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Effect of nerve growth factor on regeneration of goldfish optic axons
Brain Research, 238 (1982) 329-339
329
Elsevier Biomedical Press
E F F E C T OF N E R V E G R O W T H F A C T O R ON R E G E N E R A T I O N OF G O L D F I S H OPTIC AXONS
HENRY K. YIP and BERNICE GRAFSTEIN Department of Physiology, Cornell University Medical College, Arew York, N Y 1002l (U.S.A.)
(Accepted September 3rd, 1981) Key words': nerve regeneration - - nerve growth factor - - NGF - - trophic factors - - goldfish retina
--- optic nerve
SUMMARY Axonal outgrowth tollowing a crush of the goldfish optic nerve was enhanced if nerve growth factor ( N G F ) was administered by intraocular injection or by local application to the lesion site. Various forms of N G F (fl, 2.5S and 7S) were effective, producing a 20-40 0~ decrease in the time required for recovery of the startle reaction to a bright light. A corresponding increase in axonal outgrowth was revealed by histological examination of the optic nerves. The effect produced by a single intraocular injection given at the time of the lesion was not further increased by subsequent injections. Up to 14 days after the lesion, the size of the retinal ganglion cell bodies and the incidence of nucleoli detectable by light microscopy were not affected by the N G F treatment.
INTRODUCTION It has been known for some time that nerve growth factor ( N G F ) stimulates neurite outgrowth in sympathetic postganglionic neurons, in embryonic dorsal root ganglion cells, and possibly in noradrenergic neurons in the CNSZ,5,1a, 2°,26. Recently, it has also been reported that N G F promotes regeneration of optic axons in lower vertebrates. For example, after optic nerve transection in the newt, the proportion of the optic nerve cross-section occupied by regenerated axons was found to be larger in NGF-treated animals than in controls, and the changes elicited in the retinal ganglion cell bodies by axotomy were accelerated and intensified by N G F treatment zl,24,25. In goldfish, N G F was reported to have a dramatic effect on the size and ultrastructure of axotomized retinal ganglion cells, and these cells showed significantly increased neurite outgrowth in tissue culture if they had been treated with N G F before explanta0006-8993/82/0000-0000]$02.75 © Elsevier Biomedical Press
330 tion 23. A n o t h e r study o f goldfish retina explants showed that the effect of N G F in increasing ornithine decarboxylase activity was m o r e marked in retinas in which the retinal ganglion cells were regenerating at the time o f explantation than in controls~L These results are interesting not only because they may throw a light on the mechanism o f action o f N G F , but also because they may have a bearing on the mechanisms that make optic nerve regeneration so succvssfut in lower vertebrates, in contrast to m a m m a l s TM. In the present study, we have further analyzed the regeneration-promoting effects o f N G F using a behavioral technique to assay the rate o f regeneration following a crush of the goldfish optic nerve. In order to determine whether enhanced axonal outgrowth is accompanied by changes in the cell body, we have examined the effect o f N G F on the incidence o f nucleoti detectable by light microscopy, a parameter that increases dramatically in goldfish retinal ganglion cells following a x o t o m y t6. We have also re-examined the effect o f N G F on cell size. We have found that N G F can indeed enhance axonal outgrowth in this system, but that this effect is not necessarily accompanied by changes in cell size or nucleolar incidence. MATERIALS AND METHODS Goldfish 5-8 cm in body length (nose to base o f tail), 6-12 g in weight, were used. They were obtained from O z a r k Fisheries, Stoutland, MO, and were kept in thc laboratory at 20 °C. F o r surgical procedures, they were anesthetized by immersion in ice. In most experiments, the right optic nerve was crushed with .jewellers' forceps at the back o f the orbit; in some cases both optic nerves were ctushed, N G F from 3 different sources was tested in the course o f these experiments TABLE I N G F preparations tested
For some of the ~ and 2.5S preparations, the injection solution remaining at the end of the experiment was re-assayed and was found to contain only about I0% of the expected biological activity. It is not clear whether this loss, which may have been due to deterioration of the material in storage or loss by binding to glass surfaces, occurred before or after the injection. Sources: (1) Dr. Doughs Ishii, Dept. of Pharmacology, Columbia University College of Physicians and Surgeons; (2)Collaborative Research, Waltham, MA; (3) Dr. Robert Stach, Dept. of Biochemistry, SUNY Upstate Medical Center. BSA, bovine serum albumin (Sigma). Code
Type
Nominal biological unit
Nominal biological units per mjeetion
Vehicle
Source
A B C D E
/J [:~ 2.5S 2.5S 7S
1 ng 1 ng 0.4 ng 0.34 ng 30 ng
1000 1000 1000 t790 900
1 ~ BSA 0.9 ~ saline 2 ~ BSA 0.1% BSA Tris buffer*
(1) (1) (2) (2) (3)
* 0.05 M Tris buffer, pH 7.0. with l0 6 M ZnCl2.
331 (Table I). For intraocular application, N G F in a suitable vehicle was injected into the vitreous humor of the operated eye of the experimental fish, while control fish received a similar injection of the vehicle alone. The injections were 4-6 /A in volume and were made with a 10 #l Hamilton syringe. Local applications to the site of the nerve crush were made by injecting 4 - 6 / d cf the N G F or vehicle solution into a 5 cm length of silastic tubing which opened onto the optic nerve through a cuff made of medical grade Silastic. The cuff with the attached tubing was placed around the nerve at the time of the optic nerve crush. Testing for the startle reaction was carried out as described by Edwards et al. 7, usually beginning at 4 days after nerve crush, with the unoperated eye in each case blinded by injection of 0.025-0.05/~g tetrodotoxin or 150/~g xylocaine.
