Tissue sections from the mature rat brain and spinal cord as substrates for neurite outgrowth in vitro: Extensive growth on gray matter but little growth on white matter

EXPERIMENTALNEUROLOGY

104,39-54(1989)

Tissue Sections from the Mature Rat Brain and Spinal Cord as Substrates for Neurite Outgrowth in Vitro: Extensive Growth on Gray Matter but Little Growth on White Matter KEITH A. CRUTCHER Department

of Neurosurgery,

University

The failure of axons to regenerate within the brain and spinal cord of mature mammals has been attributed to the absence of growth-promoting substances, especially extracellular matrix components, or to the presence of growth-inhibiting substances, particularly components associated with CNS myelin. The ability of mature mammalian CNS tissue to support neurite regeneration was tested by growing explants of embryonic chick lumbar sympathetic ganglia on fresh frozen sections of the mature rat brain and spinal cord. The extent of neurite outgrowth was quantified using morphometric analysis for explants grown on sections that included most of the major anatomical divisions of the CNS. Extensive, but variable, regeneration was present on gray matter regions, whereas major white matter tracts showed poor support, if any, for neurite growth. The results are consistent with the presence of growth-inhibiting factors associated with CNS white matter but also indicate that most gray matter regions of the mature mammalian brain and spinal cord will support axonal regeneration in tissue culture in spite of the absence of known extracellular matrix components. 0 1989 Academic Press, Inc.

of Cincinnati,

Cincinnati,

Ohio 45267-0515

sis is supported by the presence of neurite-promoting extracellular matrix components, e.g., laminin, fibronectin, and heparan sulfate proteoglycan, in tissues that support axonal growth and their absence in the mature CNS (10,16,17,20,50,57,60,85). The second hypothesis is supported by the growth-inhibiting influence of oligodendrocytes and CNS myelin components on neurite growth from embryonic sympathetic and sensory neurons in tissue culture (l&19,86,87). Several investigators have reported results of experiments in which neurons were grown on CNS tissue sections (17, 29, 83) and in two of these studies neuronal explants were reported to exhibit little neurite growth on tissue sections from the mature rat CNS (17, 83). However, the tissues used for these studies were the optic nerve and spinal cord, both highly myelinated. In light of the growth-inhibiting nature of CNS myelin, the present study was undertaken to determine the in vitro neurite growth-promoting potential of tissue sections taken from areas of the mature CNS that contain gray matter as well as myelinated white matter tracts. The results clearly show that gray matter regions of the mature rat CNS support extensive neurite regeneration, whereas the major white matter tracts generally do not support neurite growth.

INTRODUCTION MATERIALS

Axonal regeneration is rare in the mature brain and spinal cord of mammals yet occurs readily within the mammalian peripheral nervous system as well as within the CNS of lower vertebrates (21, 22, 27,46, 54,66, 79, 104). The lack of axonal growth in the CNS does not reflect a limited growth potential of intrinsic neurons since peripheral nerve grafts permit extensive regeneration when inserted into the brain or spinal cord (1,3, 7, 21,22,34,80,81,92, 94,95, 100). The responsibility for abortive regeneration must therefore lie with the CNS environment. Two hypothetical mechanisms could underly this lack of environmental support. One is the absence of growth-promoting factors and the other is the presence of growth-inhibiting factors. The first hypothe-

AND

METHODS

TissuePreparation Brains and spinal cords were rapidly removed from mature (3-5 month old) female Sprague-Dawley rats following decapitation and immediately frozen at -20°C. Fresh frozen cryostat sections (10 pm in thickness) were cut on the horizontal or coronal plane and thaw-mounted (three to five per dish) onto the bottom of 35-mm tissue culture dishes (Falcon). The dishes were kept at -20°C for a period ranging from a minimum of 1 h to several weeks. Sections left for less than 1 h tended to float off the plastic following the addition of medium. Embryonic Day 9 chick embryos (White Leghorn or Rhode Island Reds obtained from the Mt. Healthy

39 All

0014-4&36/89 $3.00 Copyright 0 1989 by Academic Press, Inc. rights of reproduction in any form reserved.

