[Combined Kocher-Langenbeck and Stoppa approach in the treatment of complex acetabulum fractures]. / Abordaje combinado Kocher-Langenbeck y Stoppa en el tratamiento de fracturas complejas de acetábulo.

INTRODUCTION: Complex acetabulum fractures are a challenge for orthopedic surgeons. An evaluation of the radiographic reduction and functional result of the patients with complex fracture of the acetabulum who underwent the combined Kocher-Langenbeck and Stoppa approach was carried out. MATERIAL AND METHODS: Cross-sectional, descriptive, ambispective design. Patients with complex acetabulum fracture who underwent the combined Kocher-Langenbeck approach plus Stoppa between 2016 and 2020 were included. The clinical records were reviewed, and the quality of the radiographic reduction was evaluated according to Matta criteria. In addition, a functional evaluation was performed with the Merle d'Aubigne and Postel scale at least 12 months after the injury. RESULTS: Of the 31 patients, the average time between the date of fractures and the surgical intervention was 13.7 days (3-38 days). In the radiographic evaluation according to Matta criteria, 21 anatomical patients (67.7%), 7 almost anatomical (22.5%), 3 imperfect (9.6%). Functional results according to the Merle d'Aubigne and Postel scale resulted in 8 (25.8%) with excellent results, 16 (51.6%) with good results, 5 (22.5%) moderate and 2 (16.1%) poor patients. There was a statistical correlation between the age of the patient and the functional result (p = 0.029), also between the body mass index and blood loss (p = 0.027). CONCLUSION: The combined Kocher-Langenbeck plus Stoppa approaches are a valid alternative in these lesions, mostly with anatomical and almost anatomical radiographic results according to the Matta radiographic scale, and with excellent and good functional results according to the Merle d'Aubigne and Postel scale.

INTRODUCCIÓN: Las fracturas de acetábulo complejas son un desafío para los cirujanos ortopedistas. Se realizó una evaluación de la reducción radiográfica y resultado funcional de los pacientes con fractura compleja de acetábulo sometidos a abordaje combinado Kocher-Langenbeck y Stoppa. MATERIAL Y MÉTODOS: Diseño transversal, descriptivo, ambispectivo. Se incluyeron pacientes con fractura compleja de acetábulo que se sometieron a abordaje combinado Kocher-Langenbeck y Stoppa entre 2016 y 2020. Se revisaron los expedientes clínicos y se evaluó la calidad de la reducción radiográfica según criterios de Matta. Además, se realizó evaluación funcional con la escala de Merle d'Aubigné y Postel pasados por lo menos 12 meses de la lesión. RESULTADOS: De los 31 pacientes, el tiempo promedio entre la fecha de fractura y la intervención quirúrgica fue de 13.7 días (de tres a 38 días). En la evaluación radiográfica según criterios de Matta, 21 pacientes tuvieron resultados radiográficos anatómicos (67.7%), siete casi anatómicos (22.5%) y tres imperfectos (9.6%). Los resultados funcionales según la escala Merle d'Aubigné y Postel dieron como resultado ocho pacientes (25.8%) con resultados excelentes, 16 (51.6%) con resultado bueno, con resultado moderado cinco (22.5%) y con resultado malo dos (16.1%). Hubo correlación estadística entre la edad del paciente con el resultado funcional (p = 0.029), también entre el índice de masa corporal y pérdida sanguínea (p = 0.027). CONCLUSIÓN: Los abordajes combinados Kocher-Langenbeck y Stoppa son una alternativa en estas lesiones, en su mayoría con resultados radiográficos anatómicos y casi anatómicos, según la escala radiográfica de Matta, y con excelentes y buenos resultados funcionales, según la escala de Merle d'Aubigné y Postel.

The effect of a novel intravenous fluid (Oxsealife®) on recovery from haemorrhagic shock in pigs.

