PREFACE FOR POSTER ABSTRACTS: At the same Symposium On Neural Regeneration (December 6-10, 1995) 65 groups
of highly trained scientists in neural regeneration presented these abstracts
along with their respective displays for the benefit of all who are interested.

P1 DIFFERENTIAL PROPERTIES OF VOLTAGE-DEPENDENT Na+ and K+ CHANNELS
FOLLOWING AXOTOMY IN CUTANEOUS AFFERENT DRG NEURONS OF
ADULT RAT
B. Everill, M.A. Rizzo, S.G. Waxman and J.D. Kocsis, Yale School of Medicine, New Haven,
CT and Neuroscience Research Center, VA Medical Center, West Haven, CT

Studies of the behaviour of Na+ and K+ currents particular to cutaneous afferent dorsal root
ganglion (DRG) neurons following chronic axonal injury should increase our understanding of
hyperexcitability of these neurons following such injury. Cutaneous afferent DRG neurons in
short-term cultures were identified by retrograde fluorescence labelling of the sciatic nerve via
cutaneous injection of calf skin one week prior harvesting. Whole cell patch recordings indicate
the presence of both transient outward current, sensitive to 4-aminopyridine and TEA sensitive
outward current, sharing kinetics of A and K current, respectively. In controls amplitude of A
currents varied between cells, but also within a single cell. These currents are presently being
examined in injured neurons. Kinetically slow, TTX-resistant Na+ currents were prevalent in
control neurons. Predominantly kinetically fast, TTX-sensitive Nat currents emerge post-
axotomy. Na+ currents in axotomized neurons tended to be blocked by TTX, and by relatively
higher concentrations of lidocaine. Steady-state activation and inactivation curves constructed
from Naf current recordings of somatic membrane blebs (fragments of membrane 3-8 um
diameter corresponding to 0.3-2 pF) excised from cutaneous afferent DRG neurons revealed
that conductances post-axotomy had a decreased slope factor, broader window currents, and
splay of steady-state inactivation in comparison to slow Na+ conductances in control neurons.
The results are consistent with the hypothesis that, following axotomy, new subpopulations of
Na+ channels occur on cutaneous afferent DRG neurons. Injury does not appear to induce a
large population shift of K+ channels as occurs for Na+ channels. These subpopulations may
confer altered neuronal excitability, contribute to ectopic impulse generation, and underly pain
and paresthesias following chronic axonal injury. Supported in part by Medical Research
Services of the VA and the NIH.

P2 TKE INFLUENCE OF PULSED MAGNETIC FIELDS ON NEURITE OUTGROWTH
FROM CHICK DORSAL ROOT GANGLIA IN THE PRESENCE OF NERVE GROWTH FACTOR
C.H. Sutton*, B. GreenebaumS, B.F. Sisken#, M.S. Vadula* and I.H. Battocletti* *Medical
College of Wisconsin and Research Service, VA Medical Center, Milwaul;ee. WI, iSchool of
Science and Technology, Biomedical Research Institute, University of Wisconsin-Parkside,
Kenosha, WI and #Center for Biomedical Engineering, University of Kentucky, Lexington, KY

Pulsed electromagnetic fields (PEMF) have been demonstrated to facilitate healing of non-
united fractures in the orthopaedic clinical literature, as well as initiating angiogenesis in
endothelial cell cultures and promoting healing of soft tissue wounds with fibroblasts in vivo.
Previous studies in our laboratories have documented the stimulation of neurite outgrowth from
dorsal root ganglia (DRG) in vitro when PEMF and nerve growth factor (NGF) are used in
combination (Vadula et al., In Electromagnetics in Medicine and Biology, San Francisco Press,
1991, pp 145-151). The methods used to cultivate embryonic chick DRG have been described
elsewhere (Sutton et al., In Electricity and Magnetism in Biology and Medicine, San Francisco
Press, 1993, pp 351-354.). Culture dishes containing 20 DRG explants from'l-day-old embryos
in media were placed midway between and parallel to a pair of 5-in. diameter Helmholz coils
of 16 turns each. Twenty-microsecond duration rectangular voltage pulses produced triangular
AC coil currents of 24 amperes peak and a peak magnetic field through the dish of40 G. A burst
of 22 pulses was applied overa period of 4.8 msec. The bursts were repeated 1 5 times per second
in one series of experiments and 25 times per second in another. There were no significant
differences between the two groups. More than 900 DRGs were studied. We reproduced our
previous findings that (1) NGF and 4 mT-peak fields induced significantly (p<0.05) greater
outgrowth than NGF alone, (2) fields without NGF do not significantly alter outgrowth
compared to unexposed controls, and (3)4 mT fields and NGF can induce asymmetric
outgrowth which is not seen with NGF alone, but which has no preferred orientation with respect
to the PEMF-induced electric field. NCF alone is known to produce radial outgrowth. It was
concluded that this combination of physical and chemical modalities may have clinical
application for providing optimal survival of neurons and for enhancing the directional growth
of neurites.

P3 IN NEUROMUSCULAR ACTIVITY BENEFICIAL TO MOTOR AXON SPROUTING?
S.L. Tam, P. van der Sloot, B. Jasser, V. Archibald, N. Tyreman and T. Gordon, University of
Alberta, Edmonton, Canada

Using a rat partial denervation model, we tested the hypothesis that increased neuromuscular
activity overloads the metabolic capacity of sprouting axons during the acute phase of
motoneuron disease and contributes to the destabilization ofenlarged motor units (MU) during
the chronic phase. Four hindlimb muscles were partially denervated (PD) by cutting the L4 or
L5 spinal root and subjected either immediately (acute) or 11 (chronic) months later to increased
activity: daily functional electrical stimulation (FES, 8hr 20Hz) or natural stimulation (8hr
running on exercise wheels 1200m/day). Within a month, MU force and number of sprouts in
animals which experienced normal cage activity increased in proportion to the extent of PD to
an upper limit of 3-5 times normal. However, terminals were withdrawn from enlarged MUs
in extensively denervated muscles such that MU size was significantly reduced at 1 2 month after
PD. A one month period of increased neuromuscular activity reduced MU enlargement in
extensively denervated muscles (PD>75%) during the acute phase but did not further compro-
mise the time-related reduction in MU size in the chronic phase. These results show that normal
physiological activity conducive for maximal sprouting during the acute phase while non-
physiological activation can be detrimental. The loss of terminals from enlarged MUs can
account for the muscle weakness characteristic of the progressive weakness experienced many
years following recovery from polio virus infection or the spinal root trauma. Although a one
month duration of increased activity does not impact the withdrawal of terminals, our results
suggest a longer interval would have detrimental effects. Thus for clinicians in an era when the
prevailing thought is "exercise can do no harm," these results suggest that exercise regimes must
be used with caution. Supported by AHFMR, Rick Hansen Man in Motion Legacy Fund,
MDAC and MRC of Canada.

P4 AXOTOMY, COLCHICINE BLOCK AND ELECTRICAL STIMULATION OF THE
HYPOGLOSSAL NERVE IN RATS AND THE AXON REACTION
K. Mader, J. Andermahr, D.N. Angelov, E. Stennert and W.F. Neiss, Institut I fiir Anatomie und
Klinik fiir Hals- Nasen- und Ohrenheilkunde der Universitlt zu Kiiln, Kbln, Germany

Transection of the hypoglossal nerve affects the perikarya of hypoglossal motoneurons in two
major ways. First, it interrupts the bidirectional axoplasmic transport, notably that of trophic
factors from muscle to neuron. Second, it severely disturbs the electric membrane potential and
activity of lesioned nerve fibers and cell bodies. In summary, both factors result in the well
known nerve cell body response or axon reaction. We tried to separate the contribution of both
factors to the axon reaction. For this aim we compared axotomy to colchicine block (interrupts
axoplasmic transport but not electric conductivity) and to antidromic electric stimulation (does
not impede transport) of the peripheral hypoglossal nerve. After prelabelling of the hypoglossal
neurons with Fluorogold, we performed in Wistar rats either resection, colchicine block or
electrostimulation ofthe hypoglossal nerve. The rats were fixed by perfusion 1, 3, 5, 7, 10, 15,
25, 35 or 50 days post operation (DPO) and microglia (OX-42, OX-18, OX-6, EDI, ED2),
astroglia (anti-GFAP) and neurons (anti-CGRP) were immunostained in the lower brainstem.
Electrically stimulated animals showed microglial activation 1-7 DPO, CGRP was upregulated
for 1-7 DPO and astroglial GFAP-expression was elevated 3-15 DPO. Calchicifle block caused
strong activation of microglia 5-25 DPO, CGRP was upregulated for 3-15 DPO, GFAP
expression was elevated 3-35 days. Nerve resection caused microglial activation 2-35 DPO,
CGRP expression above baseline 2-15 DPO and elevated GFAP expression 3-35 DPO.
Doublelabelling of ED2 and Fluorogold in perivascular microgliocytes as a sign of
neurophagocytosis occurred in axotomized and colchicine-treated animals, but not after
electrostimulation. We conclude that electrical disturbances after axotomy contribute to the
early phase of the axon reaction and that lack of axonal transport causes the later responses,
including neuronal cell death and neurophagocytosis by microglia. Supported by the Jean
Uhrmacher Foundation.

P5 DELAYED HYPOGLOSSAL-FACIAL ANASTOMOSIS AFTER FACIAL NERVE INJURY
WITH PREDEGENERATION OF THE DISTAL FACIAL NERVE STUMP
O. Guntinas-Lichius, D.N. Angelov, M. Streppel, E. Stennert and W.F. Neiss, Klinik fiir Hals-
Nasen-Ohrenheilkunde und Institut I fuir Anatomie der Universitat zu Koln, Cologne, Germany

Hypoglossal-facial anastomosis (HFA) immediately after facial nerve axotomy in the rat is
followed by a reinnervation of the whisker-pad muscles by hypoglossal motoneurons and by
spontaneous resprouting of facial motoneurons causing a permanent motor hyperinnervation of
the target (J. Comp. Neurol., 338:214-224, 1993). In contrast to this and other experimental
designs in animals, the facial nerve repair after peripheral injury in man is nearly always only
possible after a time delay. The standard surgical method for the treatment of facial palsy after
destruction of the proximal portion of the facial nerve is the HFA. Therefore, we performed in
Wistar rats a HFA with a delay of 0, 7, 10, 14 or 56 days after facial nerve axotomy, and counted
42 days after nerve suture all motoneurons in the brainstem labeled by the injection of horseradish
peroxidase into the whisker-pad. In untreated animals, HRP injection into the whisker-pad labels
1254+or-54 (mean+or-SD; n =6) motoneurons exclusively in the lateral facial subnucleus. After
immediate HFA 566+or-62 neurons are labeled in the hypoglossal nucleus and 268 + 35 neurons
in the facial nucleus at 42 days post suture(dps). HFA with 7 days delay leads to an increasing
number of marked cells in both nuclei 42 dps: we counted 734+or-145 neurons in the hypoglossal
and 645+or-234 cells in the facial nucleus. With a delay time of 10 days, the number oflabeled cells
in the hypoglossal nucleus increased to 1102+or-203 neurons, whereas in the facial nucleus less
neurons, 496+or-285, appeared at 42 dps. After longer delay times, i.e. 14 and 56 days after axotomy
the numberoflabeled cells decreased in both nuclei, but was still higher than after immediate nerve
repair. We counted 933+or-304 cells at 14 days delay time and 788+or-230 neurons at 56 days delay
time in the hypoglossal nucleus, and 460+or-263 and 454+or-212 neurons respectively in the facial
nucleus. Delayed suture 7-1O days after nerve injury with predegeneration of the peripheral nerve
increases the number of hypoglossal neurons that reach the target muscles within 6 weeks after
nerve repair.

P6 ANDROGEN RECEPTOR (AR) REGULATION IN HAMSTER FACIAL MOTOR NEURONS
(FMN): EFFECTS OF AXOTOMY ANDI OR TESTOSTERONE PROPIONATE
S.M. Drengler, R.J. Handa, L.L. DonCarlos and K.J. Jones, Loyola Univ.-Chicago, Maywood, LL

Unilateral crush axotomy of the facial nerve in adult hamsters results in facial paralysis and triggers
a series of regenerative events, which culminates in full recovery of function. In previous studies,
it has been established that after axotomy, rates of FMN axon regeneration, as well the time
required for full functional recovery, differ with the sex of the animal. Females have a faster
inherent facial nerve regeneration rate than males after crush axotomy. Further, sex differences in
the ability of testerone propionate (TP) to augment the regenerative response in FMN also exist.
Regeneration rates are increased 30% in intact or gonadectomized (gdx) males after TP treatment,
whereas, the regeneration rates in females are increased only 10% by TP treatment. Behavioral
studies indicate that testosterone treatment had a significant stimulatory effect on functional
recovery in males but not females. These discoveries have led to the hypothesis that sex differences
in nerve regeneration could be due to inherent differences in AR gene expression or regulation after
injury. In the present study, this hypothesis was tested using in siru hybridization in conjunction
with computerized image analysis to quantify levels and regulation of AR mRNA in individual
FMN of gdx male hamsters after facial nerve injury. Adult gdx male hamsters were subjected to
right facial nerve axotomy at the level of the stylomastoid foramen. After axotomy, 1 IO-mm
Silastic capsule containing exogenous TP was implanted for 1, 2 or 7 days. In situ hybridization
studies using a cRNA probe selective for the ligand binding domain (AR4) demonstrated a down-
regulation of AR mRNA at all time points after axotomy, as compared to the uninjured left side.
Surprisingly, treatment with TP did not reverse the axotomy-induced down-regulation of AR
mRNA levels, even though TP treatment of uninjured gdx animals upregulates AR mRNA in FMN.
These findings suggest that axon injury can regulate the expression of androgen receptor mRNA
in motor neurons. We have also shown, in a separate immunocytochemical study using the PG-
21 antibody to the AR (a gift of Dr. Call Prins), that TP treatment increases the number of FMN
expressing AR immunoreactivity (IR) and the intensity of nuclear labeling. Experiments are in
progress to determine effects of axotomy with and without TP on AR-IR in FMN. Supported by
NIH NS28238, NTMH 48794 and NSF IBN 9408890.

P7 CYCLICAL DEGENERATION AND REGENERATION OF SYMPATHETIC NERVES
IN RAT UTERUS
E.V. Zoubina, Q. Fan, R.J. Flietstra and P.G. Smith, University of Kansas Medical Center,
Kansas City, KS

Sympathetic innervation of the uterus is an excellent system for studies of nerve regeneration
in the intact animal. During pregnancy sympathetic innervation undergoes profound changes
including widespread degeneration, with regeneration occurring after parturition. However, the
results of studies examining whether such changes occur during normal estrous cycle remain a
matter of controversy. The objective of the present study was to determine if sympathetic r
innervation of the rat uterus varies with the stages of the estrous cycle and within different
regions of the uterine horn. Uteri were taken from 16 two month old virgin Sprague-Dawley
rats after assessment of stages of the estrous cycle. Fresh frozen cryostat sections were cut from
ovarian, middle and cervical regions of uterine horns. Sympathetic innervation was evaluated
using immunostaining for dopamine D-hydroxylase (DBH), a highly efficient and selective
marker for noradrenergic sympathetic axons. To obtain information about target tissues being
innervated the same sections were stained also for smooth muscle myosin heavy chain, a smooth
muscle marker. Innervation density of coded specimens was estimated and blindly scored from
0 (no innervation) to 6 (robust innervation). 2-way ANOVA was used to determine statistical
significance. Sympathetic innervation was present within longitudinal and circular smooth
muscle bundles, major blood vessels between muscle layers and endometrial blood vessels. The
density of innervation varied with regions of uterine horn, being highest in the ovarian region
for all targets throughout the estrous cycle(P<0.001). Variations were found among stages of
the estrous cycle. The sympathetic innervation of uterine blood vessels was highest in diestrous,
diminished significantly in proestrous (P<0.05), and increased again during metestrous.
Adrenergic innervation oflongitudinal and circular smooth muscle followed the same pattern,
but density increased more gradually and remained lower in estrous than in diestrous (P<0.05).
Those observations indicate that there is a significant decrease in sympathetic innervation ofthe
uterus with transition from luteal to follicular stage (from diestrous to proestrous and estrous),
followed by gradual increase thereafter. The present findings are most consistent with neural
degeneration during proestrous followed by regeneration, with density achieving a maximum
in diestrous. We conclude that uterine sympathetic innervation of the cycling rat may provide
a useful model for studying mechanisms controlling neural degeneration and regeneration under
physiological conditions. Supported by NIH NS23502.

