| 1. |
- Ekström, Per, et al.
(author)
-
A calmodulin inhibitor with high specificity, compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules.
- 1991
-
In: Acta physiologica Scandinavica. - 0001-6772. ; 142:2, s. 181-9
-
Journal article (peer-reviewed)abstract
- The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 micrograms ml-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 micrograms ml-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 micrograms ml-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 micrograms ml-1 of compound 48/80 and slightly inhibited by 50 micrograms ml-1. At higher concentrations (greater than 100 micrograms ml-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport.
|
|
| 2. |
- Edbladh, Magnus, et al.
(author)
-
Early regeneration in vitro of adult mouse sciatic axons is dependent on local protein synthesis but may not involve neurotrophins
- 1994
-
In: Neuroscience Letters. - : Elsevier BV. - 0304-3940. ; 168:1-2, s. 37-40
-
Journal article (peer-reviewed)abstract
- The sensory axons of the adult mouse sciatic nerve were shown to regenerate after a local test crush lesion in vitro in a serum-free medium. The average outgrowth distance of the leading axons after culturing for 3 days was 2.8 ± 0.1 mm, which was shorter than in vivo (3.8 ± 0.2 mm). With the use of a compartmentalised culture system we could show that regeneration was partially dependent on local protein synthesis in the injury region. The initial stages of regeneration did not seem to involve neurotrophins since both K252a and K252b, selective and nontoxic inhibitors of the neurotrophin actions, failed to inhibit axonal growth. The present in vitro model system offers favourable conditions to investigate the early events of the regeneration process in an adult mammalian peripheral nerve.
|
|
| 3. |
- Edbladh, M., et al.
(author)
-
Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons
- 1994
-
In: Brain Research. - : Elsevier BV. - 0006-8993. ; 641:1, s. 76-82
-
Journal article (peer-reviewed)abstract
- We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological dose (10 ng/ml, ≈ nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner suggesting effects via their individual receptors. The inhibition, about 30%, was observable after 4 but not 11 days in culture. It is not clear if this reflects a stimulated differentiation of some cells. By contrast, IGF-I lacked effects on both regeneration and proliferation. In conclusion, the results suggest that insulin and IGF-II are involved in the regulation of peripheral nerve regeneration.
|
|
| 4. |
- Edström, Anders, et al.
(author)
-
Adenosine inhibition of the regeneration in vitro of adult frog sciatic sensory axons
- 1992
-
In: Brain Research. - 0006-8993. ; 570:1-2, s. 35-41
-
Journal article (peer-reviewed)abstract
- The sensory axons of the adult frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.
|
|
| 5. |
- Edström, A., et al.
(author)
-
Axonal outgrowth and neuronal apoptosis in cultured adult mouse dorsal root ganglion preparations : Effects of neurotrophins, of inhibition of neurotrophin actions and of prior axotomy
- 1996
-
In: Neuroscience. - : Elsevier BV. - 0306-4522. ; 75:4, s. 1165-1174
-
Journal article (peer-reviewed)abstract
- Dorsal root ganglia (L4 and L5) with attached spinal roots and nerve stumps were isolated from young adult mice and cultured in a layer of extracellular matrix material (matrigel). Within one day, a large number of axons grew out from the cut ends of the nerve and the dorsal root. The average outgrowth length was more than doubled by nerve growth factor, which also strongly increased the number of fibres, showing extensive branching. There was also a significant outgrowth stimulation by neurotrophin-3, but no observable effect by brain-derived neurotrophic factor. In preparations isolated and cultured six days after peripheral nerve transection in vivo, there was an increase in both the outgrowth length (about 1.5- to 2-fold) and in the number of axons. Stimulation of axonal outgrowth, which concerned outgrowth from both the peripheral nerve and the dorsal root, could be further enhanced by the addition of nerve growth factor to the culture. K-252a, a selective inhibitor of neurotrophin receptor-associated tyrosine kinase activity, did not affect either the normal outgrowth or the increased outgrowth in pre-axotomized preparations, at a concentration which abolished the stimulating effects by exogenous nerve growth factor and neurotrophin-3. Under the culturing conditions used, spontaneous apoptosis occurred, but none of the neurotrophins tested, nor K-252a, affected the number of apoptotic neuronal cells analysed by nick-labelling DNA breaks at the end of a 48-h culturing period. Altogether, the present data suggest that for most dorsal root ganglia neurons, signalling through the trk receptors does not influence the apoptosis in vitro and is not required for either the spontaneous axonal outgrowth in matrigel or the increased outgrowth which occurs after prior axotomy in vivo.
|
|
| 6. |
- EDSTRÖM, Anders, et al.
(author)
-
Is protease activity involved in fast axonal transport?
- 1984
-
In: Acta Physiologica Scandinavica. - : Wiley. - 0001-6772 .- 1365-201X. ; 121:4, s. 379-384
-
Journal article (peer-reviewed)abstract
- N‐a‐p‐Tosyl‐L‐Lysine Chloromethyl Ketone (TLCK), a protease inhibitor, was found to irreversibly inhibit rapid axonal transport of protein in vitro in the frog sciatic nerve. TLCK exerted its action at the axonal level and seemed to depress the rate rather than the amount of transported protein. The efficiency of TLCK as a protease inhibitor was demonstrated by polyacrylamide gel electrophoresis, which showed that degradation of high molecular weight proteins (presumably neurofilament subunits) into a 25 000 dalton protein could be induced by exposing the frog nerves to triton‐X and prevented by the presence of TLCK. Findings that TLCK, at a transport inhibiting concentration (0.1 mM), had little or no effects on either protein synthesis or ATP levels, suggest that TLCK did not affect transport due to general cytotoxic properties. The effects of TLCK is discussed in relation to possible roles of protease activity in axonal transport.
|
|
| 7. |
- Edström, Anders, et al.
