M.D. Fiorella Malchiodi-Albedi
email: fiorella.malchiodi@iss.it

Valentina Contrusciere

Carla Raggi

Katia Fecchi

Gabriella Rainaldi

Silvia Paradisi

Andrea Matteucci

Maria Teresa Santini

Massimo Sargiacomo

Claudio Frank

Maria Cristina Gaudiano

Marco Diociaiuti

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity

Keywords: amyloid proteins, neurotoxicity, calciton oligomers, gangliosides, lipid rafts, protein misfolding

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid β oligomers.

Immature neurons and the other cell types showed no cytotoxicity. CTOs caused cytosolic Ca 2+ rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

A C C E P T E D M A N U S C R I P T INTRODUCTION

A number of degenerative disorders are pathogenetically linked to a process of misfolding and aggregation of specific proteins, the large family of the so-called amyloid proteins [START_REF] Bucciantini | Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases[END_REF][START_REF] Glabe | Common mechanisms of amyloid oligomer pathogenesis in degenerative disease[END_REF][START_REF] Stefani | Generic cell dysfunction in neurodegenerative disorders: role of surfaces in early protein misfolding, aggregation, and aggregate cytotoxicity[END_REF].The process of aggregation that leads to the characteristic cytotoxic properties generally proceeds through the formation of dimers, oligomers and protofibrils, before finally assuming an insoluble fibrillar conformation, characterized by β-structures. It is now generally agreed that the cytotoxic species are the soluble, oligomeric aggregates, while the insoluble fibrils seem to be relatively non-harmful. Small oligomeric assemblies of Amyloid β (Aβ) for example have been specifically shown to impair long-term potentiation and dendritic spine structure in the hippocampus and to disrupt cognition in animals (For a review see Nimmrich and Ebert, 2009 [4]). Several findings suggest that a common pathologic pathway exists in the case of amyloid associated neurodegenerative diseases, which is independent from the peptide primary sequence [START_REF] Glabe | Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis[END_REF][START_REF] Lashuel | Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins? Q[END_REF]. According to this hypothesis, neurotoxicity of extracellular amyloid aggregates is due to the formation of Ca 2+ -permeable channels by incorporation of amyloid protein oligomers in the plasma membrane, resulting in an imbalance in Ca 2+ inflow and consequent cell death [START_REF] Glabe | Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis[END_REF][START_REF] Kagan | Amyloid peptide channels[END_REF][START_REF] Lal | Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm[END_REF][START_REF] Quist | Amyloid ion channels: a common structural link for proteinmisfolding disease[END_REF].

Calcitonin is a polypeptidic hormone secreted by the thyroid gland and, due to its typical aggregation behaviour, belongs to the amyloid family [START_REF] Gaudiano | Effects induced by hydroxyl radicals on salmon calcitonin: a RP-HPLC, CD and TEM study[END_REF][START_REF] Gaudiano | Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes[END_REF]. Even if it is not involved in any neurodegenerative diseases, aggregated CT is toxic to neurons in vitro [START_REF] Gilchrist | Amyloid formation by salmon calcitonin[END_REF][START_REF] Schubert | Amyloid peptides are toxic via a common oxidative mechanism[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects[END_REF], like other amyloid proteins.

Salmon Calcitonin (sCT), found to be neurotoxic as CT from other species [START_REF] Gilchrist | Amyloid formation by salmon calcitonin[END_REF][START_REF] Schubert | Amyloid peptides are toxic via a common oxidative mechanism[END_REF][START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF], is characterized by a slower aggregation rate [START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF] and this peculiarity is at the basis of its pharmacological use. This observation that may provoke concern, since the possibility that

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protein aggregates may reach the brain from the blood circulation and trigger a neurotoxic reaction should be taken into consideration, especially considering CT is often delivered as a nasal spray [START_REF] Malchiodi-Albedi | Calcitonin therapy and oligomer neurotoxicity: an underestimated risk?[END_REF]. We have recently shown that sCT Oligomers (sCTOs) forms Ca 2+ -permeable pores in liposomes [START_REF] Diociaiuti | Calcitonin forms oligomeric pore-like structures in lipid membranes[END_REF], highly reminiscent of the ion channels formed by other amyloid proteins, such as Aβ and α synuclein [START_REF] Lashuel | Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins? Q[END_REF][START_REF] Quist | Amyloid ion channels: a common structural link for proteinmisfolding disease[END_REF][START_REF] Lashuel | Neurodegenerative disease: amyloid pores from pathogenic mutations[END_REF][START_REF] Lashuel | Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils, including amyloid pores[END_REF]. We have also shown that sCTO insertion in model membranes occurs only in the presence of GM1 ganglioside and cholesterol, which are typical components of lipid rafts [START_REF] Simons | Lipid rafts and signal transduction[END_REF]. A large body of evidence sustains a role for lipid rafts in amyloid-induced neurotoxicity. Lipid rafts have been shown to be involved in Aβ formation [START_REF] Simons | Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons[END_REF][START_REF] Ehehalt | Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts[END_REF]. Of particular interest is their involvement in the mechanisms by which amyloid oligomers interact with neuronal plasma membrane and trigger a neurotoxic response. It has been reported that the neurotoxicity of some amyloid proteins, such as Aβ peptides and calcitonin, is strongly reduced by the depletion of ganglioside and/or cholesterol [START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects[END_REF][START_REF] Wang | Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides[END_REF]. In addition, it has been shown that Aβ binds to sialic acid-containing gangliosides [START_REF] Ariga | Characterization of high-affinity binding between gangliosides and amyloid betaprotein[END_REF][START_REF] Kakio | Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid[END_REF]. The aims of the present study were i) to compare the cytotoxic response to sCTOs of cultured hippocampal neurons to that observed in different cell types with the aim of determining if a diverse sensitivity exists; ii)

to investigate if sCTO-induced neurotoxic response is related to an imbalance of intracellular Ca 2+ ; iii) to characterize the role played by lipid rafts in sCTO toxicity.

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MATERIALS AND METHODS

Preparation of sCTO and Aβ solutions

Lyophilized sCT (European Pharmacopoeia, EDQM, France) was stored at -18°C before use. sCTO 1 mM solution was prepared by dissolving the protein in 5 mM phosphate buffer, pH 7.4 and incubated at room temperature for 3 h. No stock solutions of protein were employed.

After 3 h, sCT solutions, containing sCTOs, were diluted to a final concentration of 80 µM for CD, TEM, neurotoxicity and Ca 2+ influx experiments and 25, 60, 80 or 120 µM for growth curves. To analyze sCT effects in its monomeric form, sCT was diluted in deionized water (1mM) immediately before use. Aβ oligomers were prepared as described by Stine et al. [START_REF] Stine | In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis[END_REF].

Briefly, lyophilized 1-42 Aβ peptide, (Bachem, Switzerland), was dissolved in 1,1,

hexafluoro-2-propanol (HFIP, Sigma, USA) and dried. Before use, an aliquot was dissolved in dimethyl sulfoxide (5mM), brought to 1mM in Minimum Essential Medium (MEM, Invitrogen, Italy), incubated at 4°C for 24 h. The final dilution was 20 µM.

Transmission Electron Microscopy

Negative staining was obtained using a phosphotungstic acid (PTA) 2% w/v solution, buffered at pH 7.3 with NaOH. To avoid salt precipitation from PTA and/or NaOH, which can be misinterpreted as actual structures, the staining solution was filtered before each preparation through polycarbonate 0.2 µm pore filters. A droplet of protein solution was deposited onto 400mesh copper grids for electron microscopy and covered with a thin amorphous carbon film (about 20 nm). When the grid was dried, a droplet of PTA was deposited and let dry. The samples were studied at a Zeiss 902 Transmission Electron Microscope, operating at 80 kV,

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equipped with an Electron Energy Loss filter. In order to enhance the contrast, the microscope was used in the Electron Spectroscopy Imaging mode filtering at ∆E=0 eV. The image acquisition was performed by a digital HSC2 CCD camera, 1k for 1k pixels, (Proscan gmbh, Germany), thermostatted by a Peltier cooler model WKL 230 (Lauda gmbh, Germany). Image analysis and quantification was performed by the digital image analyzer analySIS 3.0 (Sis gmbh, Germany). This software allows to enhance contrast and sharpness of the acquired images and to perform morphological quantification and statistics. The dimensional measurements were performed after a careful magnification calibration of the whole imaging system based on reference standards.

Circular Dichroism (CD) measurements

CD measurements were performed on a Jasco J-715 spectropolarimeter (Jasco Corporation, Japan) in the far-UV region (260-190 nm). All spectra, mean of 4 different scans, were blank subtracted. Quartz cells of 0.1 cm path-length were employed. The spectral resolution was 0.1 nm and the speed scan 50 nm/min. All CD spectra were reported as De = [Θ] / 3300, where [Θ] is equal to (θ x 100)/(l x C) (θ is the measured ellipticity, C is the molar amino acidic concentration (=8x10 -5 x 32M) and l is the pathlength in cm). CD spectra were analyzed quantitatively by a fitting procedure performed using the CDSSTR software available at the website of the Birkbeck College of the University of London.

