The association between genome-wide polymorphisms and chronic postoperative pain: a prospective observational study.

Chronic postoperative pain is common and can have a negative impact on quality of life. Recent studies show that genetic risk factors are likely to play a role, although only gene-targeted analysis has been used to date. This is the first genome-wide association study to identify single-nucleotide polymorphisms associated with the development of chronic postoperative pain based on two independent cohorts. In a discovery cohort, 330 women scheduled for hysterectomy were genotyped. A case-control association analysis compared patients without chronic postoperative pain and the 34 who had severe chronic postoperative pain 3 months after surgery. No single-nucleotide polymorphisms reached genome-wide significance, but several showed suggestive associations with chronic postoperative pain (p < 1 × 10-5 ). Single-nucleotide polymorphisms with significance p < 1 × 10-5 were followed up in a replication cohort consisting of 203 men and women scheduled for orthopaedic or abdominal surgery. Ten of these patients developed severe chronic postoperative pain. A single-nucleotide polymorphism in NAV3 was significantly replicated with chronic postoperative pain in the replication cohort (p = 0.009). Meta-analysis revealed that two loci (IQGAP1 and CRTC3) were significantly associated with chronic postoperative pain at 3 months (IQGAP1 p = 3.93 × 10-6 ß = 2.3863, CRTC3 p = 2.26 × 10-6 , ß = 2.4209). The present genome-wide association study provides initial evidence for genetic risk factors of chronic postoperative pain and supports follow-up studies.

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 Parkinson's 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.

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.

Effect of spinal cord stimulation in an animal model of neuropathic pain relates to degree of tactile "allodynia".

Spinal cord stimulation (SCS) is an established treatment for chronic neuropathic pain. However, in recent studies conflicting results regarding the effect of SCS were noted in a selected group of patients suffering from complex regional pain syndrome and mechanical allodynia. In the present study we investigated the pain relieving effect of SCS in a rat experimental model of neuropathic pain as related to the severity of mechanical allodynia. Adult male rats (n=45) were submitted to a unilateral sciatic nerve ligation. The level of allodynia was tested using the withdrawal response to tactile stimuli with the von Frey test. A portion of these rats developed marked tactile hypersensitivity in the nerve-lesioned paw (von Frey test), similar to "tactile allodynia" observed after nerve injury in humans. Prior to SCS treatment the rats were subdivided into three groups based on the level of allodynia: mild, moderate and severe. All allodynic rats were treated with SCS (n=27) for 30 min (f=50 Hz; pulse width 0.2 ms and stimulation at 2/3 of motor threshold) at 16 days post-injury. Our data demonstrate a differential effect of SCS related to the severity of the mechanical allodynia. SCS leads to a faster and better pain relief in mildly allodynic rats as compared with the more severely allodynic rats. Thus, we suggest that the selection and subdivision of patient groups similar to those defined in our experimental setting (mild, moderate and severe allodynic) may provide better pre-treatment prediction of possible therapeutic benefits of SCS.

Sexual differences in onset of disease and response to exercise in a transgenic model of ALS.

Transgenic mice that overexpress the mutant human SOD1 gene (hSOD1) serve as an animal model for amyotrophic lateral sclerosis (ALS). Age and sex are recognized as risk factors for ALS, but physical activity remains controversial. Therefore, we investigated the effect of exercise on the phenotype of male and female hSOD1 mice. Onset of disease, progression of disease and survival were measured in low-copy and high-copy hSOD1 mice that were randomized to an exercise or sedentary group. We found that onset of disease was different for the two sexes: significantly earlier in male than in female hSOD1 mice. Exercise delayed the onset of disease in female but not in male hSOD1 mice. Also, exercise delayed the total survival time in female high-copy hSOD1 mice. Muscle morphometry and motor neuron counts were similar in all experimental groups at the end of training. Sedentary female hSOD1 mice showed more frequently irregular estrous cycles suggesting a higher estrogen exposure in exercising female mice. These results suggest a possible neuroprotective effect of female sex hormones and support the view that ALS patients should not avoid regular exercise.