Histological methods At 3-14 days after nerve crush, the fish were decapitated and the heads were immediately fixed in Bouin's solution, or, in the experiments in which the optic nerves were te be silver-stained, the unfixed nerves were removed with a small portion of optic tectum attached and were fixed on a card to keep them straight. For nucleolar counts TM and measurements of retinal ganglion cell size the eyes were embedded in paraffin, sectioned at 15/~m and stained with toluidine blue. (In one experiment ( F | N in Table 111), the retinas from half the fish in each group were dissected out, fixed by immersion in 2 ~ glutaraldehyde, embedded in Epon, sectioned at 0.5/~m, and stained with toluidine blue. Size measurements in this case were made on all cell profiles that contained nuclei, up to a total of 15 per retina. The retinas from the other fish in each group were fixed and processed in the usual way for nucleolar counts.) In each retina nucleolar counts were usually made on 105 cells and cell size measurements on 40 cells. The measurements were made on cells in at least 3 separate histological sections of each retina, beginning at different points on each section in order to ensure that cells from all over the retina were sampled. Every fifth cell profile encountered in a sequential examination of the section was taken for measurement, unless that profile contained an incomplete nucleus, in which case the next profile in sequence that fulfilled this requirement was taken. The presence of nucleoli was determined with a 100 > oil immersion microscope objective. For cell size measurements the cell profiles were drawn with a camera lucida using a 40 × objective, and the area of each drawing determined by means of a computerized graphic analyzer (Numonics, Lansdale, PA). For the optic nerves, serial longitudinal paraffin sections were made and stained with Bodian's silver technique 4. With the use of a 40 × microscope objective determinations were made of the length of the longest detectable bundle of axons, measured from the site of initiation of outgrowth (which was usually about 0.2 m m back from the site of the lesiona4), and of the number of axons seen at various distances from the lesion. For each animal, the values obtained were the means of measurements made on 5-6 separate sections that contained the complete length of nerve from the lesion area to the tectum. In both the behavioral and the histological experiments, the experimenter was uninformed of the treatments given until the experiment was concluded. Statistically significant differences were determined by Student's t-test (2-tailed).
332 INTRAOCULAR
i j
H%LE ~'"'-~
,
' ~
,~
T-z' o
TIME AFTER OPTIC NERVE CRUSH (days)
Fig. 1. Cumulative response curves for the recovery of the startle reaction after optic nerve crush: There were 10 animals per group, and each point represents the total number of animals in each group that had responded by the day of testing. The mean recovery time was 7.9 ~ 0.7 days for the N G F treated animals and 12.9 i 0.8 for the control animals (Table tI~ experiment 7SDR). Thedifference is statistically significant (P < 0.05). RESULTS
Axonal outgrowth The time required for recovery of visual function was determined by testing for the startle reaction, since it has been shown that this test is sensitive to the time required for axonal outgrowth% It was found that intraocular injection of N G F caused a 20--40 ~o decrease ;n mean recovery time (Fig. h Table I1). All 3 types of N G F tested, i.e. fl-, 2.5S and 7S N G F 5, were effective when injected in amounts of about 1000 BU (Table II), but only a small effect, which was not statistically significant, was seen when the 7S preparation was diluted 10-fold or more (Fig. 2). (The effectiveness TABLE 1l
Effect o f N G F on time o f recovery o f startle reaction following nerve crush Each value is the mean : S.E.M. for a group o f 8-10 animals. 0 injection time : 1 h or less after nerve crush. In experiment N G X only, mean recovery times for 70 % of animals in each group are given. Experiment NFBT was performed at 25 °C; mean recovery times are for animals responding by 7 days, obtained by combining the 2 N G F groups and the 2 vehicle groups.
Exp,
NGF Injection Prep. time (Table 1) (days)
lntraocular injection NGX A NFBT NFBT 7SDR
B D E
Local application NFLA B NFLA D
0 0 and 3 ~ 0 and 3 0 0 and 3 0 and 3
Mean recovery time (days) ± S.E.M. Vehicle
NGF
Difference
11.0 ~ 0.5
8.6 m 0.6*
21%
5.8 :r 0.4
4.6 ± 0.2*
21
12.9 = 0.8
7.9 :L 0.7*
---39%
14.5 m 1.7 12.0 m 1.5
11.0 ~ 1.2 8.3 ~ 0.9*
24%~ 31
12.7 ~ 1,2
3%
Intraocular injection into contralateral eye 7SSE E 0 13.1 • 1.9
* P < 0.05 for difference between N G F - and vehicle-injected animals.
333 Injections: B 0 day 0 & 3 days l~a every ?- days
15 ¸
v
-~ ,o t>rY W
0 (.9 w ¢,,, z ,< w 0 (veh.) AMOUNT
9O
900
9
OF N G F PER I N J E C T I O N
(B.U.)
Fig. 2. Effects of different doses and dosage schedules of N G F on mean recovery time of startle reaction after optic nerve crush. N G F (7S) was administered by intraocular injection at the time of crush (0 day), at the time of crush and 3 days later, or at the time of the crush and every 2 days thereafter. Control animals received an injection of vehicle (0.05 M Tris buffer) at the corresponding times. Each value is the mean ± S.E.M. for 12 animals. The decrease in mean recovery time due to N G F is statistically significant (P -< 0.001) only with the 900 BU doses. For each dose the change produced by a single injection is not significantly different from that produced by multiple injections.
of the 7S N G F relative to that of/3- or 2.5S material cannot be estimated, since the fland 2.5S preparations are more labile under storage conditions and more easily lost in handling.) The effect produced by a single injection of 900 BU NGF given at the time of the lesion was just as great as if this injection was followed by a second one 3 days later (Fig. 2). To ensure that this result was not due to a saturating effect of the large dose of N G F given in the first injection, we also tested multiple injections of lower N G F doses, including injections every 2 days throughout the recovery period, but no significant effect was seen (Fig. 2). Local application of NGF to the site of the nerve crush was also effective in decreasing the recovery time by about the same percentage as intraocular injection (Table lI, experiment NFLA). In some of these experiments the injection vehicle contained enough BSA so that the total amount of protein in the
E E
A
B
LONGEST AXON BUNDLE
DISTRIBUTION OF AXONS ~, '°°
3
~
VEHICLE
[~
NGF
p <: 0 . 0 5
0~ 50 z
O
0
0 VEH.
NGF
0.5
1.0
1.5
2.0
DISTANCE FROM CRUSH SITE (ram)
Fig. 3. The effect of N G F on (A) the longest bundle of axons and (B) the number of axons seen at various distances from the lesion in silver-stained sections of goldfish optic nerve 7 days after optic nerve crush (6 animals in the NGF-treated group (3 with fl-NGF, 3 with 2.5S NGF) and 5 animals in the vehicle-injected group). All values for the NGF-treated group are significantly higher than for the corresponding control group (in A, P < 0.05; in B, P < 0.001-0.002). Vertical lines represent S.E.M.
14 14
Local application NFLA B NITLA D
0and3 0and 3
8 7 7 6 3 3 3
NGF InjeCtion Sac. prepatime time ration (daysJ (days/ (Table 1)
lntraocular injection NGX A 0 NFBT B 0and 3 NFBT D 0and 3 FI/q C 2 and 4 NFNC B 2 NFNC B 0 NFNC B --1
Exp.
