Jos Ijkema-Paassen

Transection of peripheral nerves, bridging strategies and effect evaluation

Disruption of peripheral nerves due to trauma is a frequently occurring clinical problem. Gaps in the nerve are bridged by guiding the regenerating nerves along autologous grafts or artificial guides. This review gives an overview on the different methods of nerve repair techniques. Conventional suturing techniques are discussed as well as the use of e.g. biological, synthetic, non-degradable or degradable nerve guides. Functional assessment showed that repair of a gap with a bio-degradable guide is superior to that with autologous grafts. But still, long lasting changes were observed in the Sciatic Function Index (SFI), abnormal walking patterns, disturbed Electro Myo Graphic (EMG) patterns, next to shifts in the histochemical properties of the muscles and longlasting abnormalities in neuromuscular contacts. These phenomena are explained by an at-random reinnervation. When transecting the nerve at young ages, this did not lead to enhanced recovery. Rearing rats operated at adult age in an enriched environment, also had no beneficial effect. Future research should aim at developing longer guides, possibly lined with Schwann cells, or additives, improving specific reinnervation of the former target areas.

Sciatic nerve transection in the adult rat: Abnormal EMG patterns during locomotion by aberrant innervation of hindleg muscles

The effects of lesions in the sciatic nerve were studied in adult rats. In the left hindleg, a segment 12 mm long was resected from the proximal part of the nerve, before the bifurcation into the peroneal and tibial nerves. This segment in a reversed orientation was used as a nerve graft. EMG patterns in the tibialis anterior and the gastrocnemius muscles at both sides were recorded during locomotion in six rats after recovery periods varying from 15 to 21 weeks. The specificity of axonal outgrowth was studied in nine rats by retrogradely labeling the motoneurons with unconjugated Cholera Toxin subunit B (CTB) after injections into the gastrocnemius, the soleus, and the tibialis anterior muscles at both sides. EMG patterns at the operated side were irregular and we often observed coactivation of the gastrocnemius and tibialis anterior muscle. Moreover, burst activity was badly adjusted to the phases of the stepcycle. Retrogradely labeling indicated that the pools of motoneurons innervating the respective muscles at the left side had increased in volume. Neuronal diameters were slightly decreased but a considerable decrease was observed in dendritic branching and dendrite bundles in the pools of the SOL and in the GC were absent. No consistent trends in neuronal numbers at the affected side in comparison to the right side were detected. We conclude that axons, sprouting from the proximal stump of the sciatic nerve, innervate the muscles aselectively and that the motoneurons of origin maintain their original activation pattern.

Functional assessment of sciatic nerve reconstruction: Biodegradable poly (DLLA-epsilon-CL) nerve guides versus autologous nerve grafts

The aim of this study was to compare functional nerve recovery after reconstruction with a biodegradable p(DLLA‐ϵ‐CL) nerve guide filled with modified denatured muscle tissue (MDMT), or an autologous nerve graft. We evaluated nerve recovery using walking track analysis (measurement of the sciatic function index [SFI]) and electrostimulation tests. Functional nerve recovery after reconstruction with a biodegradable p(DLLA‐ϵ‐CL) nerve guide filled with MDMT was faster when compared with nerve reconstruction using an autologous nerve graft. We conclude that in case of a short nerve gap in the rat, reconstruction can best be carried out using a p(DLLA‐ϵ‐CL) biodegradable nerve guide filled with MDMT.

Reinnervation of muscles after transection of the sciatic nerve in adult rats

Functional recovery after transection of the sciatic nerve in adult rats is poor, probably because of abnormalities in reinnervation. Denervation and reinnervation patterns were studied morphologically in the lateral gastrocnemius (LGC), tibialis anterior (TA), and soleus (SOL) muscles for 21 weeks after nerve transection (motor endplates by acetylcholinesterase staining; nerves by silver impregnation). Motor endplates in the TA showed improving morphology with age, and, at 21 weeks, three‐quarters of these were normal. Poorest recovery was observed in the SOL, as, at 21 weeks, only one‐third of the motor endplates had a normal morphology. Polyneuronal innervation initially was more pronounced in the SOL, but, at 21 weeks, 10% of the motor endplates in all three muscles were still polyneuronally innervated. Our results indicate important differences in the reinnervation of these three hindleg muscles, and, even at 5 months, abnormalities were still present. These factors may in part explain the abnormal locomotion in rats as well as the limited recovery of function observed clinically in humans after nerve transection.

