Mejora de la profilaxis antibiótica en cirugía colo-rectal mediante el análisis de las infecciones incisionales: estudio observacional / Improving antibiotic prophylaxis in colorectal surgery through the analysis of incisional infections: observational study

INTRODUCCIÓN. Las infecciones del sitio quirúrgico son muy frecuentes tras cirugía colo-rectal. El objetivo de este estudio es analizar cuantitativa y cualitativamente dichas infecciones en nuestro servicio. MATERIAL Y MÉTODOS. Se realizó un estudio observacional en 23 enfermos sometidos a resecciones colo-rectales programadas a los que se administró la profilaxis antibiótica habitual de nuestro servicio, gentamicina y metronidazol (G+M). Se analizó la presencia de infecciones incisionales superficiales o profundas y los gérmenes causantes. Ante los resultados, y guiados por los cultivos, se decidió cambiar la profilaxis por amoxicilina y ácido clavulánico (AMC), y continuar el estudio en 38 enfermos. RESULTADOS. Los enfermos que recibieron como profilaxis G+M tuvieron un índice de infecciones incisionales del 48%. En el 90% de esas infecciones había Escherichia coli, y en un 80% enterococos o estreptococos. En el grupo de AMC hubo un índice de infecciones incisionales del 19%, siendo la diferencia con el grupo de G+M estadísticamente significativa (p=0,021). En los cultivos de sus heridas no había enterococos ni estreptococos. DISCUSIÓN. El índice de infecciones incisionales del grupo G+M es superior al comunicado en cirugía colo-rectal programada. El predominio de Escherichia coli en estas infecciones es habitual, no así la elevada presencia de cocos positivos, especialmente enterococos. Estos resultados exigen un cambio en nuestra profilaxis antibiótica, para cubrir estreptococos y enterococos, además de bacilos negativos y anaerobios. AMC parece la opción más lógica. Nuestros resultados corroboran esta hipótesis (AU)

BACKGROUND. Surgical site infections are very common after colorectal surgery. The objective of this study is to analyze quantitatively and qualitatively such infections in our service. METHOD. An observational study was performed in 23 patients undergoing elective colorectal resections who received the usual antibiotic prophylaxis of our service, gentamicin and metronidazole (G+M). Superficial and deep incisional infections, as well as microbes that cause them, were analyzed. Given the results, and guided by the cultures, it was decided to change the prophylaxis to amoxicillin and clavulanic acid (AMC), and continue the study in 38 patients. RESULTS. Patients who were given G+M for prophylaxis had incisional infection rate of 48%. Escherichia coli was present in 90% of these infections, enterococci or streptococci were present in 80% of these infections. In the AMC group there was an incisional infection rate of 19%. The observed difference with the G+M group is statistically significant (p = 0.021). Enterococci and streptococci were not isolated in their incisions. CONCLUSION. The rate of incisional infections in the G+M group is higher than the usually reported in elective colorectal surgery. The predominance of Escherichia coli is usual in these infections, but not the high presence of positive cocci, especially enterococci. These results call for a change in our antibiotic prophylaxis to cover streptococci and enterococci, as well as gram-negative bacilli and anaerobes. AMC seems the most logical choice. Our results support this hypothesis (AU)

An avian model of genetic reflex epilepsy.

The Fayoumi strain of chickens (Fepi) carries a recessive autosomal gene mutation in which homozygotes are afflicted with a photogenic and audiogenic reflex epilepsy. Seizures consist of stimulus-locked motor symptoms followed by generalized self sustained convulsions. EEG recordings show spikes and spike and waves patterns at rest which are suppressed during seizures and replaced by a desynchronized pattern of activity. Neurones of the prosencephalon discharge in bursts at rest, while neurones of the mesencephalon are bursting during seizures. Living neural chimeras were obtained by replacing specific embryonic brain vesicles in a normal chicken embryo with equivalent vesicles from a Fepi donor. These chimeras show that the epileptic phenotype can be totally or partially transferred from the Fepi to the normal chickens. Total transfer of photogenic and audiogenic seizures was obtained by substitution of both the prosencephalon and mesencephalon, while substitution of the prosencephalon alone resulted in transfer of interictal paroxysmal activity and substitution of the mesencephalon alone resulted principally in transfer of ictal motor symptoms. Increased expression of the c-fos protooncogene, as revealed by the western blot technique, confirmed the distinct encephalic localizations of the symptoms of the photogenic and audiogenic reflex epilepsy of the Fepi shown with the methods of electrophysiology and brain chimeras. We conclude that the Fepi is a good model of brain stem reflex epilepsy and suggest that the brain stem is a generator of some other animal and human genetic reflex "epileptic syndromes".

