E.A.J. Joosten

Collagen Containing Neurotrophin-3 (NT-3) Attracts Regrowing Injured Corticospinal Axons in the Adult Rat Spinal Cord and Promotes Partial Functional Recovery ☆

During development, neurotrophic factors play an important role in the guidance and outgrowth of axons. Our working hypothesis is that neurotrophic factors involved in the development of axons of a particular CNS tract are among the most promising candidates for stimulating and directing the regrowth of fibers of this tract in the lesioned adult animal. The neurotrophin NT-3 is known to be involved in the target selection of outgrowing corticospinal tract (CST) fibers. We studied the capacity of locally applied NT-3 to stimulate and direct the regrowth of axons of the CST in the lesioned adult rat spinal cord. We also studied the effect of NT-3 application on the functional recovery of rats after spinal cord injury, using the gridwalk test. NT-3 was applied at the site of the lesion dissolved into rat tail collagen type I. Four weeks after spinal cord injury and collagen implantation, significantly more CST fibers had regrown into the collagen matrix containing NT-3 (22 +/- 6%, mean +/- SEM) than into the control collagen matrix without NT-3 (7 +/- 2%). No CST fibers grew into areas caudal to the collagen implant. Despite the absence of regrowth of corticospinal axons into host tissue caudal to the lesion area, functional recovery was observed in rats with NT-3 containing collagen implants.

Effects of macrophage transplantation in the injured adult rat spinal cord: A combined immunocytochemical and biochemical study

Early and robust invasion by macrophages may be one of the reasons why axonal regeneration is more effective in the PNS than in the CNS. Therefore, we have grafted autologous peritoneal macrophages labeled with fluorescent latex microspheres into spinal cord compression lesions. At various survival times, we have studied their effect on the expression of neuronal (neurofilaments [NF], calcitonin gene‐related peptide [CGRP], 5‐hydroxytryptamine [5‐HT]) and nonneuronal markers (myelin‐associated glycoprotein [MAG], glial fibrillary acidic protein [GFAP], laminin) by using semiquantitative Western blot and immunohistochemical techniques. After 1 month, we observed a significant decrease of the expression of MAG as well as an important invasion of the lesion site by neurites, chiefly peptidergic axons of presumed dorsal root origin, in macrophage‐grafted animals compared with controls. In addition, angiogenesis and Schwann cell infiltration were more pronounced after macrophage grafts, providing an increase in laminin, a favorable substrate for axonal regrowth. By using reverse transcription‐polymerase chain reaction (RT‐PCR), mRNAs for tumor necrosis factor‐alpha (TNF‐α) were detected in the transplanted cells, whereas results were negative for nerve growth factor (NGF), neurotrophin‐3 (NT‐3), brain‐derived neurotrophic factor (BDNF), or acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Thus, macrophage grafts may represent an interesting strategy to promote axonal regeneration in the CNS. Our study suggests that they may exert their beneficial effects by degrading myelin products, which inhibit axonal regrowth, and by promoting a permissive extracellular matrix containing notably laminin. No evidence for a direct synthesis of neurotrophic factors by the transplanted macrophages was found in this study, but resident glial cells could secrete such factors as a result of stimulation by macrophage‐released cytokines. J. Neurosci. Res. 51:316–327, 1998.

Chronic mitochondrial inhibition induces selective motoneuron death in vitro: a new model for amyotrophic lateral sclerosis.

Evidence is increasing that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis, a neurodegenerative disease characterized by selective motoneuron death. To study the role of mitochondrial dysfunction in the pathways leading to motoneuron death, we developed an in vitro model of chronic motoneuron toxicity, based on malonate‐induced inhibition of complex II in the mitochondrial electron transport chain. Treatment with malonate resulted in a dose‐dependent decrease in cellular ATP levels. We observed that motoneurons were significantly more vulnerable to mitochondrial inhibition than control neurons in the dorsal horn. We could reproduce this dose‐dependent phenomenon with the complex IV inhibitor sodium azide. The free radical scavenger α‐phenyl‐N‐tert‐butylnitrone, the AMPA/kainate receptor blocker 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and riluzole, a drug that is currently used for the treatment of amyotrophic lateral sclerosis, were protective against malonate‐induced motoneuron death. Furthermore, the caspase inhibitors N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone and z‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone were both protective against malonate toxicity. Our model shows that chronic mitochondrial inhibition leads to selective motoneuron death, which is most likely apoptotic.

