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Characterisation of cell damage and death in embryonic mesencephalic tissue: a study on ultrastructure, vital stains and protease activity.

Emgård-Mattson, Mia LU ; Blomgren, K and Brundin, Patrik LU (2002) In Neuroscience 115(4). p.1177-1187
Abstract
Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was... (More)
Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was dissected, and before it was mechanically disrupted. After 2 h at room temperature, 90% of the remaining cells had intact outer membranes. However, when estimating cells lost acutely in the tissue dissociation, in addition to cells exhibiting condensed chromatin and organellar changes, we suggest that only around 14% of the cells initially dissected in the mesencephalic tissue pieces remained healthy after 2 h. There was a peak in calpain activity (specific cleavage of fodrin) immediately following tissue dissociation, and it subsided during the next few hours. Caspase-3 activity was initially low, but increased almost 20-fold 4 h after tissue disruption. Interestingly, extensive degradation of caspase-3 occurred already directly after dissection and was at least partly calpain-dependent. Our data suggest that, in addition to cells undergoing primary necrosis, some cells undergo apoptotic or related changes soon after tissue harvesting, and eventually undergo a secondary necrosis. In summary, embryonic mesencephalic cells exhibit multiple degenerative changes very early on in the neural transplant/tissue culture preparation protocol. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
caspase-3, neural transplantation, Parkinson’s disease, apoptosis, necrosis, calpain
in
Neuroscience
volume
115
issue
4
pages
1177 - 1187
publisher
Elsevier
external identifiers
  • wos:000179668400017
  • pmid:12453489
  • scopus:0037121673
ISSN
1873-7544
DOI
10.1016/S0306-4522(02)00480-3
language
English
LU publication?
yes
id
10bc41f8-47a3-404c-8ec6-8af99305ea53 (old id 110956)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12453489&dopt=Abstract
date added to LUP
2007-06-26 09:44:30
date last changed
2017-01-01 05:19:04
@article{10bc41f8-47a3-404c-8ec6-8af99305ea53,
  abstract     = {Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was dissected, and before it was mechanically disrupted. After 2 h at room temperature, 90% of the remaining cells had intact outer membranes. However, when estimating cells lost acutely in the tissue dissociation, in addition to cells exhibiting condensed chromatin and organellar changes, we suggest that only around 14% of the cells initially dissected in the mesencephalic tissue pieces remained healthy after 2 h. There was a peak in calpain activity (specific cleavage of fodrin) immediately following tissue dissociation, and it subsided during the next few hours. Caspase-3 activity was initially low, but increased almost 20-fold 4 h after tissue disruption. Interestingly, extensive degradation of caspase-3 occurred already directly after dissection and was at least partly calpain-dependent. Our data suggest that, in addition to cells undergoing primary necrosis, some cells undergo apoptotic or related changes soon after tissue harvesting, and eventually undergo a secondary necrosis. In summary, embryonic mesencephalic cells exhibit multiple degenerative changes very early on in the neural transplant/tissue culture preparation protocol.},
  author       = {Emgård-Mattson, Mia and Blomgren, K and Brundin, Patrik},
  issn         = {1873-7544},
  keyword      = {caspase-3,neural transplantation,Parkinson’s disease,apoptosis,necrosis,calpain},
  language     = {eng},
  number       = {4},
  pages        = {1177--1187},
  publisher    = {Elsevier},
  series       = {Neuroscience},
  title        = {Characterisation of cell damage and death in embryonic mesencephalic tissue: a study on ultrastructure, vital stains and protease activity.},
  url          = {http://dx.doi.org/10.1016/S0306-4522(02)00480-3},
  volume       = {115},
  year         = {2002},
}