40.7 ± 0:9 40.7 :± 0.9
42.7 :~ 3.0 42.8 & 2.1
38:3 _;: 0.5
Normal or shamoperation
± ± ± :~ ±
5.2 0.2 1.9 4.7 5.4
74:9 -~: 3.t 81.1 ± 4:8
48.1 76.1 76.t 53.3 43.8
77.4 ± 2.5 84.5 ~ 5.6
46.8 ± 1.4 78.2 ± 1.2 71.8 _q: 3.5 56.9 ± 3.9 48.1 ± 4.6
.......... Vehicle NGF
Cell size (/~m ~)
~3~ -4~o
--3~ +3~ --6°Jo ~ 7~ ÷9~o
0.56 2_ 0.12 0.51 ± 0.08
0.20 ± 0.02
Difference Normal or between shamNGF and operation vehicle
1.09 ± 0 . 0 5 1.10 ~ 0.16 1.33 ~ 0.05 1.18 ~: 0~05 0.71 ± 0 . 0 5 0.62 ± 0.09 0.51 ± 0.04
Vehicle
Difference between NGF and vehicle
0 0.68 ± 0.03 - - 4 ~ 0.63 + 0,04 t 2 ~ o 0.53 ± 0.05 ~ 4 ~
1.18 5:0.05
1.01 ± 0.06 --7~o 1.22 ~ 0 . 0 7 -1l~o 1.26 fi: 0.06 ---5 ~o
NGF
Nucleolar incidence (no./cell)
Each value is the mean 4- S.E.M. for 3-8 retinas (see Materials and Methods). The identification codes of the individual experiments correspond to those in Table I1.0 injection time = 1 h or less between nerve crush and injection; --1 day injection time = injection 1 day before crush. Experiment NFBT was performed at 25 °C.
Effects o f NGF on retinal ganglion cell size and nucleolar incidence .]bllowing nerve crush
TABLE 111
GO GO
335 injection was greatly in excess of that contributed by the N G F . This eliminates the possibility that the observed shortening of recovery time was due to a non-specific effect of protein. In one experiment with intraocular injection of N G F (Table 1I, experiment NFBT) the behavioral study was terminated at 7 days in order to make histological measurements of axonal outgrowth in silver-stained sections of the optic nerve (Fig. 3). Measurements on the length of the longest regenerating axon bundle (Fig. 3A) and the mean number of axons per nerve section seen at various distances from the site of the lesion (Fig. 3B) showed enhanced outgrowth as a result of the N G F treatment, confirming the validity of the decrease in recovery time as an index of an increase in axonal outgrowth. The mean values (-- S.E.M.) for the total number of axons in the whole nerve at 1 mm from the lesion was 980 ~ 250 (n -- 6) for the vehicle-treated animals and 3740 ::L 460 (n = 7) for the NGF-treated animals, i.e. there were nearly 4 times as many axons in the NGF-treated animals as in the controls (difference significant at a level of P < 0.001). The magnitude of the increase in outgrowth distance of the longitudinal axon bundles, about 25 '},o ~ (Fig. 3A), was very close to the magnitude of the decrease in recovery time seen in the corresponding behavioral experiment. The actual rates of outgrowth cannot be determined from these data because the initial delays before outgrowth began are not known. Injection of N G F into the eye on the side contralateral to the lesion had no effect on the time required for recovery of the startle reaction (Table II, experiment 7SSE). This shows that the N G F was not acting by a systemic route.
Cell body characteristics In accord with previous observations 16 an increase in retinal ganglion cell size was evident by 6 days after nerve crush and an increase in the incidence of nucleoli by 3 days (Table Ill). In animals examined up to 14 days after the lesion, however, the changes in the N G F - and vehicle-treated animals were not significantly different (Table Ill), even in experiments in which axonal outgrowth had been enhanced by N G F treatment (as indicated by behavioral or histological evidence). This was true regardless of whether the N G F was applied by intraocular injection or locally to the lesion site. DISCUSSION Our results show that axonal outgrowth in regenerating goldfish optic axons can be enhanced by either intraocular injection of N G F or local application of N G F at the site of the lesion. At least for the intraocular injections, N G F treatment at the time of the lesion was effective, and subsequent applications did not increase the effect. The outgrowth-enhancing effect of N G F was evident both from the decreased time required for recovery of one kind of visual function and from the increased distance of axonal outgrowth revealed by histological examination. It still remains to be determined whether N G F affects the time of initiation of axonal outgrowth or the rate of advance of the axons, or both.
336 The site and mechanism of action of the N G F are not yet known. In the present study, intraocular and local applications of N G F were about equally effective, but the site of the optic nerve crush was only a few millimeters from the eye, so that intraocularly injected N G F might have been effective primarily because of the relatively large amount reaching the lesion site by extraceHular diffusion, and conversely, a significant amount of the locally applied N G F might have reached the retinal ganglion cell bodies. One or more of the following mechanisms might therefore be operating: (a) N G F was taken up at the site of the lesion and retrogradety transported to the cell bodies of the retinal ganglion cellsl°,tl,19: (b) N G F was acting directly on the cell bodies: (c) N G F was acting locally at the site of the lesion, either on the axons 6,9,1s or on the glial cells or other supporting elements. It remains to be determined whether other procedures that enhance axonal outgrowth in goldfish optic axons, e.g. a prior conditioning lesion 14 or application of the calcium ionophore A231878, involve the same mechanisms as those underlying the N G F effect In spite of the outgrowth-enhancing effect of N G F that was observed repeatedly with various N G F preparations and routes of administration, we have been unable to confirm the original observations by Turner et al. ''z that the cell bodies of the regenerating retinal ganglion cells show a large increase in size in response to N G F . Instead, we have found that the size of the NGF-treated cells was no different from normal regenerating cells for at least 14 days after the lesion, even in an experiment (NFBT in Tables II and IIl) in which the conditions of the experiment (including temperature, survival time and N G F preparation used) were quite similar to those used by Turner et al. s'z. Our experiment differed from theirs, however, in the processing of the retinas for histology and in the selection of cells for measurement. Our method of fixation and paraffin-embedding would undoubtedly have led to some tissue shrinkage and distortion, but these should have affected the control and N G F treated retinas equally. (In one experiment I F I N in Table lll), in which the retinas were excised, then fixed and embedded as for electron microscopy, there was also no observable effect of N G F on cell size. In this experiment, however, the injections were not begun until 2 days after nerve crush, which may not be optimal for the N G F effect.) To eliminate any bias in selection of cells for measurement, we made all observations and measurements with the materials coded to conceal which had come from NGF-treated animals and which from controls, and in each case we adhered to an arbitrary schedule in selecting cell profiles for measurement lsee Methods). These conditions ensured that a random population of cells would be sampled. The cell profiles on which Turner et al. made their measurements, on the other hand. were selected by other criteria (appropriate to the fact that they were dealing with electron microscopic material), namely that each cell profile should contain a nucleolus, a procedure that might tend to favor cells with larger nucleoli. This raises the possibility that a sub-population of the retinal ganglion cells, possibly those largest in size, may be selectively sensitive to NGF. With this in mind, we examined the sizes of the 8 largest cells in the sample from each retina, but we still did not find any enlargement attributable to N G F (B. Grafstein and H. K. Yip, unpublished results~. Another hypothesis that might account for the difference between our results and those of
