Survival of melanopsin expressing retinal ganglion cells long term after optic nerve trauma in mice.

In this study we have compared the response to optic nerve crush (ONC) and to optic nerve transection (ONT) of the general population of retinal ganglion cells in charge of the image-forming visual functions that express Brn3a (Brn3a+RGCs) with that of the sub-population of non-image forming RGCs that express melanopsin (m+RGCs). Intact animals were used as control. ONT and ONC were performed at 0.5 mm from the optic disk, and retinas dissected 3, 5, 7, 14, 30, 45 or 90 days later (n = 5/injury/time point). In all the retinas, Brn3a+RGCs and m+RGCs were identified and their survival analyzed quantitatively and topographically. There were no differences in the course of RGC loss between lesions. The decrease of RGCs was significant at short time points (3 or 5 days for Brn3a+ or m+ RGCs, respectively) and, up to 14 days, the course of loss of both RGC populations was similar, surviving at this time point between 20 and 22% of their original population. However, while the loss of Brn3a+RGCs continues steadily up to 90 days when only 5-6% of them still remain, the loss of m+RGCs stops at 14 days, and the proportion of surviving m+RGCs remains constant up to 90 days (26-30%). In conclusion, m+RGC do not respond to axotomy in the same way than the rest of RGCs, and so whilst image-forming RGCs die in two exponential phases a quick one and a slow protracted one, non-image forming RGCs die only during the first quick phase.

Nerve fibre layer degeneration and retinal ganglion cell loss long term after optic nerve crush or transection in adult mice.

We have investigated the long term effects of two different models of unilateral optic nerve (ON) lesion on retinal ganglion cells (RGCs) and their axons, in the injured and contralateral retinas of adult albino mice. Intact animals were used as controls. The left ON was intraorbitally crushed or transected at 0.5 mm from the optic disk and both retinas were analyzed at 2, 3, 5, 7, 14, 30, 45 or 90 days after injury. RGCs were immunoidentified with anti-Brn3a, and their axons with anti-highly phosphorylated axonal neurofilament subunit H (pNFH). After both lesions, RGC death in the injured retinas is first significant at day 3, and progresses quickly up to 7 days slowing down till 90 days. In the same retinas, the anatomical loss of RGC axons is not evident until day 30. However, by two days after both lesions there are changes in the expression pattern of pNFH: axonal beads, axonal club- or bulb-like formations, and pNFH+RGC somas. The number of pNFH+RGC somata peak at day 5 after either lesion and is significantly higher than in intact retinas at all time points. pNFH+RGC somata are distributed across the retina, in accordance with the pattern of RGC death which is diffuse and homogenous. In the contralateral retinas there is no RGC loss, but there are few pNFH+RGCs from day 2 to day 90. In conclusion, in albino mice, axotomy-induced RGC death precedes the loss of their intraretinal axons and occurs in two phases, a rapid and a slower, but steady, one. Injured retinas show similar changes in the pattern of pNFH expression and a comparable course of RGC loss.

Utilidad de la resonancia magnética cerebral con secuencia de difusión y valor b alto en el diagnóstico de la enfermedad de Creutzfeldt-Jakob / Usefulness of high b-value diffusion-weighted MRI in the diagnosis of Creutzfeldt-Jakob disease

Introducción: los criterios diagnósticos actuales de la enfermedad de Creutzfeldt-Jakob (ECJ) probable incluyen la combinación de datos clínicos, electroencefalográficos y analíticos. En los últimos años se ha demostrado que la RM craneal con el uso de secuencias FLAIR y difusión (DWI) puede ser una herramienta útil en el diagnóstico de esta enfermedad. Describimos nuestra experiencia en la utilización de la DWI convencional (b: 1000s/mm2) y DWI con valor b alto (3000s/mm2) en el diagnóstico de la ECJ probable o definitiva. Pacientes y métodos: realizamos un análisis retrospectivo de los pacientes atendidos en nuestro hospital diagnosticados de ECJ probable o definitiva, desde el año 2002 al 2008. A todos ellos se les realizó una RM craneal con un protocolo que incluyó secuencias potenciadas en T1, T2, FLAIR y dos secuencias DWI, una con valor b convencional (1000s/mm2) y otra con valor b alto (3000s/mm2). Resultados: se atendieron a 7 pacientes con diagnóstico de ECJ probable o definitiva. En tres de ellos (43%) la secuencia FLAIR mostró cambios de señal compatibles con ECJ. En todos los pacientes en la secuencia DWI con valor b alto se observaron alteraciones características de la enfermedad, incluyendo dos casos (28%) en los que todas las secuencias realizadas, incluida la DWI convencional, fueron normales. Adicionalmente en los 7 casos (100%) las alteraciones radiológicas fueron más fáciles de identificar y más extensas con valores altos b de DWI (AU). Conclusión: la utilización de un valor b alto (3000s/mm2) en la secuencia DWI puede aumentar la sensibilidad de la RM craneal en el diagnóstico de la ECJ, permitiendo la detección de casos en los que la DWI convencional es normal

