Florence Sjögren

A microdialysis technique for routine measurement of macromolecules in the injured human brain.

OBJECTIVE: To evaluate a new intracerebral microdialysis catheter with a high-cutoff membrane and its potential for the study of macromolecules in the human brain. METHODS: Paired intracerebral microdialysis catheters were inserted in 10 patients who became comatose after subarachnoid hemorrhage or traumatic brain injury and were then treated in our neurosurgical unit. The only differences from the routine use of microdialysis in our clinic were the length (20 mm) and cutoff properties of the catheter membranes (100 kD) and the perfusion fluids used (standard perfusion fluid, 3.5% albumin, or Ringer-dextran 60). Samples were weighed (for net fluid fluxes) and analyzed at bedside (for routine metabolites) and later in the laboratory (for total protein and interleukin-6). The in vitro recovery of glucose, glutamate, and glycerol were also investigated under different conditions. RESULTS: Even brief perfusion with standard perfusion fluid resulted in a significant loss of volume from the microdialysis system. For albumin and Ringer-dextran 60 fluid, recovery was comparable to standard settings. Interleukin-6 (highest value close to 25,000 pg/ml) was sampled from all catheters, and total protein was analyzed from catheters perfused with Ringer-dextran 60 (average concentration, 234 &mgr;g protein/ml). There were detectable patterns of variations in the concentration of interleukin-6, seemingly related to concomitant variations in intracerebral conditions. In the present study, no direct comparison was made with the standard CMA 70 catheter (CMA Microdialysis, Stockholm, Sweden), but in vivo, the measured mean concentrations of glucose, glycerol, lactate, and pyruvate were comparable to those previously reported from standard catheters. In vitro, the recovery of metabolites was better when using Ringer-dextran 60 compared with albumin. CONCLUSION: Microdialysis catheters with high-cutoff membranes can be used in routine clinical practice, allowing for sampling and analysis of cytokines and other macromolecules.

Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis.

Summary Background  Cutaneous microdialysis in vivo in human skin is demonstrably of use in the study of skin metabolism, percutaneous absorption and skin inflammation. A promising area for cutaneous microdialysis is the measurement of cytokines. This requires catheters equipped with membranes permeable to molecules of high molecular weight.

Changes in extracellular concentrations of some cytokines, chemokines, and neurotrophic factors after insertion of intracerebral microdialysis catheters in neurosurgical patients.

OBJECTIVEThe extracellular levels of eight different inflammatory agents were analyzed during the initial 36 hours after insertion of microdialysis catheters in patients. METHODSCerebral extracellular fluid from 38 patients who were treated in a neurosurgical intensive care unit for severe brain injury was collected every 6 hours for 36 hours. The concentration of interleukin (IL)-1β, IL-6, IL-8, macrophage inflammatory protein-1β, regulated on activation, normal T-cell expressed and secreted (RANTES), fibroblast growth factor-2, and vascular endothelial growth factor was determined by a multiplex assay, and IL-10 was determined by enzyme-linked immunosorbent assay. RESULTSThis is the first report regarding the presence of IL-10, IL-8, macrophage inflammatory protein-1β, regulated on activation, T-cell expressed and secreted, vascular endothelial growth factor, and fibroblast growth factor-2 in the tissue level proper of the living human brain. The study also provides new information regarding the response of IL-1β and IL-6 after insertion of a microdialysis catheter. The study confirms that the intriguing patterns of interplay between different components of the inflammatory response studied in laboratory settings are present in the human brain. This was most clearly observed in the variations in response between the three different chemokines investigated, as well as in the rapid and transient response of fibroblast growth factor-2. CONCLUSIONThe data presented illustrate the opportunity to monitor biochemical events of possible importance in the human brain and indicate the potential of such monitoring in neurosurgical intensive care. The study also underlines that any analysis of events in the brain involving mechanical invasiveness needs to take into account biochemical changes that are directly related to the manipulation of brain tissue.

Differences in cerebral extracellular response of interleukin-1β, interleukin-6, and interleukin-10 after subarachnoid hemorrhage or severe head trauma in humans.

BACKGROUND:Microdialysis has become a routine method for biochemical surveillance of patients in neurosurgical intensive care units. OBJECTIVE:To analyze the intracerebral extracellular levels of 3 interleukins (ILs) during the 7 days after major subarachnoid hemorrhage or traumatic brain injury). METHODS:Microdialysate from 145 severely injured neurosurgical intensive care unit patients (88 with subarachnoid hemorrhage, 57 with traumatic brain injury) was collected every 6 hours for 7 days. The concentrations of IL-1β and IL-6 were determined by fluorescence multiplex bead technology, and IL-10 was determined by enzyme-linked immunosorbent assay. RESULTS:Presented are the response patterns of 3 ILs during the first week after 2 different types of major brain injury. These patterns are different for each IL and also differ with respect to the kind of pathological impact. For both IL-1β and IL-6, the initial peaks (mean values for all patients at day 2 being 26.9 ± 4.5 and 4399 ± 848 pg/mL, respectively) were followed by a gradual decline, with IL-6 values remaining 100-fold higher compared with IL-1β. Female patients showed a stronger and more sustained response. The response of IL-10 was different, with mean values less than 23 pg/mL and with no significant variation between any of the postimpact days. For all 3 ILs, the responses were stronger in subarachnoid hemorrhage patients. The study also indicates that under normal conditions, IL-1β, IL-6, and IL-10 are present only at very low concentrations or not at all in the extracellular space of the human brain. CONCLUSION:This is the first report presenting in some detail the human cerebral response of IL-1β, IL-6, and IL-10 after subarachnoid hemorrhage and traumatic brain injury. The 3 ILs have different reaction patterns, with the response of IL-1β and IL-6 being related to the type of cerebral damage sustained, whereas the IL-10 response was less varied.