337 Turner et al. is that the time course of the cellular enlargement in response to axotomy may have been different in the two cases. Thus if the measurements were made when the cells in the vehicle-injected (normally regenerating) animals were close to their maximal size, little if any effect of N G F might have been detectable, whereas if only a modest increase was present in the vehicle-injected animals, a prominent N G F effect might have been seen. Our findings on the nucleolar response to axotomy also appear to differ somewhat from those by Turner et al. As has previously been shown by Murray and Grafstein 1~, the axotomy-evoked increase in nucleolar incidence, as observed by light microscopy, correlates very closely with the onset of an increase in nucleolar size 16 and an increase in incorporation of RNA precursors 15. Although Turner and Delaney 21 reported that the incidence of nucleoli was unaffected by N G F treatment, their method of cell profile selection (based on the presence of a nucleolus) would invalidate their calculation of nucleolar incidence. However, they did report an increase in nucleolar size with NGF. We did not measure nucleolar size directly, but our finding that nucleolar incidence does not change with N G F treatment suggests that nucleolar size also does not change. This would be true particularly in those cases, e.g. at 3 days after nerve crush, in which a substantial proportion of cells are counted as being without nucleoli, presumably because their nucleoli are too small to be detectable by the light microscope. In this case, a significant increase in nucleolar size would be expected to reveal itself in a higher proportion of cells with nucleoli above the threshold size. Regardless of the reasons for the differences between our results and those of Turner et al., the fact that we found that axonal outgrowth was enhanced by N G F in the absence of changes in the nucleoli and in the size of the cell body suggests that this enhancement of outgrowth may not require a significant increase in total RNA or protein synthesis above that which is normally seen during regeneration. This has been confirmed by pilot experiments which have shown that labeled amino acid incorporation during regeneration is not affected by N G F (H. K. Yip and B. Grafstein, unpublished results). Our findings on the outgrowth-enhancing effect of N G F on optic axons, together with the earlier observations by Turner et al. 21-z5 on the opposing effects of N G F and N GF-antibody on regenerating retinal ganglion cells, suggest that N G F may normally play a role in the regeneration of the optic axons. The fact that goldfish brain contains significant amounts of N G F or a closely related substancO, ",27 would be consistent with this hypothesis. It is not yet clear whether this hypothesis will need to be modified to take account of our present observations suggesting that N G F may be effective only if it is given at the time of the injury. ACKNOWLEDGEMENTS We wish to thank Dr. Douglas Ishii and Dr. Robert Stach for providing us with the N G F preparations, and Dr. Lloyd A. Greene for assaying some of the preparations. We are grateful to Mr. Jonathan Javitt, Mr. Peter Mandelson, Ms. Roberta Alpert and Dr. Mark Whitnall for their participation in some of the experiments and
338 t o D r . H a m u t a l M e i r i f o r m a k i n g t h e n e r v e c u f f t e c h n i q u e a v a i l a b l e t o us. This work was supported by USPHS Grants NS-0915 and NS-14967 to BG and a Fellowship from the National Spinal Cord Injury Foundation
to H.K.Y.
REFERENCES 1 Benowitz, L. I. and Greene, L. A., Nerve growth factor in the goldfish: biological assay studies using pheochromocytoma cells, Brain Research, 162 (1979) 164-168. 2 Benowitz, L. I. and Shashoua, V. E., Immunoreactive sites for nerve growth factor (NGF) in the goldfish brain, Brain Research, 172 (1979) 561-565. 3 Bjerre, B., Bj6rklund, A. and Stenevi, U., Stimulation of growth of new axonal sprouts from lesioned monoamine neurones in adult rat brain by nerve growth factor. Brah~ Research, 60 (1973) 161-176. 4 Bodian, D., A new method for staining nerve fibers and nerve endings in mounted paraffin sections, Anat. Rec., 65 (1936) 89-97. 5 Bradshaw, R. A., Nerve growth factor, Ann. Rev. Biochem., 47 (1978) 191-216. 6 Campenot, R. B., Local control of neurite development by nerve growth factor. Proc. ttat. Acad. Sci. (U.S.A.), 74 (1977) 4516-4519. 7 Edwards, D. L., Alpert, R. M. and Grafstein, B.. Recovery of vision in regeneration of goldfish optic axons: enhancement of axonal outgrowth by a conditioning lesion, Exp. NeuroL. 72 (198I) 672-686. 8 Grafstein, B. and Meiri, H., Increased rate of regeneration in goldfish optic axons resulting from intraocular injection of calcium ionophore A-23187, Neurosci. Abstr., 6 (1980) 387. 9 Gundersen, R. W. and Barrett, J. N., Characterization of the turning response of dorsal root neurites toward nerve growth factor, J. Cell Biol., 87 (1980) 546-554. t0 Hendry, I. A., Stoeckel, K. and Thoenen, H.. The retrograde axonal transport of nerve growth factor, Brain Research, 68 (1974) 102-121 I1 Johnson, E. M., Jr., Andres, R. Y. and Bradshaw, R. A., Characterization of the retrograde transport of Nerve Growth Factor (NGF) using high specific activity [125I]NGF, Brain Research, 150 (1978) 319-331 12 Letourneau, P. C., Chemotactic response of nerve-fiber elongation to nerve growth factor Develop. Biol., 66 (1978) 183-196. 13 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev.. 48 (/968) 534-569. 14 McQuarrie, I. G. and Grafstein, B., Effect of a conditioning lesion on optic nerve regeneration in goldfish, Brain Research, 216 (1981) 253-264. 15 Murray, M., 3H-uridine incorporation by regenerating retinal ganglion cells of goldfish, Exp. Neurol., 39 (1973) 489-497. 16 Murray, M. and Grafstein° B., Changes in the morphology and amino acid incorporation of regenerating goldfish optic neurons, Exp. Neurol., 23 (1969) 544-560. 17 Schwartz, M., Kohsaka, S. and Agranoff, B. W., Nerve growth factor increases ornithine decarboxylase activity in goldfish retinal exptants, Neurosci. Abstr., 6 (1980) 687. 18 Sperry, R. W., Functional regeneration in the optic system, In W. F. Windle tEd.J, Regeneration m the Central Nervous System, Thomas, Springfield, IL, 1955, pp. 66-76. 19 Stoeckel, K., Guroff, G., Schwab, M. and Thoenen, H., The significance of retrograde axonal transport for the accumulation of systemically administered nerve growth factor (NGF) i n t h e rat superior cervical ganglion, Brain Research, 109 (1976) 271-284. 20 Thoenen, H and Barde, Y.-A.. Physiology of nerve growth factor. Physiol. Rev.. 60 t1980) 1284-1335. 21 Turner, J. E. and Delaney, R. K.. Retinal ganglion cell response to axotomy aud nerve growth factor in the regenerating visual system of the newt (Triturus viridescens): An ultrastructural morphometric analysis, Brain Research, 171 (1979) t 97-212. 22 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to nerve growth factor in the regenerating and intact visual system of the goldfish (Carasshts auratus), Brain Research, 197 (1980) 319-330. 23 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish ,'Carassius auratus) : an m vivo and in vitro analysis, Brain Research, 204 (1981) 283--294.
