Search

BackgroundCharacterizing immune cells and conditions that govern their recruitment and function in autoimmune diseases of the nervous system or in neurodegenerative processes is an area of active investigation. We sought to analyze the origin of antigen presenting cells associated with the induction of retinal autoimmunity using a system that relies on spontaneous autoimmunity, thus avoiding uncertainties associated with immunization with adjuvants at remotes sites or adoptive transfer of in vitro activated T cells.MethodsR161H mice (B10.RIII background), which spontaneously and rapidly develop severe spontaneous autoimmune uveoretinitis (SAU), were crossed to CD11cDTR/GFPmice (B6/J) allowing us to track the recruitment to and/or expansion within the retina of activated, antigen presenting cells (GFPhicells) in R161H+/− × CD11cDTR/GFPF1mice relative to the course of SAU. Parabiosis between R161H+/− × CD11cDTR/GFPF1mice and B10.RIII × B6/J F1(wild-type recipient) mice was done to explore the origin and phenotype of antigen presenting cells crucial for the induction of autoimmunity. Analysis was done by retinal imaging, flow cytometry, and histology.ResultsOnset of SAU in R161H+/− × CD11cDTR/GFPF1mice was delayed relative to B10.RIII-R161H+/−mice revealing a disease prophase prior to frank autoimmunity that was characterized by expansion of GFPhicells within the retina prior to any clinical or histological evidence of autoimmunity. Parabiosis between mice carrying the R161H and CD11cDTR/GFPtransgenes and transgene negative recipients showed that recruitment of circulating GFPhicells into retinas was highly correlative with the occurrence of SAU.ConclusionsOur results here contrast with our previous findings showing that retinal antigen presenting cells expanding in response to either sterile mechanical injury or neurodegeneration were derived from myeloid cells within the retina or optic nerve, thus highlighting a unique facet of retinal autoimmunity.

The diagnosis of polyneuropathy (PNP) is based on the anamnesis and description of complaints of the patient and clinical findings. The type of distribution as well as known diseases and drug toxic factors can provide indications. Electromyography and electroneurography can be used to differentiate between axonal and demyelinating PNP. The laboratory examinations are initially directed towards frequent and treatable causes. These are then expanded depending on the suspected diagnosis. Analysis of cerebrospinal fluid (CSF) is facultative and should be carried out when there is a suspicion of a certain form of PNP with CSF findings indicative of the diagnosis. Nerve biopsy is indicated when the etiology of a severe or progressive PNP cannot be clarified by less invasive means and can have consequences for the treatment. A genetic investigation can be meaningful with a positive family anamnesis or with typical signs of hereditary PNP. Depending on the neuropathy and context, the diagnostic approach is structured differently. The special diagnostics for small fiber neuropathy and amyloid neuropathy as well as for diabetes and alcohol abuse are dealt with in detail in this article. Numerous cases of polyneuropathy remain unexplained and regularly have a favourable prognosis.

Background Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFP hi cells possessed antigen presentation function. Subsequent studies found that these high GFP hi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFP hi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65 −/− knockout mice induced the GFP hi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival. Methods Rpe65 −/− mice were bred to CD11c GFP mice on the B6/J background. CD11c GFP Rpe65 −/− mice were also backcrossed to CX3CR1 YFP-creER ROSA DTA mice so that CX3CR1 + mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry. Results Elevated numbers of GFP hi cells were concentrated in photoreceptor cell layers of CD11c GFP Rpe65 −/− mice coinciding with the peak of cone death at 2 to 4 weeks of age, and persisted for at least 14 months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFP hi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFP hi cells and MG in Rpe65 −/− mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFP hi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFP hi cells were well segregated in the dystrophic retina; GFP hi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina. Conclusions The expression of GFP on a subset of retinal mononuclear cells in CD11c GFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFP hi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG.

Purpose We previously demonstrated that passive transfer of lymphocytes from glaucomatous mice induces retinal ganglion cell (RGC) damage in recipient animals, suggesting a role for immune responses in the multifactorial pathophysiology of glaucoma. Here we evaluate whether absence of an adaptive immune response reduces RGC loss in glaucoma. Methods Elevated intraocular pressure (IOP) was induced in one eye of C57BL/6J (B6) or T- and B-cell-deficient Rag1-/- knockout mice. After 16 weeks RGC density was determined in both the induced and the normotensive contralateral eyes. Data were compared to mice having received injections of "empty" vector (controls). The number of extravascular CD3+ cells in the retinas was determined using FACS. Results Retinas of eyes with elevated IOP contain significantly more extravasated CD3+ cells than control retinas (46.0 vs. 27.1, P = 0.025). After 16 weeks of elevated IOP the average RGC density in B6 mice decreased by 20.7% (P = 1.9 × 10-4). In contrast, RGC loss in Rag1-/- eyes with elevated IOP was significantly lower (10.3%, P = 0.006 vs. B6). RGC loss was also observed in the contralateral eyes of B6 mice, despite the absence of elevated IOP in those eyes (10.1%; P = 0.008). In RAG1-/- loss in the contralateral eyes was minimal (3.1%) and significantly below that detected in B6 (P = 0.02). Conclusions Our findings demonstrate that T Rag1-/- mice are significantly protected from glaucomatous RGC loss. In this model, lymphocyte activity contributes to approximately half of all RGC loss in eyes with elevated IOP and to essentially all loss observed in normotensive contralateral eyes.

Extrathymically derived regulatory T cells (iTregs) protect against autoimmunity to tissue-specific antigens (Ag). However, whether Ag-specific iTreg generation and function is limited to secondary lymphoid tissue or whether it can occur within the tissue-specific, local environment of the cognate Ag remains unresolved. Mice expressing beta-galactosidase (βgal) on a retina specific promoter (βgal mice) in conjunction with mice expressing green fluorescent protein (GFP) and diphtheria toxin (DTx) receptor (DTR) under control of the FoxP3 promoter, and βgal-specific T cell receptor transgenic (BG2) mice were used to examine this question. Local depletion (ocular DTx), but not systemic depletion (intra-peritoneal DTx), of βgal-specific iTregs enhanced experimental autoimmune uveoretinitis induced by activated βgal-specific effector T cells. Injections of small amounts of βgal into the anterior chamber of the eye produced similar numbers of βgal-specific iTregs in the retina whether or not the mouse was depleted of pre-existing, circulating Tregs. Together these results suggest that protection from tissue-specific autoimmunity depends on the function of local Ag-specific iTregs, and that the retina is capable of local, “on-demand” iTreg generation that is independent of circulating Tregs.

