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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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Differentially expressed genes within red and Clarke's nuclei. Non-radioactive ISH illustrates the differential expression of clone U41 seven days after spinal cord hemisection. A: Dramatic up-regulation of mRNA of clone U41 within axotomized Clarke's nucleus neurons (small arrows) ipsilateral and caudal to the lesion. Clarke's nucleus neurons of the control, non-operated side (large arrow) show only very faint expression. Bar, 100 μm B: High magnification of axotomized Clarke's nucleus neurons (arrows) demonstrates increased mRNA expression of clone U41. Bar, 20 μm C: Negative control for ISH using a sense probe. Bar, 100 μm D: Low magnification of the midbrain at the level of red nucleus. Up-regulation of clone U41 has been detected within axotomized red nucleus neurons, contalateral to the lesion (OP). Red nucleus neurons of the control side (CON) show only faint expression. Bar, 400 μm E: High magnification of red nucleus contralateral to the lesion reveals increased mRNA expression of clone U41 within axotomized neurons. Bar, 100 μm F: Red nucleus neurons of the control side showing weak mRNA expression. Bar, 100 μm
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Figure 3: Differentially expressed genes within red and Clarke's nuclei. Non-radioactive ISH illustrates the differential expression of clone U41 seven days after spinal cord hemisection. A: Dramatic up-regulation of mRNA of clone U41 within axotomized Clarke's nucleus neurons (small arrows) ipsilateral and caudal to the lesion. Clarke's nucleus neurons of the control, non-operated side (large arrow) show only very faint expression. Bar, 100 μm B: High magnification of axotomized Clarke's nucleus neurons (arrows) demonstrates increased mRNA expression of clone U41. Bar, 20 μm C: Negative control for ISH using a sense probe. Bar, 100 μm D: Low magnification of the midbrain at the level of red nucleus. Up-regulation of clone U41 has been detected within axotomized red nucleus neurons, contalateral to the lesion (OP). Red nucleus neurons of the control side (CON) show only faint expression. Bar, 400 μm E: High magnification of red nucleus contralateral to the lesion reveals increased mRNA expression of clone U41 within axotomized neurons. Bar, 100 μm F: Red nucleus neurons of the control side showing weak mRNA expression. Bar, 100 μm

Mentions: To facilitate the selection of genes for future functional analysis, we have performed comparative studies in a number of different lesion paradigms. ISH analysis of the gene expression in regenerating and non-regenerating models allows the identification of those genes which are either exclusively regulated in a regenerating or a non-regenerating system or show a different regulation pattern between the two systems. To date, we have compared the expression pattern of a few selected genes in different models. In particular, all gene fragments matching with the mouse genome (Table 4) have been analyzed by ISH in our different lesion paradigms. Four of those gene fragments were exclusively regulated in one lesion model. All 3 gene fragments which have been identified using the facial nucleus model such as clone U27 showed dramatic up-regulation in the axotomized facial nucleus neurons (Fig. 2B,2C), but not in axotomized red or Clarke's nucleus neurons. One gene fragment which has been obtained after spinal cord lesion is up-regulated in axotomized red nucleus neurons, but not in facial or Clarke's nucleus neurons after injury (data not shown). One gene fragment (Clone U41), which has been obtained from the Clarke's nucleus after spinal cord hemisection, showed a different regulation pattern between regenerating and non-regenerating neurons. No differential expression could be observed within axotomized facial nucleus neurons (Fig. 2E,2F). In contrast, a dramatic mRNA up-regulation of clone U41 could be detected within axotomized neurons either of the Clarke's nucleus (Fig. 3A,3B) or red nucleus (Fig. 2D,2E,2F). These results demonstrate that clone U41 is exclusively regulated in non-regenerating CNS neurons.


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)

Differentially expressed genes within red and Clarke's nuclei. Non-radioactive ISH illustrates the differential expression of clone U41 seven days after spinal cord hemisection. A: Dramatic up-regulation of mRNA of clone U41 within axotomized Clarke's nucleus neurons (small arrows) ipsilateral and caudal to the lesion. Clarke's nucleus neurons of the control, non-operated side (large arrow) show only very faint expression. Bar, 100 μm B: High magnification of axotomized Clarke's nucleus neurons (arrows) demonstrates increased mRNA expression of clone U41. Bar, 20 μm C: Negative control for ISH using a sense probe. Bar, 100 μm D: Low magnification of the midbrain at the level of red nucleus. Up-regulation of clone U41 has been detected within axotomized red nucleus neurons, contalateral to the lesion (OP). Red nucleus neurons of the control side (CON) show only faint expression. Bar, 400 μm E: High magnification of red nucleus contralateral to the lesion reveals increased mRNA expression of clone U41 within axotomized neurons. Bar, 100 μm F: Red nucleus neurons of the control side showing weak mRNA expression. Bar, 100 μm
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Figure 3: Differentially expressed genes within red and Clarke's nuclei. Non-radioactive ISH illustrates the differential expression of clone U41 seven days after spinal cord hemisection. A: Dramatic up-regulation of mRNA of clone U41 within axotomized Clarke's nucleus neurons (small arrows) ipsilateral and caudal to the lesion. Clarke's nucleus neurons of the control, non-operated side (large arrow) show only very faint expression. Bar, 100 μm B: High magnification of axotomized Clarke's nucleus neurons (arrows) demonstrates increased mRNA expression of clone U41. Bar, 20 μm C: Negative control for ISH using a sense probe. Bar, 100 μm D: Low magnification of the midbrain at the level of red nucleus. Up-regulation of clone U41 has been detected within axotomized red nucleus neurons, contalateral to the lesion (OP). Red nucleus neurons of the control side (CON) show only faint expression. Bar, 400 μm E: High magnification of red nucleus contralateral to the lesion reveals increased mRNA expression of clone U41 within axotomized neurons. Bar, 100 μm F: Red nucleus neurons of the control side showing weak mRNA expression. Bar, 100 μm
Mentions: To facilitate the selection of genes for future functional analysis, we have performed comparative studies in a number of different lesion paradigms. ISH analysis of the gene expression in regenerating and non-regenerating models allows the identification of those genes which are either exclusively regulated in a regenerating or a non-regenerating system or show a different regulation pattern between the two systems. To date, we have compared the expression pattern of a few selected genes in different models. In particular, all gene fragments matching with the mouse genome (Table 4) have been analyzed by ISH in our different lesion paradigms. Four of those gene fragments were exclusively regulated in one lesion model. All 3 gene fragments which have been identified using the facial nucleus model such as clone U27 showed dramatic up-regulation in the axotomized facial nucleus neurons (Fig. 2B,2C), but not in axotomized red or Clarke's nucleus neurons. One gene fragment which has been obtained after spinal cord lesion is up-regulated in axotomized red nucleus neurons, but not in facial or Clarke's nucleus neurons after injury (data not shown). One gene fragment (Clone U41), which has been obtained from the Clarke's nucleus after spinal cord hemisection, showed a different regulation pattern between regenerating and non-regenerating neurons. No differential expression could be observed within axotomized facial nucleus neurons (Fig. 2E,2F). In contrast, a dramatic mRNA up-regulation of clone U41 could be detected within axotomized neurons either of the Clarke's nucleus (Fig. 3A,3B) or red nucleus (Fig. 2D,2E,2F). These results demonstrate that clone U41 is exclusively regulated in non-regenerating CNS neurons.

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