Retinal ganglion cell response to axotomy and nerve growth factor in the regenerating visual system of the newt (Notophthalmus viridescens): An ultrastructural morphometric analysis

Brain Research, 171 (1979) 197-212 © Elsevier/North-Holland Biomedical press

197

R E T I N A L G A N G L I O N CELL RESPONSE TO A X O T O M Y A N D NERVE G R O W T H F A C T O R IN T H E R E G E N E R A T I N G VISUAL SYSTEM OF T H E N E W T (NOTOPHTHALMUS 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 November 23rd, 1978)

SUMMARY

Nerve growth factor (NGF) treatment, given as a single 200 BU intraocular injection at the time of optic nerve transection, was found to significantly accelerate the retinal ganglion cell response to axotomy in the newt (Notophthalmus~iridescens). In the control series the per cent of neurons in the retinal ganglion layer demonstrating nuclear reactivity (i.e. chromatin changes) reaches a peak by 14 days post axotomy (14 DPA), plateaus through 21 DPA and falls thereafter, returning to control levels by 90 DPA. N G F treatment is shown to significantly accelerate the entrance of responding retinal ganglion cells into the reactive nuclear phase between 1 and 7 DPA, and by 7 DPA nuclear reactivity has reached a peak, in contrast to 14 DPA for control values. Consequently, N G F treatment causes retinal ganglion cells to be in the nuclear reactive state a week longer than controls but reactivity diminishes after 21 DPA as in controls. Electron microscopic morphometric analysis further substantiates these observations by demonstrating that N G F treatment can elicit certain cellular organelle changes a week earlier (i.e. at 7 DPA) than they would normally occur (i.e. at 14 DPA) in response to axotomy. In addition to eliciting cellular hypertrophy at 7 DPA, N G F treatment significantly increases Golgi field densities in the neuronal perikaryal cytoplasm as well as a doubling of the number of nucleoli per nucleus and stimulating a significant increase in nucleolar cross-sectional areas. A dose-response relationship exists between the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 DPA and various N G F concentrations which compares favorably with the dose response study involving the number of regenerating axons per nerve cross-section at 14 DPA. Studies to determine if the N G F mediated responses were a specific effect elicited by this protein molecule or whether they are also produced by other peptides

198 which share some properties in common with N G F demonstrate that only N G F is capable of eliciting these responses.

INTRODUCTION There is increasing evidence for the role of nerve growth factor (NGF) or an NGF-like substance in central nervous system (CNS) development and regeneration. Specific binding sites for [12aI]NGF have been shown to be present in the brains of new born and adult rats11,12. In addition, N G F receptor sites have been shown to appear in significant number very early in the development of the chick embryo brain, suggesting that N G F may play a role in the development of the CNS a4. The appearance of early (days 6-8) and late (days 13-18) receptors led Szutowicz et al. a2 to speculate that N G F may serve more than one type of function during the development of the chick brain. In a separate but related study, Merrel et al. 24 have shown that the temporal changes in tectal cell surface specificity, which occur in vivo between days 7-8 in the developing chick brain, can be demonstrated to be dependent on N G F or a structural analog. If N G F or an NGF-like substance does play an important role in the development and maintenance of the CNS, then an endogenous source(s) most likely exists within the CNS. Numerous studies strongly point to glial cells as a source of NGF 4,~°,21,2~,26,29. The potential importance of N G F in regenerative processes in the CNS is suggested by its stimulatory effects on the regeneration of axons in the rat dorsal catecholamine bundle~,7, 8, rabbit vestibular neurons, in vitro, and the dorsal funiculus of young kittens'5, 30. In addition, N G F has been shown to enhance recovery of the feeding response after induced hypothalamic damage in rats 5. Ebbott and Hendry 9 have reported evidence for retrograde transport of N G F in the rat central nervous system. Also, N G F has been shown to increase ornithine decarboxylase in the rat brain 2°. More recently, studies from our laboratory have demonstrated a pronounced effect of N G F and its antiserum on optic nerve regeneration in the newt 18m,42. Most importantly, we have demonstrated that the newt visual system, as a successfully regenerating model, presents an ideal opportunity for studying NGF-mediated regenerative responses in the CNS 35-40. The present paper is an attempt to further characterize our model system and deals with various morphological and ultrastructural aspects of retinal ganglion cell perikaryal response to N G F after axotomy. MATERIALS AND METHODS

