Brain Research, 177 (1979) 35 47 ,~ Elsevier/North-Holland Biomedical Press

35

RETINAL G A N G L I O N CELL RESPONSE TO AXOTOMY AND NERVE G R O W T H FACTOR ANTISERUM IN T H E R E G E N E R A T I N G VISUAL SYSTEM OF T H E NEWT ( N O T O P H T H A L M U S VIRIDESCENS): AN U L T R A S T R U C T U R A L M O R P H O M E T R I C ANALYSIS

JAMES E, TURNER and REBECCA K. DELANEY Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, N.C. 27103 (U.S.A.)

(Accepted March 8th, 1979)

SUMMARY One 3.0 mg dose of the nerve growth factor antiserum (anti-NGF) injected into the vitreous chamber of the eye at the time of optic nerve transection elicits significant changes in the normal newt (Notophthalmus viridescens) retinal ganglion cell body response to axotomy at 7 and 14 days postaxotomy (DPA). Light microscopic observations indicate that anti-NGF treatment significantly reduces the per cent of retinal ganglion cells demonstrating nuclear chromatin reactivity (ie., homogeneous to a more heterogeneous state) from 33.36 J: 3.02 to 22.82 ~ 2.98 ~. In addition, the per cent of retinal ganglion cells demonstrating prominent nucleoli is dramatically decreased from 32.08 ~ 1.64 to 18.20 :~ 1.79~ at 7 DPA. It is also important to note that the number of prominent nucleoli in the 7 DPA group is reduced to such an extent by anti-NGF treatment that the value is not significantly different from that of intact controls. Intact controls will routinely exhibit approximately half the number of prominent nucleoli that are normal for the untreated 7 DPA group. A definite dose-response relationship can be shown to exist between the per cent of nuclear reactive ganglion cells demonstrating prominent nucleoli and various antiN G F concentrations at 14 DPA. There does not appear to be a dose-response relationship between various anti-NGF concentrations and the per cent of retinal ganglion cells demonstrating nuclear reactivity at 14 DPA. However, the degree of nuclear chromatin reactivity appears to be less at the higher anti-NGF concentrations (ie., ~: 3.0 rag/eye) at 14 DPA. Electron microscopic morphometric analysis reveals that anti-NGF treatment significantly reduces the cell perikaryal area at 7 and 14 DPA while the nuclear area remains unchanged. Therefore, there is a significant decrease in the cytoplasmic/nuclear ratios at botil 7 and 14 DPA in response to anti-NGF treatment which appears more pronounced by 14 DPA. Anti-NGF treatment also significantly reduces

36 the mitochondrial and nucleolar densities, as well as the nucleolar areas of cells at 7 and 14 DPA. There are no significant changes in Golgi field densities in response to anti-NGF treatment.

INTRODUCTION Since its isolation by Cohen 1°, the nerve growth factor antiserum (anti-NGF) has been used extensively to test for the dependence of certain neuronal populations on NGF. For example, it has been shown that serial injections o f a n t i - N G F cause selective and permanent destruction of as much as 97-99 ~ of the sympathetic nerve cells in newborn rats, mice and kittens lz. This points to a key role for N G F in the development of sympathetic neurons. In adult mice, anti-NGF treatment causes some regression of the sympathetic nervous system but does not cause the almost total destruction seen in newborn animals a,2,4,7,9. In addition, anti-NGF has been shown to inhibit the regenerative response in the adult peripheral 4 and centralS,6,s, 23 nervous systems. It has been demonstrated in our laboratory that both N G F and anti-NGF have dramatic and opposing effects on optic nerve regeneration in the newt (Notophthalmus viridescens) 12,29,3°. We have also demonstrated the newt retinal ganglion cell response to axotomy and axotomy plus N G F through light and electron microscopic morphometric analysis31, z2. These results are most intriguing since the optic nerve is considered to be a central nervous system (CNS) tract. Very little is known about the possible role of N G F or an NGF-like molecule in the developmental, maintenance and regenerating situations within the CNS, although supportive evidence in this area is beginning to accumulate3,5,~,s, la,17,2°. We have also worked extensively with this model in the past to characterize the degenerative and regenerative phenomena in response to nerve transectionZ4,z7, 30-39. The present paper is an attempt to further assess the possible role of N G F in CNS regeneration and deals with various morphological and ultrastructural aspects of the newt retinal ganglion cell perikaryal response to anti-NGF treatment after axotomy. MATERIALS AND METHODS Adult newts (Notophthalmus viridescens), obtained from Lee's Newt Farm, Oak Ridge, Tennessee, were anesthetized in dilute Chloretone solution. Animals in the dose-response study received one 4/~1 intraocular injection of either 10, I00, 1500 or 3000 #g of rabbit anti-NGF which was prepared against the highly purified 2.5S N G F (Collaborative Research, Inc.). Eyes were prepared for electron microscopy at 14 days postaxotomy (14 DPA). In a second study animals received one 4 /A intraocular injection of 3000 #g anti-NGF at the time of nerve transection. At 7 DPA half the group were sacrificed and the remaining animals received a second 4 #I intraocular injection of 3000/~g anti-NGF and were allowed to survive to 14 DPA at which time eyes were prepared for electron microscopy. Controls for these studies received either