Phosphoprotein 8-50, also termed growth-associated protein GAP43, is a membrane-bound phosphoprotein expressed in neurons. It is particularly abundant during periods of axonal outgrowth in development and regeneration of the central and peripheral nervous systems. Recently the expression of phosphoprotein 8-50 in regenerating muscle fibers was reported and it was assumed that phosphoprotein 8-50, in muscles, plays a role in the determination of the growth morphology of regenerating muscle fibers. Thus, phosphoprotein 8-50 no longer can be regarded as a neuron-specific molecule. In this paper we study the subcellular localization of phosphoprotein 8-50 in regenerating human skeletal muscle fibers by electron immunohistochemistry. Phosphoprotein 8-50 immunoreactivity is randomly distributed over the nuclear and perinuclear area of regenerating muscle fibers. Previously, by light-microscopy phosphoprotein 8-50 immunoreactivity was demonstrated on the inner face of the sarcolemma in hypotrophic type 1 fibers in congenital fiber type disproportion. It is this distribution of phosphoprotein 8-50 in developmentally disordered myocytes in particular which allows an analogy to the corresponding results found for growing axons. But we did not find subsarcolemmal expression of 8-50 in regenerating muscle fibers. Probably this indicates that there is a difference in 8-50 expression between regeneration of muscle fibers and developmentally retarded/immature myofibers. The presented data suggest that phosphoprotein 8-50 is inserted only stage-dependent in the extending sarcolemma of the growing muscle fiber. Analogously to the nervous system, phosphoprotein 8-50 may serve a local function involving transmembrane signalling by means of calmodulin binding ofphosphoprotein 8-50 and/or phosphoprotein 8-50 phosphorylation and dephosphorylation. [Neural Res 1998; 20: 360-364].

Interest in the identities, properties, functions, and origins of local APC in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of APC activity of DC from quiescent retina showed that they promoted generation of Foxp3+ T cells and inhibited activation of naive T cells by splenic DC and Ag. Conversely, in vitro assays of DC purified from injured retina demonstrated an enhanced ability to activate T cells and reduced induction of Foxp3+ T cells. These findings were supported by the observation that in situ activation of DC before adoptive transfer of β-galactosidase–specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of Ag in vivo showed that quiescent retina promoted development of parenchymal Foxp3+ T cells, but assays of preinjured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

Regulatory T cells (Tregs) are generated to antigens (Ag) found in the retina. Some Tregs are the result of ectopic expression of the retinal Ags in the thymus, where developing T cells are committed to enter the regulatory lineage. However, the generation of retinal Ag-specific Tregs independent of the thymus was uncertain. Our studies show that Tregs can be generated from mature, peripheral T cells based on exposure to retinal Ags. These peripherally induced Tregs limited immune responses and experimental autoimmune disease induced by retinal Ags and thus constitute a crucial component of retinal immune privilege.

Dermatomyositis and vasculitic neuropathies are disorders with immune mediated ischemic injuries. Cellular responses to hypoxia include the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in angiogenesis. To study their possible roles in those disorders, the immunohistochemical expression of HIF-1alpha, HIF-1beta, HIF-2alpha, vascular endothelial growth factor (VEGF), VEGF-receptor (VEGF-R) and erythropoietin-receptor was investigated. Cases of normal nerves, diabetic neuropathy, normal muscles, polymyositis and inclusion-body-myositis served as controls. The latter were chosen because they represent comparable inflammatory disorders, however, in these ischemia/hypoxia is not supposed to play such a prominent pathogenetic role. Hypoxia-related proteins were not detected in normal controls. In polymyositis and inclusion-body-myositis, there was VEGF-R-expression in muscle fibers and HIF-2alpha reactivity in endothelial cells. In dermatomyositis, HIF-1alpha and HIF-1beta were found in endothelial cells, whereas HIF-2alpha, erythropoietin-receptor, VEGF and VEGF-R additionally were observed in muscle fibers. In vasculitic and diabetic neuropathies, a variable focal expression of hypoxia-inducible factors, VEGF, VEGF-R and erythropoietin-receptor was seen in vessels. These observations suggest that the upregulation of hypoxia-related proteins may represent an adaptation mechanism of neuromuscular tissues to immune mediated deprivation of the blood supply.

The contribution of peripheral expression of tissue-specific CNS Ags to the generation of tolerance is uncertain. To study this question, we examined mice transgenic (Tg) for expression of β-galactosidase (βgal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4+ T cells specific for βgal (βgalTCR). Several strategies were used to test the hypothesis that βgal expressed in the retina supported thymus-independent tolerance and regulatory T cell development. Retinal expression generated an immunoregulatory response that depressed development of immune responses to βgal following systemic immunization with βgal. This regulation was transferable to naive mice by CD3+4+25+ T cells from naive retinal βgal+ donors. Experiments that removed the βgal+ retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25− βgalTCR T cells into retinal βgal Tg mice on the Rag−/− background led to regulatory activity that limited lymphopenia-induced proliferation of βgalTCR T cells in mice with retinal expression of βgal and inhibited the ear-swelling assay for delayed type hypersensitivity. These results show that retinal expression of very small amounts of a tissue-specific Ag can generate tolerance that includes regulatory T cells.

