s2fieldsofstudy: Biology Medicine Engineering

text:
Dynamic Computational Model of Symptomatic Bacteremia to Inform Bacterial Separation Treatment Requirements

The rise of multi-drug resistance has decreased the effectiveness of antibiotics, which has led to increased mortality rates associated with symptomatic bacteremia, or bacterial sepsis. To combat decreasing antibiotic effectiveness, extracorporeal bacterial separation approaches have been proposed to capture and separate bacteria from blood. However, bacteremia is dynamic and involves host-pathogen interactions across various anatomical sites. We developed a mathematical model that quantitatively describes the kinetics of pathogenesis and progression of symptomatic bacteremia under various conditions, including bacterial separation therapy, to better understand disease mechanisms and quantitatively assess the biological impact of bacterial separation therapy. Model validity was tested against experimental data from published studies. This is the first multi-compartment model of symptomatic bacteremia in mammals that includes extracorporeal bacterial separation and antibiotic treatment, separately and in combination. The addition of an extracorporeal bacterial separation circuit reduced the predicted time of total bacteria clearance from the blood of an immunocompromised rodent by 49%, compared to antibiotic treatment alone. Implementation of bacterial separation therapy resulted in predicted multi-drug resistant bacterial clearance from the blood of a human in 97% less time than antibiotic treatment alone. The model also proposes a quantitative correlation between time-dependent bacterial load among tissues and bacteremia severity, analogous to the well-known ‘area under the curve’ for characterization of drug efficacy. The engineering-based mathematical model developed may be useful for informing the design of extracorporeal bacterial separation devices. This work enables the quantitative identification of the characteristics required of an extracorporeal bacteria separation device to provide biological benefit. These devices will potentially decrease the bacterial load in blood. Additionally, the devices may achieve bacterial separation rates that allow consequent acceleration of bacterial clearance in other tissues, inhibiting the progression of symptomatic bacteremia, including multi-drug resistant variations.

Introduction
Symptomatic bacteremia is a common cause of severe sepsis [1]. In the United States, Gramnegative bacteria cause approximately 70% of hospital acquired infections in intensive care units [2], while up to half of all bloodstream infections are caused by Gram-negative bacilli [3]. Infections caused by Gram-negative bacteria are of particular concern because these organisms are highly efficient at acquiring and up-regulating various mechanisms that promote multidrug resistance (MDR) [4]. Global concern revolves around the ever-increasing number of infections caused by MDR Gram-negative bacteria, in particular Acinetobacter baumannii and Klebsiella pneumoniae [5].
A. baumannii and K. pneumoniae are both Gram-negative bacilli with MDR phenotypes. These bacteria are commonly found in the hospital setting and reside in internal parts of the human body [6,7]. One of the strongest risk factors for developing a Gram-negative bloodstream infection is a compromised immune system. More specifically, those suffering from neutropenia, or lack of neutrophils in the blood, run a significantly higher risk for developing a bloodstream infection than healthy individuals [8,9]. A. baumannii and K. pneumoniae infections can lead to multiple consequences, such as widespread inflammation, blood clotting, multiple organ failure, pneumonia, septic shock, and death. The crude mortality rate associated with an A. baumannii or K. pneumoniae bloodstream infection in an immunocompromised patient can exceed 70% [8,10,11].
Treatment of a bloodstream infection requires eradication of bacteria from the blood, but also from other tissues, including the source of infection. The clearance of bacteria by the host immune response is similar to the pharmacokinetic clearance of drugs. Therefore, the clearance of bacteria from the host may be interpreted using a multi-compartment model with each compartment representing a tissue into which bacteria may enter and exit, replicate, or be eliminated by treatment. Bacteria can be transferred among tissues and eventually be eliminated through an immune response, therapeutic treatment, or a combination of both. Unlike drugs, bacteria proliferate prior to host recognition, and bacteria continue to proliferate in the case of an unsuccessful host defense.
Bacterial clearance from the bloodstream in a physiologically based pharmacokinetic (PBPK) model was first described in 1983 by Cheewatrakoolpong et al. [12]. The group analyzed the kinetics of bacterial clearance from the blood and mesenteric lymph nodes of mice using a two-compartment computational model. More recently, Kang et al. developed a mathematical model that predicts bacterial clearance solely from the bloodstream [13]. In the Kang et al. model, the group incorporated the effects of an extracorporeal bacterial separation device that continuously removes bacteria from flowing blood using a magnetic nanoparticle-based separation technique. The mathematical model successfully predicted the optimal magnetic nanoparticle sizes required for removal of bacteria from whole blood. This model enabled predictions of particle-pathogen collision and magnetophoresis rates, which allowed for determination of how these factors influence magnetic pathogen separation from blood under flow using a microfluidic magnetic separation device. The biological impact of bacterial separation in the host, however, was not incorporated into this model.
The work by Kang et al. can be expanded into a five-compartment model that accounts for physical and immunological interactions, bacterial net growth, transport among tissues (lungs, spleen, liver, blood), antibiotic treatment, and extracorporeal removal of bacteria from the blood. This type of expanded, dynamic model can be used to characterize time-dependent, tissue-specific bacterial load in symptomatic bacteremia. Also, such a model could be used to characterize the biological impact of bacterial separation from the blood, and, potentially, predict the utility as a therapeutic treatment option in conjunction with antibiotic treatment.
Consequently, we developed a pharmacokinetic model of A. baumannii and K. pneumoniae symptomatic bacteremia in order to provide a quantitative and flexible framework useful for both experimental and analytical work in this area.
A five-compartment dynamic model, consisting of five first-order homogenous ordinary differential equations, was developed to study the biodistribution of A. baumannii and K. pneumoniae during symptomatic bacteremia in mammals. This pharmacokinetic model was used to assess the combination of broad-spectrum colistin antibiotic treatment and extracorporeal bacteria separation from the blood on the overall time-dependent bacterial burden in infected living systems. Colistin, a naturally occurring cationic decapeptide isolated from Paenibacillus polymyxa var. colistinus [14], is a potent broad-spectrum antimicrobial. This antibiotic is commonly used for the treatment of challenging Gram-negative pathogens [15], including A. baumannii.
The time course of a bacterial infection within important tissues of immunologically normal rodents was described using the mathematical model. The same measures were then used to examine the impact of suppressed immunity. The efficacy of antibiotic administration in immunosuppressed rodents exposed to a bacterial challenge was also analyzed. Finally, the potential benefit of extracorporeal bacterial isolation and removal from blood, in terms of bacterial load by compartment, was assessed in both rodent and human mathematical models.
Analyzing results from this model of multi-organ infection with a PBPK mathematical approach provides an opportunity to evaluate the efficacy of extracorporeal bacterial separation from the bloodstream in combination with various antibiotic treatment regimens. For the first time, a modeling approach based on experimentally obtained data allows quantitative exploration of the impact of extracorporeal bacterial separation in combination with antibiotic treatment.

