Effects of epidural anaesthesia-analgesia on intravenous anaesthesia with propofol.

The main purpose of this study was to demonstrate that the use of epidural anaesthesia-analgesia reduces the amount of propofol necessary to maintain surgical anaesthesia in dogs during ovariohysterectomy. The study was carried out on 28 bitches undergoing ovariohysterectomy with general anaesthesia using an intravenous infusion of propofol. Dogs were allocated to one of two groups. Group 1 received systemic buprenorphine together with epidural analgesia using lidocaine and buprenophine, and in Group 2 systemic buprenorphine only was given. The mean propofol infusion rate was significantly lower in dogs with epidural analgesia (P < 0.0001). In addition, the mean endotracheal extubation time was significantly longer in dogs with epidural analgesia (P < 0.0001). No significant differences were detected in any of the physiological parameters measured.

Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.

Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.

Results of an orthopaedic survey in young patients with severe haemophilia in Spain.

This paper outlines the results obtained in a cross-sectional study of a group of young patients with severe haemophilia A and B. The primary aim of the study was to ascertain the level of orthopaedic complications in the group, the effects that these complications have on quality of life, and the medical resources used on these patients. The secondary aim was to relate their current orthopaedic state to the type of treatment received before the study. The study was carried out in 11 hospitals in Spain, where 70 severe haemophilia patients (factor VIII [FVIII] < 2%), with an average age of 21.6 and a median age of 22, and no inhibitors, were monitored. The percentage of patients suffering from articular complaints was 84.3% and 85.7% according to the Gilbert and Petterson scales, respectively. Furthermore, pain was reported in 16.1% of joints, most frequently in ankles. Using the SF-36 Health Survey, patients were observed to have a poorer quality of life relative to healthy controls. Despite high levels of both the coagulant factor given to patients and the financial resources dedicated to their treatment, the type of treatment (on demand and/or as secondary prophylaxis) provided to the young adults was found to be incapable of preventing haemophilic arthropathy and subsequent negative quality of life consequences.

Pilot testing of the 'Haemo-QoL' quality of life questionnaire for haemophiliac children in six European countries.

In a multinational working group, an instrument (Haemo-QoL) to assess quality of life in children/adolescents with haemophilia and their parents has been developed. In co-operation with haemophilia treatment centres in six European countries, approximately 10 children/adolescents with haemophilia per country and their parents were asked to participate in the pilot-testing. Both self-reported and parent-reported questionnaires were provided for two age-groups of children (4-16 years). Medical data was collected from physicians from patient files. Answers to open questions from participants (58 children and 57 parents) confirmed the content of 116 of the preliminary items. Cognitive debriefing revealed that the majority of the Haemo-QoL was rated favourably, but 29 questions were recommended to be omitted and several items to be reformulated. Preliminary psychometric testing of the revised 77 item questionnaire in the same sample showed acceptable reliability and validity, which will be examined in a subsequent study with a larger patient sample.

Role of calmodulin in the differentiation/activation of microglial cells.

In the present work the role of calmodulin (CaM) in regulating lipopolysaccharide (LPS)-induced microglial activation and in the spontaneous microglial differentiation has been investigated. We used pure rat microglial cell cultures to examine the effects of W13, a specific inhibitor of CaM, on microglial activation produced by LPS and the effect of CaM inhibition on microglial proliferation induced by the macrophage colony-stimulating factor (M-CSF). Microglial morphological transformation, inducible nitric oxide synthase (iNOS) activity and proliferating cell nuclear antigen (PCNA) immunostaining were determinate. Results show that CaM does not participate in the microglial increase of iNOS produced by LPS. In contrast, it is involved in spontaneous microglial ramification and in the activation of proliferation from quiescence. Multiple second-messenger pathways are involved in the transduction of signals initiated by LPS. The study of these mechanisms may allow us to extend our knowledge of the signals controlling the expression of these mediators.

The Ca2+/calmodulin system in neuronal hyperexcitability.

