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Axonal regeneration on mature human brain tissue sections in culture.

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Axonal Regeneration on
Mature Human Brain
Tissue Sections in Culture
Keith A. Crutcher, PhD, and
Michael Privitera, MD
Fresh frozen sections of the temporal lobe from patients
undergoing surgical resection for the control of epilepsy
were used as a substrate in tissue culture to test for neurite growth-promoting activity. Robust neurite growth
from ganglia of embryonic chicks was observed on gray
matter regions of the sections and little growth was observed on white matter regions. These results indicate
that the dichotomy between gray and white matter regions of the mature central nervous system in supporting neurite growth is present in human brain tissue and
supports the general hypothesis that white matter in the
mature central nervous system inhibits neurite regeneration but that gray matter will support neurite growth.
Crutcher KA, Privitera M. Axonal regeneration on
mature human brain tissue sections in culture.
Ann Neurol 1989;26:580-583
Axonal regeneration within the mature mammalian
central nervous system (CNS) is usually abortive yet
CNS neurons will regenerate within a peripheral nerve
environment 111. The failure of axons to regenerate
within the mature mammalian brain and spinal cord
has been attributed to the lack of suitable growth factors, especially extracellular matrix components E2-41.
The optic nerve, for example, contains little extracellular matrix and does not support axonal regeneration in
v i m {2-43. In addition, cryostat-cut sections of the
spinal cord, when used as in vitro substrates o n which
embryonic neuronal explants were cultured, were
found to support minimal neurite outgrowth when
compared with tissue from the peripheral nervous system (PNS) [2). However, the tissue used for these
studies contained significant amounts of myelin (white
matter), and other workers have shown that CNS myelin contains factors that inhibit neurite outgrowth [ S ,
61. Recently, the potential for in vitro neurite regeneration was tested o n tissue sections from mature rat
Prom the Departments of Neurosurgery and Neurology, University
of Cincinnati College of Medicine, Cincinnati, OH.
Received Jan 12, 1989, and in revised form Mar 7, 1989. Accepted
for publication Mar 31, 1989.
Address correspondence to Dr Crutcher, Department of Neurosurgery, University of Cincinnati College of Medicine, Cincinnati,
OH 45267-0515.
brain areas containing large areas of gray matter (areas
containing neurons and glial cells but little myelin) as
well as myelin-rich white matter tracts [73. Extensive
neurite regeneration occurred o n gray matter but not
on white matter. The present study was undertaken to
determine whether human brain tissue exhibits similar
properties in vitro.
The overall approach was similar to that used in previous
studies of animal tissue C2, 3, 71. Samples were obtained
from the temporal cortex of patients undergoing elective
temporal lobe resection for the treatment of epilepsy. The
tissue was immediately frozen on dry ice following removal
and stored at - 70°C. Fresh frozen sections (16 pm in thickness) were cut on a cryostat and thaw-mounted onto the
bottom of 35-mm tissue culture dishes (Falcon, Becton Dickinson, Lincoln Park, NJ). Sections were cut perpendicular to
the cortical surface in order to include the cortical gray matter as well as portions of the underlying white matter in the
same section.
Chick embryos (age, embryonic day 9) were dissected in
Ham’s F12 medium (Sigma Chemical, St Louis, MO) to obtain lumbar sympathetic chain or dorsal root sensory ganglia.
The ganglia were placed on the sections and grown with 1.5
ml of Ham’s F12 medium (supplemented with antibiotics)
with 5% horse serum (HS) and nerve growth factor (NGF;
at a concentration of 60 or 240 ndml). Cultures were incubated in a humidified environment (5% carbon dioxide) at
37°C for 1 to 6 days without additional changes of medium.
The explants, neurites, and tissue sections were visualized
by staining the entire culture with a modification of the
Sevier-Munger silver stain [7, 8). Measurements of the explants were made with a HiPad digitizing tablet and BioQuant (R&M Biometrics, Nashville, TN) morphometric
software running on an Apple IIe microcomputer (Cupertino, CA). The explants and neurite perimeters were measured with the HiPad digitizing tablet and the resulting areas
were calculated by the BioQuant program. A total of 74
explants (29 on white matter and 45 on gray matter) were
measured from 39 sections (average of 2 explants per section) taken from 10 culture dishes (average of 4 sections per
dish). Only explants that were located primarily on white or
gray matter were included for morphometric analysis. The
perimeter of the neuritic halo was arbitrarily defined as the
radial boundary enclosing the majority of silver-stained neurites. The only neurites that were not encompassed by the
boundary were single neurites that extended well beyond the
main halo perimeter. 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 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.
Statistical comparisonsbetween two groups of measurements
were made using an unpaired t test with significance accepted
at a p value of less than 0.05. Tracing of the explants was
done with an Olympus microscope with an attached drawing
tube and all photographs were taken with a Nikon microscope.
