Ronald Deumens

Modeling Parkinson's disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway.

Human idiopathic Parkinsons disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by degeneration of the dopaminergic neurons of the nigrostriatal pathway. Different 6-OHDA rat models of PD have been developed in which this toxin has been injected into different parts of the nigrostriatal pathway: (a) the medial forebrain bundle which leads to extensive dopamine (DA) depletion; (b) the substantia nigra pars compacta, which leads to more specific and moderate DA depletions; and (c) subregions of the caudate-putamen complex (CPu), which also leads to specific DA depletions. In this article we review the dopaminergic depletion and behavioral consequences of 6-OHDA lesions in the rat. It was examined whether the relation between DA depletion and behavioral deficits mimic idiopathic PD. In addition, it was evaluated which model most closely approximates the human situation, especially in relation to the stage of this progressive disease. It was concluded that with respect to the site of the lesion, rats with partial lesions of the ventrolateral CPu are the most appropriate models to study early and late stages of PD. The choice of the behavioral parameters determines the use of unilateral or bilateral lesions, although it is obvious that the bilateral model mimics the human situation more closely.

Repairing injured peripheral nerves: Bridging the gap.

Peripheral nerve injuries that induce gaps larger than 1-2 cm require bridging strategies for repair. Autologous nerve grafts are still the gold standard for such interventions, although alternative treatments, as well as treatments to improve the therapeutic efficacy of autologous nerve grafting are generating increasing interest. Investigations are still mostly experimental, although some clinical studies have been undertaken. In this review, we aim to describe the developments in bridging technology which aim to replace the autograft. A multi-disciplinary approach is of utmost importance to develop and optimise treatments of the most challenging peripheral nerve injuries.

The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection.

Functional repair of neurotmesis has been proven most challenging in regenerative medicine. Progress in this field has shown that functional repair not only requires axon regeneration, but also selectivity in target reinnervation. Although selectivity in target reinnervation still involves relatively unexplored avenues, evidence-based medicine, in the end, requires behavioral proof of repair. Therefore, there is a need for tests assessing behavioral deficits after neurotmesis. To date, behavioral tests for detecting both dynamic and static parameters are limited. The CatWalk gait analysis has been shown to detect a multitude of speed-controlled dynamic and static gait deficits after experimental spinal cord injury. Therefore, we here evaluated its use in detecting both dynamic and static gait deficits after neurotmesis. After rat sciatic nerve resection CatWalk testing was performed for 8 weeks. A large amount of dynamic and static gait parameters were detected to be immediately and severely affected in the ipsilateral paw, sometimes reaching levels of only 15% of those of the unaffected paw. We conclude that the CatWalk objectively detects dynamic and static gait impairments after sciatic nerve resection and future experiments are now required to prove which of these parameters are of particular interest to detect functional repair.

CatWalk gait analysis in assessment of functional recovery after sciatic nerve injury.

Following peripheral nerve injury repair, improved behavioural outcome may be the most important evidence of functionality of axon regeneration after any repair strategy. A range of behavioural testing paradigms have been developed for peripheral nerve injury research. Complete injury of the adult rat sciatic nerve is frequently used in combination with walking track analysis. Despite its wide-spread use, these walking track analyses are unsuitable for the simultaneous assessment of both dynamic and static gait parameters. Conversely, a novel automated gait analysis system, i.e. CatWalk can simultaneously measure dynamic as well as static gait parameters and, importantly, its easy to control for the speed of locomotion which can strongly affect gait parameters. In a previous study, CatWalk was already successfully used to examine deficits in both dynamic and static gait parameters using the sciatic nerve lesion model with a 1cm gap characterized by absence of recovery [Deumens R, Jaken RJ, Marcus MA, Joosten EA. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection. J Neurosci Methods 2007;164:120-30]. Using the sciatic nerve crush injury model (validated with the static sciatic index) and a follow-up period of 12 weeks, we now show that CatWalk can also measure behavioural recovery. In particular dynamic gait parameters, coordination measures, and the intensity of paw prints are of interest in detecting recovery as far as these parameters completely return to pre-operative values after crush injury. We conclude that CatWalk can be used as a complementary approach to other behavioural testing paradigms to assess clinically relevant behavioural benefits, with a main advantage that CatWalk demonstrates both static and dynamic gait parameters at the same time.