Calmodulin (CaM) is a major Ca2+-binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2+ concentration. Calmodulin modulates numerous Ca2+-dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2+/CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2+/CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2+ concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2+ levels described in different pathological situations.

Estudio epidemiológico de la hemofilia en España / Epidemiological study of hemophilia in Spain

El objetivo de este estudio epidemiológico fue la caracterización de un grupo representativo de pacientes del territorio español afectos de hemofilia con relación a su situación diagnóstica, clínica, terapéutica y a las complicaciones de la enfermedad y de su tratamiento. Se llevó a cabo por medio de un cuestionario que se envió a todos los centros de tratamiento. Resultados: fueron remitidos 825 cuestionarios evaluables, lo que corresponde aproximadamente a un tercio de la población hemofílica española. El 88 por ciento de los hemofílicos padecían hemofilia A y el 12 por ciento hemofilia B. El 36 por ciento graves (nivel inferior al 2 por ciento). La gran mayoría de pacientes estaban siguiendo un programa de tratamiento a demanda (79 por ciento). Un reducido número seguía, en el momento del registro, un tratamiento de profilaxis secundaria (17 por ciento), y tan solo un 3,4 por ciento seguía un programa de profilaxis primaria. El consumo medio de factores por paciente durante el año 1995, fue de 45.945 UI, lo que correspondería a una media de 706 Ul/kg/año. El 30 por ciento de los pacientes presentaban una o más articulaciones con algún grado de afectación. Los pacientes graves presentaban un total de 627 articulaciones afectadas de media por paciente. El 7,3 por ciento del total presentaba o habían presentado inhibidor. El 27 por ciento tenían anticuerpos anti-VIH. Los anticuerpos de la hepatitis C estaban presentes en el 61 por ciento. El antígeno HBs fue positivo en el 4 por ciento. (AU)

The orthopaedic status of severe haemophiliacs in Spain.

This paper provides an outline of the results obtained in a cross-sectional study conducted primarily with the aim of ascertaining orthopaedic complications in a group of young severe A and B haemophiliacs, the effects which these complications have on the medical resources used with these patients, and the impact of severe haemophilia on their quality of life. Its secondary aim was to link their current orthopaedic status to the type of treatment they had received prior to the study. Eleven Spanish hospitals took part in this study, monitoring 70 severe haemophiliacs (FVIII:C <2%) without inhibitors who had a mean age of 21.6 years and a median age of 22. Retrospective data collected from birth to the conclusion of the study were used and, for certain variables, data from the last 12 months. The type of treatment given had been on-demand treatment, together with prophylaxis of variable time periods, which in 32 cases (45.7%) were prolonged (>6 months). In 40 cases (57.1%) the patients underwent one or more periods of prophylaxis. Thirty-three patients (47.8%) had over 1000 days of administration of factors VIII and IX. The analysis of the total study group reveal an average of 348 bleeding episodes per patient. The findings of this study revealed that 84.3% of these patients suffer from articular complaints on the Gilbert scale, and 85.7% on the Pettersson scale. In addition, pain was reported in 16.1% of the joints, the most frequently affected being the ankle joints. Twenty-six patients (37%) had undergone orthopaedic surgery from the time of birth to the conclusion of the study. The quality of life of the severe haemophiliacs reviewed seems to have been affected. During the last 12 months, there were 216 outpatient haematological visits and 176 orthopaedic-rehabilitation visits, as well as 12 radiological explorations and two hospitalizations. During these 12 months, medical expenditure totalled $55 473 per patient per year, the most important item being factor concentrates VIII/IX ($54 119 per patient per year). The type of treatment given to these patients (on-demand and/or as secondary prophylaxis) was found to be incapable of preventing haemophilic arthropathy or guaranteeing an acceptable quality of life, although both the administration of coagulant factor concentrate to such patients and the financial resources dedicated to their treatment was very high. Consequently, a strategic change is to be considered for the prevention of haemophilic arthropathy, based on the introduction of replacement treatment involving continuous administration of factors VIII and IX in primary prophylaxis regimens from the first years of life.

Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are Ca(2+)/calmodulin-binding proteins that are very abundant in the central nervous system. In the mammalian brain, CaMKII is composed by the association of several similar subunits at different ratios produced by four different genes. CaN is composed of two different subunits produced by two genes. We selected the most abundant subunits of each enzyme in the rodent brain, CaMKII alpha and CaN A, and compared their pattern of expression in the mouse brain by using in situ hybridization histochemistry and immunohistochemistry. We found that CaMKII and CaN were mainly expressed in cerebral cortex, hippocampus, and striatum and that low levels of expression were observed in midbrain and brainstem. CaN was also expressed in cerebellum. In the cell, the kinase and the phosphatase were detected in the perikarya, the neuronal processes, and the nucleus. The present study shows that all the regions of the mouse brain in which CaMKII is expressed also show CaN expression. This fact is consistent with the presence of common substrates for both enzymes or with a regulatory action of one versus the other. The lack of correspondence in the cerebellum could be explained by the fact that the major subunit of the kinase in this brain region is CaMKII beta.

The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells.

Ca2+ plays a critical role in the normal function of the central nervous system. However, it can also be involved in the development of different neuropathological and neurotoxicological processes. The processing of a Ca2+ signal requires its union with specific intracellular proteins. Calmodulin is a major Ca(2+)-binding protein in the brain, where it modulates numerous Ca(2+)-dependent enzymes and participates in relevant cellular functions. Among the different calmodulin-binding proteins, the Ca2+/calmodulin-dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. We present an overview on different works aimed at the study of the Ca2+/calmodulin signalling system in the neural response to convulsant agents. Ca2+ and calmodulin antagonists inhibit the seizures induced by different convulsant agents, showing that the Ca2+/calmodulin signalling system plays a role in the development of the seizures induced by these agents. Processes occurring in association with seizures, such as activation of c-fos, are not always sensitive to calmodulin, but depend on the convulsant agent considered. We characterized the pattern of expression of the three calmodulin genes in the brain of control mice and detected alterations in specific areas after inducing seizures. The results obtained are in favour of a differential regulation of these genes. We also observed alterations in the expression of the Ca2+/calmodulin-dependent protein kinase II and calcineurin after inducing seizures. In addition, we found that reactive microglial cells increase the expression of calmodulin and Ca2+/calmodulin-dependent protein kinase II in the brain after seizures.

Decreased expression of calmodulin kinase II and calcineurin messenger RNAs in the mouse hippocampus after kainic acid-induced seizures.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIalpha and CaMKIIbeta being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aalpha is the predominant form in the brain. We studied the expression of CaMKIIalpha, CaMKIIbeta, and CaN Aalpha subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIalpha and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid-treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid-treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down-regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.

Calmodulin is expressed by reactive microglia in the hippocampus of kainic acid-treated mice.

Calmodulin is a calcium-binding protein that is highly abundant in the brain, where it is involved in many essential functions. The protein is mainly expressed by neuronal cells. Calmodulin is encoded by three different genes in mammals, all of them producing an identical protein. Alterations in the expression of either calmodulin genes or protein have been reported in the rodent brain by several authors in different experimental situations. However, no mention has been made to date of possible alterations in calmodulin expression in glial cells in response to certain stimuli. In the present study, we found an increase in the expression of calmodulin in reactive microglial cells in the mouse hippocampus 24 h after an intraperitoneal administration of a convulsant dose of kainic acid. The results show that a high expression of calmodulin can be added to the list of changes described to occur in microglial cells when they become reactive microglia in response to certain kinds of stimuli, in contrast to the non-detectable level of expression of this protein observed in the resting microglial cells. It is difficult to explain such an increase due to the great number of processes in which calmodulin is involved, but the great level of calmodulin observed in the reactive microglial cells shows that calmodulin immunolabelling can be used to reveal these kinds of cells.