37 1500 or 3000 #g of rabbit serum (kindly donated by Collaborative Research, Inc.). Injections were made into the vitreous chamber of the eye according to the method of McEwen and Grafstein is. The orbital portion of the left optic nerve was severed immediately after injection according to the method of Sperry 2z and Turner and Singer 3l. After transection animals were kept iv. a moist chamber until alert and were transferred to water-filled containers and kept at a constant water temperature of 25 1 °C. Animals were kept on a constant photoperiod of a 14/10 h light/dark cycle. Eyes were prepared for electron microscopy by a microperfusion system which allowed small quantities of fixative to be delivered directly into the vitreous chamber adjacent to the retinal ganglion cell layer in a manner previously described 26. The following day eyes were halved through the optic papilla in an anteriorposterior plane, washed in cacodylate buffer, osmicated 1 h, dehydrated in an ethanol series and embedded in Epon. Thick (1 #m) and thin (600-900 nm) sections were cut with glass and diamond knives on a Porter-Blum ultramicrotome (MT-2B). Thick sections were stained with toluidine blue in borate buffer (l ~ , pH 7.4) for examination and photography with the light microscope. Thin sections were stained with uranyl acetate and lead citrate 21.

Morphometric analysis Thick and thin sections of the retinal ganglion cell layer, taken consistently from the region adjacent to the optic papilla, were quantitatively and qualitatively analyzed. For light microscopic quantitation, approximately 3000 cells from each group of at least 3 retinas each were counted using a Zeiss Universal Microscope at a magnification of 800 ×. The number of cells in the retinal ganglion layer at 7 and 14 DPA demonstrating dramatic chromatin pattern changes (ie., homogeneous to a more heterogeneous state) and prominent nucleoli were expressed as a per cent of the total number of cells counted. When matched with ultrastructural morphometric analyses, these light microscopic quantitations have been previously shown to be accurate measurements of the cell body response to axotomy25, 26. The method for determination of cells demonstrating dramatic chromatin pattern changes is reported in a previous publication 26. Electron microscopic montages of randomly selected retinal ganglion cell layer areas, consisting of 150-200 cells from each group of least 3 retinas, were prepared for analysis by photographing thin sections supported on one-hole grids covered with 0.75 °/o Parlodian films. For quantitative analysis, tissues were photographed with a Zeiss EM 9S-2 electron microscope at a primary magnification of 1600 x and montages constructed at a final magnification of 4960 x . Electron microscopic montages of neurons in the retinal ganglion cell layers were used to measure nuclear areas (sq./~m), perikaryal cytoplasmic areas (sq.#m), cell/nuclear ratios, mitochondria/sq.#m of perikaryal cytoplasm, Golgi Fields/sq./zm of perikaryal cytoplasm, nucleoli/nucleus and average nucleolar area/cell. Only neurons in the electron microscopic montages demonstrating nucleoli were analyzed according to these criteria. Morphometric analyses were performed on electron microscopic montages at 7 and 14 DPA.

