Improving the batch-to-batch reproducibility in microbial cultures during recombinant protein production by guiding the process along a predefined total biomass profile In industry Escherichia coli is the preferred host system for the heterologous biosynthesis of therapeutic proteins that do not need posttranslational modifications . In this report , the development of a robust high-cell-density fed-batch procedure for the efficient production of a therapeutic hormone is described . The strategy is to guide the process along a predefined profile of the total biomass that was derived from a given specific growth rate profile . This profile might have been built upon experience or derived from numerical process optimization . A surprisingly simple adaptive procedure correcting for deviations from the desired path was developed . In this way the batch-to-batch reproducibility can be drastically improved as compared to the process control strategies typically applied in industry . This applies not only to the biomass but , as the results clearly show , to the product titer also . Introduction Biologics are known to be rather complex products . Apparently small changes in the manufacturing processes can cause significant differences in their clinical properties . Hence , production processes for biologics are approved by authorities only with clearly defined constraints on their manufacturing procedures . Consequently , reproducibility is of utmost importance . Additionally , reproducibility is very important as it affects the downstream processing and thus quality of the final product . From the engineering point of view there are two challenges in guaranteeing batch-to-batch reproducibility . First of all , within the given constraints , the operational procedure , most robust with respect to typically appearing process fluctuations , must be found . And , secondly , while running the process along this robust path , the remaining randomly appearing disturbances must be eliminated by means of feedback control . With respect to batch-to-batch reproducibility , production processes for recombinant proteins are lagging far behind most other industrial processes . Figure 1 shows a typical example of the repeatability of biomass concentration profiles in a recombinant protein production process . The variability is quite high . Fig. 1Biomass -LRB- open circles -RRB- and target protein -LRB- open triangles -RRB- concentration profiles from 13 fed-batch fermentations for the production of a recombinant protein . Typically the batch-to-batch reproducibility of these production processes is rather low This fact was recognized by the FDA . The agency responded with a couple of measures . One essential reaction is FDA 's PAT initiative -LSB- 2 -RSB- . With PAT , improvements in pharmaceutical and biologics production processes with respect to real time automated process monitoring and control are demanded . A rigorous science-based approach to manufacturing is demanded , as better understanding is thought to lead to more efficient process control , lower process variability , thus high product quality and finally patient safety . For biologics it is particularly important to keep the processes under control early in the product synthesis process -LRB- e.g. , -LSB- 1 -RSB- -RRB- . In bioprocess engineering , process monitoring and control is being developed since many years . A review was given by Lee et al. -LSB- 8 -RSB- . The objective was to keep the process on trajectories that finally lead to a high value of the desired mass mP of the product . Within a more or less well-defined production time tP , this can be related to biomass by the following expression : where π is the specific product formation rate and x the total biomass . Both arguments of the integral are functions of time , but more importantly , both are primarily dependent on the specific biomass growth rate μ in most industrial production systems . The growth rate that a specific medium supports determines the physiological state of the cells , and particularly the cell 's protein-synthesizing machinery that is important to recombinant protein production is also under growth rate control -LSB- 12 -RSB- . Hence , the variable that rules the final outcome of the process is the specific biomass growth rate μ . Thus , design of effective cultivation processes should be based on an optimal or at least a quasi-optimal profile of the specific growth rate . This can be obtained by means of numerical optimization procedures -LRB- e.g. , -LSB- 10 -RSB- -RRB- or simply by deriving a profile from data records and experience with the production system under consideration . In order to make sure that the process follows this profile it is straightforward to control μ in the engineering sense -LRB- e.g. , -LSB- 5 -- 7 , 11 , 13 , 14 , 16 -- 18 -RSB- -RRB- . Direct control of μ works perfectly as long as there are no severe disturbances in the process . When , however , some disturbances lead to a significant deviation of the biomass from its desired path , one must correct it before one can proceed with the desired optimal or quasi-optimal μ profile . Otherwise reproducible process trajectories can not be obtained . The decisive innovation in this paper is to show that the batch-to-batch reproducibility of the production processes can be significantly enhanced when the process is controlled to a predefined profile of the biomass x . This does not mean to stay away from keeping the μ profiles found to be optimal from the physiological point of view . The x-setpoint profiles are simply derived from predefined μ profiles . Using start biomass and desired specific biomass growth rate profile it is easy to estimate total biomass profile during the cultivation : A given biomass profile xset -LRB- t -RRB- is then in close relationship with the specific biomass growth rate μ . Hence , controlling the process to an x profile should satisfy the corresponding specific growth rate profile μset -LRB- t -RRB- as well . In this case the cultivation process is more robust because the deviations in biomass concentration can be eliminated directly by controlling the integral variable x . As x can not be measured directly with sensors that work reliably at a production fermenter and provide biomass values representative for the culture , it should be measured indirectly . This is a further advantage as compared to the μ-control because we are able to estimate x much more reliably than the specific growth rate μ -LSB- 4 -RSB- . In production environments , where sufficiently many data records are available from the process under consideration , artificial neural networks -LRB- ANNs -RRB- yield very accurate estimates . Hence we use ANNs to estimate biomass x . The ANN was trained on 26 data sets measured during a process development project with the strain used in this work . Materials and methods Experiments were performed with Escherichia coli BL21 -LRB- DE3 -RRB- as the host cell . The recombinant target protein was coded on the plasmid pET 28a and expressed under the control of the T7 promoter after induction with isopropyl-thiogalactopyranosid -LRB- IPTG -RRB- . The strain was resistant to kanamycin . The product appears as inclusion body within the cytoplasm . The particular strain used did not produce measurable amounts of acetate under the cultivation conditions adjusted in the experiments reported . This was tested in the beginning by means of appropriate test kits . The main substrate was glucose . It was fed at a concentration of 600 g/kg . The other components are compiled in Table 1 . Table 1Composition of the mineral mediumMineral salt solutionTrace element solutionComponentConcentration -LRB- g/kg -RRB- ComponentConcentration -LRB- g/kg -RRB- K2HPO414.60Na2-EDTA20.10 NaH2PO4 · H2O3 .60 FeCl3 · 6H2O16 .70 -LRB- NH4 -RRB- 2SO42 .46 CaCl2 · 2H2O0 .74 Na2SO42 .00 CoCl2 · 6H2O0 .21 MgSO4 · 7H2O1 .20 ZnSO4 · 7H2O0 .18 -LRB- NH4 -RRB- 2-H-citrate1.00 CuSO4 · 5H2O0 .10 NH4Cl0 .50 MnSO4 · H2O0 .10 Kanamycin0 .10 Thiamin0 .10 Trace element solution2 mL/kg All experiments were performed within BBI Sartorius System 's BIOSTAT ® ED 15 L bioreactor with 8 L working volume -LRB- Fig. 2 -RRB- . The fermenter was equipped with standard 6-blade Rushton turbines that could be operated at up to 1,400 rpm . The aeration rate could be increased up to 24 L/min . Aeration rate and then stirrer speed were increased one after the other in order to keep the dissolved oxygen concentration at 25 % saturation . In order to suppress foam formation , increase oxygen solubility and reduce the risk of contamination , the fermenter head pressure was kept at 0.5 bar above the ambient pressure in the laboratory . Fig. 2Experimental setup of the cultivation equipment The fermentations were operated in the fed-batch mode from the early beginning with an initial volume of 5 L at pH 7 and a temperature of 35 °C . All fermentations were started in the night by automatic transfer of the inoculation biomass from a refrigerator into the reactor . Substrate feeding starts immediately after inoculation with a fixed exponential profile . According to Jenzsch et al. -LSB- 3 -RSB- , the specific growth rate setpoint was chosen as 0.5 h − 1 . Thus the glucose concentration appeared to be at a very low value just from the beginning on . Its value is in the order of the Ks value and thus below the values that can be measured during the fermentation . After the biomass concentration reached values of about 35 g/kg , the culture was induced with 1 mM IPTG . From the seventh fermentation hour on , the growth rate was kept under feedback control along an x profile that was derived from a predefined profile of the specific growth rate . The corresponding profile of the substrate feed rate was determined from the profiles of μ and x . This was taken as reference feeding profile Fref for all the fermentations described . Temperature was measured with a Pt-100 , pH with an Ingold-pH-probe , pO2 with an Ingold pO2-Clark-electrode , CO2 in the vent line with MAIHAK ® 's Unor 610 and O2 there with MAIHAK ® 's Oxor 610 . Further , the total ammonia consumption during pH control was recorded by means of a balance beneath the base reservoir . All these quantities were measured online . Additionally , enhanced foam levels could be detected with a foam sensor and , if the critical level was reached , a silicone antifoaming emulsion -LRB- ROTH ® -RRB- was added . Biomass concentrations were measured offline -LRB- via the optical density at 600 nm -RRB- with a Shimadzu ® photo-spectrometer -LRB- UV-2102PC -RRB- . In preceding experiments a correlation was established between these values and the biomass dry weights , which were determined with the standard drying and weighting technique . Glucose was determined enzymatically with a YSI 2700 Select Bioanalyzer . The product was measured with SDS-PAGE after separation of the inclusion bodies and their solubilization . Results Preliminary simulations and experiments showed that the total biomass x -LRB- t -RRB- is better suited as the controlled variable than the biomass concentration X -LRB- t -RRB- itself . The control can then be performed with a simple adaptive control algorithm comprising the following steps : Estimate the total biomass xest at the current time instant t using the artificial neural network trained before on the available data records -LSB- 4 -RSB- . A simple feedforward artificial neural network with a single hidden layer was used which makes use of the online measured signals OUR , CPR , total base consumption resulting from pH control . With an additional bias node in the input layer the network has four input nodes . Five hidden layer nodes were used where the nonlinear response function of these nodes was chosen to be tangens hyperbolicus -LRB- tanh -RRB- functions . A single output , namely the biomass x , was taken , where the weighted sum of its input signals was directly used as the output signal.Compute the deviation of estimated biomass xest from its setpoint xset : The deviation Δx in x was used to correct the growth yield value Yxs in the feed function , which itself was limited to the interval given in order to avoid too big variations in cases where the measurements are disturbed in an unusual way : The concrete limits to F were chosen by experience in control practice.In order to determine the controller variable α by which the growth yield is adapted to the current state of the process we found that it is better to make use not only of the deviation Δx but also the smoothing action of an integral correction term to the deviation : Again , the correction was limited . Here the limitation was chosen by experience in such a way that the resulting yield values are kept within reasonable limits . The parameters k1 and k2 were initially determined in simulation studies . Later , during preliminary control experiments , they were slightly adapted . Their final values are k1 = 0.1 -LRB- kg -LRB- S -RRB- -RRB- − 1 and k2 = 0.02 -LRB- kg -LRB- S -RRB- -RRB- − 1 h − 1 . The controlled biomass profiles depicted in Fig. 3 show that this simple control approach leads to a very good reproducibility of the total biomass profiles . Fig. 3Total biomass signals from five fermentations performed sequentially using the same setpoint profile As one is usually not interested in the total biomass , the corresponding biomass concentrations are depicted in Fig. 4 . As can easily be seen from both plots , the batch-to-batch variability of the trajectories is rather small , i.e. , the process total biomass control works well . Fig. 4Measurements of the biomass concentration during five experiments in which the total biomass was controlled . The induction time was tind = 11 h in all cases In order to get a better impression of the controller action , the deviations from the desired profile , i.e. , the relative deviations between the total biomass and its setpoint , are plotted in Fig. 5 . In all the experiments , the controller was switched on at t = 7 h . In the first 4 h thereafter , the relative deviations remain within a 5 % interval . The controller action then improves so that finally the relative deviation of the total biomass remains within a 2 % interval around the mean . The feed rate profiles F -LRB- t -RRB- applied in the experiments are shown in Fig. 6 . Fig. 5Relative deviations of the total biomass from the mean . The controller was switched on 7 h after the cultivation was inoculatedFig . 6Substrate feed rate profiles for the six x-controlled fed-batch experiments There is one exception . In the last experiment -LRB- S330 -RRB- feed pump was switched off from 3 to 5 h in order to test the controller performance under process conditions with an extremely hard disturbance . The controller appeared to be robust enough to cope with this disturbance . Again in the end the relative deviation from the mean remained in the 2 % interval . The next question is what does this mean for the profiles of the specific biomass growth rate , which was initially chosen to determine the setpoint profiles for the total biomass . This comparison essentially should demonstrate data consistency . The specific growth rate μ was determined from a Luedeking -- Piret type relationship between the biomass concentration , carbon dioxide production or oxygen uptake rate and the specific growth rate : Both equations can be resolved for μ . The parameters were already known from a fit of the models to the fermentation data . Hence , with the biomass concentration profiles estimated and the measured OUR -LRB- t -RRB- and CPR -LRB- t -RRB- signals , μ can easily be computed . The result is depicted in Fig. 7 . In the decisive phase of the process , the product formation phase , the resulting trajectories of the specific biomass formation rate μ -LRB- t -RRB- resemble quite well the original setpoint profiles . It should be recalled that rather high noise is to be expected when computing μ -LRB- t -RRB- . However , the results depicted in Fig. 7 nevertheless clearly show that the data are consistent . Fig. 7Specific biomass growth rate profiles determined from the measurement profiles of OUR , CPR as well as from the estimates for biomass concentration X -LRB- t -RRB- . Note that the study 330 was the test case where the substrate supply was interrupted for some period during the biomass growth phase Finally the question arises what does a high batch-to-batch reproducibility in the biomass profiles mean for the variance in the product formation profiles . Results corresponding to the data shown before are depicted in Fig. 8 . Fig. 8Product concentration profiles for the cultivation processes already mentioned . All protein data stay within the error bar ranges representing the confidential interval of the protein analysis method The corresponding protein concentration profiles are rather close together saying that the improved reproducibility in biomass profiles by means of x control also leads to an improved batch-to-batch reproducibility in the product concentration profiles . All profiles stay within the confidence interval of the analytical protein detection method . The relative error of ± 15 % of target protein analysis by SDS-PAGE was estimated from a fivefold analysis using the same fermentation samples . Discussion Previous work on controlling the fermentation processes performed to manufacture recombinant proteins focused on keeping the cultures on tracks that guaranteed a high productivity or yield . The obviously first quantity influencing the amount of protein that is produced in a cultivation run is the biomass employed , hence high-cell-density cultures are required -LRB- e.g. , -LSB- 9 , 15 -RSB- -RRB- . As the cells ' protein-synthesizing machinery that is particularly important to recombinant protein production is under growth rate control -LSB- 12 -RSB- , the performance of the cells is ruled by the specific biomass growth rate μ . Hence , much work has been put into closed loop control of fermentation processes along appropriate profiles μset -LRB- t -RRB- of the specific biomass growth rate -LSB- 5 -- 7 , 11 , 13 , 16 -- 18 -RSB- . While advantageous from the cell physiological point of view , controlling the specific growth rate directly has the disadvantage of leading to a relatively low reproducibility of the fermentations -LSB- 6 -RSB- . This is a significant disadvantage from the process quality point of view . We wished to extend the work on fermentation control towards quality assurance of process and thus product formation . This first of all requires improving the batch-to-batch reproducibility of the processes . There are two motivations for this . First , the product quality in recombinant protein manufacturing processes can be affected by changes in the fermentation operational procedure , hence the authorities link process approval with tight constraints on the process trajectories . Thus , good reproducibility increases product quality . Secondly the downstream processing can work much more efficiently when the cultivation results are highly reproducible . Therefore , having the same culture each time should be beneficial to the overall product yield as well . The results of the new control procedure discussed below show that the batch-to-batch reproducibility is significantly improved . The procedure controls the biomass along a profile that is directly derived from the specific growth rate profile that was found to be desirable . The first decisive advantage of this approach is that the control procedure is robust to distortions usually appearing in practice . This is opposite to control along a predefined profile of the specific biomass growth rate . Practically all distortions lead to changes in the biomass and this is the controlled variable . A second advantage is that the actual biomass values can be estimated with higher accuracy than the specific growth rate from the online measurement signals available at most fermenters . As shown previously -LSB- 4 -RSB- x can be representatively estimated with a root mean square error of about 0.5 g/kg by means of simple artificial neural networks . The reason for the robustness of the x control is easy to understand . The x-setpoint profile for the control was determined from a μ profile . This μ profile leads to a feed rate profile F -LRB- t -RRB- and a biomass profile x -LRB- t -RRB- . The F -LRB- t -RRB- profile is taken as the feed forward component of the process and programmed in the programmable controller at the fermenter . If due to distortion the biomass becomes smaller as expected , the cells will see more substrate than expected and respond temporarily by increasing their growth rate . If , on the other hand , the distortion leads to a higher biomass concentration , the cells see less substrate than expected and reduce their growth rate , thus correcting for their deviation in the total biomass . The feedback component of the process has thus only to correct for larger deviations . Control of the specific biomass growth rate or qualitatively similar control technique that uses the substrate feed rate F as the manipulated variable is currently not generally used in production practice . Its adoption is now supported by recent initiatives of the FDA in its PAT initiative -LSB- 2 -RSB- , but will critically depend on the ease of the control algorithm . The algorithm used in this work is an extremely simple adaptive control approach which only corrects the biomass-on-substrate yield in the basic feed rate expression . The control algorithm proved to be stable in many fermentation runs with many different organisms and several different fermenter scales up to the large production scale . It is very important to note again that the new x control approach does not require refraining from taking the μ profile of the process as the basic process control variable . This basic concept can be maintained and it is easy to derive the x profile from a given μ profile . As clearly shown in the examples , not only does this x-based control keep biomass and specific growth rate in tight limit , but also the batch-to-batch reproducibility with respect to the desired product concentration is high .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O