40

KEITH

A. CRUTCHER

Hatchery, Cincinnati, OH) were dissected in Ham’s F12 medium to obtain lumbar sympathetic chain ganglia. The ganglia were further dissected into small pieces (approx 0.1 mm2 in area) that were added to the dishes with enough medium to just cover the tissue sections (approx 200 ~1). The pool of medium over the tissue sections restricted the distribution of the explants to encourage attachment to the tissue. One hour later, 1.5 ml of Ham’s F12 medium (supplemented with antibiotics), with or without 5% horse serum (HS) or nerve growth factor (NGF; present at a concentration of 60 or 240 ngfml), was gently added to the dish in an attempt to prevent displacement of the explants from the tissue. Cultures were incubated in a humidified environment (5% CO& at 37°C for l-6 days without additional changes of medium. Tissue Staining The explants, neurites, and tissue sections were visualized by staining the entire culture with methylene blue, glyoxylic acid histofluorescence, or a modification of the Sevier-Munger silver stain (88). Methylene blue (.25% in saline) was added directly to the medium and the cultures were then incubated for 10 min at 37°C. The medium was then aspirated and replaced with PBS. The vitally stained cultures were then viewed and photographed. The cultures were subsequently air-dried and coverslipped with mineral oil. The technique of De la Torre (35) was used to visualize noradrenergic fibers. The medium was aspirated and l-2 ml of sucrose-phosphate-glyoxylic acid (SPG) solution was added to the cultures for 5-10 s. The SPG solution was decanted and the dishes were immediately dried under a cold air stream. Hot mineral oil was added to the dishes that were then placed in a hot oven (95°C) for 2.5 min. The dishes were removed and coverslipped for viewing with a fluorescence microscope. A modification of the SevierMunger silver stain (88) was used for the majority of the cultures. The medium was aspirated and the tissue was rinsed with saline twice followed by fixation with 2% glutaraldehyde (in phosphate-buffered saline) for lo-15 min. The fixative was removed and followed by three rinses with 70% ethanol. A 20% aqueous silver nitrate solution was added and the dishes were incubated at 60°C for 15 min. The dishes were then rinsed twice with water followed by an ammoniacal silver solution (10% silver nitrate, 2.5% ammonium hyroxide, and 0.6% NAzCO,) with 10 drops of 2% formalin for 15 min. The final silver solution was washed off with two rinses of water and the reaction was stopped with 5% sodium thiosulfate. The dishes were washed twice with PBS and viewed immediately or air-dried and coverslipped with mineral oil for later viewing.

Morphometric

Analysis

Measurements of the explants were made with a HiPad digitizing tablet and BioQuant (R&M Biometrics) morphometric software running on an Apple IIe microcomputer. The explants and neurite perimeters were traced on paper through the use of a drawing tube attached to an Olympus BH-2 microscope. The tracings were then measured with the HiPad digitizing tablet and the resulting areas were calculated by the BioQuant program. In order to provide some consistent measure of the degree of neurite outgrowth from each explant, the perimeter of the neuritic halo was arbitrarily defined as the point at which the majority of silver-stained neurites appeared to end. In some cases individual neurites extended beyond this arbitrary limit but the majority of neurites fell within the defined perimeter. Variability in the degree of staining of the underlying tissue section occasionally prevented the accurate identification of the neuritic perimeter (Figs. 2A and 2B shows an example). In these cases the radial extent of neurites that could be discerned was traced. Such measurements underestimate the neurite area. The measurements were made without knowledge of the culture conditions so that systematic bias was prevented in determining the amount of neurite outgrowth from different explants. Two measurements were obtained for each explant, one for the explant proper and one for the total area enclosing the explant and the neurites. The area occupied by the neurites, here referred to as the neuritic halo, was calculated by subtracting the explant area from the total area. The ratio of the area occupied by neurites (halo) to the area of the explant was also calculated in order to control for variability due to the size of the initial explant. Statistical comparisons between two groups of measurements were made using an unpaired t test with significance accepted at P < 0.05. Microscopy Living cultures were viewed with a Leitz Diavert inverted microscope equipped with differential interference contrast (DIC) optics. Stained cultures were viewed either with an Olympus BH-2 fluorescence microscope or a Nikon Microphot FX microscope equipped with epifluorescence. Tracing of the explants was done with the Olympus microscope with an attached drawing tube and all photographs were taken with the Nikon microscope. RESULTS Growth and Attachment of Explants without Tissue Sections In order to have some basis for comparison with explants grown on tissue sections, explants were grown on

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41

GROWTH ON CNS TISSUE SECTIONS TABLE

1

Explants Grown on PORN with NGF Culture

n

Explant area

3-day

21 17

0.048 0.061

I-day P value

+ 0.006 f 0.009

0.2212

Total area 0.412 k 0.05 0.640 + 0.091 0.0273*

Halo area 0.364 +- 0.045

Halo/explant ratio

0.579 zk0.085

8.025 f 0.603 9.958 + 1.201

0.0245*

0.1367

Note.Valuesareaverages f SEMs.Areasaremm*. * P < 0.05.

plain or polyornithine (PORN)-coated dishes in the presence of NGF, HS, or both. Explants did not attach to uncoated tissue culture dishes but usually attached in the presence of PORN. Explant attachment and neurite outgrowth on PORN-coated dishes was observed on the first 2 days in culture when viewed with DIC optics but the explants did not usually remain attached during the staining procedure so that subsequent quantification was not possible. Explants did not remain attached to PORN without added NGF or HS. The most neurite outgrowth was obtained in the presence of NGF without added HS. The presence of HS alone resulted in very little neurite outgrowth and, when present with NGF, appeared to increase the fasciculation of neurites so that the resulting halo was less dense in appearance than that obtained with NGF alone. Table 1 illustrates the amount of growth observed after 3 or 4 days in culture in the presence of NGF without added HS. Although the average explant size was greater on the fourth day, the difference was not statistically significant. There was a significant increase in total area and halo area between the third and fourth days but the corresponding difference in ratios was not statistically significant. The correlation coefficient between explant size and total area was 0.82 for 3-day cultures and 0.71 for 4-day cultures. Explant Growth on Tissue Sections Although it was possible to visualize the position of explants on the tissue using DIC optics, neurites growing on the tissue could not be discerned without staining. Many of the initial observations were made by staining the explants with either methylene blue (a vital stain) or glyoxylic acid (to visualize catecholamine histofluorescence). Figure 1 shows examples of explants visualized with methylene blue (Fig. lA, 1C and 1D) or glyoxylic acid (Fig. 1B). Although methylene blue provided the advantage of being able to visualize the neurites while they were still alive, the stain did not persist and did not provide a permanent record for subsequent quantification. The major disadvantages of the glyoxylic acid stain were that many of the neurites did not fluoresce under these