Objective: To describe the clinical, electrophysiologic and morphologic features of a Costa Rican family with an autosomal dominant inherited Charcot–Marie–Tooth (CMT) neuropathy. Methods: The field study took place in Costa Rica, Central America. Seven patients underwent neurological examinations and standard electrodiagnostic tests, and a sural nerve biopsy was taken from one patient. Fifteen family members were screened for gene defects associated with CMT disease. Results: Characteristic features of this family were a late age of onset (35–56 years), positive sensory symptoms and muscle cramps. Based on electrodiagnostic and morphologic data, the patients were classified as having a CMT2 neuropathy. The CMT1A duplication/HNPP deletion and point mutations in genes PMP22, MPZ, Cx32 and EGR2 implicated in the most common types of CMT disease were excluded. Subsequently, almost all known CMT loci were excluded by linkage analysis. Discussion: Features of this family were a late age of onset and positive sensory symptoms. This new autosomal dominant CMT neuropathy is associated with an unknown gene defect. [Neurol Res 2009; 31: 283–288] Objetivo: Describir las características clínicas, electrofisiológicas y morfológicas de una familia costarricense con una neuropatía de Charcot-Marie-Tooth (CMT) hereditaria autosómica dominante. Métodos: El estudio de campo se llevó a cabo en Costa Rica, Centroamérica. Siete pacientes se sometieron a exámenes neurológicos y pruebas de electrodiagnóstico estándar, ya un paciente se le tomó una biopsia del nervio sural. Quince miembros de la familia fueron evaluados por defectos genéticos asociados con la enfermedad de CMT. Resultados: Los rasgos característicos de esta familia fueron una edad de inicio tardía (35-56 años), síntomas sensoriales positivos y calambres musculares. Sobre la base de los datos morfológicos y de electrodiagnóstico, los pacientes se clasificaron con una neuropatía CMT2. Se excluyeron la duplicación de CMT1A/deleción HNPP y las mutaciones puntuales en los genes PMP22, MPZ, Cx32 y EGR2 implicados en los tipos más comunes de enfermedad de CMT. Posteriormente, casi todos los loci CMT conocidos fueron excluidos por análisis de ligamiento. Discusión: Las características de esta familia fueron una edad de inicio tardía y síntomas sensoriales positivos. Esta nueva neuropatía de CMT autosómica dominante está asociada con un defecto genético desconocido. [NeurolRes 2009; 31: 283–288] Universidad Nacional, Costa Rica. Escuela de Ciencias Biológicas

To study retinal immunity in a defined system, a CD4+ TCR transgenic mouse line (βgalTCR) specific for β-galactosidase (βgal) was created and used with transgenic mice that expressed βgal in retinal photoreceptor cells (arrβgal mice). Adoptive transfer of resting βgalTCR T cells, whether naive or Ag-experienced, into arrβgal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of βgalTCR T cells in arrβgal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrβgal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated βgalTCR T cells, even though the T cells were pathogenic if the βgal was expressed elsewhere. In vitro manipulations to increase T cell pathogenicity before transfer did not result in EAU. The only strategy that induced a high frequency of severe EAU was transfer of naive, CD25-depleted, βgalTCR T cells into lymphopenic arrβgal recipients, implicating regulatory T cells in the T cell inoculum, as well as in the recipients, in the resistance to EAU. Surprisingly, activation of the CD25-depleted βgalTCR T cells before transfer into the lymphopenic recipients reduced EAU. Taken together, the results suggest that endogenous regulatory mechanisms, as well as peripheral induction of regulatory T cells, play a role in the protection from EAU.

It is well known that immunoproteasome generates peptides for MHC Class I occupancy and recognition by cytotoxic T lymphocytes (CTL). The present study focused on evidence for alternative roles for immunoproteasome. Retina and brain were analyzed for expression of immunoproteasome subunits using immunohistochemistry and western blotting under normal conditions and after injury/stress induced by CTL attack on glia (brain) or neurons (retina). Normal retina expressed substantial levels of immunoproteasome in glia, neurons, and retinal pigment epithelium. The basal level of immunoproteasome in retina was two-fold higher than in brain; CTL-induced retinal injury further up-regulated immunoproteasome expression. Immunoproteasome up-regulation was also observed in injured brain and corresponded with expression in Purkinje cells, microglia, astrocytes, and oligodendrocytes. These results suggest that the normal environment of the retina is sufficiently challenging to require on-going expression of immunoproteasome. Further, immunoproteasome up-regulation with retinal and brain injury implies a role in neuronal protection and/or repair of damage.

Motoneurons release the heparansulfate proteoglycan agrin and thereby activate the muscle-specific receptor tyrosine kinase (MuSK), which is the main organizer of subsynaptic specializations at the neuromuscular junction. Recently, we showed that (1) the protein kinase CK2 interacts with the intracellular region of MuSK; (2) the CK2 protein is enriched and co-localized with MuSK at postsynaptic specializations; (3) CK2-mediated phosphorylation of serine residues within a specific MuSK epitope, named the kinase insert, regulates acetylcholine receptor (AChR) clustering; (4) muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function (see Genes Dev 20(13):1800-1816, 2006). Here, we investigated for the first time if CK2 is modulated in biopsies from human patients. To this end, we measured transcript amounts of the subunits CK2alpha and CK2beta and determined holoenzyme CK2 activity in 34 muscle biopsies of human patients with different muscle pathologies.

Background: Thymic expression of a photoreceptor cell antigen, interphotoreceptor retinoid-binding protein, is known to generate regulatory T cells (Treg) that prevent spontaneous autoimmune disease of the retina. However, the contribution of other endogenous, tissue-specific antigens (Ags) expressed in the retina to the generation of Treg is uncertain. Methods: Transgenic mice that express β-galactosidase (β-gal) in photoreceptor cells, together with β-gal-specific T cell receptor transgenic mice, were used to study the induction of Treg in vivo. Results: Transgenic expression of β-gal on the arrestin promoter led to a spontaneous immunoregulatory response that inhibited the development of immune responses to β-gal. The regulation was transferred by CD3+4+25+ Treg. Several strategies were then used to show that β-gal expressed in the retina supported spontaneous, thymus-independent Treg development. The endogenous Treg also differed from the Treg induced by Ag inoculation into the anterior chamber of the eye. Conclusion: These results demonstrate that retinal expression of very small amounts of a tissue-specific Ag can generate Treg in the periphery.