pat
reproducibility
recombinant proteins


Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities . In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord . Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level . Gap junctions in the adult spinal cord are constituted principally by connexin36 -LRB- Cx36 -RRB- . However , the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown . Here , we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat . For this purpose , double immunostaining against Cx36 and choline acetyltransferase -LRB- ChAT -RRB- was performed on transverse sections -LRB- 20 μm -RRB- taken from spinal segments T6 -- T8 . Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive -LRB- - ir -RRB- neurons from lamina VII -LSB- intermediolateral cell column -LRB- IML -RRB- and intercalated cell group -LRB- IC -RRB- -RSB- , lamina X -LSB- central autonomic nucleus -LRB- CA -RRB- -RSB- and in ventral horn neurons from laminae VIII and IX . Cx36 puncta were distributed in the neuronal somata and along dendritic processes . The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord . 1 Introduction Gap junctions are specialized cell -- cell contacts that provide direct intercellular communication . They often occur as plaques that can contain thousands of individual gap junction channels . Each channel is formed by the association of 12 connexin -LRB- Cx -RRB- proteins . Eleven of the 20 members connexin family have been identified so far in the mammalian central nervous system -LRB- Nagy et al. , 2004 -RRB- , but only Cx36 is expressed almost exclusively by neural cells -LRB- Belluardo et al. , 2000 ; Condorelli et al. , 2000 ; Rash et al. , 2000 -RRB- . Previous studies have shown that gap junctions play an important role in the synchronization of neuronal activity in various areas of the neonatal and adult rodent brain , including the cortex -LRB- Deans et al. , 2001 -RRB- , hippocampus -LRB- Hormuzdi et al. , 2001 ; Buhl et al. , 2003 -RRB- , thalamic reticular nucleus -LRB- Landisman et al. , 2002 -RRB- , inferior olive -LRB- Long et al. , 2002 ; De Zeeuw et al. , 2003 ; Leznik and Llinas , 2005 -RRB- and olfactory bulb glomeruli -LRB- Christie et al. , 2005 -RRB- . Such junctions enable the intercellular flow of electric current -LRB- electrotonic transmission -RRB- which can result in the generation of coordinated activity within populations of neurons . In the rat spinal cord , electrotonic and dye coupling have been shown to occur between somatic motoneurons -LRB- Gogan et al. , 1977 ; Walton and Navarrete , 1991 ; Chang et al. , 1999 ; Mentis et al. , 2002 -RRB- and between sympathetic preganglionic motoneurons -LRB- Logan et al. , 1996 ; Nolan et al. , 1999 -RRB- . It has been suggested that synchronization of electrical activity through gap junctions plays an important role in the generation of somatomotor and sympathetic motor rhythms induced by NMDA and/or 5-HT -LRB- Marina et al. , 2006 ; Tresch and Kiehn , 2000 -RRB- . In the spinal cord of adult rats , gap junctions are composed mainly by Cx36 and are distributed in the white matter and the grey matter -LRB- Rash et al. , 2000 ; Lee et al. , 2005 -RRB- . However , the expression of Cx36 protein in specific neural populations of the spinal cord remains unknown . In this study we used immunofluorescence to identify Cx36-like immunoreactivity in ChAT-positive putative sympathetic preganglionic and somatic motoneurons within thoracic spinal segments . 2 Materials and methods 2.1 Tissue preparation Experiments were performed in accordance with the UK Animals -LRB- Scientific Procedures -RRB- Act 1986 and associated guidelines . Six male Sprague -- Dawley rats -LRB- 210 -- 250 g , bred in-house at UCL -RRB- were anaesthetized deeply with 20 % urethane -LRB- 1.6 g kg − 1 i.p. -RRB- and perfused through the ascending aorta with 100 ml of 0.9 % saline at room temperature , followed by 500 ml of 4 % paraformaldehyde in 0.1 M phosphate buffered saline -LRB- PBS , pH 7.4 -RRB- at 4 °C . The thoracic spinal cord and the brain stem were removed , postfixed for 1 h and cryoprotected in 10 % sucrose in 0.1 M PBS overnight at 4 °C . Transverse slices -LRB- 20 μm thick -RRB- from spinal segments T6 -- T8 and caudal medulla were collected with a freezing microtome and stored in cryo-protectant -LRB- 30 % ethylene glycol and 20 % glycerol in 0.05 M PBS -RRB- at − 20 °C . Caudal medulla slices were taken to assay specificity of the Cx36 antibody and to determine optimal antibody concentration using the inferior olive nucleus -LRB- ION -RRB- as a positive control . Cx36-immunostaining was analyzed in spinal segments T6 -- T8 because previous studies have shown evidence of electrotonic coupling among sympathetic preganglionic motoneurons and expression of Cx36 mRNA in somatic motoneurons from thoracic spinal cord slices -LRB- Logan et al. , 1996 ; Parenti et al. , 2000 -RRB- . 2.2 Immunohistochemistry All series of tissue slices from all animals -LRB- 20 -- 30 slices/animal -RRB- were processed together in the same assay to ensure uniformity of immunostaining . After slices were washed in 0.1 M PBS with 0.1 % Triton X -LRB- PBS-T -RRB- , non-specific binding was blocked with 10 % normal donkey serum -LRB- Sigma-Aldrich -RRB- in PBS-T -LRB- blocking medium -RRB- for 60 min . Tissue was then incubated in Cx36 antibody -LRB- 1:100 dilution in blocking medium -RRB- at 4 °C overnight . The antibody was obtained by immunizing rabbits with a synthetic peptide corresponding to amino acids LQNTETTSKETEPDC of the murine Cx36 protein . The peptide sequence was compared to protein sequence databases from the Basic Local Alignment Search Tool -LRB- BLAST 2.0 , NCBI , National Institutes of Health , USA -RRB- , and showed local alignments with murine Cx36 protein only . The specificity of the Cx36 antibody was shown in Western blots and in preabsorption control experiments -LRB- Bittman et al. , 2002 -RRB- . The slices were then washed again with PBS-T and incubated with fluorescent secondary antibody -LRB- Alexa Fluor 488 donkey anti-rabbit IgG , Molecular Probes , 1:500 dilution in blocking medium -RRB- for 60 min at room temperature , and washed again with PBS . Choline acetyltransferase -LRB- ChAT -RRB- immunodetection was employed to identify putative sympathetic preganglionic and somatic motoneurons -LRB- Marina et al. , 2002 -RRB- . Following staining for Cx36 , slices were incubated in goat anti-ChAT affinity purified polyclonal antibody -LRB- Chemicon , 1:100 dilution in 1 % normal donkey serum -RRB- overnight at 4 °C . Tissue was washed and incubated with Alexa Fluor 568 donkey anti-goat IgG antibody -LRB- Molecular Probes , 1:500 dilution in 1 % normal donkey serum -RRB- for 60 min at room temperature , washed again in PBS and cover-slipped with Vectashield hard set mounting medium with the fluorescent nuclear dye DAPI -LRB- Agar Scientific , Stansted , Essex -RRB- . 2.3 Image analysis The anatomical distribution of Cx36 immunostaining was determined by visual inspection through 63 × and 100 × objectives with an Olympus BH2-RFCA epifluorescence microscope equipped with light filters -LRB- exciter 380 -- 490 nm , emitter 519 nm for the green channel -RRB- . ChAT-Immunoreactive -LRB- ir -RRB- neurons were considered positive for Cx36 if punctate staining was observed within the cell body or along the dendritic processes . The cellular distribution of Cx36 in sympathetic preganglionic and somatic motoneurons was analyzed using a Leica TCS NT SP laser scanning confocal microscope with argon , krypton , and helium-neon lasers . Green and red channels were each scanned sequentially through a 63 × objective . Alexa Fluor 488 labelling was observed by exciting at 488 nm and collecting at 515 -- 550 nm . Alexa Fluor 568 labelling was visualized by using excitation at 568 nm and collecting at 590 -- 626 nm . Serial optical sections were taken at intervals of 0.5 μm starting at the upper cell surface , i.e. , the first optical section where cytoplasmic ChAT staining became visible , and all the way down through the cell to the lower cell surface , i.e. , where ChAT staining was no longer detected . Composite figures were created for each optical section by merging both channels using Image J 1.37 c -LRB- National Institutes of Health , USA -RRB- . Colocalization of Cx36 -LRB- green -RRB- and cytoplasmic ChAT immunofluorescence -LRB- red -RRB- appeared as yellow . Thus , green puncta on the cell surface , i.e. , surrounding ChAT-ir cytoplasm , suggested the presence of Cx36 on the plasma membrane ; yellow intracellular puncta in ChAT-positive regions were indicative of cytoplasmic Cx36 . Serial sections were projected as a single image to create a 2D reconstruction of 3D data set from the sections of the cell bodies and dendritic processes , using Leica Confocal Software -LRB- Leica Microsystems Heidelberg GmbH -RRB- . Both the individual and projected images were saved directly to a computer as TIFF files . Images were minimally adjusted for brightness and contrast with Adobe Photoshop ® 6 running on a Dell Pentium 4 PC . Images were then imported into Adobe illustrator ® CS2 where groups of images were assembled and labelled . 