P8 EXPRESSION OF 160KD NEUROFILAMENT, GAP43 AND C-MYC IN THE OVINE
SUPERIOR CERVICAL GANGLION FOLLOWING DEAFFERENTATION
Bhagat and K.R. Lapwood, Massey University, Palmerston North, New Zealand

Changes in the expression of neurotropic factors/neurotransmitters in the superior cervical
ganglion (SCG) after surgical transection of afferent input or efferent outflow have previously
been used as indices of neuronal plasticity. In the sheep SCG, about which little has been
published, unilateral transection of preganglionic sympathetic fibers (decentralization) results
in an altered milieu of proteins and peptides. Of the three proteins examined immunocytochemi-
cally, the cytoskeletal 160kD neurofilament polypeptide and nerve growth associated protein
(GAP43) showed a marked elevation three days after surgery in the ipsilateral ganglion as
compared to its unoperated control counterpart. On the other hand, the intensity ofimmunostaining
for c-mye oncoprotein, a differentiation regulating transcription factor, was reduced. The 160
kD neurofilament polypeptide was localized in neuronal cell bodies and fibers: a majority of the
cells were intensely stained. Cells showed moderate levels of diffuse staining for GAP43 in
contrast to the intense punctate staining of neurites, which form a dense web around the cell
bodies. c-myc too, was localized in the cytoplasmic region of the cell bodies and in fibers, but
not all fibers were immunoreactive. With all the three antisera satellite cells, fibroblasts and
the cells associated with the vasculature showed no immunoreactivity. By 28 days after surgery,
the levels of 160 kD neurofilament and GAP43 had reduced, but the number of positive fibers
and the intensity of staining was still higher than the control. c-myc levels were similar to those
in the unoperated ganglion. The results clearly implicate a role of neuropeptides in the structural
and functional reorganization of neurons following interruption in their signalling pathways.

P9 A NEWLY SYNTHESIZED PYRIMIDINE COMPOUND, MS-818, ACTIVATED
MIGRATORY SCHWANN CELLS IN PERIPHERAL NERVE REGENERATION
K. Torigoe and A. Awaya*, Fukui Medical School, Fukui and *Institute of Biological Science,
Mitsui Pharmaceuticals, Inc., Tokyo, Japan

A newly synthesized pyrimidine compound, MS-818 (MPI & Nitsui Petrochemicals, Tokyo,
Japan), was administered intraperitoneally to adult male ddY strain mice (1mg/kg/day) after
axotomy of a common peroneal nerve, and the length of regenerating neurites was measured
by using a film model (Torigoe et al., 1993) in which the proximal stump of a transected nerve
was sandwiched between two thin plastic films. The individual mean length of the regener-
ating neurites in MS-818 group was significantly greater than that in controls at each day after
the operation. MS-818, therefore, contributed to a neural elongation. As calculated from the
mean lengths in MS-818 group, the velocity showed two phases: an initial slow phase up to
the 3rd post-operative day (day 3) followed by a faster phase. In the initial slow phase,
regenerating neurites consisted of naked axons, while in the second phase, Schwann cells
migrated from a proximal stump of a transected nerve and attached closely with regenerating
axons. It suggested that migratory Schwann cells played a role in promoting axonal
elongation. In addition, when a proximal stump was placed on the film containing many
migratory Schwann cells, the length of regenerating neurites in MS-818 groups was signifi-
cantly greater than that in controls. Migratory Schwann cells, therefore, might be activated by
MS-818 and stimulate axons so much as to extend for a longer distance. Furthermore, the
activity of migratory Schwann cells treated with MS-818 was gradually up-regulated, until it
reached at the same level as that of Schwann cells which migrated from a distal nerve stump,
meaning that migratory Schwann cells from a proximal stump can obtain the same potential
level as that from a distal stump by some treatments. In conclusion, MS-818 contributes to a
neural regeneration mainly as an activator of migratory Schwann cells.

P10 EFFECTS OF GLUCOSAMINE ON JS-l SCHWANN CELLS
L. Li, A.P. Mizisin and H.C. Poweu, Veterans Administration Research Service and Univer-
sity of California-San Diego, La Jolla, CA

For many years the polyol pathway has been implicated in the development of diabetic
complications in insulin-independent tissues such as nerve, eye and kidney. While the focus
of interest has been on the consequences of increased flux through the polyol pathway due to
hyperglycemia, less attention has been paid to potentially toxic products of polyol metabolism
such as amino sugars. This presentation is concerned with the effects of hexosamines,
potentially toxic polyol pathway byproducts, on JS-I cells derived from a rat Schwannoma
line. Cells were grown on DME media containing either 5mM glucose, 5mM glucosamine,
or 5mM galactosamine and were harvested 1, 3, 6, 12 and 24 hours after treatment. All cells
were prepared for light and electron microscopic examination (EM) and immunocytochem-
istry. Addition of 5mM glucosamine decreased cell numbers by more than half (3.02+or-0.39x
10 to the 5th versus 7.82+or-0.82)compared to controls at 24h. Cell viability,as determined by
trypan blue exclusion, decreased from 96-98% in cells grown in DMEM supplemented with 5mM glucose
of galactosamine compared to 69-75% in cells exposed to DMEM containing 5mM glu-
cosamine. DMEM supplemented with 0.05 or O.5mM g]ucosamine had no effect. Normal
cells showed bipolar profiles with prominent nuclei and many cell to cell contacts. In contrast
to the normal cell, glucosamine treated cells had a rounded profile, with vacuolated cytoplasm
and detached more readily. EM showed 'bleb' like cytoplasmic protrusions from the cell
surface appearing 12 and 24 h after exposure to 5mM glucosamine and immunostaining for
actin showed abnormal localization of this cytoskeletal protein. These findings may provide
some insight into mechanisms of polyol pathway mediated Schwann cell injury and demyeli-
nation occurring in human diabetic neuropathy. Supported by funds from the NIH, NS14162
and the VA Research Service.

P11 PMP-22 EXPRESSION DURING IN VITRO MYELINATION
L. Notterpek and E.M. Shooter, Stanford University School of Medicine, Stanford, CA

Peripheral myelin protein 22 (PMP-22) was initially described as a minor component of
peripheral myelin. Deletions, duplications and mutations in the PMP-22 gene are associated
with demyelinating neuropathies supporting a role for the protein in peripheral nervous system
(PNS) myelination. Furthermore, the protein carries the HNK-1/L2 carbohydrate epitope
suggesting an adhesion/recognition function. Despite advances in characterizing the PMP-22
gene, to date the specific role(s) of the protein in myelin remains unknown. The aim of our r
studies has been to determine the expression pattern and biochemical properties of PMP-22
during PNS myelinogenesis using a well established in vitro model system. Myelinating
cocultures of dorsal root ganglion (DRG) neurons and Schwann cells were established with
the addition of ascorbic acid (50 ~g/ml) and progesterone (20nM) to the growth medium.
Developmental expression patterns of PMP-22 were exarained by immunocytochemical and
biochemical methods and compared to that of known myelin markers, such as myelin
associated glycoprotein (MAG), protein zero (PO) and myelin basic protein (MBP). In purified
Schwann cell cultures, PMP-22 is expressed at low levels, the majority of which is localized
to the endoplasmic reticulum (ER) and Golgi compartments as indicated by double
immunolabeling. Upon extended (> 2 weeks) coculture of Schwann cells with DRG neurons
under myelinating conditions the protein is found in well-myelinated regions that are also PO
and MBP reactive. In comparison, PMP-22 does not colocalize with MAG to the nodes of
Ranvier or to the Schmidt-Lanterman clefts. Western analysis of parallel cultures also
indicates that PMP-22 expression pattern is similar to MBP, a late component of PNS
myelination. The amount of PMP-22 and MBP protein continue to increase in long term
myelinating cocultures. Based on endoglycosidase H (endo H) and N-glycosidase F (PNGase)
digestions, in vitro glycosylation pattern of PMP-22 in well-myelinated cocultures is similar
to that which has been observed in vive. The protein is endo H resistant and an 18kD form is
generated upon cleavage with PNGase. These results suggest that PMP-22 is a structural,
rather than an adhesion/recognition molecule in PNS myelination and support a role for the
protein in the maintenance of peripheral myelin.

P12 AXONAL REGENERATION TN DISSOCIATED NEURON CULTURES OF ADULT
HUMAN RETINA
S.U. Kim and H. Takahashi, University Hospital, University of British Columbia, Vancouver,
Canada

We have previously shown that surviving retinal neurons including retinal ganglion neurons
(RGN) were found in explant cultures of adult human retina after more than 2 months in vitro
(Kim and Takahashi, Invest. Ophthal., 29:, 1988). These results indicate that adult
human CNS neurons can survive in vitro when favorable conditions are provided. In orderto
establish a tissue culture model of human CNS axonal regeneration, we have isolated and
grown single cell cultures obtained from normal adult human retina. Retina tissues were
isolated from the eyes of 6 donors (donated for corneal transplantation, donor age range 63-
78 years), incubated in papain solution, and dissociated into single cells. Dissociated cells
were plated onto coverslips coated with several culture substrates including rat tail collagen,
gelatin, fibronectin, laminin, vitronectin, extracellular matrix (GIBCO), matrigel (Collabora-
tive Research), and poly-L-lysine. Dissociated cells did not attach very well on any of these
substrates. After repeated experiments with these substrates, we finally settled with pure
astrocyte cultures derived from fetal human brain. Most of the dissociated neurons isolated
from adult human retina attached to the underlying layer of astrocytes within 3-4 hrs and
started to regenerate their axons. Regenerating axons of adult human retina neurons could
readily be identified under phase contrast microscopy. Following immunolabeling with cell-
type specific markers such as neurofilament protein, MAP2, tyrosine hydroxylase, and
rhodopsin, interneurons, RGN, and photoreceptors were identified. Several large neurons
tentatively identified as RGN were found to regenerate their axons as long as 1-2 mm. These
results indicate that the dissociated neuron culture prepared from adult human retina could
serve as an experimental model to investigate the conditions favorable for axonal regeneration
in adult human CNS neurons. Supported by grants from the Medical Research Council of
Canada.

P13 THE NEURITE PROMOTING EFFECT OF LAMININ IS MEDIATED BY DIFFERENT
MECHANISMS IN EMBRYONIC AND ADULT REGENERATING MOUSE
OPTIC AXONS IN VITRO
C.A. Bates and R.L. Meyer, University of California, Irvine, CA

We have previously shown that both adult and embryonic (E15) retinal explants respond to
laminin with increased axonal outgrowth. In orderto further study the response of retinal axons
to laminin. we used antibodies to the cross arm region of the lalninin molecule (2ES) and to
the neurite promoting globular region at the base of the molecule (Clone III). We also used an
antibody which blocks the B1/B3 integrin subunit(GP140). Retinas were removed from adult
and embryonic day 15 (E15) mice, cut into 400 um squares and incubated at 37"C, 5% C02
in serum free media (DME-N2 or -N2.1) containing the 2E8, Clone III or GP140 antibody.
Numbers of neurites from El5 explants were determined 2 days post explant (DPE). Numbers
of neurites from adult explants were counted 2-5 DPE. Control explants were incubated in
serum free media alone. Clone III severely inhibited axonal outgrowth from E15 explants,
while adult explants were unaffected. Antibody 2E8 had no effect on outgrowth from E15 or
adult explants. GP140 blocked axonal outgrowth from E15 retinal explants, but had no effect
on adult explants. This suggests that the neurite promoting effect of laminin on adult retinal
axons is mediated through receptors other than the integrins. In addition, blocking the globular
domain ofthe 1aminin molecule, a fragment which promotes neurite outgrowth, has no effect
on outgrowth from adult explants, indicating that adult retinal axons interact with a different
portion of the laminin molecule. Supported by NS 26750 to RLM.

P14 DIFFERENTIAL REGULATION OF a-1 AND a-2 TUBULIN RNAs DURING GOLDFISH
OPTIC NERVE REGENERATION
M.B. Foreman, V.C. Hieber, N. Goburdhun and D. Goldman, Mental Health Research
Institute and University of Michigan, Ann Arbor, MI

The goal of this project is to identify cis-acting regulatory elements that mediate tubulin gene
induction during optic nerve regeneration in fish. RNAs for or tubulin increase in retinal
ganglion cells shortly after optic nerve crush. We have cloned the goldfish a-i and a-2 tubulin
genes. These genes are linked in the goldfish genome and encode a tubulins that differ by a
single amino acid. Using RNase protection assays and in situ hybridizations, we have
confirmed that foliowing optic nerve crush, a-i tubulin RNA increases while a-2 RNA
remains unchanged. We have isolated the a-1 gene's promoter region in order to identify cis-
acting regulatory elements that mediate its induction during optic nerve regeneration.
Expression studies in transfected cells and zebrafish have identified a functional promoter in
a piece of a-1 DNA extending 2 Kb upstream from the translational start site. Sequence
analysis indicates that this DNA contains CAAT and TATA boxes along with a number of
putative regulatory elements that are conserved between fish and mammals. Aided by grants
from the Paralyzed Veterans of America Spinal Cord Research Foundation and the Lucille P.
Markey Charitable Trust.

P15 REGULATION OF CYTOSKELETAL PROTEINS AMD THEIR mRNAs DURING
TRANSNEURONAL CELL DEATH IN THE AVIAN COCHLEAR NUCLEUS
M.S. Kelley, D.I. Lurie and E.W. Rubel, Virginia Merrill Bloedel Hearing Research Center,
University of Washington, Seattle WA

Afferent activity is important for the survival and maintenance of normal structural properties
of many neurons in the developing central nervous system. Following unilateral cochlea
removal (CR) in developing chickens, approximately 30 percent of the neurons in the avian
brain stem cochlear nucleus, nucleus magnocellularis (NM), do not survive, and the remaining
neurons atrophy. The intracellular mechanisms that lead to these transneuronal alterations are
not well understood. We have previously reported that a number of changes take place in NM
neurons within the first several hours following unilateral afferent deprivation, including
increases in calcium, decreases in overall transcription and protein synthesis, and decreases
in immunostaining forsome cytoskeletalproteins (Rubel et al.,J.Neurobiol., 21(1):169-196,
1990). The intracellular mechanisms linking these events with alterations of neuronal
structure are likely to involve changes in the cytoskeleton. To determine how afferenl
deprivation affects the neuronal cytoskeleton, the levels of several cytoskeletal proteins and
their mRNAs were evaluated in NM neurons following a unilateral CR. We have used
immunocytochemistry to examine levels oftubulin, actin, and microtubule-associated protein
2 (MAP2) within the first 6 hours following CR. Furthermore, we have used immunocy-
tochemistry and in situ hybridization to quantify levels of tubulin staining and mRNA
expression in NM neurons between 30 minutes and 5 weeks following CR. Immunocy-
tochemical results show that staining for tubulin, actin, and MAP2 decreases by 6 hours
following CR. Quantification of tubulin immunostaining indicates that levels decrease by
approximately 18 percent at 3 hours following CR and remain depressed for at least 2 days.
However, quantification of tubulin mRNA expression suggests that levels of the mRNA do not
decrease consistently until 6 hours following CR, and these decreases are not as substantial or
as prolonged as the decreases in immunoreactivity. Ultimately, the effects of afferent
deprivation on levels of tubulin mRNA may play a role in the failure of NM neurons to
maintain a normal cytoskeletal framework. However, the rapid changes that occur in tubulin
immunoreactivity do not appear to be a direct result of depletion of tubulin mRNA. Rather,
the finding that levels of tubulin immunoreactivity decrease rapidly following CR suggests
that afferent deprivation may cause a loss of cytoskeletal integrity through active degradation
of cytoskeletal proteins and/or overall decreases in protein synthesis. Supported by NIH grants
DC00520 and DC00018.