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis.

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.

Dorsal dermis development depends on a signal from the dorsal neural tube, which can be substituted by Wnt-1.

To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or of cells producing Shh led to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, triggered the formation of a feather-inducing dermis. These results show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least mimicked by Wnt-1.

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.

[Regression of Hensen's node and axial growth of the embryo]. / Recul du noeud de Hensen et croissance axiale de l'embryon.

Hensen's node is the gastrulation center in the avian embryo. It is the homologue of the amphibian dorsal blastopore lip and the zebrafish shield. It contains the progeny of all midline cells (floor plate of the neural tube, notochord and dorsal endoderm). However, microsurgical experiments on Hensen's node allow to think that organizer function is due to an extremely limited region situated in the caudal part of Hensen's node which corresponds to the boundary between prospective axial mesoderm rostrally and paraxial mesoderm caudally. This interface is essential for Hensen's node regression and organization of the caudal part of the body.

Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras.

Two apparently different mechanisms successively contribute to the formation of the neural tube in the avian embryo: bending of the neural plate during the primary neurulation in the cephalo-cervico-thoracic region and cavitation of the medullary cord during the secondary neurulation in the lumbo-sacral region. During both these processes, gastrulation continues by the caudal regression of Hensen's node--also called cordoneural hinge in the secondary neurulation. Labeling of Hensen's node or cordoneural hinge by the quail chick marker system revealed that this structure, which is the equivalent of the dorsal blastoporal lip of the Amphibian embryo, i.e., of the Spemann's organizer, gives rise to the midline cells of the three germ layers: the floor plate of the neural tube, the notocord and the dorsal cells of the intestinal endoderm. Caudally to the organizer, both in primary and secondary neurulation, the presumptive territory of the alar plates of the future neural tube overlies the precursors of the paraxial mesoderm. Regression of Hensen's node bisects the ectoderm in two bilateral neural plates leaving in its wake the floor plate, the notocord and the dorsal endoderm.

The relationships between notochord and floor plate in vertebrate development revisited.

By using the quail-chicken chimera system, we have previously shown that during development of the spinal cord, floor plate cells are inserted between neural progenitors giving rise to the alar plates. These cells are derived from the regressing Hensen's node or cordoneural hinge (HN-CNH). This common population of HN-CNH cells gives rise to three types of midline descendants: notochord, floor plate, and dorsal endoderm. Here we find that HNF3beta, an important gene in the development of the midline structures, is continuously expressed in the HN-CNH cells and their derivatives, floor plate, notochord, and dorsal endoderm. Experiments in which the notochord was removed in the posterior region of either normal chicken or of quail-chicken chimeras in which a quail HN had been grafted showed that the floor plate develops in a cell-autonomous manner in the absence of notochord. Absence of floor plate observed at the posterior level of the excision results from removal of HN-CNH material, including the future floor plate, and not from the lack of an inductive signal of notochord origin.

Species-specific immunostaining of embryonic corneal nerves: techniques for inactivating endogenous peroxidases and demonstration of lateral diffusion of antibodies in the plane of the corneal stroma.