77ssue preparation and NGF treatment Adult newts (Notophthalmus viridescens) obtained from Lee's Newt Farm, Oak Ridge, Tennessee, were anesthetized in dilute chloretone solution. Animals in the experimental groups, except those in the dose response studies, received one 2 #1 intraocular injection of 200 BU (i.e. 400 ng) NGF. Animals in the dose response series received one 2 #1 injection of either 10, 20, 200, 400 or 2000 BU of NGF. The control

199 groups received one 2 #1 injection of either the N G F diluent (i.e. phosphate buffer with NaC1), sterile saline, bovine serum albumine (Sigma) cytochrome C (Sigma), lysozyme (Sigma), insulin (Sigma) or epidermal growth factor (Collaborative Research). Injections were made into the vitreous chamber of the left eye according to the method of McEwen and Grafstein z3. The orbital portion of the left optic nerve was severed immediately after injection according to the method of Sperry 32 and Turner and Singer 38. After transection animals were kept in 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 light/dark cycle. Eyes were prepared for electron microscopy at 1, 2, 4, 7, 14, 21, 30, 45 and 90 days post axotomy (DPA). A microperfusion system was devised which allowed small quantities of fixative to be perfused directly into the vitreous chamber adjacent to the retinal ganglion cell layer. The procedure is as follows: (1) approximately 0.3 ml of cold fixative was injected into the orbital space surrounding the eye; (2) a small incision was made through the anteriolateral surface of the eye close to the ora serrata which allowed for efflux of perfused fixative; (3) a 30-gauge needle attached to a 1 ml syringe containing fixative was passed behind the cornea and lens into the vitreous chamber; (4) approximately 0.3 ml of cold fixative was perfused through the eye which was followed by fixation in situ for 15 rain. Corneas and lenses were removed and eyes were fixed over night at 4 °C. Of the various fixatives used, the best was found to be a glutaraldehyde-paraformaldehyde preparation containing acrolein and dimethyl sulfoxide buffered at pH 7.4 with 0.3 M cacodylate buffer 16. The following day eyes were halved through the optic papilla in an anteriorposterior plane, washed in cacodylate buffer, osmicated for 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 (1 ~, pH 7.4) for examination and photography with the light microscope. Thin sections were stained with uranyl acetate and lead citrate 31. Nerve growth factor was obtained from the Wellcome Research Labolatories 7S N G F ) and from Collaborative Research (2.5S NGF). With the exception of the dose-response comparison experiment (7S vs 2.5S NGF), all results from this study concerning N G F treatment involve the use of the 7S form of the molecule. The biological activity of N G F was retested in our laboratory according to the method of Levi-Montalcini iv.

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 1000-2000 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 0-90 DPA demonstrating dramatic chromatin pattern changes were expressed as a per cent of the total number of cells counted. The determination of cells demonstrating dramatic chromatin pattern changes has been reported in a previous publication 40.

200 Electron microscopic montages of randomly selected retinal ganglion cell layer areas, consisting of 150-200 cells from each group of at least 3 retinas, were prepared for analysis by photographing thin sections supported on one-hole grids covered by 0.75 ~ 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 . In addition, light microscopic montages of randomly selected areas of the central retina consisting of from 900-1200 cells were prepared for quantitation at a magnification of 1120 ×. The light microscopic montages were utilized to measure nuclear areas of all cells within the selected portion of the retinal ganglion cell layer. This information was used to construct histograms of nuclear distributions according to their size (cross-sectional areas). Electron microscopic montages of neurons in the retinal ganglion cell layer were used to measure nuclear areas (sq./~m), perikaryal cytoplasmic areas (sq. #m), cell/nuclear ratios, mitochondrial/sq. #m of perikaryal cytoplasm, Golgi fields/sq. #m of perikaryal cytoplasm, nucleoli/nucleus and average nucleolar area/cell. All neurons in the electron microscopic montages demonstrating nucleoli were analyzed according to these criteria. Morphometric analyses were performed on light and electron microscopic montages at 0 (intact controls), 7 and 14 DPA. Quantitation of the neuronal response to axotomy was accomplished by means of a Ladd Graphic Digitizer interfaced with a Monroe 1830 calculator. This analytical system allows one to follow specific structures with a cursor with resulting x-y coordinates being continuously fed into the programmed calculator by the digitizer. The high accuracy allows for good resolution of the structures under investigation. Before processing with the digitizer, structures were first outlined to facilitate quantitation. The data were grouped for statistical analysis according to the experimental treatment and the significance of difference was tested by either the Student's t-test or an analysis of variance. Since no variations in the studied parameters were observed between central and peripheral ganglion cells, values were pooled for both populations. For all analyses, roughly equivalent numbers of cells were analyzed in each animal within each group of at least 3 retinas. RESULTS