Background: current diagnostic criteria of probable Creutzfeldt-Jakob disease (CJD) include a combination of clinical, EEG and analytic data. Recent data indicate that brain MRI including fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging (DWI) sequences can be a valid and reliable tool for the diagnosis of CJD. We describe our experience with high b-value (3000s/mm2) diffusion-weighted imaging (DWI) in patients with probable or definite CJD and compare it with standard b-value (1000s/mm2) DWI. Methods: we performed a retrospective analysis of patients admitted to our Hospital Service between 2002 and 2008 with a final diagnosis of probable or definite CJD. Patients were examined using either a 1.5 Tesla or a 3 Tesla MRI. The MRI protocol included T1-weigthed spin-echo sequences, T2-weighted fast spin-echo, FLAIR and DWI sequences with high b-value and standard b-value. Results: during the study period there were 7 patients with probable or definite CJD. Only 3 patients (43%) showed changes on FLAIR sequence consistent with CJD. All the cases were detected with high b-value DWI, including 2 cases (28%) that would have been missed using standard b-value (1000s/mm2) DWI. In all the patients the changes were more conspicuous and extensive at high b-value DWI (b=3000s/mm2). Conclusion;: our data indicate that high b-value DWI may improve the sensitivity of brain MRI for the diagnosis of CJD, allowing the detection of some cases that would have been overlooked by conventional sequences (AU)

Brain derived neurotrophic factor maintains Brn3a expression in axotomized rat retinal ganglion cells.

The transcription factor Brn3a has been reported to be a good marker for adult rat retinal ganglion cells in control and injured retinas. However, it is still unclear if Brn3a expression declines progressively by the injury itself or otherwise its expression is maintained in retinal ganglion cells that, though being injured, are still alive, as might occur when assessing neuroprotective therapies. Therefore, we have automatically quantified the whole population of surviving Brn3a positive retinal ganglion cells in retinas subjected to intraorbital optic nerve transection and treated with either brain derived neurotrophic factor or vehicle. Brain derived neurotrophic factor is known to delay retinal ganglion cell death after axotomy. Thus, comparison of both groups would inform of the suitability of Brn3a as a retinal ganglion cell marker when testing neuroprotective molecules. As internal control, retinal ganglion cells were, as well, identified in all retinas by retrogradely tracing them with fluorogold. Our data show that at all the analyzed times post-lesion, the numbers of Brn3a positive retinal ganglion cells and of fluorogold positive retinal ganglion cells are significantly higher in the brain derived neurotrophic factor-treated retinas compared to the vehicle-treated ones. Moreover, detailed isodensity maps of the surviving Brn3a positive retinal ganglion cells show that a single injection of brain derived neurotrophic factor protects retinal ganglion cells throughout the entire retina. In conclusion, Brn3a is a reliable retinal ganglion cell marker that can be used to accurately measure the potential effect of a given neuroprotective therapy.

Usefulness of high b-value diffusion-weighted MRI in the diagnosis of Creutzfeldt-Jakob disease.

BACKGROUND: current diagnostic criteria of probable Creutzfeldt-Jakob disease (CJD) include a combination of clinical, EEG and analytic data. Recent data indicate that brain MRI including fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging (DWI) sequences can be a valid and reliable tool for the diagnosis of CJD. We describe our experience with high b-value (3000s/mm(2)) diffusion-weighted imaging (DWI) in patients with probable or definite CJD and compare it with standard b-value (1000s/mm(2)) DWI. METHODS: we performed a retrospective analysis of patients admitted to our Hospital Service between 2002 and 2008 with a final diagnosis of probable or definite CJD. Patients were examined using either a 1.5 Tesla or a 3 Tesla MRI. The MRI protocol included T1-weigthed spin-echo sequences, T2-weighted fast spin-echo, FLAIR and DWI sequences with high b-value and standard b-value. RESULTS: during the study period there were 7 patients with probable or definite CJD. Only 3 patients (43%) showed changes on FLAIR sequence consistent with CJD. All the cases were detected with high b-value DWI, including 2 cases (28%) that would have been missed using standard b-value (1000s/mm(2)) DWI. In all the patients the changes were more conspicuous and extensive at high b-value DWI (b=3000s/mm(2)). CONCLUSION: our data indicate that high b-value DWI may improve the sensitivity of brain MRI for the diagnosis of CJD, allowing the detection of some cases that would have been overlooked by conventional sequences.