3 Results 3.1 Connexin 36 immunostaining in the inferior olivary nucleus Caudal medulla slices were immunostained for Cx36 to be used as a positive control . Cx36-immunostaining could only be detected using high magnification lenses -LRB- at least 63 × -RRB- and appeared as minuscule round immunofluorescent puncta distributed in restricted regions within the inferior olivary nucleus -LRB- Fig. 1A -RRB- . Cx36 expression in other medullary nuclei was extremely low , as illustrated in Fig. 1B , where Cx36-like-ir puncta are virtually undetectable in the trigeminal spinal nucleus . No staining was observed in negative controls where the primary antibody was omitted -LRB- data not shown -RRB- . 3.2 Connexin36 immunostaining in the spinal cord Under low magnification -LRB- 40 × or less -RRB- , Cx36 immunostaining is very hard to detect as immunofluorescent puncta are small and sparsely distributed in the ventral horn -LRB- Rash et al. , 2000 ; Lee et al. , 2005 -RRB- . Under high magnification -LRB- 63 × and higher -RRB- , several Cx36-like-ir puncta were revealed within the soma of spinal neurons -LRB- Fig. 2 -RRB- . 3.3 Choline acetyltransferase immunostaining in the spinal cord Sympathetic preganglionic and somatic motoneurons were identified by immunohistochemical staining of ChAT in transverse spinal cord slices from thoracic spinal segments T6 -- T8 . Intracellular ChAT immunostaining -LRB- red immunofluorescence -RRB- was detected extranuclearly in the cytoplasm and in the dendritic projections -LRB- Figs. 2 and 3 -RRB- . ChAT-ir neurons located in lamina VII -LRB- IML and IC -RRB- and lamina X -LRB- CA -RRB- were considered as sympathetic preganglionic motoneurons , according to the spatial arrangement described by Cabot -LRB- 1990 ; see Fig. 2A -RRB- . Lateral and medial ChAT-ir projections were easily identified in all sympathetic preganglionic motoneurons within the IML ; dorsal and ventral dendritic projections were not identified in transverse spinal slices stained for ChAT . On the other hand , ChAT-ir neurons and their respective dendritic projections located in laminae VIII and IX were considered as somatic motoneurons , according to the anatomical boundaries described by Paxinos and Watson -LRB- 1998 ; see Fig. 3 -RRB- . 3.4 Connexin 36 immunostaining in sympathetic preganglionic motoneurons The presence of Cx36-like-ir puncta in transverse spinal cord slices taken from T6 -- T8 spinal segments immunostained for ChAT was analyzed with wide field epifluorescence microscopy . The cellular distribution of Cx36-like-ir puncta in putative sympathetic preganglionic and somatic motoneurons was analyzed in individual optical sections scanned sequentially along the cell body -LRB- z series -RRB- under high magnification . Optical sections were stacked to show a three-dimensional reconstruction of the neurons . Figs. 2 and 3 show examples of somal and dendritic labelling in ChAT-ir neurons taken from a representative spinal slice from animal number 5 . Co-expression of Cx36 -LRB- green -RRB- and ChAT -LRB- red -RRB- immunofluorescence appears as yellow . Cx36 immunostaining in ChAT-ir neurons was similar in all the slices taken from all six animals used in this study . All ChAT-ir neurons located in the different sympathetic nuclei , i.e. , IML , IC and CA , showed Cx36-like-ir puncta -LRB- See Fig. 2A and A1 -RRB- . Both , somal and dendritic puncta were observed in putative sympathetic preganglionic motoneurons . A detailed analysis of each optical section revealed that membrane labelling was very scarce ; this was indicated by the small amount of green puncta surrounding ChAT-ir cytoplasm -LRB- Figs. 2A1 -RRB- . In contrast , moderate intracellular punctate staining was detected in ChAT-ir regions , i.e. , perinuclearly , where the Cx36 is generated in the Golgi-ER complex , suggesting a substantial cytoplasmic production of Cx36 protein . On the other hand , Cx36 staining was consistently detected in ChAT-ir dendrites projected from sympathetic preganglionic neurons within the IML , i.e. , lateral and medial projections . Dendritic labelling was characterized by fine puncta distributed along the primary dendrites and thin filaments . Robust Cx36-like staining was often observed in medially projecting bundled dendrites -LRB- Fig. 2B1 -RRB- . In some cases , fine puncta were also detected in areas where fine dendrites were seen in close proximity to a cell body -LRB- Fig. 2B1 -RRB- . As mentioned before , dorsal dendritic projections were not detected in transverse spinal slices stained for ChAT , and therefore , the presence of Cx36-like-ir puncta in dorsal dendrites could not be determined . 3.5 Connexin 36 immunostaining in somatic motoneurons Cx36-immunoreactive puncta were consistently observed in somatic motoneurons from laminae VIII and IX . Cx36 labelling in somatic motoneurons was characterized by moderate punctate staining distributed perinuclearly in the cytoplasm and to a lesser extent on the plasma membrane . Punctate staining was also distributed along the dendritic arbour and it was often observed in dendro-dendritic and somato-dendritic sites of close apposition -LRB- Fig. 3 -RRB- . 4 Discussion 4.1 Methodological considerations In the present study , connexin 36-immunoreactivity was identified in putative sympathetic preganglionic motoneurons and somatic motoneurons from the lower thoracic spinal cord of adult rats . The staining pattern obtained was similar to that described previously in the spinal cord and in the ION -LRB- Rash et al. , 2000 ; Placantonakis et al. , 2006 -RRB- . Even though the peptide used to immunize the rabbits showed local alignments with murine Cx36 protein only , it is important to mention that the specificity of the antibody has only been partially tested in preabsorption control experiments and in Western blots -LRB- Bittman et al. , 2002 -RRB- . Further experimental evidence is required to fully determine the specificity of this antibody using tissue from Cx36 knock-out animals . It is important to consider that connexin expression can only be estimated at the subcellular level using high magnification objectives . Cx 36-ir puncta are quite small , with an average diameter of 0.61 μ -LRB- Placantonakis et al. , 2006 -RRB- and Cx36 protein levels in the adult spinal cord are distinctly low . Therefore , visualization at low magnification can be extremely difficult ; this point can also be illustrated by the apparent difference in the expression of Cx36mRNA levels in the adult spinal cord described in previous studies . Showing a panoramic photograph of the spinal cord at low magnification -LRB- judged by the scale bars -RRB- , Lee et al. -LRB- 2005 -RRB- showed that Cx36 mRNA is strong in the neonatal rat spinal cord but in the adult spinal cord , expression is very low and barely above background . In contrast , Cx36 mRNA expression in the adult rat spinal cord can be clearly seen in higher magnification images by Chang et al. -LRB- 2000 -RRB- . In the present study , it was also difficult to identify Cx36-like-ir puncta in panoramic images of the spinal cord -LRB- see Fig. 1A -RRB- . However , a closer inspection revealed the presence of Cx36 within ChAT-ir neurons . 