Light microscopic analysis In a previous study 40 we reported that retinal ganglion cell nuclei undergo dramatic changes in chromatin patterns in response to axotomy and are easily observed at the light microscopic level. Fig. 1 contrasts the retinal ganglion cell nuclear response to axotomy at 7 DPA with or without N G F treatment. These light micrographs demonstrate that there are many more neurons with 'reactive nuclei' demonstrating chromatin changes (i.e. heterochromatic to more euchromatic state) in the 7 DPA + N G F group than in the 7 DPA series without N G F treatment. Also, cells in the 7 DPA + N G F series appear to have larger and often multiple nucleoli when compared with the 7 DPA control group. The number or per cent of cells demonstrating nuclear chromatin changes in response to axotomy can be plotted temporally as

VC

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Fig. 1. Light micrographs of retinal ganglion cell layer 7 days after axotomy showing contrasts in the retinal ganglion cell nuclear response to axotomy with or without N G F treatment. VC, vitreous chamber; IPL, inner plexiform layer. A: control retinal ganglion cell layer (RGL) showing only a few cells with reactive nuclei (R) and the majority showing no signs of reactivity (NR). Also, note that nucleoli (N) within these cells are not very prominent. B : NGF-treated retinal ganglion layer (RGL) showing a majority of cells with reactive nuclei (R). Note the large, prominent nucleoli (N) in some of the cells, x 400.

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Fig. 2. A graph demonstrating the temporal sequence of events relating to the per cent of cells within the retinal ganglion layer demonstrating nuclear chromatin changes (i.e. heteroehromati¢ to euchromati¢ state) in response to axotomy and NGF treatment. Vertical lines at each point represent the S.E.M. Values in parentheses indicate number of retinas evaluated and total cells analyzed respectively.

indicated in Fig. 2 and in a previous study 40. In the control series the per cent of neurons in the retinal ganglion cell layer demonstrating nuclear reactivity (i.e. chromatin changes) reaches a peak by 14 DPA, plateaus through 21 D P A and falls thereafter returning to control levels by 90 DPA. N G F treatment is shown to significantly accelerate the entrance of responding retinal ganglion cells into the reactive nuclear phase between 1 and 7 DPA, and by 7 D P A nuclear reactivity has reached a peak, in contrast to 14 D P A for control values. Consequently, N G F treatment causes retinal ganglion cells to remain in the nuclear reactive state a week longer than controls but reactivity diminishes after 21 DPA like control values. As reported previously 4°, approximately 50 % of the neurons within the retinal ganglion cell layer do not demonstrate nuclear or cytoplasmic responses to axotomy and are hence referred to as

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nonreactive neurons. In addition, these nonreactive neurons do not degenerate in response to axotomy and are most likely ectopic amocrine cells 40. Observations at the light microscopic level also indicate significant changes in nuclear cross-sectional area distributions of reactive and nonreactive ganglion cell layer neurons with respect to N G F treatment (Fig. 3). N G F treatment causes a significant shift to an increase in the number and size of nuclei entering the reactive phase at 7 DPA (Fig. 3C) when compared to controls (Fig. 3B) such that the distributions are identical to the 14 DPA group (cf. Fig. 3C and D). N o evidence of cell division or cell death was observed.