conditions and that the underlying tissue could not be readily visualized. The use of the silver stain, however, was found to provide good visualization of the explant, neurites, and underlying tissue and, therefore, was the best material for quantification and for relating the neurite growth to the cytoarchitectural features of the tissue section. In order to observe attachment and growth on tissue sections without support from the underlying plastic, explants were grown on tissue sections placed on untreated culture dishes. Under these conditions, explants were never found attached to the tissue culture plastic except when sections from the spinal cord were used (see below). Occasionally, explants would attach to the edge of brain sections and extend neurites on the tissue but not on the adjacent plastic (Figs. 1A and lB), indicating that the brain tissue did not permit explant attachment to untreated plastic. Since some attachment to the plastic was observed in the presence of spinal cord tissue, however, the morphometric results from the spinal cord cultures were not included in the quantitative analysis. Explants grown on tissue sections remained attached throughout the staining procedure by the second day and usually exhibited neurite outgrowth by this time in the presence of NGF, HS, or both. Explants also attached to tissue in the absence of NGF or HS and showed some neurite outgrowth although usually much less than that observed with NGF or HS supplement. In some cases a halo of nonneuronal cells was visible in the immediate region of the explant, particularly in methylene bluestained cultures (Fig. 1C). These cells did not appear to extend beyond the inner region of the neuritic halo and could not, therefore, account for the neurite outgrowth. Figure 2 shows examples of explants growing on the amygdala under various culture conditions. The first explant (Fig. 2A) was grown in the presence of HS and NGF. Due to high background staining of the tissue, the extent of neurite growth is hard to visualize. Figure 2B shows the same explant photographed with incidental light so that the extent of neurite outgrowth can be discerned. The second explant (Fig. 2C) was grown in the presence of NGF without HS and exhibits a dense neuritic halo. The third

42

KEITH

A. CRUTCHER

FIG. 1. Sympathetic explants on sections of the rat brain grown in the presence of NGF and horse serum. (A) Two explants stained with methylene blue extending neurites in the region of the hypothalamus and along the edge of the section (arrowhead) but not onto the adjacent plastic. (B) Explant stained for norepinephrine histofluorescence with the glyoxylic acid technique exhibiting extensive radial neurite outgrowth on the hypothalamus but not onto the adjacent plastic. (C) Methylene blue-stained explant on the hippocampal formation with extensive radial neuritic outgrowth. The explant is surrounded by a zone of presumed fibroblasts which does not extend as far as the neuritic halo. (D) Methylene blue-stained explant in the region of the stria medullaris (SM), third ventricle (III), and fimbria (F). Neurites have extended on the hippocampal gray matter but avoided the white matter of the stria medullaris and fimbria. Scale bars = 0.1 mm.

e+plant was grown without NGF or HS and exhibits sparser outgrowth than the other two explants. The amyg&la was one of the few areas where significant outgrowth was observed in the absenceof NGF or HS. Table 2 showsthe change in explant size, total area, halo area, and halo/explant ratio for explants grown on tissue sections for 2-4 days in the presence of NGF and HS. The most striking change over time was in the size of the halo area. This increase resulted in a greater halo/explant ratio but the difference did not reach statistical significance, possibly because no distinction was made in the analysis

between explants on different brain regions. Table 3 shows morphometric data for explants grown on brain sections in the presence of NGF without added HS. In this casethe increase in explant size was not accompanied by a corresponding increase in the halo area so that a significant decrease occurred in the halo/explant ratio. Comparison between Explant Growth on PORN and Brain Sections Table 4 shows the results of a comparison between explants grown on brain sections, regardless of location,

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CNS

TISSUE

SECTIONS

43

FIG. 2. Explants on the amygdala under various culture conditions after 3 days. (A) Explant (grown with horse serum) adjacent to the ventral surface of the amygdala with radial neurite extension that is only visible in the immediate region of the explant due to the darkly stained tissue. (B) Same explant as in A illuminated with incidental light to show the extent of neurite outgrowth. Neurites are present as far away as the arrow and the hippocampal formation (HF). The arrowhead indicates a piece of dust on the section. (C) Explant on the amygdala grown in the presence of NGF exhibiting a dense halo of neurites. (D) Explant within the amygdala grown in F12 medium alone. The radial outgrowth is not as extensive as that seen with NGF or HS but the inhibitory effect of the white matter, in this case the optic tract (OT), is still present. Scale bars = 0.1 mm.