Blood transfusion is given according to haemoglobin thresholds aimed at restoration of arterial oxygen-carrying capacity. Patient survival after severe haemorrhagic shock depends on restoration of microvascular perfusion, tissue oxygen delivery, endothelial function and organ integrity. We investigated a novel crystalloid fluid designed for tissue oxygen delivery, Oxsealife® , with components that generate microvascular nitric oxide and scavenge reactive oxygen species generated during ischaemia-reperfusion injury. The amount of dissolved oxygen in blood progressively increased during step-wise in vitro haemodilution with this fluid, suggesting that the oxygen solubility coefficient of blood is dynamic, not static. We performed a pilot safety and efficacy study to compare resuscitation with this novel crystalloid vs. whole blood transfusion in a swine haemorrhagic shock model with animals bled to an arterial lactate oxygen debt target. Despite contributing no haemoglobin, viscosity nor oncotic potential, resuscitation with Oxsealife after severe haemorrhagic shock restored central haemodynamic parameters comparable to stored allogeneic blood transfusion. Tissue perfusion, oxygenation and metabolic outcomes were equivalent between treatment groups. Increased consumption of bicarbonate in animals given Oxsealife suggested greater capillary recruitment and enhanced clearance of acidic tissue metabolites. Serum markers of organ function, animal activity during recovery and histological analysis of tissue morphology and endothelial glycocalyx integrity confirmed functional recovery from haemorrhagic shock. We conclude that recovery of tissue oxygen delivery and organ function after haemorrhagic shock may not be dependent on treatments that increase haemoglobin levels. Oxsealife shows promise for treatment of severe haemorrhagic shock and may reduce the requirement for allogeneic blood products.

Potential of mesenchymal stem cell in stabilization of abdominal aortic aneurysm sac.

BACKGROUND: In their origin, abdominal aortic aneurysms (AAAs) are related to an inflammatory reaction within the aortic wall, which can lead to weakness and degeneration of this structure. One of the most widely accepted treatment modalities for AAAs is the placement of stent grafts. Nevertheless, in some patients blood re-enters the aneurysm sac, creating so-called leaks, which constitute a renewed risk of rupture and death.This study explores the possibility of filling aneurysm sacs treated by endovascular aneurysm repair with adipose tissue-derived mesenchymal stem cells (ASCs) in a porcine model. METHODS: We developed a porcine model using 22 animals by creating an artificial AAA made with a Dacron patch. AAAs were then treated with a coated stent that isolated the aneurysm sac, after which we introduced allogeneic ASC into the sac. Animals were followed-up for up to 3 mo. The experiment consisted of the aforementioned surgical procedure performed first, followed by computed tomography and echo-Doppler imaging during the follow-up, and finally, after sacrificing the animals, histologic analysis of tissue samples from the site of cell implantation by a blinded observer and the detection of implanted cells by immunofluorescence detection of the Y chromosome. RESULTS: Our findings demonstrate the survival of ASCs over the 3 mo after implantation and histologic changes associated with this treatment. Treated animals had less acute and chronic inflammation throughout the study period, and we observed increasing fibrosis of the aneurysm sac, no accumulation of calcium, and a regeneration of elastic fibers in the artery. CONCLUSIONS: The combination of endovascular aneurysm repair and cell therapy on AAAs has promising results for the stabilization of the sac, resulting in the generation of living tissue that can secure the stent graft and even showing some signs of wall regeneration. The therapeutic value of such cell-based therapy will require further investigation.

[Technical aspects of experimental intestinal transplant]. / Aspectos técnicos del trasplante intestinal experimental.