P16 Low LEVEL LASER IRRADIATION RESCUES AXOTOMIZED RETINAL GANGLION
CELLS IN ADULT HAMSTERS
M.C.P. Leung, K.-F. So and D. Tay, The University of Hong Kong, Hong Kong

The effect of low level laser irradiation on the degeneration of axotomized retinal ganglion
cells (RGCs) of adult hamsters was studied. S odi um pentobarbitone was used as the anesthesia
for all of the operations. Cell death was induced in the RGCs by transection of the optic nerve
at 2mm from the optic disc. The animals were then divided into two groups. The experimental
group received irradiation of Helium-neon laser (660 nm, 15 mW) through the eye for 7 or 14
consecutive days right after axotomy. The periods of daily irradiation were 1, 5 or 1 0 min. The
control group did not receive laser irradiation. All the animals were scarified at one or two
weeks post axotomy (wpa) and 3 days before killing the animals, granular blue (GB) was
applied to the proximal stump of the transected optic nerve to retrogradely label the surviving
RGCs. There were 99317+or-756 (mean +or- SD, n=5) RGCs in normal hamsters. In the control
animals, this number decreased to 11137+or-1407 (n=2) at 1 wpa and 1925+or-543 (n=2) at 2 wpa.
Following a daily laser treatment for 10 min, the number of surviving RGCs (1 wpa:
14160+or-3304 [n=3] and 2 wpa: 1330+or-339 [n=3]) were similar to that of the control group.
However, after 1 or 5 min laser treatment, the number of surviving RGCs was statistically (t-
test p<0.05) higher than that of the control group. The number of RGCs in the 5 min laser
treatment group was 90984+or-13458 (n=3) at 1 wpa and 11167+-2243 (n=4) at 2 wpa and the
number in the 1 min group was 22021+or-5019 (n=3) at 1 wpa and 3563+or-633 (n=3) at 2 wpa.
Thus, our results show that a I or 5 min daily irradiation oflower level laser through the eye
can rescue or at least delay the death of RGCs following axotomy.

P17 THE NEURONAL CELL-BODY RESPONSE FOLLOWING AXOTOMY iS RE-
LATED TO THE "STRESS RESPONSE" FOLLOWING UV-IRRADIATION
T. Buschmann, M. Zimmermann and T. Herdegen, II. Institute of Physiology, University of
Heidelberg, Heidelberg, Germany

Nerve fiber transection evokes the early and lasting expression of the c-Jun transcription factor
in those neurons which respond with the so-called axon or cell body response (CBR) to
axotomy. C-Jun acts as killer protein in perinatal neurons in vitro whereas its presence can be
related to neuronal survival in adult neurons in vive (Prog. Brain Research, 103:153-171). We
studied the hypothesis that the signal cascades and the changes at the transcriptional level are
related between the CBR and the cellular stress response. (1) Axotomy induces c-Jun and
represses the activating transcription factor 2 (ATF-2) in peripheral and central neurons, a
pattern also found following UV-irradiation of cultured adult DRG neurons. (2) Axotomy and
UV-irradiation do not increase phosphorylation of CREB indicating that PKA and CaMK
pathways are not involved. (3) The negative relation between c-Jun and ATF-2 expression
indicates a novel effect of c-Jun. The role of JNK/SAPK kinases, major mediators of the stress
response and activators of c-Jun and ATF-2, is under current research.

P18 THE EFFECT OF CONDITIONING HEAT SHOCK ON PROTEIN SYNTHESIS
AND HSP 70 SYNTHESIS IN CULTURED RAT CORTICAL NEURONS
R.N. Nishimura, S.-T. Fu and B.E. Dwyer*, VA Medical Center, Sepulveda, CA and *VA
Medical Center, White River Junction, VT

Heat shock proteins and specifically HSP 70 are thought to protect cells from immediate injury
and any subsequent injury. We have performed experiments using rat cortical neurons (E16)
which involve a conditioning mild heat shock of 43 degrees C for 15 minutes 24 and 6 hours prior to
a subsequent severe heat shock of 45 degrees C for 20 minutes (second heat shock). The conditioning
heat shack did not cause morphological changes or significant cell death. [35S]-methionine
labeling of cells after the second heat shock revealed a fall in protein synthesis but this was not
as severe as that noted in unconditioned heat treated cells. Western immunoblot analysis of
the conditioned neurons revealed an increased HSP 70 content above that noted by the
conditioning or second heat shock alone. These results are different than those found in
cultured rat cortical astrocytes where no increase in HSP 70 was noted with conditioning heat
shock. The effect of conditioning heat shock on the synthesis of HSP 32 or heme oxygenase-
1 is presently being examined. We conclude that conditioning heat shock can increase the
synthesis of HSP 70 and also partially protect neurons from inhibition of protein synthesis due
to heat stress. The role of HSP 70 in the induction of conditioning and neuronal protection is
still unknown.

P19 PROTECTION OF STRIATAL NEURONS AGAINST EXCITOTOXIC DEGENERATION
BY ANTIOXIDANT TREATMENT
N. Nakao, E.M. Grasbon -Frodl, 11. Widner and P. Erundin, University of Lund, Lund, Sweden

It has been proposed that oxidative stress and excitotoxicity cooperate to induce neuronal
degeneration. We have therefore investigated the protective effects of antioxidants against
excitotoxic injury on striatal neurons both in vitro and in vivo. In an in vivo study, we
determined whether two different types of antioxidants, the spin trapping agent, a-phenyl-
tertbutyl nitrone (PBN) and an inhibitor of lipid peroxidation, U-83836E, could protect
cultured striatal neurons against either hypoglycemic injury orN-methyl-D-aspartate-induced
excitotoxicity. We chose Dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-
32), which is enriched in medium-sized spiny neurons, as a marker for striatal neurons. PBN
and U-83836E both significantly reduced cell death induced by these insults as indicated by
an increased number of surviving DARPP-32-positive neurons. The two antioxidants also
protected neurons from progressive death in low cell density, serum-free cultures. In an in vivo
experiment, it was determined whether systemically administered PBN may exert
neuroprotective effects in the rat striatum following injection of the excitotoxic quinolinic
acid. Apomorphine-induced rotation tests revealed that PBN-treated animals were signifi-
cantly less asymmetric in their motor behavior than control rats. Treatment with PBN
significantly reduced the size of the quinolinic acid-induced striatal lesions, as assessed by the
degree of sparing of DARPP-32-positive and nicotinamide adenine dinucleotide phosphate-
diaphorasepositive neurons, and of microtubule-associated protein-2-immunoreactive areas.
Furthermore, morphological changes in the substantia nigra pars reticulata secondary to the
striatal lesion, i.e., loss of DARPP-32-positive afferent fibers and atrophic changes due to
transsynaptic degeneration, were also less extensive in the PBN-treated animals. The results
support the hypothesis that oxygen free radicals contribute to excitotoxic neuronal degenera-
tion. The in vivo neuroprotective effects of PBN against striatal excitotoxic lesions raise the
possibility that antioxidants could be used as neuroprotective agents in Huntington's disease,
which has been thought to involve excitotoxicity.

P20 INHIBITORS OF MONO-ADP-RIBOSYLATION PROTECT SPINAL DORSAL
HORN NEURONAL RESPONSE AGAINST OXIDATIVE STRESS
S.M. Douglas, K.L. Panizzon and R.A. Wallis, SepulvedaVAMC, Sepulveda, CA
and UCLA Medical Center, Los Angeles, CA

Injury to the spinal cord induces a series of biochemical events which appear to include
oxidative stress. Results from recent studies suggest that ADP-ribosylation plays a significant
role in the evolution of neuronal injury. We recently demonstrated that poly-ADP-ribosylation
inhibitors protect against oxidative injury in the adult rat spinal cord. Additionally, these
inhibitors protect against direct hydrogen peroxide injury in lymphocytes. Thus, to clarify the
role of ADP-ribosylation in oxidative injury to spinal neurons, we tested the effect of
novobiocin, a mono-ADP-ribosylation inhibitor, upon dorsal horn response in adult spinal
cord slices exposed to hydrogen peroxide-induced injury. For this study, spinal cord slices
were taken from the cervical-thoracic region and placed in a recording chamber perfused with
artificial cerebral spinal fluid. Dorsal horn responses were recorded with dorsal root
stimulation. Exposure to 50 uM hydrogen peroxide for 12 min produced major loss of dorsal
horn response, yielding a mean population spike amplitude recovery of 14%+or-5. Treatment
with 500 uM novobiocin, a mono-ADP-ribosylation inhibitor, provided robust protection,
yielding a mean recovery of 97%+or-3. Likewise, the mono-ADP-ribosylation inhibitor, meta-
iodobenzylguanidine (MIBG), also provided excellent protection against oxidative injury.
Treatment with 20 uM MIBG improved mean recovery of dorsal horn response to 91% +or-5.
These results suggest that oxidative injury to dorsal horn spinal neurons can be prevented by
inhibitors of mono-ADP-ribosylation and that mono-ADP-ribosylation may play an important
role in oxidative stress. They also demonstrate that the adult spinal cord slice preparation is
a useful model for the screening of potential neuroprotective agents in the treatment of spinal
cord injury. Supported by the VA Brain Trauma Fellowship and the VA Research Service.

P21 DIFFERENTIAL HEME OXYGENASE/NADPH DIAPIHORASE STAINING IN THE RAT CNS
B.E. Dwyer, R.N. Nishimura and S.-Y. Lu, VA Medical Centers, White River Junction, VT
and Sepulveda, CA.

Heme oxygenase (HO) catalyzes the rate-limiting step in the catabolism of heme to bilirubin.
Bilirubin has been suggested to be both neurotoxic as well as a potent antioxidant. Carbon
monoxide (GO), a putative neuroregulatory molecule believed to have properties similar to
nitric oxide (NO) is also released. We have begun to map the distribution of HO in rat CNS
as a first step in our efforts to define the importance of HO in CNS function. Little if any HO-
1 is found in normal brain tissue by western blot; HO-2 is abundant; HO-L theme oxygenase-
like immunoreactivity) which is detected by our anti-HO-1 antibody (Dwyer et al., Mol. Brain
Res., 30:3747,1995) was also abundant. HO-2 and HO-L (which is as yet unidentified)
strongly immunostained spinal cord neurons including anterior horn motorneurons. NADPH
diaphorase histochemistry used as a marker of nitric oxide synthase (NOS) activity stained far
fewer neurons. In cerebellum, Purkinje cells stain strongly for HO-2 and for HO-L, whereas
these cells stain negligibly for NADPH diaphorase. Conclusions: The differential distribution
of CO-producing heme oxygenase and NO-producing NOS raises the possibility that CO may
complement the function of NO in some regions of the CNS. Secondly, the presence of HO
in neurons raises the possibility that its activity could contribute to neuroprotection (HO-1 is
a heat shock protein) or exacerbate CNS injury. The importance of HO reaction products in
the context of normal and injured CNS function is unknown. However, recent experimental
results which show that HO inhibitors can be neuroprotective (Panizzon et al., 1995) highlight
the potential of HO activity for harm. Supported by the Research Service of the Department
of Veterans Affairs and by the United Cerebral Palsy Research Foundation.

P22 TWO ELECTROGENIC GLUTAMATE UPTAKE TRANSPORTERS WERE FOUND
IN SECONDARY CULTURES OF RAT ASTROCYTES EXPOSED TO INJURY
D.L.Eng, P.G.Lal and Y.L.Lee, VA Medical Center, Pale Alto, CA and Stanford University,
Stanford, CA

A variety of secondary cultured astrocytes were tested for the expression of 3 different
electrogenic glutamate uptake transporters. Astrocyte cultures were prepared from 1 day old
rat pups according to the methods of McCarthy and DeVellis and grown to confluence which
is specified as time Day O. The cultures were divided into 4 groups: Group A - untreated; Group
B-astrocytes injured using a scratch wound model; Group C-astrocytes treated with dbcAMP,
to induce morphological and cytoskeletal differentiation; Group D-astrocytes treated with
dbcAMP and scratch wound injury. Astrocytes from each group were harvested, and total
RNA was prepared from each group at the specified time points of Day O, Day 1, Day 2, Day
4, and Day 7, after confluence. The total mRNA was reverse transcribed using Molony
leukemia virus reverse transcriptase. Reverse transcriptase reaction was performed in
duplicate for each Group A-D at each sample time point. cDNA samples were tested for 2 glial
electrogenic glutamate uptake transporters GLT-1 and GLAST, and 1 neuronal form, EAAC-
1, using PCR. PCR primers were designed so that they would specifically amplify GLT-I,
GLAST, and EAAC-I. The 3 sequences were obtained from the genbank and aligned using
the program "pileup" and "Bestfit." The regions of the aligned sequences with no homology
to each other were chosen to design primers. The primers were designed using the program
"Prime" to yield ~ 1.5kB products. All these programs are offered online by the Genetics
computer group. The PCR reaction products were analyzed using electrophoresis on 1.5%
agarose gels. All samples from Groups A-D expressed mRNA for the 2 glial forms of the
transporters, while none of the cultures expressed the neuronal glutamate uptake transporter.
Of interest is the observation that all of these astrocyte cultures seemed to have expressed
GLAST in greater proportion than GLT-1, whereas in vivo the GLT-1 is predominantly found
in the astrocytes of the cortical hemispheres. By day 7, Group B, the scratch wound model,
displayed an increase in the expression of glutamate transporter mRNA compared to untreated
controls (Group A), however further quantitative analysis is being performed to confum this
finding. Supported by The Council for Tobacco Research and the Department of Veterans
Affairs.

P23 EFFECTS OF THE 21 AMINOSTEROID U-74389F ON GLIAL FIBRILLARY
ACIDIC PROTEIN-EXPRESSING ASTROCYTES IN THE SPINAL CORD OF
CONTROL AMD WOBBLER MICE
M.C. Gonzalez Deniselle, 8.1,. Gonzalez, G.G. Piroli, A.E. Lima and A.F. De Nicola,
University of Buenos Aires and Institute of Biology and Experimental Medicine - National
Research Council, Buenos Aires, Argentina

Wobbler (wr) mice suffer an autosomal recessive mutation producing severe motoneuron
degeneration and dense astrogliosis with increased levels of glial fibrillary acidic protein
(GFAP) in spinal cord and brain stem. They have been considered animal models of
amyotrophic lateral sclerosis and infantile spinal muscular atrophy. Using wr mice and normal
littermates, we investigated the effects of the membrane active steroid Lazaroid (LZD) U-
74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR).
Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial
in cases of CNS injury and ischemia. Four days after 50 mg pellet implantation of U-74389F
into wr mice, hyperplasia and hypertrophy of GFAP-expressing astrocytes was apparent in
spinal cord ventral (VH) and dorsal horn (DH), areas showing an already intense astrogliosis
in untreated wr mice (VH: wr: 264.66+or-122.34 cells/mm2 vs. wr+LZD: 437.52+or-60.05, p<0.01
and DH: 247.45+or-15.44 vs. 341.54+or-20.92, p<O.O1). In control mice, U-74389F also produced
astrocyte hyperplasia and hypertrophy in dorsal horn and hyperplasia in ventral-lateral
funiculi (VF-LF) of the cord (DH: control: 35.69+or-6.84 vs. control+LZD: 112.28+or-22.04,
p<0.05; VF-LF: 76.06+or-9.17 vs. 118.76+or-12.73, p<0.05). Given in vivo, U-74389F did not
change GR in spinal cord of wr or control mice, in line with the concept that it is active in
membranes but does not bind to intracellular GR (control: 43.6+or-17.0 fmol/mg prot,
control+LZD: 38.5+or-l1.0, wr: 41.6+or-4.4, wr+LZD: 48.7+or-9.7, p=NS). Besides, U-74390F did
not compete for (3H)-dexamethasone binding when added in vitro (ICSo dexamethasone: 1.4
x10 to the-8 M). In the last study, serum corticosterone levels in wr mice were identical to that of
control mice and did not change after treatment with LZD. The results suggest that stimulation
of proliferation and size of GFAP-expressing astrocytes by U74389F may be a novel
mechanism of action of this compound. The wr mouse may be a valuable animal model for
further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in
neurodegenerative diseases. Supported by NlH NS 20 866-08.