Time Course of Bacterial Infection in Immunonormal Rodents
Non-immunocompromised rodents challenged intratracheally with a prepared inoculum of either A. baumannii 10 7 CFU/mL or K. pneumoniae 10 7 CFU/mL [16,17] were modeled. The overall bacterial burden for both Gram-negative species decreased over time in all compartments, although at different rates (Fig 1A and 1B). The blood compartment of the non-immunocompromised rodent mathematical model required the least amount of time to clear bacteria to a negligible amount ( 1 CFU/mL), followed by the spleen, lungs, and liver. The bacterial clearance trends shown in Fig 1A and 1B were compared to experimental studies, in which total bacterial burden was reported for the liver, spleen, lungs, and blood of non-immunocompromised rodents following intratracheal bacterial challenge [18][19][20][21][22][23]. Within 24 hours of intratracheal bacterial challenge, the total bacterial uptake by the liver was the largest, and was followed by the spleen, lungs, and blood [19]. The results of our model were in agreement with experimental results previously described [18][19][20][21][22][23]. Bruhn et al. experimentally demonstrated that an A. baumannii bacterial load of 10 4 CFU/mL within the blood compartment of a normal rodent model was reduced to negligible bacterial densities at a rate of -0.06 log 10 CFU/ mL/h [24]. This A. baumannii clearance rate, characteristic of the non-immunocompromised rodent blood compartment, was in agreement with in our model predictions (Fig 1A). The blood compartment bacterial load dropped to half maximal concentration within approximately 40 hours of the onset bacterial challenge. This rate of bacterial clearance within the blood compartment was equivalent to -0.06 log 10 CFU/mL/h. Also, Guo et al. have reported that normal rodents infected intratracheally with an A. baumannii bolus clear the infection from the lungs at a rate of -0.1 log 10 CFU/mL/h [20]. The average A. baumannii clearance rate within the lung compartment of the dynamic model was also -0.1 log 10 CFU/mL/h (Fig 1A), corresponding to published experimental data. Furthermore, the A. baumannii bacterial burden within the liver of a normal rodent decreased at a rate of -0.06 log 10 CFU/mL/h [21], which was the same liver clearance rate produced by the mathematical modeling results.
The mathematical model predictions were in quantitative agreement with experimental studies conducted by other laboratories. The model includes numerous physiological and microbiological parameters, which were chosen from first principles, not mathematical estimations. The agreement of model predictions with experimental data supports the validity of the model structure as representative of bacteremia. The kinetic profiles of A. baumannii and K. pneumoniae concentration by tissue were similar for each set of assumptions evaluated in this work. Therefore, the remaining analysis of pathogenesis kinetics is described only for A. baumannii. The similar K. pneumoniae data is available in the supplementary data. Fig 1 suggests that an immunonormal rodent suppresses bacterial growth, with bacterial clearance from each compartment following a pattern of exponential decay. Model initial conditions were 3 hours post-inoculation, focusing the modeling effort on the slower processes of bacterial removal rather than the rapid distribution of bacteria [28]. Bacteria rapidly spread from the lungs to the bloodstream, which resulted in bacterial transport to other organs. The hematogenous spread of bacteria to other organ sites initially caused a rapid decrease in the bacterial burden of the blood compartment, but spread of infection increased the time required to suppress the infection.

Time Course of Bacterial Infection in Immunocompromised Rodents
A. baumannii and K. pneumoniae Gram-negative bacteria frequently cause sepsis in immunocompromised, neutropenic, elderly, and chronically ill individuals [29]. To better evaluate such cases, we modeled the onset of symptomatic bacteremia in neutropenic, immunocompromised rodents. Without treatment, the overall bacterial burden increased in neutropenic rodents until reaching a bacterial load associated with death [30,31]. This experimental condition was modeled in Fig 2. The fitted model agreed with published experimental data regarding bacterial burden over time in an untreated, immunocompromised rodent model [20]. Previously published experimental data demonstrated that a pulmonary A. baumannii bacterial burden within an immunocompromised rodent increased at a rate of +0.04 log 10 CFU/mL/h [20,28], which was consistent with the mathematical modeling results (Fig 2). Modeling results also suggest that the A. baumannii bacterial burden associated with the blood compartment increased at a rate of +0.05 log 10 CFU/mL/h, which was consistent with the experimental data published by Immunonormal rodents intratracheally inoculated with (A) A. baumannii (10 7 CFU/mL) or (B) K. pneumoniae (10 7 CFU/mL) were modeled. The median numbers of bacteria in each compartment observed experimentally were used as the initial conditions for these simulations [21,[24][25][26][27], and trajectories were generated using the parameter estimates described in the Materials and Methods. Bruhn et al [24]. Approximately 100 hours following intratracheal exposure to 10 7 CFU/mL A. baumannii, neutropenic rodents succumb to the bacterial challenge [32,33]. This was consistent with the lethal bacterial concentration of 10 10 CFU/mL at 96 hours predicted by mathematical modeling [31][32][33][34].
The bacterial burden increases within the lungs, blood, and spleen compartments of Fig 2 are consistent with changes in bacterial transport out of the bloodstream, not simply suppressed bacteria clearance. Predictions obtained for the untreated, immunocompromised rodent model generated bacterial concentrations known to be lethal. Blood flow through the bacterial separation device compartment was set to zero in this scenario. All modeling parameters acquired and implemented were gathered directly from published experimental results and can be found in the Materials and Methods. Solutions from this multi-compartmental model were obtained in the absence of fitting parameters and mimicked experimental data, supporting the validity of the model.

Efficacy of Antibiotic Administration in Infected, Immunosuppressed Rodents
The most common treatment for symptomatic bacteremia is empiric, broad-spectrum antibiotic therapy. Therefore, colistin methanesulfonate antibiotic administration was incorporated into our mathematical model following the presentation of symptomatic bacteremia, which was presumed to occur when the bacterial concentration in the lungs reached 10 7 CFU/mL [28] and bacteria were completely distributed throughout all compartments. Bacteria rapidly distribute throughout the blood, liver, lungs, and spleen following exposure [18].
Following simultaneous intratracheal infusion of 10 7 CFU/mL A. baumannii and 3 mg/kg colistin methanosulfate, A. baumannii was cleared from the blood of the neutropenic rodent mathematical model within 55 hours (Fig 3). The rate constants pertaining to bacterial clearance following colistin methanosulfate administration were based on experimental data gathered experimentally by Pantopoulou et al. and Montero et al [32,36], as described in the Materials and Methods. The mathematical modeling (Fig 3) agreed with experimental data regarding the effects of colistin methanosulfate administration (3 mg/kg) on an A. baumannii bacterial burden in neutropenic rodent models, clearing the bacterial infection from the blood at a rate of -0.09 log 10 CFU/mL/h [32,36].
The area under the curve (AUC) was computed for each compartment displayed in Fig 3. The AUC reflects the total, time-dependent bacterial load experienced by each compartment of our model. Of note, the rodent mathematical model suggests that the liver consistently experienced the highest total bacterial burden, approximately an order of magnitude greater than the lungs, as indicated in Table 1. This result is consistent with the severe side effects observed during antibiotic treatment for symptomatic bacteremia, especially in immunocompromised and chemotherapy patients.

Extracorporeal Bacterial Separation from Blood
Kang et al. have described the development of an extracorporeal bacterial separation device that can rapidly remove bacteria from blood for sepsis treatment [37]. The dialysis-like device employs magnetic nanoparticles functionalized with a bacteria-targeting ligand to magnetically capture pathogens, including multi-drug resistant bacteria, from flowing blood in a microfluidic device. Experimental results using this magnetic nanoparticle-based bacterial separation device suggested that it significantly reduced the levels of bacteria in the bloodstream of a rodent model. It was hypothesized that the spread of bacteria to distal organs would be significantly lowered with the decrease in bacterial concentration in the bloodstream [37]. Also, it was postulated that broad-spectrum antibiotic therapy, such as colistin methanosulfate, could be coadministered with this bacterial separation therapy, resulting in faster bacterial clearance rates.  Magnetic nanoparticle-based extracorporeal bacterial separation could prove to be an effective adjuvant therapy for sepsis treatment. To test this hypothesis, an extracorporeal bacterial separation device was incorporated into the kinetic mathematical model, integrating predictions of magnetic bacteria separation under microfluidic flow in combination with colistin methanosulfate antibiotic treatment. The approach was used to explore the possible benefits of magnetic nanoparticle-based bacterial separation from blood in conjunction with antibiotic treatment on the time-dependent bacterial load among organs during a symptomatic bacteremia episode.

Bacterial Separation Combined with Antibiotic Therapy
The predicted effect of extracorporeal bacterial separation for the purpose of clearing A. baumannii from the blood is demonstrated in Fig 4. The addition of magnetic nanoparticle-based bacterial separation operating at an ideal 100% efficiency and processing one-fifth of the total blood volume of a rodent per hour, in conjunction with antibiotic treatment, resulted in A. baumannii clearance from the blood compartment in 27 hours less time than antibiotic treatment alone (Fig 4). Therefore, the bacterial clearance rate associated with this combined treatment was 49% faster than antibiotic treatment alone ( Table 2). This is a significant result because for each hour that a septic patient is not effectively treated, the risk of mortality increases by 7.6% [38].  The AUCs of Fig 4 were also evaluated. The model suggested that the liver experienced a 52% reduction in AUC with the addition of bacterial separation in the presence of antibiotic (Table 3). With bacterial separation occurring at 100% efficiency, the extracorporeal bacterial separation device had a significant impact on overall bacterial clearance and diffusion rates. The total bacterial burden in the blood compartment decreased by an order of magnitude compared to antibiotic treatment alone. Also, the incorporation of the 100% effective bacterial separation device into the model inhibited bacterial proliferation and diffusion from the initial inoculation point of the lungs, thereby lowering the overall bacteria burden in all compartments.