PURPOSE: Our objective is to analyze the variables that influence the outcome of Small Bowel Transplantation (SBT) in rats in an experimental microsurgery program. The surgical technique and perioperative care are described in detail. METHODS: Retrospective study of the SBT in rats conducted in our experimental surgery laboratory from 2002 to 2010. The animals were divided into group A (those who survived more than 48 hours) and group B (those who died earlier without justificable cause). We compared in both groups: number of transplants performed by the surgeon, warm ischemia time, cold ischemia time and duration of the procedure. RESULTS: Five surgeons with different degrees of microsurgical training participated in the study. A total of 521 SBT were performed with an overall survival of 48%. The first successful transplant was performed after a median of 46 (25-68) transplants. Total procedure time (3.5 vs 2.9 hours) and warm ischemia time (51 vs 35 minutes) were higher in group B (p < 0.05). DISCUSSION: The number of transplants required for learning the technique is high. However, survival is acceptable when the time needed for vascular anastomosis is reduced. The SBT in rats is a valuable model for surgical training and research of the phenomena related to SBT.

Aspectos técnicos del trasplante intestinal experimental / Technical aspects of experimental intestinal transplant

Introducción. Nuestro objetivo es analizar las variables que in-fluyen en los resultados del trasplante de intestino delgado (TID) en ratas en un programa de microcirugía experimental. Se describe con detalle la técnica quirúrgica y los cuidados perioperatorios con objeto de favorecer el aprendizaje de la técnica. Material y métodos. Estudio retrospectivo de los TID en ratas realizados en nuestro laboratorio de cirugía experimental desde el año 2002 al 2010. Dividimos los animales en grupo A (los que sobrevivieron más de 48 horas) y grupo B (los que fallecieron precozmente sin causa justificable). Comparamos en ambos grupos: número de trasplantes realizados por el cirujano, tiempo de isquemia caliente, tiempo de isquemia fría y duración total del procedimiento. Resultados. Cinco cirujanos con distinto grado de formación microquirúrgica participaron en el estudio. Se realizaron en total 521 TID con una supervivencia global del 48%. El primer trasplante con éxito se realizó tras una mediana de 46 (25-68) trasplantes. El tiempo total del procedimiento (3,5 vs. 2,9 horas) y el tiempo de isquemia caliente (51 vs. 35 minutos) fueron superiores en el grupo B (p<0,05). Discusión. El número de trasplantes necesarios para el aprendizaje de la técnica es elevado. Sin embargo, la supervivencia es aceptable al reducir el tiempo empleado en las anastomosis vasculares. El TID en ratas constituye un modelo muy valioso para la formación del cirujano y para la investigación de los fenómenos relacionados con el TID (AU)

Purpose. Our objective is to analyze the variables that influence the outcome of Small Bowel Transplantation (SBT) in rats in an experimental microsurgery program. The surgical technique and perioperative care are described in detail. Methods. Retrospective study of the SBT in rats conducted in our experimental surgery laboratory from 2002 to 2010. The animals were divided into group A (those who survived more than 48 hours) and group B (those who died earlier without justificable cause). We compared in both groups: number of transplants performed by the surgeon, warm ischemia time, cold ischemia time and duration of the procedure. Results. Five surgeons with different degrees of microsurgical training participated in the study. A total of 521 SBT were performed with an overall survival of 48%. The first successful transplant was performed after a median of 46 (25-68) transplants. Total procedure time (3.5 vs 2.9 hours) and warm ischemia time (51 vs 35 minutes) were higher in group B (p<0.05).Discussion. The number of transplants required for learning the technique is high. However, survival is acceptable when the time needed for vascular anastomosis is reduced. The SBT in rats is a valuable model for surgical training and research of the phenomena related to SBT (AU)

Protective effect of supercritical fluid rosemary extract, Rosmarinus officinalis, on antioxidants of major organs of aged rats.