An anterograde tracer study of the developing corticospinal tract in the rat: three components

Light microscopic analysis of anterogradely transported wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) has been used to study the developing corticospinal tract (CST) in the rat. This study was carried out to examine the relationship between the site of injection within the cortex and the pattern of labelling of the developing CST in the spinal cord from postnatal day 1 (P1) through postnatal day 10 (P10). For this purpose the cortex was subdivided into 3 equal areas along the rostrocaudal axis: anterior, intermediate and posterior. After the operation the animals were allowed to survive for 24 h. The caudal extension of labelled CST axons originating in the anterior cortical area was restricted (L1 at P7 or P10) as compared with that of the CST fibres originating in the intermediate cortical area (S3 at P10). The axons of the posterior corticospinal (CS) neurones reach their most caudal extension in the spinal cord (T5) at P7 but then gradually disappear up till P14. Quantitative analysis of the amount of label along the length of the outgrowing CST fibres revealed the formation of a large stable peak at the level of the cervical enlargement after labelling of either the anterior or the intermediate cortical area. The formation of a second running peak which moves caudally from mid-thoracic levels at P5 to mid-lumbar levels at P10 was only accomplished by labelling the intermediate cortical area and is probably caused by the accumulation of label in the growth cones at the distal ends of the outgrowing CST fibres. After labelling the posterior cortical area, no peaks could be detected, neither at the cervical nor at the lumbar intumescence. The major spinal grey termination field of the anterior CS neurones appeared to be the cervical intumescence, whereas the major spinal grey termination field of the intermediate CS neurones is the lumbar enlargement. By contrast, axons of posterior CS neurones never showed any outgrowth into the spinal grey matter at any level. Concluding, the developing CST in the rat consists of 3 components: the first having its originating neurones in the anterior part of the cortex and its termination field in the cervical intumescence; the second with its originating neurones in the intermediate part of the cortex and its termination field predominantly in the lumbar enlargement, and a third transient one, originating in the posterior cortex and gradually disappearing from spinal cord levels. Research using anterograde tracing techniques in combination with electron microscopy is necessary to further analyse these 3 different components.

Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions

Voluntary locomotor training as induced by enriched housing of rats stimulates recovery of locomotion after spinal cord injury (SCI). Generally it is thought that spinal neural networks of motor‐ and interneurons located in the ventral and intermediate laminae within the lumbar intumescence of the spinal cord, also referred to as central pattern generators (CPGs), are the ‘producers of locomotion’ and play a pivotal role in the amelioration of locomotor deficits after SCI. It has been suggested that locomotor training provides locomotor‐specific sensory feedback into the CPGs, which stimulates remodeling of central nervous system pathways, including motor systems. Several molecules have been proposed to potentiate this process but the underlying mechanisms are not yet known. To understand these mechanisms, we studied the role of insulin‐like growth factor (IGF) I in functional recovery from SCI under normal and enriched environment (EE) housing conditions. In a first experiment, we discovered that subcutaneous administration of IGF‐I resulted in better locomotor recovery following SCI. In a second experiment, detailed analysis of the observed functional recovery induced by EE revealed full recovery of hindlimb coordination and stability of gait. This EE‐dependent functional recovery was attenuated by alterations in the pre‐synaptic bouton density within the ventral gray matter of the lumbar intumescence or CPG area. Neutralization of circulating IGF‐I significantly blocked the effectiveness of EE housing on functional recovery and diminished the EE‐induced alterations in pre‐synaptic bouton density within the CPG area. These results support the use of IGF‐I as a possible therapeutic aid in early rehabilitation after SCI.

Attempted endogenous tissue repair following experimental spinal cord injury in the rat: involvement of cell adhesion molecules L1 and NCAM?

