Long-Term Spinal Cord Stimulation Alleviates Mechanical Hypersensitivity and Increases Peripheral Cutaneous Blood Perfusion in Experimental Painful Diabetic Polyneuropathy.

OBJECTIVES: This study utilizes a model of long-term spinal cord stimulation (SCS) in experimental painful diabetic polyneuropathy (PDPN) to investigate the behavioral response during and after four weeks of SCS (12 hours/day). Second, we investigated the effect of long-term SCS on peripheral cutaneous blood perfusion in experimental PDPN. METHODS: Mechanical sensitivity was assessed in streptozotocin induced diabetic rats (n = 50) with von Frey analysis. Hypersensitive rats (n = 24) were implanted with an internal SCS battery, coupled to an SCS electrode covering spinal levels L2-L5. The effects of four weeks of daily conventional SCS for 12 hours (n = 12) or Sham SCS (n = 12) were evaluated with von Frey assessment, and laser Doppler imaging (LDI). RESULTS: Average paw withdrawal thresholds (PWT) increased during long-term SCS in the SCS group, in contrast to a decrease in the Sham group (Sham vs. SCS; p = 0.029). Twenty-four hours after long-term SCS average PWT remained higher in the SCS group. Furthermore, the SCS group showed a higher cutaneous blood perfusion during long-term SCS compared to the Sham group (Sham vs. SCS; p = 0.048). Forty-eight hours after long-term SCS, no differences in skin perfusion were observed. DISCUSSION: We demonstrated that long-term SCS results in decreased baseline mechanical hypersensitivity and results in increased peripheral blood perfusion during stimulation in a rat model of PDPN. Together, these findings indicate that long-term SCS results in modulation of the physiological circuitry related to the nociceptive system in addition to symptomatic treatment of painful symptoms.

Functional recovery, serotonergic sprouting, and endogenous progenitor fates in response to delayed environmental enrichment after spinal cord injury.

Environmental enrichment (EE) is a way to induce voluntary locomotor training that positively affects locomotor recovery after acute spinal cord injury (SCI). The beneficial effect on SCI outcome is thought to be based on enhanced plasticity in motor pathways, triggered by locomotor-specific sensory feedback to the spinal cord circuitry for locomotion (central pattern generators [CPGs]). In view of chronic SCI, we tested the hypothesis that EE improves motor outcome after SCI in the rat when started after a clinically relevant delay of 3 weeks. At the CPG level (i.e., the spinal L1-L2 level), where EE-related sensory feedback is processed, two key mechanisms of anatomical plasticity were examined: (1) serotonergic innervation, and (2) survival and differentiation of spinal cord progenitor cells. Delayed EE improved interlimb coordination, which was associated with an increased serotonergic innervation of the ventro-lateral grey matter within the L1-L2 segments. Although spinal cord progenitor cells were found to differentiate into both neurons and glial cells, EE did not affect their survival. These results show that EE induces a substantial improvement of motor outcome after SCI when commenced after a clinically-relevant delay. Increased serotonergic innervation of the lumbar CPG area is therefore suggested to play an important role in the EE-induced recovery of interlimb coordination.

Preoperative housing in an enriched environment significantly reduces the duration of post-operative pain in a rat model of knee inflammation.

The influence of the environment on clinical post-operative pain received recently more attention in human. A very common paradigm in experimental pain research to model the effect of housing conditions is the enriched environment (EE). During EE-housing, rats are housed in a large cage (i.e. social stimulation), usually containing additional tools like running wheels (i.e. physical stimulation). Interestingly, only postsurgical housing effect on post-operative pain was developed during clinical and experimental studies while little is known on the influence of preoperative housing. In this study, our aim was to investigate the influence of housing conditions prior to an operation on the development of post-operative pain, using a rat model of carrageenan-induced inflammatory pain. Four housing conditions were used: a 3-week pre-housing in standard conditions (S-) followed by a post-housing in an EE; a 3-week pre-housing in EE followed by a post-operation S-housing; a pre- and post-housing in EE; a pre- and post-S-housing. The development of mechanical allodynia was assessed by the means of the von Frey test, preoperatively and at day post-operative (DPO) 1, 3, 7, 10, 14, 17, 21, 24 and 28. Our results show that a 3-week preoperative exposure to EE leads to a significant reduction in the duration of the carrageenan-induced mechanical allodynia, comparable with a post-operative exposure to EE. Strikingly, when rats were housed in EE prior to as well as after the carrageenan injection into the knee, mechanical allodynia lasted only 2 weeks, as compared to 4 weeks in S-housed rats.