P24 OLIGODENDROCYTES DO NOT PROLIFERATE IN RESPONSE TO TRAUMATIC
INJURY IN VITRO
P.E. Knapp, University of Kentucky, Lexington, KY

When oligodendrocytes (OLs) are grown in the absence of neurons they are able to survive and
elaborate extensive, myelin-like membranes which contain myelin specific proteins and lipids.
Mature OLs both in the CNS and in vitro are commonly thought to be non-proliferative and non-
motile. However, our studies and those of others have shown that small numbers of mature OLs
can proliferate, and some in vivo findings suggest that division may be stimulated by trauma and
demyelination. In vitro, OLs also undergo a continual restructuring process. Cell processes are
constantly being withdrawn and lengthened, new processes are formed, existing processes
disappear, others are rerouted or changed in thickness. In some cases these dynamic changes
in morphology result in an actual movement of the cell body by 1OOum or more over the course
of several days. Although it is not known whether Ols in vivo undergo similar types of structural
plasticity, there is no a piori reason that they should not. In Fact, the ability of myelinating OLs
to reach out and ensheath new areas, or withdraw from others, might allow the cell to respond
to challenges such as growth, regeneration or axon loss during aging. Since remyelination is
necessary for functional recovery after CNS injury, we have been interested in how OLs respond
to such trauma and whether behaviors associated with remyelination (i.e., proliferation, process
outgrowth, motility) are changed after injury. We have developed an in vitro injury system
which mimics traumatic injury to the CNS. Mature OLs in purified cultures are injured using
a low strength laser beam to transect cell processes. Over 70% of the OLs injured in this manner
recover and survive for at least 1 week. They are photographed before injury and at 4, 24, 48
and 72 hrs afterwards. Transection injury did not prevent OLs from displaying the same types
of dynamic structural changes and movements seen in uninjured cells. Time-lapse photography
identified some injured OLs which divided during this period (<1%), but the rate of division was
not different from uninjured OLs. Addition of bFGF (20 ng/ml) at the time of injury or 24 hrs
before had no effect on the actual division of control or injured OLs, although uptake of BrdU
was unexpectedly increased. We conclude that a traumatic lesion by itself does not stimulate
or alter remyelinating behaviors in mature OLs. Although these results disagree with some in
vivo findings, the discrepancy is likely due to the fact that OLs in our studies are isolated from
possible influences of other cells in an injured area. Injury studies using OL-neuron co-cultures
and additional growth factors are in progress. Supported by NMSS RG2461A1/T.

P25 VASOACTIVE INTESTINAL PEPTIDE: MEDIATOR OF LAMININ SYNTHESIS
IN CULTURED SCHWANN CELLS
Q.-L. Zhang, P.-X. Lin, D. Shi*, H. Xian, J. Liu and H. deF. Webster, NINDS and *NIDDK,
National Institutes of Health, Bethesda MD

To learn more about neuropeptide-induced glial responses which accompany axon regenera-
tion, we studied effects of vasoactive intestinal peptide (VIP) on laminin production by
cultured Schwann cells. Schwann cells were isolated from sciatic nerves of neonatal mice,
purified and incubated for 5 d in either control medium (DMEM + 15% FCS) or control
medium containing 10 to the-7 -10-to the-11 M VIP. At 10 to the-7 and 10 to the-8 M VIP, laminin
levels measured by ELISA were significantly higher (55% and 35%) than those in control cultures.
Lower VIP concentrations (10 to the-9 -10 to the-11 M) produced smaller increases which were not
significant. Low affinity VIP receptors which mediated this effect were demonstrated on Schwann
cells by radioligand binding studies. The increased Schwann cell synthesis of laminin induced by
VIP was blocked when either a VIP antagonist or a VIP receptor antagonist was added to the
VIP containing incubation medium. In Schwann cells loaded with fura-2, VIP did not increase
cytosolic Ca2+, suggesting that VIP receptor activation in Schwann cells differs from that
observed in astrocytes and some other cell types. We suggest that the VIP-induced increase
in laminin synthesis which we have observed in cultured Schwann cells may also occur in vivo
and might be an important component of axon-Schwann cell interactions during nerve
regeneration.

P26 COMPARISON OF FUNCTIONS OF PERITONEAL MACROPHAGES AND
MICROGLIA INCUBATED WITH MYELIN IN VITRO
M.E. Smith, VA Medical Center, Pale Alto and Stanford University School of Medicine,
Stanford, CA

In experimental allergic encephalomyelitis and multiple sclerosis myelin is phagocytized by
microglia residing within the CNS, and by peripheral macrophages which migrate into the
CNS through the blood-brain barrier. These two kinds of cells are not easily distinguished
within the CNS because the markers are very similar. Both kinds of cells are upregulated
within the CNS by infiltrating activated T cells which release interferon-y (IFN-y), tumor
necrosis factor-a(TNF-a), and TNF-B. In addition to phagocytizing myelin, these macroph-
age-like cells also release proteases, reactive oxygen free-radicals, and nitric oxide, which may
predamage myelin before phagocytosis. In order to determine the effects of activating
cytokines on these cells and their functions, we preincubated macrophages and microglia with
various cytokines and activating factors, then fed myelin, and after 30 hours, measured their
proteolytic activity, oxidation of membrane lipids, carbonyl groups on membrane proteins,
and amount of myelin phagocytized. Factors used for preincubation included IFN-y, TNF-a,
granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage-colony stimulat-
ing factor(M-CSF), lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and forskolin.
Generally, much more proteolytic activity was released from peritoneal macrophages than
from microglia, while more lipid oxidation, as measured by thiobarbituric acid-reactive
substances (TBARS), was seen from microglia. Protein oxidation and phagocytosis were not
significantly different between the two kinds of cells. Although macrophages released more
proteolytic activity, they could be further stimulated by IFN-y, LPS, and PMA to secrete about
twice as much protease, while microglia showed little or no increase with these agents. Lipid
oxidation was increased in both kinds of cells by GM-CSF, and TNF-a, while LPS stimulated
only macrophages and PMA was effective only on microglia. Protein oxidation was increased
mostly in microglia by GM-CSF and PMA. None of the agents stimulated the macrophages
to phagocytize more myelin, but GM-CSF and PMA increased levels of phagocytosis by a
factor of four with microglia, and M-CSF doubled the rate. These results indicate that injurious
agents released by phagocytic cells and the degree of phagocytosis are regulated by cytokines
and protein kinase C activation. These cytokines ate those likely to be released by activated
T cells and astrocytes.

P27 SYNAPTOGENESIS BY CELLS DERIVED FROM THE HUMAN NT2 CELL LINE
M. Margulis*, R. Hartleyf, V.M.-Y. Leet. and C.-M. Tang*, *University of Maryland and
University of Pennsylvania

NT2 cells are believed to be immortalized equivalent of neural progenitor cells in the
developing human nervous system. Treatment with retinoic acid can induce NT2 cells to
commit to terminal differentiation into neurons(NT2N). While NT2N cells under current in
vitro culture conditions can express a sequence of neuronal markers that recapitulates the
maturational steps in the developing nervous system, they do not express some late events in
neuronal maturation (i.e., functional synapses). We examined the maturation of NT2N cells
under different culture conditions. When NT2N cells were co-cultured with astrocytes, patch
clamp recordings from them revealed synaptic activity that was not previously seen. These
synaptic currents exhibited the biophysical and pharmacological characteristics of glutamergic
synaptic transmission. Synaptic currents suggestive of GABAsubA-mediated synaptic signals
were also observed. These electrophysiological data were supported by positive immunohis-
tochemical staining for synaptophysin, a synaptic vesicle protein. When NT2N cells were co-
cultured with striate muscle they formed functional synapses that are reversibly blocked by
curare. These findings suggest that NT2N cells have the capacity to express functional
synapses and a variety of neurotransmitters .

P28 A NORMAL NEURAL PRECURSOR CELL LINE CAPABLE OF NEURONAL AND
GLIAL DIFFERENTIATION
R. Li and J.P. Mather, Genentech, Inc., South San Francisco, CA

Isolation and expansion in vitro of normal neuroepithelial cells capable ofneuronal differen-
tiation has long been an objective for neurobiologists. Such cells can provide new insight into
the development of the nervous system. Here we report a method ofisolating, and expanding
in vitro, a neuroepithelial cell type from E9 rat neuralplate. This has led to the establishment
of a neural epithelial precursor(NEP) cell line from normal E9 rat neural plate in a serum free
medium. These precursor cells were grown in serum free medium in coculture with a rat
embryonic Schwann cell line (ESC, see Li et al., abstract) or with ESC cell conditioned
medium. Continuous cultures could thereafter be maintained by passaging at a high plating
density without ESC cell conditioned medium. No apparent cell senescence was observed
throughout the culture period. The cell line expresses the neural precursor cell-specific
marker, nestin, an intermiate filament protein. Neural cell differentiation can be induced in
the NEP cells by exposure to basic fibroblast growth factor and forskolin. Specific neuronal
markers, Map-2, neurofilament proteins, neuron specific enolase and protein kinase C,
synaptophysin, Tau, tubulin-beta, choline acetyltransferase and the glial markers GFAP and
Galc were induced depending on the culture conditions. When injected into neonatal rat brain,
the NEP cell line gave rise to several distinct neuronal phenotypes.

P29 VARIABLE DIFFERENTIATION OF NEURONAL PRECURSORS AFTER TRANS-
PLANTATION INTO THE LESIONED SPINAL CORD
S.M. Onifer, V.P. Grover, A.B. Cannon, L.C. Than and S.R Whittemore, The Miami Project
to Cure Paralysis and University of Miami School of Medicine, Miami, FL

RN33B is a clonal neuronal cell line that was generated from dissociated, embryonic rat raphe
nuclei following infection with a retrovirus encoding the temperature-sensitive allele of SV40
large T-antigen (Brain Res., 615:27-40, 1993). After transplantation into the normal adult rat
spinal cord or hippocampal formation, RN33B cells differentiate with a morphology indistin-
guishable from that of endogenous neurons at the transplantation site (Exper. Neurol.,
122:130-142,1993). These results suggest that RN33B cell differentiation in the adult CNS
can be variably regulated by local epigenetic signals. The present study examines the
differentiation of RN33B cells after transplantation into the adult spinal cord under different
lesion conditions. Adult rats underwent transection at the T10 vertebral level or unilateral
lesion of interneurons and motor neurons at the T13-L1 vertebral level with 0.5 % kainic acid
InJectlon. Two weeks later, 2 X 10(to the 5th) E. coli lacZ-labeled RN33B cells (Cell Trans-
plantation, 2:131-149,1993) were transplanted into the grey matter of injured and naive rats
at the T13-LI vertebral level. At 2 weeks post-transplantation, B-galactosidase immunohistochemistry
revealed relatively undifferentiated RN33B cells throughout the grey matter of the kainic acid-
injured spinal cord. This observation contrasts with the finding of differentiated RN33B cells
in the grey matter of the transected or normal spinal cord. This indicates that either the signal(s)
which regulates RN33B cell differentiation is derived from interneurons, motorneurons, non-
neural cells, and/or is suppressed by molecules released by the injured CNS tissue. Supported
by The Miami Project to Cure Paralysis, General Reinsurance, and NS26887.

P30 CHARACTERIZATION OF A TRANSMITTER SYSTEM, THE 5-HT, 5-HT TRANS-
PORTER AND 5-HT1A RECEPTOR IN THE NON-PASSAGE EGF-RESPONSIVE
NEUROSPHERES
Y.H. Chiang and F.C. Zhou, Indiana University School of Medicine, Indianapolis, IN

Multipotent neuroepithelial stem cells, under the influence of epidermal growth factor (EGF),
can be procured from developing rodent brain (Reynolds et al., 1992) and human sympathetic
ganglia (Silani et al., 1994), from which neuronal and glial precursor cells derive. The aim of
this study is to examine the expression of a single neurotransmitter system, serotonin (5-HT)
as well as its transporter (5-HTT) and receptor (5-HT1A) on differentiated cells derived from
non-passage EGF-responsive neurospheres. Dissociated single-cells from cerebral cortex and
brainstem of gestational day 14 Sprague Dawley rats were obtained after enzymatic and
mechanical dissociation and cultured in DMEM/F12 medium with N2 and EGF (20 ng/ml).
No further passage of neurospheres was made for over 6 months. The 3- and 6-month-old
neurospheres were subplated on chamber slides with Neurobasal medium containing 2% fetal
calf serum for 14 days before being characterized. The expression of 5-HT, 5-HTT and 5-
HT1A receptor was examined by immunocytochemistry (with antibodies against 5-HT, 5-
HTT-N terminal fusion protein, and 5-HT1A170 peptide). The entity of proliferating
neuroepithelium was evidenced by antibodies against nestin and PCNA (proliferation cell
nuclear antigen). Glial cells and neurons were identified by immunocytochemistry with glial
fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP-2) antisera,
respectively. Immunoreactivity (IR) of 5-HT was observed on MAP-2-positive cells (with
neuronal morphology) derived from brainstem neuropsheres. 5-HTT-IR and 5-HT1A recep-
tor-IR were found on both neurons (MAP-2- positive) and glial cells (GFAP-positive) derived
from cortical and brainstem neuropsheres. Current results indicate that (a) the population of
cells derived from 3- and 6-month old non-passage neurospheres contain nestin-positive and
PCNA-positive neuroepithelial cells, and (b) the 5-HT related systems, 5-HT, 5-HTT and 5-
HT1A receptor exist among these cells.

P31 ADENO-ASSOCIATED VIRUS VECTORS FACILITATE NEURON-SPECIFIC
EXPRESSION OF GREEN FLUORESCENT PROTEIN IN THE ADULT MAMMA-
LIAN SPINAL CORD
A.L. Peel, S. Zolotukhin, G. Schrimsher, N. Muzyczka and P.J. Reier, University ofmorida,
Gainesville, FL

The central nervous system presents unique challenges in the development of gene delivery
systems. Adeno-associated virus (AAV) vectors are able to overcome many of these
challenges by their ability to deliver foreign genes to post-mitotic neurons without subsequent
pathology. A recent study by Kaplitt et al. demonstrated the potential for these vectors to
facilitate long-term expression of both beta-galactosidase and tyrosine hydroxylase in the
forebrains of adult rats. To assess the usefulness of this system as a model for spinal cord gene
therapy. the gene for green fluorescent protein (GFP) was selected as an unambiguous
demonstration of foreign gene expression. A "humanized" form of this gene, in which the
codons were altered to more closely resemble mammalian codons, was placed in two separate
vectors, either under the control of the CMV promoter or the neuron-specific enolase (NSE)
promoter. These vectors were packaged and injected into the mid-cervical to mid-thoracic
regions of adult rat spinal cords. At ten to fourteen days expression was detected in all animals.
This expression was easily distinguished from any autofluorescence using Zeiss fluorescein
optics, and no crossover was detected when visualized with rhodamine filters. The CMV
construct was preferentially expressed in cells with neuronal morphology and long processes,
although expression was also noted in a few epithelial cells. Expression from the NSE
promoter was observed in neurons only, with many more cells expressing the protein. This
study lays groundwork for the delivery of more functional genes to spinal cord neurons to
participate in either spinal cord repair following injury or in the correction of genetic defects
affecting the spinal cord.

P32 PROPERTIES OF RAT SPINAL NEUROEPITHELLAL CELLS: IN VIVO AND IN
VITRO STUDIES
P.G. Bannerman, T.M. Oliver, Z. Xu, A. Shieh and D.E. Pleasure, Abramson Pediatric
Research Center, Children's Hospital of Philadelphia, Philadelphia, PA

In the developing spinal cord, neuroepithelial cells are a transient population of cells that give
rise to neuronal and glial cell progenitors. In the embryonic rat spinal neural tube we report
the expression of GD3 immunoreactivity by radially oriented cells morphologically identifi-
able as neuroepithelial cells. The expression of GD3 immunolabeling proceeds in both a
ventral/dorsal and rostral/caudal gradient during embryonic days 12 and 13 (E12 and E13),
then disappears in a ventra/dorsal and rostal/caudal progression between E15 and E16. To
date, little is known concerning the effects of soluble growth factors on the survival,
proliferation or motility of spinal neuroepithelial cells. We have studied the effects of three
members of the FGF family of protein growth factors, namely FGF-1, FGF-2 and FGF-7 on
the outgrowth of neuroepithelial cells from the ventral aspect of explanted spinal neural tubes
using GD3 expression as a marker for neuroepithelial cells. This culture system also provides
a source of neural crest cells that derive from the dorsal surface of the explants and which
express strong Immunoreactivity for low affinity nerve growth factor receptor (p75-LNGFER),
the majority of which do not express the GD3 ganglioside. Thus, we have been able to compare
and contrast the effects of these growth factors on the development of cells present in either
the neuroepithelial or neural crest outgrowth zones. We have previously shown that FGF-1
and FGF-2 increase the survival but not the migration or mitotic index of cultured neural crest
cells. In this paper we provide evidence that FGF- I and FGF-2 increase the rate of migration
and mitotic index but not the survival afthe neuroepithelial cells, thereby contrasting starkly
with their effects on neural crest cells. FGF-7 did not mimic any of the above effects of FGF-
1 or FGF-2. Finally, we show for the first time that a subpopulation of mammalian
neuroepithelial cells is dye coupled, implicating a role for connexins in relaying developmen-
tal signals within the differentiating spinal neural tube of higher vertebrates.