Copyright 0 1989 by the American Neurological Association
Explants did not attach to the culture dish unless polyornithine or tissue sections were present, indicative of
the fact that the untreated tissue culture plastic does
not permit explant attachment. Explants that landed on
the edge of a tissue section or on breaks in the tissue
would often attach but only extend neurites onto the
tissue and not on the adjacent plastic (Fig {A]). Most
of the explants that landed on white matter appeared
to attach but many washed away during the staining
procedure. Explants that washed away left holes in the
section so that it was possible to identify their original
location (see Fig [B)). Some of the explants on the
white matter remained attached throughout the staining and were subsequently measured. Usually these
explants exhibited very little neurite outgrowth.
Most of the explants that persisted through the
staining procedure proved to be located on gray matter
regions of che section (see Fig). These explants extended neurites with a topography and extent that
varied according to their location in relationship to
other features such as adjacent white matter, the edge
of the section, or the presence of vascular and meningeal profiles. Explants that attached to the gray matter
without interference from the latter features exhibited
extensive radial neurite outgrowth (see Fig). Explants
that overlapped both gray and white matter showed
good neurite outgrowth on the gray matter but poor
outgrowth on the white matter (see Fig {D, El). Explants that encountered the pial surface or blood vessel
profiles exhibited extensive neurite outgrowth along
these surfaces, even greater than that observed on the
gray matter (see Fig {GI).Although it was not possible
to determine whether synapses were formed, some of
the neurites exhibited distinct varicosities along their
length (see Fig {I-I}).
In order to provide a quantitative comparison between the growth on white matter with that on gray
matter, the area of neurite outgrowth was measured.
The results are shown in the Table. The explants were
slghtly larger on gray matter compared with white
matter but exhibited 3 times as much neurite outgrowth as measured by the total area covered by the
neuritic halo. This difference would no doubt be
greater if the explants that did not survive staining
were included in the analysis, since the vast majority of
such explants were situated on white matter without
any obvious neurite outgrowth prior to staining.
Two principal conclusions emerge from this study. The
first is that white matter regions of the mature human
CNS weakly support neurite growth from embryonic
chick ganglia in tissue culture. This is consistent with
the general hypothesis that mature mammalian white
matter in the CNS provides a relatively nonpermissive
or inhibitory environment {5-7, 91 and also correlates
with the poor regenerative potential observed in white
matter tracts in vivo (e.g., the spinal cord). The second
conclusion is that gray matter in the mature human
brain is able to support neurite regeneration from embryonic chick ganglia in vitro. This is consistent with
the hypothesis that anatomical plasticity, most likely in
the form of collateral sprouting, continues into maturity and that the CNS retains the capacity for neuronal
rearrangements beyond the normal period of development. Examples of suspected anatomical plasticity in
human brain have been reported {lo].
The tissue used in this study was obtained from patients undergoing surgical resection for the treatment
of epilepsy. One possible limitation in interpreting
these results is that the growth-promoting potential
observed may not reflect the results that would be
obtained with normal tissue since the samples used in
this study were taken from pathological brain tissue. It
seems highly unlikely, however, that the difference between gray and white matter in supporting neurite
growth in tissue culture is restricted to epileptic tissue,
especially since the same dichotomy is present in rat
CNS tissue {7]. Another possible limitation is that regional variations might exist in the support of neurite
growth by human brain tissue in vitro as was observed
for sections of the rat brain 171. It may be possible
in future studies to use postmortem brain tissue in
this assay to determine regional variations in neuritegrowth promotion as well as to compare differences
between normal and pathological material. Preliminary
results indicate that the dichotomy between neurite
growth on gray and white matter persists on tissue
sections from postmortem-derived brain samples
(Crutcher, unpublished observations, 1989).
The molecular interactions that mediate the neurite
growth on gray matter, and its inhibition by white matter, are not known. Since recent studies have shown
that CNS myelin from the mature rat brain contains
neurite growth-inhibiting factors that can be blocked
by specific antibodies IS}, it seems likely that the poor
outgrowth obtained on human white matter is also due
to similar factors associated with central myelin in the
human CNS. It is also possible that nonspecific adhesive interactions contribute to the present results.
White matter may simply provide a less adhesive substrate than gray matter. The ability of embryonic chick
neurons to attach and grow on a human brain tissue
substrate suggests either that nonspecific adhesion is
involved or that cross-species recognition of homologous growth factors is occurring. If specific molecular
interactions are involved, it might be possible to affect
the growth with antibodies raised against putative
growth-regulating molecules. Regardless of the actual
molecules involved, the difference between gray and
white matter regions of the mature human brain in
Brief Communication: Crutcher and Privitera: Axonal Growth on Human CNS Tissue Sections 581
Silver-stained explants on tissue sectionsfrom human temporal
lobe. All were grown in the presence of nerve growth factor. (A)
Three sympathetic explants on the gray matter of temporal lobe
cortex, one of which is adjacent to a pialfissure and has sent
neun'tes to the vascular profiks in the fissure (arrowhead). The
other two explants exhibit radial neurite outgrowth. The explants were grown for 4 days. (B) Three sympathetic explants
(4-duy culture) situated on the gray matter of a temporal lobe
section. The lower two explants are situated adjacent to the
white matter. The arrows indicate two holes where explants had
been attached to the white matter but were lost during staining.
