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Human peripheral nerves hold the potential to regenerate after injuries; however, whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. The axolotl salamander is a regenerative model where nerves always regenerate quickly and fully after all types of injury. Here, de- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. We used coherent anti-Stokes Raman scattering to visualize myelin fragmentation and axonal regeneration. The presence of axons at the lesion site corresponded to onset of functional recovery in both lesion models. In addition, we detected axonal regrowth later in the double injury model in agreement with a higher severity of injury. Moreover, endogenous two-photon excited fluorescence visualized macrophages and revealed a similar timecourse of inflammation in both injury models, which did not correlate with functional recovery. Finally, using the same techniques, axonal structure and status of myelin were visualized in vivo after sciatic nerve injury. Label-free imaging is a new experimental approach that provides mechanistic insights in animal models, with the potential to be used in the future for investigation of regeneration after nerve injuries in humans.

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.

Background: Spinal cord injury (SCI) and the consecutive devastating neurological sequelae have an enormous individual and economic impact. Implantation of functionalized hydrogels is a promising approach, because they can serve as a matrix for the regenerating tissue, carry and release bioactive molecules and various cell types. We already demonstrated that non-functionalized soft alginate hydrogel supported axonal outgrowth and protected neurons against oxidative stress in vitro. Here, we investigated the effects of such soft alginate hydrogels on locomotor recovery in small and large spinal cord lesions., Method: Hemimyelonectomy of 2 mm or 4 mm length was performed in rats and soft alginate hydrogel was implanted. Functional recovery of the hindlimbs was assessed in the open field [Batto Beattie Bresnahan (BBB) score] and using swimming test [Louisville Swim score (LSS)] for 140 days post injury (DPI). Reference histology was performed., Results: Rats that received an alginate implant into 2 mm spinal cord lesions demonstrated significantly improved locomotor recovery compared to controls detectable already at 10 DPI. At 140 DPI, they reached higher LSS and BBB scores in swimming and open field tests, respectively. However, this beneficial effect of alginate was lacking in animals with larger (4 mm) lesions. Histological examination suggested that fibrous scarring in the spinal cord was reduced after alginate implantation in comparison to controls., Conclusions: Implantation of soft alginate hydrogel in small spinal cord lesions improved functional recovery. Possible underlying mechanisms include the mechanical stabilization of the wound, reduction of secondary damage and inhibition of fibrous scarring.

Spinal cord injury (SCI) triggers several lipid alterations in nervous tissue. It is characterized by extensive demyelination and the inflammatory response leads to accumulation of activated microglia/macrophages, which often transform into foam cells by accumulation of lipid droplets after engulfment of the damaged myelin sheaths. Using an experimental rat model, Raman microspectroscopy was applied to retrieve the modifications of the lipid distribution following SCI. Coherent anti-Stokes Raman scattering (CARS) and endogenous two-photon fluorescence (TPEF) microscopies were used for the detection of lipid-laden inflammatory cells. The Raman mapping of CH2 deformation mode intensity at 1440  cm(−1) retrieved the lipid-depleted injury core. Preserved white matter and inflammatory regions with myelin fragmentation and foam cells were localized by specifically addressing the distribution of esterified lipids, i.e., by mapping the intensity of the carbonyl Raman band at 1743  cm(−1), and were in agreement with CARS/TPEF microscopy. Principal component analysis revealed that the inflammatory regions are notably rich in saturated fatty acids. Therefore, Raman spectroscopy enabled to specifically detect inflammation after SCI and myelin degradation products.

Spinal cord injury (SCI) induces complex biochemical changes, which result in inhibition of nervous tissue regeneration abilities. In this study, Fourier-transform infrared (FT-IR) spectroscopy was applied to assess the outcomes of implants made of a novel type of non-functionalized soft calcium alginate hydrogel in a rat model of spinal cord hemisection (n = 28). Using FT-IR spectroscopic imaging, we evaluated the stability of the implants and the effects on morphology and biochemistry of the injured tissue one and six months after injury. A semi-quantitative evaluation of the distribution of lipids and collagen showed that alginate significantly reduced injury-induced demyelination of the contralateral white matter and fibrotic scarring in the chronic state after SCI. The spectral information enabled to detect and localize the alginate hydrogel at the lesion site and proved its long-term persistence in vivo. These findings demonstrate a positive impact of alginate hydrogel on recovery after SCI and prove FT-IR spectroscopic imaging as alternative method to evaluate and optimize future SCI repair strategies.

Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

Diagnosis of Creutzfeldt-Jakob Disease (CJD) is based on neurological signs associated with characteristic electroencephalographic activity or detection of 14-3-3 protein in cerebrospinal fluid. However, the time course of 14-3-3 protein release during sporadic CJD is unknown. We report two observations in which the level of the detected 14-3-3 protein increased significantly with time. These preliminary cases suggest that there may be an increased release of 14-3-3 protein during the course of CJD as already proven for iatrogenic CJD., (Copyright 2002 S. Karger AG, Basel)

Chronic fatigue syndrome or benign myalgic encephalomyelitis has been extensively described and investigated. Although numerous immunological abnormalities have been linked with the syndrome, none have been found to be specific. This article describes the detection of delayed-type hypersensitive responses to certain common environmental antigens in almost fifty per cent of patients with this syndrome. Such hypersensitivity can be detected by the intradermal administration of antigens derived from commensal organisms like the yeast Candida albicans albicans, and then monitoring for a systemic reaction over the following six to forty-eight hours. This approach can be consolidated by performing lymphocyte activation tests in parallel and measuring in vitro T-cell activation by Candida albicans albicans antigens by three-colour flow cytometry based on CD3, CD4 and either CD69 or CD25. Another useful parameter is the kinetics of neopterin excretion in the urine over the course of the skin test. The results showed that the intensity of the DTH response correlated with the number of T-cells activated in vitro. Various factors have been implicated in the fatigue of many patients, notably lack of sleep. However, it remains difficult to establish causality in either one direction or the other. This work is in the spirit of a multifactorial approach to the group of conditions referred to as "chronic fatigue syndrome".

The aim of this study was to develop, for the first time in the human spinal dorsal horn (DH), an in vivo method for the study of amino acids (AAs). A microdialysis technique was used to sample AAs in the extracellular fluid of the DH apex in eight patients in whom surgery in the dorsal root entry zone (DREZ) was performed. Before making microsurgical lesions, specific concentric-type microdialysis probes were implanted over a 60-minute period in the DREZ and directed to the DH apex (10 implantations). The AA concentrations in the dialysates were determined using high-performance liquid chromatography with fluorescence detection. The concentrations of excitatory AAs (glutamate and aspartate) and inhibitory AAs (gamma-aminobutyric acid and glycine) decreased and were stabilized by 45 minutes after probe implantation, whereas the levels of nonneurotransmitter AAs (alanine and threonine) were not stabilized at 60 minutes. The ability of the probe to track the changes of extracellular AAs was demonstrated. Neither intra- nor postoperative microdialysis-related complications were observed (with a follow up of 18 months). The present study demonstrates that microdialysis can be performed safely in the human DH during DREZ lesioning. Despite technical and analytical limitations related to the intraoperative conditions, this technique offers new possibilities for clinical research on neurotransmitters involved in some relevant pathological states, especially in chronic pain and spasticity.

A retrospective study was carried out at the Neurological and Neurosurgical Hospital of Lyon in order to evaluate the interest of detecting IgG oligoclonal bands by isoelectric focusing with IgG immunorevelation for the early diagnosis of multiple sclerosis (MS). Patients have been grouped according to their disorders: multiple sclerosis (281 cases), definite (182 cases) and possible (99 cases), others inflammatory neurological diseases (63 cases), various non-inflammatory neurological disorders (180 cases) and indefined neurological disorders (664 cases). The following examinations were performed: CSF cell count and cytology after concentration and cytocentrifugation, CSF and serum determination of albumin and IgG with CSF/serum ratios, agarose gel electrophoresis and isoelectric focusing of oligoclonal IgG. The technique used was isoelectric focusing using agarose gel, transfer onto PVDF membrane and then IgG immunorevelation with biotinylated anti-human IgG antibodies. Isoelectric focusing with IgG immunorevelation is the most sensitive (94%) and specific (96%) technique. Isoelectric focusing with immune detection can be recommended as the most efficient test (gold standard) for the detection of chronic CNS inflammation.

We retrospectively evaluated measurement data and clinical relevance of autoantibodies to gangliosides in peripheral neuropathies (PN). The IgG and IgM antiganglioside autoantibodies were determined by our own immunodot-blot assay on membrane and by enzyme-linked immunosorbent assay (Elisa) in sera of 1,342 patients with peripheral neuropathies. Anti-GM1 and anti-GD1b autoantibodies formed a part of the normal autoantibody repertoire and were common place in 12% of normal subjects and in 14% of disease control groups. Polyclonal IgM antiganglioside autoantibodies were detected in chronic PN, polyclonal IgG antiganglioside autoantibodies were detected in acute PN. Polyclonal IgM anti-GM1 and anti-GD1b autoantibodies were detected in 35 patients out of 48 with treatable multifocal motor neuropathy with persistent conduction blocks. These autoantibodies well discriminated between suspected motor peripheral neuropathies and motor neuron diseases (sensitivity 73%, specificity 83%, positive predictive value 60%, negative predictive value 91%). Monoclonal IgM autoantibodies reacted strongly with gangliosides in 15 patients out of 77 with M-IgM neuropathy (19%). M-IgM autoantibodies differed in their fine specificities with different principal target antigens as demonstrated with cross-reactivity. Such findings provide further evidence for a relationship between neurological syndromes and antiganglioside antibody profiles and also suggest that different gangliosides could be principal target antigens such as GM1, GD1b, GT1b, GD1a or GM2. Polyclonal IgG anti-GM1 and anti-GD1b autoantibodies were detected in 21 patients out of 22 with acute motor axonal Guillain-Barré syndrome with antecedent of infection by Campylobacter jejuni, polyclonal IgG anti-GQ1b autoantibodies in 9 patients out of 10 with Miller-Fisher syndrome. Detection of antiganglioside autoantibodies by immunodot-blot assay which is simple and quick in testing a large panel of gangliosides has become very important in the diagnosis and in the choose of expensive therapeutic strategies in chronic or acute autoimmune neuropathies.