and explants grown on PORN in the presence of NGF without HS. Average explant size was significantly greater on tissue on both days but the total area and halo area were only significantly greater on tissue on the third day. The continued increase in explant size on the fourth day without a corresponding increase in halo area resulted in a significantly smaller halo/explant ratio for tissue explants as compared to PORN explants on the fourth day. Analysis of the combined 3- and 4&y results showed that the difference between explant size and halo/explant ratio was still significant. From these data it appears that the average neurite growth of explants on brain sections is at least as good as that occurring on PORN. Regional Growth on CNS TissueSections Since explants rarely attached to white matter regions, no direct comparison can be made between gray

and white matter explants. However, explants often attached to gray matter areas adjacent to white matter areas. In such cases the extent of neurite outgrowth on gray matter was much greater than that on white matter. In fact, the major white matter tracts (corpus callosum, internal capsule, cerebral peduncle, fimbria, stria medullaris, etc.) were generally nonpermissive substrates for axonal growth (Fig. 3). In some caseslimited growth was observed on white matter tracts (Fig. 3C) but in many instances it was clear that such growth was actually occurring along vascular profiles or glial septae within the white matter tract (Fig. 3D). In contrast to the limited growth on white matter, extensive growth occurred on most gray matter regions (Fig. 4). In addition, neurites grew well on pial and vascular surfaces (Figs. 3B-3D, and 4F). Two technical lim-

44

KEITH

A. CRUTCHER TABLE

Explants

Grown

on Brain

2

Sections

with NGF and HS

Day

n

Explant area

Total area

Halo area

2 3 4

17 23 20

0.06 + 0.011 0.067 + 0.005 0.07 f 0.007

0.507 + 0.102 0.789 rt 0.071 0.987 + 0.049

0.447 -t 0.094 0.722 zk 0.068 0.917 f 0.046

11.078 +- 2.663 11.454 f 1.227 15.496 + 1.68

0.5286 0.1306 0.7381

0.0247* 0.0001* 0.0306*

0.0195* 0.0001* 0.0254:

0.8897 0.0012+ 0.0548

P value 2vs3 2vs4 3vs4

Halo/explant

ratio

Note. Values are averages f SEMs. Areas are mm’. * P < 0.05.

itations prevented accurate comparisons between some brain regions: (i) the size of the explants limited the resolution of the assay in detecting regional differences in neurite growth, and (ii) the size of the brain region was directly correlated with the number of explants that landed on it so that the sample size was greater for larger brain regions. However, some statistical comparisons were still possible (see below). For other areas the description of regional outgrowth is necessarily qualitative and further analysis must await additional studies. For the present study the general findings for the major divisions of the CNS will be described according to region with emphasis on the extent to which differences were observed between gray and white matter areas.

campal fields, i.e., subiculum, CA1, CA,, and the dentate gyrus. No obvious regional variations in the pattern of outgrowth were observed although neurite growth out of the hippocampal formation was restricted by the alveus, fimbria, and fornix. Examples of outgrowth on the hippocampal formation are shown in Figs. lC, lD, and 3C. Amygdala. Explants on the amygdala exhibited extensive radial outgrowth that was usually limited by the ventral surface of the brain, the optic tract, or the cerebral peduncle (Fig. 2). The amount of growth was greater on the amygdala than on any other brain region that was examined. Table 5 shows the results of a comparison between explants grown on the amygdala and explants grown on the cortex in the presence of NGF with or without added HS. The total area and the halo area were both significantly greater on the amygdala. The same was true when comparisons were made with explants grown on the thalamus and hypothalamus (data not shown). Basal ganglia. The striatal explants showed irregular outgrowth that was regionally restricted by the white matter bundles passing through the striatum. In some cases, fascicles of neurites extended in a pattern that reflected the underlying gray-white boundaries, Explants situated on the striatum rarely showed outgrowth extending beyond the striatal borders, being limited by the corpus callosum dorsally and laterally and the internal capsule medially.

Telencephalon Neocortex. Radial outgrowth was present from explants on neocortical tissue, regardless of the particular region. The growth was limited by the cortical surface and by the underlying corpus callosum. In several cases, explants situated on deep cortical layers showed growth on more superficial layers but restricted growth in the direction of the corpus callosum. Examples of growth on the cortex are shown in Figs. 3A, 4A, 4B, and 4F. Hippocampal formation. Extensive growth was observed from explants situated on all of the major hippo-

Explants

Grown

TABLE

3

on Brain

Sections with NGF

Culture

n

Explant area

Total area

Halo area

3-day I-day

20 31

0.071 + 0.005 0.111 k 0.008

0.577 + 0.053 0.687 f 0.076

0.506 + 0.052 0.576 2 0.07

7.456 t- 0.688 5.041+ 0.452

0.0008*

0.295

0.4722

0.0035’

P value

Note. Values are averages + SEMs. Areas are mm’. * P < 0.05.

