Frequency and predictors of acute ischaemic lesions on brain magnetic resonance imaging in young patients with a clinical diagnosis of transient ischaemic attack.

BACKGROUND AND PURPOSE: Acute lesions in patients with transient ischaemic attack (TIA) are important as they are associated with increased risk for recurrence. Characteristics associated with acute lesions in young TIA patients were therefore investigated. METHODS: The sifap1 study prospectively recruited a multinational European cohort (n = 5023) of patients aged 18-55 years with acute cerebrovascular event. The detection of acute ischaemic lesions was based on diffusion-weighted imaging (DWI). The frequency of DWI lesions was assessed in 829 TIA patients who met the criteria of symptom duration <24 h and their association with demographic, clinical and imaging variables was analysed. RESULTS: The median age was 46 years (interquartile range 40-51 years); 45% of the patients were female. In 121 patients (15%) ≥1 acute DWI lesion was detected. In 92 patients, DWI lesions were found in the anterior circulation, mostly located in cortical-subcortical areas (n = 63). Factors associated with DWI lesions in multiple regression analysis were left hemispheric presenting symptoms [odds ratio (OR) 1.92, 95% confidence interval (CI) 1.27-2.91], dysarthria (OR 2.17, 95% CI 1.38-3.43) and old brain infarctions on MRI (territories of the middle and posterior cerebral artery: OR 2.43, 95% CI 1.42-4.15; OR 2.41, 95% CI 1.02-5.69, respectively). CONCLUSIONS: In young patients with a clinical TIA 15% demonstrated acute DWI lesions on brain MRI, with an event pattern highly suggestive of an embolic origin. Except for the association with previous infarctions there was no clear clinical predictor for acute ischaemic lesions, which indicates the need to obtain MRI in young individuals with TIA.

Association of aneurysms and variation of the A1 segment.

BACKGROUND AND PURPOSE: Previous studies have described a correlation between variants of the circle of Willis and pathological findings, such as cerebrovascular diseases. Moreover, anatomic variations of the anterior cerebral artery (ACA) seem to correspond to the prevalence of aneurysms in the anterior communicating artery (ACoA). The aim of this study was to assess the prevalence of aneurysms in patients with anatomical/morphological variations of the circle of Willis. METHODS: We retrospectively analyzed 223 patients who underwent cerebral angiography between January 2002 and December 2010 for aneurysm of the ACoA. Diagnostic imaging was reviewed and statistically evaluated to detect circle of Willis anomalies, aneurysm size, and rupture. 204 patients with an unrelated diagnosis served as the control group. RESULTS: Variations of the A1 segment occurred significantly more frequently in the aneurysm group than in the control group. Mean aneurysm size in patients with grades I and III hypoplasia or aplasia was 6.58 mm whereas in patients with grade II hypoplasia it was 7.76 mm. CONCLUSIONS: We found that variations in the A1 segment of the ACAs are correlated with a higher prevalence of ACoA aneurysms compared with patients with a symmetric circle of Willis.

Accessory eye muscle in a young boy with external ophthalmoplegia.

A seven-year-old boy presented to the neuropediatric clinic with right-sided ptosis, external ophthalmoplegia, and mental retardation. Orbital magnetic resonance imaging (MRI) revealed anomalous soft tissue within the intraconal region, which was interpreted as an atavistic retractor bulbi muscle. In patients with external ophthalmoplegia and ptosis, anatomic variations observed in MRI should be given due consideration. In fact, knowledge about rare anatomical anomalies may contribute to a more accurate diagnosis of such ocular symptoms.

Beta-endorphin (1-31) in the plasma of male volunteers undergoing physical exercise.

beta-Endorphin is an opioid peptide representing the C-terminal 31 amino acid residue fragment of proopiomelanocortin (POMC). The release of beta-endorphin from the pituitary into the cardiovascular compartment under physical or emotional stress has been frequently reported. However, besides beta-endorphin (1-31), nine acetylated or non-acetylated beta-endorphin analogues exist - in addition to N-terminally elongated beta-endorphin derivatives such as beta-lipotropin (beta-LPH). Since conventional radioimmunoassays (RIAs) and even commercially available two site-RIAs pick up at least some of those beta-endorphin derivatives, only "beta-endorphin immunoreactive materials" and not authentic beta-endorphin have been determined in those studies. We have developed a highly specific two site-RIA for beta-endorphin (1-31), which does not cross-react with all beta-endorphin derivatives known to occur as yet. Using this RIA as well as further assays for determination of beta-endorphin (1-31), beta-endorphin immunoreactive material (IRM), ACTH and Cortisol in the plasma of 14 volunteers upon intensive physical exercise, we found authentic beta-endorphin only in about 50% of the plasma samples, representing therein only a minor portion of the beta-endorphin IRM.