Differential response of calmodulin genes in the mouse brain after systemic kainate administration.

In the central nervous system, many of the effects resulting from an increase in the intracellular levels of calcium are mediated by calmodulin, a major calcium-binding protein in the mammalian brain. Calmodulin is expressed by three different genes, namely CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the expression of calmodulin in the mouse brain at different times after the administration of a convulsant dose of kainate, a potent neuroexcitotoxic agent. We detected the presence of the different calmodulin messenger RNAs and of the protein itself in brain sections by in situ hybridization histochemistry and immunocytochemistry respectively. In addition, we determined the calmodulin content in brain regions by radioimmunoassay. Kainate-treated animals did not show areas of neuronal death at the different times following administration considered. An increase in the hybridization signal for CaM I messenger RNAs was observed from 5 h after kainate administration in the different brain regions tested. In contrast, the CaM II messenger RNA signal decreased gradually to a minimum 24 h after treatment in the hippocampus, while the CaM III messenger RNA signal was mostly unaffected. Calmodulin immunoreactivity also increased in the hippocampus. Nevertheless, we did not detect any significant difference in calmodulin content between brain regions of control and treated animals by radioimmunoassay. Kainate treatment induced modifications in the expression of calmodulin at the level of both messenger RNAs and protein. The results suggest a differential regulation of the three calmodulin genes in the adult mouse brain and a post-transcriptional or a post-translational regulation of calmodulin expression.

Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain.

Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.

Reserpine potentiates NMDA-induced c-fos mRNA expression in the mouse brain.

The systemic administration of a non-convulsant dose of N-methyl-D-aspartate (NMDA; 75 mg/kg i.p.), which was associated with motor activation, induced a regional c-fos mRNA expression in the mouse brain. The NMDA-induced c-fos mRNA expression was predominant in the dentate gyrus and in the medial mammillary nucleus and less pronounced in other hippocampal areas, cortical areas, bed nucleus of the stria terminalis and posterior amygdaloid nuclei. It is suggested that the hippocampus and/or the extended amygdala might be involved in the previously hypothesized dopamine-independent NMDA-mediated motor activation mechanism. No increase in c-fos mRNA expression was observed 21 h after reserpine treatment (5 mg/kg s.c.). However, reserpinization induced a significant potentiation of the NMDA-induced c-fos mRNA expression. These results show the existence of a strong and selective amine-dependent modulation of NMDA neurotransmission in the brain.

Convulsant agents activate c-fos induction in both a calmodulin-dependent and calmodulin-independent manner.

Calcium acts as a second messenger and can enter neurons through several types of calcium channel. We sought to determine whether the calcium-dependent mechanisms inducing c-fos expression are identical following activation, by appropriate drugs, of L-type voltage-sensitive calcium channels or NMDA and non-NMDA receptors or following inhibition of the GABAergic system. We used primary cortical neurons and OF1 mice, and the levels of c-fos protein and c-fos mRNA were detected after treatment with the drugs by means of immunocytochemistry and in situ hybridization. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished gamma-hexachlorocyclohexane-, Bay K 8644-, pentylenetetrazole-, and kainic acid-induced increases in c-fos expression in cultured neurons. Conversely, W-7 did not affect either NMDA- or picrotoxinin-mediated increases in c-fos expression. In mice, the pattern of protooncogene expression displayed some differences compared with cultured neurons, depending on the treatment. W-7 administered before gamma-hexachlorocyclohexane, Bay K 8644, or pentylenetetrazole blocked the expression of c-fos elicited by these compounds. However, W-7 was not able to abolish c-fos expression induced by picrotoxinin. In the animals treated with W-7 before kainic acid or NMDA administration, c-fos expression was inhibited in cerebral cortex, but it was still present in hippocampus. These results agree with the existence of diverse mechanisms transducing the calcium signals to the nucleus. Calmodulin may mediate neuronal responses depending on the route by which calcium enters the neuron, resulting in activation of different enzymes.