Rosemary leaves, "Rosmarinus officinalis", possess a variety of antioxidant, anti-tumoral and anti-inflammatory bioactivities. We hypothesized that rosemary extract could enhance antioxidant defenses and improve antioxidant status in aged rats. This work evaluates whether supplementing their diet with supercritical fluid (SFE) rosemary extract containing 20% antioxidant carnosic acid (CA) reduces oxidative stress in aged rats. Aged Wistar rats (20 months old) were included in the study. Rats were fed for 12 weeks with a standard kibble (80%) supplemented with turkey breast (20%) containing none or one of two different SFE rosemary concentrations (0.2% and 0.02%). After sacrifice, tissue samples were collected from heart and brain (cortex and hippocampus). Enzyme activities of catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) and nitric oxide synthase (NOS) were quantitatively analyzed. Lipid peroxidation and levels of reactive oxygen species (ROS) were also determined. Rosemary decreased lipid peroxidation in both brain tissues. The levels of catalase activities in heart and cortex were decreased in the rosemary-treated groups. The SFE rosemary-treated rats presented lower NOS levels in heart and lower ROS levels in hippocampus than the control rats. Supplementing the diet of aged rats with SFE rosemary extract produced a decrease in antioxidant enzyme activity, lipid peroxidation and ROS levels that was significant for catalase activity in heart and brain, NOS in heart, and LPO and ROS levels in different brain tissues. These observations suggest that the rosemary supplement improved the oxidative stress status in old rats.

Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma.

Ewing's sarcoma (ES) is characterized by specific chromosome translocations, the most common being t(11;22)(q24;q12). Additionally, other type of genetic abnormalities may occur and be relevant for explaining the variable tumour biology and clinical outcome. We have carried out a high-resolution array CGH and expression profiling on 25 ES tumour samples to characterize the DNA copy number aberrations (CNA) occurring in these tumours and determine their association with gene-expression profiles and clinical outcome. CNA were observed in 84% of the cases. We observed a median number of three aberrations per case. Besides numerical chromosome changes, smaller aberrations were found and defined at chromosomes 5p, 7q and 9p. All CNA were compiled to define the smallest overlapping regions of imbalance (SORI). A total of 35 SORI were delimited. Bioinformatics analyses were conducted to identify subgroups according to the pattern of genomic instability. Unsupervised and supervised clustering analysis (using SORI as variables) segregated the tumours in two distinct groups: one genomically stable (< or =3 CNA) and other genomically unstable (>3 CNA). The genomic unstable group showed a statistically significant shorter overall survival and was more refractory to chemotherapy. Expression profile analysis revealed significant differences between both groups. Genes related with chromosome segregation, DNA repair pathways and cell-cycle control were upregulated in the genomically unstable group. This report elucidates, for the first time, data about genomic instability in ES, based on CNA and expression profiling, and shows that a genomically unstable group of Ewing's tumours is correlated with a significant poor prognosis.

DNA profiling analysis of 100 consecutive de novo acute myeloid leukemia cases reveals patterns of genomic instability that affect all cytogenetic risk groups.

We have carried out a high-resolution whole genome DNA profiling analysis on 100 bone marrow samples from a consecutive series of de novo acute myeloid leukemia (AML) cases. After discarding copy number changes that are known to be genetic polymorphisms, we found that genomic aberrations (GA) in the form of gains or losses of genetic material were present in 74% of the samples, with a median of 2 GA per case (range 0-35). In addition to the cytogenetically detected aberration, GA were present in cases from all cytogenetic prognostic groups: 79% in the favorable group, 60% in the intermediate group (including 59% of cases with normal karyotype) and 83% in the adverse group. Five aberrant deleted regions were recurrently associated with cases with a highly aberrant genome (e.g., a 1.5 Mb deletion at 17q11.2 and a 750 kb deletion at 5q31.1). Different degrees of genomic instability showed a statistically significant impact on survival curves, even within the normal karyotype cases. This association was independent of other clinical and genetic parameters. Our study provides, for the first time, a detailed picture of the nature and frequency of DNA copy number aberrations in de novo AML.

Longitudinal depolarization gradients along the somatodendritic axis of CA1 pyramidal cells: a novel feature of spreading depression.