Species-specific and species-common monoclonal antibodies (MAbs) to nerve-specific cell surface epitopes were used to compare pre-treatment techniques for nerve staining. Endogenous peroxidases were inactivated in four ways: (1) 0.3% hydrogen peroxide (H2O2); (2) 1% periodic acid (PA) (pH 1.85-1.95); (3) sodium meta-periodate (10-40 mM, pH 4.5); or (4) HCl (pH 1.80). Staining of chick and quail corneal nerves and dorsal root ganglia (DRG) nerves with the MAbs was species-specific. Staining of chick and quail corneal nerves was unaffected by pre-treatment with 0.3% H2O2, but was eliminated by pre-treatment with 1% PA. Chick and quail DRG nerve staining tolerated 0.3% H2O2, and at least one epitope also tolerated 1% PA. Corneal nerves of both chick and quail displayed concentration-dependent sensitivity to pre-treatment with sodium meta-periodate; DRG nerves were not sensitive to such pre-treatment. Corneal nerves tolerated pre-treatment with HCI (pH 1.80), whereas DRG nerves did not. These findings indicate sensitivity of corneal nerve epitopes to oxidation, in contrast with sensitivity of DRG nerve epitopes to low pH. Results also indicate that tissue trimming regulated whole-mount staining of corneal nerves, suggesting that antibodies cannot diffuse across corneal basement membranes, even after detergent extraction. However, antibodies are able to diffuse laterally into the stroma from any cut edge.

Early- and late-migrating cranial neural crest cell populations have equivalent developmental potential in vivo.

We present the first in vivo study of the long-term fate and potential of early-migrating and late-migrating mesencephalic neural crest cell populations, by performing isochronic and heterochronic quail-to-chick grafts. Both early- and late-migrating populations form melanocytes, neurons, glia, cartilage and bone in isochronic, isotopic chimeras, showing that neither population is lineage-restricted. The early-migrating population distributes both dorsally and ventrally during normal development, while the late-migrating population is confined dorsally and forms much less cartilage and bone. When the late-migrating population is substituted heterochronically for the early-migrating population, it contributes extensively to ventral derivatives such as jaw cartilage and bone. Conversely, when the early-migrating population is substituted heterochronically for the late-migrating population, it no longer contributes to the jaw skeleton and only forms dorsal derivatives. When the late-migrating population is grafted into a late-stage host whose neural crest had previously been ablated, it migrates ventrally into the jaws. Thus, the dorsal fate restriction of the late-migrating mesencephalic neural crest cell population in normal development is due to the presence of earlier-migrating neural crest cells, rather than to any change in the environment or to any intrinsic difference in migratory ability or potential between early- and late-migrating cell populations. These results highlight the plasticity of the neural crest and show that its fate is determined primarily by the environment.

A spinal cord fate map in the avian embryo: while regressing, Hensen's node lays down the notochord and floor plate thus joining the spinal cord lateral walls.

The spinal cord of thoracic, lumbar and caudal levels is derived from a region designated as the sinus rhomboidalis in the 6-somite-stage embryo. Using quail/chick grafts performed in ovo, we show the following. (1) The floor plate and notochord derive from a common population of cells, located in Hensen's node, which is equivalent to the chordoneural hinge (CNH) as it was defined at the tail bud stage. (2) The lateral walls and the roof of the neural tube originate caudally and laterally to Hensen's node, during the regression of which the basal plate anlage is bisected by floor plate tissue. (3) Primary and secondary neurulations involve similar morphogenetic movements but, in contrast to primary neurulation, extensive bilateral cell mixing is observed on the dorsal side of the region of secondary neurulation. (4) The posterior midline of the sinus rhomboidalis gives rise to somitic mesoderm and not to spinal cord. Moreover, mesodermal progenitors are spatially arranged along the rest of the primitive streak, more caudal cells giving rise to more lateral embryonic structures. Together with the results reported in our study of tail bud development (Catala, M., Teillet, M.-A. and Le Douarin, N.M. (1995). Mech. Dev. 51, 51-65), these results show that the mechanisms that preside at axial elongation from the 6-somite stage onwards are fundamentally similar during the complete process of neurulation.

Brain chimeras in birds: application to the study of a genetic form of reflex epilepsy.

A strain of chicken, called here FEpi (for Fayoumi epileptic), bearing an autosomal recessive mutation, exhibits a form of reflex epilepsy with EEG interictal paroxysmal manifestations and generalized seizures in response to either light or sound stimulations. By using the brain chimera technology, we demonstrate here that the epileptic phenotype can be partially or totally transferred from an FEpi to a normal chick by grafting specific regions of the embryonic brain. The mesencephalon contains the generator of all epileptic manifestations whether they involve visual or auditory neuronal circuits, with the exception of the abnormal EEG which is transmitted exclusively by telencephalic grafts. This analysis supports the hypothesis that certain forms of human and mammalian epilepsies have a brainstem origin.