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.

Local acute application of BDNF in the lesioned spinal cord anti-inflammatory and anti-oxidant effects.

We studied the early anti-inflammatory and anti-oxidant effects of local application of BDNF after dorsal spinal cord transection in the adult rat. Both the distribution and accumulation of neutrophils and microglial cells in and around the lesion site (inflammatory response) and the accumulation of lipid peroxidation product 4-hydroxynonenal (HNE; oxidative damage) around the lesion was examined using immunohistochemical techniques. We demonstrate that BDNF application affects the microglial response in and around the lesion and results in a reduced lipid peroxidation as shown by HNE-immunoreactive staining around the lesion 48 h post-injury. The early anti-inflammatory and anti-oxidant effects of local BDNF-application into the lesioned spinal cord may contribute to the observed decreased loss of locomotor function of the hindlimbs 2 days after injury.

Successful pain relief in non-responders to spinal cord stimulation: the combined use of ketamine and spinal cord stimulation.

Although spinal cord stimulation (SCS) is an established therapy for chronic neuropathic pain, still 30% of patients do not respond adequately to trial stimulation. These so called "non-responders" do not receive a permanent implantation for pain relief. The induction and maintenance of central sensitization plays a pivotal role in (chronic) neuropathic pain and is thought to be the resultant of the activation of the N-methyl-d-aspartate (NMDA) receptor in the dorsal horn. Blocking the NMDA receptor through the use of the non-competitive blocker ketamine has shown to attenuate neuropathic pain, although the undesirable side effects limit its use. The present study was performed to examine whether the combination of SCS with an individually determined sub-effective dose of intrathecal (i.t.) ketamine could convert non-responders into responders in rats with chronic neuropathic pain. Rats received a partial ligation of the sciatic nerve for the induction of neuropathic pain. Animals with tactile hypersensitivity to von Frey monofilaments (n=15) received 30 min of SCS. Non-responders to SCS (n=8) received their individually determined sub-effective i.t. dose of ketamine followed by 30 min of SCS. No side effects of the sub-effective dose of ketamine could be noted. The combined treatment of SCS and sub-effective dose of i.t. ketamine in non-responders resulted in a significant reduction of the withdrawal threshold in all previous non-responders to SCS, thereby converting them into responders to SCS.

Transplacental transfer of remifentanil in the pregnant ewe.

BACKGROUND AND PURPOSE: While remifentanil can be used either during labour or fetal surgery, more should be known about the transplacental transfer of this opioid. The aim of this study was to investigate the placental transfer and haemodynamic effects of remifentanil after i.v. administration to pregnant ewes. EXPERIMENTAL APPROACH: Seven pregnant ewes received a continuous infusion of remifentanil (0.33 µg·kg⁻¹·min⁻¹) for 1 h, and maternal and fetal arterial blood samples were drawn at regular intervals during and up to 1 h after the discontinuation of the infusion. Haemodynamic parameters were monitored continuously. Blood gas samples were drawn at baseline and once during the infusion. KEY RESULTS: Peak maternal remifentanil plasma levels of 4.0 ± 0.9 ng·mL⁻¹ (mean ± SD) and peak fetal plasma levels of 0.4 ± 0.3 ng·mL⁻¹ were obtained. Remifentanil plasma levels dropped rapidly after the discontinuation of the infusion. The continuous infusion of remifentanil did not result in significant maternal or fetal haemodynamic changes. CONCLUSIONS AND IMPLICATIONS: Remifentanil rapidly passes through the placenta of pregnant ewes and although remifentanil concentrations stay significantly lower in the fetus compared with those in the mother, remifentanil can be detected in significant amounts in the fetus.

Spinal cord stimulation in a mouse chronic neuropathic pain model.

