| 1. |
- Edström, A., et al.
(författare)
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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
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Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522. ; 75:4, s. 1165-1174
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Edström, Anders, et al.
(författare)
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Moderate elevation of extracellular potassium transiently inhibits regeneration of sensory axons in cultured adult sciatic nerves
- 1995
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Ingår i: Brain Research. - 0006-8993. ; 693:1-2, s. 148-154
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Tidskriftsartikel (refereegranskat)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+.
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| 3. |
- Edström, A., et al.
(författare)
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Phospholipase A2 activity is required for regeneration of sensory axons in cultured adult sciatic nerves
- 1996
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Ingår i: Journal of Neuroscience Research. - 0360-4012. ; 43:2, s. 183-189
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Tidskriftsartikel (refereegranskat)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.
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| 4. |
- Ekström, Per
(författare)
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Increased cyclic AMP in in vitro regenerating frog sciatic nerves inhibits Schwann cell proliferation bur has no effect on axonal outgrowth
- 1995
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 0360-4012. ; 42:1, s. 54-62
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Tidskriftsartikel (refereegranskat)abstract
- In the present study the role of cAMP for axonal outgrowth and Schwann cell proliferation was studying using the cultured frog sciatic nerve. An intrinsic rise in nerve injury, both in vitro and in vivo. Treatment with 0.1‐1.0μM 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 regeneraton. 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. © 1995 Wiley‐Liss, Inc.
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| 5. |
- Ekström, Per, et al.
(författare)
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The immune modulator Linomide prevents neuronal death in injured peripheral nerves of the mouse
- 1998
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Ingår i: NeuroReport. - 0959-4965. ; 9:7, s. 1337-1341
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal death after injury or disease could result from imbalanced cytokine expression. Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulator with effects on cytokine production, suppresses autoimmune diseases of the nervous system. Here adult mice were pre-treated with 200 mg/kg/day of Linomide for 9 days, after which the sciatic nerves were crushed. After another 10 days of Linomide treatment the dorsal root ganglia were dissected out and stained for apoptosis, either immediately or after 2 days in culture, which increases cell death. Superior cervical ganglia were also cultured for 2 days. The Linomide pretreatment profoundly reduced (~60- 80%) the injury-induced apoptotic death of neurons and satellite cells in both systems. The results suggest that modulation of the inflammatory cytokine cascade is a promising road to nerve cell rescue.
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| 6. |
- Hornfelt, M., et al.
(författare)
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Upregulation of cytosolic phospholipase A2 correlates with apoptosis in mouse superior cervical and dorsal root ganglia neurons
- 1999
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Ingår i: Neuroscience Letters. - 0304-3940. ; 265:2, s. 87-90
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Tidskriftsartikel (refereegranskat)abstract
- The involvement of cytosolic phospholipase A2 (cPLA2) in apoptosis of adult mouse superior cervical and dorsal root ganglia neurons has been investigated by the use of immunohistochemistry for cPLA2 and DNA nick-end labeling for apoptotic cells, respectively, cPLA2 immunoreactivity was strongly upregulated in neurons of both preparations during in vitro culturing. By double labeling it was unequivocally demonstrated that cPLA2 was present and upregulated only in neurons undergoing apoptosis. A similar picture emerged when cPLA2 immunoreactivity was compared with staining with Fluoro-Jade, a novel fluorochrome marker for neuronal degeneration. The preferential presence of cPLA2 in apoptotic and degenerating cells suggests that the enzyme is important for some mechanism involved in or intimately coupled to neuronal cell death.
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| 7. |
- Remgård, Pär, et al.
(författare)
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Calmodulin and In Vitro Regenerating Frog Sciatic Nerves : Release and Extracellular Effects
- 1995
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Ingår i: European Journal of Neuroscience. - : Wiley. - 0953-816X .- 1460-9568. ; 7:6, s. 1386-1392
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Tidskriftsartikel (refereegranskat)abstract
- Although calmodulin (CaM) is commonly considered to be an intracellular protein, it has been suggested lately that it is released and exerts functions extracellularly. In the present investigation this was studied in in vitro regenerating adult frog (Rana temporaria) sciatic nerves. Using a multi‐compartment incubation chamber, the non‐neuronal cells in the outgrowth region of such nerves were radiolabelled with amino acid precursors. Based on immunological criteria, these cells were shown to release CaM. When the nerves were treated with CaM, both the outgrowth of sensory axons and the injury‐induced proliferation of non‐neuronal cells were partially inhibited. The inhibitory effects occurred even when the incubation medium contained as little as 30 pM CaM. Furthermore, treatment with anti‐CaM antibodies resulted in reduced outgrowth, which suggested that during normal conditions extracellular CaM is kept at an optimal concentration. Finally, conditioned medium was found to contain several CaM‐binding proteins. The present findings may reflect an earlier unknown function of extracellular CaM in controlling various growth mechanisms in integrated tissues.
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| 8. |
- Svensson, Bodil, et al.
(författare)
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Increased levels of mitogen activated protein kinase (MAP-K) detected in the injured adult mouse sciatic nerve
- 1995
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Ingår i: Neuroscience Letters. - 0304-3940. ; 200:1, s. 33-36
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Tidskriftsartikel (refereegranskat)abstract
- Adult mouse sciatic nerves (SNs) with attached dorsal root ganglia (DRG) were analysed for the presence of mitogen activated protein kinase (MAP-K) during normal and regenerative conditions. By immunohistochemistry, MAP-k was found to be present in the normal nerve at low levels in both Schwann cells and DRG nerve cell bodies, with a profoundly increased expression during regeneration. In axonal outgrowth assays, treatment with 2 mM 2-aminopurine (2-AP), a MAP-K antagonist, inhibited the regeneration of axons from the SN as well as from the cultured superior cervical ganglia. The reduced outgrowth was probably not due to toxic effects of the drug since the ganglionic protein synthesis was not inhibited. It is possible that 2-AP inteferes with regeneration-related events by inhibition of MAP-K.
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| 9. |
- Svensson, B., et al.
(författare)
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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
- 1995
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 64:3, s. 1000-1007
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: 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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| 10. |
- Tonge, David, et al.
(författare)
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Use of explant cultures of peripheral nerves of adult vertebrates to study axonal regeneration in vitro
- 1998
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Ingår i: Progress in Neurobiology. - 0301-0082. ; 54:4, s. 459-480
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Tidskriftsartikel (refereegranskat)abstract
- Explanted preparations of peripheral nerves with attached dorsal root ganglia of adult mammals and amphibia survive for several days in serum-free medium and can be used to study axonal regeneration in vitro. This review outlines the methods which we routinely use and how they may be applied to study different aspects of axonal regeneration. When the peripheral nerves are crushed in vitro, axons regenerate through the crush site into the distal stump within 1 day (mouse) or 3 days (frog). The outgrowth distance of the leading sensory axons can be determined with the use of a simple method based on axonal transport of labelled proteins. A compartmentalised system permits selective application of drugs and other agents to either ganglia or peripheral nerve containing the regenerating axons and has been used to study selected aspects of regeneration including influence of non-neuronal cells, retrograde signalling, axonal release of proteins during regeneration and the role of phospholipase A2 activity. Explanted preparations may also be cultured in a layer of extracellular matrix material (matrigel), in which spontaneous outgrowth of a large number of naked axons from the cut ends of nerves starts within 1 day and continues for several days. This provides an opportunity to study the direct effects of different agents on axonal elongation. Preparations cultured in collagen gels show sparse spontaneous axonal growth, but this can be increased by addition of certain growth factors. The phenotype of the regenerating axons can be studied using immunohistochemical methods.
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