We studied the subcellular correlates of spreading depression (SD) in the CA1 rat hippocampus by combining intrasomatic and intradendritic recordings of pyramidal cells with extracellular DC and evoked field and unitary activity. The results demonstrate that during SD only specific parts of the dendritic membranes are deeply depolarized and electrically shunted. Somatic impalements yielded near-zero membrane potential (V(m)) and maximum decrease of input resistance (R(in)) whether the accompanying extracellular negative potential (V(o)) moved along the basal, the apical or both dendritic arbors. However, apical intradendritic recordings showed a different course of local V(m) that is hardly detected from the soma. A decreasing depolarization gradient was observed from the edge of SD-affected fully depolarized subcellular regions toward distal dendrites. Within apical dendrites, the depolarizing front moved toward and stopped at proximal dendrites during the time course of SD so that distal dendrites had repolarized in part or in full by the end of the wave. The drop of local R(in) was initially maximal at any somatodendritic loci and also recovered partially before the end of SD. This recovery was stronger and faster in far dendrites and is best explained by a wave-like somatopetal closure of membrane conductances. Cell subregions far from SD-affected membranes remain electrically excitable and show evoked unitary and field activity. We propose that neuronal depolarization during SD is caused by current flow through extended but discrete patches of shunted membranes driven by uneven longitudinal depolarization.

[Tracking along the path toward ischemic neuronal death]. / Las huellas eléctricas en el camino hacia la muerte neuronal isquémica.

INTRODUCTION AND DEVELOPMENT: During episodes of ischemia/anoxia, the neurochemical and environmental changes considered toxic for nervous tissue lie behind the characteristic abrupt massive cell depolarization (MCD). A strong resemblance with other pathologic events enable us to postulate that MCD is a different state of the tissue that includes among others the anoxic depolarization and Le o s spreading depression. MCD is an active event. Neurons enter and leave MCD suddenly and synchronously, and contrary to current belief, their membrane integrity is preserved and ion gradients are only reduced. Biophysical membrane properties are not compatible with some postulates based on endotoxines. There is a direct relation between MCD susceptibility of the different neuron types/nuclei and their vulnerability to ischemia/anoxia. Two different substates can be distinguished in the associated interstitial potentials that are likely related to neuronal and glial dysfunction, respectively. The different modes and timings of anoxic neuronal death depend on the duration of MCD, the functional integrity of the glial network, and the history of previous insults. CONCLUSIONS: MCD is a cellular state of risk bridging life and death. Neurons die if they cannot exit, but may recover if they do promptly, although still have to face subtle changes as well initiated during MCD that will eventually lead them to a delayed death. Avoiding MCD is escaping death. From a clinical point of view, the relevant point is that manipulating MCD entails the simultaneous control of all toxic neurochemical concomitants. Reinforcing vulnerable neurons to avoid their falling into MCD is possible

Las huellas eléctricas en el caminohacia la muerte neuronal isquémica / Tracking along the path toward ischemic neuronal death

Introducción y desarrollo. Durante episodios de isquemia/anoxia, las alteraciones neuroquímicas y ambientales consideradas neurotóxicas se inician tras una despolarización celular masiva (DCM) abrupta y característica. Su similitud con varios procesos fisiopatológicos permite postular que la DCM es un estado tisular/ celular propio que comprende, entre otros, la despolarización anóxica y las ondas de Leão (spreading depression). La DCM es un proceso activo. Las neuronas entran y salen sincrónica y súbitamente, y contra la creencia general, conservan la integridad de membrana, y los gradientes iónicos sólo se reducen. Las propiedades biofísicas de membrana son incompatibles con algunas interpretaciones `toxicistas'. La propensión de diversos tipos/núcleos neuronales a desarrollar DCM y su vulnerabilidad a isquemia/anoxia se relacionan directamente. En los potenciales extracelulares se observan dos subestados, posiblemente asociados a disfunción neuronal y glial. La duración de este `coma' celular, el estado funcional del sincitio glial y el historial de `agresiones' previas determinan la muerte neuronal en sus distintas modalidades y tempos. Conclusiones. La DCM es un estado de riesgo que se proyecta como un puente entre vida y muerte celular. Si las neuronas no consiguen salir de él, mueren, pero si lo hacen, pueden recuperarse, aunque algunas todavía tendrán que hacer frente a cambios más sutiles iniciados también en ese período y que les llevará a una muerte retrasada. Evitar la DCM es evitar la muerte neuronal. Clínicamente, lo más relevante es que la manipulación de la DCM implica el control simultáneo de todas sus concomitantes neuroquímicas `tóxicas'. Es posible reforzar las neuronas vulnerables para impedir su entrada en DCM (AU)

