DIFFERENTIATION AND APOPTOSIS WITHOUT DNA FRAGMENTATION IN CULTURED SCHWANN CELLS DERIVED FROM WALLERLAN-DEGENERATED NERVE
Hibasami H., Hirata H., Morita A., Ohkaya S., Sasaki H., Uchida A. Department of Medical Sciences, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
The mechanism of Schwann cell death during nerve degeneration and regeneration has not been clear. So, we investigated the behavior of Schwann cells prepared from wallerian-degenerated adult rat sciatic nerve in vitro. The cultured Schwann cells showed serial changes in morphology, mitotic activity and migratory activity as they do during Schwann cell cable formation in vivo. At the final stage of differentiation, the Schwann cells became rounded and detached from the flask with extensive blebbing. Electron micrographs clearly demonstrated typical cytoplasmic changes of apoptosis, but, nuclei of most of the cells retained their size and morphology with residual nucleolar structures. An agarose gel electro-phoresis of DNA clearly demonstrated that there was not any DNA fragmentation up to 120h after detachment. In situ apoptosis detection assay of wallerian degenerated nerve segment did not show any DNA degradation despite the substantial decrease in Schwann cell number in vivo. These results indicate that supernumerary Schwann cells are removed by apoptosis, however, the death process is unusual in that it lacks DNA fragmentation. Similar apoptotic cell death has been reported recently in some cells.
INDUCTION OF CELL DIFFERENTIATION AND APOPTOSIS OF SCHWANN CELLS FROM WALLERIAN DEGENERATED NERVE BY ENDOGENEOUS NEUROTROPHINS THROUGH ITS P75 RECEPTOR
Hirata H., Hibasami H., Morita A., Ohkaya S., Uchida A. Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
The present study was conducted to investigate the mechanism of Schwann cell differentiation and cell death during wallerian degeneration. Cultured Schwann cells from adult rat wallerian degenerated nerve showed serial changes in morphology, mitotic and migratory activity followed by apoptotic cell death in 7 days. We examined serial changes in intracellular ceramide level and in the expression of NGF, BDNF, and p75 receptor. We also examined the effect of sphyngomyelin hydrolysis and neutralizing antibodies to NGF or p75 receptor on cell differentiation and cell death. Intracellular ceramide level and p75 receptor expression were increased with time. The treatment with C2 ceramide accelerated cell differentiation at low dose and induced cell death at high dose. BIM, an inhibitor of sphyngomyelinase, inhibited cell death. PDMP, an inhibitor of glucosylceramide synthnase, induced rapid cell differentiation and cell death. Neutralizing antibody to p75 receptor strongly inhibited both cell differentiation and cell death. The expression of NGF and BDNF was constant. Neutralizing antibody to NGF did not show any effect, suggesting binding by other neurotrophins than NGF to p75 receptor also induce sphingomyelin hydrolysis. These data indicate that, in contrast to development, p75 receptor signals both cell differentiation and apoptosis by mediating neurotrophin-induced ceramide production in wallerian degeneration.
ROLE OF GDNF, PERSEPHIN AND THEIR RECEPTORS IN PERIPHERAL NERVE INJURY
Höke A., Cheng C., and Zochodne D.W. Dept. of Clinical Neurosciences and the Neuroscience Research Group, University of Calgary, Calgary, Alberta, T2N 4N1 CANADA
Glial derived neurotrophic factor (GDNF) is a trophic factor for spinal cord (SC) motor and dorsal root ganglia (DRG) sensory neurons in vitro. Persephin is a new member of this family and has similar actions to GDNF. Both of these factors may be secreted by Schwann cells, and support regenerating axons after injury. In order to see the changes in the expression pattern of these growth factors and their receptors (ret, GFRa-1 and GFRa-2) we looked at mRNA levels (by semi-quantitative multiplex RT-PCR) in SC, DRG, and proximal and distal stumps of a mixed peripheral nerve (sciatic) after transection. GDNF mRNA was expressed at very low levels in the SC, DRG, intact nerve and proximal stump of transected nerve, but was upregulated in the distal denervated nerve as early as 48 hours. GFRa-1 mRNA levels were elevated in the DRG and in the distal stump at 48 hours; returned to baseline levels by 14 days in the DRG but remained elevated in the distal denervated stump. Ret mRNA levels were initially downregulated in the distal denervated stump but recovered within 14 days. There was no change in Ret mRNA levels in the SC or the DRG. GFRa-2 and Persephin mRNA levels did not change in any of the tissues examined. These findings suggest that increased GDNF mRNA expression in the distal stump and increased GFRa-1 in both DRG and distal stump may be important for the support of regenerating sensory axons. Supported by Medical Research Council of Canada, MDAC and Alberta Heritage Foundation for Medical Research
CNTF AND sCNTFRa PROMOTE MACROPHAGE CHEMOTAXIS IN MICROCHEMOTAXIS CHAMBERS.