Cell cultures

Primary hippocampal neurons were prepared from embryonic-day-18 rat brain, according to the method of Brewer et al. [START_REF] Brewer | Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination[END_REF], slightly modified [START_REF] Malchiodi-Albedi | Astrocytes contribute to neuronal impairment in beta A toxicity increasing apoptosis in rat hippocampal neurons[END_REF]. All experimental procedures were in line with the "Ethical principles and guidelines for scientific experiments on animals" of the µM. This solution was then removed and replaced with extracellular solution, and the dishes were quickly placed on the microscope stage. To measure fluorescence changes, a Hamamatsu (Shizouka, Japan) Argus 50 computerized analysis system was used, recording every 6 sec the ratio between the values of light intensity at 340 and 380 nm stimulation.

Apoptosis detection

To detect apoptosis, cells were fixed as described, stained with Hoechst 33258 and observed at the Nikon fluorescence microscope. Non-apoptotic cells exhibit a diffuse nuclear fluorescence while apoptotic nuclei appear fragmented or highly condensed. In some experiments, neurons were treated with the TUNEL technique, using the DeadEnd kit (Promega, Madison, WI) and observed at a Nikon Optiphot microscope. Differential counts of normal and apoptotic cells were performed on randomly chosen microscopic fields for a total of at least 200 cells for each coverslip. Two coverslips were scored for each condition. The values obtained for each coverslip were averaged to produce a single mean value for each experiment.

Growth curves

In order to determine cell proliferation, MG-63 and NIH-3T3 cells were seeded at 2 x 10 4 cells/ml of cell culture medium and incubated at 37°C in a 5% CO 2 atmosphere. After 24 h of incubation, cells were treated with 25, 60, 80 or 120 µM sCTO for 20 min and then left to grow in the medium in absence of sCTO for 24, 48 and 72 h. Untreated MG-63 cells were considered as control. After each time of incubation, cells were detached by first adding 10 mM EDTA (pH 7.4) and, once this solution was removed, by adding 0.25% trypsin and counted by using a Neubauer chamber. Each point was conducted in triplicate.

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Preparation of low-density Triton-insoluble (LDTI) plasma membrane domains

The different cell types were fractionated using a detergent (1% Triton X-100, Sigma)containing method [START_REF] Lisanti | Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae[END_REF]. The cells were washed in ice cold PBS twice and lysed in 750 µl MESbuffered saline (MBS: 25 mM MES pH 6.5, 150 mM NaCl), containing 1% Triton X-100, 1 mM Na3VO4 and protease inhibitor cocktail (Roche, Indianapolis, IN) for 30 min on ice. Cell lysate was Dounce homogenized, adjusted to 40% sucrose and placed at the bottom of an ultracentrifuge tube. A 5-30% linear sucrose gradient was then placed above the homogenate and the mixture was centrifuged at 45.000 rpm for 16h at 4°C in an SW60 rotor (Beckman Instruments, Palo Alto, CA). Lipid raft fractions are visible as a light-scattering band migrating at approximately 20% sucrose. Twelve 0.375 ml fractions were collected from the top to the bottom of the gradient. The lipid rafts were separated from other fractions, diluted in MBS and centrifuged at 30.000 g for 30 min at 4 °C. Protein content of total membrane and lipid rafts was determined by BCA assay (Pierce Rockford, Ill) using bovine serum albumin as a standard.

Lipid extraction and thin-layer chromatography

Total lipids were extracted according to the method of Folch et al. [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF]. Total membranes (50 µg) and lipid rafts (1.5 µg) were diluted with equal volume of deionized water. Total lipids were dissolved into chloroform/methanol (2:1 v/v) and applied to TLC plates. The spot corresponding to cholesterol was first identified by co-migration with the cholesterol standard.

Cholesterol was separated by TLC using acetic acid: hexane: ether ethyl (1:70:30 v/v/v) as the developing solvent and was visualized by copper acid (3% w/v) staining. The plate was heated for a few minutes at 180°C. The quantity of cholesterol was determined by densitometry and

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RESULTS

sCT forms amyloid-like oligomers

We first proceeded to obtain and characterize samples of sCT in an early stage of the aggregation process. For this purpose, sCT (1mM) was dissolved in phosphate buffer (5 mM) and allowed to aggregate for 3 h at room temperature, to facilitate the aggregation process. To obtain sCT monomers, sCT was dissolved in pure deionized distilled water, immediately before use, as previously suggested [START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects[END_REF][START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF]. As demonstrated by Energy Filtered-Transmission Electron Microscopy (EF-TEM) analysis, aggregated sCT (Fig. 1A) formed small isolated globules of mean diameter of about 14 nm (Fig. 1C). These features represent the classical intermediates occurring between monomers and mature fibrils and show a remarkable similarity with Aβ oligomers and other amyloid proteins [START_REF] Lashuel | Neurodegenerative disease: amyloid pores from pathogenic mutations[END_REF][START_REF] Lashuel | Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils, including amyloid pores[END_REF]. No mature fibrils were observed. When sCT was dissolved in distilled water and observed without pre-incubation (Fig. 1B), only very small white dots, of mean diameter of about 5 nm (Fig. 1C), were present, likely representing sCT monomers or small molecular weight oligomers, in analogy to what observed for bovine [START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects[END_REF] and human [START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF] CT. CD data (Fig. 1D), processed according to Whitmore and Wallace [START_REF] Whitmore | Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases[END_REF], indicated that, in sCT freshly dissolved in distilled water, the random coil component was 72% while the β-structure was 23% (5% of α-structures). It is well known that sCT in water solution and in monomeric state is mainly random coil [START_REF] Epand | Amphipathic helix and its relationship to the interaction of calcitonin with phospholipids[END_REF]. CD data from sCT dissolved in phosphate buffer and aggregated for 3 h (Figure 1D), show that, in this very early aggregation stage, the amount of β-structure rose to about 28% while the amount of random coil fell to 67 %.

The amount of α-structures did not change. This is consistent with the aggregation process

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described for the amyloid proteins, where random components tend to organize in β-structures.

This result is in good agreement with similar experiments [START_REF] Gaudiano | Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes[END_REF][START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF].

sCTOs damage fine neurites and synapses like Aβ oligomers

Neuritic damage is a well-described effect of Aβ in hippocampal neurons in culture.

Furthermore, synaptic damage, a hallmark of AD, has been put in relation to the presence of Aβ oligomers [START_REF] Lacor | Synaptic targeting by Alzheimer's-related amyloid beta oligomers[END_REF]. Since sCTOs have morphological and structural similarities to amyloid proteins, we asked if the effects induced by sCTOs on neurites and synapses in mature hippocampal neurons were similar to those induced by Aβ oligomers. We immunostained mature neurons for MAP2, a component of the dendritic cytoskeleton, to label the neuritic tree, and for synaptophysin, an integral protein of synaptic vesicles, to label synapses. When compared to control cultures, both sCTOs and Aβ oligomers induced a remarkable dendritic loss, especially in the finest branches (Fig. 2, upper panel). In addition both of them caused an evident decrease in the number of synaptic boutons (Fig. 2, lower panel).

sCTOs are cytotoxic for mature hippocampal neurons, but not for other cell types

Amyloid oligomers are thought to initiate an apoptotic cascade, leading to neuronal cell death [START_REF] Morishima | Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand[END_REF][START_REF] Hashimoto | Role of protein aggregation in mitochondrial dysfunction and neurodegeneration in Alzheimer's and Parkinson's disease[END_REF]. To verify if sCTOs had a pro-apoptotic effect, to more closely link cytotoxicity to Ca 2+ imbalance, we applied a protocol reminiscent of the "delayed treatment paradigm", described by Choi in 1988 [START_REF] Choi | Glutamate neurotoxicity and diseases of the nervous system[END_REF], where a brief (20 min) excitotoxic insult is followed by 24-h-

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recovery in cell culture medium. The overstimulation of N-methyl-D-aspartate (NMDA) receptor obtained with this treatment induces an intense increase in intracellular Ca 2+ that preferentially triggers an apoptotic pathway of cell death. Similarly, we exposed the cell cultures to 20-min treatment with sCTOs and allowed a 24-h-recovery in cell culture medium in absence of sCTOs.

We estimated the apoptotic rate by identifying nuclei showing fragmentation or condensation of chromatin, using the Hoechst stain. Mature hippocampal neurons, at DIV14, showed a marked increase of the apoptotic rate, when treated with sCTOs, with respect to controls (Fig. 3A,B). The results were confirmed by the Terminal transferase-mediated dUTP-biotin Nick End-Labeling TUNEL assay (Fig. 3B,C). We then analyzed sCTOs cytotoxicity in immature hippocampal neurons (6 DIV) and non-neuronal primary cultures (cortical astrocytes from rat embryos) or immortalized cell lines of non-neural histogenesis (MG63 osteoblasts and NIH-3T3 fibroblasts).