339 24 Turner, J. E., Delaney, R. K. and Powell, R. E., Retinal ganglion cell response to axotomy in the regenerating visual system of the newt (Triturus viridescens): an ultrastructural morphometric analysis, Exp. Neurol., 62 (1978) 444 462. 25 Turner, J. E. and Glaze, K. A., Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): effect of nerve growth factor, Exp. Neurol., 57 (1977) 687-697. 26 Varon, S., Nerve growth factor and its mode of action, Exp. Neurol., 48 (1975) 75 92. 27 Weis, J. S., The occurrence of nerve growth factor in teleost fish, Experientia, 24 (1968) 736-737.
329
Elsevier Biomedical Press
E F F E C T OF N E R V E G R O W T H F A C T O R ON R E G E N E R A T I O N OF G O L D F I S H OPTIC AXONS
HENRY K. YIP and BERNICE GRAFSTEIN Department of Physiology, Cornell University Medical College, Arew York, N Y 1002l (U.S.A.)
(Accepted September 3rd, 1981) Key words': nerve regeneration - - nerve growth factor - - NGF - - trophic factors - - goldfish retina
--- optic nerve
SUMMARY Axonal outgrowth tollowing a crush of the goldfish optic nerve was enhanced if nerve growth factor ( N G F ) was administered by intraocular injection or by local application to the lesion site. Various forms of N G F (fl, 2.5S and 7S) were effective, producing a 20-40 0~ decrease in the time required for recovery of the startle reaction to a bright light. A corresponding increase in axonal outgrowth was revealed by histological examination of the optic nerves. The effect produced by a single intraocular injection given at the time of the lesion was not further increased by subsequent injections. Up to 14 days after the lesion, the size of the retinal ganglion cell bodies and the incidence of nucleoli detectable by light microscopy were not affected by the N G F treatment.
INTRODUCTION It has been known for some time that nerve growth factor ( N G F ) stimulates neurite outgrowth in sympathetic postganglionic neurons, in embryonic dorsal root ganglion cells, and possibly in noradrenergic neurons in the CNSZ,5,1a, 2°,26. Recently, it has also been reported that N G F promotes regeneration of optic axons in lower vertebrates. For example, after optic nerve transection in the newt, the proportion of the optic nerve cross-section occupied by regenerated axons was found to be larger in NGF-treated animals than in controls, and the changes elicited in the retinal ganglion cell bodies by axotomy were accelerated and intensified by N G F treatment zl,24,25. In goldfish, N G F was reported to have a dramatic effect on the size and ultrastructure of axotomized retinal ganglion cells, and these cells showed significantly increased neurite outgrowth in tissue culture if they had been treated with N G F before explanta0006-8993/82/0000-0000]$02.75 © Elsevier Biomedical Press
330 tion 23. A n o t h e r study o f goldfish retina explants showed that the effect of N G F in increasing ornithine decarboxylase activity was m o r e marked in retinas in which the retinal ganglion cells were regenerating at the time o f explantation than in controls~L These results are interesting not only because they may throw a light on the mechanism o f action o f N G F , but also because they may have a bearing on the mechanisms that make optic nerve regeneration so succvssfut in lower vertebrates, in contrast to m a m m a l s TM. In the present study, we have further analyzed the regeneration-promoting effects o f N G F using a behavioral technique to assay the rate o f regeneration following a crush of the goldfish optic nerve. In order to determine whether enhanced axonal outgrowth is accompanied by changes in the cell body, we have examined the effect o f N G F on the incidence o f nucleoti detectable by light microscopy, a parameter that increases dramatically in goldfish retinal ganglion cells following a x o t o m y t6. We have also re-examined the effect o f N G F on cell size. We have found that N G F can indeed enhance axonal outgrowth in this system, but that this effect is not necessarily accompanied by changes in cell size or nucleolar incidence. MATERIALS AND METHODS Goldfish 5-8 cm in body length (nose to base o f tail), 6-12 g in weight, were used. They were obtained from O z a r k Fisheries, Stoutland, MO, and were kept in thc laboratory at 20 °C. F o r surgical procedures, they were anesthetized by immersion in ice. In most experiments, the right optic nerve was crushed with .jewellers' forceps at the back o f the orbit; in some cases both optic nerves were ctushed, N G F from 3 different sources was tested in the course o f these experiments TABLE I N G F preparations tested
For some of the ~ and 2.5S preparations, the injection solution remaining at the end of the experiment was re-assayed and was found to contain only about I0% of the expected biological activity. It is not clear whether this loss, which may have been due to deterioration of the material in storage or loss by binding to glass surfaces, occurred before or after the injection. Sources: (1) Dr. Doughs Ishii, Dept. of Pharmacology, Columbia University College of Physicians and Surgeons; (2)Collaborative Research, Waltham, MA; (3) Dr. Robert Stach, Dept. of Biochemistry, SUNY Upstate Medical Center. BSA, bovine serum albumin (Sigma). Code
Type
Nominal biological unit
Nominal biological units per mjeetion
Vehicle
Source
A B C D E
/J [:~ 2.5S 2.5S 7S
1 ng 1 ng 0.4 ng 0.34 ng 30 ng
1000 1000 1000 t790 900
1 ~ BSA 0.9 ~ saline 2 ~ BSA 0.1% BSA Tris buffer*
(1) (1) (2) (2) (3)
* 0.05 M Tris buffer, pH 7.0. with l0 6 M ZnCl2.
