Screening for immune biomarkers associated with infection or protection against Ehrlichia ruminantium by RNA-sequencing analysis.

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

A multi-epitope DNA vaccine co-administered with monophosphoryl lipid A adjuvant provides protection against tick transmitted Ehrlichia ruminantium in sheep.

Previously, a heartwater experimental DNA vaccine provided 100% protection following laboratory challenge with Ehrlichia ruminantium administered by needle but not against an E. ruminantium tick challenge in the field. A multi-epitope DNA vaccine incorporating both CD4+ and CD8+ cytotoxic T lymphocytes epitopes could provide a better alternative. In this study, we investigated the use of multi-epitope DNA vaccines against an E. ruminantium experimental tick challenge in sheep. The multi-epitope DNA vaccines were delivered via the intramuscular route and intradermal route using the gene gun in the presence of monophosphoryl lipid A (MPL) adjuvant, which was either applied topically to the gene gun inoculation site or co-administered with the vaccine via the intramuscular route. Initially two constructs namely, pSignal plus and pLamp were tested with MPL applied topically only and no protection was obtained in this formulation. However, when pLamp was co-administered with MPL via the intramuscular route in addition to topical application, its protective efficiency improved to protect 60% of the sheep against tick challenge. In this formulation, the vaccine induced enhanced activation of memory T cell responses both before and after challenge with variations amongst the different sheep possibly due to their different genetic backgrounds. In conclusion, this study showed that a heartwater multi-epitope DNA vaccine, co-administered with MPL adjuvant can protect sheep following a laboratory E. ruminantium tick challenge.

Screening of cardiovascular agents in plasma with LC-MS/MS: A valuable tool for objective drug adherence assessment.

Adherence to cardiovascular preventive agents is important to prevent short and long term cardiovascular events. Recently, qualitatively compound screening using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained interest for drug adherence assessment in patients at high risk of cardiovascular events. Therefore, we developed and tested an assay including 52 compounds and metabolites, covering over 95% of the antihypertensive and antithrombotic agents available worldwide. Trichloroacetic acid was used as simple and fast method for protein precipitation. The assay was validated for lower limit of quantification (LLOQ), linearity, stability for freeze/thaw, room temperature, autosampler and matrix effects. The LLOQ for each compound was targeted under the population trough concentration (PTC) as reported in literature to assure high sensitivity for adherence detection. This was accomplished for 50 of 52 compounds with a LLOQ equal or lower compared to the PTC. Linearity was confirmed for all compounds (r2 > 0.995), except for acetylsalicylic acid (r2 = 0.991). For room temperature stability, 12 compounds showed degradation over 20% after 20 h. 3 compounds suffer from matrix effect with recoveries < 50%. After analytical validation, blood samples from 91 patients with difficult-to-treat hypertension were analyzed. Patients were unaware of adherence assessment. Adherence varied largely per agent and per concentration ratio (CR) (ratio of the detected concentration with LC-MS/MS and the PTC) cut-off value. Additionally, stratification by adherence group showed that the percentage of patients classified as non-adherent increased from 6.6% for qualitative analysis (pos/neg) to 19.8% for a CR cut-off of 0.5. The data imply that using the CR cut off values has a significant and relevant effect on patient adherence classification.

Identification and characterisation of conserved epitopes of E. ruminantium that activate Th1 CD4+ T cells: Towards the development of a multi-epitope vaccine.

Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However, not much is known about Ehrlichia ruminantium epitopes, particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4+ Th1 epitopes that induce IFNγ, a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly, peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay, quantitative real-time PCR and flow cytometry. Of the 246 peptides, 23 peptides, Erum0660 (p0660-42), Erum1150 (p1150-18, p1150-19), Erum2540 (p2540-6, p2540-16, p2540-19, p2540-20, p2540-21), Erum5420 (p5420-13, p5420-14), Erum7140 (p7140-6, p7140-7, p7140-12, p7140-13, p7140-20), Erum7320 (p7320-8, p7320-9, p7320-21), Erum7350 (p7350-9), Erum7360 (p7360-8), Erum7620 (p7620-2, p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12, p7140-13, p7140-20, and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4+ T cells to rapidly proliferate and significantly increase IFN-γ production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1α, IL-2, IL-12p40, TNF-α, IFN-γ, inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro.

Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.

Innate immune transcriptomic evaluation of PBMC isolated from sheep after infection with E. ruminantium Welgevonden strain.

Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine-cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.

Spinal cord stimulation in experimental chronic painful diabetic polyneuropathy: Delayed effect of High-frequency stimulation.

BACKGROUND: Spinal cord stimulation (SCS) has been shown to provide pain relief in painful diabetic polyneuropathy (PDPN). As the vasculature system plays a great role in the pathophysiology of PDPN, a potential beneficial side-effect of SCS is peripheral vasodilation, with high frequency (HF) SCS in particular. We hypothesize that HF-SCS (500 Hz), compared with conventional (CON) or low frequency (LF)-SCS will result in increased alleviation of mechanical hypersensitivity in chronic experimental PDPN. METHODS: Diabetes was induced in 8-week-old female Sprague-Dawley rats with an intraperitoneal injection of 65 mg/kg of streptozotocin (n = 44). Rats with a significant decrease in mechanical withdrawal response to von Frey filaments over a period of 20 weeks were implanted with SCS electrodes (n = 18). Rats were assigned to a cross-over design with a random order of LF-, CON-, HF- and sham SCS and mechanical withdrawal thresholds were assessed with von Frey testing. RESULTS: Compared with sham treatment, the average 50% WT score for 5 Hz was 4.88 g higher during stimulation (p = 0.156), and 1.77 g higher post-stimulation (p = 0.008). CON-SCS resulted in 50% WT scores 5.7 g, and 2.51 g higher during (p = 0.064) and after stimulation (p < 0.004), respectively. HF-SCS started out with an average difference in 50% WT score compared with sham of 1.87 g during stimulation (p = 0.279), and subsequently the steepest rise to a difference of 5.47 g post-stimulation (p < 0.001). CONCLUSIONS: We demonstrated a delayed effect of HF-SCS on mechanical hypersensitivity in chronic PDPN animals compared with LF-, or CON-SCS. SIGNIFICANCE: This study evaluates the effect of SCS frequency (5-500 Hz) on mechanical hypersensitivity in the chronic phase of experimental PDPN. High frequency (500 Hz) - SCS resulted in a delayed effect- on pain-related behavioural outcome in chronic PDPN.

Medical specialists care and hospital costs for low back pain in the Netherlands.

BACKGROUND: To examine the organization of medical specialist care and hospital costs for low back pain (LBP) in the Netherlands. METHODS: Aggregated health insurance claims data were provided on all diagnosis treatment combination (DTC) declarations for LBP patients first referred to the hospital in the 2nd half of 2008 and retrieved from Vektis, an organization that collects health insurance claims data. Data were available up to 1 January 2012. The data included patient characteristics, DTC-specific information including: neurology, neurosurgery, orthopaedic surgery, anaesthesiology and radiology. RESULTS: In total 80,652 LBP patients were referred to the hospital for the first time in the second half of 2008 accumulating to a total of 173,620 DTC's with total costs of €194 million. Of these patients, 56% were female and 61% aged above 50 years at first referral to the hospital. The average number of DTC's and costs per patient were 2.15 and €2410, respectively (during the follow-up period of 3.0-3.5 year). Moreover, 51% of the patients needed only one DTC; less than 10% of patients needed 5 DTC's. Following the DTC number, the share of consultations to the neurologist decreased, whereas that of the anaesthesiology specialism increased. The largest portion of costs was allocated to the anaesthesiology and neurosurgery specialisms. CONCLUSIONS: In the Netherlands, LBP patients consult a variety of medical specialists, in particular, the neurology, hospital costs for LBP patients are expected to increase given the rising incidence. Optimal diagnosis and tailored treatment plans might result in reduced costs and improved patient outcomes. SIGNIFICANCE: Low back pain patients consult various specialists, with the majority first referred to the neurologist. More than half of the LBD patients require only one DTC and less than 10% needed five DTC's or more. The largest portion of the hospital costs for LBP patients is allocated to the anaesthesiology and neurosurgery specialisms.

[Multidisciplinary treatment of orofacial pain]. / Multidisciplinaire behandeling van aangezichtspijn.

The diagnosis and treatment of orofacial pain can be complex. The differential diagnosis is very extensive. Therefore, multidisciplinary diagnosis and treatment are often indicated. The diagnosis of chronic pain also entails the investigation of psychological factors. This is because psychological problems can play a role in the chronification of pain, but they can also be a consequence of chronic pain. Patients with persistent orofacial complaints should be seen by a medical team consisting of an oral and maxillofacial surgeon, a neurologist, an anaesthesiologist/pain specialist, a dentist-gnathologist, an orofacial physical therapist, and a psychologist or psychiatrist specialising in orofacial pain. Treatment options should be discussed, taking into account literature concerning their effectiveness. The general conclusion is that much research remains to be done into the causes of, and treatments for, orofacial pain.

African horse sickness virus serotype 4 antigens, VP1-1, VP2-2, VP4, VP7 and NS3, induce cytotoxic T cell responses in vitro.

It was shown in a previous study that proliferating CD8+ T cells could be detected in immune horse peripheral blood mononuclear cells (PBMC) when stimulated with African horse sickness virus serotype 4 (AHSV4). In this study the cytotoxicity of CD8+ T cells were tested by using the fluorescent antigen-transfected target cells-cytotoxic T lymphocytes (FATT-CTL) assay, for both the virus and its individual proteins expressed in Escherichia coli. This CTL assay measures the killing of viral protein expressing cells. AHSV proteins were successfully expressed in E. coli using the pET102/D-TOPO expression vector and the effector cells were stimulated with these recombinant proteins or with live viable virulent AHSV4. The AHSV genes were amplified and cloned into the pIRES-hrGFP II (pGFPempty) vector and these plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used to nucleofect PBMC, the target cells. The elimination of antigen-GFP expressing cells by CTL was quantified by flowcytometry. VP1-1, VP2-2, VP4, VP7 and NS3, antigen-specific CD8+ T cells resulted in cell lysis suggesting that CTL may play a role in the immune response induced against the AHSV4 vaccine strain.

Immune gene expression profiling of PBMC isolated from horses vaccinated with attenuated African horsesickness virus serotype 4.

Development of African horsesickness (AHS) subunit vaccines will have to include a rational approach that uses knowledge of how the virus interacts with the host immune system. The global in vivo immune response induced by attenuated AHSV serotype 4 in horses was characterised using transcriptome sequencing. PBMC were collected with 24h intervals for four days after inoculation and four days after a second boost, 21 days later. Transcriptome data were normalised to the day 0 naïve transcriptome and up- or down-regulated immune genes identified using the CLC workbench. Peak expression was observed 24h after each inoculation. Innate immunity was up-regulated after both inoculations and was characterised by type-1 interferon activation via the RIG-1/MDA5 pathway and the up-regulation of complement cascade components. After the second boost an adaptive immune response could be identified that included the production of cytokines indicative of T helper (Th)1, Th2 and Th17 responses.

Pain prevalence and its determinants after spinal cord injury: a systematic review.

Pain prevalence studies are important as they illustrate the magnitude of pain problems in a certain patient population, such as patients living with a spinal cord injury (SCI). Strikingly, reported pain prevalence rates in SCI patients are found to vary greatly, while determinants for the differences between pain prevalence reports remain unclear. We here aim to identify determinants for the differences (heterogeneity) in pain prevalence reports through a systematic review of all SCI pain prevalence reporting studies. Literature search was done using Medline, Cumulative Index to Nursing and Allied Health Literature, ISI Web of Knowledge and Embase. Data abstraction was performed while blinded and was followed by meta-(regression)-analyses. We identified 82 studies. Study design-related determinants of SCI pain prevalence reports were pain definition strictness (mild, moderate or high), primary study goal (pain study or not), data source (retrospective or not), and in a limited number of cases response/attrition rates. While correcting for these items, population characteristics correlating with pain prevalence rates were both proportion of patients with a depression and average time after injury (positive correlations). Between-study heterogeneity may remain even after the identification/correction of above-mentioned causes of heterogeneity.Pain after SCI does seem to relate to the duration of the injury and depression, yet major causes of bias in reported pain prevalence are found to be related to the primary study goal (pain study or not), choice of pain definition and the use of retrospective data.