Kobayashi H., Mizisin A.P. University of California, San Diego, La Jolla CA, USA and Meiji Seika Kaisha Ltd, Tokyo, Japan.
During Wallerian degeneration in the peripheral nervous system, macrophages recruited from the circulation invade the degenerating nerve segment, phagocytose debris and express factors that influence Schwann cell behavior. While the importance of macrophages in Wallerian degeneration is clear, much less is known about the factor(s) mediating the recruitment of these cells from the circulation. Evidence that CNTF is a Schwann-cell-derived lesion factor and that skeletal muscle sheds CNTFRa after denervation points to the possibility that CNTF and sCNTFRa provide a chemotactic stimulus promoting the recruitment of macrophages that express the gpl30/LIFRb receptor complex. Using 48-well microchemotaxis chambers, we investigated whether CNTF and/or sCNTFRa are chemo-attractants for macrophages. Various concentrations of known or potential chemoattractants in Hank's balanced salt solution containing 0.2% BSA (HBSS) were added to the bottom wells and resident macrophages, obtained from Swiss Webster mice after peritoneal lavage, were suspended in HBSS and added to each upper well (1.0 X 10(exp5) cells/well). Chambers were incubated at 37C for 4-6 hours in humidified air with 5% CO2 before removing the 5 mm pore filter separating the lower and upper wells. After staining, cells that migrated through the filter to the bottom surface were counted. Chemotactic activity, defined as the number of cells migrating in response to a known or potential chemoattractant divided by the number migrating in response to HBSS alone, was indicated by a chemotactic index (CI) greater than 1. Macrophages migrated in response to poly I, a macrophage chemoattractant, but not fMLP, a monoctye chemoattractant, or LIF, a ligand for the gpl30/LIFRb receptor complex. sCNTFRa promoted macrophage migration in a dose-dependent manner with the greatest response at 20 ng/ml (CI = 2.03 ± 0.52; N = 4; P<0.03). CNTF did not significantly promote macrophage chemotaxis except in combination with sCNTFRa (CI = 1.60 ± 0.49; N = 6; P<0.03). These data provide support for the notion that CNTF and sCNTFRa participate in the response to peripheral nerve injury by promoting macrophage chemotaxis. Supported by NIH Grant NS32339
MATURATION RATE IS INDEPENDENT OF ELONGATION IN PERIPHERAL NERVE OF CAT
Krarup C., Moldovan M., Fuglehohm K., Sørensen J. University of Copenhagen, Denmark
We studied the relationship between the rate of elongation
of regenerating axons and the rate of maturation as expressed
in the recovery of conduction velocity. Regeneration was followed
after crush of the tibial nerve just distal to the branches to
the deep toe-flexors (CN, n=5). To slow elongation (Fuglehohm
et al 1998) the nerve was in addition frozen over 30 mm (CF30,
n=5) or 40 mm (CF40, n=5). Electrophysiological studies were carried
out weekly for about 2 months using implanted cuff-electrodes
to relate axonal elongation and maturation before target tissue
reinnervation. The rates of elongation along the tibial nerves
were 3.9 in the CN, 1.8 in the CF30, and 0.2 mm/day in the CF40
group of nerves (P<0.05, ANCOVA). In contrast to the CN and
the CF30, the CF40 group axons did not regenerate through the
tibial nerve cuff within the observation period. Recovery of conduction
velocities (cv) had three phases: During the early phase the cv
was <4 m/s, followed by a rapid increase in cv to 15 m/s, finally
followed by a slower rate of recovery. This pattern was similar
in all lesion types and at all recording sites. The rate of recovery
was faster in the CF30 than in the CN groups (P<0.01), and
this did not occur because of differences in parent fiber size
as indicated by their conduction velocities. The different recovery
could be explained on the basis of maturation lagging further
behind axonal regeneration after crash than after the slower regeneration
in crush+freeze. Our findings suggest that maturation may be under
the control of separate cellular mechanisms than elongation. A
mathematical model was constructed to simulate the influence of
changes in axonal caliber and myelination on the conduction velocity
during elongation.
Fugleholin K et al. Br Res 1998;792:309-318
A MAGNETONEUROGRAPHIC EVALUATION OF THE LONG-TERM CHANGES IN PROXIMAL SEGMENTS 0F TRANSECTED AND RECONSTRUCTED PERIEPHERAL NERVES.