Device Optimization Based on Bacterial Separation Efficiency
Bacterial separation efficiency has a significant impact on the rate of bacteria removal from the blood compartment ( Fig 5). As previously displayed in Table 2, 100% separation efficiency promoted the clearance of A. baumannii from the blood in 27 hours less time than antibiotic treatment alone. To explore the role of separation efficiency on predicted bacterial load in vivo, model analysis was carried out for separation efficiencies of 60% and 20%. This approach was intended to inform the design constraints of extracorporeal bacteria separation devices for impact in complex living systems. 60% bacterial separation efficiency resulted in clearance of A. baumannii from the blood compartment in 20 hours less time that antibiotic treatment alone ( Table 4). As the separation efficiency further decreased to 20%, bacterial removal became ineffective. Therefore, device design features that impact bacterial separation efficiency and nonlinearly influence clearance must be considered for proposed applications. Total bacteria separation efficiencies produced by the mathematically modeled extracorporeal separation devices were dependent on nanoparticle size. As the nanoparticle radius increased, the bacterial separation efficiency decreased, resulting in slower clearance of bacteria from blood ( Fig 5). The collision rate constant decreased as the nanoparticle radius increased, resulting in less total nanoparticle interaction with, and binding to, bacteria [13]. Once the nanoparticle radius exceeded approximately 40 nm, the efficiency of bacterial binding and capture was not significant enough to impact the overall rate of bacterial clearance. The optimal nanoparticle size for bacteria capture and removal was estimated to be 25 nm or less, which directly corresponded to previously published literature [13]. The reduced mass of smaller nanoparticles, however, decreases the magnetic attraction force [13]. Thus, careful attention to the design of the separation device is required to achieve efficiency compatible with predicted improvement in overall bacterial clearance. Therefore, multiple, interconnected nanotechnology design features are important considerations in magnetic nanoparticlebased bacterial separation devices.
The impact of bacterial separation efficiency was evaluated by the mathematical model and reported in terms of resultant total bacterial load, or AUC, experienced by each tissue compartment of an A. baumannii infected immunocompromised rodent. As the nanoparticle radius increased above 25 nm, the overall separation efficiency characteristic of the extracorporeal bacterial separation device decreased. Table 5 demonstrated that as the nanoparticle radius increased above 25 nm, the total bacterial burden experienced by each compartment also increased. All other parameters held constant, a nanoparticle radius of 43 nm is equivalent to a 20% separation efficiency, which has no significant effect on the AUC compared to antibiotic treatment alone. Therefore, in order for magnetic nanoparticle-based bacterial separation devices to aid in the reduction of the total bacterial burden within each compartment, the efficiency of bacterial separation from the blood compartment must exceed 20%.

Bacterial Separation for Treatment of Symptomatic Bacteremia in Human Model
The use of extracorporeal bacterial separation for treatment of symptomatic bacteremia in humans has never been explored experimentally or by mathematical modeling. The successful rodent model was modified to explore the possible benefits of magnetic nanoparticle-based extracorporeal bacterial separation in humans during an episode of symptomatic bacteremia. By incorporating human parameters (Materials and Methods) into the model and adjusting for the tissue volume differences of humans compared to rodents, we were able to estimate the impact of bacterial separation, in conjunction with antibiotic treatment. The model extrapolation to humans has known, and, likely, unknown limitations. However, this approach is a first step towards an understanding of the extracorporeal bacterial clearance necessary for biological impact. Symptomatic bacteremia occurs in an adult humans when the bacterial burden reaches approximately 10 CFU/mL in the bloodstream or 10 3 CFU/mL in the lungs [39][40][41]. Therefore, our model incorporated these parameters as markers to indicate the time at which to apply extracorporeal bacterial separation and antibiotic treatment. Results suggested that A. baumannii clearance from the blood compartment was reduced by 14 hours with the addition of 100% efficient extracorporeal bacterial separation treatment, compared to antibiotic treatment alone (Fig 6). The flow rate through the bacterial separation compartment was programmed to process one-fifth of the total human blood volume per hour, similar to the blood flow rates used during kidney dialysis in humans. Based on the AUCs of Fig 6, the combination therapy approach decreased the total bacterial burden in the model compartments, with the most significant difference in the blood compartment (Table 6).
This work is the first to estimate, by mathematically models, bacterial load as a surrogate of the ultimate biological impact of infection. Bacterial load, or AUC, is directly correlated to the progression of symptomatic bacteremia and death [42]. Therefore, extracorporeal bacterial separation devices designed to operate at high efficiencies would not only significantly reduce the total bacterial load, but may also inhibit the progression of symptomatic bacteremia. Inhibition Computational Model of Symptomatic Bacteremia to Inform Treatment of disease progression would provide additional time to identify ideal therapies for infected patients, while reducing the mortality rates associated with symptomatic bacteremia.

Bacterial Separation for Treatment of Multi-Drug Resistant Bacteremia in Human Model
Bacterial species identification in the clinical setting takes one to three days, a time during which the patient is treated with broad-spectrum antibiotics. This method is not optimal, especially for patients suffering from MDR infections. Symptomatic bacteremia caused by MDR bacteria is one of the most critical public health issues, even in first-world countries [43]. The increasing frequency of MDR bacterial strains is leading into the 'post-antibiotic era' .
MDR-associated symptomatic bacteremia was mathematically modeled by assuming that the MDR A. baumannii were 50% less susceptible to antibiotic treatment than non-MDR A. baumannii. Bacterial infection that is 0% susceptible to antibiotic treatment behaves similarly to the trajectory shown in Fig 2 of Fig 3. Choosing a MDR A. baumannii susceptibility of 50% allows for quantitative evaluation of model sensitivity to the degree of bacterial antibiotic resistance. Fig 7 described the clearance of MDR A. baumannii using the additional assistance of 100% efficient extracorporeal bacterial separation, compared to antibiotic treatment alone. The addition of extracorporeal bacterial separation operating at 100% efficiency resulted in MDR A. baumannii clearance from the blood of a human mathematical model in 1 h, which Computational Model of Symptomatic Bacteremia to Inform Treatment was 97% faster than antibiotic treatment alone. Antibiotics prove less effective in the treatment of MDR symptomatic bacteremia, which highlights the need for alternative treatment methods. Bacterial separation treatment methods have been shown to bind and remove multiple clinical isolates of antibiotic-resistant organisms [37]. This supports the development of new engineering-based bacterial separation devices to combat MDR bacteria and delay progression to septic shock. Our mathematical modeling work established that extracorporeal bacterial separation reduced the total bacterial burden in the bloodstream of a bacteremic subject by an order of magnitude, compared to antibiotic treatment alone. This correlated to a 49% reduction in non-MDR bacterial exposure time due to the addition of extracorporeal bacterial separation therapy. Efficient bacterial separation also reduced the spread of bacteria to distal sites. Results further indicated that engineering-based bacterial separation devices could offer a particularly effective therapeutic strategy for patients suffering from MDR infections that render existing drug therapies inadequate. The addition of extracorporeal bacterial separation resulted in MDR bacterial clearance from the blood of a human mathematical model in 97% less time than antibiotic treatment alone. This was a notably important prediction because decreasing the length of bacterial infection significantly reduces the risk of mortality associated with symptomatic bacteremia [38]. The lack of antibiotics in the developmental pipeline, combined with the increasing rate of MDR, has created a dire need for the discovery of new therapies effective against MDR bacterial infections. Therefore, development of mathematical models is necessary to rapidly determine the effectiveness of new treatment therapies, such as extracorporeal bacterial separation. Our work was the first dynamic mathematical model to demonstrate the potential usefulness of extracorporeal bacterial separation for the treatment of symptomatic bacteremia and its MDR counterpart.

untreated infection, whereas infection that is 100% susceptible to antibiotic treatment was shown in
Engineers have had a profound impact on the development of disease treatment systems, such as kidney dialysis devices. It is clear that engineers also have a role to play in the design of extracorporeal bacterial separation devices. To aid in the optimization of bacterial separation device design, more experimental data quantifying bacterial load in living systems must be gathered to better understand the correlation between bacterial load and patient outcome.
Currently, data describing bacterial load in rodent systems is sparse, and corresponding human data is even less prevalent. These are key pieces of information that must be obtained in order to confidently design new treatment methods that reduce total bacterial burden, duration of infection, and the 'post-antibiotic era' threat. Designing extracorporeal bacterial separation devices that remove bacteria from the blood at a rate deemed biologically relevant by this information may inhibit the progression of symptomatic bacteremia, decrease overall mortality rates, and help combat MDR bacterial infections.

Kinetic Model and Parameter Values
A five-compartment kinetic model was developed to explore the pathogenesis kinetics and treatment of symptomatic bacteremia (Fig 8). It was assumed that infection occurred via intratracheal instillation of a 10 7 CFU/mL Gram-negative bacteria (Acinetobacter baumannii or Klebsiella pneumoniae) bolus into the lung compartment. This is a common bacterial concentration used to establish and study rodent bacteremia models [16]. Also, the most common site of infection leading to bacteremia in humans is the lungs [44,45]. Bacterial exchange among all five compartments, which included the lungs, spleen, liver, blood, and bacterial separation device, was programmed as specified by the model construction and parameters extracted from experimental studies of bacteremia. The lungs, liver, spleen, and blood were included because all have experimentally demonstrated the most significant and rapid uptake of bloodborne bacteria [18]. Kinetics were assumed to be first order. Bacteria proliferated (p (h -1 )) within the lungs, spleen, liver, and blood, and were cleared (c) by immunological interactions at rates (h -1 ) specific to each compartment. No experimental evidence was available to distinguish p from c in vivo, therefore, the two terms were considered as a single net bacterial growth rate (G x (h -1 )). Bacterial transport between compartments was represented as a function of the blood flow rate, Q, the compartment volume, V, and a partitioning coefficient, x [18]. The partition coefficient represented the ratio of bacteria concentration between compartments and was proportional to the concentration of bacteria in the donor compartment [18]. Bacterial separation efficiency, f(r f ), represented the percent of target bacterial cells separated by the bacterial separation compartment (CFU mL -1 ) per total number of target bacterial cells (CFU mL -1 ).
Five first-order homogenous ordinary differential equations (ODEs) were used as a model representation of the physiological features of the system. The system of equations was based on previously published pharmacokinetic models [18,46]. Each autonomous ODE represented the instantaneous rate of change of bacteria concentration in the respective compartment, Intratracheal instillation of a Gram-negative bacteria bolus initially occurred in the lung compartment, with initial concentration L 0 (CFU mL -1 ). Bacterial proliferation rates (p, h −1 ), clearance rates (c, h −1 ), and transport rates between compartments were included in the model schematic. The rate of bacterial transport between compartments was represented as a function of blood flow rate per compartment volume (Q/V, mL h -1 ), modified by an experimentally determined partitioning coefficient (x, dimensionless). doi:10.1371/journal.pone.0163167.g008 where L, S, H, E, and B were the bacterial concentrations (CFU mL -1 ), in the lungs, spleen, liver, extracorporeal bacterial separation device, and blood, respectively. The model was designed such that bacteria were transported from the spleen into the liver via hepatic portal circulation. Liver (H, hepatic) circulation was divided into splenic portal venous blood and the combination of hepatic arterial flow and its tributaries. Mouse and human organ mass values were based on published literature and were used to describe organ volumes, V [18]. Organ blood flow rates, Q, and blood:organ partition coefficients, x, were also gathered from previously published data [18]. Empirical values used to describe compartmental volumes, blood flow rates, and partition coefficients can be found in Tables 7-9. Volume, V (g or mL) Lungs (L) 1.14 [47] 1315 [48] Spleen (S) 1.97 [49] 127 [48] Liver (H) 0.97 [50] 830 [48] Extracorporeal Bacterial Separation Device (E)

Variable 90
doi:10.1371/journal.pone.0163167.t008 The rodent model was based on 58.5 ml of blood per kg of bodyweight [51]. A mouse weighing 31 g would have a total blood volume of approximately 1.80 mL. Therefore, subsequent calculations were scaled to account for the 5000 mL average total blood volume of humans.
The net bacterial growth rates, G x , in each compartment of non-immunocompromised subjects, along with immunocompromised neutropenic subjects, can be found in Tables 10 and  11. These values were derived from published literature in which the total bacterial burden was measured in each compartment of a rodent at specified time points. The bacterial burden rates of change, or net bacterial growth rates, were calculated for each compartment using the cited published literature. The calculated rates of change were then implemented as the net bacterial growth rates in the model. The rates of change were estimated by linearization of the experimental data from which rate of change can be calculated, which is equivalent to the slope of the linear approximation. An example of this calculation is shown in the S5 Fig. Similar, time dependent and organ specific bacterial levels have not been reported for human subjects. Therefore, net bacterial growth rates in human compartments were extrapolated from rodent models based on relative compartment characteristics.
Net bacterial growth rates corresponding to each compartment following colistin antibiotic treatment are displayed in Tables 12 and 13 [32,52]. Experimental evidence was not available to distinguish p from c in vivo. However, it was possible to deduce single net bacterial growth rates (G x (h -1 )) by analyzing literature that reported the total bacterial burden in each compartment before and after colistin treatment.

Magnetic Separation Component Design
The bacterial separation (E) component of the model embodied a fluidic device in which continuous magnetic separation of Gram-negative bacteria from non-Newtonian particulate blood flow occurred. The magnetic separation device consisted of two steps, together comprising the total magnetic separation efficiency, f(r f ): 1) Gram-negative bacteria-targeted magnetic nanoparticles bound to the bacterial cells and 2) the magnetic separation of nanoparticle-bacteria complexes from the blood. Total magnetic separation efficiency, f(r f ), of bacteria from the blood was dependent upon magnetic nanoparticle size, blood viscosity, and magnetic forces. The modeling parameters and equations used to calculate the value of f(r f ) were based on previous studies [13,53]. Briefly, the value of f(r f ) was calculated for various nanoparticle sizes by determining the binding kinetics between the magnetic nanoparticles and bacteria. Assuming that all bacteria bound to magnetic nanoparticles were removed by the magnetic fluidic device, the binding efficiency, x(rf), was represented by where b was the concentration of magnetic nanoparticles, k d the diffusion collision rate constant, k shear the shear collision rate constant, and c e (3.7 Ã 10 −4 <dimensionless>) an empirical constant representing the binding efficiency of bacteria to the magnetic nanoparticles in whole blood [13]. Then, the effects of the magnetophoretic separation of the magnetic nanoparticle-bound bacteria from the blood under continuous flow were determined. The magnetic forces acting on the magnetic nanoparticle-bound bacteria directly impacted the magnetic separation efficiency. Therefore, the magnetic separation efficiency was estimated by calculating the magnetic force induced by our defined theoretical parameters, where r f was the radius of a magnetic nanoparticle, μ o was the magnetic permeability of vacuum (4π Ã 10 −7 < T m A -1 >), B 2 was the magnetic field intensity (B 2 = 40 < T 2 m -1 >), N was the number of magnetic nanoparticles bound to the bacteria cell, and Δχ (<dimensionless>) was the volumetric susceptibility of the magnetic nanoparticles [53]. The following conditions were used: where r c was the effective spherical radius of a pathogen (0.5 μm, A. baumannii [54]; 0.65 μm, K. pneumoniae [55]) and it was assumed, based on previous literature, that half of the cell surface was covered by magnetic particles (ρ = 0.5 <dimensionless>) [13,53]. Assuming a quasi-static motion, where r n was the effective hydraulic radius of the bacteria-nanoparticle complex and η was the blood viscosity (4 Ã 10 −3 <N s m -2 >) [13]. The variable r n can be represented as For small particles, F mag = F drag and, therefore, the magnetophoretic velocity (v mag ) of the magnetic nanoparticle-bacteria complex can be defined as Blood flowing through the magnetic extraction fluidic component at a flow rate of Q (L h -1 ) resulted in an average linear velocity (v 1 ) of bacteria labeled with magnetic nanoparticles of where a was the cross-sectional area of the device channel (0.002 m x 0.0006 m; width x height) [13]. The characteristic residence time, t res , of the nanoparticle-bound bacteria in the magnetic fluidic device channel was approximated by where L L was the hypothetical length of the channel (L L = 0.027 m) [13]. The magnetophoretic transverse time (t mag ), the time it takes the nanoparticle-bound bacteria to be extracted from the blood by the magnets of the fluidic device, was described by where L h was the height of the magnetic fluidic device channel (0.0006 m) [13]. The magnetic separation efficiency, m(r f ), was then estimated by comparing the characteristic residence time (t res ) to the time required for the nanoparticle-bound bacteria to be extracted from the blood by the magnets of the fluidic device (t mag ) [13].
Finally, the total magnetic separation efficiency, f(r f ), for the two-step process was calculated using Maximum magnetic separation of bacteria from blood was predicted to occur when using magnetic nanoparticles with 25 nm radii. As the magnetic nanoparticle radius increased, the extraction efficiency decreased (Fig 9). Experimental and theoretical results from Kang et al. were used to validate this trend [13].