Electron microscopic analysis The electron micrographs depicted in Fig. 4 and 5 further substantiate the light microscopic observations reported from Fig. 1. The retinal ganglion cells receiving N G F treatment appear to have larger, more euchromatic nuclei with larger and often multiple nucleoli. In addition, the amount of perikaryal cytoplasm appears to be greater and the organelles more numerous in the 7 DPA + NGF-treated ganglion cells. Electron microscopic morphometric analysis indicates that the qualitative

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Fig. 4. Electron micrographs contrasting retinal ganglion cells 7 days after axotomy and N G F treatment. A: a portion of the control retinal ganglion cell layer showing cells whose nuclei (N) are predominately heterochromatic with most demonstrating only one nucleolus (Nu) per cell. Also, note the sparse rim of perikaryal cytoplasm (Xs). B: a portion of the NGF-treated retinal ganglion cell layer showing an increased number of cells with reactive, more euchromatic nuclei (N), multiple nucleoli (Nu) and an expanded perikaryal cytoplasm (Xs). Bar equals 1/~m.

205

Fig. 5. Higher magnification of a portion of expanded perikaryal cytoplasm of NGF-treated retinal ganglion cell 7 days after axotomy. Note predominant Golgi fields (G), rosettes of ribosomes (R), microtubules (MT) and mitochondria (M). Bar equals 0.5/zm.

206 o b s e r v a t i o n s r e p o r t e d f r o m b o t h light a n d e l e c t r o n m i c r o g r a p h s a r e c o r r e c t ( T a b l e s I a n d II). T a b l e I s h o w s t h a t at 7 D P A , N G F t r e a t m e n t significantly increases n e u r o n a l p e r i k a r y a l a n d n u c l e a r a r e a s in c o n t r a s t to 7 D P A

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TABLE I Morphometric analysis o f retinal ganglion cell response to axotomy and N G F treatment in the newt ( Triturus viridescens)

DPA = days post axotomy. Data represent average value 4- S.E.M. Figures in parentheses indicate number of retinas evaluated and total cells analyzed. Only cells from randomly selected sections with a nucleolus were analyzed. Treatment

Perikaryal area (sq. /~m)

7 DPA (3, 74) 137.09 ± 4.14 7 DPA ÷ N G F (3, 57) 181.64 i 6.10 14 DPA (3, 52) 186.94 4- 7.39*

Nuclear area (sq. tzm)

Perikaryal/nuclear ratio

112.29 4- 3.25 141.82 4- 4.35 142.59 i 4.27*

1.23 4- 0.02 1.30 4- 0.03 1.30 -4- 0.02*

* 14 DPA values significantly different from 7 DPA results (P < 0.01) but not significantly different from the 7 DPA 4- N G F series (P > 0.05).

TABLE II Morphometric analysis o f retinal ganglion cell response to axotomy and N G F treatment in the newt (Triturus viridescens)

DPA = days post axotomy. Data represent average value 4- S.E.M. Figures in parentheses indicate number of retinas evaluated and total cells analyzed. Only cells from randomly selected sections with a nucleolus were analyzed. Treatment

7 DPA (3, 74) 7 DPA + N G F (3, 57) 14 DPA (3, 52)

Mitochondria/ sq. /~m

Golgi fields/ sq. Izm

Nueleoli/nueleus

Nucleolar area (sq. I~m)

0.30 ± 0.03

0.004 ± 0.001

1.11 ± 0.04

0.42 ± 0.04

0.34 ± 0.03

0.019 ± 0.004

1.96 4- 0.11

0.58 4- 0.06*

0.34 -4- 0.03**

0.021 ± 0.004

2.02 4- 0.09***

0.47 4- 0.03§

* Significantly different from 7 DPA value (P < 0.01) but not significantly different from 14 DPA value (P > 0.05). ** No significant difference among values (P > 0.05). *** Significantly different from 7 DPA value (P < 0.01) but not significantly different from 7 DPA + N G F value (P > 0.05). § Not significantly different from 7 DPA or 7 DPA + N G F values (P > 0.05).