To explore pathways to neuron loss in inflammatory diseases, transgenic mice expressing beta-galactosidase (beta-gal) in either astrocytes or photoreceptor cells, or both, were inoculated with activated, beta-gal-specific cytotoxic CD8 T lymphocytes (CTLs). beta-gal-positive astrocytes in brain were rapidly attacked, with particular damage in cerebellum. Substantial loss of cerebellar granule cells was found, even though these neurons did not express beta-gal. The small number of beta-gal-positive retinal astrocytes present in these mice was also rapidly destroyed by transferred CTLs, but without detectable consequences for retinal neurons. However, in mice with photoreceptor cell-specific beta-gal expression, near-total destruction of photoreceptor cells was produced by CTL transfer. Attack on photoreceptor cells displayed minimal inflammation, and onset was a week later than onset of astrocyte-directed disease. CTL transfer into F1 mice expressing beta-gal in both astrocytes and photoreceptor cells confirmed that pathogenesis directed against antigen expressed in glia versus neurons proceeded in two distinct, independent phases. beta-gal-positive retinal astrocytes were severely affected by 5 days post-transfer, followed by rapid resolution. Photoreceptor cells in the same retina were unaffected until 12 days post-transfer. The susceptibility of photoreceptor cells was not enhanced by the prior CTL attack on beta-gal-expressing retinal astrocytes. The results demonstrate that extensive bystander killing of neurons can occur in vivo as a result of direct CTL attack on surrounding astrocytes. Antigen-expressing retinal neurons were also efficiently killed by CTLs, but by a mechanism that was substantially delayed and dissociated from the killing of retinal astrocytes.

Oxidative stress and nuclear factor-kappaB (NF-kappaB) activation are linked to the pathogenesis of many metabolic, degenerative, and chronic inflammatory diseases. Activation of the receptor for advanced glycation end products (RAGE) by its specific ligand N(epsilon)-carboxymethyllysine (CML) results in the activation of NF-kappaB and the production of proinflammatory cytokines. To determine whether engagement of RAGE contributes to the pathogenesis of inflammatory myopathies, we performed immunohistochemical studies on the presence of CML-modified proteins, RAGE and activated NF-kappaB in muscle biopsies of patients with polymyositis (PM, n=10), dermatomyositis (DM, n=10), limb girdle muscular dystrophy (LGMD, n=10) and in 10 controls with normal muscle biopsy results. In inflammatory myopathies CML, RAGE and NF-kappaB were detected in mononuclear cells and in regenerating muscle fibers. CML, NF-kappaB and, to a lesser extent, RAGE were also found in degenerating muscle fibers, but colocalization of CML, RAGE and NF-kappaB was only seen in infiltrating mononuclear cells and regenerating muscle fibers. Immunofluorescence double labeling demonstrated an expression of CML, RAGE and NF-kappaB in CD4-, CD8-, CD22- and CD68-positive mononuclear cells. Western blot analysis showed an increased immunoreactivity for CML-modified proteins in PM and DM. In LGMD, CML, RAGE and NF-kappaB were found in regenerating muscle fibers and less frequently in degenerating muscle fibers, and with lower staining intensities than in inflammatory myopathies. Our data suggests that the CML-RAGE-NF-kappaB pathway is an evident proinflammatory pathomechanism in mononuclear effector cells in PM and DM. RAGE-mediated NF-kappaB activation may be involved in muscle fiber regeneration in inflammatory myopathies and LGMD.

ZusammenfassungMyalgien können als unspezifisches Symptom bei einer Vielzahl neurologischer und anderer Erkrankungen auftreten. Häufig sind diese Schmerzen nicht durch eine primäre Schädigung des Skelettmuskels selbst verursacht, sondern Begleitsymptome unterschiedlicher Erkrankungen. Primäre muskuläre Ursachen müssen jedoch bedacht werden, um entsprechend zu beraten und eine angemessene Therapie anbieten zu können.In der vorliegenden Arbeit werden Myopathien und primär nicht muskuläre Erkrankungen im Hinblick auf die Differenzialdiagnose von Muskelschmerzen vorgestellt.

ZusammenfassungLokalisierte oder generalisierte Schmerzen der Skelettmuskulatur sind häufige Leit- oder Begleitsymptome von Erkrankungen aus verschiedenen medizinischen Fachdisziplinen. Die ätiologische Abklärung von Myalgien erfolgt anhand des Beschwerdebildes, des klinischen Untersuchungsbefundes und gegebenenfalls weiterführender Untersuchungen. Die vorliegende Arbeit stellt alle derzeitigen diagnostischen Möglichkeiten vor sowie einen Algorithmus für die zielgerichtete Diagnostik bei Myalgien.

The pathogenesis of the inflammatory myopathies is still unclear, making their treatment largely empirical. Improved understanding of the molecular mechanisms of inflammatory muscle injury may, however, lead to the development of more specific immunotherapies. To elucidate a possible pathogenic contribution of calcium-binding proteins such as the annexins, we immunohistochemically investigated muscle biopsy specimens from patients with dermatomyositis (10 cases), polymyositis (9 cases), and inclusion-body myositis (4 cases), compared to control cases comprising sarcoid myopathy (3 cases), Duchenne muscular dystrophy (DMD; 4 cases), and normal muscle (3 cases). We found expression of annexins A1, A2, A4, and A6 in the vascular endothelium of all cases. Myofibers expressed annexins A5, A6, and A7 diffusely and weakly in the cytosol, whereas annexins A5 and A7 were also particularly localized to the sarcolemma. In the inflammatory myopathies, in areas of myonecrosis in DMD, and in granulomatous lesions of sarcoid myopathy, reactivity of annexins A1, A2, A4, A5, and A6 was observed in macrophages and T-lymphocytes. Whereas the latter annexins appear to be nonspecific indicators of activation, annexin A1 upregulation may represent endogenous anti-inflammatory mechanisms that merit further investigation.

Binding of ligands to the receptor for advanced glycation endproducts (RAGE) results in activation of the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) and subsequent expression of NF-kappaB-regulated cytokines. In order to determine whether engagement of RAGE contributes to the pathogenesis of vasculitic neuropathy, we studied the presence of the RAGE ligand N(epsilon)-(carboxymethyl)lysine (CML), the receptor itself, NF-kappaB, and interleukin-6 (IL-6) in sural nerve biopsies of 12 patients with vasculitic neuropathies and 12 controls. In the patients, CML, RAGE, NF-kappaB, and IL-6 were localized in mononuclear cells, epineurial and endoneurial vessels and the perineurium. CML, RAGE, NF-kappaB, and IL-6 were expressed by CD4(+), CD8(+), and CD68(+) cells invading the nerves. Controls showed only weak staining. These data suggest that the RAGE pathway plays a critical proinflammatory role in vasculitic neuropathy.