331 (Table I). For intraocular application, N G F in a suitable vehicle was injected into the vitreous humor of the operated eye of the experimental fish, while control fish received a similar injection of the vehicle alone. The injections were 4-6 /A in volume and were made with a 10 #l Hamilton syringe. Local applications to the site of the nerve crush were made by injecting 4 - 6 / d cf the N G F or vehicle solution into a 5 cm length of silastic tubing which opened onto the optic nerve through a cuff made of medical grade Silastic. The cuff with the attached tubing was placed around the nerve at the time of the optic nerve crush. Testing for the startle reaction was carried out as described by Edwards et al. 7, usually beginning at 4 days after nerve crush, with the unoperated eye in each case blinded by injection of 0.025-0.05/~g tetrodotoxin or 150/~g xylocaine.
Histological methods At 3-14 days after nerve crush, the fish were decapitated and the heads were immediately fixed in Bouin's solution, or, in the experiments in which the optic nerves were te be silver-stained, the unfixed nerves were removed with a small portion of optic tectum attached and were fixed on a card to keep them straight. For nucleolar counts TM and measurements of retinal ganglion cell size the eyes were embedded in paraffin, sectioned at 15/~m and stained with toluidine blue. (In one experiment ( F | N in Table 111), the retinas from half the fish in each group were dissected out, fixed by immersion in 2 ~ glutaraldehyde, embedded in Epon, sectioned at 0.5/~m, and stained with toluidine blue. Size measurements in this case were made on all cell profiles that contained nuclei, up to a total of 15 per retina. The retinas from the other fish in each group were fixed and processed in the usual way for nucleolar counts.) In each retina nucleolar counts were usually made on 105 cells and cell size measurements on 40 cells. The measurements were made on cells in at least 3 separate histological sections of each retina, beginning at different points on each section in order to ensure that cells from all over the retina were sampled. Every fifth cell profile encountered in a sequential examination of the section was taken for measurement, unless that profile contained an incomplete nucleus, in which case the next profile in sequence that fulfilled this requirement was taken. The presence of nucleoli was determined with a 100 > oil immersion microscope objective. For cell size measurements the cell profiles were drawn with a camera lucida using a 40 × objective, and the area of each drawing determined by means of a computerized graphic analyzer (Numonics, Lansdale, PA). For the optic nerves, serial longitudinal paraffin sections were made and stained with Bodian's silver technique 4. With the use of a 40 × microscope objective determinations were made of the length of the longest detectable bundle of axons, measured from the site of initiation of outgrowth (which was usually about 0.2 m m back from the site of the lesiona4), and of the number of axons seen at various distances from the lesion. For each animal, the values obtained were the means of measurements made on 5-6 separate sections that contained the complete length of nerve from the lesion area to the tectum. In both the behavioral and the histological experiments, the experimenter was uninformed of the treatments given until the experiment was concluded. Statistically significant differences were determined by Student's t-test (2-tailed).
332 INTRAOCULAR
i j
H%LE ~'"'-~
,
' ~
,~
T-z' o
TIME AFTER OPTIC NERVE CRUSH (days)
Fig. 1. Cumulative response curves for the recovery of the startle reaction after optic nerve crush: There were 10 animals per group, and each point represents the total number of animals in each group that had responded by the day of testing. The mean recovery time was 7.9 ~ 0.7 days for the N G F treated animals and 12.9 i 0.8 for the control animals (Table tI~ experiment 7SDR). Thedifference is statistically significant (P < 0.05). RESULTS
Axonal outgrowth The time required for recovery of visual function was determined by testing for the startle reaction, since it has been shown that this test is sensitive to the time required for axonal outgrowth% It was found that intraocular injection of N G F caused a 20--40 ~o decrease ;n mean recovery time (Fig. h Table I1). All 3 types of N G F tested, i.e. fl-, 2.5S and 7S N G F 5, were effective when injected in amounts of about 1000 BU (Table II), but only a small effect, which was not statistically significant, was seen when the 7S preparation was diluted 10-fold or more (Fig. 2). (The effectiveness TABLE 1l
Effect o f N G F on time o f recovery o f startle reaction following nerve crush Each value is the mean : S.E.M. for a group o f 8-10 animals. 0 injection time : 1 h or less after nerve crush. In experiment N G X only, mean recovery times for 70 % of animals in each group are given. Experiment NFBT was performed at 25 °C; mean recovery times are for animals responding by 7 days, obtained by combining the 2 N G F groups and the 2 vehicle groups.
Exp,
NGF Injection Prep. time (Table 1) (days)
lntraocular injection NGX A NFBT NFBT 7SDR
B D E
Local application NFLA B NFLA D
0 0 and 3 ~ 0 and 3 0 0 and 3 0 and 3
Mean recovery time (days) ± S.E.M. Vehicle
NGF
Difference
11.0 ~ 0.5
8.6 m 0.6*
21%
5.8 :r 0.4
4.6 ± 0.2*
21
12.9 = 0.8
7.9 :L 0.7*
---39%
14.5 m 1.7 12.0 m 1.5
11.0 ~ 1.2 8.3 ~ 0.9*
24%~ 31
12.7 ~ 1,2
3%
Intraocular injection into contralateral eye 7SSE E 0 13.1 • 1.9
* P < 0.05 for difference between N G F - and vehicle-injected animals.
333 Injections: B 0 day 0 & 3 days l~a every ?- days
15 ¸
v
-~ ,o t>rY W
0 (.9 w ¢,,, z ,< w 0 (veh.) AMOUNT
9O
900
9
OF N G F PER I N J E C T I O N
(B.U.)