Immunocytochemical detection of somatostatin receptors sst1, sst2A, sst2B, and sst3 in paraffin-embedded breast cancer tissue using subtype-specific antibodies.

The long-acting somatostatin analogue octreotide (SMS 201-995) inhibits growth of certain breast cancer cell lines in vivo and in vitro. Because the antiproliferative action of octreotide depends on at least the presence of somatostatin receptors, it is crucial to determine the pattern of somatostatin receptor protein expression on the tumor cells. In the present study, we have raised polyclonal antibodies to somatostatin receptor subtypes (ssts) sst1, sst2A, sst2B, and sst3 using peptides corresponding to their COOH-terminal sequences. These antisera were used for immunocytochemical staining of paraffin sections of 33 primary breast cancers. Somatostatin receptor-like immunoreactivity (Li) was predominantly localized to the plasma membrane of the tumor cells. In the vast majority of positively stained tumors, somatostatin receptor-Li was uniformly present on nearly all tumor cells. Both the level and the pattern of expression of ssts varied greatly between individual carcinomas. sst2A-Li and/or sst2B-Li was detectable in 28 tumors (85%); among these, 14 tumors (42%) showed particularly high levels of sst2-Li. sst1-Li was found in 17 (52%) cases and sst3-Li in 16 (48%) cases. The expression of ssts was independent of patient age, menopausal status, diagnosis, histological grade, and levels of estrogen and progesterone receptors. The immunocytochemical determination of somatostatin receptor status allows direct detection of receptor protein on the tumor cells and, hence, may provide more precise information than reverse transcription-PCR for predicting response to octreotide therapy in breast cancer.

Immunolocalization of two mu-opioid receptor isoforms (MOR1 and MOR1B) in the rat central nervous system.

We have recently shown that the cytoplasmic tail of the rat mu-opioid receptor undergoes alternative splicing giving rise to two isoforms, rMOR1 and rMOR1B. These isoforms exhibit similar pharmacological profiles, however, differ in agonist-induced desensitization of coupling to adenylate cyclase. In the present study, we have raised polyclonal antibodies that specifically detect either rMOR1 or rMOR1B and used these antisera for immunocytochemical localization of the receptor proteins in the rat central nervous system. Prominent MOR1B-like immunoreactivity was found in the external plexiform layer of the main olfactory bulb localized to a dense plexus of dendrites mostly originating from mitral cells and extending into the glomerular layer. MOR1-like immunoreactivity was restricted to the perikarya of mitral cells and to distinct juxtaglomerular cells as well as their processes. While MOR1-, DOR1- and KOR1-like immunoreactivity was absent from the external plexiform layer, high densities of opioid peptides were found in this layer suggesting that MOR1B may be a targeted receptor of these peptides. MOR1-like immunoreactivity was observed in many pain-controlling brain areas including the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei and periaqueductal gray while MOR1B-like immunoreactivity was not detectable in these regions. Taken together, we provide evidence that the mu receptor isoforms, MOR1 and MOR1B, exhibit a strikingly different distribution in that MOR1 appears to be the major isoform widely distributed throughout the central nervous system and MOR1B being predominantly localized to the olfactory bulb.

Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions.

Antisera were generated against nociceptin/orphanin FQ, the putative ligand of the opioid receptor-like ORL1 receptor. Dot blot analysis showed that the antibodies selectively detect nociceptin but not dynorphin or other opioid peptides. Immunofluorescent staining of tissue sections revealed dense plexus of nociceptin-immunoreactive nerve fibres and terminals within the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei, locus coeruleus, periaqueductal grey, amygdala, habenula, hypothalamic region and septal area in mice and rats. When adjacent sections were stained either with the nociceptin antibody or the pan-opioid 3-E7 mouse monoclonal antibody, an overlapping distribution was observed in many nociceptive centres including the superficial dorsal horn, sensory trigeminal complex and periaqueductal grey. However, confocal microscopic examination of dual-labelled spinal cord and brain stem sections showed no instances of co-localization of nociceptin and opioid peptides in these regions. Intracerebroventricular administration of nociceptin has been shown to induce hyperalgesia. Thus, the present results suggest that nociceptin and opioids are released from different terminals thereby modulating pain signals in opposite ways.

Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation.