Prostaglandins (PGs) are critical mediators of physiologic processes and inflammation. They are produced by two different isoforms of the cyclooxygenase (COX) enzyme, namely COX-1 and COX-2. In particular COX-2 was demonstrated to be crucial for PG-synthesis in inflammation. Recently, inhibition of COX-2 was shown to prevent the loss of motor neurons in a model of amyotrophic lateral sclerosis (ALS). Furthermore, spinal COX-2 expression was shown to be increased in transgenic mice that produce an ALS-like syndrome. Therefore, we investigated the expression of COX-1 and COX-2 in the spinal cord of seven human sporadic ALS patients by means of immunohistochemistry. Specimens from seven patients without any neurological disease served as controls. COX-2 expression was dramatically increased in the spinal cord of patients with ALS. Its protein was found in motor neurons, interneurons and glial cells. Statistical analysis showed a significantly higher expression of COX-2 in ALS for both neurons and glia. In contrast, COX-1 expression was predominantly confined to microglia and no apparent difference was detected between controls and ALS. In addition, we studied the concentration of prostaglandin E2 (PG E2) as a marker for COX activity in the cerebrospinal fluid of nine patients diagnosed for ALS and compared the results with those from nine patients without motor neuron disease. PG E2 levels were markedly increased in ALS cases (45.8 +/- 35.1 pg/mL) compared to the non-ALS specimens (15.8 +/- 3.7 pg/mL). The results of our study corroborate a potential role for COX-2 in the pathogenesis of motor neuron death in ALS. Selective COX-2 inhibition might therefore offer a new possibility in the treatment of human ALS. However, to determine the exact role of COX-2 in human ALS will require further research.

Vasculitic neuropathies are immune mediated diseases of the peripheral nervous system, in which inflammation of the blood vessels causes damage to the nerves. We distinguish neuropathies associated with primary and secondary systemic vasculitis, with rheumatic diseases, with malignant disorders, drug-induced vasculitis and the non-systemic vasculitic neuropathies (NSVN). The typical clinical picture consists in an asymmetric or multifocal, painful sensorimotor neuropathy with an acute, sub-acute or chronic course and acute relapses. Neurophysiology reveals an active, asymmetric, axonal sensorimotor neuropathy. The disorders usually respond to immunosuppressive treatment. A diagnosis of definite vasculitis can be made with evidence of vasculitis in a biopsy specimen. The absence of positive morphological evidence, however, does not exclude the diagnosis. There is no single laboratory test that can prove or exclude vasculitis, in NSVN even an elaborate panel of blood tests can show normal findings. Systemic vasculitis has an incidence of 4/100000 per year and, untreated, has a poor prognosis, which is greatly improved by the use of immunosuppressive treatment. The prognosis of NSVN is generally better, although many patients need long term immunosuppression. Current treatment recommendations for vasculitic neuropathies are presented.

ZusammenfassungUnter vaskulitischen Neuropathien versteht man immunvermittelte Erkrankungen des peripheren Nervensystems, bei denen die Schädigung der Nerven durch eine Entzündung der Blutgefäße bedingt ist. Unterschieden werden vaskulitische Neuropathien bei primären und sekundären systemischen Vaskulitiden, bei entzündlichrheumatischer Systemerkrankung, bei malignen Erkrankungen, medikamenteninduzierten Vaskulitiden und die nicht-systemische vaskulitische Neuropathie (NSVN). Das typische klinische Erscheinungsbild einer vaskulitischen Neuropathie ist eine asymmetrische oder multifokale schmerzhafte sensomotorische Neuropathie mit akutem/subakutem oder chronischem Verlauf mit schubförmigen Verschlechterungen. Charakteristisch sind weiterhin der elektrophysiologische Nachweis einer aktiven asymmetrischen axonalen sensomotorischen Neuropathie und die Besserung durch immunsuppressive Therapie. Eine gesicherte Vaskulitis liegt vor, wird ein positiver Biopsiebefund erhoben; das Fehlen eindeutiger morphologischer Befunde schließt sie jedoch nicht aus. Es gibt keinen Labortest, der eine Vaskulitis definitiv nachweist oder ausschließt, so ist auch bei ausschließlich unauffälligen Ergebnissen von durchaus umfangreichen Blutuntersuchungen das Vorliegen einer NSVN möglich. Systemische Vaskulitiden treten mit einer Inzidenz von mindestens 4/100000 jährlich auf und haben unbehandelt eine schlechte Prognose, die durch immunsuppressive Therapie entscheidend verbessert wird. Bei der NSVN ist die Prognose deutlich besser, eine längerfristige immunsuppressive Therapie ist auch hier häufig erforderlich.

The iron Lewis acid catalyst, [(η5-C5H5)Fe(CO)2(THF)]+[BF4]−, has previously been found to catalyze a growing array of synthetic transformations. Building upon these precedents, the utility of the catalyst has now been extended to catalytic reaction involving olefins and (N-(p-tosyl)imino)phenyliodinane, PhI=NTs, which serves as a nitrenoid precursor, to produce aziridines with yields up to 85%.

We studied the occurrence of Ne-(carboxymethyl)lysine (CML), the major in vivo Maillard product in nerve and muscle in various inflammatory and noninflammatory diseases using a previously characterised specific CML antiserum. We found an increased CML staining in the perineurium of biopsies from B12-deficient, diabetic and vasculitic neuropathy and to a lesser extent, in alcoholic neuropathy and in mononuclear inflammatory cells. In diabetic neuropathy CML, the receptor for advanced glycation endproducts (RAGE) and nuclear factor kappa B (activated NFκB) were colocalised indicating possible interaction. In myositis, intracellular CML depositions were observed in infiltrating monocytes, myoblasts and necrotic fibers and Western blot analysis showed a marked increase of CML-modified proteins. To evaluate the impact of inflammation-associated cells on CML formation, we designed in vitro experiments using granulocytes and the monocytic cell line Mono-Mac6 (MM6). Oxidative burst of zymosan-stimulated granulocytes led to CML modification of extracellular proteins within 60–120 min. Predifferentiated MM6 cells, exerting NADPH oxidase activity but lacking myeloperoxidase activity, showed CML modification of distinct intracellular proteins within 1–3 days upon stimulation. Our findings suggest that CML modification occurs in vivo and in vitro when oxidative stress from e.g. inflammatory cells is present, indicating that CML may represent a marker for oxidative stress.