Effects of the gliotoxin fluorocitrate on spreading depression and glial membrane potential in rat brain in situ.

DC extracellular potential shifts (deltaVo) associated with spreading depression (SD) reflect massive cell depolarization, but their cellular generators remain obscure. We have recently reported that the glial specific metabolic poison fluorocitrate (FC) delivered by microdialysis in situ caused a rapid impairment of glial function followed some hours later by loss of neuronal electrogenic activity and neuron death. We have used the time windows for selective decay of cell types so created to study the relative participation of glia and neurons in SD, and we report a detailed analysis of the effects of FC on evoked SD waves and glial membrane potential (Vm). Extracellular potential (Vo), interstitial potassium concentration ([K+]o), evoked potentials, and transmembrane glial potentials were monitored in the CA1 area before, during, and after administration of FC with or without elevated K+ concentration in the dialysate. SD waves propagated faster and lasted longer during FC treatment. DeltaVo in stratum pyramidale, which normally are much shorter and of smaller amplitude than those in stratum radiatum, expanded during FC treatment to match those in stratum radiatum. The coalescing SD waves that develop late during prolonged high-K+ dialysis and are typically limited to stratum radiatum, also expanded into stratum pyramidale under the influence of FC. SD provoked in neocortex normally does not spread to the CA1, but during FC treatment it readily reached CA1 via entorhinal cortex. Once neuronal function began to deteriorate, SD waves became smaller and slower, and eventually failed to enter the region around the FC source. Slow, moderately negative deltaVo that mirrored [K+]o increments could still be recorded well after neuronal function and SD-associated Vo had disappeared. Glial cell Vm gradually depolarized during FC administration, beginning much before depression of neuronal antidromic action potentials. Calculations based on the results predict a large decrease in glial potassium content during FC treatment. The results are compatible with neurons being the major generator of the deltaVo associated with SD. We conclude that energy shortage in glial cells makes brain tissue more susceptible to SD and therefore it may increase the risk of neuron damage.

Neurotensin increases intestinal adaptation and reduces enteroglucagon-like immunoreactivity after large bowel resection in rats.

OBJECTIVE: To assess the effects of giving neurotensin on intestinal adaptation after colectomy and their relation to enteroglucagon-like immunoreactivity. DESIGN: Laboratory experiment. SETTING: Teaching hospital, Spain. MATERIAL: 55 Male Wistar rats. INTERVENTIONS: All animals were anaesthetised before undergoing laparotomy; 24 animals had 75% of their colon resected. Half of the animals (12 in each group) were treated with neurotensin (600 micrograms/kg body wt/day) for 14 days. MAIN OUTCOME MEASURES: Differences in the number of mitoses and in nuclear antigen staining of proliferating cells in the intestinal mucosal crypts; plasma enteroglucagon-like immunoreactivity. RESULTS: After colon resection, the proliferative status, number of mitoses (p < 0.01), and nuclear antigen staining of proliferating cells (p < 0.001) increased significantly in the jejunum of animals treated with neurotensin (p < 0.05). Less pronounced effects were observed in colon and ileum. Plasma enteroglucagon-like immunoreactivity levels fell significantly in all animals given neurotensin (p < 0.05). CONCLUSIONS: Neurotensin increases the adaptive intestinal process after colon resection and reduces plasma enteroglucagon-like immunoreactivity in rats.

Heptanol but not fluoroacetate prevents the propagation of spreading depression in rat hippocampal slices.