Fig. 2. Effects of different doses and dosage schedules of N G F on mean recovery time of startle reaction after optic nerve crush. N G F (7S) was administered by intraocular injection at the time of crush (0 day), at the time of crush and 3 days later, or at the time of the crush and every 2 days thereafter. Control animals received an injection of vehicle (0.05 M Tris buffer) at the corresponding times. Each value is the mean ± S.E.M. for 12 animals. The decrease in mean recovery time due to N G F is statistically significant (P -< 0.001) only with the 900 BU doses. For each dose the change produced by a single injection is not significantly different from that produced by multiple injections.
of the 7S N G F relative to that of/3- or 2.5S material cannot be estimated, since the fland 2.5S preparations are more labile under storage conditions and more easily lost in handling.) The effect produced by a single injection of 900 BU NGF given at the time of the lesion was just as great as if this injection was followed by a second one 3 days later (Fig. 2). To ensure that this result was not due to a saturating effect of the large dose of N G F given in the first injection, we also tested multiple injections of lower N G F doses, including injections every 2 days throughout the recovery period, but no significant effect was seen (Fig. 2). Local application of NGF to the site of the nerve crush was also effective in decreasing the recovery time by about the same percentage as intraocular injection (Table lI, experiment NFLA). In some of these experiments the injection vehicle contained enough BSA so that the total amount of protein in the
E E
A
B
LONGEST AXON BUNDLE
DISTRIBUTION OF AXONS ~, '°°
3
~
VEHICLE
[~
NGF
p <: 0 . 0 5
0~ 50 z
O
0
0 VEH.
NGF
0.5
1.0
1.5
2.0
DISTANCE FROM CRUSH SITE (ram)
Fig. 3. The effect of N G F on (A) the longest bundle of axons and (B) the number of axons seen at various distances from the lesion in silver-stained sections of goldfish optic nerve 7 days after optic nerve crush (6 animals in the NGF-treated group (3 with fl-NGF, 3 with 2.5S NGF) and 5 animals in the vehicle-injected group). All values for the NGF-treated group are significantly higher than for the corresponding control group (in A, P < 0.05; in B, P < 0.001-0.002). Vertical lines represent S.E.M.
14 14
Local application NFLA B NITLA D
0and3 0and 3
8 7 7 6 3 3 3
NGF InjeCtion Sac. prepatime time ration (daysJ (days/ (Table 1)
lntraocular injection NGX A 0 NFBT B 0and 3 NFBT D 0and 3 FI/q C 2 and 4 NFNC B 2 NFNC B 0 NFNC B --1
Exp.
40.7 ± 0:9 40.7 :± 0.9
42.7 :~ 3.0 42.8 & 2.1
38:3 _;: 0.5
Normal or shamoperation
± ± ± :~ ±
5.2 0.2 1.9 4.7 5.4
74:9 -~: 3.t 81.1 ± 4:8
48.1 76.1 76.t 53.3 43.8
77.4 ± 2.5 84.5 ~ 5.6
46.8 ± 1.4 78.2 ± 1.2 71.8 _q: 3.5 56.9 ± 3.9 48.1 ± 4.6
.......... Vehicle NGF
Cell size (/~m ~)
~3~ -4~o
--3~ +3~ --6°Jo ~ 7~ ÷9~o
0.56 2_ 0.12 0.51 ± 0.08
0.20 ± 0.02
Difference Normal or between shamNGF and operation vehicle
1.09 ± 0 . 0 5 1.10 ~ 0.16 1.33 ~ 0.05 1.18 ~: 0~05 0.71 ± 0 . 0 5 0.62 ± 0.09 0.51 ± 0.04
Vehicle
Difference between NGF and vehicle
0 0.68 ± 0.03 - - 4 ~ 0.63 + 0,04 t 2 ~ o 0.53 ± 0.05 ~ 4 ~
1.18 5:0.05
1.01 ± 0.06 --7~o 1.22 ~ 0 . 0 7 -1l~o 1.26 fi: 0.06 ---5 ~o
NGF
Nucleolar incidence (no./cell)
Each value is the mean 4- S.E.M. for 3-8 retinas (see Materials and Methods). The identification codes of the individual experiments correspond to those in Table I1.0 injection time = 1 h or less between nerve crush and injection; --1 day injection time = injection 1 day before crush. Experiment NFBT was performed at 25 °C.
Effects o f NGF on retinal ganglion cell size and nucleolar incidence .]bllowing nerve crush
TABLE 111
GO GO
335 injection was greatly in excess of that contributed by the N G F . This eliminates the possibility that the observed shortening of recovery time was due to a non-specific effect of protein. In one experiment with intraocular injection of N G F (Table 1I, experiment NFBT) the behavioral study was terminated at 7 days in order to make histological measurements of axonal outgrowth in silver-stained sections of the optic nerve (Fig. 3). Measurements on the length of the longest regenerating axon bundle (Fig. 3A) and the mean number of axons per nerve section seen at various distances from the site of the lesion (Fig. 3B) showed enhanced outgrowth as a result of the N G F treatment, confirming the validity of the decrease in recovery time as an index of an increase in axonal outgrowth. The mean values (-- S.E.M.) for the total number of axons in the whole nerve at 1 mm from the lesion was 980 ~ 250 (n -- 6) for the vehicle-treated animals and 3740 ::L 460 (n = 7) for the NGF-treated animals, i.e. there were nearly 4 times as many axons in the NGF-treated animals as in the controls (difference significant at a level of P < 0.001). The magnitude of the increase in outgrowth distance of the longitudinal axon bundles, about 25 '},o ~ (Fig. 3A), was very close to the magnitude of the decrease in recovery time seen in the corresponding behavioral experiment. The actual rates of outgrowth cannot be determined from these data because the initial delays before outgrowth began are not known. Injection of N G F into the eye on the side contralateral to the lesion had no effect on the time required for recovery of the startle reaction (Table II, experiment 7SSE). This shows that the N G F was not acting by a systemic route.
Cell body characteristics In accord with previous observations 16 an increase in retinal ganglion cell size was evident by 6 days after nerve crush and an increase in the incidence of nucleoli by 3 days (Table Ill). In animals examined up to 14 days after the lesion, however, the changes in the N G F - and vehicle-treated animals were not significantly different (Table Ill), even in experiments in which axonal outgrowth had been enhanced by N G F treatment (as indicated by behavioral or histological evidence). This was true regardless of whether the N G F was applied by intraocular injection or locally to the lesion site. DISCUSSION Our results show that axonal outgrowth in regenerating goldfish optic axons can be enhanced by either intraocular injection of N G F or local application of N G F at the site of the lesion. At least for the intraocular injections, N G F treatment at the time of the lesion was effective, and subsequent applications did not increase the effect. The outgrowth-enhancing effect of N G F was evident both from the decreased time required for recovery of one kind of visual function and from the increased distance of axonal outgrowth revealed by histological examination. It still remains to be determined whether N G F affects the time of initiation of axonal outgrowth or the rate of advance of the axons, or both.