In amyotrophic lateral sclerosis (ALS) patients, hyperintense signals at the subcortical precentral gyrus in brain fluid attenuated inversion recovery (FLAIR) MR images have been found more frequently than in controls. Quantitative analysis has revealed a significant increase of the FLAIR-magnetic resonance imaging (MRI) signal at the subcortical precentral gyrus of ALS patients compared to healthy controls. In addition, hypointense signals at the rim of the precentral gyrus in FLAIR and T2-weighted images have been shown in ALS patients. In 17 ALS patients, we evaluated hyperintense signals in T2-, T1-, proton density-weighted and FLAIR MR images, and hypointense signals in T2-weighted and FLAIR images 15.7+/-3.0 months after the initial examination by visual scoring. In FLAIR images, a quantitative analysis was added. The visual scores of hyperintense signals along the corticospinal tract did not change significantly in all sequences. However, the quantitative evaluation of FLAIR images revealed a significant increase of the signal intensity at the subcortical precentral gyrus (p

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease which has no known cure and limited symptomatic treatment. While coping strategies in more common diseases are widely assessed, coping is poorly understood in ALS.We examined 41 ALS patients using a standardised interview, a validated coping self-rating questionnaire and a self-rating depression scale. The evaluation was repeated after six months."Loss of speech", "loss of mobility" and "the poor prognosis" were the most frequent answers in the standardised interview to questions regarding the worst aspect of the disease. Pain was seldom mentioned. "Family members" were most helpful in coping with the disease, followed by "unspecific mechanisms" and "technical aids". None of our patients expressed a wish for assisted suicide. In comparison with other fatal diseases, patients with ALS had similar rankings in the coping mechanism of "rumination", but lower rankings in "search for social integration", "defence of fear", "search for information and communication". In contrast,* "search for hold in the religion" was of high importance for our ALS patients. In the follow-up examination the importance of "search for information and communication" increased.The results emphasise the importance of "loss of speech" and the importance of the caring family as well as the availability of technical aids in ALS. Coping in ALS seems to be based mainly on "rumination" and *"hold in the religion", but the increasing importance of "search for information" indicates that the sustained offer of information is essential.

Artículo científico -- Universidad de Costa Rica. Instituto de Investigaciones en Salud, 2001. Este documento es privado debido a limitaciones de derechos de autor. Autosomal recessive Charcot-Marie-Tooth disease (CMT) represents a heterogeneous group of disorders affecting the peripheral nervous system. The axonal form of the disease is designated as "CMT type 2" (CMT2), and one locus (1q21.2-q21.3) has been reported for the autosomal recessive form. Here we report the results of a genomewide search in an inbred Costa Rican family (CR-1) affected with autosomal recessive CMT2. By analyzing three branches of the family we detected linkage to the 19q13.3 region, and subsequent homozygosity mapping defined shared haplotypes between markers D195902 and D19S907 in a 5.5-cM range. A maximum two-point LOD score of 9.08 was obtained for marker 019S867, at a recombination fraction of .00, which strongly supports linkage to this locus. The epithelial membrane protein 3 gene, encoding a PMP22 homologous protein and located on 19q13.3, was ruled out as being responsible for this form of CMT. The age at onset of chronic symmetric sensory-motor polyneuropathy was 28-42 years (mean 33.8 years); the electrophysiological data clearly reflect an axonal degenerative process. The phenotype and locus are different from those of demyelinating CMT4F, recently mapped to 19q13.1-13.3; hence, the disease affecting the Costa Rican family constitutes an axonal, autosomal recessive CMT subtype (ARCMT2B). Universidad de Costa Rica. Instituto de Investigaciones en Salud UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)

Segmental muscle enlargement occurs in a variety of neurogenic conditions. We present a patient with calf hypertrophy, likely produced by continuous neuromuscular irritability and compensatory type 1 and type 2 muscle fiber hypertrophy. The underlying lesion of the SI nerve root was caused by scarring, which could be demonstrated by Cadolinum enhanced, fat saturated magnetic resonance imaging. Thus, the application of this technique is recommended in otherwise etiologically unclear cases of neurogenic muscular lesions in order to detect chronic nerve root pathology. [Neurol Res 2000; 22: 469-472]

Chronic venous insufficiency (CVI) of the lower legs may cause tissue damage, but involvement of peripheral nerves is uncertain. We examined 30 patients with CVI and 20 healthy controls using motor and sensory nerve conduction studies, vibration testing and thermotesting, quantitative sudomotor axon-reflex test, and laser Doppler flowmetry. Subjects with possible confounding factors for peripheral neuropathies were excluded. Prolongation of distal motor latency of the peroneal nerve (median, 5.4 versus 4.5 ms; P = 0.02), increased limits for warm (9.60 degrees C versus 5.20 degrees C; P = 0.016) and cold detection (3.45 degrees C versus 1.55 degrees C; P = 0.016) and reduced vibration sense (2.8925 versus 1.1075; P < 0.008) were found. The results demonstrate a disturbance of A-alpha fibers, A-beta fibers, A-delta fibers, and thermoafferent-C fibers, possibly induced by ischemia due to venous microangiopathy and increased endoneurial pressure. Analogous to neuropathic ulcers in diabetes, the CVI-associated neuropathy may also be a cofactor in the development of venous ulcers.