Kuypers P.D.L., Walbeehm E.T., Dudock v Heel M., Godschalk M., Hovius S.E.R. The Erasmus University in Rotterdam The Netherlands (Europe)
In this study the long-term changes in the proximal segment of transected and reconstructed peripheral nerves are evaluated based on Nerve Compound Action Current (NCAC) amplitudes measured by use of a quantitative magnetoneurographic (MNG) recording technique. The average axon diameter in the proximal segment of a transected and reconstructed peripheral nerve will decrease shortly after the transection and increase again when the regenerating axons make, contact with their targets. The magnetically recorded NCAC amplitude and the conduction velocity (CV) are directly related to the axon diameters. In this experiment the peroneal nerve was unilaterally transected and reconstructed in 42 rabbits. After 3, 4.5, 6, 8, 12, 24 and 36 weeks of regeneration time hindleg motor function recovery, NCAC amplitude and CV(1st peak) were studied. Our results demonstrate a significant decrease in signal amplitude and CV in the first 8 weeks after the reconstruction (P<0.05). After 8 weeks of regeneration time, motor function and the CV of the recorded signals start to recover, but the signal amplitudes do not. Based on the correlation of the CV and the signal amplitude with the axon diameter they would both be expected to increase with recovering function. As an explanation for this lack of increase in signal amplitude we suggest that, the expected increase due to axonal maturation is canceled out by a decrease in amplitude due to a number of axons which lose their signal conducting capability.
PATHOPHYSIOLOGY OF BURN NEUROPATHY
Lynch C.D.P., Nukada H. University of Otago, PO Box 913, Dunedin. New Zealand.
We investigated the role of Endothelial, Platelet and Mast Cell derived compounds in an established rat model of thermal nerve injury. Mobilised nerve was suspended on a heating cuff in vivo. Plasma, Nerve Blood Flow (NBF), and Nerve Conduction Studies (NCS) were sampled serially over 90 minutes. Temporal correlation of serotonin, histamine and free radical species were made with the physiological and morphological changes in the sciatic nerve during the nerve heat paradigm. There was no significant elevation of free radical species (TBAR method), but there was a trend toward higher plasma serotonin levels (HPLC method) in the animals receiving longer duration's of thermal injury. This was associated with a trend toward reduced nerve blood flow, and statistically significant alterations in nerve conduction parameters (conduction velocity, CMAP amplitude) in the heated animals. Early morphological changes confirmed disruption to endothelium, endoneurial mast cells, and vascular injury characterised by thrombosed vessels and oedema in subperineurial and endoneurial regions. Later changes were consistent with axonal degeneration. Longer duration of thermal injury produced pan-fascicular damage with little cellular infiltration in heated segments by 7 days. In conclusion we have reconfirmed the temporal physiological changes of thermal nerve injury, and the specific pattern of structural damage over time. Non-significant changes of specific plasma compounds during thermal injury in this study suggest their role in these changes remains speculative.
SCHWANN CELLS CAN INDUCE COLLATERAL SPROUTING FROM INTACT AXONS - AN EXPERIMENTAL STUDY OF END-TO-SIDE NEURORRHAPHY USING A Y-CHAMBER MODEL IN RAT
Matsumoto M., Hirata H., Hibasami H., Morita A., Uchida A. Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
Axonal regeneration across end-to-side neurorrhaphy has recently been reported. However, neither the mechanism by which collateral sprouting from intact axons is elicited nor the origin of the regenerating axons have not been clear. The present study was conducted to answer these questions. To avoid any mechanical damage to the donor nerve during surgery, the intact tibial nerve and distal segment of the transected peroneal nerve were placed in a Y-shaped silicone chamber with 1mm gap in stead of a direct end-to-side suture. Axonal regeneration from the tibial nerve across the gap into the peroneal nerve was assessed using a retrograde neurotracer and immunohistochemical staining. Axonal regeneration across the gap was observed in 66% of the rats. The neurotracer experiment clearly showed that all regenerating axons were sensory axons from the dorsal root ganglia. Immunohistochemical analysis revealed that collateral axonal sprouting from intact axons of the tibial nerve took place only when the Schwann cells from the degenerated peroneal nerve invaded into the epineurial layer of the tibial nerve.
THRESHOLD ELECTROTONUS IN REGENERATED MOTOR FIBERS OF CAT
Moldovan M., Sørensen J., Krarup C. University of Copenhagen, Denmark
The purpose of this study was to investigate excitability of
regenerating nerve fibers using 100-ms threshold electrotonus
(TE 100 at -40%, -20%, 20%, 40%), strength-duration time constant
(TC), and rheobase (RB) as described by Bostock et al (1998).
Eight tibial nerves from 5 adult cats were crushed just distal
to the branches to the deep toe-flexors and studied during light
anesthesia before the lesion: control nerves (CN); and in regenerated
nerves after muscle reinnervation (RN). The tibial nerve was stimulated
at the ankle via a non-polarizing surface Ag-AgCl cathode with
the anode placed 5 cm proximally; the evoked compound muscle action
potential (CM-AP) was recorded from plantar muscles through a
subcutaneous needle electrode referenced to the first toe. About
4 months (110±6 days, mean ±s.e.m.) after the nerve
lesion the CMAP amplitude had recovered to 26±2% of control.