Halo/explant

ratio

NEURITE

GROWTH

ON

CNS

TABLE

TISSUE

45

SECTIONS

4

Explant Growth on Tissue and PORN with NGF Culture 3-day 3-day

tissue PORN

n

Explant

20 21

0.071 0.048

P value 4-day 4-day

+ 0.005 -+ 0.006

0.0072* 31 17

tissue PORN

0.111 0.061

P value Combined Tissue PORN

Values

k 0.008 + 0.009

0.0004*

Total

area

Halo

area

0.577 + 0.053 0.412 r 0.05

0.506 f 0.052 0.364 f 0.045

0.0296*

0.0441*

0.687 + 0.076 0.640 + 0.091

0.576 + 0.07 0.579 + 0.085

0.7024

Halo/explant

ratio

7.456 + 0.688 8.025 rt 0.603 0.5361 5.041 zk 0.452 9.958 5 1.201

0.9808

0.0001*

3- and 4&y 51 38

0.095 + 0.006 0.054 f 0.005

P value Note.

area

0.0001* are averages

f SEMs.

Areas

0.644 t- 0.051 0.514 f 0.052 0.0833

0.549 * 0.047 0.460 f 0.048

5.988 8.890

0.2001

k 0.416 + 0.642

0.0002*

are mm’.

* P < 0.05.

Diencephulon Thalamus. Most explants on thalamic regions showed moderate outgrowth but there were regional variations that appeared to correlate with the amount of white matter present. Growth within the medial thalamic region, for example, was usually extensive (Fig. 4E) but growth in the region of the superior thalamic radiation (Fig. 3B) was more limited. Growth was also restricted by the internal capsule and the stria medullaris when explants were situated adjacent to these white matter tracts. Hypothalamus. Overall, hypothalamic regions supported extensive neurite outgrowth that was limited by the fasciculus retroflexus, fornix, cerebral peduncle, third ventricle, and ventral surface of the brain. Two explants attached in the region of the ventral hypothalamus are shown in Fig. 1A. One explant exhibits extensive neurite growth along the ventral pial surface (arrowhead). Another example of outgrowth on the hypothalamus is shown in Fig. 1B. In both cases there was no neurite growth on the adjacent tissue culture plastic. Brain Stem Due to the limited size of the brain stem relative to the other brain regions only a few explants were encountered attached to brain stem regions. One example of an explant attached to a horizontal section of the superior colliculus is shown in Fig. 4D. In general, neurite growth was limited on brain stem sections. This was probably due, at least in part, to the fact that most explants encountered white matter tracts or fascicles of white matter in most of the brain stem regions that were sampled. Cerebellum Moderate to extensive outgrowth was present from explants situated on cerebellar sections with consider-

able variability attributable to the white matter portions of the folia. An example of growth on the cerebellum is shown in Fig. 4C. Spinal Cord Figure 5 shows examples of explants grown in the presence of spinal cord sections. Unlike the situation for cultures containing brain sections, explants attached to the plastic, in addition to the tissue, when spinal cord sections were present. This usually occurred for explants situated in close proximity to a spinal cord section. Furthermore, explants on the plastic exhibited more neurite outgrowth than explants on the spinal cord sections. The latter often attached to the central gray matter and were able to extend axons along the gray matter as well as along the adjacent white matter (Fig. 5A). The growth on white matter was greater than that observed on white matter from brain sections but not as great as the growth on the plastic dish (Fig. 5B). Neurites extending from explants on the plastic grew up to the spinal cord sections but usually stopped within 20 pm of the section (Fig. 50. The few neurites that crossed this boundary region would often grow on gray matter regions of the cord (Fig. 5D). No obvious differences were present in the growth of explants on tissue sections from different spinal cord levels. DISCUSSION Technical Considerations The in vitro approach used in this study is derived from similar procedures described previously (17, 29, 83). The use of tissue sections thawed directly onto culture dishes and visualization of the neurites and tissue with silver staining are the major differences in the pres-

46

FIG. 3. to a coronal

KEITH

A. CRUTCHER

Differential neurite outgrowth on gray and white matter regions of sections from the mature rat brain. section in the region of the cerebral cortex. The upper explant is located on the outer cortical laminae

(A) Two explants att
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ent approach. Although embryonic chick sympathetic ganglia were used to probe the neurite-promoting ability of mature rat CNS tissue in this study, the same approach could be taken with other developing neural tissue. In fact, it would be feasible to test the ability of developing CNS regions to extend neurites on target and nontarget regions. Alternatively, sections of other tissue can be used as substrates. We have recently undertaken similar studies using tissue sections from mature human brain and found results comparable to those reported here (Crutcher and Privitera, in preparation). The restrictions of the system described in this study, in addition to the well-known limitations of all in vitro paradigms, stem from the limited resolution of the assay and from the fact that only one type of explant was used to test the growth-promoting ability of the CNS tissue sections. The resolution can potentially be improved by using dissociated cells (29) or smaller explants. In order to generalize the observations to other types of neurons, it will clearly be necessary to undertake additional studies similar in nature to this one. Neurite Growth

on Gray versus White Matter

The results obtained in this study indicate that tissue sections from the mature mammalian CNS can support neurite growth in vitro if the neurites are given the opportunity to grow on areas that contain primarily gray matter. The growth is due to the tissue because the underlying plastic does not support growth. This contradicts the conclusions reached in other studies (17, 83) but the apparent discrepancy is easily resolved if the growth-inhibiting nature of CNS white matter (18, 19, 86,87) is considered since previous studies used predominantly white matter-containing tissue. In fact, one of the most striking results of this study is the discrepancy between gray and white matter regions of the CNS in their ability to support neurite elongation in tissue culture. Inhibition of neurite growth has been observed in several preparations (36,48,49,51-53,65,68,69,86,93, 98, 99) and the poor support of axonal regeneration by CNS tissue in vivo has been reported by several investigators (2,8,10, 21,22,34). The specific hypothesis that CNS myelin-associated factors might inhibit axonal regeneration was proposed by Berry (9) and recently supported by the definitive studies of Caroni and Schwab (18, 19) and Schwab and Caroni (87). Berry (9) sug-

directions but the neurite growth stops callosum and exhibits very poor neurite hippocampal formation is in the upper exhibits outgrowth in all directions but the thalamus (arrowhead). (C) Explant Extensive neurite growth is present on extensive growth along the pial interface glial septum in the white matter. Scale