Die haufigste Ursache der Polyneuropathien (PNP) ist mit ca. 35% der Diabetes, ca. 22% der Falle sind ungeklarter Atiologie, ca. 11% alkoholinduziert, zusammen etwa 2 Drittel aller Falle. Unter den weiteren Ursachen der PNP finden sind Vaskulitiden in ca. 4%. Pathogenetisch resultiert als Folge von entzundlichen Infiltraten der Gefaswande, Stenosierungen der Vasa nervorum oder Einblutungen schlieslich die ischamische Lasion von Nervenfasern, die zu einer axonalen Degeneration fuhrt. Die folgende Ubersicht geht zunachst auf die Klassifikation der Vaskulitiden ein, wobei festgestellt werden muss, dass die hier aus der Sicht des Neurologen verwendete Klassifikation sich von der heutzutage ublicherweise in der Inneren Medizin bevorzugten Einteilung der Vaskulitiden entsprechend der Chapel-Hill-Consensus-Conference (nach dem Gefastyp) unterscheidet. Sodann werden wesentliche Gesichtspunkte zur Epidemiologie, Pathophysiologie, Pathomorphologie und Diagnostik beschrieben und systematisch die Nosologie der verschiedenen Formen der Vaskulitiden unter besonderer Berucksichtigung neurologischer Storungen referiert. In tabellarischer Form werden bewahrte, neue und experimentelle Therapieoptionen dargestellt. Fur die tagliche Praxis sollte festgehalten werden, dass neurologische Symptome im Sinn einer PNP immer auch an eine Vaskulitisinduzierte Ursache denken lassen sollten.

Vasculitic neuropathy shows cluster of differentiation (CD)4+ and CD8+ perivascular infiltrates. CD8+ cells kill the target by different ways. Some mechanisms require the release of granzymes and T-cell restricted intracellular antigen (TIA-1) which induce cell death by apoptosis. A nonsecretory ligand-mediated mechanism has been proposed for apoptosis and requires the interaction of Fas and Fas-ligand. We studied apoptosis by immunohistochemistry and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) in nerve biopsy specimens in 19 cases of vasculitic neuropathy and 8 controls. In vasculitic neuropathy, perivascular mononuclear cells expressed TIA-1, granzyme A, and granzyme B. Regarding the ligand-mediated mechanism, we observed Fas+ and Fas-ligand+ mononuclear cells in an epineurial-perivascular distribution. Cells undergoing apoptosis, as demonstrated by TUNEL, represented mononuclear cells. In vasculitic neuropathy, apoptosis by both a secretory and a nonsecretory mechanism is suspected. However, apoptosis seems to be restricted to inflammatory mononuclear cells, suggesting that it may play a critical role in recovery from vasculitic neuropathy.

Multiple system atrophy represents an enigmatic, clinico-pathologically defined neurodegenerative disease. According to findings of the last three decades, the historically derived, previously used terms olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager-syndrome should be avoided in clinical use because of both the clinical and morphological overlap between these syndromes. Complex neurodegenerative syndromes other than multiple system atrophy according to recently developed criteria, should rather be referred to as multiple system degenerations.

Hepatitis C virus (HCV) infection is often associated with abnormal immunological responses. We describe four patients with vasculitic neurological signs and symptoms following HCV infection. A 56-year-old woman with HCV infection developed peripheral neuropathy characterized by asymmetric distal painful hypesthesia, dysesthesia and moderate motor weakness of the lower limbs. Serological examinations revealed cryoglobulinemia and low levels of complement C4. A biopsy of the sural nerve revealed vasculitic neuropathy. HCV infection associated immunomediated vasculitis was diagnosed. While steroid therapy was ineffective, treatment with interferon-alpha improved the neuropathy considerably without, however, eliminating HCV infection. A 62-year-old man with HCV infection developed peripheral sensory neuropathy. Complement C3 was slightly diminished. Nerve biopsy revealed vasculitic neuropathy. A 71-year-old woman developed chronic symmetric sensomotor polyneuropathy. HCV hepatitis followed blood transfusions. Cryoglobulins tested positive, consistent with type II cryoglobulinemia. Complements C3 and C4 were diminished. Inflammatory infiltrates in the sural nerve biopsy specimen led to the diagnosis of chronic vasculitic disorder. A 55-year-old woman with HCV infection developed vasculitis of the skin, connective tissue, visceral organs, and kidney, leading to hemodialysis. Neurologically she developed severe apathy and drowsiness, myoclonic jerks, exaggerated deep tendon reflexes, and positive pyramidal signs. Magnetic resonance imaging of the brain showed diffuse increased signal abnormalities involving supra- and infratentorial white matter suggesting cerebral vasculitis. Cryoglobulins were positive, complements C3 and C4 slightly diminished (54 mg/dl, 4.3 mg/dl). Supportive therapy resulted in neurological improvement. Treatment with interferon-alpha was discontinued because of agranulocytosis. In patients with peripheral neuropathy or signs of leucencephalopathy, a hepatitis C associated vasculitis should be considered in the differential diagnosis.

Bartonella henselae, the causative agent of cat scratch disease and bacillary angiomatosis, is associated with an expanding spectrum of diseases. Here, we report on a 40-year-old patient suffering from chronic recurrent painful ulcers of the toes, distal axonal sensomotor polyneuropathy and Raynaud's phenomenon. Biopsy of the sural nerve demonstrated an axonal neuropathy with a neurogenic muscular atrophy. Treatment with high dose corticosteroids had no beneficial effect. A biopsy taken from a recurring ulcer 7 years after the beginning of the disease revealed superficial ulcerated hyperkeratosis with subepithelial proliferation of small vessels compatible with a diagnosis of verruca peruana, however, without detection of microorganism. Serologic analysis revealed an elevated IFT titer of 1:1,024 against B. henselae. Treatment with erythromycin induced healing of the ulcer, remission of the vasculitis and the polyneuropathy, and a decline of the IFT titer. This case illustrates that B. henselae infection should be considered in patients with vasculitis and polyneuropathic syndromes.