The spectrum of inflammatory cell response to dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), depending upon the concentration and mode of application to the skin, can induce either a non‐immunological immediate contact urticaria or an irritant reaction. The dermal cellular infiltrate after open application of varying concentrations of DMSO has been studied in an experimental guinea pig model. The composition of the dermal cellular infiltrate showed a spectrum dependent on the concentration and number of applications of DMSO. The immediate reaction infiltrate 3 h after application of 100% DMSO consisted of 50% granulocytes, basophils being predominant. On the other hand, 12% DMSO applied 3 × daily for 3 days (cumulative insult) caused histologically a cellular reaction in which 80% of the infiltrate consisted of mononuclear cells. The present findings are compared to the microscopic findings in 3 other cutaneous reactions previously studied in this animal model, namely, the Type I immediate hypersensitivity reaction, the Type IV delayed hypersensitivity reaction, and the irritant reaction. Differing cellular infiltrate patterns are discernible at the same time points. The study illustrates the spectrum of inflammatory reactions seen in the skin and provides background information for future clinical studies, for instance, on the role of the basophil granulocyte in immediate contact reactions.

Intracerebral microdialysis in neurosurgical intensive care patients utilising catheters with different molecular cut-off (20 and 100 kD)

SummaryObjective. To compare the properties of a new intracerebral micro-dialysis catheter with a high cut-off membrane (molecular cut-off 100 kDalton) with a standard catheter (CMA70, molecular cut-off 20 kDalton).Methods. Paired intracerebral microdialysis catheters were inserted in fifteen comatose patients treated in a neurosurgical intensive care unit following subarachnoid haemorrhage or traumatic brain injury. The high-cut-off catheter (D100) differed from the CMA 70 catheter by the length (20 mm) and cut-off properties of the catheter membranes (100 kDalton) and the perfusion fluids used (Ringer-Dextran 60). Samples were collected every 4–6 hours, analyzed bedside (for glucose, glutamate, glycerol, lactate, pyruvate and urea) and later in the laboratory (for total protein).Results. Fluid recovery was similar for the two types of catheters, but significantly more protein was recovered by the D100 catheter. The recovery of glycerol and pyruvate did not differ, while minor differences in recovery of glutamate and glucose were observed. The recovery of lactate was considerably lower in the D100 catheter (p < 0.01), influencing the lactate/pyruvate-ratio. The patterns of concentration changes over time were consistent for all metabolites, and independent of type of catheter.Conclusion. Microdialysis catheters with high cut-off membranes can be used in routine clinical practice in the NSICU, adding the possibility of macro-molecule sampling from the extracellular space during monitoring.

Release of VEGF and FGF in the extracellular space following severe subarachnoidal haemorrhage or traumatic head injury in humans

Microdialysate fluid from 145 severely injured NSICU-patients, 88 with subarachnoidal haemorrage (SAH), and 57 with traumatic brain injury (TBI), was collected by microdialysis during the first 7 days following impact, and levels of the neurotrophins fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) were analysed. The study illustrates both similarities and differences in the reaction patterns of the 2 inflammatory proteins. The highest concentrations of both FGF2 and VEGF were measured on Day 2 (mean (± SE) values being 47.1 ± 15.33 and 116.9 ± 41.85 pg/ml, respectively, in the pooled patient material). The VEGF concentration was significantly higher in TBI-patients, while the FGF2 showed a tendency to be higher in SAH-patients. This is the first report presenting in some detail the human cerebral response of FGF2 and VEGF following SAH and TBI. Apart from increasing the understanding of the post-impact inflammatory response of the human brain, the study identifies potential threshold values for these chemokines that may serve as monitoring indicators in the NSICU.