None of them showed modifications of the apoptotic rate, as shown by the Hoechst stain. Growth curves of MG63 osteoblasts (Fig. 4) and NIH-3T3 fibroblasts (data not shown) were also unaffected. It should be noted that apoptosis in mature hippocampal neurons was related to the presence of oligomeric structures, since when CT was dissolved in water and used without previous incubation, no significant increase in cell death was observed (Fig. 5A), as already indicated [START_REF] Schubert | Amyloid peptides are toxic via a common oxidative mechanism[END_REF][START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation[END_REF].

sCTOs increase intracellular Ca 2+ in mature hippocampal neurons, but not in other cell types

A number of findings support the hypothesis that amyloid proteins form Ca 2+ -permeable channels on cell membranes, which induce a perturbation of cytosolic Ca 2+ [START_REF] Lashuel | Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins? Q[END_REF][START_REF] Kagan | Amyloid peptide channels[END_REF][START_REF] Lal | Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm[END_REF] and trigger apoptosis [START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF]. We monitored intracytoplasmic free Ca 2+ by Fura 2AM, after administration of

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sCTOs, in mature and immature hippocampal neurons, primary cortical astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts (Fig. 3D,E). While mature hippocampal neurons had an intense and sustained increase in cytosolic Ca 2+ , which involved all the recorded cells without recovery for the duration of the experiments (8 min) (Fig. 3D,E), immature hippocampal neurons, astrocytes and NIH-3T3 fibroblasts did not show any change in intracellular Ca 2+ .

MG63 osteoblasts did show a perturbation in cytosolic Ca 2+ , but it consisted in a much lower and rapidly recovering increase, which affected only a minority of cells. As for increased apoptosis, Ca 2+ perturbation in mature hippocampal neurons was related to the presence of oligomers, since treatment with CT monomers did not induce any change in intracellular Ca 2+ (Fig. 5B). Thus the pro-apoptotic effect of sCTOs in hippocampal neurons was conceivably related to the intense and unbalanced Ca 2+ increase, which was lacking in the other cell types.

Lipid rafts are more abundant in mature hippocampal neurons than in the other cell types

We have recently shown that the presence of GM1-and cholesterol-enriched lipid rafts favors the formation of Ca 2+ -permeable, sCTO ion-channels in model membranes [START_REF] Diociaiuti | Calcitonin forms oligomeric pore-like structures in lipid membranes[END_REF]. To quantitatively investigate differences in the lipid composition of the different cell types, we evaluated the ratio between cholesterol in low-density Triton-insoluble (LDTI) plasma membrane domain and total cholesterol (weight %), as a measure of lipid raft content in the plasma cell membrane. Results clearly indicate that mature neurons show the highest content in lipid rafts (Fig. 6A), with respect to the other cell types.

Is sCTO-induced Ca 2+ rise due to preexisting ion channel stimulation?

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Some authors sustain that amyloid oligomers insert into the neuronal membrane to form a cation-conducting pore, leading to Ca 2+ dysregulation, and favor the hypothesis that stimulation of preexisting Ca 2+ -permeable ion channels is not involved [START_REF] Glabe | Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis[END_REF][START_REF] Demuro | Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers[END_REF]. Other authors have, in contrast, evidenced a role for glutamate receptor activation in the neurotoxic effect of amyloid proteins, such as Aβ [START_REF] Kelly | beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons[END_REF][START_REF] Domingues | Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits[END_REF][START_REF] Hsieh | AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss[END_REF] and HypF-N [START_REF] Pellistri | Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca 2+ increase in primary neuronal cells[END_REF]. To explore the role of glutamate receptors in sCTO neurotoxicity, we first tried to establish if the observed Ca 2+ increase in mature hippocampal neurons after sCTO treatment was due to an intracellular release or to an extracellular influx. To this aim, we used thapsigargin, a specific sarcoplasmic/endoplasmic reticulum calcium ATPase pump inhibitor, which depletes intracellular Ca 2+ stores. Two µM thapsigargin evoked only a very inconspicuous rise in intracellular Ca 2+ , consistent with Ca 2+ leakage from intracellular stores, but subsequent application of sCTOs oligomers produced a large and rapid increase, similar to that observed in control cells without thapsigargin, suggesting that sCTO-induced Ca 2+ rise was mostly due to an extracellular influx (Fig. 7A). Then we moved to analyze if the NMDA receptor (which is the glutamate receptor mostly responsible for excitotoxic Ca 2+ rise in neurons) was involved in the influx. First, we pretreated hippocampal neurons with MK801, a specific NMDA inhibitor. MK801 poorly affected sCTO-driven Ca 2+ influx (Fig. 7B), causing only a small delay in the response, while it completely blocked NMDAdriven Ca 2+ increase (data not shown). sCTOs, however, could interact with the NMDA receptor at a site different from that bound to MK801, thus modifying receptor structure and regulation.

To this purpose, we pre-treated hippocampal neurons with antibodies against the NR1subunit, which is a mandatory component of all NMDA receptors, to check if immunoglobulins specifically bound to the receptor could hinder its interaction with sCTOs and suppress Ca 2+ influx. In the presence of anti-NR1, again the response to sCTOs was slightly delayed; however,

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after a few seconds, it reached the same intensity and stability as in absence of the antibody (Fig. 7C). To further analyse sCTO response, we pre-treated mature hippocampal neurons with an antibody-anti GM1, in the attempt to block the pore formation. The presence of the antibody completely abolished sCTOs-induced Ca 2+ increase. After 10 min, an administration of 100 µM NMDA induced a classical abrupt and intense Ca 2+ rise, supporting the fact that the NMDA receptor activity had not been modified by the presence of anti-GM1 antibody (Fig. 7D). The response was reliably due to a specific interaction with GM1, since unrelated antibodies, such as anti-BSA, at the same concentration, did not affect sCTO response (Fig. 7E). These results

suggest that although NMDA receptor may be rapidly activated at the beginning of the response, the formation of sCTO Ca 2+ -permeable pores is the main mechanism responsible for Ca 2+ dysregulation.

Depletion of gangliosides affects sCTO response in mature hippocampal neurons

To further support the hypothesis that the highest content in lipid rafts in mature hippocampal neurons was related to their susceptibility to the sCTO pro-apoptotic effect, we tested if lipid raft depletion modified sCTO-induced cytotoxicity and Ca 2+ influx. In mature hippocampal neurons treated with neuraminidase (NAA), which specifically removes gangliosides, a characteristic component of lipid rafts, sCTO-driven increase in the apoptotic rate was remarkable reduced (Fig. 8 A); in parallel, cytosolic Ca 2+ increase was inhibited (Fig. 8 B).

This observation strongly supports the hypothesis that the high content in lipid rafts promotes sCTO neurotoxicity. In addition, we also pre-treated the cells with MCDX, in order to remove cholesterol from the plasma membrane. Again, we observed a significant reduction in the apoptotic rate of hippocampal neurons, although they showed a variable response to sCTOs in

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DISCUSSION

Pathologies deriving from misfolded amyloid proteins are particularly frequent in CNS.

The reasons for this peculiar vulnerability are presently unknown, but several hypotheses may be formulated. The brain may provide a particularly suitable environment for protein misfolding process, or, alternatively, neuronal cells may be particularly sensitive to the toxic potential of amyloid proteins. We used CT, a protein that undergoes a process of amyloid aggregation, to investigate if neuronal cells are more vulnerable to amyloid proteins than cells of different histogenesis, a comparative study that to our knowledge has never been performed. We chose sCT because it is known to aggregate more slowly than human CT [START_REF] Gaudiano | Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes[END_REF][START_REF] Arvinte | The structure and mechanism of formation of human calcitonin fibrils[END_REF]. To observe the early stages of oligomerization, incubation was limited to 3 h to obtain a sample rich in oligomers, but devoid of fibrils.

Consistently with its amyloid nature, in mature (14 DIV) hippocampal neurons, sCTOs induced cell damage that resembled remarkably that induced by Aβ, such as altered neuritic tree, with loss of the finest branches, and synaptotoxicity, evidenced by loss or alteration of synaptic structures. This is of particular relevance since damage of synapses is considered to be responsible for neuronal dysfunction underlying AD cognitive impairment and represents the pathologic finding that best correlates with the degree of dementia [START_REF] Nimmrich | Is Alzheimer's disease a result of presynaptic failure? Synaptic dysfunctions induced by oligomeric beta-amyloid[END_REF]. To highlight specific susceptibility of neuronal cells to amyloid toxicity, we compared sCTO toxicity in mature and immature (6 DIV) hippocampal neurons to that of two immortalized cell lines (MG63 osteoblasts and NIH-3T3 fibroblasts). In addition, we also analysed the effects on primary astrocytic cultures from rat fetal brain, to exclude that immortalized lines could present a more resistant phenotype, due to their manipulated genome. Our results show that mature, but not immature, hippocampal neurons respond to sCTOs with an evident increase in apoptosis, while the other cell types did

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not show any increase in cytotoxicity. Furthermore, sCTO administration to mature, but not immature hippocampal neurons induced an intense and sustained rise in intracellular Ca 2+ , while the other cell types were nearly unresponsive. MG63 osteoblasts did show a slight response in terms of Ca 2+ rise, but the increase was much lower that the one observed in neurons, rapidly disappeared and affected a very small proportion of cells.

