Elbert A.J. Joosten

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.

The CatWalk method: a detailed analysis of behavioral changes after acute inflammatory pain in the rat.

Experimental pain research is often complicated by the absence of an objective and detailed method to analyze behavioral changes. In the present study, acute pain was induced into the right knee of the rat (n=15) through the injection of 2mg carrageenan (CAR) in saline. A control group received vehicle injection into the knee (n=15). With the use of an automated quantitative gait analysis system, the CatWalk, it was possible to quantitatively analyze behavioral changes at post-injection time 2.5, 4, 24 and 48h. The CatWalk analysis of individual paw parameters like the intensity of the paw print or the time contact with the floor showed a significant effect after CAR injection into the knee. These CatWalk parameters were highly correlated with von Frey data and thus representative for the development of mechanical allodynia. Furthermore, detailed CatWalk analysis of the gait (i.e. coordinated interaction between left and right hindlimb) showed very fine, accurate and significant coordination changes in the experimental rats from 4h post-injection. In conclusion, the CatWalk method allows an objective and detailed detection of both pain-induced gait adaptations as well as the development of mechanical allodynia in an acute inflammatory pain model.

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.

High frequency stimulation of the subthalamic nucleus improves speed of locomotion but impairs forelimb movement in parkinsonian rats

The subthalamic nucleus (STN) plays an important role in motor and non-motor behavior in Parkinsons disease, but its involvement in gait functions is largely unknown. In this study, we investigated the role of the STN on gait in a rat model of PD using the CatWalk method. Parkinsonian rats received bilateral high frequency stimulation (HFS) with different stimulation amplitudes of the STN. Rats were rendered parkinsonian by bilateral injections of 6-hydroxydopamine (6-OHDA) into the striatum. One group of 6-OHDA animals was implanted bilaterally with stimulation electrodes at the level of the STN. Stimulations were performed at 130 Hz (frequency), 60 micros (pulse width) and varying amplitudes of 0, 3, 30 and 150 microA. Rats were evaluated in an automated quantitative gait analysis method (CatWalk method). After behavioral evaluations, rats were killed and the brains processed for histological stainings to determine the impact of the dopaminergic lesion (tyrosine hydroxylase immunohistochemistry) and the localization of the electrode tip (hematoxylin-eosin histochemistry). Results show that bilateral 6-OHDA infusion significantly decreased (70%) the number of dopaminergic cells in the substantia nigra pars compacta (SNc). Due to 6-OHDA treatment, the gait parameters changed considerably. There was a general slowness. The most pronounced effects were seen at the level of the hind paws. Due to implantation of STN electrodes the step pattern changed. STN electrical stimulation improved the general slowness but induced slowing of the forelimb movement. Furthermore, we found that HFS with a medium amplitude significantly changed speed, the so-called dynamic aspect of gait. The static features of gait were only significantly influenced with low amplitude. Remarkably, STN stimulation affected predominantly the forepaws/limbs.

Macrophages and neurons are targets of retinoic acid signaling after spinal cord contusion injury.

The physiological reactions after spinal cord injury are accompanied by local synthesis of the transcriptional activator retinoic acid (RA). RA exerts its effects by binding to retinoic acid receptors (RAR) which heterodimerize with retinoid X receptors (RXR) and then act as ligand‐activated transcription factors. To identify possible cellular targets of RA we investigated protein levels and cellular distribution of retinoid receptors in the rat spinal cord at 4, 7, 14 and 21u2003days after a contusion injury. In the nonlesioned spinal cord, immunoreactivity for RARα, RXRα, RXRβ and RXRγ was localized in the cytosol of neurons, that of RXRα and RXRβ in astrocytes and that of RARα, RXRα and RXRγ in some oligodendrocytes. After contusion injury RARα and all RXRs appeared in the cell nuclei of reactive microglia and macrophages. This nuclear staining began at 4u2003days, was most prominent at 7 and 14u2003days and had decreased at 21u2003days after injury. A similar nuclear translocation was also observed for the RARα, RXRα and RXRβ staining in neurons situated around the border of the contusion. These observations suggest that RA participates as a signal for the physiological responses of microglia and neurons after CNS injury.

Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro

Olfactory ensheathing cells (OECs) together with olfactory nerve fibroblasts (ONFs) and neonatal astrocytes are potent stimulators of neurite growth in adulthood and during development, respectively. Since it is known that alignment of glial cells is important for the correct outgrowth of axon tracts, it was hypothesized that the alignment of glial cells stimulates directional and enhanced neurite outgrowth. Adult OEC/ONF and neonatal astrocytes were cultured either on biodegradable poly(d,l)-lactide matrices or in Petri dishes for 4 days. Thereafter neonatal cerebral cortical neurons were added. After a 2-days coculture period the cultures were fixed and processed for a combined MAP-2 and phosphorylated neurofilament (RT97) staining. The neurite growth (neurite elongation and neurite formation) and the neurite direction were assessed. We show that (1). OEC/ONF cultures are more potent in stimulating the length of the longest neurite of cocultured neurons, (2). alignment of glial is achieved in vitro on our biomatrices, (3). aligned glial/biomatrix complexes do not enhance neurite growth, and (4). aligned glial/biomatrix complexes direct neurite outgrowth. These data have significant implications for in vivo experiments focusing on glial transplantation. Transplanting glial/biomatrix complexes may stimulate the directional regrowth of severed axons across a lesion site.

Chronically injured corticospinal axons do not cross large spinal lesion gaps after a multifactorial transplantation strategy using olfactory ensheathing cell/olfactory nerve fibroblast-biomatrix bridges

Transplantation of mixed cultures containing olfactory ensheathing cell (OEC) and olfactory nerve fibroblasts (ONF) has been shown to stimulate regrowth of both acutely and chronically injured corticospinal (CS) axons across small spinal cord lesion gaps. Here, we used a multifactorial transplantation strategy to stimulate regrowth of chronically injured CS axons across large spinal cord lesion gaps. This strategy combined the transplantation of aligned OEC/ONF‐biomatrix complexes, as described previously (Deumens et al. [2004] Neuroscience 125:591–604), within the lesion gap with additional OEC/ONF injections rostral and caudal to the lesion site. We show an enhanced presence of injured CS axons directly rostral to the lesion gap, with no effects on injured CS axons at or caudal to the lesion gap. Furthermore, injured CS axons did not penetrate the OEC/ONF‐biomatrix complex within the lesion gap. The enhanced presence of CS axons rostral to the lesion gap was not accompanied by any recovery of behavioral parameters assessed with the BBB locomotor rating scale or CatWalk gait analysis. We conclude that our multifactorial transplantation strategy should be optimized to create an OEC/ONF continuum in the injured spinal cord and thereby stimulate regrowth of injured CS axons across large spinal lesion gaps.

The CatWalk method: Assessment of mechanical allodynia in experimental chronic pain

The CatWalk gait analysis system has recently been suggested as a rapid and objective alternative method over the von Frey test to assess mechanical allodynia in chronic neuropathic pain models. Our results demonstrate that no correlation exists between the development of mechanical allodynia and changes in CatWalk-gait parameters in a chronic inflammatory pain model. Hence, the use of the CatWalk in assessment of experimental chronic pain is discussed.

Enriched environment and the recovery from inflammatory pain: Social versus physical aspects and their interaction

In this study, we aimed at comparing the effect of the social versus the physical enrichment of the environment on inflammatory pain. Hence, a rat model of carrageenan-induced knee inflammation was used. Four housing conditions were investigated: a physically enriched environment (PE), a socially enriched environment (SE), an enriched environment (EE) (i.e. physically and socially enriched) and a restricted environment (RE) (i.e. non-physically or socially enriched housing). Mechanical allodynia was assessed using the von Frey test preoperatively and at day post-operative (DPO) 1, 3, 7, 10, 14, 17, 21, 24 and 28. Besides, anxiety was evaluated at DPO29, using the Elevated Plus-Maze test. Results show that RE housing resulted in a duration of mechanical allodynia of 4 weeks and of only 3 weeks in EE housing. Housing in a physically enriched environment also resulted in a reduction of the duration of mechanical allodynia of 1 week. Finally, if housed in a SE, the mechanical allodynia lasted for 3 weeks and an half. From these data, we conclude that both physical and social aspects of the environment are involved in the reduction of inflammatory pain duration, although the PE has a larger effect than the SE in this experimental setting. Interestingly, an inter-dependent relationship was noted between the PE and SE. Moreover, no significant difference in the rat anxiety was measured between groups, suggesting that the pain outcomes are likely not biased by the mean of potential housing condition-induced anxiety.