P33 AGE DEPENDENT MOTOR NEURONAL SURVIVAL OBSERVED AFTER FACIAL
NERVE CRUSH IS ALTERED BY INTERACTIONS WITH GROWTH FACTORS
K. Kuzis and F.P. Eckenstein, Oregon Health Sciences University, Portland, OR

As early as 1840 it was noted that more neurons survive a peripheral nerve injury when the inj ury
occurs late in life rather than in the first two postnatal weeks. This age dependent survival has
never been explained. The suggestion that growth factors may contribute to the survival seen
in lesioned motor neuron systems comes from the observation that putative lesion factors, such
as Ciliary Neurotrophic Factor (CNTF), Acidic Fibroblast Growth Factor (aFGF), and Basic
Fibroblast Growth Factor (bFGF) are expressed at levels which appear to also be age related.
Here we present a statistical examination ofthe neuronal survival in animals whose facial nerve
was crushed at various postnatal ages (PO, 2, 4, 7, 14) and examined at various times following
the injury (0, 3, 7, 14 days). This examination reveals that the onset of neuronal death is
consistent and independent of age at the time of injury, while the extent of cell loss following
these facial nerve crushes are, as historically observed, age dependent. In addition we use
several methods to examine the ability of putative lesion factors to influence neuronal survival
following injury.

P34 MOLECULAR CHARACTERIZATION OF FIBROBLAST GROWTH FACTOR-1 AS
A NEUROTROPHIC FACTOR FOR LIMB REGENERATION IN ADULT NEWTS
Y. Wei, J.D. Nace, M.J. Botelho, I.-M. Chiu and R.A. Tassava, The Ohio State University,
Columbus, OH

A feature of newt limb regeneration is the formation of a blastema, a mass of undifferentiated
cells covered by a wound epithelium, at the end of the amputated stump. Blastema formation
requires a nerve supply, which can be fulfilled by either spinal motor or sensory nerves, provided
that the density of the nerve fibers at the amputation level is above a certain threshold. Since
blastema cell proliferation ceases upon limb denervation, it has been proposed that nerves exert
a mitogenic effect on the amputated limb by releasing neurotrophic factors (NTFs) into the
blastema milieu. Previous studies suggest that fibroblast growth factors (FGFs) are strong
candidates as NTFs, because (1) infusion of FGFs promotes the resumption of mitotic activity
in denervated newt regenerates, (2) FGF-1 peptide is present in the blastema, and (3) FGF
receptors are expressed by blastema cells. To investigate the possibility of nerves as a major
source of blastema FGF-1, a PstI/Bg/II fragment of a newt-specific FGF-1 cDNA was
subclonc=d into the pBluescript SK+ vector and used as a template to generate an antisense
riboprobe by in vilro transcription. In situ hybridizations were then carried out using this
riboprobe to examine the expression of FGF-1 by regeneration-related cell populations in adult
newts after amputation at the fibula/tibia level of the hindlimb. Results showed that the stump
tissue cells, blastema cells and wound epithelial cells of the regenerating limbs had no FGF-I
specific transcripts even 2 weeks after amputation. Similarly, the Schwann cells that myelinate
the peripheral nerves innervating the regenerating hindlimbs were inactive in transcribing FGF-
1 mRNA. In contrast, the hindlimb-associated spinal cord segments and corresponding dorsal
root ganglia demonstrated strong riboprobe signals. In the spinal cord the signal was found
mainly in the peripheral area of the gray matter, including the region where the cell bodies of
the spinal motor neurons are located. Whereas, in the dorsal root ganglia the riboprobe signal
was associated with almost all of the cell bodies of the sensory neurons. Thus FGF-1 is
synthesized by the spinal sensory and motor neurons but not by other regeneration-related cell
populations. It is therefore possible that FGF-I peptide is released from nerve terminals into
the blastema, binds to its specific receptors and stimulates blastema cell proliferation, thus
mediating nerve-dependent blastema cell proliferation.

P35 SIGNAL TRANSDUCTION MECHANISMS FOR bFGF AND EXTRACELLULAR
ATP IN ASTROCYTES
J.T. Neary, P.K. Dash* and Q. Zhu, *University of Houston, Houston, TX, and VA Medical
Center and University of Miami School of Medicine, Miami, FL

Extracellular ATP has mitogenic and morphogenic activity on astrocytes and can also act
synergistically with bFGF (Neary et al., J. Neurochem., 63:490-94, 1994). To determine the
signaling pathways underlying the trophic effects of ATP and bFGF, we have investigated the
ability of these agents to stimulate mitogen-activated protein kinase (MAPK), an enzyme
involved in cellular growth, and AP-1, a functional transcription complex implicated in the
expression of a diverse array of genes and which may be activated in part by MAPK. We found
that extracellular ATP and bFGF activate MAPK to a similar extent (3- to 4-fold) in cultured
astrocytes obtained from neonatal rat cerebral cortices. Interestingly, these agents activate
MAPK by distinct pathways because activation of protein kinase A markedly reduced the ability
of bFGF to stimulate MAPK but had no significant effect on the stimulation of MAPK by
extracellular ATP. This indicates that the bFGF/MAPK pathway is mediated by Raf-l which
is blocked by protein kinase A and suggests a novel pathway for signaling from ATP/P2
purinoceptors to MAPK. Similarly, different mechanisms were found for AP-1 activation by
ATP and bFGF. Experiments with cycloheximide indicated that bFGF-induced AP-I binding
is mainly dependent on protein synthesis, whereas the ATP-evoked AP-1 binding is mediated
by both protein synthesis-dependent and -independent pathways. This suggests that part of the
ATP-evoked increase in AP-1 complex formation is due to a post-translational mechanism such
as protein phosphorylation, perhaps as catalyzed by MAPK or related protein kinases. The
utilization of distinct mechanisms for MAPK and AP-1 activation may underlie the synergistic
interactions between ATP and bFGF. Because ATP is released following tissue injury, the
growth-related actions of ATP, alone or in concert with polypeptide growth factors such as
bFGF, may have important consequences in brain trauma and regeneration. This work was
supported by the Department of Veterans Affairs.

P36 SERUM FREE CULTURE OF RAT AND HUMAN SCHWANN CELLS:
IDENTIFICATION OF GAS6 AS A GROWTH FACTOR FOR SCHWANN CELLS
R. Li, J. Chen,G. Hammonds, H. Phillips, M. Armanini, P. Wood, R. Bunge, P. Godowski,M.X.
Sliwkowski and J.P. Mather, Genentech Inc., South San Francisco, CA and The Miami Project
to Cure Paralysis, Miami, FL

Schwann cells (SC) are one of the principal components of the peripheral nervous system. They
play a crucial role in nerve regeneration and can be used clinically in the repair of injured nerves.
One approach to obtaining normal functionally differentiated cell lines from celt types not easily
grown in culture has been to start primary cultures in serum-free medium containing factors and
attachment proteins specifically designed to favor the replication of only the cell type of interest.
We have established serum-free, defined culture conditions which rapidly expand primary
cultures of rat or human Schwann cells in vitro. By culturing SC on laminin, in serum-free
medium with hormones and growth factors, we repeatedly generate homogenous SC cultures
which yield normal SC lines from the dorsal root ganglia (DRG) of both embryonic and adult
rats. These cells maintain the phenotype of SC. With minor modifications, this approach can
be used to culture adult human SC without fibroblast growth. We find that Gas6, a ligand for
the Axl and Rse/Tyro 3 receptor protein tyrosine kinase family, stimulates human SC growth,
increasing both cell number and thymidine incorporation. Gas6 has synergistic effects with the
other known human SC mitogens, heregulin/GGF and forskolin. Addition of Gas6 causes
phosphorylation of Axl and Rse/Tyro 3 simultaneously and results in ERK-2 activation. A
combination of Cas6 with heregulin and forskolin, on a defined background, supports maxinal
SC proliferation, while preserving the typical SC morphology and expression of the SC markers
S-100, GFAP and NGF receptor. Gas6 mRNA is present in both spinal motor neurons and large
neurons of the dorsal root ganglia, while neural injury has been reported to upregulate Rse/Axl
in the SC. This is the first demonstration of a potentially important biological role forthe human
Gas6/Rse, Axl system.

P37 DETAILED STRUCTURE FUNCTION ANALYSIS OF TKE CARBOXYL TERMINUS
OF NERVE GROWTH FACTOR
A.Kruttgen, J. Heymach, P. Kahle and E.M. Shooter, Stanford University Medical School,
Stanford, CA

Nerve growth factor (NGF) is a member of the neurotrophin family and plays an important role
in the development and maintenance of the peripheral nervous system as well as of the central
nervous system. Previous work in our laboratory has shown that the C-terminus of NGF seems
to be important for its function, because deletion of residues 112-120 (C-9 aa) abolished its
bioactivity. In the present study we performed a detailed structure function analysis of this
region by deleting stepwise the nine C-terminal residues. In addition we constructed cDNAs
for the double-point-mutants, ArgLys114 115 to SerSer and ArgArgl18 119 to SerSer, as well
as a C-terminal myc-tagged form. The mutants were expressed by transient transfection of
COS-7 cells and quantification was carried out by ELISA and Western analysis of the
conditioned media. The mutants were subjected to two different bioassays: Trk-tyrosine-
autophosphorylation and neurite outgrowth in PC12 cells. Our data indicate that deletion of
residues 116-120 (C-5 aa) does not affect Trk-phosphorylation. Concerning neurite outgrowth,
deletion of residues 115-120 (C-6 aa) reduced the bioactivity to 25%, deletion of 114-120 (C-
7aa)showed only 12.5% activity while mutant 113-120(C-8aa) was completely inactive. Our
results define the residues of the NGF C-terminus that are important for bioactivity . On the
other hand, the NGF with a myc-tag on its C-terminus showed normal bioactivity in the neurite
outgrowth assay, so that it seems to be possible to attach other proteins to this neurotrophin
without affecting its bioactivity. This knowledge may be used for designing neurotrophins with
therapeutically useful properties, such as the ability to cross the blood-brain barrier.

P38 NGF, BDNF, AND NT-3 UNDERGO REGULATED SECRETION IN ATT-20 AND
PC12 NEUROENDOCRINE CELLS
J.V. Heymach, Jr., A. Kruttgen and E.M. Shooter, Stanford University Medical School,
Stanford, CA

The neurotrophins nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), and
neurotrophin-3 (NT-3) are known to be involved in a number of activity dependent processes
within the mammalian nervous system, although precise mechanisms by which they exert their
effects are poorly understood. To examine the possibility that the release of these neurotrophins
may be controlled by activity, we have investigated their secretion by AtT-20 and PC12 cells,
cell lines containing regulated secretory pathways which appear to be similar to those of
neurons. In AtT-20 cells which were stably or transiently transfected with expression plasmids
for NGF, BDNF, or NT-3, there was a 3-6 fold increase in the neurotrophin present within
conditioned media (as detected by two-site ELISA) when the cells were stimulated with 5 mM
8-BrcAMP, a known secretagogue, suggesting that the neurotrophins were being released by the
regulated secretory pathway. Furthermore, in both transfected AtT-20 and PC12 cells,
neurotrophin secretion was stimulated in less than 10 minutes by depolarization with 50 mM
KCI. The depolarization- or 8-BrcAMP-induced increases in neurotrophin release were not
abolished by a 3 hour pretreatment with cycloheximide, demonstrating that new protein
synthesis was not responsible for the increased neurotrophin release. Furthermore, treatments
which lead to an increase in neurotrophin release were accompanied by a decrease in
intracellular neurotrophin content. The regulated secretion of NGF was not affected by
deletions within the propeptide nor by the introduction of an epitope tag to the carboxy terminus
of the neurotrophin moiety. Our data indicates that the neurotrophins NGF, BDNF, and NT-3
undergo rapid regulated release in the model cell lines studied and suggests that they may be
secreted in a similar manner by neurons.

P39 ADHESIVE FUNCTION OF TRK-EXPRESSING FIBROBLASTS
H. Zhou, G.D. Yancopoulos* andE.M. Shooter, Stanford University Medical School, Stanford,
CA and Regeneron Pharmaceuticals, Inc., Tarrytown, NY

Neurotrophin receptors trkA, trkB and trkC belong to a family of transmembrane tyrosine
kinases and play crucial roles during development and maintenance of neural tissues. Upon
binding to their cognate ligands, they dimerize and become autophosphorylated, initiating a
well defined signal transduction pathway. The extracellular domain of trks consists of a leucine-
rich motif(LRM cassette) followed by two immunoglobulin-like (Ig-like) domains. Both the
LRM cassette and Ig-like domains have been shown to be involved in cell-cell recognition and
communication. Most recently, splice variants of trkB or trkC, lacking the tyrosine kinase
domain, have been isolated from neural tissues. Thus, the presence of these adhesive motifs and
the kinase-deficient trkB or trkC isoforms suggests that trks may also be involved in cell
adhesion in addition to the well established role in promoting cell survival and differentiation.
To investigate the potential adhesive function of trks, full length trkA, B, C cDNAs were stably
transfected into fibroblasts (NIH 3T3 or MG87 cells) and trk-expressing clones were isolated
and characterized by immunoblot analysis using pan-trk antibodies. Transfectant clones
producing trkA, trkB or trkC were subjected to a homotypic aggregation assay. Results showed
that parental cells were non-adhesive during the assay period of 1 h and trk-producing cells
displayed varying degrees of aggregation. Furthermore, the adhesion observed by these cells
was Ca2+-, Mg2+- and temperatute-dependent, characteristics shared by the cadherin family of
adhesion molecules. Experiments are in progress to further characterize the adhesive specificity
of these uk-producing cells.

P40 INFLUENCE OF GROWTH AM) TROPHIC FACTORS ON REGENERATION BY
CHRONICALLY INJURED NEURONS -- EFFECTS OF IMMEDIATE OR DE-
LAYED TREATMENT
J.D. Houle and J.-H. Ye, University of Arkansas for Medical Sciences, Little Rock, AR

The potential to promote axonal regeneration of supraspinal neurons by treatment with growth
or trophic factors after a longstanding spinal cord injury was studied. Supraspinal neurons were
retrogradely labeled with True Blue (TB) at the time of a C3 hemisection lesion of the adult rat
spinalcord. In one study, gel foam soaked with a specific factor was placed into the lesion cavity
immediately after injury and then replaced with fresh factor 3 days later. In a second study,
treatment was delayed until 28 days after injury, with replacement with fresh factor 3 days later.
In each experiment, an autologous peripheral nerve graft (PNG) was apposed to the rostral
surface of the cavity on day 35 after the hemisection lesion. The distal end of the PNG was
exposed to Nuclear Yellow (NY) 4 weeks after grafting to retrogradely label neurons with an
axon in the PNG, with animal sacrifice 2 days later. Chronically injured neurons capable of
regeneration were identified in vibratome sections by their content of TB and NY. For each
experiment, control animals received saline-treated gel foam and experimental animals re-
ceived gel foam treated with either a combination of brain derived neurotrophic factor and
neurotrophic factor 3 (BDNF+NT-3), ciliary neurotrophic factor (CNTF) or insulin-like growth
factor 1 (IGF-1). A significant increase in the total number of chronically injured, regenerating
neurons was observed following treatment with IGF-1 or CNTF in either an immediate or
delayed exposure, although, differences in the response of specific neuron types were found. As
examples, enhanced regeneration by reticular formation neurons occurred after all IGF-1 or
CNTF treatments, yet, vestibular neurons responded significantly only to a delayed exposure
to CNTF. Significant increase in the number of regenerating locus coeruleus or rubrospinal
neurons occurred only with IGF-1 treatment (immediate or delayed). Exposure to BDNF/NT-
3 immediately after injury resulted in a significant increase in regeneration by reticular
formation neurons, but not in any other neuronal group. No significant increases were found
with delayed treatment with BDNF/NT-3. These results demonstrate the potential to increase
the regenerative response of specific chronically injured supraspinal neurons with specific
growth or uophic factors. Variable responses after immediate or delayed treatment suggest
possibly different mechanisms of action, i.e., primarily as an injury survival factor or as an
axonal growth promoting factor. BDNF, NT-3 and CNTF were supplied by Regeneron
Pharmaceuticals, Inc. Supported by NIH Grant NS 26380.

