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Identification of regeneration-associated genes after central and peripheral nerve injury in the adult rat.

Schmitt AB, Breuer S, Liman J, Buss A, Schlangen C, Pech K, Hol EM, Brook GA, Noth J, Schwaiger FW - BMC Neurosci (2003)

Bottom Line: In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments.Twenty one genes (approximately 15%) have been demonstrated to be differentially expressed.The detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Aachen University Medical School, Pauwelsstrasse 30, 52057 Aachen, Germany. neurodoc@gmx.de

ABSTRACT

Background: It is well known that neurons of the peripheral nervous system have the capacity to regenerate a severed axon leading to functional recovery, whereas neurons of the central nervous system do not regenerate successfully after injury. The underlying molecular programs initiated by axotomized peripheral and central nervous system neurons are not yet fully understood.

Results: To gain insight into the molecular mechanisms underlying the process of regeneration in the nervous system, differential display polymerase chain reaction has been used to identify differentially expressed genes following axotomy of peripheral and central nerve fibers. For this purpose, axotomy induced changes of regenerating facial nucleus neurons, and non-regenerating red nucleus and Clarke's nucleus neurons have been analyzed in an intra-animal side-to-side comparison. One hundred and thirty five gene fragments have been isolated, of which 69 correspond to known genes encoding for a number of different functional classes of proteins such as transcription factors, signaling molecules, homeobox-genes, receptors and proteins involved in metabolism. Sixty gene fragments correspond to genomic mouse sequences without known function. In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments. Twenty one genes (approximately 15%) have been demonstrated to be differentially expressed.

Conclusions: The detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.

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Detection of differentially expressed genes after facial nerve transection Radioactive labeled DD-PCR fragments after electrophoretic separation using a polyacrylamide gel and exposition to an x-ray film. Gel image illustrates that fragments with high and low expression can be identified. Lanes 1 and 2 represent the operated side. Lanes 3 and 4 represent the un-operated side (facial nucleus). By comparing operated and control side, fragments which are considered to be differentially displayed appear in both lanes for either operated or un-operated sides (arrows). Fragments indicating either up- or down-regulation have been chosen for further analysis.
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Figure 1: Detection of differentially expressed genes after facial nerve transection Radioactive labeled DD-PCR fragments after electrophoretic separation using a polyacrylamide gel and exposition to an x-ray film. Gel image illustrates that fragments with high and low expression can be identified. Lanes 1 and 2 represent the operated side. Lanes 3 and 4 represent the un-operated side (facial nucleus). By comparing operated and control side, fragments which are considered to be differentially displayed appear in both lanes for either operated or un-operated sides (arrows). Fragments indicating either up- or down-regulation have been chosen for further analysis.

Mentions: We have performed radioactive DD-PCR using a set of arbitrary primers labeled with 35S-dATP (Table 1). This technical approach is more sensitive compared with non-radioactive DD-PCR (e.g. loading PCR products on agarose gels visualized by ethidium bromide). Even gene fragments with low expression could be identified (Fig. 1). For reducing the number of false-positives, duplicated DD-PCR runs were performed for each sample and PCR products were loaded onto the same gel. Only bands (fragments) which appeared to be differentially expressed in both of the duplicated lanes were used for further analysis (Fig. 1). We have thus isolated 180 fragments, 135 of which could be successfully reamplified. After sequencing and gene bank (NCBI) analysis, we have obtained 135 differentially expressed genes. Sixty nine of the gene fragments revealed a high homology to known genes, which encoded for proteins with a number of different functions reflecting the complexity of the process of regeneration. The known genes could be classified into several major groups (Table 2), of which a high number (39) were transcription factors, homeobox-genes and genes involved in signaling pathways or metabolism. Six gene fragments did not show homologies to sequences in the gene bank. The additional comparison with the mouse genome, which has been recently published, revealed 60 gene fragments showing homologies to different regions of several mouse chromosomes with unknown function (Table 4).