Model Analysis
All analysis was written in Matlab R2015b (The Mathworks, Inc, Natick, Mass). The model equations were solved using the appropriate ODE solver in Matlab. The parameters values were verified by fitting the solution of the differential equations to experimental data found in literature.

S1 Fig. Bacterial burden increased over time in neutropenic rodents until reaching a lethal
K. pneumoniae concentration. The median numbers of bacteria in each compartment observed experimentally in previous literature were used as the initial conditions for these simulations (25,35), and trajectories were generated using the parameter estimates shown in Table 11. (TIFF)


s2fieldsofstudy: Biology Medicine

text:
Desflurane impairs outcome of organotypic hippocampal slices in an in vitro model of traumatic brain injury

Decreased mortality and disability after traumatic brain injury is a significant medical challenge. Desflurane, a widely used volatile anesthetic has proven to be neuroprotective in a variety of in vitro and in vivo models of ischemic brain injury. The aim of this study was to investigate whether desflurane exhibits neuroprotective properties in an in vitro model of traumatic brain injury. Organotypic hippocampal slice cultures were prepared from brains of 5–7-day-old C57/BL6 mouse pups. After 14 days of culture, the slices were subjected to a focal mechanical trauma and thereafter incubated with three different concentrations of desflurane (2, 4 and 6%) for 2, 24 and 72 hours. Cell injury was assessed with propodium iodide uptake. Our results showed that after 2 hours of desflurane exposure, no significant change in trauma intensity was observed. However, 2% and 4% desflurane could reduce the trauma intensity significantly in the no trauma group than in the no desflurane and trauma group. Incubation with 4% desflurane for 24 hours doubled the trauma intensity in comparison to the trauma control group and the trauma intensity further increased after 72 hours of incubation. Furthermore, a dose-dependent increase of trauma intensity after 24 hours exposure was observed. Our results suggest that a general neuroprotective attribute of desflurane in an in vitro model of traumatic brain injury was not observed.

RESEARCH ARTICLE
cells as a result of a mechanical damage through linear translational and rotational Forces as well as a defect caused by the collision to the skull (primary injury) (Feng et al., 2015). Second, the cellular response to an injury (secondary injury) including cell membrane depolarization with a resulting release of neurotransmitters like glutamate and other excitatory amino acids. This depolarization leads to an influx and an additional release of intracellular Ca 2+ . Furthermore mechanoporation of the cell membrane and the axolemma leads to an increase of intracellular Ca 2+ concentration with a following activation of caspases and calpains as well as a generation of free radicals followed by activation of the apoptotic and necrotic pathway (McAllister, 2011).
Desflurane (1,2,2,2-tetrafluoroethyl difluoromethyl ether) is a fluorinated methyl ethyl ether, a commonly used volatile anesthetic. Desflurane acts at the level of the central nervous system interacting with the release of neurotransmitters, influences the reuptake of neurotransmitters, changes the binding of neurotransmitters on the post-synaptic receptor sites, and influences the ionic conductance of the postsynaptic membrane. Further gammaamonibutyric acid (GABA) receptors are activated, and calcium channels which prevent the release of glutamate are inhibited (Wenker, 1999). Desflurane has proven to be neuroprotective in a variety of in vitro and in vivo models of ischemic brain injury (Haelewyn et al., 2003;Erdem et al., 2005;Wang et al., 2007;Matchett et al., 2009;McAuliff et al., 2009;Yu et al., 2010). Mainly volatile anesthetics induce neuroapoptosis in several major brain regions during the synaptogenesis (Jevtovic-Todorovic et al., 2003;Bambrink et al., 2010;Briner et al., 2010;Culley et al., 2011;Cao et al., 2014). In a recently performed systematic review, Liu et al. (2014) confirmed a preponderant toxic but also an isolated protective effect of inhaled anesthetics on the developing brain of rodents, piglets and primates. In addition a neurotoxic effect of isoflurane and sevoflurane on hippocampal slice cultures is described (Wise-Faberowski et al., 2005;Brosnan and Bickler, 2013;Wise-Faberowski and Osorio-Lujan, 2013). Here we aimed to reveal the possible neuroprotective or deleterious effects of desflurane in an in vitro model of traumatic brain injury and to further assess how desflurane acts on organotypic hippocampal slices without TBI.

Ethics statement
All experiments in this study were approved by the animal protection representative at the Institute of Animal Research at RWTH Aachen University Hospital, Germany according to the German animal protection law §4 Section 3.

TBI
After 14 days of incubation the growth medium was changed to the experimental medium supplemented with 4.5 µM propodium iodide (PI). The only difference between the growth medium and the experimental medium is the exchange of horse serum for the equal portion of Eagle's minimal essential medium. Afterwards the slices were incubated for 30 minutes at a humidified atmosphere of 37°C, 95% air and 5% CO 2 again, before taking a baseline fluorescence image and dividing the slices into groups (TBI positive and TBI negative group). The CA1 region of the hippocampus was identified microscopically and subsequently traumatized with a previously described apparatus (Adamchik et al., 2000;Loetscher et al., 2009;Rossaint et al., 2009). This apparatus allows hitting the CA1 region with a metal stylus under stereomicropscopic control, by deactivating the power of an electromagnet, which retains this stylus at a height of 7 mm. This height and the weight of the stylus correlates to a force of 5.26 µJ. Thereafter, the experimental medium was exchanged for a new experimental medium supplemented with 4.5 µM propodium iodide and the slices were incubated in a pressure chamber for 2, 24 and 72 hours in different atmospheres. The pressure chambers were filled with a vapor of desflurane (D-Vapor, Dräger Medical GmbH, Lübeck, Germany) via an anesthetic machine (Sulla 808V, Dräger Medical GmbH, Lübeck, Germany) at an airflow of 8 L/min for 8 minutes. The carrier gas for desflurane (5% CO 2 , 21%O 2 , 74% N 2 containing 2 vol%, 4 vol% or 6 vol% desflurane) was mixed by Linde Gas Therapeutics, Unterschleissheim, Germany. A control group (TBI positive) without desflurane was also incubated. The chamber atmosphere was analyzed with an anesthesia monitor Krings M, et al. / Med Gas Res www.medgasres.com flurane in TBI organic hippocampal slice cultures resulted in no significant reduction in trauma intensity at concentrations of 6% desflurane (P = 0.384), 4% desflurane (P = 0.273) and 2% desflurane (P = 0.879) (Figure 1). However in the no trauma group a concentration of 2% desflurane (P = 0.033) and 4% desflurane (P = 0.004) could reduce the trauma intensity significantly in comparison to the no desflurane and trauma group (Figure 1).

Propodium iodide staining
For staining the slices were stained with propodium iodide (a fluorescence coloring agent which connects to DNA if the cell membrane is disintegrated). Inside the cell propodium iodide becomes highly fluorescent with a main emission spectrum in the red region (Macklis and Madison, 1990

Assessment of cell injury
The fluorescence images were digitalized at 8 bits allowing the distinction between a spectrum of 256 (0-255) gray scale levels. The severity of cells is manifested by a high propodium iodide uptake and a high fluorescence value. Aided by analyzing software (Image J, NIH, USA, http://rsb.info.nih.gov), a histogram of the red pixel value of each slice was created to show the absolute number of pixel with the same grey scale value. A threshold of 100 was set to eliminate the background fluorescence and the pixel value beyond was summarized (Loetscher et al., 2009;Rossaint et al., 2009;Schoeler et al., 2012). Slices with a significant cell death based on the preparation were excluded.