[Spinocerebellar ataxia type 8: the case of a Spanish family]. / Ataxia espinocerebelosa tipo 8: presentación de una familia española.

INTRODUCTION: Dominant autosomic ataxias include a group of neurodegenerative diseases characterized by the abnormal expansion of triplets. CASE REPORT: Male aged 33, with expansion of the SCA 8 gene (100 repetitions), who presented a clinical picture compatible with a pancerebellar syndrome. The patient had been diagnosed 11 years earlier as suffering from previously of histiocytosis X. A clinico genetic study was conducted on the patient and several members of his family (parents and two sisters). Both sisters and the father were found to be carriers of the expansion (110 and 150 repetitions, respectively), and are currently asymptomatic. RESULTS AND DISCUSSION: There is no relation between the number of repetitions and the age of onset of the disease. The normal interval in our population oscillates between 16 37 repetitions, and the pathological interval has not been well determined. There may be a relation between the SCA 8 form and histiocytosis X.

Ataxia espinocerebelosa tipo 8: presentación de una familia española / Spinocerebellar ataxia type 8: the case of a spanish family

Introducción. Las ataxias autosómicas dominantes son un grupo de enfermedades neurodegenerativas causadas por la expansión anormal de tripletes. Caso clínico. Varón de 33 años, con expansión en el gen SCA8 (100 repeticiones), que presentó un cuadro clínico compatible con un síndrome pancerebeloso. El paciente se había diagnosticado 11 años antes de histiocitosis X. Se ha realizado un estudio clinicogenético del paciente y varios miembros de la familia (padres y dos hermanas); han resultando portadores de la expansión las dos hermanas y el padre del probando (110 y 150 repeticiones, respectivamente), y se encuentran asintomáticos. Resultados y discusión. No existe relación entre el número de repeticiones y la edad de aparición de la enfermedad. El intervalo normal en nuestra población oscila entre 16-37 repeticiones, y el intervalo patológico no se ha determinado bien. Es posible que exista una relación entre la forma SCA8 y la histiocitosis X (AU)

[The headache in general neruological practice]. / La cefalea en la consulta de neurología general.

INTRODUCTION: Headache is one of the most frequent causes of consultation in a neurology clinic. We present a descriptive, epidemiological study about such a pathology in a general neurology clinic. We evaluated as the most relevant points the frequency of different types of headaches, the reasons for remission and diagnosis concordance with the remitting doctor, normally the General Practitioner. PATIENTS AND METHODS: The collection of data was carried out by means of structured interviews over a three month period. Apart from the epidemiological data of the subjects, the characteristics of the headache, the medication and its possible overuse, were also collected. A systematic neurological examination of all patients was carried out. RESULTS: 286 patients were included. The frequency of different diagnosis (the most frequent chronic daily headache, 39.2%), the number of patients who abused of analgesics (31.8%) and the diagnosis concordance with the referring doctor (20%) was analyzed among other variables. CONCLUSIONS: The study highlights the high number of patients suffering from daily chronic headache and the fundamental role that the abuse of analgesics plays in this diagnostic entity. The diagnosis concordance is too low. A correct initial diagnosis as a result of a better neurological training in primary care would prevent in greater numbers the abuse of analgesics and would offer a better quality of care.

La cefalea en la consulta de neurología general / Headache in a general neurology clinic

Introducción y objetivos: La cefalea es uno de los motivos de consulta más frecuente en la consulta de neurología. Presentamos un trabajo epidemiológico, descriptivo, acerca de dicha afección en una consulta de neurología general. Se valoran, como puntos más relevantes, la frecuencia de los distintos tipos de cefalea, el motivo de remisión del paciente y la concordancia diagnóstica con el médico remitente, habitualmente atención primaria. Pacientes y métodos: La recogida de datos se llevó a cabo, de forma protocolizada, durante un período de 3 meses. Además de la filiación de los pacientes se recogieron las características clínicas de la cefalea y los fármacos utilizados, así como el posible abuso de los mismos. Se realizó una exploración neurológica sistematizada de todos los pacientes. Posteriormente se emitió un juicio diagnóstico. Resultados: Se incluyeron 286 pacientes. Se calculó la frecuencia de los distintos diagnósticos (el más frecuente la cefalea crónica diaria, 39,2 por ciento), el número de pacientes con criterios de abuso de analgésicos (31,8 por ciento) y el porcentaje de concordancia diagnóstica con el médico remitente (20 por ciento), entre otros puntos. Conclusiones: Destaca el elevado número de pacientes con cefalea crónica diaria y el papel fundamental que desempeña el abuso de analgésicos en esta entidad. La concordancia diagnóstica es llamativamente baja. Un correcto diagnóstico inicial, resultante de una mejor formación neurológica de atención primaria, evitaría, en buena medida, el abuso de fármacos y ofertaría una mayor calidad asistencial (AU)

[Acute phenytoin poisoning secondary to an interaction with ticlopidine]. / Intoxicación aguda por fenitoína secundaria a interacción con ticlopidina.