4.2 Cellular distribution of Cx36 immunostaining in ChAT-ir neurons This study shows that ChAT-ir neurons from the adult rat thoracic spinal cord express the neuron-specific gap junction protein Cx36 . Such labelling was detected both in the IML , IC and CA and in laminae VIII and IX and it was distributed in neuronal somata and along dendritic processes . Somal labelling was mainly characterized by moderate cytoplasmic punctate staining with scarce labelling on the plasma membrane and cell nucleus . Like all membrane proteins , connexins are synthesized by ribosomes and are transported in small vesicles that follow the cell 's secretory pathway from the endoplasmic reticulum to the plasma membrane -LRB- Yeager et al. , 1998 ; Martin and Evans , 2004 -RRB- . Removal of gap junctions occurs through the formation of annular gap junctions by phagocytosis and lysosomal degradation -LRB- Yeager et al. , 1998 -RRB- . Therefore , the cytoplasmic labelling observed in putative sympathetic preganglionic motoneurons and in somatic motoneurons may represent the trafficking pathways of connexins leading to the formation and/or degradation of gap junction channels . Our results therefore support the contention that Cx36 is actively synthesized by the adult spinal cord . This is the first documentation of connexin 36-like immunostaining in ChAT-ir -LRB- putative sympathetic preganglionic -RRB- neurons in sympathetic nuclei of the spinal cord . Furthermore , our results are consistent with several studies , which have demonstrated that Cx36 mRNA is widely expressed in somatic motoneurons during the embryonic and neonatal life , and continues to be expressed in the adulthood -LRB- Chang et al. , 1999 ; Chang et al. , 2000 ; Parenti et al. , 2000 -RRB- . Cx36 labelling was scarce on the somatic plasma membrane of ChAT-ir neurons and it was never found in sites of close apposition between neighbouring cell bodies . However , abundant Cx36 labelling was identified in and along dendritic processes . In fact , Cx36-like-ir puncta were commonly detected in tightly bundled dendrites and in areas where fine dendrites were in close proximity with other cell bodies . The presence of Cx36 in these areas might represent a potential site for gap junctional coupling between dendrites and between the somata and the dendrites of the neighbouring sympathetic preganglionic and somatic motoneurons . This is consistent with in vitro studies using slice preparations , which have identified putative sites of coupling between dendrites and/or cell bodies in dye-coupled sympathetic preganglionic and somatic motoneurons -LRB- Becker and Navarrete , 1990 ; Shen and Dun , 1990 ; Mentis et al. , 2002 -RRB- . This has also been confirmed at the ultrastructural level , where dendro-dendritic gap junctions have been identified in somatic motoneurons -LRB- van der Want et al. , 1998 -RRB- . In fact , gap junctions in somatic motoneurons are distributed predominantly in dendro-dendritic and dendro-somatic sites and to a much lesser extent , in somato-somatic points -LRB- Matsumoto et al. , 1988 -RRB- . Further ultrastructural studies are required to determine whether dendritic Cx36 protein is involved in the formation of homotypic gap junctional plaques among sympathetic preganglionic motoneurons or even heterotypic gap junctions between sympathetic preganglionic motoneurons and interneurons or astrocytes . For instance , previous studies have shown the presence of heterotypic gap junctions in the lumbar spinal cord , composed of Cx32 in superficial dorsal horn neurons on one side , and Cx43 in astrocytic processes on the other -LRB- Qin et al. , 2005 -RRB- . Interestingly , neuronal tracing studies have proposed the presence of dendro-dendritic connections -LRB- presumably through gap junctions -RRB- between lamina V neurons from the dorsal horn and sympathetic preganglionic motoneurons from the IML -LRB- Cabot et al. , 1994 -RRB- . It will be interesting to determine whether Cx36 in sympathetic preganglionic motoneurons is involved in the formation of heterotypic gap junctions with lamina V neurons . 4.3 Physiological role of gap junctional communication between sympathetic preganglionic motoneurons Paired cell recordings have shown evidence of synchronization of action potential firing and subthreshold membrane potential oscillations between electrotonically coupled sympathetic preganglionic motoneurons -LRB- Logan et al. , 1996 ; Nolan et al. , 1999 -RRB- . Since action potential discharge in one of the sympathetic preganglionic motoneurons was almost simultaneously registered as a spikelet in the second sympathetic preganglionic neuron , it was considered that gap junctions mediating electrotonic coupling are located near the site of action potential generation -LRB- Logan et al. , 1996 -RRB- . These data are consistent with the abundance of the gap junction protein Cx36 that we found between primary dendrites in sympathetic preganglionic motoneurons and supports the idea that electrical synapses might be located in dendro-dendritic sites of close apposition . Electrical synapses in sympathetic preganglionic motoneurons behave in a similar way to a low-pass filter , allowing electrotonic transmission of sub - and suprathreshold activity -LRB- Nolan et al. , 1999 -RRB- . Therefore , it has been recently proposed that by increasing the degree of synchrony of discharges in the population of sympathetic preganglionic motoneurons , gap junctional communication might play a significant role in the generation of relatively low frequency coherent sympathetic motor rhythms induced by 5-HT in the adult rat -LRB- Marina et al. , 2006 -RRB- . 4.4 Physiological role of gap junctional communication between somatic motoneurons Several studies have shown evidence of electrical and dye coupling between somatic motoneurons in the neonatal spinal cord -LRB- Gogan et al. , 1977 ; Becker and Navarrete , 1990 ; Walton and Navarrete , 1991 ; Chang et al. , 1999 ; Mentis et al. , 2002 -RRB- . Experiments using gap junction blockers and NMDA receptor antagonists have shown that coherent somato-motor rhythms result from the interaction between membrane properties at the single cell level and the transmission of this activity through gap junctions -LRB- see Kiehn and Tresch , 2002 -RRB- . It has been suggested that gap junction coupling is a very important factor that contributes to the synchronization of individual somatic motoneurons to produce a coordinated rhythmic motor output . The dye transfer properties between neonatal somatic motoneurons change as development progresses , moving from Lucifer yellow to neurobiotin coupling -LRB- Becker and Navarrete 1990 ; Mentis et al. , 2002 -RRB- . The number and intensity of labelling of dye-coupled somatic motoneurons are substantially reduced and becomes almost absent shortly after birth -LRB- Gogan et al. , 1977 ; Becker and Navarrete , 1990 ; Walton and Navarrete , 1991 ; Chang et al. , 1999 ; Mentis et al. , 2002 -RRB- . Electrotonic coupling between somatic motoneurons in adult animals has only been observed in pathological conditions such as nerve injury -LRB- Chang et al. , 1999 -RRB- . However , Cx36 mRNA expression in axotomized somatic motoneurons remains unaffected -LRB- Chang et al. , 1999 -RRB- . In a similar way , recent evidence has shown that spinal cord injury is associated with a strong up-regulation of Cx43 in astrocytes , but in contrast , neural Cx36 expression remains unaffected -LRB- Lee et al. , 2005 -RRB- . This suggests that an increased synthesis of Cx36 protein is not involved in the reappearance of coupling in adult injured animals -LRB- Chang et al. , 1999 -RRB- . Therefore , it might be possible that the reappearance of coupling between adult somatic motoneurons following injury might be produced by different mechanisms , such as the activation of gap junctions that contain different connexins other than Cx36 or by the release of factors that affect either connexin assembly into functional gap junctions , or the open state of the channels . In fact , several studies have shown that gap junction coupling can be modified by the activation of multiple G protein-coupled receptors , by pH and intracellular calcium levels -LRB- see Bennett , 1997 ; Bennett and Zukin 2004 -RRB- . For instance , early postnatal blockade of NMDA receptors has been shown to prevent the decrease in electrotonic and dye coupling observed during the first postnatal week -LRB- Mentis et al. , 2002 -RRB- . These data show the importance of gap junction coupling regulation by neuromodulators and suggest the possibility that electrotonic coupling between somatic motoneurons could also reappear in the adult animal under different kinds of pathological states , or even in physiological conditions . To date , this possibility has not been explored . 5 Conclusions Our results confirm and expand previous observations regarding Cx36 expression in the spinal cord -LRB- Rash et al. , 2000 ; Lee et al. , 2005 -RRB- and provide anatomical evidence that suggests that gap junctions composed by Cx36 may mediate electrotonic coupling between sympathetic preganglionic motoneurons and between somatic motoneurons . Further work using different approaches , such as blockers for Cx36 gap junction channels , Cx36-knock-out animals , transfection of sympathetic preganglionic motoneurons and somatic motoneurons with dominant-negative constructs of Cx36 and Cx36-specific antisense oligodeoxynucleotides , will reveal the precise role of Cx36 in the generation of coherent sympathetic and somato-motor rhythmic activity in the spinal cord of adult animals .

O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O B O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O B I O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B I O B O B O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B I O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O B O O O B I O O O O

sympathetic
motoneurons
gap junctions
spinal cord
rhythmic


Confirmation

of

Sentinel

Lymph

Node

Identity

by

Analysis

of

Fine-Needle

Biopsy

Samples

Using

Inductively

Coupled

Plasma

Mass

Spectrometry

Background

The

sentinel

lymph

node

-LRB-

SLN

-RRB-

biopsy

technique

is

a

reliable

means

of

determining

the

tumor-harboring

status

of

regional

lymph

nodes

in

melanoma

patients

When

technetium

m-labeled

antimony

trisulfide

colloid

-LRB-

-RRB-

particles

are

used

to

perform

preoperative

lymphoscintigraphy

for

SLN

identification

they

are

retained

in

the

SLN

but

are

absent

or

present

in

only

tiny

amounts

in

non-SLNs

The

present

study

investigated

the

potential

for

a

novel

means

of

assessing

the

accuracy

of

surgical

identification

of

SLNs

This

involved

the

use

of

inductively

coupled

plasma

mass

spectrometry

-LRB-

ICP-MS

-RRB-

to

analyze

antimony

concentrations

in

fine-needle

biopsy

-LRB-

FNB

-RRB-

samples

from

surgically

procured

lymph

nodes

The

sentinel

lymph

node

-LRB-

SLN

-RRB-

biopsy

technique

introduced

in

the

early

has

made

it

possible

to

establish

the

tumor-harboring

status

of

the

regional

node

field

in

melanoma

patients

with

a

minimally

invasive

procedure

Within

years

of

the

publication

describing

the

procedure

the

accuracy

of

SLN

biopsy

was

confirmed

in

two

further

studies

in

which

the

SLN

status

was

found

to

accurately

reflect

the

status

of

the

entire

lymph

node

field

The

SLN

biopsy

technique

has

since

become

widely

accepted

as

a

method

of

staging

the

disease

of

patients

with

melanoma

who

have

clinically

negative

lymph

nodes

The

status

of

the

SLN

is

used

to

determine

whether

further

surgical

or

adjuvant

therapy

is

appropriate

and

to

assess

patient

prognosis

The

tumor-harboring

status

of

the

SLN

is

the

single

most

important

prognostic

factor

for

melanoma

patients

surpassing

Breslow

thickness

ulceration

and

mitotic

rate

To

provide

accurate

prognostic

information

and

to

guide

appropriate

management

it

is

imperative

that

all

true

SLNs

are

accurately

identified

excised

and

assessed

for

their

tumor-harboring

status

In

a

number

of

cases

a

false-negative

result

may

occur

whereby

a

patient

with

disease

originally

assessed

as

SLN-negative

subsequently

develops

recurrence

in

a

regional

node

field

It

is

disturbing

to

note

that

false-negative

rates

of

up

to

have

been

reported

from

some

major

melanoma

treatment

centers

and

cooperative

groups

Available

evidence

suggests

that

these

failures

occur

not

because

the

SLN

biopsy

concept

is

flawed

but

because

of

methodological

and

technical

shortcomings

in

nuclear

medicine

surgery

and

histopathology

In

an

attempt

to

identify

how

many

of

these

false-negative

cases

might

have

been

because

of

surgical

failures

that

is

the

removal

of

a

lymph

node

that

was

not

the

SLN

identified

by

the

preoperative

lymphoscintigraphy

we

have

developed

a

technique

for

assaying

antimony

in

tissue

sections

This

work

is

based

on

the

concept

that

technetium

m

labeled

antimony

trisulfide

colloid

-LRB-

-RRB-

particles

used

for

preoperative

lymphoscintigraphy

in

Australia

are

retained

in

the

SLN

but

are

absent

or

present

in

only

tiny

amounts

in

non-SLNs

It

has

been

shown

that

antimony

is

preferentially

retained

by

the

SLN

and

can

be

measured

in

nodal

tissue

sections

by

inductively

coupled

plasma

mass

spectrometry

-LRB-

ICP-MS

-RRB-

It

has

also

been

demonstrated

that

human

tissue

contains

only

negligible

amounts

of

antimony

ICP-MS

enables

validation

of

the

SLN

biopsy

and

can

identify

false-negative

results

attributable

to

inaccurate

SLN

removal

Although

the

morbidity

associated

with

SLN

biopsy

is

low

it

is

an

invasive

procedure

with

a

definite

risk

of

complications

and

morbidity

In

addition

the

financial

implications

of

SLN

biopsy

must

be

considered

One

estimate

of

the

cost

of

performing

a

SLN

biopsy

procedure

on

an

outpatient

basis

was

US$

a

high

cost

for

providing

melanoma

treatment

services

to

the

community

The

development

of

a

minimally

invasive

technique

for

the

identification

of

SLN

metastases

may

minimize

or

eliminate

the

morbidity

of

the

procedure

and

provide

a

potential

cost

benefit

It

should

be

possible

to

identify

SLNs

by

preoperative

lymphoscintigraphy

localize

the

SLNs

with

ultrasound

and

then

perform

a

percutaneous

fine-needle

biopsy

-LRB-

FNB

-RRB-

under

ultrasound

control

Preliminary

studies

of

proton

magnetic

resonance

spectroscopy

-LRB-

MRS

-RRB-

analysis

of

FNB

samples

from

lymph

nodes

indicate

that

this

technique

has

the

capacity

to

distinguish

nodes

containing

metastatic

melanoma

from

uninvolved

nodes

with

high

sensitivity

specificity

and

accuracy

In

addition

to

accurately

determining

the

tumor-harboring

status

of

the

lymph

node

by

this

technique

it

is

also

valuable

to

verify

that

the

sampled

lymph

node

is

a

true

SLN

particularly

if

it

has

been

assessed

as

being

free

of

metastatic

tumor

The

objective

of

this

study

was

to

develop

a

method

for

confirming

that

a

FNB

specimen

was

obtained

from

a

true

SLN

by

measuring

the

level

of

antimony

-LRB-

present

in

radiocolloid

used

during

preoperative

lymphoscintigraphy

-RRB-

in

the

FNB

sample

by

ICP-MS

Materials

and

Methods

Instrumentation

All

measurements

were

made

with

an

Agilent

Inductively

Coupled

Plasma

Mass

Spectrometer

equipped

with

a

MicroMist

glass

concentric

nebulizer

a

Quartz-Scott

spray

chamber

-LRB-

Peltier

cooled

°C

-RRB-

and

an

Agilent

three-channel

peristaltic

pump

The

operating

conditions

were

optimized

daily

to

ensure

maximum

sensitivity

Typical

operating

conditions

are

summarized

in

Table

The

mass

spectrometer

was

operated

in

spectrum

mode

with

an

integration

time

of

ms

on

each

of

the

following

isotopes

-LRB-

internal

standard/control

-RRB-

TABLE

Operating

parameters

for

Agilent

Inductively

Coupled

Plasma

Mass

SpectrometryParameterConditionRadiofrequency

WSample

mmPlasma

gas

flow

L

min

gas

L

min

pump

rpsSample

read

sRinsing

sDwell

revolution

per

second

Reagents

and

Chemicals

All

reagents

used

were

of

the

highest

purity

available

Seventy

percent

double-distilled

nitric

acid

hydrochloric

acid

-LRB-

Arastar

-RRB-

and

hydrogen

peroxide

were

obtained

from

Sigma-Aldrich

Australia

Ultra-high-purity

water

was

produced

by

passing

distilled

water

through

a

Milli-Q

deionizing

system

-LRB-

Millipore

Australia

-RRB-

Standards

and

Certified

Reference

Materials

Certified

reference

material

GBW

Human

Hair

Powder

-LRB-

Langfang

China

-RRB-

was

selected

because

of

its

certified

antimony

levels

Multiple

samples

of

the

reference

material

were

also

analyzed

with

each

batch

of

samples

and

during

method

development

to

ensure

the

accuracy

and

precision

of

the

analytic

technique

SLN

Biopsy

Procedure

Preoperative

lymphoscintigraphy

was

performed

to

identify

the

node

fields

receiving

direct

lymphatic

drainage

This

process

involved

intradermal

injections

of

around

the

primary

cutaneous

melanoma

site

followed

by

early

and

delayed

imaging

with

a

scintillation

camera

The

location

of

the

SLNs

was

marked

on

the

overlying

skin

by

the

nuclear

medicine

physician

to

assist

the

surgeon

in

locating

SLNs

during

surgery

The

SLN

biopsy

procedure

was

performed

within

hours

of

the

radiocolloid

injection

so

that

residual

radioactivity

in

lymph

nodes

could

be

measured

intraoperatively

with

a

handheld

gamma

probe

-LRB-

Navigator

GPS

RMD

Instruments

Watertown

MA

-RRB-

Fifteen

minutes

before

the

operative

procedure

multiple

intradermal

injections

of

Patent

Blue

V

dye

-LRB-

Guerbet

Roissy

France

-RRB-

were

made

around

the

primary

cutaneous

melanoma

site

SLN

identification

was

based

on

visualization

of

the

nodal

blue

dye

staining

and

results

from

the

preoperative

lymphoscintigraphy

with

gamma

probe

confirmation

The

experimental

protocols

of

this

study

were

approved

by

the

University

of

Sydney

Ethics

Review

Board

in

accordance

with

the

precepts

established

by

the

Declaration

of

Helsinki

FNB

Samples

The

FNB

collection

process

involved

puncturing

the

fresh

SLN

specimen

within

minutes

of

its

surgical

removal

with

a

needle

attached

to

a

plastic

syringe

Multiple

passes

were

then

made

through

each

quarter

of

the

specimen

A

total

of

FNB

samples

-LRB-

from

presumptive

SLNs

and

nodes

considered

to

be

non-SLNs

-RRB-

were

collected

All

samples

were

placed

in

polypropylene

vials

containing

μL

of

phosphate-buffered

saline

-LRB-

mM

of

KCl

mM

of

NaCl

mM

of

pH

-RRB-

made

up

in

perdeuterated

water

-LRB-

phosphate-buffered

saline

-RRB-

and

immediately

snap-frozen

in

liquid

nitrogen

and

stored

at

°C

Microwave

Digestion

Procedure

Each

FNB

sample

was

transferred

to

a

polypropylene

tube

and

prepared

by

the

previously

described

microwave

digestion

procedure

Briefly

the

sample

was

digested

in

a

solution

containing

μL

of

nitric

acid

μL

of

hydrochloric

acid

and

μL

of

hydrogen

peroxide

Each

sample

was

digested

five

times

in

a

W

microwave

oven

on

the

defrost

setting

for

seconds

each

time

The

digest

was

then

quantitatively

transferred

to

a

second

polypropylene

tube

made

up

to

g

with

a

nitric

acid

solution

and

assayed

by

ICP-MS

Statistical

Analysis

The

unpaired

t-test

was

used

to

analyze

differences

between

the

levels

of

antimony

in

the

FNB

digests

from

SLNs

and

non-SLNs

A

P

value

of

less

than

was

considered

statistically

significant

Results

The

certified

concentration

of

antimony

in

GBW

is

μg/g

-LRB-

mean

SD

-RRB-

Replicate

analyses

of

reference

materials

during

method

validation

and

sample

analysis

gave

a

mean

value

of

μg/g

and

a

relative

standard

deviation

of

confirming

the

accuracy

and

precision

of

the

method

The

limit

of

detection

-LRB-

LOD

-RRB-

was

evaluated

by

the

3σ

criterion

-LRB-

the

LOD

is

given

by

mb

where

mb

is

the

blank

measurement

mean

and

σb

is

standard

deviation

of

five

blank

measurements

-RRB-

and

found

to

be

parts

per

billion

-LRB-

ppb

-RRB-

The

matrix-matched

calibration

standards

were

in

the

range

of

to

ppb

and

gave

an

value

of

during

method

validation

and

sample

analysis

The

relative

standard

deviation

of

the

slopes

of

the

calibration

curves

was

found

to

be

The

concentration

of

antimony

in

the

digested

SLNs

and

non-SLNs

was

measured

and

the

results

are

presented

in

Tables

and

respectively

The

mean

and

median

concentrations

of

antimony

were

and

ppb

respectively

in

the

SLNs

-LRB-

range

LOD

-RRB-

and

and

ppb

in

the

non-SLNs

-LRB-

range

LOD

-RRB-

These

results

indicate

that

the

levels

of

antimony

in

FNBs

from

SLNs

were

significantly

greater

than

from

non-SLNs

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

TABLE

Concentration

of

antimony

-LRB-

ppb

-RRB-

in

fine-needle

biopsy

sample

digests

collected

from

sentinel

lymph

nodesSample

No.Antimony

concentration

-LRB-

ppb

-RRB-

.06666.09797.17388.29859.302010.308511.325712.380713.381214.386415.390316.404517.497118.607919.640720.724321.8582221.091231.438241.444251.724261.760271.847282.013292.150302.298312.756323.248

LOD

limit

of

detection

-LRB-

ppb

-RRB-

TABLE

Concentration

of

antimony

-LRB-

ppb

-RRB-

in

fine-needle

biopsy

sample

digests

collected

from

nonsentinel

lymph

nodesSample

No.Antimony

concentration

-LRB-

ppb

-RRB-

LOD

limit

of

detection

-LRB-

ppb

-RRB-

Fig.

Antimony

concentration

in

digested

fine-needle

biopsy

samples

from

sentinel

lymph

nodes

and

nonsentinel

lymph

nodes

Discussion

Current

standard

clinical

management

of

patients

with

primary

cutaneous

melanoma

includes

wide

local

excision

of

the

primary

tumor

and

SLN

biopsy

for

patients

considered

to

be

at

high

risk

of

having

regional

node

field

metastases

Technical

failures

of

the

procedure

could

be

attributable

to

errors

in

lymphoscintigraphy

sentinel

lymphadenectomy

or

histologic

evaluation

In

such

cases

the

potential

for

disease

recurrence

in

a

previously

mapped

lymph

node

basin

exists

A

reliable

technique

to

confirm

the

identity

of

a

SLN

has

the

potential

to

reduce

false-negative

results

attributable

to

surgical

errors

In

a

previous

study

we

demonstrated

that

the

analysis

of

antimony

in

sectioned

nodal

tissue

could

be

used

to

distinguish

SLNs

and

non-SLNs

removed

from

the

same

nodal

basin

This

technique

can

be

used

within

the

bounds

of

currently

used

histopathologic

protocols

and

has

the

advantage

of

being

applicable

to

archival

paraffin-embedded

tissue

It

has

also

been

established

that

false-negative

results

may

be

caused

by

the

removal

of

a

lymph

node

incorrectly

classified

as

a

sentinel

lymph

node

Because

neither

blue

dye

nor

radioisotope

persist

after

procedures

required

for

microscopic

examination

they

can

not

be

used

retrospectively

to

confirm

that

the

true

SLN

had

been

removed

The

only

other

reported

method

to

confirm

SLN

identity

involves

the

injection

along

with

the

blue

dye

of

carbon

particles

that

are

retained

by

the

node

However

these

large

dense

carbon

particles

may

hinder

optimal

histologic

examination

by

obscuring

metastatic

melanoma

cells

As

far

as

we

are

aware

this

method

is

not

in

routine

clinical

use

FNB

digests

from

four

nodes

presumed

to

be

SLNs

-LRB-

-RRB-

were

found

to

contain

extremely

low

levels

of

antimony

below

the

LOD

In

a

previous

study

we

also

identified

SLNs

that

contained

extremely

low

levels

of

antimony

The

most

likely

reason

for

the

low

antimony

levels

would

be

inaccurate

classification

of

the

node

that

had

been

removed

as

a

SLN

when

it

is

in

fact

a

non-SLNs

Other

possible

explanations

to

account

for

the

low

antimony

levels

include

variable

distribution

of

antimony

throughout

the

node

limited

migration

of

the

colloid

to

the

lymph

nodes

or

tumor

deposits

preventing

colloid

uptake

Haigh

et

al.

investigated

the

distribution

of

carbon

dye

in

SLNs

and

found

a

high

concentration

of

carbon

particles

around

the

point

of

entry

of

afferent

lymphatic

channels

It

is

likely

that

antimony

exhibits

an

analogous

distribution

pattern

If

so

FNB

samples

that

fail

to

include

this

region

of

the

SLN

may

contain

falsely

low

antimony

levels

In

view

of

these

factors

it

would

be

useful

to

set

a

criterion

standard

for

classifying

a

given

node

as

sentinel

or

not

on

the

basis

of

antimony

levels

assessed

by

ICP-MS

However

defining

such

a

criterion

on

the

basis

of

our

small

sample

set

is

difficult

In

a

previous

study

we

assessed

antimony

levels

in

archival

tissue

sections

of

paired

tumor-positive

SLNs

and

tumor-negative

non-SLNs

removed

from

the

same

regional

node

field

during

the

same

operative

procedure

from

individual

patients

The

aims

of

this

study

were

to

determine

whether

antimony

concentrations

could

be

used

to

confirm

whether

removed

SLNs

were

true

SLNs

and

to

differentiate

SLNs

from

non-SLNs

The

median

concentration

of

antimony

in

the

SLNs

was

ppb

and

in

non-SLNs

was

ppb

-LRB-

P

-RRB-

By

using

a

cutoff

point

of

ppb

-LRB-

the

median

concentration

of

antimony

in

all

SLNs

and

non-SLNs

-RRB-

to

differentiate

SLNs

from

non-SLNs

of

SLNs

and

of

non-SLNs

were

correctly

identified

by

the

SLN

biopsy

procedure

Although

SLN

biopsy

is

a

highly

accurate

method

for

staging

regional

lymph

nodes

it

is

an

invasive

surgical

procedure

that

is

costly

and

is

associated

with

an

inherent

risk

of

complication

and

morbidity

Pathologic

assessment

of

SLNs

is

laborious

time-consuming

and

costly

and

involves

examination

of

multiple

hematoxylin

and

eosin

stained

histopathologic

sections

as

well

as

sections

stained

with

immunohistochemical

techniques

The

development

of

a

rapid

nonsurgical

technique

that

allows

detection

of

metastatic

tumor

deposits

would

be

of

great

benefit

It

would

eliminate

the

need

for

surgical

excision

of

lymph

nodes

and

reduce

the

complexity

of

pathologic

assessment

of

SLNs

However

to

ensure

that

potential

false-negative

results

are

minimized

it

is

imperative

to

ensure

the

accuracy

of

SLN

identification

Several

studies

have

identified

proton

MRS

as

a

candidate

for

the

nonsurgical

assessment

of

lymph

nodes

The

technique

monitors

changes

in

the

chemical

composition

of

cells

during

tumor

development

and

can

identify

differences

that

are

not

morphologically

discernible

In

these

studies

the

spectra

collected

from

SLN

FNB

samples

harboring

tumor

cells

contain

choline

and

taurine

peaks

which

are

absent

in

disease-free

samples

Another

sensitive

technique

for

the

detection

of

melanoma

metastases

in

SLNs

is

the

assessment

of

tyrosinase

mRNA

by

reverse

transcriptase

polymerase

chain

reaction

-LRB-

RT-PCR

-RRB-

However

as

far

as

we

are

aware

RT-PCR

assessment

of

tyrosinase

mRNA

has

not

been

studied

in

FNB

samples

of

SLNs

A

major

potential

problem

of

RT-PCR

analysis

of

FNB

samples

of

SLNs

is

the

issue

of

false-positive

and

false-negative

results

in

such

specimens

and

this

would

need

to

be

assessed

before

the

technique

could

be

used

in

clinical

practice

The

results

of

our

preliminary

work

provide

evidence

that

determining

antimony

levels

in

FNB

specimens

by

ICP-MS

can

confirm

SLN

identity

and

can

differentiate

SLNs

from

non-SLNs

This

technique

may

be

a

useful

adjunct

to

other

techniques

assessing

tumor-harboring

status

of

SLNs

on

FNB

specimens

such

as

MRS.

Potentially

proton

MRS

analysis

of

a

FNB

from

a

node

confirmed

to

be

a

true

SLN

by

ICP-MS

could

provide

a

reliable

method

for

determining

the

tumor-harboring

status

of

SLNs

Although

the

results

are

promising

and

raise

the

possibility

that

SLN

assessment

may

be

performed

on

FNB

specimens

-LRB-

rather

than

histologic

specimens

of

excised

SLNs

-RRB-

in

the

future

further

validation

studies

are

necessary

before

such

techniques

are

used

in

widespread

clinical

practice

Furthermore

the

highly

specialized

and

expensive

nature

of

the

equipment

used

for

ICP-MS

will

likely

limit

the

technique

to

specialist

centers

O O B I I O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O 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