Statistical analysis
Each amount of the pixel values was concluded for each experimental group. The mean ± SEM (standard error of the mean) of the pixel values were calculated using SPSS20 (IBM SPSS Statistics, IBM Cooperation, Somers, NY, USA). The TBI positive group which was incubated without desflurane was set as a reference value. Statistical analyses were performed using an analysis of variance (one-way ANOVA). P-values < 0.05 were considered significant. Note: After 2-hour incubation, cell death was stained as shown on fluorescence images. Trauma intensity of the different groups is shown in relation to trauma intensity of mechanical traumatized slices (the trauma no desflurane group was set as 1 or 100%) after an incubation with 5% CO 2 , 21% O 2 ,74% N 2 for 2 hours. Significant differences to the trauma no desflurane group are marked with *. There was no significant reduction in trauma intensity at the groups with mechanical trauma (P > 0.05). In the group without TBI, a concentration of 2% desflurane (*P = 0.033) and 4% desflurane (*P =0.004) reduced the trauma intensity significantly in comparison to the group without desflurane and trauma. For each group an average of 80 with a minimum of 56 slices were analyzed.

results
After 24 hours of incubation with desflurane, an increase of trauma intensity in all groups was observed (Figure 2). In the group without TBI, the increase of trauma intensity rose from 2% deflurane (P = 0.000) to 6% desflurane (P = 0.000). In the trauma group, no significant difference was seen between 2% desflurane and the no desflurane and trauma group (P = 0.837); further an increase of trauma intensity was observed at concentrations of 4% desflurane (P = 0.006) and 6% desflurane (P = 0.000). There was no significant difference between the trauma group and group without trauma at concentration of 6% desflurane (P = 0.385).
After 72 hours of incubation, the trauma intensity in the trauma and no trauma groups increased continuously especially in the trauma groups to more than the double in comparison to the 24 hours of incubation group (Figure 3).

dIscussIon
We observed no significant change in trauma intensity in TBI organic hippocampal slice cultures after 2 hours of desflurane exposure. After 24 hours the trauma intensity increased up to the fourfold which further increased after 72 hours. Therefore, a general neuroprotective attribute of desflurane in an in vitro model of traumatic brain injury was not observed. After 2 hours of incubation with 2% and 4% desflurane in slices not mechanically damaged a significant reduction of trauma intensity could be observed. It suggests that the absolute value of the trauma intensity is low in these groups, so that the result has to be analyzed critically.
Desflurane has also shown general neuroprotective properties in a variety of in vitro and in vivo models of ischemic brain injury (Haelewyn et al., 2003;Erdem et al., 2005;Wang et al., 2007;Matchett et al., 2009;McAuliffe et al., 2009;Yu et al., 2010). Desflurane preconditioning remarkably reduces the infarct volumes in an in vivo model of middle cerebral artery occlusion of adult rats (Haelewyn et al., 2003). Delayed preconditioning of 3 hours with desflurane, isoflurane and sevoflurane provides neuroprotection in a model of neonatal hypoxia-ischemia in 9-day-old mice. This neuroprotection was assessed by a battery of behavioral tests after a carotid ligation with a resulting hypoxia of 60 minutes. An improved performance in striatal dependent functions was noted in the preconditioned test group. However, performance on the spatial memory-dependent phases which is attributed to the hippocampus function was not improved. Yet a histological evaluation of cell loss of the striatum, the dorsal hippocampus and the ventral hippocampus did not show any significant effect of preconditioning with volatile anesthetics (McAuliffe et al., 2009).
Likewise neuroprotective properties of volatile anesthetics could be observed in an in vitro model with cerebellar slice cultures of 2-3-month-old rats, which were preconditioned for 15 minutes with desflurane, isoflurane, sevoflurane or halothane. 15 minutes after preconditioning this slices where subjected to oxygen glucose deprivation for 10 minutes. A protective character of this preconditioning could be attested by staining the cell damage using spectrophotometric measurement of formazan produced by 2,3,5-triphenyltetrazolium 5 Note: After 24-hour incubation, cell death was stained as shown fluorescence images. Trauma intensity of different groups is shown in relation to trauma intensity of traumatized slices (the trauma no desflurane group after 2 hours of incubation was set as 1) after an incubation with 5% CO 2 , 21% O 2 ,74% N 2 for 2 hours. Significant differences compared with the positive control group after two hours of incubation (Trauma without desflurane) are marked with *. Important significant differences between the groups after 24 hour incubation are also marked with * on the top of the figure. After 24 hours of incubation the trauma intensity tripled as compared to the trauma intensity after 2 hours of incubation.
In the group without TBI the increase of trauma intensity rises also from 2% desflurane (*P = 0.000) to 6% desflurane (*P = 0.000). For the trauma group a significant increase of trauma intensity was observed at 4% desflurane (*P = 0.006) and 6% desflurane (*P = 0.000). For each group an average of 73 with a minimum of 62 slices were analyzed. Note: In summary on this figure slices were exposed to a concentration of 4% desflurane and to 5% CO 2 , 21% O 2 , 74% N 2 (trauma no desflurane) for 2-72 hours. Trauma intensity of the different groups is shown in relation to trauma intensity of traumatized slices after an incubation without desflurane for 2 hours. Significant differences compared with the trauma no desflurane after two hours of incubation are marked with *.
After 2 hours of incubation, there was no significant difference between the trauma no desflurane group and the trauma group with 4% desflurane (P = 0.273). After 24 hours, an increase to the double trauma intensity at the trauma group without desflurane was observed. Incubating with 4% desflurane after trauma led to an increase of trauma intensity to the four-fold. Finally after 72 hours the trauma intensity represents without desflurane and with 4% desflurane in the trauma group. For each group an average of 94 with a minimum of 73 slices were analyzed.
hours after the oxygen glucose deprivation (Wang et al., 2007). Another in vitro model could demonstrate a neuroprotective effect of sevoflurane in a neonatal asphyxia model. Neuronal glia cells were cultured from cerebral neocortices of 1-2-day-old pups and exposed to oxygenglucose deprivation for 75 minutes. 16 hours thereafter the lactat dehydrogenase (LDH) was analyzed using standardized colorimetric enzyme kit. Preconditioning with sevoflurane at concentrations of 2.7% and 3.3% reduced the LDH release, whereas at concentrations under 2% no effect was seen. A benefit of preconditioning with volatile anesthetics in pure neuronal cultures of 16 day old embryonic was only observed in a combination of sevolfurane and xenon (Luo et al., 2008). In our model we could notice that desflurane exposure lasting more than two hours in traumatic brain injury model is neurotoxic in the developing brain during the phase of synapthogenesis. Current clinical and preclinical evidence suspects that common anesthetic agents cause an impairment of the developing brain (Jevtovic-Todorovic et al., 2003Teng et al., 2005;Satomoto et al., 2009;Briner et al., 2010;Head et al., 2011;Istaphanous et al., 2011;Kodama et al., 2011;Stratmann et al., 2011;Zhang et al., 2012;Sanders et al., 2013). Wise-Faberowski et al. (2005) could confirm a neuronal degeneration of organic hippocampal slice cultures induced by isoflurane which is dependent on the age of the slices and the duration of incubation. This is similar to the effect of desflurane in our research.
The inhibition of N-methyl-D-aspartate (NMDA) receptors and the activation of the γ-aminobutyric-acid (GABA) receptors appear to be responsible for this neurotoxicity (Istaphanous et al., 2011). Moreover halogenated ethers impart their effects on both receptors with a variation in their effects on the NMDA-receptors (Kodama et al., 2011). A combination of an impact on both receptors might induce more neurotoxicity than an activation of only a single one (Fredriksson et al., 2007).
The exact mechanism by which common anesthetics mediate an injuryis not yet clear. One hypothesis is that the anesthetics can activate the intrinsic and extrinsic apoptotic pathway by suppressing neurotrophic synaptic signaling (Sanders et al., 2013). This hypothesis includes the activation of the intrinsic pathway by reducing the release of tissue plasminogen activator (tPA) into the synaptic cleft due to suppressing neuronal activity of the developing neurons by volatile anesthetics. Tissue plasminogen activator converts plasminogen to plasmin which is released from presynaptic vesicles at the time of the depolarization. On his part plasmin converts the neurothrophic factor (BDNF) from its precursor protein proBDNF to the mature BDNF (mBDNF). Mature BDNF enhances neuronal surviving whereas proBDNF induces apoptosis by stimulating the p75 neurotropin receptor (p75NTR). This stimulation of the p75NTR leads to an activation of the Ras homolog gene family, resulting in the depolymerization of the actin cytoskeleton (Teng et al., 2005;Head et al., 2011;Stratmann, 2011). Concurrently the extrinsic pathway is typically activated by external stimuli such as cytokine tumor necrosis factor (TNF-α) which is triggered by pro-inflammatory effects of sedative agents in the young (Sanders et al., 2013). Furthermore significant changes in the dendritic arbor during the synaptogenesis could be observed in rats after their exposure to volatile anesthetics. These drugs increased dendritic spine density on dendritic shafts during the peak of the synaptogenetic period (Briner et al., 2010). This impact and the change in the dendritic arbor neonatale exposure with common anesthetics may cause learning deficits, deficits in fear conditioning and abnormal social behaviors akin to autism in adulthood (Satomoto et al., 2009). The hippocampus plays an important role in learning and memory processes due to its property of long-term potentiation. It may be particularly harmed by common anesthetics during the synaptogenesis (Stratmann et al., 2011).
The clinical impact of our research is limited as anesthesia duration over 2 hours in the very young is exceptional. Furthermore, the assessment of the cell damage in context to its effect on the living organism is not reproduced in our model. In addition, it is unclear, if the applied cell destruction in the CA1 region of the hippocampus has any effect on the cognition and on the behavior of creatures. We assessed the effect of desflurane in newborn hippocampal slices. The effect in full grown neurons therefore is still to be evaluated. Equally, possible impairment of other brain regions through desflurane is not addressed here.
Adding desflurane to the fixed mixed carrier gas leads to a proportionated decrease of the oxygen concentration. Using 6 % desflurane the oxygen concentration decreases approximately to 19 percent. It remains vague if this minimal reduction of the oxygen concentration leads to an impairment of the organotypic hippocampal slices.
In conclusion, we could not observe a general protective effect of desflurane in an organotypic model of traumatic brain injury. On the contrary, incubation with desflurane for 24 and 72 hours led to a dose-and time-dependent increase in trauma. The exact pathway of how desflurane affects the organotypic slice cultures remains unclear. A potential neuroprotection could be observed in the group without trauma after 2 hours of incubation with 2% and 4% desflurane. This possible neuroprotection has to be evaluated critically due to the low absolute value of the trauma intensity.