CNS plasticity after hemicerebellectomy in the young rat. Quantitative relations between aberrant and normal cerebello-rubral projections

The double labelling technique has been applied to study the quantitative relations between aberrant and normal cerebello-rubral projections in rats hemicerebellectomized on the 2nd, 5th, 10th, 20th or 30th day after birth, respectively. In the group of rats lesioned on the 2nd day about 30% of cells in the lateral nucleus and 20% in the interpositus nuclei are labelled retrogradely by the fluorescent tracer injected in the aberrant ipsilateral projection area. These values decreased in the groups of rats operated on the 5th or 10th day and reached values of about 1-2% in rats lesioned on the 20th or 30th day. These values have been also found in control rats. Both in experimental rats and in control rats less than 0.5% of cells are double labelled from both projection areas. This implies that the aberrant cerebello-rubral fibres stem from separate parent cells and are not collaterals from normally projecting fibres.

Corticotropin-releasing factor and urocortin differentially modulate rat Purkinje cell dendritic outgrowth and differentiation in vitro

The precise outgrowth and arborization of dendrites is crucial for their function as integrators of signals relayed from axons and, hence, the functioning of the brain. Proper dendritic differentiation is particularly resonant for Purkinje cells as the intrinsic activity of this cell‐type is governed by functionally distinct regions of its dendritic tree. Activity‐dependent mechanisms, driven by electrical signaling and trophic factors, account for the most active period of dendritogenesis. An as yet unexplored trophic modulator of Purkinje cell dendritic development is corticotropin‐releasing factor (CRF) and family member, urocortin, both of which are localized in climbing fibers. Here, we use rat organotypic cerebellar slice cultures to investigate the roles of CRF and urocortin on Purkinje cell dendritic development. Intermittent exposure (12 h per day for 10 days in vitro) of CRF and urocortin induced significantly more dendritic outgrowth (45% and 70%, respectively) and elongation (25% and 15%, respectively) compared with untreated cells. Conversely, constant exposure to CRF and urocortin significantly inhibited dendritic outgrowth. The trophic effects of CRF and urocortin are mediated by the protein kinase A and mitogen‐activating protein kinase pathways. The study shows unequivocally that CRF and urocortin are potent regulators of dendritic development. However, their stimulatory or inhibitory effects are dependent upon the degree of expression of these peptides. Furthermore, the effects of CRF and urocortin on neuronal differentiation and re‐modeling may provide a cellular basis for pathologies such as major depression, which show perturbations in the expression of these stress peptides.

Corticotropin-releasing factor receptor types 1 and 2 are differentially expressed in pre- and post-synaptic elements in the post-natal developing rat cerebellum

Corticotropin‐releasing factor (CRF)‐like proteins act via two G‐protein‐coupled receptors (CRF‐R1 and CRF‐R2) playing important neuromodulatory roles in stress responses and synaptic plasticity. The cerebellar expression of corticotropin‐releasing factor‐like ligands has been well documented, but their receptor localization has not. This is the first combination of a light microscopic and ultrastructural study to localize corticotropin‐releasing factor receptors immunohistologically in the developing rat cerebellum. Both CRF‐R1 and CRF‐R2 were expressed in climbing fibres from early stages (post‐natal day 3) to the adult, but CRF‐R2 immmunoreactivity was only prominent throughout the molecular layer in the posterior cerebellar lobules. CRF‐R1 immunoreactivity was concentrated in apical regions of Purkinje cell somata and later in primary dendrites exhibiting a diffuse cytoplasmic appearance. In Purkinje cells, CRF‐R1 immunoreactivity was never membrane bound post‐synaptically in dendritic spines while CRF‐R2 immunoreactivity was found on plasmic membranes of Purkinje cells from post‐natal day 15 onwards. We conclude that the localization of these receptors in cerebellar afferents implies their pre‐synaptic control of the release of corticotropin‐releasing factor‐like ligands, impacting on the sensory information being transmitted from afferents. Furthermore, the fact that CRF‐R2 is membrane bound at synapses, while CRF‐R1 is not, suggests that ligands couple to CRF‐R2 via synaptic transmission and to CRF‐R1 via volume transmission. Finally, the distinct expression profiles of receptors along structural domains of Purkinje cells suggest that the role for these receptors is to modulate afferent inputs.