INTRODUCTION: Ticlopidine and phenytoin are two drugs which are widely used in everyday clinical practice, the first as a platelet anti-aggregant and the second as an anti-epileptic agent. Their association is not uncommon, since there is a high incidence of epileptic crises of vascular origin in adults. CLINICAL CASE: We present the case of a 77 year old man who had been treated for twenty years with phenytoin for partial crises secondary to a left frontal hematoma. He then followed treatment with ticlopidine for a femoropopliteal bypass, and the dose was later increased because of a vertebro-basilar ictus. On both occasions he presented with ataxia, dysarthria and nausea due to phenytoin intoxication. The blood levels of this drug were above the therapeutic level. The symptoms disappeared and the patient returned to normal after reduction of the dose of phenytoin and suspending treatment with ticlopidine. DISCUSSION: The mechanism of action of ticlopidine at cytochrome P450 level, inhibiting the metabolic clearance of phenytoin, was responsible for this interaction. We have found only three other cases of interaction between these drugs in the literature, and ours is the first to be published in Spain. CONCLUSION: We emphasize the importance of checking plasma phenytoin levels when starting treatment with ticlopidine, since it may be necessary to reduce the dose of phenytoin.

[Palpebral ptosis and blepharospasm secondary to hemispheric cerebral infarction]. / Ptosis palpebral y blefarospasmo secundarios a infarto hemisférico cerebral.

A case that combines cerebral eyelid ptosis and blepharospasm secondary to cerebral hemisphere infarction is shown. EMG recording from the facial and eyelid muscles revealed lack of the levator palpebrae superioris, orbicularis oculi and corrugator muscles activity. Any voluntary opening attempt lead to a simultaneous contraction of the three muscles. Blink reflex responses were normal although showed facilitation features on the right side and mild inhibition characteristics from the left side. Median nerve SEP revealed a loss of precentral components (P22-N30) as well as a delay and amplitude decrease of N20. Transcranial magnetic stimulation disclosed a complete lesion of corticospinal pathway for right upper limb. In this case, a right hemisphere lesion caused an unusual eyelid motor abnormality: cerebral eyelid ptosis and blepharospasm induced by the voluntary eyelid opening.

Influence of alpha-tocopherol incorporation on Ca(2+)-induced fusion of phosphatidylserine vesicles.

The effect of alpha-tocopherol on the Ca(2+)-induced fusion of large unilamellar phosphatidylserine vesicles has been investigated. Mixing of aqueous vesicle contents was followed continually with the terbium-dipicolinic acid (Tb-DPA) assay, while the dissociation of preencapsulated Tb-DPA complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure phosphatidylserine and phosphatidylserine containing 2, 5, and 10 mol% of alpha-tocopherol were employed at different Ca2+ concentrations. The presence of low amounts of alpha-tocopherol decreased the initial rate of fusion without changing the Ca2+ threshold concentration. The reduction of the initial rate of fusion was proportional to the amount of alpha-tocopherol present in the bilayer. An alpha-tocopherol concentration-dependent decrease of both the initial rate and the final extent of release of vesicle contents was also observed. This effect was more evident as more alpha-tocopherol was incorporated in the bilayer, so that in the presence of 10 mol% of alpha-tocopherol no significant release was observed after 5 min. The stabilization of the vesicular structure exerted by alpha-tocopherol was responsible for the apparent increase of the fluorescence intensity of the Tb-DPA complex at later stages of the process. The results reflect a perturbation of the membrane by low concentrations of alpha-tocopherol which may account for a number of biological effects of this vitamin, not related to its antioxidant role.

The interaction of alpha-tocopherol with phosphatidylserine vesicles and calcium.