P41 CNTF AND BDNF DIFFERENTIALLY DIRECT THE TERMINAL NEUROTRANS-
MITTER PHENOTYPE OF RAPHE NEURONS
M.J. Eaten, J.S. Rudge* and S.R. Whittemore, The Miami Project to Cure Paralysis, University
of Miami School of Medicine, Miami, FL and *Regeneron Pharmaceuticals, Tarrytown, NY

We have utilized RN46A cells, an immortalized serotonergic neuronal cell line derived from
E13 raphe (J. Neurosci., 14:6744-53, 1994), as a model for the maturation of embryonic raphe
neurons. RN46A cells express the serotonergic phenotype when treated with various factors,
including brain-derived neurouophic factor (BDNF) and ACTHsub4-10. Treatment with BDNF,
combined with partial membrane depolarization initiates serotonin (5HT) synthesis in RN46A
cells (Dev. Biol., 170:169-82, 1995) by increasing the expression of tryptophan hydroxylase
(TPH) and subsequent activation of TPH by opening voltage-gated Ca2+channels. An increase
in 5HT re-uptake during differentiation requires treatment of RN46A cells with ACTHsub4-10 or
S100B. Ciliary neurotrophic factor (CNTF) is able to block the effects of BDNF on TPH and
5HT synthesis in RN46A cells. This decrease in 5HT synthesis after CNTF treatment is also
seen in primary raphe cuItures, an effect not related to a loss of serotonergic neurons. In
addition, CNTF decreases 5HT re-uptake 50%. Most importantly, CNTF terminally switches these
neurons to a cholinergic phenotype by increasing choline uptake 5-fold, virtually eliminating
5HT expression while increasing ChAT activity 300-fold. Of the other members of CNTF-
cytokine family, only LIF and 1L6 have a similar effect on ChAT activity, oncostatin M has no
effect. Collectively, these data demonstrate distinct effects of BDM; and CNTF on the
phenotype of raphe neurons. As both trkB and CNTFRcr are present in the developing raphe
nucleus, these data suggest that potentially novel mechanisms regulate raphe development. This
work was supported by The Miami Project to Cure Paralysis, General Reinsurance, NS26887,
and SCRF1524-01.

P42 EXPRESSION OF IMMEDIATE EARLY GENE C-JUN IN CNS OF ADULT RATS
AFTER SPINAL CORD INJURY
E.Yakovchenko, M. McAtee and B. Bregman, Georgetown University, Washington, DC

Protein product of the immediate early gene c-jun is highly expressed in peripherally projecting
neurons during regeneration but is not expressed in central neurons which fail to regenerate. We
expect that c-jun plays an important role in induction of genes specific for regrowth of mature
CNS neurons after injury. Transplants of fetal spinal cord tissue placed into the site of a spinal
cord lesion in the adult rat elicits growth of host axons. This axonal growth is further increased
by the application of exogenous neurotrophic factors at the site of injury/transplantation. The
aim of this study was to determine if interventions which increase the amount of axonal growth
after injury are accompanied by an upregulation of c-jun expression and by an alteration in the
neuronal atrophy and/or cell loss of the axotomized neurons. Immunohistochemistry (IHC) and
quantitative image analysis (QIA) were used. The following experimental interventions were
used: spinal cord hemisection only (cervical or midthoracic); fetal spinal cord transplant at the
site of hemisection; immediate local application of neurotrophins (BDNF or NT-3) at the site
of hemisection with or without transplant. In a subset of the animals injured neurons were
labeled with FluroGold. Tissue was analyzed at 7 and 30 days after injury. By 7 days after lesion
IHC and QIA showed increased c-jun expression in axotomized red nucleus and locus coeruleus
of animals from all experimental groups compared to normal and hemisected only rats. Fetal
spinal cord transplants, exogenous neurotrophic factors (BDM; and NT3) and the combination
of both significantly affected axotomized neurons, inducing high expression of c-jun protein.
Cell number and cell size distribution among immunostained cells was obtained by QIA. We
also compared c-jun expression in dorsal root ganglion neurons after central or peripheral
axotomy. After central axotomy, the root was placed between two pieces of fetal spinal cord
transplant at the site of hemisection. The ratio of labeled to unlabeled dorsal root ganglion cells
was calculated; there were more c-jun labeled cells in the animals with transplants compared
to axotomy alone animals. Small and medium diameter DRG neurons were preferentially
affected. Our results suggest that expression of the c-jun immediate early gene in the nature
CNS neurons is elicited by interventions which also increase the extent of axonal growth after
injury, and that these interventions also preserve a number of other aspects of the morphology
of the axotomized neurons. Supported by NIH NS 19259; neurotrophins generously provided
by Regeneron.

P43 CELLULAR RESPONSES TO SPINAL CORD INJURY IN MICE THAT DO NOT
EXHIBIT PROGRESSIVE NECROSIS AND CAVITATION
M. Fujiki*, Z. ZhangS, L. Guth and 0. Steward*, *University of Virginia Health Sciences
Center, Charlottesville, VA and College of William and Mary, Williamsburg, VA

Spinal cord injury in most mammals leads to progressive necrosis and cavitation producing a
tissue environment in which degenerative events predominate and growth and repair processes
are minimally expressed. We have recently found, however, that the histopathological
sequellae following crush injury is vastly different in at least one strain of mouse (C57B16J) in
that progressive necrosis and cavitation does not occur. Instead, the lesion site is completely
filled in by a connective tissue scar in a type of wound healing response. The present study
characterizes the post-injury responses of macrophages and astrocytes in mice in order to try to
identify the differences that account for the unique wound healing response. Crush injuries were
produced at the T8 level using an extradural approach and animals were allowed to survive for
2 days-12 weeks post-injury. Reactive changes in macrophages/microglia were evaluated
immunocytochemically using the MAC1 antibody; reactive changes in astrocytes were evalu-
ated using GFAP immunohistochemistry. MAC1 positive macrophages accumulated within
and around the crush site beginning within days after the injury. There were also increases in
MAC1 immunostaining within the spinal cord parenchyma adjacent to the crush and within the
dorsal columns. A connective tissue matrix developed at the site of the injury surrounding the
clusters of macrophagcs. Similar connective tissue matrices also developed around clusters of
macrophages within the dorsal columns. The areas containing connective tissue and clusters
of macrophages were entirely unstained by GFAP. These results suggest the development of
discrete tissue environments within the injured murine spinal cord, one containing collections
of macrophages and fibrous connective tissue (a non-CNS environment) and the other
containing different types of macrophages/microglia together with reactive astrocytes (a CNS
environment). Supported by NS 32280 to O.S.

P44 QUANTITATIVE AND QUALITATIVE ANALYSIS OF INTRAPARENCHYMAL
INFLAMMATION IN A RAT SPINAL CONTUSION INJURY MODEL
P.G. Popovich and B.T. Stokes, The Ohio State University College of Medicine, Columbus, OH

The inflammatory response in traumatized spinal cord has been associated with both the
promotion of neural regeneration and secondary tissue injury. The cellular mediators of these
seemingly antagonistic processes are nor wellcharacterized. The present data describe the time
course, distribution and magni~ude of the cellular immune response following contusion injury
to the rat thoracic spinal cord using immunohistochemical and image analysis techniques.
Macrophage (M~) and microglial reactions follow a progressive time course peaking at day 7
post-lnJury (p.i.) at the lesion epicenter and become differentially distributed in both gray and
white matter over a period of 28 days post-injury. Chronic activation of white matter microglia
is associated with Wallerian degeneration in the dorsal funiculus and lateral/ventral white
matter. Blood-spinal cord barrier (BSB) integrity is also compromised in these white matter
regions. Whether or not macrophage activation promotes tissue injury or regeneration is not
clear. However, preliminary observations in macrophage-depleted animals, suggests a sparing
of white matter tracts at the lesion epicenter. T-lymphocytes infiltrate the epicenter in parallel
with peak microglial/M~ activation (7 days p.i.). The proximity of T-cells and MHC class II
(+) M~'s/microglia in a region of the spinal cord marked by widespread disruption of the BSB
may result in a compromise of the "privileged" immunological status of the host CNS. A
priming of T-cell immunity could amplify subsequent inflammatory responses in the chroni-
cally injured spinal cord (e.g., following intraspinal transplantation). The present data indicate
that, like inflammatory responses in peripheral tissues, the CNS possesses a stereotypical
inflammatory response marked by non-specific (microglial/M~ activation) and specific (T-cell
activation) cellular immunity. However, unlike peripheral tissues which are capable of
regeneration, enhanced immune function may be deleterious to spinal tissues surviving the
mechanical injury of impact trauma.

P45 EVIDENCE FOR NUCLEAR CONDENSATION SUGGESTIVE OF APOPTOSIS IN
SPINAL CORD INJURY
M.S. Beattie, M:J. Crowe, S.L. Shuman, J.N. Masters and J.C. Bresnahan, The Ohio State
University, Columbus OH

Apoptosis has been observed in several pathologic states and can be distinguished from necrosis
by the characteristic manner in which the chromatin is broken down. To determine the extent
of chromatin condensation in spinal cord injury, rats (300-400 gm) were anesthetized and the
spinal cord was contused at T10 using the NYU device. A 10gm rod was dropped 2.5 cm onto
exposed dura. The wound was closed and animals allowed to survive for 30 min, 6, 12, 24, or
48 h, 8 (n=4 per time point) or 21d (n=2). Animals were then re-anesthetized and perfused with
4% paraformaldehyde/PBS. Spinal cords were sectioned (20 um) and stained with the nuclear
dye Hoechst 33342 (10 ug/ml). Spinal cord tissue from arat with 30 min survival time showed
no evidence of chromatin condensation. Most damage to spinal cord integrity was contained
within 6 mm rostral(R) and caudal (C)to the lesion center. After 6 h, a few nuclei had condensed
chromatin typical of apoptosis. Such nuclei were found mainly in the dorsal funiculi (DF)
within +or-6 mm of the lesion center. Disruption of spinal cord integrity extended 10-11 mm R
and 17 mm C to the lesion center. After 12 h, numerous condensed nuclear profiles were evident.
Most were observed in the DF, but several were found in the dorsal and ventral hems (DH &
VH) and lateral funiculi (LF). Apoptotic nuclei were observed 6 mm R and 8-9 mm C from the
lesion center, however, cord damage extended 10 mm R and 17 mm C to the lesion center. After
24 h, apoptotic nuclei were present in DF, DH and VH and were visible 6 mm R and 13 mm C
to the lesion center. After 48 h, numerous apoptotic profiles were seen extending 8 mm R and
14 mm C to the lesion center. In rats which were allowed to survive 8 or 21 days post-injury,
the lesion center was filled with invading phagocytic cells that contained apparently fragmented
DNA. However, the majority of classically described apoptotic bodies are evident primarily in
the white matter and extend at least 17 mm from the lesion. From this study, we have observed
that a spatio-temporal gradient of cells with apoptotic nuclei is evident up to 21 days post-
contusion. Supported by NS10165 & NS07291.

P46 ALPHA-ADRENERGIC AND 5HTsub1C/2 RECEPTORS OF THE NORMAL AND
INJURED HUMAN SPINAL CORD: AN AUTORADIOGRAPHIC STUDY
A.E. Sverstiuk and S.E. Croul, Medical College of Pennsylvania, Philadelphia, PA

Data on adrenergic and serotonin receptor distribution in normal human spinal cord are
fragmentary and incomplete. Even less is known about these receptor' s response to injury. On
the other hand, some clinical and experimental observations suggest involvement ofadrenergic
spinal pathways in limited recovery and serotonin subsystem in posttraumatic spasticity. Also,
experimental evidence of close interaction between these two subsystems have been reported.
Using quantitative receptor binding autoradiography, we assayed asubl- and asub2-adrenergic, and
5HTsub1C/2 receptors in autopsy human spinal cord tissue recovered from 9 patients without any
known neurologic deficit. Heterogeneous regional distribution of asub2-adrenergic and 5HTsub1C/2
specific binding was shown at different levels of the spinal cord gray matter. In order to estimate
receptor binding response to injury similar receptor binding studies were performed on autopsy
spinal cord tissue from 7 individuals who suffered spinal cord injuries and 2 patients with
disorders leading to corticospinal tract degeneration.

P47 DEXAMETHASONE TREATMENT UP-REGULATES mRNA FOR THE Na, K-ATPase
IN SPECIFIC SETS OF NEURONS FROM RATS WITH SPINAL CORD TRANSECTION
S.L. Gonzalez, M.C. Gonzalez Deniselle, C. Grille, A. Lima and A.F. Dt:Nicola, University
of Buenos Aires and Institute of Biology and Experimental Medicine, National Research
Council, Buenos Aims, Argentina

Glucocorticoids (GC) have beneficial effects in CNS trauma and degenerative diseases. GC
hormones actions on the membrane-bound enzyme Na,K-ATPase are of increasing interest, in
view of the key role the enzyme plays in neurotransmission and membrane repolarization.
Using oligonucleotides probes for the mRNA of the asub3- and Bsub 1-subunits of the enzyme, we have
previously demonstrated that adrenalectomy decreases while Dexamethasone (DEX) restores
mRNA levels for both isoforms in motoneurons of normal spinal cords. In the present report
we describe the effects of transection and DEX treatment on mRNA biosynthesis of the
catalytic subunit using in situ hybridization. Sections of the lumbar region of spinal cords from
sham-operated, transected (TRX) and TRX rats receiving DEX, were hybridized with a 35S_
dATP labelled oligonucleotide of 42 bases coding for the alpha-sub-3-subunit. The number of silver
grains deposits and the area of individual neurons were determined using computerized image
analysis. On the basis of area, we categorized the neuronal populations from ventral horn in
arbitrary groups ranging from u2. Grain density (nO grains per u2) in smallest size
cells (u2) was similar in the 3 groups of rats. In neurons of higher areas (
u2) TRX rats showed a significant reduction in grain density (p<0.05). Percent reduction in
TRX rats was highest in neurons measuring u2 (56%, p<0.001). DEX treatment
significantly reversed this reduction, increasing grain density maximally in the u2
neuronal group (85%, p<0.05). No effect of DEX was evident in smallest neurons (<1000 u2)
in which the lesion was also ineffective. Although morphometric studies cannot ascribe these
changes in mRNA levels to a neuronal type, previous reports have shown that larger neurons
are alpha-motoneurons. whereas the smaller group are a mixture of gamma-motoneurons,
Renshaw cells and interneurons. Because severe reduction of Na,K-ATPase has been associ-
ated with CNS pathology, the stimulation ofits expression may constitute an important event
by which GC reestablish spinal cord function after injury. Supported by NIH NS 20866-08.

P48 CHRONIC EFFECTS OF SPINAL CORD INJURY ON THE BLOOD-SPINAL BARRIER
IN GUINEA PIGS
C.B. Jaeger and A.R. Blight, Purdue University, SVM, West Lafayette, iN and University of
North Carolina, Chapel Hill, NC

The blood-brain barrier(BBB) maintains ion homeostasis. It is characterized by endothelial cell
junctions with zonulae occludens, basal lamina and intimate contacts of astrocyte foot processes
that surround capillaries, but the role of glia in BBB integrity remains undefined. Spinal cord
compression causes mechanical damage to endothelial cells that opens the BBB. We studied
the fine structure of capillaries in the injury area during the course of BBB repair. Female guinea
pigs were subjected to spinal cord compression. At different times post-injury, 7 days to 5.5
months, horseradish peroxidase tracer (HRP) was injected into the circulation. Animals were
euthanized and tracer leakage in the injured segment was verified with the light microscope.
The fine structure of capillaries was also examined. Endothelial cell junctions were leaky to
HRP up to two weeks following injury. This correlated with the occurrence of non-overlapping
celljunctions which, in many instances, were separated by clefts between adjacent cells. At 7
days survival, numerous capillary profiles withjuxtaposed astrocyte foot processes were noted
in addition to altered cell associations and thick, electron dense basement lamina. Endothelial
cells were in contact with macrophages, microglia and degenerating astrocytes. Complete
sealing of the BBB against interstitial HRP leakage did not occur until 17 days post-injury.
Collagen frbrils and amorphous extracellular matrix with characteristics of elastin were
observed in enlarged perivascular spaces. At 1.7 to 5.5 months post-injury endothelial cells
formed complex junctions that prevented tracer leakage. Most of the capillaries in the injured
segment were covered by a thin basement lamina. Further expansion of interstitial spaces was
evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular
regions contained layers of assembled collagen. Reactive astrocytes with altered morphologies
lacked close contacts with endothelial cells. These data suggest that changes in perivascular cell
associations and decoupling of astrocyte foot processes from capillaries do not prevent
reformation of the blood-spinal barrier.