Objective. Development of a spinal cord stimulation (SCS) system in a mouse model of chronic neuropathic pain. Materials and Methods. Male C57BL/6 mice (N = 6) underwent a partial ligation of the sciatic nerve. Development of mechanical hyperalgesia was tested using the withdrawal response to tactile stimuli with the von Frey test. An SCS system was implanted on day 14. On day 16, the mice were stimulated for 30 min (f = 50 Hz; pulse width 0.2 msec and stimulation at 2/3 of motor threshold). Repeated measure analysis of variance (anova) and paired Student's t-test with Bonferroni correction were used to evaluate the development of mechanical hyperalgesia and the therapeutic effect of SCS. Results. Five out of six mice developed marked mechanical hyperalgesia in the nerve-lesioned paw that persisted for the duration of the study (16 days). No changes contralateral to the injury were observed. In four out of five mice, a successful implantation of the electrodes followed by stimulation was achieved. Then, SCS resulted in a fast and robust increase of withdrawal threshold back to pre-injury levels. After termination of the SCS, the withdrawal threshold of the ipsilateral paw slowly decreased. No effect of SCS on the contralateral paw was noted. Conclusion. The development of a mouse SCS system is described that is practical in use, is reproducible, and shows a comparative therapeutic effect in treatment of chronic neuropathic pain as reported in rat.

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.

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.

Intraspinal administration of an antibody against CD81 enhances functional recovery and tissue sparing after experimental spinal cord injury.

We previously demonstrated that the tetraspanin protein CD81 is up-regulated by astrocytes and microglia after traumatic spinal cord injury in rats and that CD81 is involved in adhesion and proliferation of cultured astrocytes and microglia. Since these reactive glial cells contribute to secondary damage and glial scar formation, we studied the effect of local administration of an anti-CD81 antibody in experimental spinal cord injury. Adult rats were subjected to a moderate spinal cord contusion injury and treated for 2 weeks with different doses of the anti-CD81 antibody AMP1 (0.5-5 microg/h) or non-immune IgG (5.0 microg/h). A technique was developed to infuse the antibodies directly into the lesion site via an intraspinal cannula connected to a pump. Functional recovery was monitored during 8 postoperative weeks by means of the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, the BBB subscore and Grid-walk test. At the end of the study, quantitative histology was performed to assess tissue sparing. Our data showed that by itself cannulation of the lesion site resulted in minimal functional and histological impairments. Application of 0.5 microg/h AMP1 resulted in a marked functional recovery (BBB 2 points; Grid-walk 30% less errors compared to control). This recovery was accompanied by an 18% increase in tissue sparing at the lesion epicentre. No gross histological changes in glial scarring were apparent. Our data demonstrate beneficial effects of an anti-CD81 antibody on functional recovery in spinal cord injured rats and suggest that this effect is mediated through a reduction in secondary tissue loss.

Collagen containing neonatal astrocytes stimulates regrowth of injured fibers and promotes modest locomotor recovery after spinal cord injury.

The use of collagen as a vehicle to transplant neonatal astroglial cells into the lesioned spinal cord of the adult rat allows a precise application of these cells into the lesion gap and minimizes the migration of the transplanted cells. This approach might lead to anatomical and functional recovery. In the present study, 20 adult female Wistar rats were subjected to a dorsal hemisection at thoracic spinal cord levels. Cultured cortical neonatal rat astrocytes were transplanted into the lesion with collagen as a vehicle (N = 10). Prior to transplantation, the cultured astroglial cells were labelled with fast blue. Control rats received collagen implants only (N = 10). During 1 month of survival time, functional recovery of all rats was continuously monitored. Histological data showed that the prelabelled astroglial cells survived transplantation and were localized predominantly in the collagen implant. Virtually no fast blue-labelled GFAP-positive astroglial cells migrated out of the implant into the adjacent host spinal cord. The presence of transplanted neonatal astroglial cells resulted in a significant increase in the number of ingrowing neurofilament-positive fibers (including anterogradely labeled corticospinal axons) into the implant. Ingrowing fibers were closely associated with the transplanted astroglial cells. The implantation of neonatal astroglial cells did result in modest temporary improvements of locomotor recovery as observed during open-field locomotion analysis (BBB subscore) or during crossing of a walkway (catwalk).