A new method to study regeneration in vitro of the adult frog sciatic sensory axons

Edbladh, M.; Remgård, Pär; Edström, Anders

doi:10.1016/0165-0270(90)90065-n

Cited by 17 publications

(9 citation statements)

References 9 publications

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“…It is interesting that inhibition of regeneration was independent of exposure of the ganglia to OA, which suggests that the phosphatase-coupled events of importance to regeneration take place mainly in the outgrowth region . It is clear that Schwann cells and other nonneuronal cells in this region play a part in frog nerve regeneration, because both their injury-induced division and de novo synthesis of proteins are crucial for a proper axonal outgrowth (Edbladh et al, 1990) . These cells may therefore have been targets for OA.…”

Section: Discussionmentioning

confidence: 99%

“…Adult frogs (Rana temporaria) were kept at 20°C in a semiaquatic environment. Dissection and culturing conditions of the sciatic nerves were as described in detail previously (Edbladh et al ., 1990) . In brief, sciatic nerves with attached spinal ganglia were dissected out under sterile conditions and immediately subjected to a test crush 1 mm distal to the rami cutaneus fernoris posterior.…”

Section: Methodsmentioning

confidence: 99%

“…Axonal outgrowth in the integrated nerve . The outgrowth in vitro of crush-injured sensory axons starts after an initial delay of -3 .4 days and is maintained at a constant rate of 0.8-0 .9 mm per day for several days (Edbladh et al ., 1990) . Nerves were locally crushed and cultured for 7 days .…”

Section: Regenerationmentioning

confidence: 99%

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Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Svensson

Ekström

Edström

1995

Journal of Neurochemistry

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Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation‐dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury‐induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 nM) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating‐dephosphorylating reactions are critical for regeneration of peripheral nerves.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Svensson

Ekström

Edström

1995

Journal of Neurochemistry

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“…In the present study I have sought to analyze the role of cAMP in nerve regeneration by using the in vitro regenerating adult frog sciatic nerve. This preparation displays both regrowth of sensory axons and injury-induced division of SC in a defined, serum-free culturing environment (Edstrom and Kanje, 1988;Edbladh et al, 1990;Edstrom et al, 1990). The advantages of these features are obvious.…”

Section: In Vivo Regenerationmentioning

confidence: 99%

Increased cyclic AMP in in vitro regenerating frog sciatic nerves inhibits Schwann cell proliferation bur has no effect on axonal outgrowth

Ekström¹

1995

J of Neuroscience Research

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In the present study the role of cAMP for axonal outgrowth and Schwann cell proliferation was studied using the cultured frog sciatic nerve. An intrinsic rise in nerve and ganglionic cAMP could be measured as a response to nerve injury, both in vitro and in vivo. Treatment with 0.1-1.0 microM forskolin, an activator of the cAMP-generating enzyme adenylyl cyclase, increased the cAMP content up to 13-fold, but was yet without effect on axonal outgrowth during an 8-day culturing period. HA-1004, an inhibitor of cAMP-dependent protein kinase, also lacked effect on the regeneration. In contrast, the proliferation of Schwann cells, measured as [3H]thymidine incorporation, was inhibited to about 70% of control by forskolin, whereas HA-1004 stimulated proliferation to approximately 130% of control. The results suggest that cAMP is involved in the injury-induced proliferation of Schwann cells of an adult peripheral nerve but that it lacks a central role in the regeneration of sensory axons of such nerves.

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“…The in vitro technique introduced by Edbladh et al (1990) makes it possible to selectively label the material produced by non-neuronal cells resident in the injured region of an adult frog sciatic nerve. By using a multichamber culture system we have been able to monitor the production and retrograde transport of proteins to the cell bodies.…”

Section: Introductionmentioning

confidence: 99%

Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating adult frog sciatic sensory axons

Edbladh

Ekström

Edström

1994

J of Neuroscience Research

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The local synthesis and subsequent retrograde axonal transport of [35S]methionine-labelled proteins was studied in the in vitro regenerating adult frog sciatic sensory axons. By the use of a three compartment culture system, proteins in the outgrowth region were selectively labelled. After 2 days in culture a rise in TCA-insoluble radioactivity was detected in the dorsal root ganglia, which could be prevented by the addition of vinblastine or 2,4-dinitrophenol to the nerve proximal to the crush site. Two-dimensional polyacrylamide gel electrophoresis of ganglionic proteins revealed a pattern of 35 labelled polypeptides with apparent molecular masses (Mm) ranging from < 15 to 95 kDa and with isoelectric points (pI) ranging from 4.5 to 6.5. The major ones, representing about 75% of the activity in a gel, were of Mm/pI 47/5.4, 48/6.1,. 57/6.0, 62/5.2, 65/4.9-5.0, 65/5.2, and 81/5.4 respectively. One of these polypeptides (47/5.4) was identified as actin and another (81/5.4) as a member of the heat shock protein 70 family. The spots at 65/4.9-5.0 were tubulin isoforms. There was a striking similarity between transported proteins on one hand, and proteins synthesized in the injured nerve on the other, with respect to the Mm/pI of at least 14 protein species. The results suggest that a selected set of proteins, synthesized by non-neuronal cells, e.g., Schwann cells, is transferred to the ganglionic cell bodies by retrograde axonal transport.

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A new method to study regeneration in vitro of the adult frog sciatic sensory axons

Cited by 17 publications

References 9 publications

Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Increased cyclic AMP in in vitro regenerating frog sciatic nerves inhibits Schwann cell proliferation bur has no effect on axonal outgrowth

Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating adult frog sciatic sensory axons

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