P49 SCHWANN CELLS CAN MISDIRECT REGENERATING AXONS
T.J. Sims and S.A. Gilmore, University of Arkansas for Medical Sciences, Little Rock, AR

A series of recent studies in this laboratory examined the ability of primary afferent nerve fibers
to regrow into the central nervous system (CNS) in animals in which the glial population of the
spinal cord was depleted by exposure to x-radiation. Normally the dorsal root entry zone
(DREZ) of the spinal cord acts as a barrier to the entry of regrowing primary afferent nerve fibers
into the CNS. Astrocytes are considered to play an important role in this barrier function. In
these experiments the fourth lumbar dorsal root was lesioned proximal to the dorsal root
ganglion in irradiated animals and in their non-irradiated littermate controls between three and
four weeks of age. This procedure resulted in formation of a dense mat of astrocytic processes
at the DREZ in non-irradiated animals and failure of the regrowing axons to enter the CNS
environment. In contrast, this astrocytic response did not occur in the radiation-induced, glia-
depleted animals and the regrowing axons were successful in entering the spinal cord. At the
time of root lesion, some of these animals underwent other surgical procedures for transplan-
tation of cultured central glia or the introduction ofgrowth factors into the spinal cord. In some
of these animals the HRP tracer placed on the root to follow the regrowing axons filled neurons
within the dorsal gray matter, indicating that processes from these cells grew into the roots for
distances in excess of 8 mm. Such labeled neurons were never observed in spinal cords of non-
irradiated rats subjected to the same procedures. Similar observations of axons extending from
the spinal cord environment into lesioned dorsal roots have been reported in neonatal animals.
A similarity between these two different experimental situations is that in both, the neonate and
the older but previously irradiated animal, the spinal cord lacks its full complement of mature
astrocytes. An additional consideration in the irradiated animals is that, by the time the root was
injured, some of its central processes in the spinal cord were myelinated by Schwann cells.
Subsequent to the root lesion, it is possible that vacated Schwann cell tubes within the spinal cord
serve as guides for injured central processes and route them in the wrong direction. These
observations raise a very important dilemma with respect to the use of Schwann cells to promote
CNS regeneration in that, on the one hand, they support axonal elongation; on the other hand,
however, they may guide these axons in inappropriate directions. Supported by NIH Grant NS
04761.

P50 REGENERATION OF MYELIN IN CRYOGENIC SPINAL CORD LESIONS: GENE
EXPRESSION OF THE TRANSCRIPTION FACTOR, MyTI, AND OF MYELIN-
RELATED PROTEINS BY SCHWANN CELLS AND OLIGODENDROCYTES
D.-L. Yao*, L.D. Hudson*, N. WestS, G.H Collinsl and H.deF. Webster*, *NINDS, NIH,

Bethesda, MD, and SHealth Sciences Center, State University of New York, Syracuse, NY

Regeneration of myelin was studied in a spinal cord injury model in which axons and myelin
sheaths were destroyed without producing cavitation or hemorrhage (Collins et al., 1986).
Focal cryogenic lesions were produced in dorsal columns of adult rats according to the Collins-
West procedure. One to eight wks later, mRNA distributions of myelin transcription factor I
(MyTI), a novel member of the zinc finger superfamily, were correlated with 1) distributions
and relative mRNA levels of myelin basic protein (MBP), proteolipid protein (PLP), P,
glycoprotein (Po), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). 2) numbers and
distributions of regenerating PNS and CNS myelin sheaths. In the normal spinal cord, low
mRNA levels of MyTI, or a closely related protein, were detected in scattered neurons and some
glial-like cells; higher labeling levels were detected in Schwann cells located in dorsal and
ventral roots. In 2 wk lesions, dense labeling by probes for MyTT and Po mRNA was found in
Schwann cells located in dorsal margins of lesions. Regenerating myelin sheaths were first
observed in perivascular areas of 3 wk lesions; they were Po-positive and Schwann cell-
associated. In 3-4 wk lesions, decreasing NyTI mRNA levels were associated with rising
mRNA levels of MBP, PLP, CNP and P,, and a large increase in numbers ofregenerating MBP-
positive (CNS) and Po-positive (PNS) myelin sheaths. By 8 wks, numbers of regenerating
sheaths were about 70% of those observed in controls. Of those which had regenerated,
oligodendrocyte-associated, CNS myelin sheaths accounted for more than 90% of the total. The
results suggest that expression of MyTI mRNA in lesion Schwann cells and a few oligodendro-
cyte-like cells precedes upregulation of myelin protein synthesis. Finally, our evidence shows
that massive CNS myelin regeneration is an important and characteristic feature of this model.
Further studies of growth factor distributions, angiogenesis, the extracellular matrix, and
interactions of glial cells and axons in these lesions may identify better ways to treat patients
with spinal cord injury.

P51 BEHAVIOURAL CONSEQUENCES OF DEMYELINATION AND REMYELINATLON
IN THE RAT DORSAL FUNICULUS
N.D. Jeffery and W.F. Blakemore, University of Cambridge, Cambridge, England

Although secure conduction in the spinal cord has been demonstrated to be associated with
remyelination of previously demyelinated axons, the behavioural consequences of demyelina-
tion and remyelination have not been investigated. Using the gliotoxin ethidium bromide we
created areas of demyelination in the dorsal funiculus of the spinal cord of rats. Behavioural
testing was carried out by analysis of hindfoot placement and grip as each rat crossed a series
of different diameter horizontal cylindrical wooden beams. Rats were killed at 4-5 weeks post-
lesioning for histological examination including measurement of the area of unlesioned
corticospinal tract (CST). Rats in which 90% or more of the CST had been demyelinated
exhibited deficits in function in the early post operative phase which were correlated with the
extent of demyelination. These deficits disappeared during the period when remyelination
would be expected to occur(from 14 days post lesioning), suggesting that remyelination may
be responsible for re-establishment of efficient locomotion. This method of gait analysis is
sensitive enough to detect lesions which cause dysfunction of most of the axons in the CST. The
effects of X-irradiation mediated suppression of remyelination and transplant-induced
remyelination are the subject of current experiments.

P52 HYPERREFLEXIA OF PROPRIOCEPTIVE PLACING AFTER MODERATE SPINAL
CORD CONTUSION IN RATS
D.M. Basso, S.L. Shuman, M.D. Harkness, M.S. Beattie and J.C. Bresnahan, The Ohio State
University, Columbus, OH

The study of placing responses in cats has shown that tactile/contact placing involves the motor
cortex and red nucleus while proprioceptive placing (PP) may be mediated by spinal systems.
In contrast, the motor cortex has been implicated in both tactile and PP in rats. Species variations
could account for this difference, but difficulties in the assessment of placing in rats may also
be responsible. Quantitative behavioral analysis is needed to investigate PP in rats. For this
study, we used a spinal cord contusion (SCI) model which eliminated cortical input to the
lumbar cord and used sensitive kinematic analysis techniques. Female Sprague-Dawley rats
(275-300g, n=5) had midthoracic SCI which displaced the cord surface l.lmm. Videotapes of
PP tests from 1-70 days postop (dpo) were used for kinematic analysis. Three normal rats served
as kinematic controls; interanimal variability in PP was low. Rats recovered PP more slowly
than open field locomotion, contact righting and flexor withdrawal. PP could not be elicited at
1 dpo despite near maximal joint displacement over an extended time period. By 19 dpo,
hindlimb angular excursions were nearly normal. At 70 dpo, knee extension and ankle flexion
excursion decreased and the peak knee velocity shifted from the extension to the flexion phase,
suggesting development of new motor control strategies. Although the movement time of PP
responses in SCI rats was similar to controls, the stimulus-duration threshold was greatly
reduced (0.038s vs 0.33s, respectively). These data suggest that hyperreflexia after incomplete
SCI may produce contact rather than proprioceptive placing-like behaviors. These contact
placing type behaviors do not appear to be cortically mediated. Supported by NIH NS10165,
NS07291 and APA BB29302.

P53 SKIN FIBROBLASTS GENETICALLY MODIFIED TO SECRETE NT-3 OR BDNF
STIMULATE EXTENSIVE AXONAL SPROUTING AFTER MID-THORACIC SPINAL
CONTUSION INJURY IN THE RAT
P.J. Horner, J. Ray, M.-C.C. Senut, B.T. Stokes and F.H. Gage, Salk Institute, LaJolla, CA and
The Ohio State University, Coiumbus, OH

Spinal cord trauma results in the destruction of short and long tract fiber systems which exhibit
limited post-injury regrowth. In the present experiments we utilized a spinal contusion model
to determine if the sub-chronic administration of neurotrophic factors within a developing
spinal syrinx would stimulate axonal sprouting and/or elongation. Somatic gene transfer was
utilized to create Fischer 344 skin fibroblasts that produce NT-3, BDNF or B-galactosidase (B-
gal) as a control. Transfected fibroblasts (2.5x10to the 5th cells/20ml) were grafted bilaterally into
spinal lesions created with the Ohio State contusion device. All transplants filled the central syrinx
and a healthy syncytium existed between grafted cells and the host spinal parenchyma.
Fibroblasts also were detected within the degenerating dorsal columns several segments from
the lesion epicenter. Laminar deposition of collagen, not seen in injured non-transplanted
animals, was apparent throughout grafted tissue. Neurofilament staining (RT-97) revealed
sparse numbers of axonal processes within control (B-gal) grafts. In contrast, both BDNF and
NT-3 producing transplants contained extensive neuritic ingrowth. These data demonstrate that
modified fibroblasts incorporate well into spinal contusion cavities and that both BDNF and
NT-3 producing cells stimulate robust, post-injury axonal growth. Work supported by The
Hollfelder Foundation, The American Paralysis Association, The International Spinal Research
Trust and NS 10165-21.

P54 RESCUING AXOTOMIZED NEURONS IN RAT SPINAL CORD BY TRANSPLANTATION
OF GENETICALLY MODIFIED CELLS THAT PRODUCE NEUROTROPHINS
B.T. Himes+, J. Solowska-Baird*, E.Y. Snyder#, A. TesslertS and I. Fischer*, *Medical
College of Pennsylvania and Hahnemann University and SVA Medical Center, Philadelphia,
PA, and #Harvard Medical School, Boston, MA

Our previous transplantation experiments have shown that fetal CNS regions expressing
neurotrophin mRNA prevent cell death in Clarke's nucleus (CN) of the adult rat following
axotomy. To examine the role of specific neurotrophins in CN survival, we are currently
transplanting cells genetically modified to produce neurotrophin-3 (NT-3), brain-derived
neurotrophic factor (BDNF) or nerve growth factor (NGF). DNA encoding each of the
neurouophin genes was introduced into a novel retroviral vector (provided by L. Lillien)
containing a reporter gene (alkaline phosphatase, or B-galactosidase) and an IRES sequence that
allows translation to be initiated from an internal site of a single mRNA. PCR, immunocy-
tochemistry and bioassay using embryonic chick DRG explants confirmed the expression of
biologically active neurotrophins by infected 3T3 control cells. The retroviral vectors
expressing neurotrophins were then used to infect various primary cultures and cell lines. These
cells were transplanted into a hemisection cavity that axotomizes ipsilateral CN neurons and
leaves the contralateral CN intact. CN neuron survival at L1 was determined on both sides. We
are focusing our experiments on genetically modifying and transplanting immortalized neural
progenitor cell lines C17-2 (provided by E. Snyder) and RN33B (provided by S. Whittemore).
Since these cells can differentiate in response to local cues, they are promising for enhancing
regeneration and recovery in spinalcord. We have found that these cells survive grafting into
the spinal cord, are not tumorigenic and continue to express the reporter gene for at least 2
months. Supported by The VA Research Service, NS 24707 & 09486.

P55 AUTOLOGOUS SCHWANN CELL IMPLANTS FOR REPAIR OF NON-HUMAN
PRIMATE EXPERIMENTAL CAUDA EQUINA LESIONS
P.W. Madsen, R.P. Bunge and P.M. Wood, The Miami Project to Cure Paralysis, University of
Miami School of Medicine, Miami, FL

Autologous nerve grafting is currently widely used in the repair of PNS injuries; however, a
problem with this approach is that the amount of tissue available for grafting is limited. This
problem could be circumvented in an alternative strategy in which small nerve biopsies were
used to generate large populations of Schwann cells (SCs); these cells would be used to construct
grafts in the desired lengths. Effective methods have been developed at the Miami Project for
the isolation and expansion of populations of adult derived SCs from rodent, human and non-
human primate nerves. We have explored the feasibility of this approach in primates. Four sural
nerve biopsies were obtained from adult male Cynamologous monkeys under general anesthe-
sia; epineural tissues were removed, and nerve fascicles were cut into 5 mm fragments. Using
the technique of Morrissey et al., 1991, these fragments were maintained for 14 days in
Dulbecco's modified Eagle's medium with 10% heat inactivated fetal bovine serum, 10 nM
recombinant human heregulin, 1 uM forskolin, and 50 ng/ml nerve growth factor (DHFN
medium). The fragments were then dissociated following overnight incubation in 0.05%
collagenase/0.25% dispase, and the SCs were plated onto poly-L-lysine coated culture dishes.
Initial results (fust two biopsies) revealed poor SC purity as determined by S100 staining, (27-
46%). An additional two biopsies were treated as above with the addition of choleratoxin to the
mitogen mixture with unsatisfactory results. Two additional sural nerve biopsies were treated
as above without choleratoxin and the use of on collagen coated petri dishes in DHFlV medium.
Under these conditions, SCs proliferated with an average doubling frequency of=l.l per week;
this rate of proliferation provided a thousand-fold increase in the number of SCs over an 8-9
week period of growth. SC purity remained >90% through the 8th doubling and declined
thereafter. SCs were harvested after six doublings (3 passages), and used to fill a PAN/PVC
channel for transplantation back to the original nerve donors. SCs tested at this time
demonstrated strict dependence on exogenous mitogens for proliferation, indicating transfer-
mation did not occur. These results demonstrate the feasibility of generating large numbers of
SCs for autologous grafting in primates. Supported by NS28059, the State of Florida, and The
Miami Project to Cure Paralysis.

P56 SCHWANN CELL-COATED CARBON FILAMENTS AS STIMULATORS OF CNS
AXONAL REGROWTH
G.H. De Vries, S. Sayers, K. Klein, L. Liu and T. Khan, Hines VA Hospital, Hines, 1l.

Successful regeneration in the CNS requires both a modification of the CNS environment to
permit axonal regrowth and directional guidance of axonal regrowth. We have previously
utilized small diameter (5 um) carbon filaments (Amoco Thornel TM) to provide directional
guidance for regrowth of transected axons in the CNS (Brain Res., 541:139, 1991). In the
present study, we coated the fibers with Schwann cells (SC)to provide a source oftrophic factors
and a favorable surface for CNS axonal regrowth. The SC utilized are a human cell line (NF-
1T) derived from a malignant soft tissue sarcoma in a patient diagnosed with neurofibromatosis.
These cells were shown to have an antigenic profile consistent with their SC origin. The cells
aligned themselves in fascicles in culture and demonstrated a typical SC spindle shaped bipolar
morphology. In addition, when NF-1T cells were co-cultured with dorsal root ganglion
neurites, the SC adhered to the surface of the neurites as expected for SC. The SC were co-
cultured with carbon filaments in the absence and presence of 14 day old rat fetal spinal cord
tissue for various amounts of time. Scanning electron microscopy revealed that the NF-1T cells
interacted with the surface of the carbon filaments by aligning themselves with their long axis
parallel to the long axis of the carbon filaments. The surface of the NF-1T cells was covered
with numerous filopodia which appeared to aid in the stabilization of these cells on the carbon
filaments. The presence of SC in the co-cultures stimulated the fetal spinal cord explants to
produce neurites. Experiments are in progress to maximize the ratio of SC to carbon filaments
so that the majority of the surface of the carbon filaments will be covered with NF- IT cells.
These SC-coated filaments will then be used in in vivo models of spinal cord injury to assess
their effectiveness as simulators of axonal regrowth. Supported by NS 15408 (GHDV) and
Department of Veterans Affairs, Rehabilitation R & D Service (TK).