38

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(4:1618) k

(4:19.64) _135~

35

/

:11861

T

~ 25

!

2s~

1 ~

5

5

~

.M

Intact Controls

7 DPA4- 7 DPA+ 7 DPA Anti-NGF Rabbit (3rag/eye) Serum ( :5 mg/eye)

Anti-NOF Rabbit (3rag/eye) Serum (3rag/eye)

Fig. 1. The nuclear responses of retinal ganglion cells with respect to various treatments at 7 days after a~otomy. Solid bars represent values for the per cent of retinal ganglion cells demonstrating prominent nucleoli. Open bars represent per cent retinal ganglion cells demonstrating nuclear chromatin reactivity. Numbers in parentheses indicate total retinas and ganglion cells analyzed. The significance of difference between each group is represented by * P • 0.05 and ** P , 0.01. Vertical lines at each bar represent the S.E.M. Q u a n t i t a t i o n o f the n e u r o n a l reponse to a x o t o m y was accomplished by means o f a L a d d G r a p h i c Digitizer interfaced with a M o n r o e 1830 Calculator. This analytical system allows one to follow specific structures with a cursor with resulting X and Y c o o r d i n a t e s being c o n t i n u o u s l y fed into the p r o g r a m m e d c a l c u l a t o r by the digitizer. T h e high accuracy allows for g o o d resolution o f the structures under investigation, Before processing with the digitizer, structures were first outlined to facilitate quantitation. T h e d a t a were g r o u p e d for statistical analysis a c c o r d i n g to the e x p e r i m e n t a l t r e a t m e n t a n d the significance o f difference was tested with either S t u d e n t ' s t-test or an analysis o f variance. Since no v a r i a t i o n s in the studied p a r a m e t e r s were observed between central a n d p e r i p h e r a l ganglion cells, values were p o o l e d for b o t h p o p u l a tions. F o r all analyses, r o u g h l y equivalent n u m b e r s o f cells were analyzed in each a n i m a l within each g r o u p o f at least three retinas. RESULTS

Light microscopic analysis One 3.0 m g dose o f a n t i - N G F injected into the vitreous c h a m b e r o f the eye at the

39

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6(

50

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~

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.< 40 ta p 0.05) values within 7 DPA and 14 DPA groups. a n d 14 D P A while the nuclear areas r e m a i n unchanged. Therefore, there is a significant decrease in the c y t o p l a s m i c / n u c l e a r ratios at b o t h 7 a n d 14 D P A in response to a n t i - N G F t r e a t m e n t which a p p e a r s m o r e p r o n o u n c e d by 14 D P A . There is a progressive and significant increase in the p e r i k a r y a l areas and c y t o p l a s m i c / n u c l e a r ratios between u n o p e r a t e d , 7 and 14 D P A control values in response to a x o t o m y (Table I). However, a n t i - N G F t r e a t m e n t prevents any significant increase in these values between 7 and 14 D P A . Table I I | d e m o n s t r a t e s that a n t i - N G F significantly reduces the m i t o c h o n d r i a l a n d nucleolar densities as well as the nucleolar areas of cells at 7 a n d 14 D P A . There are no significant changes in Golgi field densities in response to a n t i - N G F treatment. There is a progressive and significant increase in nucleolar densities a n d areas between unoperated, 7 and 14 D P A c o n t r o l values in response to a x o t o m y . However, a n t i - N G F t r e a t m e n t prevents any significant increase in these values between 7 and 14 D P A . DISCUSSION One o f the earliest a n d most p r o n o u n c e d a n t i - N G F effects t h a t can be observed at the light microscopic level is a d r a m a t i c reduction (ie, by 45 ~ ) in the per cent o f cells d e m o n s t r a t i n g p r o m i n e n t nucleoli at 7 D P A . This o b s e r v a t i o n was further substantiated by electron microscopic m o r p h o m e t r i c analysis which d e m o n s t r a t e d a

Fig. 4. Electron micrographs of retinal ganglion cells 14 days after axotomy. A: a typical anti-NG F control cell (x's) demonstrating a greater amount of perikaryal cytoplasm, a more heterogeneous staining nucleus and a much larger nucleolus (Nu) than in B below. B: a typical anti-NGF treated cell demonstrating a reduced amount of perikaryal cytoplasm, less nuclear heterogeneity and smaller nucleolus (Nu) than in A above.

