Functional polymorphisms in matrix metalloproteinases -1, -3, -9 and -12 in relation to cervical artery dissection.

BACKGROUND: Cervical artery dissection is a leading cause of cerebral ischemia in young adults. Morphological investigations have shown alterations in the extracellular matrix (ECM) of affected vessel walls. As matrix metalloproteinases (MMP) play a central role in the regulation of the ECM, an increased expression of these enzymes might lead to the endothelial damage in spontaneous cervical artery dissection (sCAD). Five different DNA polymorphisms in MMP-1, -3, -9 and -12 were tested for their frequency in patients with sCAD and compared with those of a control population. METHODS: Blood was sampled from 70 unrelated patients presenting consecutively in the department of neurology of the Aachen University Medical School with sCAD and from 87 control subjects living in the same area as the patients. The MMP polymorphisms were analyzed with hybridization probes using the LightCycler (Roche Diagnostics), by sequencing using the ABI 310 Genetic Analyzer (Applied Biosystems) and with the GeneScan program on a ABI 310 Genetic Analyzer. RESULTS: No statistically significant differences in the allelic distribution were found between sCAD patients and the controls. CONCLUSION: Alleles of these 5 functional polymorphisms of MMPs seem not to be associated with structural alterations in the blood vessel wall of sCAD patients. However, this does not exclude a pathogenetic role for MMPs in sCAD via secondary factors such as cytokines that are able to induce these enzymes in cervical blood vessel walls.

NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury.

BACKGROUND: A major class of axon growth-repulsive molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). Experimental spinal cord injury (SCI) has demonstrated rapid re-expression of CSPGs at and around the lesion site. The pharmacological digestion of CSPGs in such lesion models results in substantially enhanced axonal regeneration and a significant functional recovery. The potential therapeutic relevance of interfering with CSPG expression or function following experimental injuries seems clear, however, the spatio-temporal pattern of expression of individual members of the CSPG family following human spinal cord injury is only poorly defined. In the present correlative investigation, the expression pattern of CSPG family members NG2, neurocan, versican and phosphacan was studied in the human spinal cord. METHODS: An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type. RESULTS: In sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. Furthermore, phosphacan, neurocan and versican were present in the myelin sheaths of ventral and dorsal nerve roots axons. After human SCI, NG2 and phosphacan were both detected in the evolving astroglial scar. Neurocan and versican were detected exclusively in the lesion epicentre, being associated with infiltrating Schwann cells in the myelin sheaths of invading peripheral nerve fibres from lesioned dorsal roots. CONCLUSION: NG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might play an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. The present data points to the importance of such correlative investigations for demonstrating the clinical relevance of experimental data.

Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that degrade the extracellular matrix and other extracellular proteins. Studies in experimental animals demonstrate that MMPs play a number of roles in the detrimental as well as in the beneficial events after spinal cord injury (SCI). In the present correlative investigation, the expression pattern of several MMPs and their inhibitors has been investigated in the human spinal cord. METHODS: An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type. RESULTS: In the unlesioned human spinal cord, MMP and TIMP immunoreactivity was scarce. After traumatic SCI, a lesion-induced bi-phasic pattern of raised MMP-1 levels could be found with an early up-regulation in macrophages within the lesion epicentre and a later induction in peri-lesional activated astrocytes. There was an early and brief induction of MMP-2 at the lesion core in macrophages. MMP-9 and -12 expression peaked at 24 days after injury and both molecules were mostly expressed in macrophages at the lesion epicentre. Whereas MMP-9 levels rose progressively from 1 week to 3 weeks, there was an isolated peak of MMP-12 expression at 24 days. The post-traumatic distribution of the MMP inhibitors TIMP-1, -2 and -3 was limited. Only occasional TIMP immuno-positive macrophages could be detected at short survival times. The only clear induction was detected for TIMP-3 at survival times of 8 months and 1 year in peri-lesional activated astrocytes. CONCLUSION: The involvement of MMP-1, -2, -9 and -12 has been demonstrated in the post-traumatic events after human SCI. With an expression pattern corresponding largely to prior experimental studies, they were mainly expressed during the first weeks after injury and were most likely involved in the destructive inflammatory events of protein breakdown and phagocytosis carried out by infiltrating neutrophils and macrophages, as well as being involved in enhanced permeability of the blood spinal cord barrier. Similar to animal investigations, the strong induction of MMPs was not accompanied by an expression of their inhibitors, allowing these proteins to exert their effects in the lesioned spinal cord.

Growth-modulating molecules are associated with invading Schwann cells and not astrocytes in human traumatic spinal cord injury.

Despite considerable progress in recent years, the underlying mechanisms responsible for the failure of axonal regeneration after spinal cord injury (SCI) remain only partially understood. Experimental data have demonstrated that a major impediment to the outgrowth of severed axons is the scar tissue that finally dominates the lesion site and, in severe injuries, is comprised of connective tissue and fluid-filled cysts, surrounded by a dense astroglial scar. Reactive astrocytes and infiltrating cells, such as fibroblasts, produce a dense extracellular matrix (ECM) that represents a physical and molecular barrier to axon regeneration. In the human situation, correlative data on the molecular composition of the scar tissue that forms following traumatic SCI is scarce. A detailed investigation on the expression of putative growth-inhibitory and growth-promoting molecules was therefore performed in samples of post-mortem human spinal cord, taken from patients who died following severe traumatic SCI. The lesion-induced scar could be subdivided into a Schwann cell dominated domain which contained large neuromas and a surrounding dense ECM, and a well delineated astroglial scar that isolated the Schwann cell/ECM rich territories from the intact spinal parenchyma. The axon growth-modulating molecules collagen IV, laminin and fibronectin were all present in the post-traumatic scar tissue. These molecules were almost exclusively found in the Schwann cell-rich domain which had an apparent growth-promoting effect on PNS axons. In the astrocytic domain, these molecules were restricted to blood vessel walls without a co-localization with the few regenerating CNS neurites located in this region. Taken together, these results favour the notion that it is the astroglial compartment that plays a dominant role in preventing CNS axon regeneration. The failure to demonstrate any collagen IV, laminin or fibronectin upregulation associated with the astroglial scar suggests that other molecules may play a more significant role in preventing axon regeneration following human SCI.

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions.

BACKGROUND: Interruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C. RESULTS: In situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarke's and Red nucleus neurons did not show an induction of this gene. CONCLUSION: This differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.

Retrograde reactions of Clarke's nucleus neurons after human spinal cord injury.

Successful axon regeneration depends on the expression of regeneration-associated genes by axotomized neurons. Here, we demonstrate, for the first time to our knowledge, the expression of regeneration-associated genes by axotomized human CNS neurons. In situ hybridization and immunohistochemistry showed a transient induction of GAP-43 and c-jun in Clarke's nucleus neurons caudal to traumatic human spinal cord injury. These results support experimental data that nonregenerating central nervous system neurons can temporarily upregulate regeneration-associated genes, reflecting a transient regenerative capacity that fails over time.

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

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.