(C) Dorsal root ganglion explant on a portion of gray matter.
Extensive radial neurite outgrowth is present. (0)Sympathetic
Annals of Neurology Vol 26 No 4 October 1989
explant (2-day culture) attached at the border between gray and
white matter (double arrow). The top half of the explant is
enlarged in E and the lower half is enlarged in F as indicated by
the long arrows. (E) Sympathetic neun'tes extend on the gray
matter. (F)Sparse neuritic outgrowth on white matter. (G) Dwsal root ganglion explant ( 2 A y culture) attached to the edge of a
tissue section showing extensive neurite outgrowth along a pialvasnrlarfissure (arrow), kss extensive outgrowth on the gray
matter, and no outgrowth on the adjacent plastic. (H)Sympathetic neurites ( 2 A y culture) growing on gray matter. Seueral
varicmities arepment (arrow). Scale bar = 1 mm in A and
B; 100 pm in C-H.
Explant and Total Area (Mean 2 SEM) on White and Gray
Matter Regions after 4 Days in Culture
White matter
Gray matter
p value
Explant (mm2)
0.079 0.005
0.094 2 0.004
Total (mm')
0.558 2 0.099
1.543 0.091
supporting neurite regeneration might be relevant in
understanding the factors that affect the use of potentially therapeutic procedures such as neural grafting.
For example, the relative amount of gray and white
matter in the region of a neural transplant may influence the amount or topography of innervation obtained. These results also encourage the hope that
neuronal plasticity within gray matter of the mature
human brain and spinal cord can be manipulated to
combat the disabling effects of CNS injury and disease.
Nerve growth factor was kindly provided by Dr William Mobley,
University of California at San Francisco.
This work was supported by the Alzheimer's Research Center at the
University of Cincinnati, National Institute on Aging grant AG07691, and by the Clifford F. Ahlers Foundation.
The expert technical assistance of Paula Schmidt, Johanna Neaderhomer, and Jean Weingartner is gratefully acknowledged. We are
especially indebted to Dr Hwa-Shain Yeh (Department of Neurosurgery) for his cooperation in obtaining the surgical specimens.
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4. Schwab ME, Thoenen H. Dissociated neurons regenerate into
sciatic but not optic nerve explants in culture irrespective of
neurotrophic factors. J Neurosci 1985;5:2415-2423
5. Caroni P, Schwab ME. Antibody against myelin-associated inhibitor of neurite growth neutralizes non-permissive substrate
properties of CNS white matter. Neuron 1988;1:85-96
6. Schwab ME, Caroni P. Oligodendrocytes and CNS myelin are
nonpermissive substrates for neurite growth and fibroblasr
spreading in vitro. J Neurosci 1988;8:2381-2393
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Exp Neurol 1989;104:39-54
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Peripheral Neuropathy
in Amyotrophic
G. Vita, MD," S. Serra, MD,X R. Dattola, MD,"
M. Santoro, MD," A. Toscano, MD," C. Venuto, MD,"
G. Cmrrozza, MD,? and A. Baradello, MD'
We investigated involvement of the peripheral nervous
system in 6 patients with amyotrophic choreaacanthocytosis. Electromyographic and neurographic
findings, and pathological changes as demonstrated by
examination of biopsy specimens of muscle and surd
nerve indicate that most patients had an axonal sensorimotor polyneuropathy with more pronounced involvement of the distal portion of the nerves. Results
obtained in one patient raised the question of an anterior horn cell disorder.
Vita G , Serra S, Dattola R, Santoro M, Toscano A,
Venuto C, Carrozta G, Baradello A. Peripheral
neuropathy in amyotrophic chorea-acanthocytosis.
Ann Neurol 1989;26:583-587
Amyotrophic chorea-acanthocytosis (ACA) is a rare
familial disease of adult onset characterized by orafacial dyskinesia and choreic movements of the limbs,
tongue or lip biting, caudate atrophy, neurogenic muscular atrophy, and acanthocytosis without abnormalities in plasma lipoproteins { 1-41. Pathogenesis remains
obscure, but recently abnormalities of erythrocyte membrane proteins have been reported [5]. Although more than 50 patients with ACA have been
reported in the English and Japanese literature, in-
From the *Institute of Neurological and Neurosurgical Sciences and
the tDepartment of Human Pathology, University of Messina Medical School, Messina, Italy.
Received Nov 17, 1988, and in revised form Mar 7, 1989. Accepted
for publication Mar 9, 1989.
Address correspondence to Dr Vita, Clinica Neurologica 2", Polichnico Universitario, 98125 Messma, Italy.
Copyright 0 1989 by the American Neurological Association 583
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axonal, culture, section, brain, regenerative, tissue, human, mature
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