A multicenter test was conducted to investigate the effectiveness of the selective serotonin agonist sumatriptan in patients with the computerized headache diagnosis of migraine. A computer program was used for diagnostic evaluation of patients attending a general practice because of headache. The results of the analysis were taken as a direct decision on therapy. If the patients satisfied the criteria for migraine, they were given subcutaneous sumatriptan for treating three migraine attacks. The patients were able to use the study medication under outpatient conditions. The therapeutic efficacy of the medicine was recorded in a headache diary. A total of 91 patients were included in the study at 22 practices in Germany. An average of four patients per practice were recruited. In the first migraine attack treated, headache improvement was experienced by 77.7% of the patients treated. In the second and third attacks an improvement was experienced by 93.5% and 89.8%, respectively. The results show that by optimizing diagnostic reliability with the aid of the computer program a high response rate can be achieved under practice conditions using the selective serotonin agonist sumatriptan. Since the computer program described permits a specific diagnosis, it improves the prospects of effective headache therapy in the individual patient. Thus treatment based on this approach can reduce inputs of time and money in migraine therapy.

The insulin like growth factors (IGFs) are mitogenic peptides that can stimulate cell division and differentiation. In experimental studies it has been shown that there is local-production of IGFs and their mRNA in regenerating muscle. The effect of IGF-I and -II is mediated by a cell surface, membrane bound receptor (IGF-I-R). In addition, IGF-II has its own distinct type 2 receptor. But the role of IGF-II binding to the type 2 receptor is unclear, and it is now widely believed that the growth-promoting activities of IGF-II are also mediated via the IGF-I-R. So far, there is some knowledge about the regulation and effects of IGFs in muscle regeneration in vitro and in animal models in vivo. However, cell lines and in vitro experiments with myogenic precursors differ in some respects from human regenerating muscle in vivo. Thus, we performed our immunohistochemical study to investigate the potential role of IGF-II and IGF-I-R in regenerating human skeletal muscle in situ. To investigate the state of regeneration, neural cell adhesion molecule (N-CAM) and cytoskeletal protein vimentin serial sections were also performed. Light-microscopic evaluation showed that muscle fiber regeneration in inflammatory and dystrophic myopathy corresponds closely to IGF-II and IGF-I-R immunoreactivity in muscle fibers. The coexpression of IGF-II and IGF-I-R in regenerating muscle fibers corroborates the assumption that in human skeletal muscle there is a trophic pathway concerning the synthesis and autocrine action of IGF-II via the IGF-I-R. In conclusion, in human skeletal muscle, in vivo, muscle fiber derived IGF-II probably is biologically active on the cell type of origin and may play an autocrine role in muscle regeneration via the IGF-I-R.

Bei Reizung von Afferenzen des N. trigeminus wird die Willkuraktivitat der Kaumuskulatur typischerweise in 2 aufeinanderfolgenden Phasen (ES1 und ES2) gehemmt. Die erste Hemmphase wird fruhe exterozeptive Suppressionsphase (ES1), die zweite Hemmphase spate exterozeptive Suppressionsphase (ES2) genannt. Die zwischen den beiden Suppressionsperioden gelegene Phase wird als Fazilitationsperiode (FP) bezeichnet. In der Regel ist dieses Suppressionsverhalten bei exterozeptiver Reizung bei Gesunden regelmasig auslosbar. Zwar kann der Reflex auch mit nicht schmerzhaften Reizen ausgelost werden, das Antwortverhalten ist jedoch bei Einsatz von schmerzhaften Reizen maximal. Aufgrund dieses Zusammenhangs zwischen Reizintensitat, Schmerzwahrnehmung und Reflexantwort wird der Reflex als antinozizeptive Reaktion angesehen. Chronische Schmerzerkrankungen wie der chronische Kopfschmerz vom Spannungstyp und die Migrane ohne Aura gehen mit Veranderungen im normalen Ableitbild der ES einher. Ebenso last sich die Auspragung der ES durch unterschiedliche, in der Schmerztherapie angewandte Substanzen wie z. B. Serotoninagonisten-oder antagonisten, Acetylsalicylsaure oder Naloxon beeinflussen. In dieser Ubersicht werden die verschiedenen Einflusgrosen auf die ES-Reflexantwort systematisch aufgezeigt. Darauf aufbauend wird die diagnostische Bedeutung dieser Veranderungen fur pathophysiologische Vorgange in der Schmerzverarbeitung und bei Schmerzerkrankungen dargelegt.

The rationale for using essential oils to alleviate headache is based on several assumptions. Especially for peppermint oil certain analgesic mechanisms were recently described. Local application of peppermint oil generates a long-lasting cooling effect on the skin, caused by a steric alteration of the calcium channels of the cold-receptors. Further it was shown that peppermint oil inhibits non-competively 5-hydroxytryptamin (Serotonine) and substance P induced smooth muscle contraction in animal-models. It is also known that peppermint oil induces a significant increase of the skin blood flow of the forehead after local application, measured by laser doppler. Assuming that a clinical relevant analgesic action exists, this ought to be observable in experimental algesimetric human tests. Therefore the effects of peppermint oil and eucalyptus oil preparations on neurophysiological, psychological and experimental algesimetric parameters were investigated in 32 healthy subjects in a double-blind, placebo-controlled, randomized cross-over design. Four different test preparations were used: preparation 1 (LI1701) consisted of 10 g peppermint oil and 5 g eucalyptus oil plus ethanol 90% to 100 g; preparation 2 (LI1702) of 10 g peppermint oil and traces of eucalyptus oil plus ethanol 90% to 100g; preparation 3 (LI1703) of traces of peppermint oil and 5 g eucalyptus oil plus ethanol 90% to 100 g; and preparation 4 (placebo) of traces of peppermint oil and traces of eucalyptus oil plus ethanol 90% to 100 g. The test preparations were applied to large areas of the forehead and temples using a small sponge. The treatment effect of the preparations was evaluated by comparing baseline and treatment measurements. The combination of peppermint oil, eucalyptus oil and ethanol can increase cognitive performance while having a muscle-relaxing and mentally relaxing effect, but has little influence on pain sensitivity. A significant analgesic effect with a reduction in sensitivity to headache is however produced by the combination of peppermint oil and ethanol. The essential plant oil preparations can thus be shown by laboratory tests to exert significant effects on mechanisms associated with the pathophysiology of clinical headache syndromes. Further, to investigate the efficacy of essential oil preparations compared to usual analgesics (paracetamol or acetylsalicylicacid) a double blind, placebo controlled, randomized study should be performed.