Throughout the hyperpolarizing TE100 the RN showed a markedly
increased threshold compared with the CN. During depolarizing
TE100 the RN showed a more pronounced threshold reduction than
the CN especially during the first 20 ms (F+S1 segments). The
TC in RN (279±17ms) was similar
to the TC in CN (267±19ms).
However, the RB was significantly (Wilcoxon, p<0.05) higher
in RN (2.7±0.4 mA) than CN (1.17±0.1 mA). The data
indicate that the membrane properties of regenerated motor nerve
fibers at about 60 days after muscle reinnervation deviate markedly
from control nerve.
See review by Bostock H et al. Muscle Nerve 1998;21:137-158
ROLE OF TENASCIN-C IN SCHWANN CELL CABLE FORMATION
Morita A., Hirata H., Hibasami H., Matsumoto M., Uchida A. Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
Both in-vivo and in-vitro study was carried out to investigate the role of tenascin-C on Schwann cells during wallerian degeneration. Immunohistochemical analysis was performed to study the expression of tenascin-C and Schwann cell proliferation during wallerian degeneration. Schwann cells started to proliferate 3 days after nerve transection, The mitotic activity peaked at Day5 and decreased gradually thereafter down to the baseline level by Day7. As in the normal nerve, tenascin-C was expressed in the epineurium and at the nodes of Ranvier for the first 4 days after nerve transection. On Day5, weak tenascin-C expression appeared within the endoneurial space. The expression increased gradually thereafter and peaked at Day14, suggesting that tenascin-C play some roles after the completion of Schwann cell tube formation. In vitro study was carried out to examine the effect of tenascin-C on mitotic and migratory activity of Schwann cells prepared from wallerian degenerated adult rat nerve. Tenascin-C treatment did not show any effect on cell proliferation. In contrast tenascin-C inhibited Schwann cell migration. Western blotting demonstrated inverted correlation between tenascin-C production by Schwann cells and mitotic activity of the cell. These data suggest that tenascin-C was produced by Schwann cells during wallerian degeneration to maintain Schwann cell cables by inhibiting the cells from moving out of the endoneurial space.
THE REPAIR OF SHORT NERVE GAP WITH THE ELONGATION OF WALLERIAN DEGENERATED NERVE
Ohkaya S., Hirata H., Hibasami H., Morita A., Sasaki H., Matsumoto M., Uchida A. Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
In the present study, we investigated the usefulness of the elongation of wallerian degenerated nerve for the repair of short nerve gap. Adult Wistar rats of which left sciatic nerve were transected divided into the following 4 groups; Groupl (no coaptation), Group2 (direct neurorrhaphy), Group3 (elongation of the diatal nerve segment follow by neurorrhaphy), Group4 (nerve graft). Rats were kept for 12 weeks to evaluate the following subjects; functional assessment, muscle weight, blood flow in the sciatic nerve, histology with regard to Schwann cell proliferation and angiogenesis in the distal nerve segment. Rats in group3 showed as good functional recovery as rats in group2. As for the muscle weight recovery, group 3 was better than any other group. As for the blood flow of sciatic nerve, group 3 showed statistically significant increase compared to other groups. Histological assessment revealed enhanced Schwann cell proliferaton and angiogenesis in the elongated nerve segment.
TROPHIC EFFECTS OF DENERVATED SKELETAL MUSCLE EXTRACTS
Okajima S., Fujiwara H., Takimoto Y., Hirasawa Y. Fushiki S.(1) Dept. of Orthopedic Surgery, (1)Dynamic Pathology, Research Institute for Neurological Diseases and Geriatrics, Kyoto Prefectural University of Medicine, Kyoto City and Prefecture, JAPAN
Skeletal muscle, which is the target organ for motor neurons, influences the survival, growth, and differentiation of nerve cells. The aim of this study is to examine the effects of denervated muscle extracts on promoting rat's peripheral nerve regeneration. To prepare denervated muscle extracts, the limb skeletal muscles of Wister rats that were innervated by sciatic nerve were removed 1 week after denervation. Then they were homogenized, centrifuged, and dialyzed through cellulose membrane for 24hrs at 4°C. In same process, intact muscle extracts were prepared. 32 male Wister rats weighing about 200g were used. T-shaped silicone tube was implanted between 15mm gap of the left sciatic nerve. The animals were divided into four groups. Group A was administrated with 0.2 mg/ml denervated muscle extracts. Group B was administrated with 0.2 mg/ml intact muscle extracts. Group C was control group administrated with saline. Group D was normal group without tube. After 3 months, 1,1'-dioctadecyl-3,3,3',3',-tetramethlindocarbo-cyamine perchlorate (DiI) was injected to the left anterior tibial muscle of all rats. Then 3 weeks later, all animals were perfused with 4% paraformaldehyde/PBS. The spinal cord and the dorsal root ganglion (DRG) were excised, frozen, and sectioned 20µm thick on a cryostat. We examined and counted the labeled cells by a fluorescent microscope. The motor neurons in segments L4-L5 of anterior horn and DRG cells in part of L4, 5 root were labeled with DiI retrogradely in Group A, B, and D. The number of labeled motor neurons was about 170 in Group A, about 30 in Group B, 0 in Group C, and about 520 in Group D. The number of labeled DRG cells in each Group was counted about 130, 120, 0, and 1000 respectively. There was significant difference in the numbers of labeled anterior horn cells between Group A and group B (t-test, P<0.001), but no significant difference in labeled DRG cells. The results of this study demonstrated that the denervated skeletal muscle extracts of adult rats have the specific effects on the regeneration of motor nerve in vivo.