Organization and development of the tail bud analyzed with the quail-chick chimaera system.

After closure of the posterior neuropore, the caudal part of the embryo designated as the 'tail bud' forms a mass of undifferentiated cells from which the lumbosacral and caudal parts of the body develop. It has been proposed that the tail bud is a homogeneous structure comparable to a blastema (Holmdahl, 1925; Griffith et al., 1992). Another view is that morphogenesis of the tail bud is merely the continuation of the gastrulation process (Pasteels, 1937, 1943). In order to try to solve this controversy, we have studied the fate of definite and discrete regions of the tail bud at the 25-somite stage by using the quail-chick marker system. We found that the tail bud is composed of different domains endowed with a definite fate. A ventro-rostral region equivalent to the chordo-neural hinge defined by Pasteels gives rise to the notochord and floor plate and thus corresponds to the Hensen's node which in the tail bud pursues its rostrocaudal movement. The presumptive territory of the lateral walls of the lumbo-sacro-caudal neural tube is located caudally to the Hensen's node as it stands at the 25-somite stage. Material destined to form the sacral and caudal somites is still located in the dorsal midline in the caudalmost part of the tail bud. We thus show that the movements of invagination and divergence which characterize gastrulation are still going on in the tail bud after the 25-somite stage. Thus the somitic material located medio-dorsally diverges laterally and contributes by apposition to the growth of the trunco-caudal part of the body. The parallel between tail bud development in Amniotes and Amphibians as described recently by Gont et al. (1993) is striking and points to the unity in the development mechanisms within the Vertebrate phylum.

Steel and c-kit in the development of avian melanocytes: a study of normally pigmented birds and of the hyperpigmented mutant silky fowl.

We describe here the expression of c-kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso-laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c-kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c-kit-positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c-kit positive cells are present in the Steel factor-producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111-2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c-kit expressing melanoblasts migrate initially in the dorso-lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant.

Brain chimeras for the study of an avian model of genetic epilepsy: structures involved in sound and light-induced seizures.

The epileptic homozygotes of the Fayoumi strain of chickens (Fepi) are affected by photogenic reflex epilepsy with complete penetrance. Here we demonstrate that they are equally affected by audiogenic reflex epilepsy induced by intense sound stimulation. All the Fepi display sound-induced seizures from hatching to adulthood consisting of initial 'ictal arousal' and running fits usually followed by generalized clonico-tonic convulsions. A running fit is the preconvulsive motor symptom specifically induced by auditory stimulation while neck myoclonus is the preconvulsive motor symptom specifically induced by photic stimulation. The EEG interictal spikes and spike and waves are suppressed and replaced by a desynchronized trace during the seizures of both kinds. Viable neural chimeras were obtained by graft of embryonic brain vesicles from Fepi donors into normal chick embryos. Transfer of the complete audiogenic and photogenic phenotypes was obtained in chimeras resulting from embryonic substitution of both the prosencephalon and mesencephalon. The substitution of the prosencephalon alone resulted in transfer of interictal paroxysmal EEG activity accompanied by the sound and light-induced desynchronization and 'ictal arousal' with no motor seizures. Chimeras with embryonic substitution of the mesencephalon alone displayed running fits and convulsions induced by sound stimulation but only neck myoclonus following light stimulation. The conclusions are reached that: (i) the Fepi is a model of audiogenic and photogenic reflex epilepsy; (ii) in both types, the seizure initiator and the convulsion generator are localized in the brainstem, although reinforcement from telencephalic visual structures is needed to trigger photogenic generalized convulsions.

Reflex epilepsy of the fowl and its transfer to normal chickens by brain embryonic grafts.

The genetic photosensitive epilepsy of the Fayoumi chicken was transferred to normal chickens by in situ grafts at 2 days of incubation, of both the prosencephalic and mesencephalic brain vesicles taken from epileptic embryos. However, mesencephalic graft is sufficient to allow convulsions under sound stimulation. Typical EEG patterns are recorded in chimeras having the prosencephalon plus or not the mesencephalon. We conclude that, in this mutant, the whole neural tissue is affected, but the seizure generator is localized inside the mesencephalon, and specific sensory pathways are necessary for seizures to occur.