When considering the pathogenesis of amyloid protein neurotoxicity, two main possibilities are presently considered: i) oligomers of small molecular weight insert into membrane bilayers to create cytotoxic pores in a relatively non-specific mechanism [START_REF] Glabe | Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis[END_REF][START_REF] Kagan | Amyloid peptide channels[END_REF][START_REF] Lal | Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm[END_REF][START_REF] Quist | Amyloid ion channels: a common structural link for proteinmisfolding disease[END_REF][START_REF] Demuro | Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers[END_REF];

ii) oligomers bind to particular membrane targets as highly specific ligands [START_REF] Lacor | Synaptic targeting by Alzheimer's-related amyloid beta oligomers[END_REF]. In favour of the first hypothesis is the observation that several disease-related amyloid proteins share a common mechanism for oligomer-mediated toxicity, notwithstanding their different primary sequence [START_REF] Glabe | Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis[END_REF][START_REF] Demuro | Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers[END_REF]. According to this theory, the toxicity of amyloid oligomers is intrinsically related to their aggregation state and point to the formation of pores, with consequent dysregulation of intracellular Ca 2+ , as a primary pathogenetic mechanism.

In our experiments, we found that mature hippocampal neurons were more susceptible to sCTO-induced Ca 2+ dysregulation, at difference with the other examined cell types. Which characteristic may be at the basis of this peculiar reactivity? A large body of evidence sustains a role for lipid rafts in amyloid-induced neurotoxicity. Lipid rafts have been put in relation to Aβ formation [START_REF] Simons | Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons[END_REF][START_REF] Ehehalt | Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts[END_REF]. Of particular interest is their involvement in the mechanisms by which amyloid oligomers interact with neuronal plasma membrane and triggers a neurotoxic response.

It has been reported that the neurotoxicity of some amyloid proteins, such as Aβ peptides and calcitonin, is strongly reduced by the depletion of ganglioside and/or cholesterol [START_REF] Wang | The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects[END_REF][START_REF] Wang | Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides[END_REF]. In addition, it has been shown that Aβ binds to sialic acid-containing gangliosides [START_REF] Ariga | Characterization of high-affinity binding between gangliosides and amyloid betaprotein[END_REF][START_REF] Kakio | Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid[END_REF][START_REF] Mclaurin | Structural transitions associated with the interaction of Alzheimer beta-amyloid peptides with gangliosides[END_REF]. The neuronal plasma membrane is particularly rich in lipid rafts. Thus, content in lipid rafts higher

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than the other cell types could render neurons more vulnerable to amyloid toxicity. This hypothesis was confirmed by our results, where mature neuronal cells showed a much more elevated content in lipid rafts of the other cells types examined. It has also been demonstrated that lipid rafts increase in the plasma membrane during in vitro maturation in hippocampal neurons [START_REF] Ledesma | Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes[END_REF]. This could explain why mature neurons were sensitive to sCTO toxicity, but immature neurons were not. In agreement with this hypothesis, we found that a much higher content in lipid rafts was present in the plasma membrane of 14 DIV hippocampal neurons, with respect to 6 DIV neurons, primary astrocytic cell cultures, MG63 osteoblasts and NIH-3T3 fibroblasts.

We have recently shown that the presence of GM1-and cholesterol-enriched lipid rafts favors the formation of Ca 2+ -permeable, sCTO ion-channels in model membranes [START_REF] Diociaiuti | Calcitonin forms oligomeric pore-like structures in lipid membranes[END_REF]. The high content in lipid rafts could facilitate the formation of sCTO Ca 2+ -permeable pores in mature hippocampal neurons, a mechanism hypothesized for other amyloid proteins [START_REF] Demuro | Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers[END_REF] that implies a nonspecific interaction, independent from the stimulation preexisting Ca 2+ -permeable plasma membrane ion channels. This view has, however, been challenged by authors who evidenced a role for glutamate receptors in Aβ [START_REF] Kelly | beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons[END_REF][START_REF] Domingues | Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits[END_REF][START_REF] Hsieh | AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss[END_REF] and HypF-N, the N-terminal domain of the Escherichia coli hydrogenase maturation factor, a protein that is not associated with any amyloid disease but shows an aggregative behavior [START_REF] Pellistri | Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca 2+ increase in primary neuronal cells[END_REF]. It has been hypothesized at this regard that oligomers may act at two steps, separated in time [START_REF] Pellistri | Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca 2+ increase in primary neuronal cells[END_REF]. A first step, very rapid (in the order of seconds) where Ca 2+ increases following glutamate receptor stimulation by the oligomers, followed by a second, delayed step where oligomers permeabilize nonspecifically the cell membrane, possibly via the formation of pores. Our results show that sCTO-induced Ca 2+ entry was poorly affected by the presence of MK801, an NMDA receptor blocker, or pretreatment with an anti-NR1 antibody, used to mask possible sites of interactions between sCTOs and the

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NMDA receptor, the responses in these cases being only slightly delayed. On the contrary, pretreatment of neurons with an antibody against GM1 completely suppressed sCTO-driven Ca 2+ rise, without altering NMDA receptor activity. On the basis of the delayed Ca 2+ rise observed when NMDA receptor was blocked, we cannot exclude that a component due to an interaction between sCTOs and the NMDA receptor is present, or that the AMPA type of glutamate receptors, which have not been examined in the present study, may be involved. However, the block of Ca 2+ response obtained with an anti-GM1 antibodies strongly supports the conclusion that the intense and protracted Ca 2+ dysregulation observed after sCTOs treatment is reliably due to the pore formation. As further support to the role of lipid rafts in sCTO toxicity, we observed that lipid raft disruption obtained by NAA, which removes sialic acid from gangliosides, a characteristic component of lipid rafts, inhibited Ca 2+ rise and protected against sCTO neurotoxicity, probably modifying the plasma membrane area susceptible to the insertion of the pore-like structures. This perspective is, in our opinion, pretty innovative, since it does not focus on the inhibition of the amyloid oligomer formation but to the hindrance of its interaction with the neuronal cell membrane. MCDX, which removes cholesterol, also reduced both sCTO toxicity and Ca 2+ rise, but with a less clear-cut effect than NAA. Actually, the role of cholesterol in amyloid toxicity is quite controversial. A comparative study on the susceptibility of different cell lines to toxicity of the amyloid protein HypF-N showed inverse relation to membrane content in total cholesterol [START_REF] Cecchi | Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates[END_REF]. On the other hand, it has recently been demonstrated that membrane cholesterol levels in mature neuron are significantly higher than those detected in young ones and that decreasing membrane cholesterol in mature neurons reduces their susceptibility to Aβ-induced cell death [47].

The present findings show that our previous results on the mechanism of Ca Aβ for 20min, washed, fed with conditioned medium and kept in culture for 24h. After fixation, cells were immunostained for MAP2 (red) and synaptophysin (green). Nuclei were counterstained with Hoechst 33258 (blue). In control cultures, anti-MAP2 antibody evidences a rich dendritic tree (upper panel, bar=50 µm), while synapses appear as green dots along the dendrite (lower panel, bar=10 µm). After sCTO or Aβ treatments, the extension of the dendritic tree was evidently reduced (upper panel) and the number of synapses decreased (lower panel).

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evoked an extremely small rise in intracellular Ca 2+ , consistent with Ca 2+ leakage from intracellular stores, while sCTOs, administered after a few minutes, produced a much more rapid and intense increase, similar to that observed in control cells, suggesting that sCTO-induced Ca 2+ rise was mostly due to an extracellular Ca 2+ influx (Fig. 7A). To analyze if the NMDA receptor (which is the glutamate receptor mostly responsible for excitotoxic Ca 2+ rise in neurons) was involved in the influx, we pretreated hippocampal neurons with MK801, a specific NMDA inhibitor. MK801 did not affect sCTO-driven Ca 2+ influx (Fig. 7B), while it blocked NMDAdriven Ca 2+ increase (data not shown). To rule out the hypothesis that sCTOs could bind to the NMDA receptor at a site different from that blocked by MK801, we pre-treated hippocampal neurons with antibodies against the NR1subunit, to mask possible sites of interaction between sCTOs and the receptor. Even in the presence of anti-NR1, after a slight delay, the response to sCTOs reached the same intensity and stability as in absence of the antibody (Fig. 7C). On the contrary, pre-treatment with an antibody-anti GM1, aimed at blocking the pore formation, did abolish sCTOs-induced Ca 2+ increase. After 10 min, an administration of 100 µM NMDA induced a classical abrupt and intense Ca 2+ rise, supporting the fact that the NMDA receptor activity had not been modified by the presence of anti-GM1 antibody (Fig. 7D). Pre-treatment with anti-BSA IgGs, an unrelated antibody, did not affect sCTO response (Fig. 7E).

Fig. 1 .

 1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. sCTOs and Aβ oligomers similarly damage the neuritic tree and synapses in mature hippocampal neurons. Mature hippocampal neurons were treated with 80 µM sCTOs or 20 µM

Fig. 3 .Fig. 5 .Fig. 6

 356 Fig. 3. sCTO induces increase in apoptosis and intracellular Ca 2+ levels in mature hippocampal

Fig. 7

 7 Fig. 7 Stimulation of NMDA receptors does not play an important role in sCTO-driven Ca 2+ influx. Changes in intracellular Ca 2+ were evaluated by optical fluorimetric recordings with Fura-

Fig. 8 .Fig. 8

 88 Fig. 8. Lipid raft depletion in the neuronal plasma membrane affects sCTO-induced neurotoxicity and Ca 2+ perturbation. . A: Mature hippocampal neurons were given sCTOs as

ACCEPTED MANUSCRIPTcomparison with 20 µg of standard compound using the imaging densitometer GS -700 (Bio-Rad).Statistical AnalysisAll data were represented as mean + SEM and analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison (Fig.4,5) or by unpaired t-test analysis (Fig.3) using with the Prism program (GraphPad). p<0.05 was considered significant.

