 1928). S. David and A. I. Aguayo, Science 214, 931 (1981). . A. I. Aguayo, S. David, P. M. Richardson, G. M. Bray,Aav. Cell. Neurobiol. 3, 215 (1982), 4. P. I. Reier, L. I. Stensaas, L. Guth, in Spinal Coral Reconstruction, C. C. Kao, R. P. Bunge, P. I. Reier, Eds. (Raven, New York, 1983), pp. 163—195. 5. E. I, H. Nathaniel and D. R, Nathaniel, Exp. Neurol. 40, 333 (1973). 6. L. I. Stensaas, P, R. Burgess, K. W. Horch, Soc. NeurosciAhstr. S, 684 (1979). 7. L. I. Stensaas, L. M. Partlow, P. W. Burgess, K. W. Horch, in Neural Regeneration, F. I, Seil, E. Her- bert, B. M. Carlson, Eds, (Elsevier, New York, 1987), pp. 457—468. 8. T. Carlstedt, Brain Rex. 347, 188 (1985). 9. C. C. Kao, L. W. Chang, I. M. B. Bloodworth, Ir., Exp. Neurol. 54, 591 (1977). See figures 1 F and 13. 10. R. P. Rees, M. P. Bunge, R. P. Bunge,]. Cell Biol. 68, 240 (1976). 11. R. I. Lasek and P. Hofl'man, Cold Spring Harbor Confi Cell Proliferation 3, 1021 (1976). WP 7 AUGUST I987 12. R. I. Lasek and M. I. Katz, in Neural Regeneration, F. I. Seil, E. Herbert, B. M. Carlson, Eds. (Elsevier, New York, 1987), pp. 49—60. 13. R. I. Lasek and M. M. Black, in Mechani...
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