336 The site and mechanism of action of the N G F are not yet known. In the present study, intraocular and local applications of N G F were about equally effective, but the site of the optic nerve crush was only a few millimeters from the eye, so that intraocularly injected N G F might have been effective primarily because of the relatively large amount reaching the lesion site by extraceHular diffusion, and conversely, a significant amount of the locally applied N G F might have reached the retinal ganglion cell bodies. One or more of the following mechanisms might therefore be operating: (a) N G F was taken up at the site of the lesion and retrogradety transported to the cell bodies of the retinal ganglion cellsl°,tl,19: (b) N G F was acting directly on the cell bodies: (c) N G F was acting locally at the site of the lesion, either on the axons 6,9,1s or on the glial cells or other supporting elements. It remains to be determined whether other procedures that enhance axonal outgrowth in goldfish optic axons, e.g. a prior conditioning lesion 14 or application of the calcium ionophore A231878, involve the same mechanisms as those underlying the N G F effect In spite of the outgrowth-enhancing effect of N G F that was observed repeatedly with various N G F preparations and routes of administration, we have been unable to confirm the original observations by Turner et al. ''z that the cell bodies of the regenerating retinal ganglion cells show a large increase in size in response to N G F . Instead, we have found that the size of the NGF-treated cells was no different from normal regenerating cells for at least 14 days after the lesion, even in an experiment (NFBT in Tables II and IIl) in which the conditions of the experiment (including temperature, survival time and N G F preparation used) were quite similar to those used by Turner et al. s'z. Our experiment differed from theirs, however, in the processing of the retinas for histology and in the selection of cells for measurement. Our method of fixation and paraffin-embedding would undoubtedly have led to some tissue shrinkage and distortion, but these should have affected the control and N G F treated retinas equally. (In one experiment I F I N in Table lll), in which the retinas were excised, then fixed and embedded as for electron microscopy, there was also no observable effect of N G F on cell size. In this experiment, however, the injections were not begun until 2 days after nerve crush, which may not be optimal for the N G F effect.) To eliminate any bias in selection of cells for measurement, we made all observations and measurements with the materials coded to conceal which had come from NGF-treated animals and which from controls, and in each case we adhered to an arbitrary schedule in selecting cell profiles for measurement lsee Methods). These conditions ensured that a random population of cells would be sampled. The cell profiles on which Turner et al. made their measurements, on the other hand. were selected by other criteria (appropriate to the fact that they were dealing with electron microscopic material), namely that each cell profile should contain a nucleolus, a procedure that might tend to favor cells with larger nucleoli. This raises the possibility that a sub-population of the retinal ganglion cells, possibly those largest in size, may be selectively sensitive to NGF. With this in mind, we examined the sizes of the 8 largest cells in the sample from each retina, but we still did not find any enlargement attributable to N G F (B. Grafstein and H. K. Yip, unpublished results~. Another hypothesis that might account for the difference between our results and those of
337 Turner et al. is that the time course of the cellular enlargement in response to axotomy may have been different in the two cases. Thus if the measurements were made when the cells in the vehicle-injected (normally regenerating) animals were close to their maximal size, little if any effect of N G F might have been detectable, whereas if only a modest increase was present in the vehicle-injected animals, a prominent N G F effect might have been seen. Our findings on the nucleolar response to axotomy also appear to differ somewhat from those by Turner et al. As has previously been shown by Murray and Grafstein 1~, the axotomy-evoked increase in nucleolar incidence, as observed by light microscopy, correlates very closely with the onset of an increase in nucleolar size 16 and an increase in incorporation of RNA precursors 15. Although Turner and Delaney 21 reported that the incidence of nucleoli was unaffected by N G F treatment, their method of cell profile selection (based on the presence of a nucleolus) would invalidate their calculation of nucleolar incidence. However, they did report an increase in nucleolar size with NGF. We did not measure nucleolar size directly, but our finding that nucleolar incidence does not change with N G F treatment suggests that nucleolar size also does not change. This would be true particularly in those cases, e.g. at 3 days after nerve crush, in which a substantial proportion of cells are counted as being without nucleoli, presumably because their nucleoli are too small to be detectable by the light microscope. In this case, a significant increase in nucleolar size would be expected to reveal itself in a higher proportion of cells with nucleoli above the threshold size. Regardless of the reasons for the differences between our results and those of Turner et al., the fact that we found that axonal outgrowth was enhanced by N G F in the absence of changes in the nucleoli and in the size of the cell body suggests that this enhancement of outgrowth may not require a significant increase in total RNA or protein synthesis above that which is normally seen during regeneration. This has been confirmed by pilot experiments which have shown that labeled amino acid incorporation during regeneration is not affected by N G F (H. K. Yip and B. Grafstein, unpublished results). Our findings on the outgrowth-enhancing effect of N G F on optic axons, together with the earlier observations by Turner et al. 21-z5 on the opposing effects of N G F and N GF-antibody on regenerating retinal ganglion cells, suggest that N G F may normally play a role in the regeneration of the optic axons. The fact that goldfish brain contains significant amounts of N G F or a closely related substancO, ",27 would be consistent with this hypothesis. It is not yet clear whether this hypothesis will need to be modified to take account of our present observations suggesting that N G F may be effective only if it is given at the time of the injury. ACKNOWLEDGEMENTS We wish to thank Dr. Douglas Ishii and Dr. Robert Stach for providing us with the N G F preparations, and Dr. Lloyd A. Greene for assaying some of the preparations. We are grateful to Mr. Jonathan Javitt, Mr. Peter Mandelson, Ms. Roberta Alpert and Dr. Mark Whitnall for their participation in some of the experiments and
338 t o D r . H a m u t a l M e i r i f o r m a k i n g t h e n e r v e c u f f t e c h n i q u e a v a i l a b l e t o us. This work was supported by USPHS Grants NS-0915 and NS-14967 to BG and a Fellowship from the National Spinal Cord Injury Foundation
to H.K.Y.
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339 24 Turner, J. E., Delaney, R. K. and Powell, R. E., Retinal ganglion cell response to axotomy in the regenerating visual system of the newt (Triturus viridescens): an ultrastructural morphometric analysis, Exp. Neurol., 62 (1978) 444 462. 25 Turner, J. E. and Glaze, K. A., Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): effect of nerve growth factor, Exp. Neurol., 57 (1977) 687-697. 26 Varon, S., Nerve growth factor and its mode of action, Exp. Neurol., 48 (1975) 75 92. 27 Weis, J. S., The occurrence of nerve growth factor in teleost fish, Experientia, 24 (1968) 736-737.