PMID- 21778686
OWN - NLM
STAT- MEDLINE
DCOM- 20120402
LR  - 20111004
IS  - 1421-9859 (Electronic)
IS  - 0378-5866 (Linking)
VI  - 33
IP  - 2
DP  - 2011
TI  - Human retinal development in an in situ whole eye culture system.
PG  - 110-7
LID - 10.1159/000328170 [doi]
AB  - Phenotypic characterization of human retinogenesis may be facilitated by use of 
      the tissue culture system paradigm. Traditionally, most culture protocols involve 
      isolation of retinal tissue and/or cells, imposing various degrees of trauma, 
      which in many cases leads to abnormal development. In this paper, we present a 
      novel culture technique using whole embryonic eyes to investigate whether the 
      retina in situ can develop normally in vitro. All procedures were carried out in 
      accordance with the Declaration of Helsinki. Human embryos were obtained from 
      elective abortions with the informed consent of the women seeking abortion. A 
      total of 19 eyes were enucleated. The ages of the embryonic retinas were 6-7.5 
      weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The 
      remaining 17 eyes were placed on culture plates and divided into 3 groups kept 
      for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the 
      specimens were processed for hematoxylin and eosin staining, immunohistochemistry 
      and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling 
      (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; 
      rod bipolar cells) and vimentin (Muller cells) were used. TUNEL was used to 
      detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control 
      retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. 
      Specimens kept 7-14 DIV had a similar appearance, while 28-day specimens 
      consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured 
      retinas displayed completely normal lamination without rosettes or double folds. 
      Pyknotic cells were found at the inner margin of the retinas, and the proportion 
      of these cells increased with time in vitro. TUNEL staining revealed a few 
      scattered cells in 7-DIV specimens, and the amount of stained cells in the inner 
      part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin 
      labeling showed cells arranged in a vertical pattern in all retinas. Labeling 
      with recoverin revealed photoreceptors in 4 of the retinas kept for 14 DIV, and 
      in all retinas kept for 28 DIV. After 28 DIV, 2 of the eyes labeled with PKC 
      contained rod bipolar cells with minimal axons. Here we showed that human 
      embryonic retinas can be kept in culture in situ within the eye for at least 4 
      weeks. Abnormal lamination is not as frequent as in isolated full-thickness 
      retinas, indicating that physical and biochemical contact with surrounding 
      tissues is vital for proper development. Several types of the retina-specific 
      neuronal and glial cells were seen to differentiate according to the in vivo 
      schedule. The results are important for future studies of retinal development, 
      and the technique can also be used for testing the effects of various drugs on 
      the immature retina.
CI  - Copyright (c) 2011 S. Karger AG, Basel.
FAU - Engelsberg, Karl
AU  - Engelsberg K
AD  - Department of Ophthalmology, University Hospital, Lund, Sweden. 
      karl.engelsberg@med.lu.se
FAU - Ghosh, Fredrik
AU  - Ghosh F
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20110721
PL  - Switzerland
TA  - Dev Neurosci
JT  - Developmental neuroscience
JID - 7809375
RN  - 0 (Culture Media)
RN  - 0 (Vimentin)
RN  - 135844-11-0 (Recoverin)
SB  - IM
MH  - Apoptosis
MH  - Cell Differentiation/physiology
MH  - Cells, Cultured
MH  - Culture Media
MH  - Female
MH  - Gestational Age
MH  - Humans
MH  - Immunohistochemistry
MH  - In Situ Nick-End Labeling
MH  - *Neuroglia/cytology/metabolism
MH  - Organ Culture Techniques/*methods
MH  - Pregnancy
MH  - Recoverin/metabolism
MH  - *Retina/embryology/growth & development
MH  - *Retinal Cone Photoreceptor Cells/cytology/metabolism
MH  - *Retinal Rod Photoreceptor Cells/cytology/metabolism
MH  - Time Factors
MH  - Vimentin/metabolism
EDAT- 2011/07/23 06:00
MHDA- 2012/04/03 06:00
CRDT- 2011/07/23 06:00
PHST- 2010/11/26 00:00 [received]
PHST- 2011/04/05 00:00 [accepted]
PHST- 2011/07/23 06:00 [entrez]
PHST- 2011/07/23 06:00 [pubmed]
PHST- 2012/04/03 06:00 [medline]
AID - 000328170 [pii]
AID - 10.1159/000328170 [doi]
PST - ppublish
SO  - Dev Neurosci. 2011;33(2):110-7. doi: 10.1159/000328170. Epub 2011 Jul 21.