CNS

TISSUE

SECTIONS

47

gested that growth inhibition was due to degradation products of myelin but the present results, along with the results of Caroni and Schwab (18, 19) suggest that the growth-inhibiting factors are associated with normal CNS myelin or other white matter factors. The general conclusion that CNS gray matter supports neurite growth and that white matter inhibits such growth is tempered by the fact that the terms “gray” and “white” matter are not absolute and the identification of a brain region as white or gray is arbitrary, especially when significant mixing occurs, e.g., in the striatum. Some myelinated fibers are present in most gray matter regions but are overwhelmed by the abundance of unmyelinated fibers, neurons, and glial cells. By the same token, major white matter tracts contain unmyelinated axons, astrocytes, and blood vessels. In spite of these semantic limitations, the dramatic difference in neurite growth-promoting potential between the brain areas that contain an abundance of myelin and areas that are myelin-poor is consistent with the hypothesis that myelin-associated factors inhibit neurite regeneration. In addition, the results of this study support the conclusion that growth inhibition is mediated through close contact (87) since axons were able to grow in areas adjacent to white matter. In addition to the clear difference between gray and white matter regions in supporting neurite growth, regional variations in the growth-promoting potential of various gray matter regions were also present. These could be explained, in part, by the presence of variable amounts of myelin-associated growth inhibitors. On the other hand, other factors may influence the amount of growth observed. For example, the extensive growth along vascular profiles and pial surfaces is consistent with the known growth-promoting effects of several extracellular matrix factors such as laminin, fibronectin, and heparan sulfate proteoglycan (45, 47, 57, 67, 82), molecules normally restricted to pial and vascular surfaces in the mature brain and spinal cord (10,16,20,60). Variations in the density of blood vessels from one brain region to another could, therefore, theoretically influence the extent of neurite growth. Regional variations in other neuronal growth factors could also influence the amount of regeneration. Nerve growth factor, for example, is present in high concentrations in some CNS areas but is virtually absent in others

at the edge of the tissue section in the upper part of the field. The lower explant is attached to the corpus outgrowth. (B) Explant in the region of the dorsolateral thalamus adjacent to the fimbria (F). The part of the field and the lateral (LT) and ventral (VT) thalamic nuclei are to the right. The explant does not extend onto the fimbria. The neurites extend along the pial interface between the fimbria and situated in the pial interface between the hippocampal formation (HF) and the cerebral peduncle (CP). the hippocampal tissue but not on the white matter tract. (D) Higher magnification of C, showing the and around a blood vessel section as well as a fascicle of neurites (arrowhead) that have grown along a bars = 0.1 mm.

48

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A. CRUTCHER

FIG. 4. Explants on different brain regions. (A) Several explants attached to a silver-stained coronal section that includes the dorsal hippocampal formation (D), corpus callosum (CC), and striatum (S). Explants are situated on the cingulate cortex, hippocampal formation, and thalamus. The arrowhead indicates an artifact in the section. Five days with horse serum. (B) Explant attached to the cingulate cortex just anterior to the corpus callosum (CC) on a horizontal section. The arrowhead indicates the location of the midline and a point where neurites have failed to cross a gap in the tissue. Four days with NGF. (C) Explant on a horizontal section through the cerebellum with some neuritic growth on the white matter (W). Four days with NGF. (D) Explant on a horizontal section through the level of the superior colliculus. Four days with NGF. (E) Two explants in the medial region of the thalamus. The upper one exhibits sparse outgrowth, whereas the lower one exhibits very dense outgrowth. Three days with NGF. (F) Explant (right side) situated on a section of the cerebral cortex. Extensive neurite growth is present along a penetrating cortical blood vessel (arrowheads). Two days with NGF and horse serum. Scale bars = 1 mm (A) and 0.1 mm (B-F).

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ON

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TABLE

TISSUE

49

SECTIONS

6

Explants on Amygdala and Cortex with NGF Culture Amygdala Cortex

n

Explant

11 50

0.096 0.088

P value Note. Values * P < 0.05.

area k 0.004 T!Z0.006

0.59 are averages

+ SEMs.

Areas

Total 0.972 0.733

area + 0.09 20.052

o.o47f3*

Halo

area

Halo/explant

ratio

0.876 + 0.095 0.644 + 0.049

10.477 z!r 1.315 8.458 f 0.829

0.0439*

0.2864

are mm’.