44 TABLE ili

Morphometric analysis of retinal ganglion cell response to axotomy and anti-NGF treatment in the newt ( Notophthalmus viridescens) Treatment

Mitochondria/ sq.l~m

Unoperated controls(3:60)**0.21 7 D P A * * * rabbit serum controls, 3mg/eye(3:56) 0.28 7 DPA + AntiN G F , 3 mg/eye (3:70) 0.21 14 DPA rabbit serum controls, 3 rag/eye(3:64) 0.53 14 DPA -t AntiNGF, 3 mg/eye (3:70) 0.44

Golgi fields/ sq.l~m

Nucleoli/nucleus

Nucleolar area (sq./~m)

± 0.03*

0.008 i 0,003

1.00 ± 0.00

0.24 } 0.02

± 0.02

0.003 :~ 0.001

1.61 -e 0.09

0.72 " 0.05

± 0.02

0.005 _k 0,002

1.37 J:. 0.07

0,57 _[ 0.05

± 0.03

0.010 ± 0,002

1.83 ~ 0.09

1.16 ! 0.09

± 0.04§

0.007 :± 0.002§§

1.47 ± 0.07§

0.59 :~ 0.04§

* Average values ± S.E. ** Indicates numbers of retinas evaluated and total cells analyzed. *** DPA, days postaxotomy. § Indicates that anti-NGF treatment significantly reduces (P 0.001) values within 7 DPA and 14 DPA groups. §§ Indicates that anti-NGF treatment does not significantly reduce (P 0.05) values within the 7 DPA and 14 DPA groups.

significant decrease in nucleolar size (area, by 49 %) and number per cell at both 7 and 14 DPA. It has been previously reported that one of the earliest signs of anti-NGF treatment in the superior cervical ganglia of newborn mice were ultrastructural changes in the nuclear compartment, specifically nucleolar degeneration!< Anti-NGF treatment also significantly reduces the per cent of retinal ganglion cells demonstrating nuclear reactivity in response to axotomy (ie, homogeneous to a more heterogeneous state) at 7 DPA. It is presumed that this anti-NGF mediated, diminished response to axotomy in the nuclear compartment ultimately represents a decrease in the cell's capacity to synthesize RNA. In addition, it is possible that anti-NGF treatment affects the general metabolic level of the retinal ganglion cell since a significant reduction in mitochondrial density is observed at both 7 and 14 DPA. With apparent decreases in the synthetic and metabolic machinery of the anti-NGF treated ganglion cells, it is not surprising to find a significant decrease in the cytoplasmic/nuclear ratio of these cells. A reduction in the size and number of various organelles as well as in synthetic and metabolic rates of mouse superior cervical ganglion cells in response to anti-NGF treatment has also been reported 2,x4,16. Results from the present study suggesting a diminished cell activity in response to axotomy and anti-NGF tend to correlate well with our previous study in which we reported that anti-NGF treatment causes more than a 50~o reduction in the number of regenerating axons per nerve cross-section at 14 DPA r'.