LAMININ REVERSES NEUROBLASTOMA CELL APOPTOSIS INDUCED BY SERA FROM PATIENTS WITH NEUROPATHY
Pittenger G.L., Burcus N.I., Vinik A.I. The Strelitz Diabetes Institutes, Eastern Va. Medical School, Norfolk, VA, USA
Sera from patients with 'neuropathies' exert neurotoxicity on NlE-115 neuroblastorna cells (NB) by an autoimmune induction of apoptosis that correlates with nerve dysfunction. We have previously shown there is a down-regulation of laminin gene expression in dorsal root ganglion neurons in streptozotocin diabetic rats. Since upregulation of both laminin and its ß2 subunit are associated with enhanced neurite outgrowth, we tested the possibility that laminin might reverse the NB neurotoxicity of sera from patients with neuropathy. NB were cultured for 4 days in DMEM supplemented with 10% of either pooled human serum (Sigma) or patient test sera selected for their neurotoxicity. Another set of identical cultures was treated with 20 µg/ml of laminin. There was no significant effect of laminin on pooled human serum-treated cultures (88,833±76,314, mean increment ± SEM on day 4 of culture). Of 18 patients' sera tested, neurotoxicity was reversed in 14 cultures treated with toxic sera, including sera from Type 1 (663,685 ± 132,970, n=7), Type 2 (744,688 ± 29,688, n=5) or non-diabetic neuropathy (707,250 ± 101,829, n=6) patients (p< 0.02, ANOVA). Sera that failed to reverse toxicity with 20µg/ml were tested with doses of 100 and 50 µg/ml of laminin to test whether higher doses might overcome toxicity. A dose-dependent response was detected in cultures treated with these sera, although there was not a return to the levels found in either controls or those responding to 20µg/ml. Tests with equimolar amounts of a peptide from the ß1 chain of laminin showed an ability to reverse neurotoxicity in a subset of "responders", but was not able to completely mimic the effects of the whole laminin molecule. These data indicate that serum-mediated apoptosis is correctable in vitro with intact exogenous laminin but not by ß subunit peptides. Thus, laminin might be an important neurotrophic factor able to overcome immune-mediated nerve damage and may have therapeutic potential for neuropathy.
DELIVERY OF THERAPEUTIC PROTEINS INTO TIIE NERVOUS SYSTEM: THERAPEUTIC BENEFIT OF PUTRESCINE-MODIFIED ANTIOXIDANT ENZYMES IN FALS TRANSGENIC MICE
Poduslo J.F. Molecular Neurobiology Lab-oratory, Mayo Clinic and Mayo Foundation, Rochester, MN USA
Modification of antioxidant enzymes (catalase and superoxide
dismutase) with naturally-occurring polyamines (such as putrescine,
PUT) results in an increased permeability coefficient-surface
area product at the blood-brain and blood-nerve barriers with
retained enzymatic activity compared to the native enzymes. More
recently, we have shown that putrescine-modified catalase (PUT-CAT)
has a 2.4 fold increase in BBB permeability in the spinal cord
compared to CAT and is delivered in intact form with increased
enzyme activity after parenteral administration. We have utilized
this technology to test the therapeutic benefit of PUT-CAT to
remove hydrogen peroxide and hence modify the time course of the
SOD1 mutation-induced motor neuron disease in a transgenic mouse
line of familial ALS (G93A). The mechanism by which SOD1 mutations
results in motor neuron degeneration in FALS mice partly involves
oxidative damage and an increased peroxidase activity of the mutant
SOD1, which explains in part the dominant cytotoxic gain of function.