(32, 5589, 102). Since NGF clearly affects sympathetic neurite regeneration in tissue culture (13-15), it is possible that regional variations in NGF concentration in the tissue sections used for this study influence the rate or amount of axonal growth. The extensive outgrowth observed on the hippocampal formation, neocortex, and amygdala is consistent with the fact that generally high levels of NGF have been reported for these regions (32, 55, 89, 102). It is difficult to test this directly because the NGF concentrations used to supplement most of the cultures, in this study as well as in others (17), are far in excess of those normally present in the tissue. Furthermore, since substrate-bound NGF has been shown to affect neurite growth in tissue culture (44,84), it is possible that the NGF in the tissue or in the medium exerts an effect as a substrate-bound factor. It seems unlikely, however, that NGF is present in sufficient amounts to account for the results obtained with explants grown in the absence of added NGF. NGF is not the only factor that could influence neurite growth. Other putative growth factors have been identified within the mature brain including brain-derived growth factor (6), acidic and basic fibroblast growth factor (96,97), and other uncharacterized growth factors (5, 43, 62, 71, 101). Various membrane adhesion molecules (37, 40, 41, 70) are likely to be present as well. Any of these could theoretically contribute to the growth-promoting potential of CNS gray matter regions but since regional concentrations of these putative growth factors have not been reported no direct correlations can be made. Various neurotransmitters have also been shown to exert growth-inhibiting or growth-promoting effects (48,49, 63,68,69) and could theoretically contribute to the observed results although the concentrations of such compounds in culture are also likely to be very low. In addition, nonspecific adhesive interactions could influence the extent of outgrowth as has been shown in previous work (58). This latter possibility might also account for the fact that very few explants attached to white matter portions of the sections. If adhesive interactions contribute to differential outgrowth in vitro similar effects might operate in uivo as well. At this point, however, it is difficult to draw definitive conclusions on the relative

roles these various interactions have in permitting neurite extension. It does seem likely, however, that most of the extracellular matrix factors that have been implicated in supporting regeneration in vivo and in vitro (45, 47, 57, 67, 82,105) cannot account for the growth observed on CNS tissue sections. The absence of neurite growth in the study by Carbonetto et al. (17) was interpreted as being most consistent with the hypothesis that extracellular matrix components, which are lacking in the mature brain, are essential for successful axonal regeneration. Other investigators have also postulated that extracellular matrix factors are required for successful axonal elongation (83). It is possible that such factors are released by the explants and deposited on the tissue sections. However, since the tissue culture conditions used in this study do not permit attachment or neurite outgrowth without the presence of tissue sections (excluding spinal cord tissue) it seemslikely that the tissue itself ultimately exerts a growth-promoting effect. In any event, the discrepancy between growth on white and gray matter regions must be due to differences in the ability of the tissue to promote neurite growth, either by itself or in combination with factors in the medium. Either way, the substantial growth occurring on gray matter regions indicates that the “nonpermissiveness” of mature CNS, at least in vitro, applies only to the white matter regions. Implications for Understanding kvonal Growth Regulation in Vivo Perhaps the most important implications of the present results relate to our understanding of the potential for axonal growth within the mature mammalian brain and spinal cord. The long-standing dogma that CNS neurons cannot regenerate was effectively removed by the definitive nerve graft experiments of Aguayo and coworkers (1,3, 7,34,80,81,95). Attention is now focused on the factors present in the CNS that either inhibit or promote axonal growth. That such growth is possible in vivo under certain circumstances has been known for some time. Just as the growth-inhibiting influence of

50

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FIG. 6. Explants grown with spinal cord sections for 4 days in the presence of NGF. (A) Two explants on a thoracic spinal cord section. The upper one is on the dorsal horn region (the contralateral dorsal horn is labeled DH) and extends neurites onto the adjacent dorsal column and onto another spinal cord section. The lower explant is situated on the intermediate gray and extends neurites on the ventral horn and adjacent lateral, ventral, and dorsal funiculi. (B) Explant attached to the edge of a spinal cord section extending neurites on the plastic as well as on the spinal cord ventral funiculus. More extensive growth is present on the plastic. (C) Explant attached to the plastic with radial neurite outgrowth. Some of the neurites have grown toward a spinal cord section (DH, dorsal horn) but do not extend onto the tissue (arrowhead). (D) Higher magnification of the explant-spinal cord interface shown in C. A boundary region separates most of the neurites from the adjacent tissue (arrowhead) although some of the neurites have grown into the dorsal horn region (arrow). Scale bars = 0.1 mm.

mature CNS white matter in vitro correlates with the abortive regeneration commonly observed in vivo, the growth-promoting potential of gray matter regions in vitro is entirely consistent with the numerous examples of anatomical plasticity that have been described following tissue transplantation (12,25,38,56,74,91) or denervation in vivo (4, 8, 9, 11, 27, 28, 31, 42, 46, 54, 64, 7578, 104). In most of these cases, however, the observed growth is restricted to gray matter regions. It is difficult to draw direct comparisons between in vitro results, such as those obtained in this study, and in vivo examples of anatomical plasticity. The growth of intrinsic sympathetic axons into the rat hippocampal formation following septal denervation (31), however, provides one parallel to the in vitro growth from sympa-

thetic explants observed in the present study. In both cases, sympathetic axons are able to grow in association with mature CNS brain tissue. In the case of in vivo sympathetic sprouting, however, the growth is only observed following septal denervation of the hippocampal formation, whereas the tissue sections were taken from normal (unlesioned) animals. In addition, sympathohippocampal fibers are restricted to the dentate and CA3 region of the hippocampus in vivo but sympathetic explants grown on sections of the hippocampal formation do not show this topographic specificity. The differences between the in vivo and in vitro sympathetic growth may be due to several factors including the following: (i) the use of embryonic (developing) chick sympathetic neurons in vitro versus the mature sympa-