It is widely accepted that the devastating consequences of spinal cord injury are due to the failure of lesioned CNS axons to regenerate. The current study of the spontaneous tissue repair processes following dorsal hemisection of the adult rat spinal cord demonstrates a phase of rapid and substantial nerve fibre in‐growth into the lesion that was derived largely from both rostral and caudal spinal tissues. The response was characterized by increasing numbers of axons traversing the clearly defined interface between the lesion and the adjacent intact spinal cord, beginning by 5u2003days post operation (p.o.). Having penetrated the lesion, axons became associated with a framework of NGFr‐positive non‐neuronal cells (Schwann cells and leptomeningeal cells). Surprisingly few of these axons were derived from CGRP‐ or SP‐immunoreactive dorsal root ganglion neurons. At the longest survival time (56u2003days p.o.), there was a marked shift in the overall orientation of fibres from a largely rostro‐caudal to a dorso‐ventral axis. Attempts to identify which recognition molecules may be important for these re‐organizational processes during attempted tissue repair demonstrated the widespread and intense expression of the cell adhesion molecules (CAM) L1 and N‐CAM. Double immunofluorescence suggested that both Schwann cells and leptomeningeal cells contributed to the pattern of CAM expression associated with the cellular framework within the lesion.

CORTICOSPINAL TRACT REGROWTH

The natural ability of the adult central nervous system of higher vertebrates to recover from injury is highly limited. This limitation is most likely due to an inhospitable environment and/or intrinsic incapacities of the neurons to re-extend their neurites after injury or axotomy. The rat corticospinal tract is the largest tract leading from brain to spinal cord and is often used as a model in developmental and regeneration studies. The extensive know-how of factors involved in the development of the corticospinal tract did provide the foundation for many studies on corticospinal tract regrowth after injury in the adult spinal cord. The results of these experiments, as discussed in this review, have led to important contributions to the further understanding of central nervous system regeneration.

Oxidant treatment causes a dose-dependent phenotype of apoptosis in cultured motoneurons

Evidence is growing that reactive oxygen species (ROS), by‐products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose‐dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose). J. Neurosci. Res. 54:778–786, 1998.

Axon guidance of outgrowing corticospinal fibres in the rat

This review is concerned with the development of the rat corticospinal tract (CST). The CST is a long descending central pathway, restricted to mammals, which is involved both in motor and sensory control. The rat CST is a very useful model in experimental research on the development of fibre systems in mammals because of its postnatal outgrowth throughout the spinal cord as well as its experimental accessibility. Hence mechanisms underlying axon outgrowth and subsequent target cell finding can be studied relatively easily. In this respect the corticospinal tract forms an important example and model system for the better understanding of central nervous system development in general.

Prevention of apoptotic motoneuron death in vitro by neurotrophins and muscle extract

In this study, it is shown that rat motoneurons in culture are highly dependent on trophic support and die in the absence of such support via active cell death, or apoptosis. This apoptotic death occurs in their in vitro life (within 24 h) and can be partially prevented by treating the cultures with neurotrophic substances. The most effective support comes from an extract prepared from embryonic chick muscle: without muscle extract, no healthy, neurite-bearing motoneurons are present, but with 0.3 and 1.2% muscle extract, their numbers increase dose-dependently. Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), cannot replace muscle extract and keep motoneurons alive on their own, but in the presence of muscle extract (0.3%) they have a significant effect on survival (increase up to four times, compared to 0.3% muscle extract). At the same time, they prevent apoptotic cell death. Selective motoneuron death has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Despite strong evidence that motoneurons in ALS die via apoptosis, this has not been shown unequivocally in post mortem material. This study shows that motoneurons are very sensitive to conditions that initiate apoptosis, and that cell death can be prevented to a large degree with relatively low concentrations of neurotrophic factors. In view of the fact that several patient trials with neurotrophic factors already are underway, studies with motoneurons in culture may prove important in understanding the mechanism of cell death and the efficacy of drugs such as neurotrophic factors, but also other types of drug.