Continuous s.c. administration of PUT-CAT significantly delayed
the age at which onset of clinical disease occurred (indicated
by loss of splay and/or tremors of hindlimbs) in a high-expressor
line of FALS transgenic mice. Intraperitoneal injection of PUT-CAT
given two times per week also significantly delayed the onset
of clinical disease in a low-expressor line of FALS mice. PUT-CAT
also significantly delayed the age at which clinical weakness
developed (quantified by measuring the shortening of stride length)
in both lines of FALS mice. No significant changes were observed
in survival times of the high-expressor FALS mice; however, a
trend toward a prolongation of survival was observed in the PUT-CAT
treated low-expressor FALS mice. These results support the role
of free radical-mediated damage in the initiation of motor neuron
degeneration in FALS and indicate that PUT-CAT interacts with
this critical initiation step to delay the onset of clinical disease
as well as the development of clinical weakness in FALS transgenic
mice.
ROLE OF ACTIVATION OF ORNITHINE DECARBOXYLASE IN SCHWANN CELL MIGRATION
Sasaki H., Hirata H., Hibasami H., Morita A., Uchida A. Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu-city, Mie, Japan
We investigated the role of polyamins in Schwann cell cable formation during wallerian degeneration. Sciatic nerves were cut at three levels, forming two isolated wallerian degenerating nerve segments of 5mm, and the nerve segments were bridged with a silicone chamber, leaving a 3mm nerve gap. On Day3, the nerve gap had been connected by fibrin bridge and Schwann cells in the nerve segments started to proliferate and maintained high mitotic activity thereafter. Schwann cells in the nerve segments accelerated migration toward the gap forming regeneration fronts on Day7. By Dayl0 the fibrin bridge had been replaced by a cellular tissue which showed gradual conformation change from granulation tissue like structure to Schwann cell cable like structure between Dayl0 and 21. Schwann cells in the nerve bridge started to proliferate on Dayl0 and maintained high mitotic activity thereafter In the both nerve stumps, ornithine decarboxylase (ODC) activity was increased temporarily between Day3 and 10 with a peak at Day7. In the bridge, ODC activity started to increase at Day 7, reached the maximum activity (2.5-fold increase) at Day 10 and maintained high activity by Day2l. These findings suggests that activation of ODC may play an important role in the initiation of cell proliferation, cell migration, and formation of Schwann cell cable structure.
NO LOSS OF DORSAL ROOT GANGLION CELLS AFTER NERVE CRUSH IN P75 KNOCKOUT MICE.
Sørensen B., Tandrup T., Jakobsen J. Department of Neurology, University Hospital of Aarhus, Denmark
HYPOTHESIS: The p75 low affinity receptor mediates the NGF
signals and therefore is thought to play a role in neuronal survival.
We hypothesized that neuronal loss after axotomy is more pronounced
in adult mice lacking the p75 low affinity receptor. METHODS:
A crush lesion was performed on the right sciatic nerve during
30s in p75 mice (n=7) and Balb mice (n=7). After 14 days the fifth
lumbar dorsal root ganglion (DRG) on both sides were removed,
the contralateral DRG being used as control. The number and mean
volume of pericarya were estimated using assumption free stereological
techniques including systematic sampling techniques, the optical
fractionater and the planar vertical rotator. The present results
include 3 Balb and 5 p75 mice. RESULTS: A and B cell numbers were
2467±503 and 5604±193 in non-crushed Balb mice and
1291±231 and 2295±263 in non-crushed p75 mice, respectively.
In the crushed condition A and B cell numbers were 2376±130
and 4159±46 in Balb mice and 1334±253 and 2120±103
in p75 mice, respectively. In Balb mice the B-cell loss on the
crushed side was l445±164 (p<0.01) whereas no B-cell
loss occurred in p75 on the crushed side (175± 255).There
was no relative difference of perikaryal volume in response to
crush between the two groups. CONCLUSIONS: Results obtained
were unexpected. It is possible that the p75 receptor triggers
an apoptotic response in B-cells of the dorsal root ganglion.
THE ROLE OF VASCULAR DISEASE IN THE DEVELOPMENT OF CHRONIC IDIOPATHIC AXONAL POLYNEUROPATHY
Teunissen. L.L.(1), Notermans, N.C.(1) , van der Graaf, Y.(1), Franssen, H.(1), Linssen, W.H.J.P.(2), Banga, J.D.(1), Eikelboom, B.C.(1), Laman, D.(2), Wokke, J.H.J. (1)University Hospital Utrecht, (2) St Lucas-Andreas Ziekenhuis Amsterdam
Chronic idiopathic axonal polyneuropathy (CIAP) is a polyneuropathy with an unknown cause. It has a male predominance and an onset in older age. In this age group vascular disease is common. In patients with peripheral arterial disease (PAD), neuropathic changes have been described. To evaluate if vascular disease plays a role in the development of CIAP, we compared the pre-valence of vascular disease and risk factors in 97 patients with CIAP and the prevalence of polyneuropathy in 96 patients with PAD with 97 age and sex matched controls. Electrophyiologic examination was performed in 43 patients with PAD and 48 controls. Manifest vascular disease (i.e. stroke or myocard infarction) was present in 40 patients with CIAP and 18 controls, OR 3,0 95% confidence interval 1,6-5,8. Risk factors for vascular disease were more often present in CIAP in comparison with controls. Clinical signs of a polyneuropathy were present in 22 patients with PAD and 8 controls (OR 2,2, CI 1,1-8,5). Electrophysiological examination was abnormal in 14 patients with PAD and 1 control (OR 3,2, CI 1,4-7,7). Since vascular disease is more common in CIAP and neuropathy is more common in patients with PAD, we conclude that vascular disease may play a role in the development of CIAP.