Exogenous opioids can produce localized opioid receptor-mediated antinociception in peripheral inflamed tissue. Previous studies show that activation of endogenous opioids by a cold water swim in rats with hind paw inflammation results in a similar local antinociceptive effect but suggest that pituitary-adrenal opioid pools are not directly involved in producing this effect. Here we show increased amounts of opioid peptides in immune cells infiltrating the inflamed tissue. Furthermore, we demonstrate immunoreactive opioid receptors on peripheral terminals of sensory neurons. The local administration of antibodies against opioid peptides or receptors or systemic pretreatment with the immunosuppressant cyclosporine blocks cold water swim-induced antinociception. These findings suggest that antinociception in inflammation can be brought about by endogenous opioids from immune cells interacting with opioid receptors on peripheral sensory nerves.

Intrinsic mechanisms of antinociception in inflammation: local opioid receptors and beta-endorphin.

This study examined antinociception induced through the activation of local opioid receptors in inflammation by endogenous opioids. Rats developed a unilateral localized inflammation upon injection of Freund's adjuvant into one hindpaw. Four to 6 d later they were subjected to cold water swim (CWS), an environmental stimulus known to activate intrinsic opioid systems. Following CWS (1 min) the animals' withdrawal threshold to noxious pressure applied onto the paws increased significantly more on the inflamed paw than on the noninflamed paw. This unilateral antinociceptive effect in inflamed paws was dose-dependently and stereospecifically reversible by intraplantar (i.pl.) but not systemic (i.v. or s.c.) administration of the opioid antagonist naloxone (18 micrograms). This suggested that CWS-induced antinociception in inflamed tissue was brought about by the activation of local opioid receptors. Antiinflammatory or vasoconstrictive events, as measured by paw volume and temperature, did not contribute to this unilateral antinociception. Receptor-selective antagonists indicated the involvement of mu- and delta- but not kappa-receptors. Intravenous application of a universal antibody to endogenous opioid peptides (3-E7) and a specific antibody to beta-endorphin, but not antisera against metenkephalin or dynorphin, abolished the CWS effect. Finally, the i.pl. injection of synthetic beta-endorphin (1-31) produced an antinociceptive effect in inflamed paws which was reversible by i.pl. naloxone and selective mu- and delta-receptor antagonists. These findings suggest that antinociception in inflamed tissue can be induced through the activation of local opioid receptors by endogenous beta-endorphin released during CWS.

Beta-endorphin in the brainstem and the cerebellum of the human infant: regional levels' profile assessed with immunoaffinity chromatography and solid phase radioimmunoassay.

The regional levels' profile of human beta-endorphin (beta h-EP) was studied in the brainstem and the cerebellum of 16 infant victims of "Sudden Infant Death Syndrome" and other death causes. An immunoaffinity chromatography procedure based on a monoclonal antibody directed specifically against the N-terminus of beta-EP was used to extract this peptide from the tissue samples. Beta-EP was then assessed quantitatively by means of a very sensitive solid phase radioimmunoassay (using a polyclonal antibody specific for the C-terminus of beta-EP) developed especially for the study presented here.

Cerebral beta-endorphin levels in a woman with Prader-Labhart-Willi syndrome.

By means of a specific two-site immunoradiometric assay, we explored the beta-endorphin levels in various brain regions of a patient affected by Prader-Labhart-Willi Syndrome. The rank of the beta-endorphin levels of five cerebral zones (hypothalamus, substantia grisea centralis, pons dorsalis, medulla oblongata dorsalis medialis, thalamus medialis) of the patient was homologous to that of subjects without the syndrome, except for the medulla oblongata dorsalis medialis. In patient with the Prader-Labhart-Willi Syndrome this region had a higher ranking level than in subjects without it. However, a functional meaning cannot be attributed to such difference because the patient of this study did not exhibit neurological disturbances relating to elevated beta-endorphin levels in the medullary region investigated.

Immunocytochemical demonstration of opioid receptors in selected rat brain areas and neuroblastoma x glioma hybrid (NG108-15) cells using a monoclonal anti-idiotypic antibody.

A monoclonal anti-idiotypic opioid receptor antibody was used for the light-microscopic visualization of opioid receptors in several brain structures and monolayer cultures of a neuroblastoma x glioma hybrid cell-line (NG108-15). The antibody proved to be specific, displaying affinity for mu greater than delta much greater than kappa opioid receptors. Receptor distribution in the brain areas studied was in agreement with previous autoradiographic analyses; of particular interest, high densities of immunoreactive opioid receptors were found in the perikarya and in the initial parts of the axons and dendrites; light microscopy did not allow an exact determination of the subcellular localization of opioid receptors, but the immunoreactivity seemed to be associated with the plasma membrane and to be present within the cytoplasm as well. Similar observations were made for the cell bodies and neurites of NG108-15 cells. The methodology described potentially permits the study of opioid receptor distribution in discrete brain areas under different physiological and pharmacological conditions and of the ontogeny of these receptors; in addition, it may help to find a morphological basis for events such as receptor internalization and recycling.