207 controls, N G F t r e a t m e n t is also shown t o : (1) significantly increase G o l g i field densities in the n e u r o n a l p e r i k a r y a l c y t o p l a s m ; (2) d o u b l e the n u m b e r o f nucleoli p e r nucleus; a n d (3) stimulate a significant increase in n u c l e o l a r cross-sectional areas (Table II). H o w e v e r , m i t o c h o n d r i a l densities are n o t c h a n g e d by N G F treatment. A s shown in Tables I a n d II, the 7 D P A ÷ N G F g r o u p values are n o t significantly different f r o m 14 D P A results except t h a t N G F a p p e a r s to cause a significant increase in the n u c l e a r cross-sectional area. Consequently, results f r o m Tables I a n d II indicate t h a t N G F t r e a t m e n t (i.e. a single 200 B U i n t r a o c u l a r injection given at the time o f A. 7 DAYS AFTER AXOTOMY

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Fig. 6. Dose-response curves comparing the retinal ganglion cell perikaryal (A) and axonal regeneration (B) responses to various N G F concentrations. Note that 200 BU is equivalent to approximately 400 ng of NGF. Vertical lines at each point represent S.E.M. In A, values in parentheses represent number of retinas evaluated and total cells analyzed. In B, values in parentheses indicate numbers of optic nerve cross-sections analyzed. The C, values on the abscissas represent control values (i.e. nerve transection without N G F treatment).

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Fig. 7. A dose-response study demonstrating that very similar curves are elicited by either 7 S or 2.5 S NGF concentrations with respect to the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 days after axotomy. Also, note that the per cent of prominent nucleoli appearing in the 2.5 S-treated retinal ganglion cells shows a dose-response relationship at 7 days after axotomy.Vertical lines at each point represent the S.E.M. P values indicate significant difference from control (C) means. Mean values represent an analysis of at least 3 retinas and 1000-2000cells/group were analyzed.

optic nerve transection) can elicite certain cellular organelle changes a week earlier than they would normally occur.

Dose response study Results from an earlier study 41 indicate a dose response relationship between N G F amounts ranging from 2-2000 BU (i.e. N G F given as a single intraocular injection at the time of nerve transection) and the number of regenerating axons per nerve cross-section at 14 D P A (Fig. 6B). The half maximal response for that study was reported to be elicited by an N G F amount of 10 BU and values plateaued between 200-2000 BU. Under conditions identical to those in Fig. 6B, results from the present study indicate that a very similar dose response relationship exists between various N G F amounts and the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 D P A (Fig. 6A). As in the previous study the half maximal response was elicited by an N G F amount of 10 BU and values also plateaued between 200-2000 BU (c.f. Fig. 6A and B). Fig. 7 shows that very similar dose-response relationships exist between various 7S and 2.5S N G F concentrations and the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 DPA. The maximum response for both forms of N G F is elicited by approximately 400 ng/eye; however, the 7S growth factor is capable of generating this response at concentrations as low as 20 lag/eye (Fig. 7).

209 TABLE III Per cent retinal ganglion cells demonstrating nuclear reactivity at 7 days post axotomy in response to various treatments

Treatment involved one intraocular injection given at time of axotomy. Parentheses under Treatment give weight and volume of substance injected. Data represent average value 4- S.E.M. Parentheses in data column indicate number of retinas evaluated and total cells analyzed. Treatment

% Cells demonstrating nuclear reactivity

200 B.U. NGF (0.4/~g/2/~1) Saline controls (2 #I) BSA (0.4/~g/2/zl) Cytochrome C (0.4/~g/2 bd) Lysozyme (0.4/zg/2 #1) Insulin (0.4 ~tg/2/~1) EGF (0.4 t~g/2 ~tl)

50.01 ± 1.50" (6,2895) 24.88 ± 3.67 (5,2842) 29.49 4- 2.06j (3,970) 27.85 ± 1.22 (5,2026) 25.37 ~ 0.90 (4,1332) 31.90 ± 2.41 (6,2008) 27.87 ± 2.81 4,2445)

* Only value significantly different from controls (P < 0.01).

Specificity o f N G F response

Studies to determine if the N G F mediated responses were a specific effect elicited by this protein molecule or whether they are also produced by other peptides which share some properties in common with N G F were undertaken and results are indicated in Table III. The peptides assayed share some properties in common with N G F , such as the isoelectric point which is respectively 9.8-10.1 and 11.0-11.2 for cytochrome C and lysozyme22, or are endowed with similar biological activity (EGF and insulin) 1 and antigenic properties (EGF and BSA) 1. The results from Table III indicate that of these protein molecules only N G F is capable of eliciting a significant elevation in the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 DPA. Both the 7S and 2.5S forms of N G F were found to be effective in this system (Fig. 7). DISCUSSION Light and electron microscopic analyses indicate that N G F treatment, given as a single 200 BU intraocular injection at the time of optic nerve transection, significantly accelerates the retinal ganglion cell response to axotomy. More specifically, our observations demonstrate that N G F treatment can elicite a number of retinal ganglion cell organelle changes a week earlier (i.e. at 7 DPA) than would normally occur (i.e. at 14 DPA). The earliest changes that could be detected as a result of N G F treatment