NGF MODULATES EXPRESSION OF NITRIC OXIDE SYNTHASE IN INJURED DRG NEURONS
Verge V.M.K., Wilson T.D., Johnston J. Cameco MS Neuroscience Research Center and University of Saskatchewan, Saskatoon,SK, CANADA S7K OM7
A long term objective of our laboratory is to better understand the role that neurotrophins play in the response of sensory neurons to axotomy and to apply this information to improve regeneration and restoration of function. Peripheral nerve injury induces a multitude of phenotypic changes in lumbar DRG, including a dramatic increase in neuronal expression of nitric oxide synthase (NOS) involved in the generation of nitric oxide, a potent intercellular messenger. As NOS is expressed predominantly in small to medium sized DRG neurons following injury, a population which includes the nerve growth factor (NGF) responsive neurons, we examined whether expression of NOS is limited to this subpopulation and whether it is modulated by exogenous NGF. Quantitative in situ hybridization was performed on serial sections to examine the degree of colocalized expression of NOS mRNA and trkA mRNA (the NGF receptor) in neurons injured for 4, 7 or 21 days. This approach was also used to examine the the ability of immediate or delayed infusion of NGF to counteract or prevent increased NOS expression in injured neurons. Results indicate that approximately 2/3 of axotomized neurons which express NOS also express trkA, and not surprisingly delayed infusion of NGF was effective in mitigating increased expression of NOS in this population of neurons, but not in those NOS positive neurons which do not express trkA. However, if NGF was infused immediately at the time of injury it was effective in mitigating NOS expression in both the trkA and non-trkA populations, suggesting that a trophic cascade may have been altered. Supported by the Canadian Medical Research Council
MAGNETO-NEUROGRAPHY (MNG) AND MORPHOMETRIC ANALYSIS IN THE REGENERATING PROXIMAL NERVE SEGMENT.
Walbeehm E.T., Dudok van Heel E.B.M., Kuypers P.D.L., Terenghi G., Hovius S.E.R. Institute of Plastic Surgery, EUR, Rotterdam, Netherlands
Previous experiments evaluating peripheral nerve regeneration after transection and reconstruction of the common peroneal nerve in rabbits using MNG have shown a consistent decrease in peak-peak amplitude of 50-60% in the proximal stump. Several explanations are given in literature for this electrophysiological phenomenon. Five rabbits received a standardized transection and reconstruction of the common peroneal nerve. After 12 weeks regeneration MNG was performed and histological specimens were taken, 5 cm proximal to the lesion from operated and contralateral control nerves. Results showed a consistent decrease in peak-peak amplitude 57%, conduction velocity showed a non- significant trend towards a decrease (101.2 m/s to 85.1 m/s ). Morphometric results demonstrated a significant decrease of larger myelinated axons (10-15 µm; 284 versus 82, p=0.02). The fibre diameter (axon + myelin) showed similar, but non-significant changes. The maximal measured axonal diameter changed significantly from 12.85 µm to 12.02 µm. Changes in peak-peak amplitude correlated very well with changes in larger myelinated axons (corr. coeff.: 0.85, p<0.01) and with the maximal axon diameter (corr. coeff.: 0.77, p<0.05). Axonal diameter of larger myelinated axons is controlled by the amount of neurofilament per cross-section. After injury neurofilament synthesis decreases, and axonal diameter diminishes. Amplitude is approximately the square of diameter, and larger axons have a stronger contribution to amplitude, therefore the decrease in peak-peak amplitude can largely be explained by the decrease in axonal diameter.
DEVELOPMENT OF AN ENTRAPMENT NEUROPATHY IS A TWO-STAGE PROCESS.
Weerasuriya A., Nelson S.L., Crowley, N.R. Mercer University School of Medicine, Macon, GA, USA.