(author)
-
Moderate elevation of extracellular potassium transiently inhibits regeneration of sensory axons in cultured adult sciatic nerves
- 1995
-
In: Brain Research. - 0006-8993. ; 693:1-2, s. 148-154
-
Journal article (peer-reviewed)abstract
- The adult frog dorsal root ganglia (DRG) together with the sciatic nerve (ScN) has previously been shown to survive in organ culture for several days. If a local test crush is made at the beginning of culturing, there is an initial delay of about 3 days before the sensory axons start to grow into the distal nerve stump at a rate of about 0.6-0.9 mm/day. The present results showed that axonal growth was unaffected in preparations maintained for 8 days in medium containing 10 mM K+ (5 mM is the physiological level). In contrast, the outgrowth was markedly reduced by 15 mM K+ and still more by 20 and 25 mM K+. The growth inhibition was partially counteracted by nifedipine, a Ca2+-channel antagonist. Other experiments clearly showed that high K+ exerted its effects during the early phase of the regeneration and lacked effects at later stages. The possibility that Ca2+-binding proteins, e.g., calbindin, which showed immunohistochemical reactivity in different structures, contribute to the growth adaptation to high K+ will be considered. The generality of the findings was supported by inhibition of axonal outgrowth of adult mouse sciatic sensory axons by high K+.
|
|
| 8. |
- Edström, A., et al.
(author)
-
Phospholipase A2 activity is required for regeneration of sensory axons in cultured adult sciatic nerves
- 1996
-
In: Journal of Neuroscience Research. - 0360-4012. ; 43:2, s. 183-189
-
Journal article (peer-reviewed)abstract
- The adult frog dorsal root ganglia (DRGs) and their sciatic nerves (ScN) survive in organ culture for several days. About 3 days after a local test crush, the sensory axons start to regenerate into the distal nerve stump at a rate of approximately 0.6-0.9 mm/day. The axonal outgrowth is inhibited in a non-toxic way by low concentrations of three different phospholipase A2 (PLA2) inhibitors: 4-bromophenacyl bromide (BPB), aristolochic acid, and oleyl-oxyethyl-phosphoryl-choline (OOPC). In contrast, the outgrowth was slightly stimulated by 0.2 μM melittin, a PLA2 activator. Most experiments refer to the effects of BPB, which was shown to almost completely inhibit outgrowth at a concentration which did not affect either ganglionic protein synthesis or axonal transport. Using a compartmental system it could clearly be shown that BPB exerted its action in the outgrowth region. Other experiments showed that the initial period (about 3 days), which precedes the outgrowth, was unaffected by BPB. Several structures, including axonal ones, showed immunoreactivity for the low molecular form of PLA2 (sPLA2). The results suggest that PLA2 activity plays an important role in nerve regeneration and exerts its action at a local level, where the growth cones move forward.
|
|
| 9. |
- Edström, Anders, et al.
(author)
-
Regeneration in vitro of the adult frog sciatic sensory axons
- 1990
-
In: Restorative Neurology and Neuroscience. - 0922-6028. ; 1:3-4, s. 261-266
-
Journal article (peer-reviewed)abstract
- The adult frog sciatic nerve offers several advantages as an in vitro model to study nerve regeneration. The nerve with the attached dorsal root ganglia can easily be isolated and incubated in a culture medium for several days. If the nerve is subjected to a crush immediately after dissection there is a delay of 3.4 days after which the sensory axons start to regenerate into the distal nerve stump at a constant rate of about 1.1 mm · day−1 in serum-containing and 1.0 mm · day−1 in serum-free medium. Serum-free cultures may be used in future studies to examine the effect of various neurotrophic factors. The existence of an accurate method for examining the outgrowth distance, based on axonal transport of labelled proteins, contributes to the attractiveness of the model. A compartmental culture system permits separate exposure of the ganglia and the nerve to different agents. Taking advantage of this, pharmacological studies suggest that Schwann cells produce signals, dependent on newly transcribed RNA, which transform the preparation into a growth state. The present model system offers favourable conditions to learn more about the early events and also the subsequent steps of the regeneration process.
|
|
| 10. |
- Edström, Anders, et al.
(author)
-
The use of the regenerating frog sciatic nerve for pharmacological studies of orthograde and retrograde axonal transport
- 1987
-
In: Brain Research. - : Elsevier BV. - 0006-8993. ; 401:1, s. 34-42
-
Journal article (peer-reviewed)abstract
- The outgrowth region of the regenerating frog sciatic nerve shows an increased permeability for various drugs, which has been utilized for pharmacological studies of axonal transport. Six days after a bilateral crush lesion, the nerves, including the spinal ganglia, were incubated in a compartmented chamber. Orthograde transport was assessed from the proximodistal distribution and the accumulation of labelled proteins in the nerve growth region. Retrograde transport was examined by allowing orthogradely transported materials to reverse at the regenerating region and then to accumulate at a ligature during a second incubation period. The distribution of radioactivity along the nerve was assayed by fluorography of whole-mount nerve preparations or by scintillation counting. Fluorography made it possible to increase the spatial resolution and to demonstrate effects in the elongating part of the regenerating nerve. Colchicine at low concentrations (10-100 μM) only inhibited axonal transport in the outgrowth region (6 mm long at 6 days after crush) and along some mm of the nerve proximal to the crush. Compound 48/80 (50 μg/ml), the most specific calmodulin inhibitor so far described, inhibited the transport along the same part of the nerve. Cytochalasin B (10 μg/ml) inhibited transport by effects limited to the outgrowth region. Both orthograde and retrograde transport showed sensitivity to these and some other drugs. The regenerating frog sciatic nerve seems to have significant advantages for pharmacological studies of axonal transport.
|
|