Polymers from functional macrolactones as potential biomaterials: enzymatic ring opening polymerization, biodegradation, and biocompatibility.

We systematically investigated a series of polymers derived from macrolactones, namely, pentadecalactone, hexadecalactone, and their unsaturated analogues ambrettolide and globalide as potential biomaterials. By enzymatic ring-opening polymerization these monomers can conveniently be polymerized to high molecular weight. The polymers are highly crystalline with melting points around 95 degrees C for the saturated polymers and lower melting points for the unsaturated polymers (46-55 degrees C). All polymers are nontoxic as measured by an MTT assay for metabolic cell activity of a 3T3 mouse fibroblast cell line. Degradation studies showed no hydrolytic or enzymatic degradability of the polymers, which was ascribed to the high crystallinity and hydrophobicity of the materials. The unsaturated polymers were cross-linked in the melt, yielding fully amorphous transparent materials with a gel content of 97%.

GRK2: A Novel Cell-Specific Regulator of Severity and Duration of Inflammatory Pain

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2+/− mice with an ∼50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre–Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Nav1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2+/− mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2+/− mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor α (TNF-α) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-α activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2+/− mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.

Cytocompatibility of a novel, longitudinally microstructured collagen scaffold intended for nerve tissue repair.

Traumatic injury to the nervous system induces functional deficits as a result of axonal destruction and the formation of scar tissue, cystic cavitation, and physical gaps. Bioengineering bridging materials should ideally act as cell carriers for the implantation of axon growth-promoting glia, as well as supporting integration with host cell types. Here, we describe the cytocompatibility of a novel, micro-structured porcine collagen scaffold containing densely packed and highly orientated channels that, in three-dimensional (3D) tissue culture, supports attachment, proliferation, aligned process extension, and directed migration by populations of glial cells (olfactory nerve ensheathing cells and astrocytes) and orientated axonal growth by neurons (differentiated human SH-SY5Y neuroblastoma cell line). The seeded glia required several weeks to penetrate deeply into the highly porous scaffold, where they adopted an orientated morphology similar to that displayed in simple 2D cultures. The direct interaction between SH-SY5Y-derived nerve fibers and the collagen scaffold also resulted in highly orientated axonal growth. It is likely that biocompatible scaffolds that are capable of promoting glial cell attachment, migration, and highly orientated process outgrowth will be important for future repair strategies for traumatically injured nervous tissues.

Olfactory ensheathing cells, olfactory nerve fibroblasts and biomatrices to promote long-distance axon regrowth and functional recovery in the dorsally hemisected adult rat spinal cord.

Cellular transplantation, including olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF), after experimental spinal cord injury in the rat has previously resulted in regrowth of severed corticospinal (CS) axons across small lesion gaps and partial functional recovery. In order to stimulate CS axon regrowth across large lesion gaps, we used a multifactorial transplantation strategy to create an OEC/ONF continuum in spinal cords with a 2-mm-long dorsal hemisection lesion gap. This strategy involved the use of aligned OEC/ONF-poly(D,L)-lactide biomatrix bridges within the lesion gap and OEC/ONF injections at 1 mm rostral and caudal to the lesion gap. In order to test the effects of this complete strategy, control animals only received injections with culture medium rostral and caudal to the lesion gap. Anatomically, our multifactorial intervention resulted in an enhanced presence of injured CS axons directly rostral to the lesion gap (65.0 +/- 12.8% in transplanted animals versus 13.1 +/- 3.9% in control animals). No regrowth of these axons was observed through the lesion site, which may be related to a lack of OEC/ONF survival on the biomatrices. Furthermore, a 10-fold increase of neurofilament-positive axon ingrowth into the lesion site as compared to untreated control animals was observed. With the use of quantitative gait analysis, a modest recovery in stride length and swing speed of the hind limbs was observed. Although multifactorial strategies may be needed to stimulate repair of large spinal lesion gaps, we conclude that the combined use of OEC/ONF and poly(D,L)-lactide biomatrices is rather limited.

The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves.

The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.

Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury

We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere-loaded hydrogels. Intense GFAP (astrocytes), low βIII tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals.