Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

BACKGROUND: Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating pain condition. The mechanisms of CRPS-I still remain poorly understood. We aim to explore expression profiles of genes relevant to pain and neuroinflammation mechanisms involved in CRPS-I. METHODS: The rat chronic post-ischemic pain (CPIP) model that mimics human CRPS-I was established. RNA-sequencing (RNA-Seq), qPCR, Western blot, immunostaining, and pharmacological studies were used for profiling gene changes in ipsilateral spinal cord dorsal horn (SCDH) of CPIP model rat and further validation. RESULTS: CPIP rats developed persistent mechanical allodynia in bilateral hind paws, accompanied with obvious glial activation in SCDH. RNA-Seq identified a total of 435 differentially expressed genes (DEGs) in ipsilateral SCDH of CPIP rats. qPCR confirmed the expression of several representative genes. Functional analysis of DEGs identified that the most significantly enriched biological processes of upregulated genes include inflammatory and innate immune response. We further identified NLRP3 inflammasome expression to be significantly upregulated in SCDH of CPIP rats. Pharmacological blocking NLRP3 inflammasome reduced IL-1ß overproduction, glial activation in SCDH as well as mechanical allodynia of CPIP rats. CONCLUSION: Our study revealed that immune and inflammatory responses are predominant biological events in SCDH of CPIP rats. We further identified NLRP3 inflammasome in SCDH as a key contributor to the pain and inflammation responses in CPIP rats. Thus, our study provided putative novel targets that may help to develop effective therapeutics against CRPS-I.

Complex regional pain syndrome type I after diphtheria-tetanus (Di-Te) vaccination.

Complex regional pain syndrome type I (CRPS I) is a disorder of one or more extremities characterized by pain, abnormal sensitivity (allodynia), swelling, limited range of motion, vasomotor instability, fatigue and emotional distress. The symptoms may be aggravated by even minor activity or weather change. It is usually provoked by injury, surgery or injection but in a small proportion of patients CRPS I develops without a clear causative event. There are several literature reports on CRPS after rubella and hepatitis B vaccination. We present a case of CRPS I affecting the left arm after diphtheria and tetanus (Di-Te) vaccination in the left deltoid muscle in a young girl having experienced profound emotional stress before the vaccination procedure. History data on previous minor trauma at the site of vaccination or emotional stress may necessitate temporary vaccination delay due to their proneness to impaired local or systemic immune response and CRPS as a complication of vaccination. If a child or an adult has prominent swelling and severe pain after vaccination, the diagnosis of CRPS I should be considered and if confirmed, the multidisciplinary treatment should start as soon as possible.

Status of immune mediators in complex regional pain syndrome type I.

Complex regional pain syndrome type I (CRPS-I) can affect an extremity after minor trauma or operation. The pathogenesis of this syndrome is unclear. It has clinical signs of severe local inflammation as a result of an exaggerated inflammatory response, but neurogenic dysregulation is also a contributor. Several studies investigated the role of inflammatory mediators and cytokines thus far; however, the results are heterogeneous and vary between different settings. This review summarizes recent study results that show a clear underlying inflammatory response at the local site, where systemic responses seem to be inconsistent. An induction of CRPS-like symptoms by application of neuroinflammatory mediators was shown recently. Local inflammation is involved in the pathophysiology of CRPS-I. We must expand our knowledge of pathophysiologic mechanisms, and we are still far away from using inflammatory markers in diagnosis and follow-up of CRPS-I.

Lymphocyte subsets and the role of TH1/TH2 balance in stressed chronic pain patients.

BACKGROUND: The complex regional pain syndrome (CRPS) and fibromyalgia (FM) are chronic pain syndromes occurring in highly stressed individuals. Despite the known connection between the nervous system and immune cells, information on distribution of lymphocyte subsets under stress and pain conditions is limited. METHODS: We performed a comparative study in 15 patients with CRPS type I, 22 patients with FM and 37 age- and sex-matched healthy controls and investigated the influence of pain and stress on lymphocyte number, subpopulations and the Th1/Th2 cytokine ratio in T lymphocytes. RESULTS: Lymphocyte numbers did not differ between groups. Quantitative analyses of lymphocyte subpopulations showed a significant reduction of cytotoxic CD8+ lymphocytes in both CRPS (p < 0.01) and FM (p < 0.05) patients as compared with healthy controls. Additionally, CRPS patients were characterized by a lower percentage of IL-2-producing T cell subpopulations reflecting a diminished Th1 response in contrast to no changes in the Th2 cytokine profile. CONCLUSIONS: Future studies are warranted to answer whether such immunological changes play a pathogenetic role in CRPS and FM or merely reflect the consequences of a pain-induced neurohumoral stress response, and whether they contribute to immunosuppression in stressed chronic pain patients.

HLA-DQ1 associated with reflex sympathetic dystrophy.

Reflex sympathetic dystrophy (RSD) is a relatively common disabling disorder of unknown pathophysiology. From a cohort of 52 patients, carefully selected to fulfill the recently formulated diagnostic criteria for RSD, venous blood samples were taken for typing of class I and II major histocompatibility antigens. The frequency of HLA-DQ1 was found to be significantly increased compared with control frequencies. The association provides an indication for an organic basis of RSD.

Mast cells are involved in inflammatory reactions during Complex Regional Pain Syndrome type 1.

BACKGROUND: The Complex Regional Pain Syndrome type 1 (CRPS1) is a complication of surgery or trauma but spontaneous development is also described. Although the pathogenesis remains debatable, afferent, efferent and central nervous system mechanisms are proposed. Recently we showed involvement of the proinflammatory cytokines IL-6 and TNFalpha which is direct evidence for an inflammatory process. Many types of cells, such as activated T lymphocytes, monocytes, macrophages and skin resident cells like mast cells, could contribute to the production of cytokines. Involvement of mast cells is relatively easy to detect by measurement of tryptase. AIM: To establish whether mast cells are involved in the inflammatory reactions during CRPS1. METHODS: Twenty patients fulfilling the Bruehl criteria with CRPS1 in one extremity were studied. Impairment was assessed by registration of pain and measurement of differences in temperature, volume and mobility between the involved and uninvolved extremity. Blisters were made with a suction method in order to determine cytokines and mast cell derived tryptase in the involved and uninvolved extremity. RESULTS: In the blister fluid a significant difference (median +/- interquartile range, Wilcoxon signed-ranks test P < 0.05) was found between the involved and uninvolved extremity in IL-6 [53.5 (17.3-225) versus 6.2 (2-20.3) pg/ml], TNFalpha [31 (15.5-131.5) versus 8 (4-39) pg/ml], and tryptase [37 (20.5-62.3) versus 12.5 (6.7-23.5) ng/ml]. There was a significant correlation (0.455) between the intensity of pain and tryptase levels in the involved extremity (Spearman's test, P < 0.05). CONCLUSION: Mast cells are involved in inflammatory reactions during the CRPS1. Mast cells could play a role in the production of cytokines such as TNFalpha.

Neuroimmune alterations in the complex regional pain syndrome.

This review focuses on some clinical aspects of the complex regional pain syndrome, such as oedema, local temperature changes and chronic pain, as a result of supposed neurogenic inflammation. Involvement of the immune system could imply the subsequent release of neuropeptides, pro-inflammatory cytokines and eicosanoids, which in turn leads to a complex cross-talk of primary and secondary generated mediators of inflammation. The development and application of drugs that act through selective receptor antagonism or enzymatic synthesis inhibition to prevent further stimulation of this cascade that could inevitably lead to chronicity of this disease are extensively discussed.

Neurogenic inflammation in an animal model of neuropathic pain.

Loose ligation of a rat sciatic nerve (chronic constriction injury (CCI) model) provokes signs and symptoms like those observed in reflex sympathetic dystrophy (RSD) patients. Primary afferent nociceptive C-fibers seem to be involved in an afferent orthodromic as well as in an efferent antidromic manner. In this study we hypothesize that consequent to development of antidromic impulses in C-nociceptive afferents, neuropeptides released from peripheral endings of these fibers, increase skin blood flow (SBF), vascular permeability, and tissue accumulation of polymorphonuclear leukocytes (PMNs). Collectively, these phenomena have been referred to as neurogenic inflammation. To investigate the presence of neurogenic inflammation in the CCI-model, we assessed skin blood flow (SBF) as well as the level of edema and accumulation of PMNs in muscle tissue obtained from the affected hindpaw. SBF was measured, by means of laser Doppler flowmetry, before ligation as well as at day 4 after ligation. At day 4, SBF measurements were performed before and after abolition of the capability of C-fibers to mediate a vasodilator response. To this end, capsaicin was applied perineurally. Increased vascular permeability was inferred from the level of edema of muscle tissue as determined by assessment of wet/dry weight ratios of muscle biopsies. PMN accumulation was investigated by enzymatic detection of myeloperoxidase (MPO) activity in muscle biopsies. Compared with preligation values, at day 4 SBF was increased more than twofold (p < 0.05). The latter response was annihilated by capsaicin application. Compared with sham operated controls, wet/dry ratios were higher in the ligated animals (1.104 vs. 1.068; p < 0.05). Likewise, when compared with sham operated controls, MPO activity was found to be increased in the ligated hindpaw (Optic Density 0.15 vs. 0.89; p < 0.001). In conclusion, the findings of this study indicate that loose ligation of a sciatic nerve induces an inflammatory response in the ipsilateral hindpaw, which most likely is mediated by release of neuropeptides from the peripheral endings of antidromically acting nociceptive C-fibres.

Evidence for immune system involvement in reflex sympathetic dystrophy.

Skin biopsies from patients with reflex sympathetic dystrophy were immunostained using a variety of antisera. An incidental finding with S100 staining was the presence of numerous Langerhans cells in the epidermis. All patients had significant pain at the time of biopsy, and all had symptoms refractory to treatment. The potential implications of this finding are discussed.

Tumor necrosis factor-alpha and interleukin-6 are not correlated with the characteristics of Complex Regional Pain Syndrome type 1 in 66 patients.

It was previously shown in a group of 9 patients with complex regional pain syndrome type 1 (CRPS1) that levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are higher in blister fluid from the involved side. We hypothesize that local inflammation is responsible for the characteristics of CRPS1. The aim of this study was to confirm the previous observation in a large group of CRPS1 patients, repeating the measurement of TNF-alpha and IL-6 in blister fluid. Furthermore, we sought to determine whether these cytokines are responsible for the characteristics of CRPS1 and characterize the relationship between cytokine levels and duration of the disease. Sixty-six patients with CRPS1 participated. Skin blisters were artificially induced for measurement of cytokines in both extremities. The following disease characteristics were assessed: pain and differences in temperature, volume, and mobility between the extremities. TNF-alpha and IL-6 levels were significantly higher in blister fluid from the involved side. However, cytokine levels did not correlate with the characteristics or duration of the disease. Our findings confirm the presence of local inflammation in a population of 66 patients in the first 2 years of CRPS1. Proinflammatory cytokines seem to be only partly involved in the pathophysiology of CRPS1, as indicated by the lack of coherence between TNF-alpha and IL-6 levels and the signs and symptoms of inflammation and disease duration. Other inflammatory mediators and mechanisms, such as central sensitization, are probably involved in the early stages of CRPS1.

Effect of anti-NGF antibodies in a rat tibia fracture model of complex regional pain syndrome type I.

Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia resembling the clinical characteristics of patients with complex regional pain syndrome type I (CRPS I). Nerve growth factor (NGF) has been shown to support nociceptive and other types of changes found in neuropathic pain models. We hypothesized that anti-NGF antibodies might reduce one or more of the CRPS I-like features of the rat fracture model. For our studies one distal tibia of each experimental rat was fractured and casted for 4 weeks. The rats were injected with anti-NGF or vehicle at days 17 and 24 post-fracture. Nociceptive testing as well as assessment of edema and hindpaw warmth were followed during this period. Molecular and biochemical techniques were used to follow cytokine, NGF and neuropeptide levels in hindpaw skin and sciatic nerves. Lumbar spinal cord Fos immunostaining was performed. Bone microarchitecture was measured using microcomputed tomography (microCT). We found that tibia fracture upregulated NGF expression in hindpaw skin and tibia bone along with sciatic nerve neuropeptide content. We also found nociceptive sensitization, enhanced spinal cord Fos expression, osteopenia and enhanced cytokine content of hindpaw skin on the side of the fracture. Anti-NGF treatment reduced neuropeptide levels in sciatic nerve and reduced nociceptive sensitization. There was less spinal cord Fos expression and bone loss in the anti-NGF treated animals. Conversely, anti-NGF did not decrease hindpaw edema, warmth or cytokine production. Collectively, anti-NGF reduced some but not all signs characteristic of CRPS illustrating the complexity of CRPS pathogenesis and NGF signaling.

Three brothers with algodystrophy of the hip.

We describe the clinical features of algodystrophy of the hip in 3 brothers, probably the first familial presentation of this disease to be reported. The symptoms and evolution of the disease are as usually described. The familial presentation suggests a genetic predisposition. HLA typing showed an identity of antigenic formula in the 3 brothers, a rare coincidence.

Reflex sympathetic dystrophy: is the immune system involved?

OBJECTIVE: Evaluation of immune system function in patients with reflex sympathetic dystrophy (RSD). DESIGN: Survey on blood samples obtained from RSD patients and from a randomly selected control group. The lymphocyte populations (T, B, NK cells), and the activated T cells (CD25, and HLA-Dr-positive CD4 and CD8 cells) were analyzed by flow cytometry with dual-color direct immunofluorescence after whole-blood lysis. Clinical chemistry parameters were analyzed in additional serum samples. SETTING: Tertiary care center (outpatient rehabilitation clinic). SUBJECTS: Thirteen patients (nine women) with RSD and a control group of 21 healthy individuals. MAIN OUTCOME MEASURES: The results of the flow cytometry analysis of RSD patients were related to those of the control subjects. Means were analyzed, and confidence intervals for differences of the means were calculated. The means of the clinical chemical analysis were related to local reference values. RESULTS: The flow cytometry analysis did not differ between RSD patients and healthy controls. Although in some patients an individual parameter of clinical chemical analysis differed from its reference value, all of the mean values were within reference limits. Stratification on medications with immunomodulatory effects and on probability of a definite diagnosis of RSD had no influence on the results. CONCLUSION: No association between immunologic indices and RSD was found. This finding is relevant, because recent theories stress that it is not the sympathetic nervous system but a local inflammatory reaction that is fundamental in the pathogenesis of RSD. The results of this study do not support this theory.