45 There is a dose-response relationship between anti-NGF treatment and retinal ganglion cell response to axotomy. The dose-response study indicates that the minimally effective dose of anti-NGF necessary to elicit a significant reduction in the number of cells demonstrating prominent nucleoli in response to axotomy is 1.5 nag/eye when evaluated at 14 DPA. This was also the anti-NGF concentration determined to be minimally effective with respect to our earlier study concerning newt optic nerve regeneration 12. The dose response curve appears to begin to plateau at 6.0 rag/eye at which point there is a 42 °/o reduction in the number of cells demonstrating prominent nucleoli in response to axotomy. The 6.0 mg dose was given in two equal but separate doses, one at the time of optic nerve transection and one at 7 DPA. Although the 6.0 mg values are lower than those of the 3.0 mg group (40.18 ~- 2.24 vs. 37.57 :+ 1.23 %) the two values are not significantly different as are those of the 1.5 and 3.0 mg groups. This may mean that the critical period for eliciting the most pronounced anti-NGF effects is during the first week after nerve transection. This hypothesis would agree with previous reports that the N G F mediated stimulation of regenerative growth of central noradrenergic neurons in the rat is only apparent if N G F is administered at the time of nerve transection, but not 2 or 4 days after the lesion"3. We have previously reported the stimulatory effect of N G F on newt optic nerve regeneration and retinal ganglion cell response to axotomy ')5,26,~s,29. In addition, we have reported that anti-NGF treatment causes a significant reduction in the number of regenerating axons per optic nerve cross-section 12. Our previous findings added to those of the present study provide strong support for the hypothesis that N G F or an NGF-like molecule is instrumental in the successfully regenerating newt visual system. This hypothesis gathers considerable strength from our anti-NGF work since recent evidence strongly suggests that anti-NGF exerts its primary effect by removal of N G F from the immediate environment in which the two substances are associatedl.% Most recently, it has been reported that N G F and anti-NGF exert strong stimulatory and inhibitory effects, respectively, on forebrain regeneration in another urode[ amphibian, the axolotl 13. The N G F and anti-NGF work in the rat model also adds considerably to the idea of the importance o~" N G F or an NGF-like substance in successful CNS regeneration ~3. ACKNOWLEDGEMENTS This work was supported by a Basil O'Connor Starter Research Grant from the National Foundation-March of Dimes; a grant from the National Society for the Prevention of Blindness made possible through the Adler Foundation and an N I H Grant NS 12070 awarded to Dr. Turner. Dr. Turner is also the recipient of an N I H Research Career Development Award NS 00338.