Polyneuropathies associated with IgM monoclonal gammopathy (IgM-MG) are the most frequent types of peripheral neuropathies associated with monoclonal gammopathy. The pathogenic relevance of IgM-MG in peripheral neuropathies is supported by several arguments. The more significant are pathological data on nerve biopsies and the demonstration of IgM antibody activity to peripheral nerve antigens, mainly myelin-associated glycoprotein (MAG) and, sulfated 3-glucuronyl paragloboside (SGPG) and sulfated 3-glucuronyl lactosaminyl paragloboside (SGLPG). The objective of this study was to determine the performance characteristics of 3 immunoassays for detection of anti-myelin antibodies in neuropathy associated with monoclonal IgM gammopathy. Monoclonal IgM may show antibody activity to carbohydrate epitopes shared by several nerve specific antigens. Results obtained with three reliable assay systems, indirect immunofluorescence for antibodies to myelin sheaths (33%), thin layer chromatography for anti-SGPG/SGLPG antibodies (54%) and Western-blot for anti-MAG antibodies (39%). Comparing the different assay systems for carbohydrate structure present on both MAG and SGPG/SGLPG on myelin sheath, the thin layer chromatography appeared to be more sensitive and reliable than Western blot.

The introduction of the CRM 470 in 1993 (certified reference material for 14 serum proteins) and its utilization by industrial companies for cross-calibrating their commercial standards has been an important break-through in protein standardization. This improvement has been clearly illustrated by the last national quality control survey performed in France in may 1995. At this time, about 60% of the 1,870 participants have already adopted the new standardization. The between-run precision (interlaboratory and intertechnique) has been considerably improved by the use of the new international standard (5.8 to 12.2% versus 10 to 24.1% before standardization); the same is true for accuracy. These results should convince the last reluctant laboratories to adopt the new standardization. Thus, it seems now possible to define reference ranges for serum proteins: this is the new task assigned to the Committee for Plasma Protein Standardization of the IFCC.

We compare the precision of immunochemical techniques for determining immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), transferrin and haptoglobin. Precision was found satisfactory. Results of samples containing monoclonal Ig, showed substantial discrepancies, especially the serum containing monoclonal IgM. In this case, the nephelometric analysers gave the best results. In conclusion, when different techniques are used for monitoring protein immunoassays, a good knowledge of the performance of the analyser is necessary for correct interpretation and to avoid potentially misleading results.

This study demonstrates the advantages and limitations of normalizing results for five serum proteins (IgG, IgA, IgM, transferrin and haptoglobin), analysed in liquid phase on ten different systems (open clinical chemistry and dedicated protein analysers). Seven sets of results from normal and pathological sera (without monoclonal proteins) were compared using: - calibrators supplied by each manufacturer; - - serial dilutions of a single stabilized pool of liquid serum. In addition to validating the quality of the stabilised serum, we have been able to identify: - significant variations in results using different analytical systems for the same sample; - a major reduction in these variations, often greater than 50%, through normalization using a common serum pool. In some two thirds of the cases, this reduction brought the degree of variation into an acceptable range.

This study is the third part of a multicenter evaluation carried out with ten analysers. Five proteins (IgG, IgA, IgM, transferrin, haptoglobin) were assayed in three sera, each containing one monoclonal Ig (IgG, IgA, or IgM). The expected agreement was not obtained with these particular sera, except in the case of haptoglobin. The marked imprecision of the monoclonal Ig quantification is highlighted, including the fact that many erroneous results are due to the antigen excess phenomenon. It is also shown that in the serum containing monoclonal IgM, quantification of the other proteins, remaining polyclonal Ig, as well as transferrin and haptoglobin, is difficult. The authors recall that electrophoresis and immunoelectrophoresis, which are indispensable for the characterization of the monoclonal components, are of great help in solving such difficulties.

Several national and international quality assurance schemes clearly demonstrated the discrepancies between the results given by different analytical systems designed for the immunochemical measurement of proteins. Thus, the need for the preparation of a new international secondary reference material appeared urgent to the International Federation for Clinical Chemistry (IFCC). In this paper, the authors describe the different steps of the preparation of the new standard CRM 470. This work financed by the Bureau Communautaire de Référence (BCR) has been done in collaboration with the College of American Pathologists (CAP). The physicochemical properties of the proteins present in the CRM 470 and the various controls undertaken on the preparation are described. The CRM 470 material (40,000 lyophylised 1 ml vials) is now certified for the 14 plasma proteins of major interest in clinical chemistry. The worldwide introduction of this material will considerably improve the accuracy of immunochemical proteins' determination and will allow the determination of the reference values as a function of age and sex.