We investigated whether heptanol and other long-chain alcohols that are known to block gap junctions interfere with the generation or the propagation of spreading depression (SD). Waves of SD were triggered by micro-injection of concentrated KCl solution in stratum (s.) radiatum of CA1 of rat hippocampal tissue slices. DC-coupled recordings of extracellular potential (V0) were made at the injection and at a second site approximately 1 mm distant in st. radiatum and sometimes also in st. pyramidale. Extracellular excitatory postsynaptic potentials (fEPSPs) were evoked by stimulation of the Schaffer collateral bundle; in some experiments, antidromic population spikes were evoked by stimulation of the alveus. Bath application of 3 mM heptanol or 5 mM hexanol completely and reversibly prevented the propagation of the SD-related potential shift (delta V0) without abolishing the delta V0 at the injection site. Octanol (1 mM) had a similar but less reliably reversible effect. fEPSPs were depressed by approximately 30% by heptanol and octanol, 65% by hexanol. Antidromic population spikes were depressed by 30%. In isolated, patchclamped CA1 pyramidal neurons, heptanol partially and reversibly depressed voltage-dependent Na currents possibly explaining the slight depression of antidromic spikes and, by acting on presynaptic action potentials, also the depression of fEPSPs. Fluoroacetate (FAc), a putative selective blocker of glial metabolism, first induced multiple spike firing in response to single afferent volleys and then severely suppressed synaptic transmission (confirming earlier reports) without depressing the antidromic population spike. FAc did not inhibit SD propagation. The effect of alkyl alcohols is compatible with the idea that the opening of normally closed neuronal gap junctions is required for SD propagation. Alternative possible explanations include interference with the lipid phase of neuron membranes. The absence of SD inhibition by FAc confirms that synaptic transmission is not necessary for the propagation of SD, and it suggests that normally functioning glial cells are not essential for SD generation or propagation.

Is glia disfunction the initial cause of neuronal death in ischemic penumbra?

The supportive role of glial cells on neuronal function and survival has been studied in anesthetized rats by using the selective gliotoxin fluorocitrate. Disabling glia operation reproduced many features of ischemic penumbra. An initial mild acidosis and increased interstitial potassium but not glutamate was followed after 3-4 h by repetitive spreading depression waves. These gradually provoked higher levels of acidosis, potassium and glutamate, gradual neuronal function decay and finally, neuron death. We conclude that neurons become highly vulnerable to spreading depression waves only in absence of normal glia operation. Our findings directly associate early glial disfunction to neuronal loss and lead to new insights for the understanding of ischemic pathology.

The effect of depressing glial function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival.

The supporting role of glial cells in maintaining neurons and in ion homeostasis has been studied in situ by perfusing the gliotoxin fluorocitrate (FC) through a microdialysis fiber in the CA1 area of urethane-anesthetized rats. Extracellular direct current potential, extracellular potassium concentration ([K+]o) and amino acid levels, extracellular pH (pHo), and evoked field activity were studied. Histology verified the swelling of glial cells after 4 hr of FC treatment. Massive neuron damage was evident after 8 hr. FC dialysis caused the rapid decrease of glutamine, pHo became progressively more acid, and [K+]o moderately elevated. Orthodromic transmission was variably blocked within 30 min to 4 hr. After 4 hr, spreading depression (SD) waves that originated from the neocortex invaded hippocampal CA1, [K+]o increased to higher levels, pHo became very acid, and there were steep increases in taurine, glutamate, and GABA levels. Simultaneously, the antidromic population spike (a-PS) became depressed and eventually disappeared. When a shorter dialysis probe that spared cortex was used to sample CA1, no SD was seen, a-PS was not abolished, and ion homeostasis was altered less markedly. Repeated SD provoked in hippocampus in the absence of FC caused only mild depression of a-PS. Dialysis of high-K+ solution in healthy neocortex or hippocampus caused only slight elevation of [K+]o at distances of 200-400 microns from the dialysis membrane. After treatment with FC, similar high-K+ dialysis raised [K+]o much more. We conclude the following: (1) recurrent SD waves injure neurons if and only if glial function has failed; (2) neurons can regulate [K+]o, albeit imperfectly; (3) glia is required for the normal fine tuning of [K+]o and particularly for the recovery of pathologically elevated [K+]o; and (4) glia are required for the regulation of pHo. The similarities between glial poisoning by FC and the reported changes in the penumbra of ischemic infarcts suggest that the extension of neuron loss into the penumbral region might depend on failure of glial protection.