The Cerebral Extracellular Release of Glycerol, Glutamate, and FGF2 Is Increased in Older Patients following Severe Traumatic Brain Injury

Old age is associated with a poor recovery from traumatic brain injury (TBI). In a retrospective study we investigated if the biochemical response following TBI is age dependent. Extracellular fluids were continuously sampled by microdialysis in 69 patients admitted to our NSICU following severe TBI. The concentrations of glycerol, glutamate, lactate, pyruvate, and eight different cytokines (IL-1β, IL-6, IL-10, IL-8, MIP-1β, RANTES, FGF2, and VEGF) were determined by fluorescence multiplex bead technology. Patients in the oldest age group (≥65 years) had significantly higher microdialysate concentrations of glycerol and glutamate compared to younger patients: the mean microdialysate concentration of glycerol increased from 55.9 μmol/L (25-44 year) to 252 μmol/L (≥65 years; p<0.0001); similarly glutamate increased from 15.8 mmol/L to 92.2 mmol/L (p<0.0001). The lactate-pyruvate ratio was also significantly higher in the patients ≥65 years of age (63.9) compared with all the other age groups. The patterns of cytokine responses varied. For some cytokines (IL-1b, IL-10, and IL-8) there were no differences between age groups, while for others (MIP-1b, RANTES, VEGF, and IL-6) some differences were observed, but with no clear correlation with increasing age. For FGF2 the mean microdialysate concentration was 43 pg/mL in patients ≥65 years old, significantly higher compared to all other age groups (p<0.0001). Increased concentrations of glycerol and glutamate would indicate more extensive damaging processes in the elderly. An increase in concentration of FGF2 could serve a protective function, but could also be related to a dysregulation of the timing in the cellular response in elderly patients.

SULFATIDE-INDUCED L-SELECTIN ACTIVATION GENERATES INTRACELLULAR OXYGEN RADICALS IN HUMAN NEUTROPHILS : MODULATION BY EXTRACELLULAR ADENOSINE

The sulfated form of galactocerebrosides (sulfatides) have recently been established as ligands for L-selectin. In this study we show that exposure of human neutrophils to sulfatides induces a transient generation of oxygen radicals, revealed by the luminol-enhanced chemiluminescence (CL) technique. The CL response was mainly located intracellularly, and was dependent on sulfation of the galactose ring, since non-sulfated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular generation of oxygen metabolites. Removal or blocking of L-selectin with chymotrypsin and monoclonal antibodies, respectively, markedly reduced the effects of sulfatides. Furthermore, sulfatides amplified the CL response triggered by ionomycin, whereas the response induced by phorbol-12-myristate-13-acetate was slightly reduced. The tyrosine kinase inhibitor, genistein, markedly inhibited the oxygen radical production induced by sulfatides, and totally abolished the potentiating effects of sulfatides in fMLP- and ionomycin-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca2+]i. Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca(2+)-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominately released from the vascular endothelium which might suppress an inappropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidative burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-100 times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent as the adenosine-mediated inhibition of the fMLP-induced response. This difference in sensitivity to adenosine could be explained by various expression of extracellular adenosine deaminase (ADA), since we found that the ADA-inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) markedly reduced the oxygen radical production caused by sulfatides and almost totally abolished the potentiating effects of sulfatides on the fMLP-induced respiratory burst. In contrary, EHNA only slightly reduced the fMLP-triggered CL response. We suggest that the initial activation of L-selectin prepare the neutrophil for an effective microbicidal activity in the extravascular space. This process might be dependent on a L-selectin-mediated increase in the expression and activity of ADA, which locally reduces the extracellular level of adenosine.

Decreased Th1-Type Inflammatory Cytokine Expression in the Skin Is Associated with Persisting Symptoms after Treatment of Erythema Migrans

BACKGROUND Despite the good prognosis of erythema migrans (EM), some patients have persisting symptoms of various character and duration post-treatment. Several factors may affect the clinical outcome of EM, e.g. the early interaction between Borrelia (B.) burgdorferi and the host immune response, the B. burgdorferi genotype, antibiotic treatment as well as other clinical circumstances. Our study was designed to determine whether early cytokine expression in the skin and in peripheral blood in patients with EM is associated with the clinical outcome. METHODS A prospective follow-up study of 109 patients with EM was conducted at the Åland Islands, Finland. Symptoms were evaluated at 3, 6, 12 and 24 months post-treatment. Skin biopsies from the EM and healthy skin were immunohistochemically analysed for expression of interleukin (IL)-4, IL-10, IL-12p70 and interferon (IFN)-γ, as well as for B. burgdorferi DNA. Blood samples were analysed for B. burgdorferi antibodies, allergic predisposition and levels of systemic cytokines. FINDINGS None of the patients developed late manifestations of Lyme borreliosis. However, at the 6-month follow-up, 7 of 88 patients reported persisting symptoms of diverse character. Compared to asymptomatic patients, these 7 patients showed decreased expression of the Th1-associated cytokine IFN-γ in the EM biopsies (p=0.003). B. afzelii DNA was found in 48%, B. garinii in 15% and B. burgdorferi sensu stricto in 1% of the EM biopsies, and species distribution was the same in patients with and without post-treatment symptoms. The two groups did not differ regarding baseline patient characteristics, B. burgdorferi antibodies, allergic predisposition or systemic cytokine levels. CONCLUSION Patients with persisting symptoms following an EM show a decreased Th1-type inflammatory response in infected skin early during the infection, which might reflect a dysregulation of the early immune response. This finding supports the importance of an early, local Th1-type response for optimal resolution of LB.