The objective of this study was to investigate the causative factors of entrapment neuropathies. In the first series of experiments, the endoneurial hydrostatic pressure (EHP) was measured as a function of age in the sciatic nerves of rats ranging in age from 2 days to 18 months. EHP was 1.4 mm Hg at 2 days, rose to 2.5 mm Hg by 2 weeks and remained in the 2.5 to 3.2 mm Hg range until 14 weeks of age (details in Soc. Neurosci. Abstr., 22, 772, 1996). Then, EHP increased during the next 12 weeks to about 4.8 mm Hg and remained at that level through 18 months of age. Rats reach sexual maturity at about 6-7 weeks of age. These data clearly demonstrate that EHP, after reaching a plateau in the sub-adult to young adult stage, rises to another plateau in the mature adult to later stages. From these data, we concluded that older sciatic nerves were more susceptible to injury arising from increases in EHP induced from inside as well as outside the endoneurium. In the second series, consistent with this conclusion, the compliance of the perineurium of older nerves was found to be about 30% less than that of young adult nerves. In the third series, to understand the contribution of convective endoneurial fluid flow (EFF) to EHP, we measured EHP in two sets of rats with either ligated or crushed sciatic nerves. EHP was measured both proximal and distal to the site of lesion, and from 90 minutes to 12 weeks after lesioning. The largest increases in EHP (22-28 mm Hg) was observed within 4 days of ligation (details in Soc. Neurosci. Abstr, 24, 267, 1998). Furthermore, a significant proximo-distal EHP gradient was observed across the site of ligation. In crushed nerves, EHP peaked at 15 mm Hg one day after crush and a proximo-distal EHP gradient was not observed. It is concluded that obstruction of EFF by the ligatures contributes to the significantly higher proximal EHPs in ligated nerves, and the proximo-distal EHP gradient seen in these nerves. Based on these conclusions, it is hypothesized that the initial event in the evolution of an entrapment neuropathy is the limitation and reduction of EFF due to externally applied mechanical forces. Elevation of EHP due to continued application of these external forces and obstruction of EFF leads to endoneurial ischemia and its attendant pathology. This hypothesis is also consistent with the observations that arcade-game-playing teenagers do not necessarily develop CTS, and pregnancy, diabetes and other conditions with increased tissue water content are more likely to be associated with CTS.
EXPERIMENTAL ONION-BULB FORMATION AFTER A SINGLE CRUSH NERVE INJURY IN THE MACROPHAGE SCAVENGER RECEPTOR KNOCKOUT MICE
Yoshikawa H., Naba I. Osaka University Graduate School of Medicine, Suita, JAPAN
After crush injury to peripheral nerve fibers, axonal degeneration
with secondary demyelination and clearance of myelin debris by
Schwann cells and macrophages occur. Recent studies have shown
that oxidized LDL is taken up by macrophages via the macrophage
scavenger receptors (MSRs) , but anti-MSR class-A (MSR-A) antibody
inhibits phagocytosis of myelin in vitro. We therefore investigated
the role of MSR in regeneration of nerve fibers after a single
crush injury using the MSR-A knockout mice. The immumohistochemical
reaction with anti-oxidized phosphatidylcholine showed dense staining
of the crushed myelinated fibers, whereas non-crushed nerve fibers
showed no staining at all. A morphometric examination of the transverse
epoxy section at 21 days after the injury showed no statistically
significant difference in the remaining nerve fiber density between
the MSR-A knockout mice and the wild-type mice, but there were
numerous small onion-bulbs at the crush site in the knockout mice.
These results suggested that anti-oxidized phospholipid antibody
reacted with degenerated myelin, and that dysfunction of phagocytosis
by macrophages might be responsible for onion-bulb formation.
ANATOMICAL BASIS OF OSSEOPERCEPTION
Ysander M.(1), Brånemark R.(1), Myers R.R.(2) (1)Department of Orthopedics, Gothenburg University, Gothenburg, Sweden, (2)Department of Anesthesiology, VA Healthcare System and the University of California, San Diego, La Jolla, CA, U.S.A.
Direct skeletal anchoring of amputation prostheses using osseointegrated
titanium implants produces a sensory perception ability not seen
with traditional socket prostheses. This phenomenon is called
osseoperception and is of great importance in the rehabilitation
of the amputated patient by facilitating improved feedback control
of the prosthesis and enhanced psychological acceptance of the
artificial limb. The anatomical basis of osseoperception is believed
to be associated with increased sensory nerve fiber density in
bone as well as structural and functional changes in sensory and
sympathetic neurons. We have developed an experimental model in
which titanium implants are installed in the intramedullary space
of the rat femur. Healing time and loading conditions have been
altered to observe neural changes throughout the period of bone
remodeling. Changes in nerve fiber density are studied with the
PGP 9.5 antibody technique using fluorescent secondary antibodies
and confocal microscopy. Immunohistochemical characteristics of
neural elements in remodeled bone are studied by staining for
neuropeptides and cytokines. The sensory neuropeptide CGRP is
of special interest because of its role in regulating bone remodeling.
Preliminary results show changes in DRG neuropeptide expression
paralleling CGRP increases and axonal sprouting in remodeling
bone, and that the rat is a suitable model for osseointegration
and osseoperception research.