The interaction of alpha-tocopherol with dimyristoylphosphatidylserine (DMPS) has been studied in the presence and in the absence of Ca2+ by using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and 45Ca2+-binding. In the absence of Ca2+, DSC showed that alpha-tocopherol decreases the temperature of the lamellar gel to lamellar liquid crystalline phase transition as well as it decreases delta H of this transition. Two different peaks were detected at 10 mol% of alpha-tocopherol and probably one of the peaks correspond to pure or nearly pure DMPS and the other to DMPS incorporating most of the alpha-tocopherol. The phase transition was totally abolished at 30 mol% of alpha-tocopherol. In the presence of Ca2+ this L(beta) to L(alpha) phase transition of DMPS was even more perturbed by alpha-tocopherol, so that it was totally abolished by only 7 mol% of alpha-tocopherol, at Ca2+ concentrations which were clearly non-saturating, like those giving DMPS/Ca2+ molar ratio of 4:1 and 10:1. Furthermore, the transition of the DMPS/Ca2+ complex observed at 91.6 degrees C was perturbed by the presence of alpha-tocopherol, indicating a change in the structure of the crystalline complex. The FT-IR analysis of the effect of alpha-tocopherol on DPMS phase transition confirmed the decrease in the phase transition temperature of the phospholipid, and also that alpha-tocopherol increases the number of gauche isomers in the gel state but has no effect in the liquid crystalline state. The binding of 45Ca2+ was also affected by the presence of alpha-tocopherol, so that the number of binding sites was decreased, and this may be interesting for situations in which phosphatidylserine and Ca2+ are simultaneously implicated in biological functions, such as membrane fusion and enzyme activation.

Interaction between alpha-tocopherol and heteroacid phosphatidylcholines with different amounts of unsaturation.

Differential scanning calorimetry was used to study the influence of a alpha-tocopherol on the thermotropic properties of model membranes composed by a series of heteroacid phosphatidylcholines with different amount of unsaturation in the sn-2 chain. The effect of alpha-tocopherol on 1,2-distearoylglycerophosphocholine (18:0,18:0), 1-stearoyl-2-oleoylgylcerophosphocholine (18:0,18:1), 1-stearoyl-2-linoleoylglycerophosphocholine (18:0,18:2), 1-stearoyl-2-linolenoylglycerophosphocholine (18:0,18:3), and 1-stearoyl-2-arachidonoylglycerophosphocholine (18:0,20:4) was determined. The addition of alpha-tocopherol perturbed the thermotropic gel to liquid-crystalline phase transition of these phospholipids. alpha-Tocopherol broadened the endotherm, lowered the transition temperature and decreased the associated enthalpy change. Partial phase diagrams showed the presence of fluid immiscibilities giving rise to lateral phase separation of domains containing different amounts of alpha-tocopherol. We suggest that, in these alpha-tocopherol-rich domains, the influence exerted by the vitamin on the phospholipids is strong enough to alter their thermotropic properties such that an additional endotherm appears in the thermogram, a characteristic not observed in homoacid phosphatidylcholines. alpha-Tocopherol caused a concentration-dependent removal of the detectable phase transition in all cases. The magnitude of the influence of alpha-tocopherol on phospholipid was dependent on the degree of unsaturation of the sn-2 acyl chain. These results are explained on the basis of the effect of alpha-tocopherol which will reduce the differences between gel and liquid crystalline states, the magnitude of these differences depending on the type of phospholipid considered, which are probably related to the change of molecular shape of phosphatidylcholines containing a polyunsaturated acyl chain.

The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed.

Role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes.

The role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes was investigated in vitro, monitoring the mixing of membrane lipids with the octadecyl rhodamine B fluorescence dequenching assay. Polylysine was able to induce fusion of chromaffin granule ghosts with plasma membrane vesicles in the absence of Ca2+. This fusion was maximal at 1.6 microM polylysine and the kinetics were dependent on the amount of plasma membrane added. Polylysine lowered the Ca2+ threshold concentration for inducing fusion, increased the extent of fusion at a given Ca2+ concentration and acted synergistically with Ca2+ when added prior to the cation. Removal of membrane proteins by trypsinization of chromaffin granule ghost and/or plasma membranes increased the extent of polylysine induced fusion. Incorporation of diacylglycerol into the plasma membrane promoted Ca(2+)-induced fusion. Chromaffin granule ghosts were induced to fuse with model membranes of different complexities from one resembling the inner monolayer of the erythrocyte membrane to one composed solely of pure phosphatidylserine. The characteristics of the fusion with model membranes were qualitatively similar to that observed with target plasma membrane, although differences in kinetics and stoichiometries were found. Interestingly, considerably low (microM) Ca2+ concentrations were able to induce fusion of chromaffin granule ghosts with diacylglycerol containing phosphatidylserine vesicles in the presence of polylysine. Our results indicate that acidic phospholipid-like phosphatidylserine may have an important role in the process of fusion and suggest that diacylglycerol, as a destabilizing lipid, may have a role by itself in promoting exocytosis in chromaffin cells.