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thetic neurons that sprout in the rat; (ii) the fact that regeneration is presumably occurring in vitro as opposed to nonregenerative growth (sprouting) in uiuo (31); (iii) trophic support, e.g., NGF concentration, is optimal in vitro but may be limiting in uiuo (24); and (iv) growth in vitro occurs on tissue sections, whereas sympathetic ingrowth in uiuo occurs in living tissue where the interactions are more dynamic and complex. The first point recognizes the presumably greater growth potential of developing tissue relative to that of mature neurons. The second consideration arises from the assumption that most of the neurites observed in culture represent regenerating axons, although this has not been demonstrated vigorously. Some of the fibers could be newly grown. In any event, this explanation for the difference between in vitro and in uiuo growth seems less important in light of the fact that regenerating axons from mature rat superior cervical ganglia transplants depend on septal denervation in order to extensively innervate the hippocampal formation (11). The third possibility, that of trophic support, may well be relevant since NGF has been implicated as part of the signal eliciting sympathetic sprouting following septohippocampal denervation (24, 31, 32). In particular, the increase in hippocampal NGF following septal denervation occurs with a regional and temporal specificity that is consistent with a role in the in uiuo sprouting response (24). In uitro, NGF availability is presumably not restricted spatially or temporally. Finally, the interaction between growing axons and living tissue is obviously more dynamic than the growth of axons on tissue sections. It would be difficult to enumerate the potential influences that living tissue provides that are not present in tissue sections, but some obvious candidates are electrical activity, release of neurotransmitters, and interactions with glia, cellular elements that have been the focus of much recent work (5,26,39,41,59,61,62,70,73, 90,91). In particular, astrocytes have been implicated in the regulation of axonal growth both during development (90,91) and during regeneration (65,103). One possible influence that astrocytes might exert on growing axons in uiuo is the induction of synapse formation (65). Since most documented examples of anatomical plasticity in gray matter regions of the mature brain involve synapse formation, it is possible that the restriction on extensive axonal elongation in uiuo is due to synaptogenesis. In some examples of in uiuo sprouting, the rate of axonal growth is slow (4, 42) and may occur through a process of successive synaptogenesis that would depend on the availability of synaptic sites. Such “synaptic creep” would not be expected to limit the growth of axons that do not normally form synaptic specializations, e.g., sympathetic fibers (33). It is not known to what extent synaptogenesis may be occurring in the tissue culture system used in this study but synaptogen-

CNS

TISSUE

51

SECTIONS

esis could theoretically influence the rate and extent of neurite outgrowth. The interpretation of the results obtained with spinal cord tissue is complicated by the attachment and neurite extension of explants situated on the plastic. Since the plastic is not normally permissive, either for attachment or neurite extension, the spinal cord tissue must be releasing growth-promoting material that binds to the plastic and permits subsequent explant attachment and neurite outgrowth. The material may also modify the growth-promoting ability of the tissue section since explants on the tissue sections showed better-than-expected outgrowth on the spinal cord white matter. The growth-inhibiting nature of the white matter was evident, however, by the border established between the white matter and neurites growing in the vicinity of the tissue section (Figs. 5C and 5D) and by the reduced growth on white matter relative to the adjacent plastic from explants overlapping both the tissue and the plastic. The presence of substrate-bound neuronal growth factors was first demonstrated by Collins (23) and has since been documented by several other laboratories ((30), for review). The identity of the activity in the spinal cord cultures is unknown but indicates the presence of both growth-promoting and growth-inhibiting substances in the spinal cord tissue. In spite of the theoretical distance between the tissue culture dish and the intact organism, the dramatic discrepancy between gray and white matter regions of mature CNS tissue in supporting axonal growth in uiuo and in uitro suggests that there are fundamental differences between the two types of CNS tissue. These differences may be explained teleologically by the need to restrict anatomical plasticity in maturity to brain regions mediating behavioral plasticity, such as learning and memory. Major pathways that serve to connect one brain region to another, however, would provide effective barriers to the establishment of anomalous connections once essential pathways have developed and the basic plan of the CNS is established. In order to restore function to the damaged brain and spinal cord, advantage should be taken of the growth-promoting nature of gray matter as well as deriving strategies to overcome the growth-inhibiting influence of white matter. ACKNOWLEDGMENTS NGF was kindly provided by Dr. William Mobley, University of California San Francisco. The work described here was supported by the NIH (NS 17131) and by the Clifford F. Ahlers Trust, University of Cincinnati. The expert technical assistance of Paula Schmidt and Jean Weingartner is gratefully acknowledged.

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