Identification of regeneration-associated genes after central and peripheral nerve injury in the adult rat.

Schmitt AB, Breuer S, Liman J, Buss A, Schlangen C, Pech K, Hol EM, Brook GA, Noth J, Schwaiger FW - BMC Neurosci (2003)

Detection of differentially expressed genes after facial nerve transection Radioactive labeled DD-PCR fragments after electrophoretic separation using a polyacrylamide gel and exposition to an x-ray film. Gel image illustrates that fragments with high and low expression can be identified. Lanes 1 and 2 represent the operated side. Lanes 3 and 4 represent the un-operated side (facial nucleus). By comparing operated and control side, fragments which are considered to be differentially displayed appear in both lanes for either operated or un-operated sides (arrows). Fragments indicating either up- or down-regulation have been chosen for further analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161801&req=5

Figure 1: Detection of differentially expressed genes after facial nerve transection Radioactive labeled DD-PCR fragments after electrophoretic separation using a polyacrylamide gel and exposition to an x-ray film. Gel image illustrates that fragments with high and low expression can be identified. Lanes 1 and 2 represent the operated side. Lanes 3 and 4 represent the un-operated side (facial nucleus). By comparing operated and control side, fragments which are considered to be differentially displayed appear in both lanes for either operated or un-operated sides (arrows). Fragments indicating either up- or down-regulation have been chosen for further analysis.
Mentions: We have performed radioactive DD-PCR using a set of arbitrary primers labeled with 35S-dATP (Table 1). This technical approach is more sensitive compared with non-radioactive DD-PCR (e.g. loading PCR products on agarose gels visualized by ethidium bromide). Even gene fragments with low expression could be identified (Fig. 1). For reducing the number of false-positives, duplicated DD-PCR runs were performed for each sample and PCR products were loaded onto the same gel. Only bands (fragments) which appeared to be differentially expressed in both of the duplicated lanes were used for further analysis (Fig. 1). We have thus isolated 180 fragments, 135 of which could be successfully reamplified. After sequencing and gene bank (NCBI) analysis, we have obtained 135 differentially expressed genes. Sixty nine of the gene fragments revealed a high homology to known genes, which encoded for proteins with a number of different functions reflecting the complexity of the process of regeneration. The known genes could be classified into several major groups (Table 2), of which a high number (39) were transcription factors, homeobox-genes and genes involved in signaling pathways or metabolism. Six gene fragments did not show homologies to sequences in the gene bank. The additional comparison with the mouse genome, which has been recently published, revealed 60 gene fragments showing homologies to different regions of several mouse chromosomes with unknown function (Table 4).

Bottom Line: In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments.Twenty one genes (approximately 15%) have been demonstrated to be differentially expressed.The detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Aachen University Medical School, Pauwelsstrasse 30, 52057 Aachen, Germany. neurodoc@gmx.de

ABSTRACT

Background: It is well known that neurons of the peripheral nervous system have the capacity to regenerate a severed axon leading to functional recovery, whereas neurons of the central nervous system do not regenerate successfully after injury. The underlying molecular programs initiated by axotomized peripheral and central nervous system neurons are not yet fully understood.

Results: To gain insight into the molecular mechanisms underlying the process of regeneration in the nervous system, differential display polymerase chain reaction has been used to identify differentially expressed genes following axotomy of peripheral and central nerve fibers. For this purpose, axotomy induced changes of regenerating facial nucleus neurons, and non-regenerating red nucleus and Clarke's nucleus neurons have been analyzed in an intra-animal side-to-side comparison. One hundred and thirty five gene fragments have been isolated, of which 69 correspond to known genes encoding for a number of different functional classes of proteins such as transcription factors, signaling molecules, homeobox-genes, receptors and proteins involved in metabolism. Sixty gene fragments correspond to genomic mouse sequences without known function. In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments. Twenty one genes (approximately 15%) have been demonstrated to be differentially expressed.

Conclusions: The detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.

Show MeSH
Related in: MedlinePlus