The effect of Spinal Cord Stimulation in mice with chronic neuropathic pain after partial ligation of the sciatic nerve.

The effect of Spinal Cord Stimulation (SCS) in chronic neuropathic pain is inversely related to the severity of mechanical allodynia and the underlying mechanisms are poorly understood. To understand these mechanisms further we aimed to develop a model of SCS in a neuropathic mouse. Further, the CatWalk analysis, which is claimed to be an improved test for mechanical allodynia and therapeutic intervention, was used to analyze the effect of SCS on mechanical allodynia. Male C57BL/6 mice (N=31) underwent partial ligation of the sciatic nerve. After 14days an electrode was implanted and the effect of SCS (N=22) on mechanical allodynia was tested. Unligated mice (N=8) also received SCS. Behavioral analysis was performed using von Frey filaments and the CatWalk system. The withdrawal threshold showed a significant decrease which remained over time. Changes in CatWalk parameters were observed after 2days, but tended to diminish during the next 14days. Thirty minutes of SCS resulted in a 100% response and return to pre-neuropathy levels of the withdrawal threshold. The effect of SCS on the withdrawal threshold was comparable for the most severe and milder allodynic animals. SCS did not affect any of the CatWalk parameters in all mice. In conclusion, we developed a model of SCS in a chronic neuropathic pain C57BL/6 mouse. The CatWalk gait analysis does not result in the detection of behavioral changes to SCS in mice with chronic neuropathic pain and control animals. This model allows future molecular-genetic studies on the mechanisms of SCS in chronic neuropathic pain.

Environmental housing affects the duration of mechanical allodynia and the spinal astroglial activation in a rat model of chronic inflammatory pain.

In this study, we aimed at investigating the effect of an enriched environment (EE) on the recovery from chronic inflammatory pain. Inflammatory pain was induced by the injection of 2 mg of carrageenan (CAR) into the right knee of male Sprague-Dawley rats (n=34). Rats were housed either singly (S-housed) or in an EE (EE-housed). The EE consisted of a large cage (L x W x H=2.0 x 1.0 x 0.8 m) containing various attributes (e.g. running wheels, shelter house, climbing frame). Withdrawal response to von Frey filament was used to assess mechanical allodynia at days post-operative (DPO) -1, 1, 7, 14, 21 and 28. S-housed animals showed a marked tactile sensitivity in the ipsilateral paw from DPO1 to DPO21. Four weeks after the CAR injection, S-housed rats were no longer allodynic. In contrast, EE-housed rats showed a significantly faster recovery: already at DPO21, they were no longer allodynic. In a first attempt to analyse the possible role of astroglial cells in the EE-induced effect, histological analysis at DPO21 was performed. Immunohistochemical staining of the spinal dorsal horn at L3-L5 indeed showed that spinal levels of astroglial activation are different between the two housing groups and therefore may play a role in the EE-induced effect on the duration of mechanical allodynia. In conclusion, our results showed that EE-housing results in a reduced duration of mechanical allodynia in chronic inflammatory pain in rats. Astroglial activation is suggested to be involved in this housing effect.

Acute rolipram/thalidomide treatment improves tissue sparing and locomotion after experimental spinal cord injury.

Traumatic spinal cord injury (SCI) causes severe and permanent functional deficits due to the primary mechanical insult followed by secondary tissue degeneration. The cascade of secondary degenerative events constitutes a range of therapeutic targets which, if successfully treated, could significantly ameliorate functional loss after traumatic SCI. During the early hours after injury, potent pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) are synthesized and released, playing key roles in secondary tissue degeneration. In the present investigation, the ability of rolipram and thalidomide (FDA approved drugs) to reduce secondary tissue degeneration and improve motor function was assessed in an experimental model of spinal cord contusion injury. The combined acute single intraperitoneal administration of both drugs attenuated TNF-alpha and IL-1beta production and improved white matter sparing at the lesion epicenter. This was accompanied by a significant (2.6 point) improvement in the BBB locomotor score by 6 weeks. There is, at present, no widely accepted intervention strategy that is appropriate for the early treatment of human SCI. The present data suggest that clinical trials for the acute combined application of rolipram and thalidomide may be warranted. The use of such "established drugs" could facilitate the early initiation of trials.

Spinal cord stimulation induces c-Fos expression in the dorsal horn in rats with neuropathic pain after partial sciatic nerve injury.