extfieldsofstudy: Biology Medicine

text:
Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

INTRODUCTION
isceral leishmaniasis (VL), or Kala-Azar, is a zoonotic disease transmitted by female sand flies and caused by leishmania species. The parasites are transmitted by the bite of female phlebotomine sand flies to the vertebrate host, where the infecting promastigotes differentiate into amastigotes; this is fatal if left untreated. Leishmania species causing VL have been shown to be capable of infecting humans as well as domestic and wild animals in the Old and New Worlds [1,2] . VL is endemic in northwestern and southern parts of Iran with about 100-300 new cases reported annually. Domestic dogs (Canis familiaris) are considered to be the main reservoir host [3][4][5][6] , some of which are asymptomatic, while others represent clinical signs ranging from mild popular skin diseases to sever forms characterized by cachexia, alopecia, keratoconjunctivitis, anorexia, weight loss, and death [7][8][9] . A close contact between dogs and human populations increases the risk of parasite transmission from dogs to sand flies and then to humans [10] . Human VL is the most severe form of the disease characterized by prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anemia, pancytopenia, and hyper gammaglobulinemia. Human VL is nearly always fatal [11] , unless treated; therefore, early diagnosis of human VL is essential for prevention of mortality and morbidity. A variety of methods have been currently used for the diagnosis of VL, based on aspirates or biopsy specimens of tissues (such as spleen, liver, and bone marrow), in which bone marrow samples display less specificity while spleen samples exhibit more specificity [9,11] . The specimens are subjected to microscopic examination and cultured on Novy-McNeal-Nicolle and RPMI-1640 media supplemented with 20% fetal calf serum, 2 mM L-glutamine, 55 μg/ml penicillin and 125 μg/ml streptomycin [12] . The growth of promastigotes typically takes between 2-14 days.
Due to the high risk of bleeding in splenic aspiration, serological assays are used for the detection of antibodies in serum samples derived from humans and dogs. Indirect fluorescent antibody (IFA), indirect hemagglutination assay, complement fixation, direct agglutination test (DAT) and enzyme-linked immunosorbent assay (ELISA) are some of these assays with different specificities and sensitivities [13][14][15] . In these tests, antigens are derived from either whole parasite or total parasite lysate. However, development of a recombinant Leishmania antigen would be a valuable platform for serological diagnosis because the production of such an antigen would be parasite growth-independent and more standardized [16] . This review seeks to survey the application of different recombinant proteins for the serodiagnosis of VL in both humans and dogs.

Recombinant proteins for ELISA
ELISA is a serological test used to diagnose the majority of infectious diseases including leishmaniasis. Although the technique is sensitive, its specificity depends on the applied antigen. Several antigens have been already used for immunodiagnostic methods but most commonly is crude soluble antigen (CSA) [2,5,11,13] . To prepare CSA, promastigotes of L. infantum are disrupted by ultrasonic treatment, and the supernatant will be used as a soluble antigen to coat ELISA plates [11] . The CSA ELISA shows a considerable overlapping in antibody titers between the controls and patients with active VL. On the other hand, the application of CSA as a diagnostic antigen can suffer from cross-reactivity with other diseases, such as leprosy, malaria, and tuberculosis, where these diseases are endemic [17,18] . To this end, emphasis has been focused on leishmania antigenic components so that recombinant antigens have been evaluated to be substituted with native parasite antigens, including rkE16 (recombinant kE16), rk26, rK39, and rA2 for serological tests [15,19,20] .
rK39, an antigen for VL diagnosis, exhibits a repetitive immune-dominant epitope in Kinesin-related proteins and is highly conserved among VL species [21] . Several studies carried out in VL-endemic areas have demonstrated that the rK39 ELISA is a sensitive and a specific method for the serodiagnosis and prognosis of human VL [21][22][23][24][25][26] . Additionally, preliminary studies have shown that the rK39 ELISA test is most amenable for the serodiagnosis of canine leishmaniasis [27] . In a study performed by Taran et al. [28] , rK39 dipstick test was used to evaluate the performance of K39 sub recombinant antigen from L. infantum LON49, which its sensitivity and specificity were found to be 90.7% and 95.6%, respectively. Results from their study suggested that rK26 is a specific antigen for the detection of antibodies in sera from patients with VL infection [29] . In addition, Farajnia et al. [30] evaluated the performance of rK26 by ELISA and demonstrated that the sensitivity of this antigen among VL-confirmed patients was 96.8%. Pattabhi et al. [31] produced rK28 and used it in ELISA as an antigen. They also evaluated VL-confirmed sera isolated from Sudanese patients and observed the sensitivity and specificity of 96.8% and 96.2%, respectively. KE16 from L. donovani is another kinesin gene derived from leishmania species that has been successfully cloned and expressed. The rKE16 was used [1,9] in ELISA to evaluate the serodiagnostic capacity of Indian Kala-Azar [32,33] . Results from those studies showed that the expressed Ld-rKE16 antigen can serve as a highly specific and sensitive tool for VL diagnosis [32,33] .
KMP11, rH2A and the Q protein are the antigens expressed in L. donovani complex (L. donovani, L. infantum syn. and L. chagasi). The recombinant antigen "Q" protein is a chimeric protein formed by the genetic fusion of five antigenic determinants from four Leishmania proteins. The fragments forming Q antigen are highly immunogenic during L. infantum infection. In a study, when KMP11, rH2A, and the Q protein were used as antigens in ELISA for the diagnosis VL patients, the sensitivity of ELISA was demonstrated to be high [34] . The A2 proteins of L. donovani are a stagespecific protein present in the amastigote stage, appearing to facilitate visceralization of parasites in the mammalian host. In L. donovani, these proteins range in size from 42 to 100 kDa and have highly conserved repetitive elements located on chromosome 22 in tandem arrays [35][36][37][38] . In a previous study, we demonstrated that A2 proteins are present in amastigote form of L. infantum isolated from dogs in Meshkin-Shahr, Ardebil Province, North-Western Iran [38] . It is well-established that the A2 protein is overexpressed in the amastigote stage in L. donovani as well as L. mexicana species complex, including L. amazonensis, but not in L. tropica or L. braziliensis species complexes. At the same time, Anti-A2 antibodies were detected in human and dog sera suffering from active VL [36,39] . In a study carried out by Porrozzi et al. [17] , ELISA was used for serodiagnosis of symptomatic and asymptomatic VL in dogs based on crude and recombinant proteins (rA2, rK26, and rK39).
In addition, it was found that rA2 is more sensitive to asymptomatic dogs, when compared to the soluble antigens, rK39, and rK26.
In a study performed by Souza et al. [40] , the recombinant antigen, ATP diphosphohydrolase (rLic-NTPDase-2), was produced and validated for use in the immunodiagnosis of VL. In brief, a microplate was coated with the purified rLic-NTPDase-2 antigen for the ELISA assay. Next, serum samples from naturally infected dogs in Brazil were tested for VL detection.
Although showing high sensitivity and specificity (100%), this test was not able to distinguish VL. Therefore, it seems that rLic-NTPDase-2 may not be a useful marker for VL prognosis.
ELISA-rHsp83 was used as an antigen to detect antileishmania antibodies, which showed significantly high sensitivity [16] . In a study performed by Boarino et al. [41] , a combination of K9, K26, and K39 sub recombinant chimeric antigens was coated in a single well and used to diagnose human and canine VL by an indirect ELISA assay. A total of 384 human and 609 canine serum samples were tested, in which the specificity was found to be suitable for both human and canine samples (99%) but the sensitivity of chimeric ELISA was higher in canine VL (96%) than in human VL (82%).
To determine antibodies in the control and VL patients in Sudan, an immunodominant kinesin protein (rKLo8) from an autochthonous L. donovani strain was expressed, used as an ELISA coating antigen, and compared with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16). Among 106 parasitologically VL-confirmed patient sera, the sensitivity and specificity of rKLo8coated ELISA were 98.1% and 96.1%, respectively, as compared to 96.2% sensitivity of rk39 [42] . In a study done by Islam et al. [43] , rKRP42, the recombinant kinesin-related protein of L. donovani, with a molecular weight of 42 kDa, was produced and evaluated in urine-based ELISA test for the diagnosis of VL. The rKRP42 urine ELISA showed a high sensitivity of 94% and specificity of 99.6% among VL samples.
More recently, two novel recombinant antigens, the multiepitope proteins PQ10 and PQ20, were used in canine VL ELISA, which were able to identify asymptomatic dogs (80%). PQ10 consists of a 10codon sequence present in antigenic peptides, while PQ20 includes a 20-codon sequence exist in T-and Bcell epitopes, respectively. In addition, a flexible linker was used as a spacer between epitope sequences. These antigens was used for VL ELISA and demonstrated to be sensitive enough for the identification of asymptomatic dogs (80%) [44] .