ACCEPTED MANUSCRIPTterms of Ca 2+ influx. The majority of treated cells rapidly reverted to the initial Ca 2+ level (Fig.8B).

Acknowledgements

The authors wish to thank Maria Rosaria Domenici for helpful comments. This work was partially supported by a FIRB grant (RBNE03FMCJ_002) from the Italian Ministry for University and Research to M.S. K.F. is recipient of a postdoctoral fellowship from FIRB.

A C C E P T E D

M A N U S C R I P T


U Schmidt

J Neumann

G Pratschke

Schumacher

MD Robert M Eisele
email: robert_m.eisele@charite.de

J Zhukowa

S Chopra

S C Schmidt

U Neumann

J Pratschke

G Schumacher

Results of liver resection in combination with radiofrequency ablation for hepatic malignancies

Results of liver resection in combination with radiofrequency ablation for hepatic malignancies

Introduction

Radiofrequency ablation (RFA) of liver tumors has been established for the treatment of irresectable liver tumors with limited size [START_REF] Curley | Radiofrequency ablation of malignant liver tumors[END_REF][START_REF] Sutherland | Radiofrequency ablation of liver tumors: a systematic review[END_REF][START_REF] Lam | Impact of radiofrequency ablation on the management of patients with hepatocellular carcinoma in a specialized centre[END_REF]. The approach of RFA may be percutaneous, laparoscopic, or via laparotomy. The decision for a specific approach is based among others on tumor site, number of nodules and general health condition. The percutaneous and laparoscopic approaches are minimally invasive and are used successfully under certain conditions. [START_REF] Kettenbach | Percutaneous radiofrequency ablation of liver cell carcinoma: a current overview[END_REF][START_REF] Yokoyama | Percutaneous and laparoscopic approaches of radiofrequency ablation treatment for liver cancer[END_REF][START_REF] Lencioni | Percutaneous radiofrequency ablation of hepatic colorectal metastases: technique, indications, results, and new promises[END_REF][START_REF] Lencioni | Percutaneous ablation of hepatocellular carcinoma: state-ofthe-art[END_REF]. RFA via laparotomy offers many advantages compared to the percutaneous and laparoscopic approach. The placement of the RFA electrode is more accurate and safer than using percutaneous or laparoscopic approaches, which may be the reason for improved tumor control in case of open surgical approach [START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF]. Intraoperative staging is made possible by the use of intraoperative ultrasound. Furthermore, RFA may extend the feasibility of surgery in case of multiple tumors [START_REF] Hanna | Radiofrequency ablation of primary and metastatic hepatic malignancies[END_REF][START_REF] Poon | Recent advances in techniques of liver resection[END_REF]. With a multimodal approach including hepatic resection, RFA and chemotherapy, a median survival of 36 months could be achieved in technically unresectable patients with colorectal liver metastases [START_REF] Elias | Hepatectomy plus intraoperative radiofrequency ablation and chemotherapy to treat technically unresectable multiple colorectal liver metastases[END_REF].

These results double the survival rate compared to other treatment modalities in this group of patients. In patients with bilobar metastases or advanced cirrhosis, major liver resections are impossible because of the postoperative liver failure. We show here an extension of resectability of otherwise irresectable tumors using the combination of liver resection and RFA.

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Patients and Methods

Indication for liver resection, RFA and the combination of both, was made individually. Criteria for the decision were extent of tumor, number of tumor nodules, presence and degree of cirrhosis, and expected loss of functional liver parenchyma.

RFA was performed instead of resection to prevent loss of functional liver parenchyma, which could lead to fatal liver failure. Whether to treat an individual tumor nodule by resection or RFA was usually not determined until the procedure; the decision was only made intraoperatively. In cirrhotic livers, RFA of technically resectable HCC nodules was occasionally preferred rather than loss of large parenchymal masses due to anatomic resection, e.g. in case of advanced cirrhotic impairment of functional capabilities, whereas in metastatic disease, RFA was performed in case of bilobar disease after resection of one liver lobe and residual tumor deep in the parenchyma of the remaining; superficial lesions were rather resected.

Technical features

Pretherapeutic diagnostic work-up included cross-sectional imaging as well as blood values reflecting liver function. All ablations were performed under intraoperative ultrasound guidance. Cool-tip single needles and a 200 W generator (Radionics, Burlington, MA) were used in 12 procedures, and the RITA medical device Starburst XL 15 cm and the model 1500 generator (RITA medical Ltd., Mountain View, CA) in [START_REF] Poon | Recent advances in techniques of liver resection[END_REF]. In general, it was intended to treat one tumor with a single ablation. Overlapping ablation areas were considered in tumor sizes exceeding 4.0 cm (n=2). Immediate postoperative cross-sectional imaging was performed only in selected cases (n=3), if intraoperative ultrasound was not conclusive in confirming complete ablation of the tumor. Indication for a surgical treatment was confirmed by a multi-disciplinary tumor board. Informed consent was obtained prior to RFA in all procedures.

Follow-up

Follow-up was performed every three months during the first year, every six months during the second year and thereafter annually. As is usual in oncological practice, the screening method of choice was transabdominal ultrasound supplemented in case of suspicious ultrasound findings by computed tomograms or magnetic resonance imaging, usually enhanced by contrast media, besides basic clinical

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examinations and assessment of laboratory parameters (e.g. tumor markers). If suspicious findings remained, the patients received further treatment; histopathological confirmation of tumor recurrence was not compulsory.

Presentation of the data

Continuous data were expressed as median with ranges or mean +/-standard deviation and compared using the Mann-Whitney U test after exclusion of a Gaussian distribution by Kolmogorov-Smirnov testing. Categorical data were compared using the Chi square test or Fisher's exact test where appropriate. The statistical analyses were performed using commercially available SPSS 14.0 statistical software (SPSS Company, Chicago, Illinois, U.S.A.). Differences were considered to be statistically significant if the p value was less than 0.05. Survival data were counted regardless of the censored cases; cumulative analysis of overall and disease-free survivals for different groups were however calculated by the Kaplan-Meier method, and statistical comparisons were based on the log-rank test.

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Results

Indications

From 2001 to 2005, 22 liver resections, combined with RFA, were performed. None of the procedures were combined with extrahepatic surgery. Age of the patients was 61 +/-12 years. 15 patients were male, seven female. The patients suffered from hepatocellular carcinoma (HCC) (n=10), cholangiocellular carcinoma (CCC) (n=2), or metastases (n=10), which derived from colorectal cancer (n=8), malignant melanoma

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Survival and outcome

Outcome and survival is shown on a patient-by-patient basis in table 1. Tumor recurrence was observed in 20 patients. Truly local recurrence after RFA was seen in seven patients (32% on a per-patient-, 27% on a per-tumor-basis). Tumor recurrence with new tumor nodules in other parts of the liver was present in 12 patients. After a follow up of 31 months, eight patients are still alive, two without tumor burden and six with tumor recurrence. Four of the latter patients received further treatment comprising of a liver transplantation in one case and repeated resection in the other three patients. Seven patients were treated with chemotherapy due to systemic tumor progression. An explicit analysis of the success rates was not performed for the heterogeneity of the patient population and the different chemotherapeutic regimens applied. Kaplan-Meier estimations of survival and disease free survival are displayed separately for metastases and primary liver tumors in figure 1. No statistically significant difference in survival was found in the log-rank comparison of the results of both entities (p=0.815 and p=0.841 for overall and disease free survival, respectively).

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Discussion

The presented series shows a successful extension of the feasibilty of surgical treatment of liver tumors using the combination of liver resection and RFA. These findings resemble some results reported in the literature [START_REF] Elias | Hepatectomy plus intraoperative radiofrequency ablation and chemotherapy to treat technically unresectable multiple colorectal liver metastases[END_REF][START_REF] Elias | Usefulness of intraoperative radiofrequency thermoablation of liver tumours associated or not with hepatectomy[END_REF][START_REF] Oshowo | Radiofrequency ablation extends the scope of surgery in colorectal liver metastases[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Evrard | Surgical treatment of liver metastases by radiofrequency ablation, resection, or in combination[END_REF][START_REF] Tepel | Intraoperative radiofrequency ablation (RFA) for irresectable liver malignancies[END_REF][START_REF] Abdalla | Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases[END_REF][START_REF] Mateo | Optimal cytoreduction after combined resection and radiofrequency ablation of hepatic metastases from recurrent malignant ovartian tumors[END_REF][START_REF] Raut | Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis[END_REF][START_REF] Fioole | Combining partial liver resection and local ablation of liver tumours: a preliminary Dutch experience[END_REF][START_REF] Hildebrand | Radiofrequency-ablation of unresectable primary and secondary liver tumors: results in 88 patients[END_REF][START_REF] Choi | Combined hepatectomy and radiofrequency ablation for multifocal hepatocellular carcinomas: long-term follow-up results and prognostic factors[END_REF][START_REF] Kornprat | Role of intraoperative thermoablation combined with resection in the treatment of hepatic metastasis from colorectal cancer[END_REF] on the feasibility of this treatment approach with limited complications and increased survival. Table 2 provides an overview of reports on such a combined treatment. The total population encompassed accounts for 631 patients. Approximately one third of the publications consist of case reports; only two reports on larger series (> 100 patients) are available. The patient population in ten of the 15 reports included patients with predominantly metastastic, mostly colorectal liver disease with an assumed normal liver function. In contrary, our patient groups included not only metastatic liver tumors with normal liver function, but also primary liver tumors (HCC and CCC). Seven out of a total of ten patients with HCC presented with liver cirrhosis. Two of these patients had an advanced liver disease (stage B according to the Child-Pugh-classification).