Spinal cord stimulation (SCS) is an established treatment for intractable neuropathic pain, especially CRPS-1. The mechanisms of action of SCS have only been partly elucidated and include suppression of the hyper-excitability of the Wide Dynamic Range neurons and a GABA increase in the dorsal horn. In the present study we demonstrate an increase of c-Fos immunoreactive cells in the dorsal horn after SCS, suggesting early cellular activation that may preclude earlier described electrophysiological and biochemical changes in the dorsal horn after SCS. In a rat model of neuropathic pain, allodynia was induced and quantified using the von Frey test. In 11 rats a SCS device was implanted and spinal cord stimulation performed. Withdrawal threshold were measured every 15 min up to 90 min. A sham group (n=6) also had a SCS device implanted, but did not receive SCS. After SCS the animals were perfused and histology was performed for quantification of c-Fos immunoreactivity in the dorsal horns. We found a significant increase in c-Fos in the SCS group compared to our sham group and control tissue, indicating late cellular activity in the dorsal horn after SCS.

Strain and locomotor speed affect over-ground locomotion in intact rats.

A variety of animal models for neurological disease and injury exist and locomotor performance is an important outcome parameter in studies employing these models. The CatWalk, an automated quantitative gait analysis method is a method to study over-ground locomotor performance in large groups of animals. In the present study, we used the CatWalk which allowed us to investigate strain differences in over-ground locomotion in three commonly used strains of laboratory rat (i.e. Lewis, Wistar and Sprague-Dawley rats) based on objective data-analysis in a large number of animals. The present results revealed marked strain differences on the static paw parameters; base-of-support, and the relative paw position. Furthermore, strain differences were noted on the static parameter stride length and the dynamic parameters stance-, swing- and stepcycle duration, which are due logically to morphological differences between strains. The parameters related to coordination did not reveal any differences between the strains. Furthermore, the swing duration and the cruciate and alternate patterns i.e. regular step patterns Ca ("cruciate" pattern type a) and Ab ("alternate" pattern type b) were shown to be differentially affected by the locomotor speed. We conclude that differences in gait traits exist between the three laboratory rat strains investigated and several of the examined gait parameters showed strain dependent interdependency with locomotor speed.

Mice lacking L1 have reduced CGRP fibre in-growth into spinal transection lesions.

Repair strategies for spinal cord injury often focus on promoting regeneration of injured axons and stimulating subsequent functional recovery. Although many of these strategies have proven their merits, less is known about potential unwanted side-effects, such as sprouting of nociceptive CGRP immunoreactive axons, which may bring about pain-related behavior. Sprouting of CGRP axons into lesion sites spontaneously occurs after spinal cord injury (SCI). Using L1-deficient mice we show a reduction of such CGRP growth response. This reduction was specific for CGRP axons since the overall neurofilament positive fibre in-growth into the spinal lesion site was not affected. Our results may have important implications on the development and assessment of repair strategies that should not only stimulate functional recovery, but also prevent the development of pain or autonomic dysreflexia.

Limitations in transplantation of astroglia-biomatrix bridges to stimulate corticospinal axon regrowth across large spinal lesion gaps.

Regrowth of injured axons across rather small spinal cord lesion gaps and subsequent functional recovery has been obtained after many interventions. Long-distance regeneration of injured axons across clinically relevant large spinal lesion gaps is relatively unexplored. Here, we aimed at stimulating long-distance regrowth of the injured corticospinal (CS) tract. During development, an oriented framework of immature astrocytes is important for correct CS axon outgrowth. Furthermore, a continuous growth promoting substrate may be needed to maintain a CS axon regrowth response across relatively large spinal lesion gaps. Hence, we acutely transplanted poly(D,L)-lactide matrices, which after seeded with immature astrocytes render aligned astrocyte-biomatrix complexes (R. Deumens, et al. Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro. Neuroscience 125 (3) (2004) 591-604), into 2-mm long dorsal hemisection lesion gaps. In order to create a growth promoting continuum, astrocyte suspensions were also injected rostral and caudal to the lesion gap. During 2 months, locomotion was continuously monitored. Histological analysis showed that astrocytes injected into host spinal tissue survived, but did not migrate. None of the astrocytes on the biomatrices survived within the lesion gap. BDA-labeled CS axons did not penetrate the graft. However, directly rostral to the lesion gap, 120.9+/-38.5% of the BDA-labeled CS axons were present in contrast to 12.8+/-3.9% in untreated control animals. The observed anatomical changes were not accompanied by locomotor improvements as analyzed with the BBB and CatWalk. We conclude that although multifactorial strategies may be needed to stimulate long-distance CS axon regrowth, future studies should focus on enhancing the viability of cell/biomatrix complexes within large spinal lesion gaps.

