Fig.2c. Longitudinal sections o f rat sciatic nerve, showing loss o f CGRP immunostaining distal to the lesion, unless reinnervation is allowed.
The top section shows the distal stump 48 hours after transection, the bottom section shows the proximal and distal sides around the crush lesion, 7 days after injury.
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Fig.2d. Longitudinal sections o f rat sciatic nerve, showing continued presence o f CGRP immunostaining at the proximal stump nerve front, at 7 and 14 days after nerve transection
Fig.2e. Longitudinal sections o f rat sciatic nerve, showing continued presence o f CGRP immunostaining at the regenerating nerve front, at 7 and 14 days after nerve crush, allowing regeneration into the distal degenerating nerve.
CGRP AND cAMPi
The effect of CGRP on cAMPi elevation and DNA synthesis of cultured non neuronal cells derived from rat sciatic nerve, was examined. For these studies we prepared cultures of pure Schwann cells or fibroblasts using a novel adaptation of immunoselection by panning (Barres et al., 1988).
CGRP was added to cultures for a 10 minute period following which cAMPi levels were determined. CGRP stimulates a dose-dependent elevation of cAMPi in both Schwann cells (Fig. 3) and fibroblasts (Fig. 4b) in vitro, which is potentiated by the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX) (Fig. 4a,b).
45 40 35
cAMPi
finol/1 O'* CELLS 3 0
25
2 0
- 1 0 - 6 - 4
Fig.3. Dose response curve for CGRP stimulation of Schwann cell cAMPi.
Schwann cells were plated at 10,000 cells per well of a 24-well plate in serum-free defined medium, and were 24 hours old in culture at the time of assay. Cells were exposed to CGRP for 10 minutes, at varying doses, and cAMPi assayed. Values are mean +/- s.e. of triplicate wells. Where no error bars are shown, in this and
cAMPi finol/10^ CELLS 60 0.3mM IBMX DEFINED MEDIUM 50 40 30 20 10 - 1 0 - 6
Fig.4a. Schwann cell cAMPi dose response to CGRP + / - IBMX
Schwann cells were plated at 10,000 cells per well of a 24-well plate in serum-free defined medium. Cells were exposed to CGRP for 1 0 minutes, at varying doses, and
cAMPi assayed. IBMX, where used, was added 30 mins. before CGRP. Values are mean +/- s.e. of triplicate wells. Where error bars are shown with only positive or negative deflection this is to facilitate graphical representation.
cAMPi fin o l/10 CELLS 4 0 0 DEFINED MEDIUM 350 IBMX 0.5mM 300 250 2 0 0 150 1 0 0 50 — 1 0 LOG,. [CGRP] - 8 [CGRP] - 6 - 4
Fig.4b. Fibroblast cAMPi dose response to CGRP + / - IBMX
Endoneurial fibroblasts were plated at 10,000 cells per well of a 24-well plate in serum-free defined medium. Cells were exposed to CGRP for 1 0 minutes, at varying
doses, and cAMPi assayed. IBMX, where used, was added 30 mins. before CGRP. Values are mean +/- s.e. of triplicate wells.
SCHWANN CELL PROLIFERATION
Two methods were used to assess Schwann cell proliferation; the incorporation of ^^^IdU into newly synthesised DNA as a marker of replication, and a non-radioactive cell viability / proliferation assay based on the conversion of the tétrazolium salt MTT to a formazan-containing dye by the mitochondrial enzyme succinyl dehydrogenase. The MTT assay was used because criticisms have been levelled at techniques that measure only DNA synthesis as a marker of proliferation (Oliver et al., 1989); errors have arisen due to arrest of cells in S or G2 phases of the cell cycle, whereby DNA
synthesis progresses without subsequent cell division and proliferation. The MTT assay has been shown to correlate closely with cell number in many systems, but must also be interpreted with caution as it is dependent on the metabolic activity / viability of cells; a decrease in mitochondrial activity is an early sign of cell death by apoptosis.
Two distinct signals are required for Schwann cell proliferation. Firstly, a growth factor such as PDGF, FGF or IGF-I, and secondly, a permissive signal that elevates cAMPi. Forskolin, which elevates cAMPi via activation of adenylate cyclase, is widely used in experiments to elevate cAMPi.
Growth factor activity is mostly dependent on the activation of receptor tyrosine kinases, but may be mediated indirectly via phospholipase Cy (PLCy) followed by activation of PKC; in fact, activators of PKC can substitute for growth factors in many mitogen assays. The phorbol ester PMA is mitogenic for Schwann cells in defined medium but, as is the case with growth factors, only if cAMPi is elevated (Fig.Sa). PDGF added to cultures already stimulated with PMA, in the presence of cAMPi elevation, promotes some additional activity (Fig.Sa), but this is barely significant, unless PMA is used at submaximal doses (Fig.Sb). Inhibiting PKC with either H-7 or staurosporine, prevents Schwann cell proliferation to mitogenic combinations such as PDGF and forskolin (Fig.Sb). In cultures where basal proliferation was high, this was also inhibited. Schwann cell growth factor mitogens, such as PDGF, are known to act via receptor tyrosine kinases in other systems. Genistein, which inhibits tyrosine kinases, abolishes all Schwann cell mitogenic activity and leads to the rapid onset of cell death, probably by apoptosis. The activation of tyrosine kinase would therefore seem of prime importance in mediating growth factor action on Schwann cell proliferation and survival. Tyrosine kinases are not involved in the generation of cAMPi, and growth factors such as PDGF, do not elevate Schwann cell cAMPi. I suggest that PKC activation is at least one important downstream mediator of growth factor tyrosine kinase activation, which in itself is sufficient for promoting DNA replication if cAMPi is also elevated. It is not yet
possible to conclude that downstream PKC activation is sufficient to mediate the growth promoting activity of tyrosine kinases; where tyrosine kinases were inhibited with genistein, Schwann cells failed to proliferate and died, and no survival action / increase in cell number could be shown with phorbol esters, but DNA incorporation was increased above controls in the presence of both PMA and forskolin. Further experiments are required to distinguish whether tyrosine kinases promote cell survival and proliferation by distinct signalling systems. PKC seems sufficient to stimulate proliferation in forskolin-treated cultures, but may not be sufficient to prevent apoptosis. Inhibition of PKA prevents the permissive action of forskolin (Fig.Sb), suggesting that PKA is important for enabling growth factor activity, and probably mediates the action of cAMPi on proliferation. Pertussis toxin which inhibits Gi thereby increasing basal cAMPi levels, also enables the growth factor activity of PMA. Eventually, cultures incubated with inhibitory doses of any of these three classes of protein kinase inhibitors, die, probably via apoptosis; interestingly, only with tyrosine kinase inhibitors does this occur within 24-48 hours, whereas a longer time is required for other inhibitors.
1 4 . 0 0 0 f - 12.000 1 0,000 8, 000 C . P . M . 6, 000 4 , 0 0 0 2,0 0 0 S A T O S D E F I N E D M E D I U M P M A (1 H M )
1
T
C O N T . P D G F C G R P F O R S . P D G F + F O R S . CONT. = CONTROL; FORS. = FORSKOLINFig.Sa. Incorporation o f by pure cultured Schwann cells, in the presence or absence o f PMA.
Cells were plated in serum-free defined medium, at 10,000 cells per w ell o f laminin coated 96-w ell plates. Cells were 24 hours post-nerve dissociation at the start o f incubations. M itogens were added to cultures for a total o f 48 hours, with additions repeated at 24 hours. Enzyme inhibitors and PM A were added 4 hours prior to mitogens Values are mean +/- s.e. o f triplicate cultures
CPM