Only limited resections were performed in this group of patients. However, in two of the five patients with Child A cirrhosis, a left sided hemihepatectomy was performed in combination with RFA for a tumor in the remaining right liver lobe. Resection of tumors in cirrhotic livers is an established approach [START_REF] Yanaga | Current status of hepatic resection for hepatocellular carcinoma[END_REF]. RFA in cirrhotic livers has also been described by several authors [START_REF] Santambrogio | Radiofrequency of hepatocellular carcinoma in patients with liver cirrhosis: a critical appraisal of the laparoscopic approach[END_REF]. The combination of both resection and RFA for HCC in cirrhotic livers may have an increased risk of postoperative liver failure. However, there was no such postoperative deterioration of liver function in the presented series. In contrary, even the postoperative hospital stay seems to be shorter or at least comparable to non-cirrhotic patients in this subgroup of patients. A present report on a single center experience with simultaneous resection and RFA for HCC by Kim et al. [START_REF] Kim | Intraoperative radiofrequency ablation for hepatocellular carcinoma: longterm results in a large series[END_REF] confirms data provided by Choi et al. [START_REF] Choi | Combined hepatectomy and radiofrequency ablation for multifocal hepatocellular carcinomas: long-term follow-up results and prognostic factors[END_REF], who found a 3 and 5 year-survival of 73% and 47%, respectively, compared to a similar rate of complications and a slightly better survival (80% for 3 years and 55% for 5 years) [START_REF] Kim | Intraoperative radiofrequency ablation for hepatocellular carcinoma: longterm results in a large series[END_REF]. In contrast, Raut et al. [START_REF] Raut | Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis[END_REF] report on inferior survival rates (45% for 3 and 5 years) and more complications compared to the Korean experience [START_REF] Choi | Combined hepatectomy and radiofrequency ablation for multifocal hepatocellular carcinomas: long-term follow-up results and prognostic factors[END_REF]. Our own data approach those figures (survival 50% after 3 years for HCC patients only, see fig. 1). However, liver transplantation (LTX) for HCC reveals a better long-term survival of up to 71% after 5 years [START_REF] Jonas | Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis[END_REF], if appropriate selection criteria are adopted.

CCC is generally not a good indication for LTX, however, there is a highly selective group in which LTX may be superior to other therapies [START_REF] Pascher | Intrahepatic cholangiocarcinoma: indication for transplantation[END_REF]. In conclusion, after

Complications

Similar to reports in the literature [START_REF] Elias | Hepatectomy plus intraoperative radiofrequency ablation and chemotherapy to treat technically unresectable multiple colorectal liver metastases[END_REF][START_REF] Elias | Usefulness of intraoperative radiofrequency thermoablation of liver tumours associated or not with hepatectomy[END_REF][START_REF] Oshowo | Radiofrequency ablation extends the scope of surgery in colorectal liver metastases[END_REF][START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF][START_REF] Evrard | Surgical treatment of liver metastases by radiofrequency ablation, resection, or in combination[END_REF][START_REF] Tepel | Intraoperative radiofrequency ablation (RFA) for irresectable liver malignancies[END_REF][START_REF] Abdalla | Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases[END_REF][START_REF] Raut | Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis[END_REF][START_REF] Fioole | Combining partial liver resection and local ablation of liver tumours: a preliminary Dutch experience[END_REF][START_REF] Choi | Combined hepatectomy and radiofrequency ablation for multifocal hepatocellular carcinomas: long-term follow-up results and prognostic factors[END_REF][START_REF] Kim | Intraoperative radiofrequency ablation for hepatocellular carcinoma: longterm results in a large series[END_REF], a considerable rate of complications ranging from 10 to 33% was observed in our experience. However, no postoperative liver failure was observed. Notably, neither heat injury of adjacent organs nor intrahepatic abscess formation, or biliary duct damage, occurred in the presented series, which indicates, that intraoperative RFA can be performed safely and without adding intolerable risk to the patient. Frequent complications were biliary leakages inherent in hepatic resection. Most of them could be managed by endoscopic interventions, and were eventually treated successfully. No death occurred.

Liver metastases

Liver resection for colorectal metastases seems to provide a five-year survival of 25 to 40%. Neoadjuvant chemotherapy may even improve resectability. Thirteen to sixteen percent of unresectable patients could be rendered resectable. The most important factor is to achieve a tumor-free status after resection. Liver resection of metastases from other primary tumors such as breast, stomach, pancreas, melanoma, or others has to be decided individually, if extrahepatic tumor is disclosed. The effectiveness of RFA versus liver resection for colorectal liver metastases has been previously shown already [START_REF] Abdalla | Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases[END_REF]. Compared to RFA, liver resection appears to lead to prolonged overall survival. Thus, open surgery and subsequent liver resection should be performed whenever possible, which can then be combined with RFA during the same procedure if necessary. In such a setting RFA can be performed safely and efficiently without loss of large parts of functional liver parenchyma. Moreover, RFA during laparotomy causes significantly better longterm tumor control than laparoscopic or percutaneous RFA, because of a more precise placement of the RFA electrode [START_REF] Eisele | Open surgical is superior to percutaneous access for radiofrequency ablation of hepatic metastases[END_REF]. The result is an expected better local tumor control [START_REF] Eisele | Open surgical is superior to percutaneous access for radiofrequency ablation of hepatic metastases[END_REF]. The comparably high local recurrence rate of 27% on a per-tumor basis in the presented series is partly explained by the presence of large nodules (≥ 3 cm, n = 7 [approximately one third of all RF-ablated nodules]) treated with RFA.

Tumor size exceeding 3 cm is known to be a significant risk factor for increased local

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recurrence rates [START_REF] Kuvshinoff | Radiofrequency ablation of liver tumors: influence of technique and tumor size[END_REF]. Furthermore, propensity score methods applied by Gleisner et al. demonstrated, that a poor survival following combined resection and RFA may be the result of different tumor biologic profiles rather than inappropriate treatment strategies including RFA [START_REF] Gleisner | Colorectal liver metastasesrecurrence and survival following hepatic resection, radiofrequency ablation, and combined resection-radiofrequency ablation[END_REF]. With a multimodal approach including (major and minor) hepatic resection, RFA and chemotherapy, a median survival of 36 months could be achieved in technically irresectable patients with colorectal liver metastases [START_REF] Elias | Hepatectomy plus intraoperative radiofrequency ablation and chemotherapy to treat technically unresectable multiple colorectal liver metastases[END_REF]. This figure doubles the survival of any other treatment modality in this group of patients. Our results confirm these data regarding the low rate of complications; median overall survival was slightly inferior with 26 months in a mixed population with various indications. However, in selected cases, intraoperatively found irresectability can be converted into an operable condition using RFA additionally to hepatic resections.

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  (n=1) or neuroendocrine disorders (n=1). All patients presented with bifocal (n=6) or multilocular tumor disease (n=16). While patients with metastases and CCC had no liver cirrhosis, seven out of ten patients with HCC suffered from Child A (n=5) or Child B (n=2) cirrhosis. Three patients with HCC had no cirrhosis. Four patients had received chemotherapy prior to the combined surgical procedure. Procedures The largest resection was a hemihepatectomy in eight patients. No extended resection was performed in combination with RFA. Other resections included left lateral bisegmentectomies (n=6) or atypical resections (n=18, sum beyond 22 due to procedures including various types of resections [n=5]). RFA was performed to ablate one (n=18) or two (n=4) nodules. Tumor size of ablated nodules ranged from 1.0 to 4.2 cm in diameter. Seven RF-ablated tumors had a diameter of 3 cm or were larger. Complications According to the classification of Clavien, the majority of the complications were classified as grade I. Four patients underwent endoscopic treatment of biliary leakage as a grade III complication with uneventful further clinical course. Two grade IV complications occurred: One patient needed temporary hemodialysis for acute renal failure due to formation of ascites, and the other spent 142 days in the hospital, among them 81 in the intensive care unit, for septic complications. No grade V complication, i.e. death of the patient, occurred. Patients were dismissed after an average hospital stay of 23 days. Patients with liver cirrhosis did not stay significantly longer than the average; in contrary, their hospital stay tended towards a shorter duration (15 days, p=0.581 [Mann-Whitney-U-test]).

  thorough patient selection and correct preoperative staging, an individual decision for this approach may lead to the indication for a combined treatment.