210 involve nuclear chromatin and nucleolar alterations. An acceleration of the per cent of cells entering the nuclear reactive phase in response to axotomy, which involves a dramatic change from a predominately heterochromatic to euchromatic state and an early increase in the size and number of nucleoli, was observed as a result of N G F treatment. It is possible that these observations reflect the ability of exogenous N G F to stimulate a D N A dependent R N A synthesis in the regenerating retinal ganglion cell. N G F treatment given to newborn and adult mice has been shown to also increase the size of nucleoli in the intact sympathetic nervous system 3. In turn, one of the earliest cytotoxic effects of the N G F antiserum is in the nucleus 27, particularly in the nucleolus 2,19 of sympathetic neurons. Furthermore, the stimulatory effect of N G F on sympathetic neurons has been shown to be blocked by actinomycin D treatment 17 and N G F antiserum administration causes a dramatic reduction in R N A synthesis in these neurons 19. In addition to chromatin and nucleolar changes, we also reported cellular and nuclear hypertrophy as well as dramatic increases in Golgi field densities and in unbound ribosomes in response to N G F treatment. Cellular hypertrophy, nuclear enlargement3,14,18,~°8 and increases in unbound ribosomes 3,1s,2s have also been reported to be elicited by N G F treatment in the intact sensory and sympathetic neurons. No increases in mitochondria have been reported 3,zs. However, both an increase in size3,18, 2s and number 18 of the Golgi apparatus has been observed in the intact peripheral nervous system in response to N G F treatment. In addition, we found no evidence of increases in the neurofibrillar-neurotubular material of retinal ganglion cells as has been reported for NGF-treated peripheral neurons3,18; however, Schwab and Thoenen 2s also do not report increases in these organelles of superior cervical ganglion neurons at lower N G F concentrations. Most significantly, these N G F mediated responses in regenerating retinal ganglion cells have been shown in the present study to be a specific effect produced by either the 7S or 2.5S form of the molecule and not by those substances which share some properties in c o m m o n with N G F . Both the 7S and 2.5S forms of N G F elicit very similar dose-response curves with a maximum response at approximately 400 ng/eye; however, the 7S growth factor is capable of generating this response at concentrations as low as 20 rig/eye. A dose response relationship was shown to exist between the per cent of retinal ganglion cells demonstrating nuclear reactivity at 7 D P A and various N G F concentrations. This compares favorably with the dose response study involving the number of regenerating axons per nerve cross-section at 14 D P A 41. In both studies the halfmaximal response was elicited at 10 BU and the maximal response was obtained at 200 BU N G F . At this maximal concentration twice the number of retinal ganglion cells were in the nuclear reactive phase by 7 D P A than controls, and at this same concentration one week later (i.e. 14 DPA), twice the number of regenerating axons per nerve cross-section were observed 41. It is very likely that these two relationships are closely related. More specifically, the single intraocular injection of N G F given at the time of nerve transection, causes the normal retinal ganglion cell perikaryal response to axotomy (i.e. preparation tor regeneration of an axon) to be accelerated by a week, which may in turn result in early and/or more numerous fiber outgrowth that is eventually reflected by the marked increase in regenerating fibers a week later.

211 ACKNOWLEDGEMENTS This work was supported by a Basil O ' C o n n o r Starter Research G r a n t f r o m the N a t i o n a l F o u n d a t i o n M a r c h of Dimes; a grant from the N a t i o n a l Society for the P r e v e n t i o n of Blindness made possible t h r o u g h the Adler F o u n d a t i o n a n d a n N I H G r a n t NS 12070 awarded to J. E. T. ; J. E. T. is also the recipient of a n N I H Research Career D e v e l o p m e n t A w a r d NS 00338. W e w o u l d like to gratefully acknowledge the expert technical assistance of Ms. R o d y Spivey.

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