Role of neuronal synchronizing mechanisms in the propagation of spreading depression in the in vivo hippocampus.

To detect what initiates spreading depression (SD), the early prodromal events were investigated in hippocampal CA1 of urethane-anesthetized rats. SD was provoked by microdialysis or focal microinjection of high-K+ solution. Extracellular DC potentials and extracellular potassium concentration ([K+]o) were recorded, and spontaneous and evoked potentials analyzed for current source-density (CSD). In the front of an approaching SD wave, several seconds before the onset of the typical sustained negative potential shift (delta Vo) and the increased [K+]o, fast electrical activity was detected. This consisted initially of small rhythmic (60-70 Hz) sawtooth wavelets, which then gave way to a shower of population spikes (PSs) of identical frequency. Prodromal wavelets and PSs were synchronized over considerable distances in the tissue. Sawtooth wavelets were identified as pacemakers of the prodromal PS burst. Simultaneous recording at three depths revealed that the spontaneous prodromal PSs occurred exactly in phase in dendrites and somata whereas synaptically transmitted PSs arose first in the proximal dendrites and were conducted from there into the soma membrane. During a spike burst, stratum (st.) pyramidale served as current sink, while in the proximal sublayer of st. radiatum spike-sinks gave way to spike sources that grew larger as the sinks in st. pyramidale began to subside. Blocking synaptic transmission did not abolish the prodromal spike burst, yet repetitive orthodromic activation inhibited it without altering the subsequent SD waveform. Complex changes in cell excitability were detected even before fast spontaneous activities. We concluded that, in the initial evolution of SD, changes in neuron function precede the regenerating depolarization by several seconds. We propose that the opening of normally closed electric junctions among neurons can best explain the long-distance synchronization and the flow current that occurs in the leading edge of a propagating wave of SD.

Neuroprotective effect of acidic fibroblast growth factor on seizure-associated brain damage.

The neuroprotective effect of acidic fibroblast growth factor (aFGF) has been analysed in a rat model of seizures-associated brain damage. We report that after treatment with a convulsivant dose of Kainic acid, systemically administered aFGF prevents neuronal degeneration in specific brain areas, mainly in the hippocampal formation. Our findings extend the potential pharmacological use of fibroblast growth factors and afford new data to understand the neurophysiology of these proteins.

Unusual organization of the ilvIH promoter of Escherichia coli.

Analysis of plasmids containing ilvIH-galK fusions indicated that the Escherichia coli ilvIH promoter and sequences sufficient to cause leucine repression lie within 363 base pairs (bp) of ilvI. Experiments designed to locate the promoter and regulatory sequences more precisely gave the following results. The positions of the 5' endpoints of both unlabeled and pulse-labeled ilvIH mRNAs transcribed in vivo lie 30 bp upstream of ilvI. By contrast, the major in vitro RNA endpoints lie at positions further upstream. Several mutations which increase the expression of ilvIH lie 40 to 50 bp upstream of ilvI, within a putative promoter termed P1. Deletion of a 50-bp region immediately upstream of ilvI, which includes P1, resulted in the loss of all ilvIH promoter activity. Deletion of sequences more than 200 bp upstream of ilvI reduced ilvIH promoter activity by more than 80%. These results suggest that transcription of the ilvIH operon is initiated from promoter P1 but that sequences more than 200 bp upstream are required for optimal transcription of the operon.