Recombinant proteins for the rapid immunechromatographic dipstick test
The rapid immunochromatographic dipstick test is a qualitative test able to detect anti-Leishmania circulating antibodies by the leishmanial recombinant antigen, such as rK39 and is adapted for use under field conditions. The rK39 antigen is the product of a gene cloned from L. chagasi containing a 39-amino-acid repeat. Taken together, K39 is a part of a 230-kDa protein that is encoded by a kinesin-like gene of L. chagasi and conserved among viscero-tropic Leishmania species [19,43] . The rapid rK39 immunochromatic dipstick test is both sensitive (67-100%) and specific (70-100%) [42,[45][46][47][48][49] . However, the crossreactivity with malaria, enteric fever, disseminated tuberculosis and Toxoplasma gondii may occur. In a study carried out in Iran, the sensitivity and specificity of the rK39 strip test were compared with the immunofluorescent antibody test in 47 children suspected to VL. The sensitivity and specificity of the strip test were 82.4% and 100%, while those of the immunofluorescent antibody test were 100% and 92.7%, respectively [50] . Mohebali et al. [51] compared the rK39 dipstick test with DAT to detect L. infantum infection in dogs in endemic areas in Iran. The tests were carried out on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil Province, North-Western Iran. A sensitivity of 70.9% and specificity of 84.9% were found for the rK39 dipstick test.
In a literature search for VL detection, 31 studies have used dipstick form of rK39 in dogs. Their results revealed that the overall sensitivity was 86.7% in symptomatic dogs. The reasons for such variability are not clear; in addition, the number of studies for such analysis is too small for a detailed comparison of different tests and geographical areas [47] . In parallel, a clinical cohort study in Sudan was designed to evaluate the rK28-based RDT for diagnosis of L. donovani VL. The serum analysis of VL-suspected individuals possesses a reasonable specificity and sensitivity of 97.6% and of 94.5% [53] .
The rkE16 dipstick test is a one-step rapid immunochromatographic test for diagnosis of Kala-Azar. The test is based on a 39-amino-acid protein, the rKE16 antigen, derived from the C-terminus of the kinesin protein from an Indian isolate of L. donovani [32] . In our previous study, the average of positive cases among asymptomatic dogs was 32.4% with rKE16 [9] .

Recombinant proteins for the latex agglutination test
In the majority of immunoagglutination assays, latex is the main particle for antigen absorption. The attachment of molecules to latex particles can be achieved through either physical adsorption or covalent coupling. For visual slide agglutination, a particle diameter range of 0.2-0.9 mm is needed [54] . The only recombinant antigen used for the latex test is rk39, which is generally used in latex agglutination at a concentration of 1 mg/ml in glycine-buffered saline. The sensitivity of the latex test among parasitologically proven index cases was only 80%, which was due to a lower power of antibody detection by this test [55] .

Recombinant proteins for other serological assays
Several serological tests are used for the diagnosis of VL, including IFA, DAT, ELISA, and dot-ELISA. The mentioned tests are mostly based on purified, watersoluble antigen fractions of promastigote or amastigote stages or on recombinant antigens. It is notable that recombinant proteins are only used in ELISA, latex agglutination test, and rapid immunochromatographic dipstick test, but not in other assays. On the basis of publications, there are no report indicating that recombinant antigens can be used for IFA and DAT assays.

Conclusion
The technological advances in recombinant antigens as reagents for the serological diagnosis of VL have resulted in high sensitivity and specificity of the serological tests. Several recombinant proteins have been shown to be useful for the diagnosis of leishmania infection in dogs. It should be noted that dogs are considered as a major reservoir of L. infantum and, therefore, promote the spread of human leishmaniasis. Several proteins able to generate substantial titers of antibodies and are detectable in canine VL, including KMP11, LiP, rk39, rk26, rA2, rk9, rKE16, and histones such as H 2 A. Early diagnosis and control of canine VL can reduce the transmission of infection from asymptomatic dogs to sand flies and humans. Different types of antigens with variable sensitivity and specificity have been used in ELISA and in rapid immunochromatographic dipstick tests, depending on both the kind of recombinant antigens and the areas under study.
ELISA is a sensitive and specific test for the detection of antibodies in humans and dogs infected with VL. Anti-A2 antibodies are easily detected by a rA2 protein containing a tag of six histidine residues (A2-HIS), in 77% of the patient sera with symptomatic VL and in 87% of the sera from dogs, which were positive for Leishmania immunofluorescent antibody test (IFAT) or parasitological evaluation. Anti-A2 antibodies have been also detected in symptomatic and asymptomatic dogs [17,20,41] .
Data from such studies have indicated that using a combination of recombinant proteins in a single-well test would be better for diagnosis of human and canine VL by an indirect ELISA. The rapid immunochromatographic dipstick test, for example rK39 strip test, is a simple and cost-effective assay that uses chromategraphic strips impregnated with the rK39 antigen and can be easily used in the field. Several studies have shown that the rK39 strip test is a reliable test for VL diagnosis with no false positives. In comparison to DAT and ELISA, dipstick rK39 is a simple, more practical, sufficiently sensitive and highly specific method for the diagnosis of active VL in humans as well as in dogs. Furthermore, the test can be performed with a few drops of blood, serum, or plasma in field conditions [2] . There are several serological tests used for the diagnosis of VL in humans and dogs. Moreover, reasonable levels of sensitivity and specificity are particularly desirable to avoid false negative and false positive reactions. This issue can lead to disease transmission and unnecessary euthanasia of healthy dogs. The problems with the conventional assays (such as IFAT, DAT, and ELISA) may be overcome by using recombinant polypeptides containing a specific epitope that is able to elicit an immune response in the majority of dogs and humans with VL. Results indicate that each antigen carries immunodominant epitopes, the combination of which may further increase the sensitivity of currently available serological tests [17] . Although several recombinant proteins with high efficacy end up with reasonable results for the diagnosis of human and canine VL, more optimization is still required for appropriate antigens to provide high-throughput performance.