,5/6,7/8 2,7/8 5/6 12 5 no no no

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Conflict of interest statement

There are no conflicts of interest. None of the authors have any conflict of interest in the submitted manuscript EXTENDED INDICATION


* Mehra
email: g.mehra@doctors.org.uk

MRCOG Mr G Mehra

Laparoscopic assisted radical vaginal hysterectomy for cervical carcinoma : morbidity and long-term follow-up

Keywords: Cervical cancer, laparoscopic assisted radical vaginal hysterectomy, morbidity, Recurrence-free survival

Objectives: To study the feasibility, morbidity and outcome of cervical cancer patients treated with laparoscopic assisted radical vaginal hysterectomy (LARVH).

Methods:

The study group included 53 women with cervical cancer (stage Ib). They included women undergoing LARVH at the joint cancer-centres between 1994 -2002.

Data was collected on operating-time, nodal-yield, hospital-stay, complications recurrence rate and survival rate. The group was followed up until 2006.

RESULTS:

Of 53 women who were selected for LARVH, in 2 women LARVH was abandoned when nodes were positive at frozen section. The median age was 42 years while the operating-time was 210min with a nodal yield of 23 and a hospital-stay of 5 days. Final histology revealed 10 women with lympho-vascular invasion, 1 nodal metastases and invasion of parametrium / vagina in 2 women. 7 received adjuvant radiotherapy. 3 had chemo-radiation. Complications included voiding-difficulty (6), urinary tract infection (5), pyrexia (4), haemorrhage (2), pain (1), port-site haematoma (1) and nerve injury (1). Late complications included lymphoedema (4), urinary incontinence (4), voiding-problems (2), lymphocyst (1), venous-thrombosis (1) and rectocele (1). The median follow-up was 41 months. 4 women had recurrence, of which 3 women died. The five year survival was 89%.

CONCLUSIONS:

Vaginal radical hysterectomy with laparoscopic pelvic lymphadenectomy is feasible and safe with regards to mortality and has low morbidity.

INTRODUCTION

The surgical management of cervical cancer has undergone considerable changes over the past few years. The aim has been to reduce the morbidity associated with surgery without compromising the survival.

In the past, the vaginal route for radical hysterectomy did not become popular because of the need for an abdominal incision for pelvic lymphadenectomy. The vaginal approach has been revived as a result of the development of laparoscopic pelvic lymphadenectomy, an approach considered to be not only less invasive but also achieving excellent lymph node clearance [START_REF] Querleu | Laparoscopic pelvic lymphadenectomy in the staging of early carcinoma of the cervix[END_REF][START_REF] Childers | The role of laparoscopic lymphadenectomy in the management of cervical carcinoma[END_REF][START_REF] Possover | Laparoscopic para-aortic and pelvic lymphadenectomy : Experience with 150 patients and review of the literature[END_REF] . In 1987, Dargent was the first to describe the technique which combined Schauta's operation with laparoscopic removal of the lymphnodes [START_REF] Dargent | A new future for schauta operation through presurgical retroperitoneal pelviscopy[END_REF] .

In the recent years there has been a trend to evaluate the morbidity, recurrence rates and survival following laparosopic assisted radical vaginal hysterectomy after the initial studies on feasibility and different techniques [START_REF] Hertel | Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer[END_REF][START_REF] Querleu | Modified radical vaginal hysterectomy with or without nerve-sparing dissection : a comparative study[END_REF][START_REF] Sardi | Laparoscopically assisted schauta operation: learning experience at the gynaecologic oncology unit, Buenos Aires University Hospital[END_REF][START_REF] Yoo | Comparitive study of laparoscopico-vaginal radical hysterectomy and abdominal radical hysterectomy in patients with early cervical cancer[END_REF][START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF] . The overall morbidity in cervical cancer patients seems to be a cumulation of surgical and adjuvant interventions and reducing surgical morbidity by adopting the least invasive route for surgery is quite promising.

We undertook this study to report the long term morbidity, recurrence and survival outcome in our series of laparoscopic assisted radical vaginal hysterectomy (LARVH).

(ii) Inclusion Criteria:

All cases of FIGO stage ≤Ib1 (selected after thorough clinical, histolopathological and radiological assessment with MRI) and no radiological evidence of lymphnode involvement or metastatic disease were selected for LARVH.

(iii) Surgical Technique:

We followed the previously described technique of modified radical hysterectomy by Querleu D and Dargent D et al [START_REF] Querleu | Laparoscopic surgery in gynaecological oncology[END_REF] . Our extent of radical dissection corresponded to Type-II Piver-Rutledge classification of extended hysterectomy where the medial half of the uterosacral and cardinal ligaments were removed with uterine vessels being divided medial to the ureter. A laparoscopic pelvic lymphadenectomy was performed through a transperitoneal or a retroperitoneal approach using the technique previously described in the literature [START_REF] Querleu | Laparoscopic pelvic lymphadenectomy in the staging of early carcinoma of the cervix[END_REF][START_REF] Childers | The role of laparoscopic lymphadenectomy in the management of cervical carcinoma[END_REF][START_REF] Dargent | A new future for schauta operation through presurgical retroperitoneal pelviscopy[END_REF] .

(iv) Data collection & Follow-up:

Operating-time was calculated from anaesthetic charts (incision to completion of surgery). The nodal-yield was determined at histological assessment. The hospitalstay and complications were recorded.

All women had their catheter removed 48 hours postoperative. Those with residual volume of urine >100mls were re-catheterised. Where the final histology revealed invasion beyond cervix or in the presence of two or more poor prognostic factors {poorly differentiated tumour, close margins and lymphatic/vascular space invasion (LVSI)} or nodal metastases, women received adjuvant radiotherapy or chemoradiation. We maintained a prospective follow-up by interview and clinical examination. All women were questioned for any urinary or bowel symptoms. Those Of the 53 women 51 completed LARVH. Two women had positive nodes at frozen section and therefore radical hysterectomy was not carried out. They both had chemoradiation. Their median age, parity and histology details are shown in table 1.

(ii) Surgical Details:

Laparoscopic lymphadenectomy was performed one week prior to radical vaginal hysterectomy in 15 women. Paraaortic lymphnodes were sampled in one woman where there was suspicion of gross enlargement. The nodes were negative on histology.

The surgical and postoperative data are shown in table 1. The operating time was prolonged in 3 cases due to difficult vaginal access, intra-operative bleeding and extensive adhesions respectively.

(iii) Histopathology:

Of 51 women who had LARVH, none had positive nodes on histology. 2 women had occult vaginal and parametrial invasion on histology. 11 women had LVSI while one woman had tumour involvement of the surgical margins.

(iv) Complications:

The complications in our series are listed in table 2. One case of urinary incontinence presented 12 days after surgery but this resolved following pelvic floor exercises. One case of secondary haemorrhage from the vault following infection and another one with pelvic collection were managed with antibiotics. A large pelvic lymphocoele diagnosed two years after surgery needed laparoscopic drainage. There was no long-term gastrointestinal morbidity attributed to surgery alone. One woman receiving external beam radiotherapy had radiation-induced sub-acute bowel obstruction followed by an entero-colic fistula at 32 months after surgery.

(v) Survival and Outcome:

Table 3 gives details of follow-up, adjuvant therapy and survival outcome. 4 women in our series were lost to follow-up. 4 women had recurrence. Three of these recurrences occurred in the pelvis. Two of them had had adjuvant radiotherapy with their primary treatment and now received chemotherapy. They later developed distant metastatic disease and died. The third woman who had pelvic recurrence received chemoradiation. There was one woman with distant recurrence who later died.

Another woman succumbed due to an unrelated cause.

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8

DISCUSSION

Radical vaginal hysterectomy was first described in 1901 by Schauta [START_REF] Schauta | Die eiwerterte vaginale totalexstirpation des uterus beim kollumkazinom Wein[END_REF] . The apparent advantages of vaginal route include a short hospital stay, early recovery and less blood loss at surgery [START_REF] Yoo | Comparitive study of laparoscopico-vaginal radical hysterectomy and abdominal radical hysterectomy in patients with early cervical cancer[END_REF][START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF][START_REF] Malur | Laparoscopically assisted radical vaginal vs radical abdominal hysterectomy type II in patients with cervical cancer[END_REF][START_REF] Roy | Vaginal radical hysterectomy versus abdominal radical hysterectomy in the treatment of early-stage cervical cancer[END_REF][START_REF] Morgan | Is laparoscopically assisted radical vaginal hysterectomy for cervical carcinoma safe? A case control study with follow up[END_REF][START_REF] Sharma | Laparoscopically assisted radical vaginal hysterectomy (Coelio-Schauta): A comparison with open Wertheim/Meigs hysterectomy[END_REF][START_REF] Jackson | Laparoscopically assisted radical vaginal hysterectomy vs. radical abdominal hysterectomy for cervical cancer: a match controlled study[END_REF] . Whether it has any additional benefits for overall morbidity or survival is still unclear.

(i) Operative Parameters & Histology:

Our nodal yield and the operative parameters of LARVH are similar to other authors [8,9] . The latter third of our group had histology of pelvic nodes before vaginal surgery.

Two women deferred surgery and received chemoradiation instead. This practise avoids delay in starting adjuvant therapy.

Our initial staging by examination, MRI and laparoscopy at the time of lymphadenectomy failed to pick up two cases with invasion into the parametrium or vagina. These were identified at final histology. Such cases where there is microscopic spread beyond the cervix are occasionally difficult to identify during clinical or radiological staging. When they finally get detected on histology then they get completion treatment with chemoradiation.