Neurite outgrowth promoting effects of enriched and mixed OEC/ONF cultures.

Olfactory ensheathing cell (OEC) transplants stimulate axon regeneration and partial functional recovery after spinal cord injury. However, it remains unclear whether enriched OEC or mixed transplants of OEC and olfactory nerve fibroblasts (ONF) are optimal for stimulating axon regrowth. The neurite outgrowth stimulating effects of enriched OEC, ONF, and mixed OEC/ONF cultures on neonatal cerebral cortical neurons were compared using co-cultures. We show that (1) OEC are more neurite outgrowth promoting than ONF, and (2) ONF do not enhance the neurite outgrowth stimulating effects of OEC in mixed OEC/ONF cultures. Hence, our data indicate that there is no preference for the use of enriched OEC or mixed OEC/ONF cultures with respect to stimulation of neurite growth in vitro.

The assessment of locomotor function in spinal cord injured rats: the importance of objective analysis of coordination.

The Basso, Beattie and Bresnahan (BBB) locomotor rating scale is the most widely used open field test and has been accepted as a valid way to assess locomotor function after spinal cord contusion injury in the rat. A limitation within the BBB locomotor rating scale is the correct assessment of forelimb (FL)-hindlimb (HL) coordination. This limitation can have major implications for the final assessment of locomotor function. In the present study, we show an objective method to assess coordination based on the regularity index (RI), achieved through the use of the CatWalk method. The RI grades the degree of coordination as the result of the number of normal step sequence patterns multiplied by four and divided by the total amount of paw placements. Using the RI, single walkway crossings can be objectively analyzed on coordination. Integration of the CatWalk based coordination into the BBB scale indicates that objective analysis of coordination results in reliable and more sensitive assessment of locomotor function. This new method has been tested successfully in determination of positive effects of enriched housing on functional recovery after spinal cord injury (SCI).

Pulsed and continuous radiofrequency current adjacent to the cervical dorsal root ganglion of the rat induces late cellular activity in the dorsal horn.

BACKGROUND: Pulsed radiofrequency treatment has recently been described as a non-neurodestructive or minimally neurodestructive alternative to radiofrequency heat lesions. In clinical practice long-lasting results of pulsed radiofrequency treatment adjacent to the cervical dorsal root ganglion for the management of chronic radicular spinal pain have been reported without neurologic complications. However, the mode of action is unclear. An early (3 h) effect of pulsed radiofrequency as measured by an increase of c-Fos in the pain-processing neurons of the dorsal horn of rats has been described in the literature. This effect was not mediated by tissue heating. The authors investigated a possible late or long-term effect of three different radiofrequency modalities. METHODS: Cervical laminectomy was performed in 19 male Wistar rats. The cervical dorsal root ganglion was randomly exposed to one of the four interventions: sham, continuous radiofrequency current at 67 centigrades, or pulsed radiofrequency current for 120 s or 8 min. The animals were sacrificed and the spinal cord was prepared for c-Fos labeling 7 days after the intervention. RESULTS: The number of c-Fos immunoreactive cells in the dorsal horn was significantly increased in the three different radiofrequency modalities as compared with sham. No significant difference was demonstrated between the three active intervention groups. CONCLUSIONS: The authors demonstrated a late neuronal activity in the dorsal horn after exposure of the cervical dorsal root ganglion to different radiofrequency modalities, which was not temperature dependent.

cGMP, but not cAMP, in rat hippocampus is involved in early stages of object memory consolidation.

The present study investigates the role of cGMP and cAMP on the memory performance in the object recognition task in rats. The analogue 8-Br-GMP or 8-Br-cAMP was administered bilaterally into the hippocampus (0, 1, 3 and 10 microg in 0.5 microl saline/site) immediately after the exposure to two identical objects. After 24 h, saline-treated animals spent equal times exploring a new and the familiar object demonstrating that they did not recognize the familiar one. However, a dose-dependent improvement in object recognition was found after injection of 8-Br-cGMP. In contrast, 8-Br-cAMP did not improve the memory performance at the doses tested. These results indicate that hippocampal cGMP but not cAMP is involved in early stages of consolidation of object memory.