The effect of spinal cord stimulation frequency in experimental painful diabetic polyneuropathy.

BACKGROUND: Spinal cord stimulation (SCS) has been shown to be an effective treatment for painful diabetic polyneuropathy (PDP). An increase of efficacy is needed since only 67% of patients benefit from SCS. This study aimed to develop an animal model for SCS in PDP and study the effect of various stimulation frequencies on the functional outcome. As the pathophysiology of PDP is complex, including vasoconstriction and nerve injury, the frequency of SCS may result in different outcomes. METHODS: Diabetes mellitus was induced by an intraperitoneal injection of streptozotocin in 8-week-old female Sprague-Dawley rats (n=76; glucose >15 mmol/L; n=51). A SCS device was implanted at level Th13 4 weeks later. SCS of the dorsal columns was applied for 30 min and the effect on mechanical hypersensitivity was evaluated. RESULTS: Mechanical hypersensitivity developed in 26 rats, which were included (low-frequency, n=6; mid-frequency, n=8; high frequency, n=9; and sham, n=3). SCS of the dorsal columns was applied for 40 min, and the effect on mechanical hypersensitivity was evaluated. In all treatment groups, SCS resulted in reversal of mechanical hypersensitivity and a clinically relevant reduction was achieved in 70% of animals. No differences in efficacy were found between the different treatment groups. CONCLUSIONS: The pain-relieving effect of SCS in PDP was studied in an experimental model. Our study shows that SCS on mechanical hypersensitivity in PDP rats is equally effective when applied at low, mid and high frequency.

Clinical course of non-specific low back pain: a systematic review of prospective cohort studies set in primary care.

BACKGROUND AND OBJECTIVE: Non-specific low back pain is a relatively common and recurrent condition for which at present there is no effective cure. In current guidelines, the prognosis of acute non-specific back pain is assumed to be favourable, but this assumption is mainly based on return to function. This systematic review investigates the clinical course of pain in patients with non-specific acute low back pain who seek treatment in primary care. DATABASES AND DATA TREATMENT: Included were prospective studies, with follow-up of at least 12 months, that studied the prognosis of patients with low back pain for less than 3 months of duration in primary care settings. Proportions of patients still reporting pain during follow-up were pooled using a random-effects model. Subgroup analyses were used to identify sources of variation between the results of individual studies. RESULTS: A total of 11 studies were eligible for evaluation. In the first 3 months, recovery is observed in 33% of patients, but 1 year after onset, 65% still report pain. Subgroup analysis reveals that the pooled proportion of patients still reporting pain after 1 year was 71% at 12 months for studies that considered total absence of pain as a criterion for recovery versus 57% for studies that used a less stringent definition. The pooled proportion for Australian studies was 41% versus 69% for European or US studies. CONCLUSIONS: The findings of this review indicate that the assumption that spontaneous recovery occurs in a large majority of patients is not justified. There should be more focus on intensive follow-up of patients who have not recovered within the first 3 months.

Pain relief and quality-of-life improvement after spinal cord stimulation in painful diabetic polyneuropathy: a pilot study.

BACKGROUND: Painful diabetic polyneuropathy (PDP) is associated with high pain scores and is difficult to treat. Therefore, spinal cord stimulation (SCS) has been suggested as second-line treatment. In this study, the feasibility and efficacy of SCS in PDP were investigated, as well as the predictive value of clinical sensory testing for the treatment outcome. METHODS: Fifteen patients with intractable PDP in the lower limbs were recruited. During lead implantation, the feasibility of achieving adequate paraesthesia coverage using one stimulation lead was investigated. If trial stimulation was successful, a definitive neurostimulator was implanted. Pain intensity was scored using an 11-point numeric rating scale and patients' global impression of change scale. Additionally, neuropathic pain characteristics, quality of life, sleep quality and mood were assessed. The predictive value of clinical sensory testing for the treatment outcome was analysed. RESULTS: Adequate paraesthesia coverage was achieved in 14 out of 15 patients. Clinically relevant pain relief was present in 11 patients after trial stimulation and 10 patients at 12 months. The quality of life was significantly increased at 2 weeks and 3 months in patients with successful SCS treatment. Several neuropathic pain characteristics and quality of sleep were improved at 2 weeks and 12 months. Preoperative clinical sensory testing did not differentiate between treatment responders from non-responders. CONCLUSIONS: SCS seems to be an efficacious and feasible treatment for intractable PDP. In this exploratory study, it was not possible to predict the treatment outcome using clinical sensory testing. These results justify performing a randomized clinical trial.

Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy.

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.

Decreased intracellular GABA levels contribute to spinal cord stimulation-induced analgesia in rats suffering from painful peripheral neuropathy: the role of KCC2 and GABA(A) receptor-mediated inhibition.

Elevated spinal extracellular γ-aminobutyric acid (GABA) levels have been described during spinal cord stimulation (SCS)-induced analgesia in experimental chronic peripheral neuropathy. Interestingly, these increased GABA levels strongly exceeded the time frame of SCS-induced analgesia. In line with the former, pharmacologically-enhanced extracellular GABA levels by GABA(B) receptor agonists in combination with SCS in non-responders to SCS solely could convert these non-responders into responders. However, similar treatment with GABA(A) receptor agonists and SCS is known to be less efficient. Since K⁺ Cl⁻ cotransporter 2 (KCC2) functionality strongly determines proper GABA(A) receptor-mediated inhibition, both decreased numbers of GABA(A) receptors as well as reduced KCC2 protein expression might play a pivotal role in this loss of GABA(A) receptor-mediated inhibition in non-responders. Here, we explored the mechanisms underlying both changes in extracellular GABA levels and impaired GABA(A) receptor-mediated inhibition after 30 min of SCS in rats suffering from partial sciatic nerve ligation (PSNL). Immediately after cessation of SCS, a decreased spinal intracellular dorsal horn GABA-immunoreactivity was observed in responders when compared to non-responders or sham SCS rats. One hour later however, GABA-immunoreactivity was already increased to similar levels as those observed in non-responder or sham SCS rats. These changes did not coincide with alterations in the number of GABA-immunoreactive cells. C-Fos/GABA double-fluorescence clearly confirmed a SCS-induced activation of GABA-immunoreactive cells in responders immediately after SCS. Differences in spinal dorsal horn GABA(A) receptor-immunoreactivity and KCC2 protein levels were absent between all SCS groups. However, KCC2 protein levels were significantly decreased compared to sham PSNL animals. In conclusion, reduced intracellular GABA levels are only present during the time frame of SCS in responders and strongly point to a SCS-mediated on/off GABAergic release mechanism. Furthermore, a KCC2-dependent impaired GABA(A) receptor-mediated inhibition seems to be present both in responders and non-responders to SCS due to similar KCC2 and GABA(A) receptor levels.

Spinal cord stimulation of dorsal columns in a rat model of neuropathic pain: evidence for a segmental spinal mechanism of pain relief.

Although spinal cord stimulation (SCS) of the dorsal columns is an established method for treating chronic neuropathic pain, patients still suffer from a substantial level of pain. From a clinical perspective it is known that the location of the SCS is of pivotal importance, thereby suggesting a segmental spinal mode of action. However, experimental studies suggest that SCS acts also through the modulation of supraspinal mechanisms, which might suggest that the location is unimportant. Here we investigated the effect of the rostrocaudal location of SCS stimulation and the effectiveness of pain relief in a rat model of chronic neuropathic pain. Adult male rats (n=45) were submitted to a partial ligation of the sciatic nerve. The majority of animals developed tactile hypersensitivity in the nerve lesioned paw. All allodynic rats were submitted to SCS (n=33) for 30 minutes (f=50 Hz; pulse width 0.2 ms). In one group (n=16) the electrodes were located at the level where the injured sciatic nerve afferents enter the spinal cord (T13), and in a second group (n=17) the electrodes were positioned at more rostral levels (T11) as verified by X-ray. A repositioning experiment of electrodes from T12 to T13 was performed in 2 animals. Our data demonstrate that SCS of the dorsal columns at the level where the injured fibers enter the spinal cord dorsal horn result in a much better pain-relieving effect than SCS at more rostral levels. From this we conclude that SCS in treatment of neuropathic pain acts through a segmental spinal site of action.

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.