We appreciate that our study group comprised of a significant proportion of women diagnosed with adenocarcinoma. This may either be a coincidence or perhaps a reflection of the trend already witnessed in the UK where the proportion of adenocarcinoma has risen in recent years. Cancer screening programme seems to have more false negative test results with glandular abnormalities and detect predominantly squamous type lesions.

(ii) Surgical Complications:

The majority of our complications occurred in the first half of our series. It is postulated that it takes 20 cases to get skilled to perform this procedure [START_REF] Sardi | Laparoscopically assisted schauta operation: learning experience at the gynaecologic oncology unit, Buenos Aires University Hospital[END_REF] .

Complication rates decrease with experience and are similar to abdominal route [START_REF] Yoo | Comparitive study of laparoscopico-vaginal radical hysterectomy and abdominal radical hysterectomy in patients with early cervical cancer[END_REF][START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF][START_REF] Malur | Laparoscopically assisted radical vaginal vs radical abdominal hysterectomy type II in patients with cervical cancer[END_REF][START_REF] Roy | Vaginal radical hysterectomy versus abdominal radical hysterectomy in the treatment of early-stage cervical cancer[END_REF][START_REF] Jackson | Laparoscopically assisted radical vaginal hysterectomy vs. radical abdominal hysterectomy for cervical cancer: a match controlled study[END_REF] . Wound infection, febrile morbidity and the incidence of paralytic ileus may be lower with LARVH [START_REF] Roy | Vaginal radical hysterectomy versus abdominal radical hysterectomy in the treatment of early-stage cervical cancer[END_REF] . We had no case of wound infection or paralytic ileus. Port site haematoma in one woman was managed conservatively.

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9 With experience majority of intraoperative complications can be dealt at laparoscopy.

In our series, in a woman who had haemorrhage from the ureterovesical angle, this was controlled by laparoscopic internal iliac ligation. The rate of conversion to laparotomy has been reported in the literature to be less than 1 in 20 cases and is most likely to happen during the initial learning phase [START_REF] Hertel | Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer[END_REF]17,18] . Table 4 lists the reported long-term complications with LARVH.

(iii) Urinary tract morbidity:

We did not encounter any bladder or ureteric injury in our series. LARVH may have a lower risk of bladder dysfunction when compared to abdominal route [START_REF] Sharma | Laparoscopically assisted radical vaginal hysterectomy (Coelio-Schauta): A comparison with open Wertheim/Meigs hysterectomy[END_REF][START_REF] Jackson | Laparoscopically assisted radical vaginal hysterectomy vs. radical abdominal hysterectomy for cervical cancer: a match controlled study[END_REF] . We encountered voiding problems in 6 women. Steed et al [START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF] reported the median time to normal residual volume (<100ml) as 10 days (2-71 range), but he assessed bladder function in majority of their patients at 10 days after surgery. Others have quoted the mean time for restoration of bladder function to be around 11 days (1-50 range) [START_REF] Hertel | Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer[END_REF] . In our series 6 women required re-catheterisation. 4 were discharged with catheters left in-situ for 7 days and no one required long-term catheterisation beyond a week.

Hence, majority women were able to void normally following removal of catheter 48 hours after surgery. 3 women had underlying urinary tract infection. We recommend routine screening for urinary infection preoperatively and that one could remove urinary catheter with bladder assessment as early as 48 hours after LARVH.

The rate of urinary incontinence, especially detrusor over-activity, seems to be significantly higher following radical hysterectomy [19] . Significant incontinence occurs in approximately 1:4 women following abdominal radical hysterectomy [20,21] .

Nerve-sparing during LARVH does not seem to decrease the urinary tract morbidity and approximately 1:6 women reported symptoms of stress incontinence on interview after a mean follow-up period of 38 months following LARVH [START_REF] Querleu | Modified radical vaginal hysterectomy with or without nerve-sparing dissection : a comparative study[END_REF] . In our series, 5 women reported urinary incontinence during follow-up.

(iv) Survival and Outcome:

The survival after LARVH seems to be similar to that after traditional abdominal surgery. The meta-analysis by Magrina [22] concluded an overall 5 year survival rate of 85.7% (80-90%) for stage-Ib cervical cancers who underwent radical abdominal hysterectomy. In our series the 5 year survival rate was 89%. Similar 5 year survival

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10 rate was reported by Sardi et al [START_REF] Sardi | Laparoscopically assisted schauta operation: learning experience at the gynaecologic oncology unit, Buenos Aires University Hospital[END_REF] . The projected 5 year survival rate by Hertel et al [START_REF] Hertel | Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer[END_REF] was 83% following LARVH. In a comparative study between LARVH and abdominal modified radical hysterectomy by Steed et al [START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF] , the 2 year recurrence free survival was 94% and there was no difference between the two groups. The 2 year survival in our group was similar (96.7%).

(v) Conclusion:

We conclude that in carefully selected women LARVH seems to be a safe procedure associated with a reasonably low short and long-term morbidity. al [START_REF] Steed | A comparison of laparoscopic-assisted radical vaginal hysterectomy and radical abdominal hysterectomy in the treatment of cervical cancer[END_REF] Nam et al [START_REF] Yoo | Comparitive study of laparoscopico-vaginal radical hysterectomy and abdominal radical hysterectomy in patients with early cervical cancer[END_REF] Hertel et al [START_REF] Hertel | Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer[END_REF] Park et al [18] Sardi et al [START_REF] Sardi | Laparoscopically assisted schauta operation: learning experience at the gynaecologic oncology unit, Buenos Aires University Hospital[END_REF] Renaud et al [18] Our

  Our study group included 53 women with cervical cancer (stageIb) undergoing LARVH over a period of eight years (July 1994 to July 2002) in the cancer network comprising of St Bartholomew's NHS Hospital (London) and Barking, Havering & Redbridge NHS Hospitals (Essex) in the United Kingdom.

  had urodynamic assessment. The recurrence-free survival data was calculated from the date of surgery and Kaplan and Meier method was used to calculate the overall survival in the LARVH group.

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  women experienced problems with the urinary tract. 2 had voiding difficulty. The first presented at one month but her symptoms resolved spontaneously. The other presented at 12 months and required urethral dilatation. 5 women developed urinary incontinence. The median time interval from surgery to presentation of symptoms of incontinence was 12 months (1-58 range). Three had detrusor over-activity, one had urodynamic stress incontinence and another had combined detrusor over-activity and M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 7 urodynamic stress incontinence. The symptoms resolved in all women with pelvic floor exercise and bladder drill.

  . Park PC, Lim K.T, Chung H.W, Lee K.H, Seong S.J, Shim J.U et al. Clinical evaluation of laparoscopic-assisted radical vaginal hysterectomy with pelvic and/or paraaortic lymphadenectomy. J. Am. Assoc. Gynecol. Laparosc 9 1 (2002), 49-53. [18]. Renaud M.C, Plante M and Roy M, Combined laparoscopic and vaginal radical surgery in cervical cancer. Gynecol Oncol 79 (2000), 59-63. [19]. Butler-Manuel S.A, Summerville K, Ford A, Blake P, Riley A.J, Sultan H, Monga A.K et al. Self assessment of morbidity following radical hysterectomy for cervical cancer. Journal of Obstetrics and Gynaecology 19 (2) (1999), 180-183. [20]. Ralph G, Tamussino K, Litchenegger W. Urological complications after radical abdominal hysterectomy with or without radiotherapy for cervical cancer. Arch Gynecol Obstet 248 (1990), 61-65. [21]. Naik R, Nwabinelli J, Mayne C, Nordin A, de Baros Lopes A, Monaghan J.M, et al. Prevalence and management of (non-fistulous) urinary incontinence in women following radical hysterectomy for early stage cervical cancer. Eur J Gynaecol Oncol 22 (2001), 22-30. [22]. Magrina J.F. Primary surgery for stageIb-IIa cervical cancer, including shortterm and long-term morbidity and treatment in pregnancy. J Natl Cancer Inst Monogr 21 (1996), 53-59. M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

Table 1 .

 1 General characteristics and operative data

	Number of Women :		51
	Mean Age (yrs)	:		39 (25-69)
	Mean Parity	:		02 (0-7)
	FIGO Stage	:	Stage Ib1	51
	Histological Types :	Squamous Cell	30
			Adenocarcinoma	21
	Median Operating Time (min)	210 (100-340 range)
	Median Nodal Yield (nodes)		23 (6-62 range)
	Median Fall in Haemoglobin (g/dL)	2.4 (0.2-7.0 range)
	Rate of Blood Transfusion (%)	7.5
	Median time to urine residual		2 (2-7 range)
	<100ml (days)
	Median Hospital Stay (days)		05 (1-18 range)

Table 3 .

 3 Follow-up data and adjuvant treatment

	Total number followed	51
	Median duration of follow-up (months)	41 (10-104)
	Women receiving adjuvant radiotherapy	07
	Women receiving adjuvant chemo-radiation	03
	Median disease-free interval (months)	30.5	-
	Mean survival time (months)	96 (88-103 ; 95% CI)
	Overall mortality	5.9%
	5 year survival rate	89%	-

Table 4 .

 4 Late Complications of LARVH reported in the literature Steed et

Conflict of interest

The authors declare that they have no conflict of interest.

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