46 REFERENCES 1 Aguayo, A. J., Martin, J. B. and Bray, G. M., Effects of nerve growth factor antiserum on peripheral unmyelinated nerve fibers, Acta neuropath. (Berl.), 20 (1972) 288-298. 2 Angeletti, P. W., Levi-Montalcinin, R. and Caramia, F., Analysis of the effects of the antiserum to the nerve growth factor in adult mice, Brain Research, 27 (1971) 343-355. 3 Berger, B. D., Wise, C. D. and Stein, L., Nerve growth factor: enhanced recovery of feeding after hypothalamic damage, Science, 180 (1973) 506-508. 4 Bjerre, B., Bjorklund, A. and Edwards, D. C., Axonal regeneration of peripheral adrenergic neurons: Effects of antiserum to nerve growth factor in mouse, Cell Tiss. Res., 148 (1974) 441-476. 5 Bjerre, B., Bjorklund, A. and Stenevi, V., Stimulation of growth of new axon sprouts from lesioned m onoamine neurons in adult rat brain by nerve growth factor, Brain Research, 60 (1973) 161-176. 6 Bjerre, B., Bjorklund, A. and Stenevi, V., Inhibition of regenerative growth of central noradrenergic neurons by intracerebrally administered anti-NGF serum, Brain Research, 74 (1974) 1-18. 7 Bjerre, B. and Rosengren, E., Effect of nerve growth factor and its antiserum of axonal regeneration of short adrenergic neurons in the male mouse, Cell Tiss. Res., 150 (1974) 299-322. 8 Bjorklund, A. and Stenevi, V., Nerve growth factor: stimulation of regenerative growth of central noradrenergic neurons, Science, 175 (1972) 1251-1253. 9 Bray, G. M. Aguayo, A. J. and Martin, J. B., Immunosympathectomy - - Late effects on the composition of rat cervical sympathetic trunks and influence on axonal regeneration, Acta neuropath. (Berl.), 26 (1973) 345-352. 10 Cohen, S., Purification of a nerve growth promoting protein from the mouse salivary gland and its neurocytotoxic antiserum, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 302-311. 11 Ebbott, S. and Hendry, I., Retrograde transport of nerve growth factor in the rat central nervous system, Brain Research, 139 (1978) 160-163. 12 Glaze, K. A. and Turner, J. E., Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): Effect of nerve growth factor antiserum, Exp. Neurol., 58 (1978) 500--510. 13 Hall, K., Adams, P. M., Mellinger, M. W. and Perez-Polo, J. R., Effects of nerve growth factor, antibodies to nerve growth factor and d-amphetamine on forebrain regeneration in the axolotl, Soc. Neurosci. Abstr., 4 (1978) 531. 14 Larrahee, M. G., Early effect of antiserum to the nerve growth factor on retinal ganglion metabolism and synaptic transmission, In E. Zaimis and J. Knight, (Eds.), Nerve Growth Factor and Its Antiserum, Anthlone Press, University of London, 1972, pp. 177-184. 15 Levi-Montalcini, R. and Booker, B., Destruction of the sympathetic ganglia in mammals by an antiserum to nerve-growth protein, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 384-~391. 16 Levi-Montalcini, R., Caramia, F. and Angeletti, P. U., Alterations in the fine structure of nucleoli in sympathetic neurons following NGF-antiserum treatment, Brain Research, 12 (1969) 54-73. 17 Lewis, M. E., Lokshmanan, J., Nagiah, K., MacDonnelly, P. C. and Guroff, G., Nerve growth fa~.tor increases activity of ornithine decarboxylase in rat brain, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1021-1023. 18 McEwen, G. and Grafstein, B., Fast and slow components in axonal transport of protein, J. Cell Biol., 38 (1974) 494-508. 19 Nagaiah, K., Lakshmana, Montgomery, P., Uh, M. W. and Guroff, G., Characteristics of the purified nerve growth factor antibody, J. Neurochem., 31 (1978) 647-655. 20 Scott, D. and Lin, C. N., Factors promoting regeneration of spinal neurons: positive influence ol nerve growth factor, Progr. Brain Res., 13 (1965) 127-150. 21 Smiley, G. R. and Dixon, D., Fine structure of midline epithelium in the developing Falate cf the mouse, Anat. Rec., 161 (1968)293-310. 22 Sperry, R. A., Visuomotor coordination in the newt (Triturus viridescens) after regeneraticn cf the optic nerve, J. comp. Neurol., 79 (1943) 33-55. 23 Stenevi, V., Bjerre, B., Bjorklund, A. and Mobley, W., Effects of localized intracerelzral injection of nerve growth factor on the regenerative growth of lesioned central noradrenergic neurons, Brain Research, 69 (1974) 217-234. 24 Turner, J. E., Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens), J. exp. Zool., 193 (1975) 87-98. 25 Turner, J. E. and Delaney, R. K., Retinal ganglion cell response to axotomy and nerve growth factor in the regenerating visual system of the newt (Notophthalmus viridescens), Brain Research, in press.

47 26 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. 27 Turner, .l.E. and Glaze, K.A., The early stages of Wallaterian degeneration in the severed optic nerve of the newt (Triturus viridescens), Anat. Rec., 187 (1977) 291-310. 28 Turner, J. E. and Glaze, K. A., Regenerative repair in the several optic nerve of the newt (Triturus viridescens) : Effect of nerve growth factor, Exp. Neurol., 57 (1977) 687-697. 29 Turner, J. E. and K. A. Glaze, Glial reaction to nerve growth factor in the regenerating optic nerve of the newt (Triturus viridescens), Exp. NeuroL, 59 (1978) 190-201. 30 Turner, J. E. and Singer, M., An electron microscopic study of the newt (Triturus viridescens) optic nerve, J. comp. NeuroL, 156 (1974) 1-18. 31 Turner, J. E. and Singer, M., The ultrastructure of regeneration in the severed newt optic nerve, J. exp. Zool., 190 (1974) 249-268. 32 Turner, J. E. and Singer, M., The ultrastructure of Wallerian degeneration in the severed optic nerve of the newt (Triturus viridescens), Anat. Rec., 181 (1975) 267 287.