P57 METHYLPREDNISOLONE ADMINISTRATION IMPROVES AXONAL
REGENERATION INTO SCHWANN CELL GRAFTS IN TRANSECTED ADULT
RAT SPINAL CORD
A. Chen, X.M. Xu, N. Kleitman and M.B. Bunge, The Miami Project to Cure Paralysis,
University of Miami School of Medicine, Miami, FL

Schwann cell (SC) grafts support regeneration of axons of spinal cord neurons when the grafts
are transplanted into transected adult rat mid-thoracic spinalcord. Clinically, methylpredniso-
lone (MP) is considered neuroprotective if administered within eight hours after spinal cord
injury. We investigated whether axonal regrowth into SC grafts is enhanced when MP is
administered at the time of transplantation. SCs from adult rat sciatic nerves were purified in
culture, suspended in Matrigel, and drawn into semipermeable polymeric channels. MP(30 mg/
kg) or vehicle (control) was administered intravenously at 5 min, 2 h and 4 h to adult Fischer
rats after transection at T8 and removal of 1-3 caudal segments. The rostral cord stump was
inserted 1 mm into the channel; the distal end of the channel was capped in initial experiments.
Thirty to 45 days later, three times more myelinated axons (1159+or-308) were present in SC/MP
grafts (n=6) than in SC/vehicle cables (355+or-108, n=5). The MP group, compared with vehicle
controls, also showed larger cables, better survival of host cord next to the graft, four times
greater propriospinal neuronal response, and growth of serotonin- and dopamine Bhydroxylase-
immunoreactive axons at least 2 mm into the graft. Very significantly, supraspinal brainstem
neurons extended axons into the graft only when MP was administered (means=46 vs. O, n=3).
When grafts were placed in channels open at both ends, a substantial bridge connecting the two
stumps of the spinal cord had Formed by 30 days. Propriospinal axonal regeneration into grafts
was further enhanced and brainsrem neuronal response was again detected when open channel
grafts were studied in combination with MP. Anterograde tracing with PHA-L to detect growth
of regenerated fibers from the SC graft into the distal host cord showed that only in combination
with MP was there some growth into the distal cord. These results indicate that MP improves
axonal regeneration from both spinal cord and brainstem neurons into distant thoracic SC grafts
and modifies the SC graft-distal cord interface. Supported by NM NS09923 and NS28059 and
The Miami Project. Guidance channels were gifts from Dr. P. Aebischer(Lausanne);XMX was
a Daniel Heumann International Scholar.

P58 DIFFERENT ROUTES OF SPINAL CORD INGROWTH FROM PERIPHERALLY
GRAFTED YOUNG AND MORE MATURE HUMAN EMBRYONAL DORSAL ROOT
GANGLION CELLS
E.N. Kozlova, A. Seiger, I. Stromberg, M. Bygdeman and H. Aldskogius, Karolinska Institute,
Stockholm,Sweden

The regenerative capacity is different in the peripheral and central nervous system. Injured
dorsal root ganglion (DRG) cell axons regenerate successfully in the peripheral part of the dorsal
root, but cease to grow when they encounter the dorsal root entry zone (DREZ), i.e., the
boundary between the peripheral and central nervous system. We have recently shown that this
"barrier" can be overcome by growing axons from human embryonic DRGs (9.5-12 weeks of
age), grafted in place of native lumbar DRGs in adult rat hosts (Kozlova et al., NeuroReport,
6:169-272, 1995). However, when approaching the host DREZ, fibers from these grafted DRG
cells appeared to avoid contact with the astrocytes (which extended long processes in a distal
direction), changed their direction, and entered the spinal cord along the pia and blood vessels.
In contrast, fibers originating from similarly grafted 5-6.5 weeks old embryonic grafts, did not
change their direction at the DREZ, but grew into the host spinal cord in contact with DREZ
astrocytes. In the grey matter the ingrowing fibers from young as well as more mature grafts
ramified extensively. These findings suggest that grafted young human embryonic DRG cells
are either not sensitive to the inhibitory properties of the mature DREZ or are able to neutralize
these properties. Furthermore, our findings indicate that growing frbers may find an alternative
route to the DREZ into the grey matter of the mature spinal cord.

P59 ELECTROPHYSIOLOGICAL IMPROVEMENT FOLLOWING SPINAL CORD
INJURY USING CARBON FILAMENT IMPLANTS AND ORG 2766
T. Khan and S. Sayers, Hines VA Hospital, Hines, IL

Melanocortins have been found to exert a trophic influence on both the central and peripheral
nervous systems. Previous studies from our laboratory have shown that carbon filaments are
capable of supporting neurite growth by providing a favorable attachment surface and
directionaiity to regrowing axons. The purpose of this study was to determine whether the
implantation of carbon filaments and/or the administration of Org 2766 (a tri-substituted ACTH
4-9 analog) would have any beneficial effect after spinal cord injury. Rats were anesthetized
and a total transection of the spinal cord was performed at the T8-T9 level. For animals receiving
carbon filament implants, a bundle of approximately 10,000 carbon filaments of 5 um diameter
were placed into the transection gap. For animals receiving Org 2766 (N.V. Organon, The
Netherlands), a 10 ug subcutaneous injection was given after surgery, and one ug subcutaneous
injections at 48 hr intervals for a two week period. At the end of the eight-week survival period,
the return of sensory function was evaluated by somatosensory evoked potentials (SSEPs) and
the return of motor function by motor evoked potentials (MEPs) using an electromagnetic
stimulator. No SSEP or MEP responses were present in the spinal cord transected animals. All
of the animals in which the spinal cord was transected and which received carbon filament
implants and Org 2766 showed both SSEP and MEP responses. Among the group of animals
in which the spinal cord was transected and which received Org 2766, without carbon filament
implants, 66% showed SSEP responses and 33% showed MEP responses recorded across the
lesion site. This preliminary study demonstrated that Org 2766 had a beneficial effect after
spinal cord injury, as evaluated by electrophysiological methods. This research was supported
by funds from the Rehabilitation R&D Center, Department of Veterans Affairs.

P60 AXON REGENERATION IN NEONATAL MUSCLE FOLLOWING NERVE INJURY
M.D.K. Naidu, K. Subramaniam and G. Vrbova', University of Malaya, Kuala Lumpur,
Malaysia and *University College London, London, United Kingdom

Disruption of normal nerve-muscle interaction in neonates causes several changes which
include death of motoneurons and proportionate loss of muscle fibers. After reinnervation, the
recovery of the reinnervated muscle is also poor. The aim of this study was to investigate the
pattern of reinnervation of the target muscle following temporary denervation during this
criticalperiod. Three day old neonatal rats had their common peroneal nerve crushed in the
popliteal fossa 3 mm proximal to the Extensor Digitorum Longus (EDL) muscle. At weekly
intervals of 1-4 weeks the EDL muscles were removed, weighed, sectioned, visualized using the
silver cholinesterase staining method to study the reinnervation pattern; the number of muscle
fibers were also counted. The results showed that bands of Biingner were absent, a proportion
of endplates disintegrated with a reduction in the number of muscle fibers. This study has shown
that interruption of normal nerve-muscle interaction during the early neonatal period is very
detrimental to the subsequent growth and development of muscle. This research was supported
by grants from University of Malaya (Research Grant #: PJP 336/92, F 10/95), the China
Medical Board and Commission of European Communities.

P61 NIMODIPINE SPEEDS UP REGENERATION BUT REDUCES FINAL MOTORIC
HYPERINNERVATION AFTER PERIPHERAL NERVE SUTURE
W.F. Neiss, D.N. Angelov, M. Streppel, J. Andermahr, K. Mader and E. Stennert, Institut I fiir
Anatomie und Klinik fiir Hals-, Nasen- und Ohrenheilkunde der UniversitPt zu Kijin, Kijln,
Germany

There are no pharmaceuticals in clinical use that could accelerate the resprouting of axons after
peripheral nerve disruption and suture. Following hypoglossal-facial cross anastomosis in the
rat the Ca2+-entry blocker nimodipine quite remarkably speeded up reinnervation of the mimetic
muscles of the whisker pad (J. Neurotrauma, 10 Suppl 1:55,1994). This effect had been so
dramatic that we repeated these experiments with the different model of facial-facial reanastomosis
(FFA). The right facial nerve was cut and sutured in 120 adult female Wistar rats. Postopera-
tively, half of the animals were treated with 1000 ppm nimodipine in food pellets, the other half
received placebo. 48 hours before sacrifice, horseradish peroxidase (HRP) was injected into
both whiskerpads. 1O to 112 days post operation (dpo) six rats per group were fixed by perfusion
and the number of HRP-labeled motoneurons counted in both facial nuclei. On the unoperated
side, HRP labelled 1283+or-86 motoneurons (mean+or-SD; n=60) in placebo and 1256+or-81 in
nimodipine treated rats. At 14 days after FFA, 0-12 facial neurons contained HRP in both
groups. At 16 dpo 171+or-9 nerve cells (mean+or-SD, n=6 rats) were retrogradely labeled with
placebo, but 386+or-34 with nimodipine. This difference proved significant (p<0.05) until 24 dpo,
when 367+or-43 neurons were labeled in placebo, but 657+or-194 in nimodipine-treated rats. No
differences were found at 28-42 dpo and in the final stage the situation was reversed. Wirhour
nimodipine, 1600+or-31, i.e., 32.6% more neurons projected into the whiskerpad than on the
unoperated side. With nimodipine this pathological hyperinnervation was reduced to 1354+or-33,
i.e., 12.1%. This second nimodipine effect was also significant (p<0.001), it might be the more
imponant one in facilitating a better functional reorganization of the muscle innervation.
Supported by Bayer AG.

P62 MOTONEURONS EXPRESS MHC CLASS I ANTIGEN FOLLOWING PERIPH-
ERAL NERVE SUTURE: AN EFFECT INHIBITED BY NIMODIPINE
C. Krebs, D.N. Angelov, M. Walther, O. Guntinas-Lichius, E. Stennert and W. F. Neiss, Institut
I fur Anatomie und Klinik fur Hals-, Nasen- und Ohrenheilkunde der Universitat zu Koln, Koln,
Germany

Axotomy of a motoric nerve with subsequent suture causes a broad variety of reactions on
neuronal and glial level. Suture immediately following transection of the hypoglossal nerve
almost fully prevents neuronal cell death (Neiss et al., Ann. Anat., 174:419-433, 1992). Under
these conditions of survival we studied the immunological reaction of the neuron and the effect
of the Ca2+-entry blocker nimodipine. The hypoglossal nerve in adult female Wistar rats was
axotomized and subsequently sutured. Half of the animals were fed with Nimodipine (1000
ppm in food pellets), half with placebo. 7, 14, 21, 28 and 35 days post operation (dpo) the
animals were perfused with periodate-lysine-paraformaldehyde. We studied the MHC class I
expression with the mAb OX-18 and the CGRP expression with a polyclonal antibody. Under
physiological conditions motoneurons do not express MHC class i. Following axotomy we
were able to prove OX-18 staining of neurons at all dpo on the operated side. The intensity of
staining continuously decreased from 7 dpo to 35 dpo. Nimodipine-treated animals showed
significantly lower levels of MHC class I at all dpo. Nimodipine had no effect on CGRP
expression which was increased in the operated nucleus and decreased slowly and continuously
from 7 dpo to 35 dpo. Neurons thus seem to signal their lesion by expressing MHC class I
antigen while upregulating CGRP expression as a neuroprotective factor. The accelerative
effect of nimodipine on neuronal regeneration may also be due to its inhibition of MHC class
I. Supported by Bayer AG.

P63 IMMUNOREACTIVITY FOR PLASMA MEMBRANE CALCIUM ATPASE IN
EDINGER WESTPHAL SYNAPTIC TERMINALS
J.T. Fujii, Wayne State University School of Medicine, Detroit, MI

The plasma membrane calcium ATPase pump (PMCA) is a high affinity calcium pump that is
typically associated with the plasma membrane. Using the monoclonal antibody 5F10 (Affinity
Bioreagents), we have examined PMCA expression in the neurons of the chick Edinger
Westphal (EW) nucleus. Immunoreactivity for PMCA, associated with the plasma membrane,
is extremely weak in cell bodies located in the EW nucleus. However, PMCA immunoreactivity
is clearly visible in EW synaptic terminals located in the ciliary ganglion. PMCA immunore-
activity resembles immunoreactivity for the synaptic vesicle antigen SV-2 and co-localizes with
immunoreactivity for substance P and enkephalin. While PMCA immunoreactivity is most
striking in EW calyciform terminals, it is also present in boutons. The presence of PMCA
immunoreactivity in EW synaptic terminals was confirmed using transmission electron micros-
copy. Ultrathin sections were cut of tissue stained as frozen sections using a biotinylated
secondary antibody, HRP avidin complex, DAB, and silver intensification. Surprisingly,
reaction product was primarily associated with clusters of synaptic vesicles rather than plasma
membrane. Synaptic vesicles isolated from Torpedo electric organ also stain positive for PMCA
immunoreactivity in immunodot assays. Control experiments show that Torpedo vesicles are
immunoreactive for SV-2 and negative for SERCA (sarcoplasmic/endoplasmic reticulum
calcium ATPase). Supported by NIH ROI EY09768.

P64 DIVERSITY OF GENE EXPRESSION BETWEEN a- AND B-TUBULIN OR B- AND y-
ACTIN DURING NERVE REGENERATION
S.-3. Can, M. Fan, R.-F. Mi and S.-H. Liu, Institute of Basic Medical Sciences, Beijing, China

It was well known that microtubule and microfilament are the major components for axonal
reconstruction and involved in the mechanism of axonal transport. After crushing rat sciatic
nerve, we have found that both the transport rate and transport quantity of their subunits, tubulin
and actin raised in the regenerating nerve. For determing whether the increased subunits could
come from cell body,we fr!rther found that the mRNAs of a-, B-tubulin and B-, y-actin increased
in the spinal motoneurous by in situ hybridization and image analysis. As we know, the a- and
B-tubulin or B- and y-actin must polymerize a heterodimer then the heterodimers can assemble
microtubule or microfilament respectively. Interestingly the time course of individual subunit
gene expression of both heterodimers appeared in significant differences with each other during
regeneration. For example, a-tubulin mRNA increased from 20 h to day 5 after crushing nerve
but B-tubulin increased merely in 1-7 days. Similarly, the y-actin mRNA increased by 4h and
lasted to day 10 while y-actin mRNA increased by 3-7 days only. Moreover, the asynchronous
increasl^s of a- and B-tubulin in the spinal motoneurous and regenerating nerve were further
proved by immunohistochemistry . Why the time courses of gene expression of individual
subunit genes appeared asynchronously? By which mechanism it could be carried out and what
is its role? However, results indicated that the soma of motoneurons did possess the potency to
upregulate cytoskeletal proteins for axon regeneration.

P65 ASYMMETRY IN PLANARIAN NERVOUS SYSTEM REGENERATION AS RE-
VEALED BY NEUROPEPTIDES
I.M. Sheiman, E.N. Zavada, C.P. Tiras and E.V. Zoubina, Institute of Biophysics of Cell,
Puschino, Russia

Functional, neurochemical and morphological asymmetry of the nervous system had been
shown for humans and animals. In the present study asymmetrical aspect of regeneration was
analyzed for the first time. For experiments was chosen simple biological model of morpho-
logical and functional recovery of nervous system: reestablishing of feeding behavior in
planarian Dugesia tigrina after injury. Animals were cut along the body axis (pharynx
amputated) and recovery of feeding capability was assessed. Several groups of operated
animals were placed in solutions of Neuropeptides (NP), known for their regulatory properties
for regeneration. Were used Dalargin (10to the-9M), Vasopressin (10-to the-7M), Hydra Head Activator
(HHA)(10to the-7M,10to the-9M ), Luliberin Releasing Hormone (LHRH)(10to the-7M, 10to the-9M),
Melanostatin(MST)(l0 to the-9M), and Thyroliberin Releasing Hormone (TRH)(10to the-9M). Feeding had
been reestablished along with regeneration of nervous system and pharynx in course of 5-8 days. For
animals of control groups there was no difference between the speed of regeneration of left and
right halves. All neuropeptides affected regeneration process. Dalargin, Vasopressin and HHA
stimulated and LHRH, MST and TRH inhibited regeneration. Effect of NP was different on left
and right regenerates. Stimulators affected more strongly regeneration of right halves, and
inhibitors - regeneration of left ones. We conclude that the hidden asymmetry in regulation of
nervous system regeneration was revealed by the regulatory NPs affecting regeneration.