Peptide neuroanatomy of adjuvant-induced arthritic inflammation in rat.

The influence of adjuvant-induced arthritis of the rat on central and peripheral peptide neuroanatomy was investigated by immunohistochemistry. The most striking feature of arthritic rats was the differential intensification of neuronal proenkephalin- and prodynorphin-related staining in dorsal horn. Changes were ipsilateral in monoarthritic and bilateral in polyarthritic rats as compared to controls. Opioid responsive neurons were target of substance P (SP) and calcitonin gene-related peptide (CGRP) fibers. Changes of SP and CGRP predominated in peripheral inflamed tissue and consisted of intensified immunostaining and an apparent sprouting of sensory fibers particularly around venules, in the epidermis and in areas infiltrated by immunocompetent cells. Opioid staining was absent from primary afferents but present in some immune cells of inflamed tissue. Endogenous antinociceptive opioids and pro-nociceptive/pro-inflammatory SP and CGRP may be crucial in the concerted response of the neuroimmune system to chronic inflammatory pain.

Monoclonal anti-idiotypic antibodies to opioid receptors.

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., Höllt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.

A novel two-site immunoradiometric assay for beta-endorphin using nitrocellulose as solid phase.

A two-site immunoradiometric assay for the highly specific direct quantitation of nonacetylated beta h-EP in crude brain tissue samples has been developed with a detection limit of 10 fmol per well. The assay used two different antibodies with distinct specificities: a polyclonal rabbit anti-beta h-EP antibody binding between the middle portion and the C-terminal end of beta h-EP was bound to nitrocellulose membrane discs, a solid phase with a high protein binding capacity. In the following two incubation steps, the beta h-EP containing crude tissue extract--or the beta h-EP-standard--and, subsequently, the 125I-labeled monoclonal 3-E7 mouse antibody directed against the N-terminus of beta h-EP were added. Binding of beta h-EP to the solid phase antibody in the first incubation step was not affected by the addition of cross reacting opioid peptides derived from beta h-LPH up to 10 pmol per disc. Nonspecific binding of the labeled antibody to the solid phase could be lowered to 3% of total counts by the use of PBS containing nonfat dry milk as blocking solution and incubation buffer, a procedure that did not reduce maximum specific binding. Dilution studies performed with extracts sampled from the anterior hypothalamus excluded the interference of tissue factors in the assay.

Cerebral distribution of beta-lipotropin and beta-endorphin in infantile progressive spinal muscular atrophy of Werdnig and Hoffman disease.

The regional distribution's profile of beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) was determined in the brain of an infant who died from Werdnig-Hoffmann's disease. Regional levels of beta-endorphin-like immunoreactivity (beta-ELIR), resulting from beta-EP and beta-LPH, were generally low in comparison to the homologous levels found in victims dying of other diseases.

Opiates induce long-term increases in prodynorphin-derived peptide levels in the guinea-pig myenteric plexus.

The subcutaneous administration of a single dose of an opiate agonist (levorphanol) or antagonist (naloxone) to guinea pigs results in an at least 3-fold elevation of dynorphin and alpha-neoendorphin-immunoreactivity in the longitudinal muscle myenteric plexus preparation. The effects are time- and dose-dependent, significant elevations first being observed 6 h after treatment and lasting for up to 24 h. Pretreatment levels of opioid peptides were observed after 8 days. Combined injection of the narcotic agonist and antagonist, at sufficiently high doses, resulted in an additive effect of the individual drugs. The respective stereoisomers dextrorphan and (+)-naloxone did not affect prodynorphin-derived peptide concentrations. An increase of endogenous opioids was also observed after administration of the nonopiate clonidine, a compound which, like opiates, alters the activity of the myenteric plexus. It is suggested that feedback mechanisms in the myenteric plexus are responsible for the elevation of endogenous opioid peptides following exposure to exogenous opiates. Using a monoclonal antibody (3-E7), which recognizes virtually all endogenous opioid peptides, it was found that levels of higher molecular material were also increased upon opiate challenge. This suggests that a single dose of an exogenous opiate results in an increase in peptide synthesis.