Development of Postural Muscles and Their Innervation

Control of posture is a prerequisite for efficient motor performance. Posture depends on muscles capable of enduring contractions, whereas movements often require quick, forceful muscle actions. To serve these different goals, muscles contain fibers that meet these different tasks. Muscles with strong postural functions mainly consist of slow muscle fibers with a great resistance against fatigue. Flexor muscles in the leg and arm muscles are mainly composed of fast muscle fibers producing relatively large forces that are rapidly fatigable. Development of the neuromuscular system continues after birth. We discuss in the human baby and in animal experiments changes in muscle fiber properties, regression from polyneural into mononeural innervation, and developmental changes in the motoneurons of postural muscles during that period. The regression of poly-neural innervation in postural muscles and the development of dendrite bundles of their motoneurons seem to be linked to the transition from the immature into the adult-like patterns of moving and postural control.

Muscle differentiation after sciatic nerve transection and reinnervation in adult rats

Reinnervation after peripheral nerve transections generally leads to poor functional recovery. In order to study whether changes in muscles might be a contributing factor in this phenomenon we studied muscle morphology and fibre type distributions after sciatic nerve transection in the rat hind limb. Proximally, before the bifurcation in the tibial and common peroneal nerve, a 12 mm segment of the sciatic nerve was resected, reversed and re-implanted as an autologous nerve graft. After survival periods of 7, 15 and 21 weeks the lateral gastrocnemius, tibialis anterior and soleus muscles were dissected, stained with mATP-ase, and fibre type distributions were studied. In addition, numbers of muscle fibres were counted, and cross sectional areas were calculated. After 7 weeks, cross sectional areas were decreased in all muscles. In the gastrocnemius and tibialis anterior muscles the fibre number remained unaltered but the hypotrophy had been reversed at later ages. The number of muscle fibres in the soleus muscle remained decreased over the entire period of observation. The percentages of type II fibres in the gastrocnemius and tibialis anterior muscles were decreased at 7 and 15 weeks but these again approached normal values at 21 weeks. The type I fibres, however, remained arranged in groups. In the soleus muscle a large increase in the percentage of type II muscle fibres was observed and this remained until 21 weeks. We conclude that a non-selective reinnervation and later readjustments by regression of polyneural innervation may in part explain the changes in distributions of various fibre types.

Regression of polyneural innervation in the human psoas muscle

During the early stages of mammalian ontogeny muscle fibres are innervated by more than one axon. This polyneural innervation is replaced by mononeural innervation in the course of development. The regression of polyneural innervation in the psoas muscle in the human is the topic of the present study. Innervation patterns were studied in fetuses from 15 1/2 weeks of post menstrual age (PMA) and in babies until 80 weeks PMA (40 weeks after term age) and compared to data from two adults. Motor endplates were stained by a combined acetylcholinesterase stain. Innervation patterns and motor endplate morphology were studied and the sizes of endplates were measured. As a main result of our study polyneural innervation of the psoas muscle remains at a level of about 2 endings per endplate (range 1-5 terminals) until 18-25 weeks PMA and decreases thereafter. From 52 weeks PMA (12 weeks post term) onwards, muscle fibres are predominantly mononeurally innervated. During development the morphology of the terminal patterns of the nerve endings becomes more complex and the size of endplates increases, implying that the adult pattern of muscle innervation is reached at the age at which a major functional transformation in the neurobehavioural repertoire occurs (i.e. the end of the second and the beginning of the third month.